key: cord- -ocp yodg authors: swaan, corien m; appels, rolf; kretzschmar, mirjam ee; van steenbergen, jim e title: timeliness of contact tracing among flight passengers for influenza a/h n date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ocp yodg background: during the initial containment phase of influenza a/h n , close contacts of cases were traced to provide antiviral prophylaxis within h after exposure and to alert them on signs of disease for early diagnosis and treatment. passengers seated on the same row, two rows in front or behind a patient infectious for influenza, during a flight of ≥ h were considered close contacts. this study evaluates the timeliness of flight-contact tracing (ct) as performed following national and international ct requests addressed to the center of infectious disease control (cib/rivm), and implemented by the municipal health services of schiphol airport. methods: elapsed days between date of flight arrival and the date passenger lists became available (contact details identified - ci) was used as proxy for timeliness of ct. in a retrospective study, dates of flight arrival, onset of illness, laboratory diagnosis, ct request and identification of contacts details through passenger lists, following ct requests to the rivm for flights landed at schiphol airport were collected and analyzed. results: requests for ct were identified. three of these were declined as over days had elapsed since flight arrival. in out of requests, contact details were obtained within days after arrival ( %). the average delay between arrival and ci was , days (range - ), mainly caused by delay in diagnosis of the index patient after arrival ( , days). in four flights ( %), contacts were not identified or only after > days. ci involving dutch airlines was faster than non-dutch airlines (p < , ). passenger locator cards did not improve timeliness of ci. in only three flights contact details were identified within days after arrival. conclusion: ct for influenza a/h n among flight passengers was not successful for timely provision of prophylaxis. ct had little additional value for alerting passengers for disease symptoms, as this information already was provided during and after the flight. public health authorities should take into account patient delays in seeking medical advise and laboratory confirmation in relation to maximum time to provide postexposure prophylaxis when deciding to install contact tracing measures. international standardization of ct guidelines is recommended. aircrafts can function as transport vehicle for patients infected with influenza, leading to introduction of a new virus strain to non-endemic areas [ , ] . although the risk is small, passengers might be infected by a contagious patient during the flight [ ] [ ] [ ] [ ] , as well as during public transport [ ] . transmission during the flight increases the possibility of further transmission in the area of destination. for these reasons, during the initial phase of the influenza a/h n pandemic, many countries initiated contact tracing among flight passengers of flights where contagious patients with laboratory confirmed influenza a/h n were notified. a risk assessment guideline for infectious diseases transmitted on aircrafts has been developed by the european centre for disease prevention and control (ecdc) [ ] , which includes influenza. literature study revealed on-board transmission in flights with a duration of less than h. the majority of infected contacts during these flights were seated on the same row, or one or two rows in front of behind the index [ ] [ ] [ ] [ ] . contacts up to and rows distance from the index were infected in one study [ ] . as these contacts also had personal contact with the index during the flight, transmission across a distance of so many rows is not proven. the guideline concludes that it is difficult to design a single contact tracing algorithm for influenza. due to the short incubation period of influenza, it is almost impossible to provide contacts with postexposure prophylaxis (pep) within the time that it is most effective, which is h after exposure [ ] . therefore, the main aim of contact tracing might be to interrupt the chain of transmission by alerting contacts for early diagnosis and treatment. although the world health organization (who) developed technical advice for case management of influenza a/h n in air transport during the pandemic [ ] , no international standardized protocol for contact tracing for this pathogen was available. in line with the ecdc guideline [ ] and the dutch guideline for 'incidental introduction of a new influenza strain' [ ] , in the netherlands close contacts of a patient with laboratory confirmed pandemic influenza were identified. in case the index had been contagious during a flight with a duration of ≥ h, passengers and cabin crew were to be informed on signs and symptoms of the disease and to seek medical care in case they would occur. in addition, close contacts, defined as passengers seated on the same row, two rows in front and two rows behind the index case, as well as the cabin crew working in this compartment, were traced by public health authorities to provide a day prophylactic course of oseltamivir as soon as possible (preferably within h after exposure). schiphol airport is the only airport in the netherlands where trans-atlantic flights arrive. its municipal health services (mhs, ggd kennemerland) and the center for infectious disease control (cib-rivm) frequently experienced that, despite all efforts, the time period elapsing from exposure to administration of the first oseltamivir dose exceeded the required h. acquiring contact details from airlines was time consuming, and contact details on passenger lists were often minimal, so that contacts were difficult to trace. in this study, we assess the time delay in contact tracing of flight passengers for influenza a/h n as performed in the netherlands during the initial phase of the pandemic. our data show that despite all efforts the effectiveness of this control measure in daily practice is minimal. from april th until june nd , contact tracing among flight passengers in the netherlands was indicated for laboratory confirmed influenza a/h n cases, who traveled on a flight for h or longer while being contagious, defined as day before, until days after disease onset. these criteria were installed by the cib, which also functions as national focal point (nfp). the procedure for contact tracing is complex, see figure . requests for contact tracing to the cib for dutch index patients originate from any dutch mhs which identifies a patient who traveled by plane while being contagious for an infectious disease which requires contact tracing. other nation's health authorities will make a request to the cib in case they diagnosed a patient which arrived at schiphol airport for transit while being infectious. requests for ct in the last group are submitted to the national focal point (nfp) or through the early warning and response system of the eu (ewrs). the cib verifies laboratory confirmation, and the indication for contact tracing regarding flight duration. the mhs of the airport where the specific flight landed coordinates contact tracing for flight passengers. in case of schiphol, mhs kennemerland approaches the involved airline company requesting the passenger list. the airline provides passenger lists with at least passenger names, seat numbers and booking or contact details. mhs kennemerland then completes contact details through booking offices or using other search methods. close contacts living in the netherlands are traced by the respective dutch mhs's. for tracing foreign contacts, the cib sends a notification with contact details to the nfp of the country of final destination, or through the ewrs system for eu countries. during the pandemic, ct requests were turned down if more than days had elapsed after flight arrival, as contact tracing was not considered to have additional value. during the study period, passenger locator cards (plc) only were used on direct flights from mexico during the initial phase of the pandemic. these flights were all run by dutch airlines. for each contact investigation performed in the period april th until june nd , the following data were collected: flight arrival date, first day of illness of index patient, date of laboratory diagnosis, date of contact tracing request and the date passenger lists were obtained and contact details were completed ('contacts details identified'). from these data, time intervals (in days) between flight arrival and date of diagnosis (interval i), between diagnosis and request dates (interval ii) and between request and contact details identified dates (interval iii) were calculated, see figure . date of actual contact tracing and oseltamivir administration was not available in this study, but is inherently always hours if not days later. as the airline company traces contacts amongst crewmembers, these are not included in this study. data were analyzed using spss software (version , usa). the influence of availability of plc's on timeliness and the origin of the airline company (dutch or non-dutch) were statistically analyzed. in the period april th until june nd , indications for ct were identified. three international requests concerning ct for influenza patients diagnosed outside the netherlands were declined as already more than days had elapsed since flight arrival. in out of the remaining contact investigations, passenger lists with contact details were obtained within days after arrival ( %), see table . in total contact details of close contacts were identified, of which contacts lived in the netherlands, and contacts abroad. the average number of close contacts per flight was (range: - ). in four contact investigations ( %), contact details were not obtained, or provided later than days after flight arrival and ct was stopped. these ct were all related to non-dutch airlines, and total delay *:. in the beginning of the pandemic one request for contact tracing was accepted after days **: these late ct requests were accepted as the passenger lists of the concerned flights already were available from earlier contact investigations ***: date of diagnosis not known ª: passenger locator card was stated days for further data processing. of the requests, the total delay between request and contact detail identification was longer for non-dutch airlines (mean , sd , ) compared with dutch airlines (mean . days, sd , )( -sided mann-whitney test, p = , ). for the completed contact investigations, interval i was the largest interval in the contact tracing procedure (mean , days, range - , % ci , - , , n = ). the other intervals ii and iii were shorter, with a mean of , days and , days respectively, see table . figure shows the medians of the described intervals. since / index cases were already ill before, or during the day of arrival of the flight, the delay in interval i is mainly caused by delay in seeking medical advice and diagnostic procedure itself. after acceptance of the request for ct by the cib, ggd kennemerland needed on average , days (range - , % ci , - , days) to collect the passenger list from the airlines and complete contact details (interval iii). the total delay between flight arrival and identification of contact details was on average , days (range - days, % confidence interval , - , days), see table . in only out of contact investigations ( %), contacts were identified within days after arrival. in out of these contact investigations, plc's were available. interval iii of the ct with plc's available was shorter ( , days, sd , ) than for ct's without plc ( , days, sd , ), this was not significant however (p: , ). overall delay in ct with plc's also was shorter (mean , , sd , ), but not significant, when compared to ct without plc's (mean , , sd , ) (p: , ). in this study we evaluated the timeliness of contact tracing (ct) of flight contacts in daily practice. we conclude that the prevailing policy to provide close contacts antiviral pep during the early phase of the influenza pandemic is very difficult to implement effectively and therefore has little effect to control disease spread. active case finding through contact tracing of exposed persons is an important procedure during the containment phase of an emerging communicable disease. however, our data show that, even in a small-industrialized country with modern communication tools, tracing of flight contacts exceeds the required maximum of h after exposure. for influenza, close contacts of contagious index cases are entitled to receive antiviral pep within h after exposure to prevent them from becoming ill and further spreading of the disease. starting oseltamivir within h does not prevent disease but shortens the disease period, mitigates symptoms and might decrease further transmission. awareness among contacts to seek medical evaluation when influenza-like (ili) symptoms occur, for both proper antiviral treatment and (home-) isolation advice, reduces further spreading. as influenza has a relative short latent period, for influenza a(h n )/ varying between , - , days [ , ] , contacts ideally should be informed within day. oseltamivir postexposure prophylaxis for this pandemic strain is reported to be effective even when administrated more than h after exposure in household settings [ ] , however, delays in administration are not specified. we cannot exclude the possibility that in our study, even delayed administration of oseltamivir prophylaxis may have prevented some people from becoming ill, although we anticipate the effectiveness of the intervention overall to be less in this setting than in households. our study among contact investigations showed an average total delay of , days between flight arrival and identification of contacts by passenger list, which is too late for effective pep, and late for alerting on first symptoms of disease. only in three contact investigations ( %), contact details were obtained within h. however, after identification of passenger details, health authorities need time to actually trace the contact and administer pep. it is highly unlikely that this was achieved within the same h. we therefore conclude that contact investigation for provision of pep as conducted here was ineffective. regarding the awareness of ili symptoms, schiphol airport handed all passengers on flights arriving from mexico information leaflets on influenza a/h n with information on early symptoms and requesting them to seek medical advice in case of fever and respiratory symptoms such as coughing. posters with this information were placed in passenger halls, to inform passengers arriving indirectly from mexico via transit through other airports, or arriving from non-endemic areas with higher transmission (e.g. usa). as contact details were identified on average . days after exposure, however not contacted yet, we conclude that ct did not have additional value for timely achievement of increased awareness. it is not a new finding that contact tracing of flight passengers is a time-consuming procedure [ ] . in one study among flight passengers during the pandemic in , % ( / ) of the contacts were reached within h [ ] . in a measles contact investigation, % ( / ) of responding passengers were contacted within h. in this study however, the diagnosis of measles was already suspected during the flight, and laboratory confirmation was initiated immediately after landing [ ] . it also helped that many contacts were tourists staying at the same hotels, which facilitated tracing them. our study shows that the longest delay before identification of contact details for an influenza index case is caused by the time between arrival and laboratory diagnosis (interval i, , days). this delay is a result of patients delay in seeking medical care, and doctor's delay, including laboratory confirmation. for influenza, the indicated laboratory test was polymerase chain reaction, which takes several hours to obtain the result and in the beginning of the pandemic, the pcr test was not yet available in many laboratories. patients delay was considerable however. it even took the seven passengers with date of onset before the flight, and therefore symptomatic during the flight, to days after arrival before laboratory confirmation was made. also, none of the airline reported that these patients already were identified during the flight, nor that infection control measures were taken. for the indexes that became ill on the day of arrival, delay until laboratory confirmation still lasted days (range - days). a prepandemic study by sharangpani et al. among flight passengers showed that they are more willing to seek physicians care in case they developed flu-like symptoms when the perceived the pandemic as serious [ ] . leggat et al. demonstrated during the pandemic that only a minority ( , %) of australian citizens would cancel their air travel in case of cough and fever lasting more than day. this was higher among persons who were more concerned about the pandemic [ ] . in the netherlands, the perceived severity of the disease decreased significant during this study period [ ] . we expect that the delay until laboratory diagnoses in this study considerably is affected by patients delay seeking medical care, which might be better in diseases experienced as more threatening. collecting passenger details from foreign airlines also caused considerable delay because of differences in time zones and the need to convince the concerned airline companies about the urgency to collect and hand-over passenger lists with contact details. sometimes official request letters were necessary for legal reasons to release personal contact details. dutch companies were easier to convince by dutch health authorities to hand over passenger details. our data show that contact details that were identified too late or not at all, indeed more often originated from non-dutch than from dutch airline companies. an internationally standardized contact tracing protocol, communicated with the international civil aviation organization (icao) and international air transport association (iata), would facilitate the timeliness, and therefore effectiveness of contact tracing. although one might expect differently, timeliness of ct for flights where plc's were available, was not better than ct for flights without plc. however, plc's reduces the effort, in terms of staff support for airline companies and the municipal health service to collect useful passenger information considerably. plc's were only used by dutch airlines, who already were able to provide passenger lists relatively quickly. this also explains the limited attributed shortening in timeliness. contact details on plc's might be more accurate to trace the passenger than details provided by the passenger list or booking station. this is further investigated. this study has several limitations. as available data were recorded in days, and not in hours, it was not possible to determine the time intervals more precisely. as this was both with first and last date of the intervals, we expect no negative or positive bias. secondly, the arrival date was used for date of exposure, while the actual exposure might have already taken place the day before at departure of the flight. this would imply an increase in delay and decrease the effectiveness of contact tracing. also, we have no data if, and when contacts were actually reached and oseltamivir was administered. since several steps were still required to reach the contacts after they were identified through passenger lists, this only would have lead to further delay in administrating prophylaxis. further investigation into the timeliness of administration of prophylaxis among these contacts is initiated, to have insight in the delay of this last interval to facilitate future decisions on the effectiveness and necessity of contact tracing among flight passengers. lastly, this study includes ct initiated at only one airport. ct procedures might be different at airports in other countries, which influences interval iii. as this is not causing the main delay, we do not expect that in other countries ct would be much faster. we conclude that tracing close contacts among flight passengers during the initial phase of pandemic a/ h n was not effective, as timely provision of pep could not be achieved in most cases. most contacts came from an endemic area (mexico) or areas with well known increased transmission during the first months of the pandemic. the additional risk for those travelers of being a close contact during a long haul flight is small ( , %) [ ] . furthermore, airline companies and/or schiphol airport already provided contacts with information on the disease and its symptoms by. the benefit to inform them of the fact that they were contacts of a laboratory confirmed case did not justify the extra effort health authorities invested in contact tracing, especially during a period where public health officials, airports and airline companies were absorbed by efforts of other pandemic related control measures. in hindsight, the limited burden of disease of influenza a/h n did not justify contact tracing efforts. the main reason for flight contact tracing is raising alertness for possible exposure to uncommon infectious diseases, enabling early recognition and treatment of the disease and timely installation of control measures (e.g. sars and viral hemorrhagic fevers). for some diseases, pep is indicated as well. the risk assessment upon which the decision to install contact tracing is based should incorporate -apart from an evaluation of the severity and rarity of disease -an assessment of the required timeliness of effective control measures [ ] . the expected time for laboratory confirmation of index cases and identification and tracing of contacts should be related to the maximum period during which quarantine, pep or other control measures are effective in order to decide on the benefit of this time consuming procedure. lastly, also cabin crew should be aware of their role of signaling infectious patients. in consultation with medical professionals, direct control measures can be installed, as well as medical evaluation after landing. empirical evidence for the effect of airline travel on inter-regional influenza spread in the united states spread of a novel influenza a (h n ) virus via global airline transportation calculating the potential for withinflight transmission of influenza a (h n ) transmission of infectious diseases during commercial air travel transmission of pandemic a/h n influenza on passenger aircraft: retrospective cohort study lack of airborne transmission during outbreak of pandemic (h n ) among tour group members, china is public transport a risk factor for acute respiratory infection? ecdc: risk assessment guidelines for infectious diseases transmitted on aircraft an outbreak of influenza a/taiwan/ / (h n ) infections at a naval base and its association with airplane travel outbreak of influenza-like illness [corrected] related to air travel an outbreak of influenza aboard a commercial airliner mixed outbreak of parainfluenza type and influenza b associated with tourism and air travel antiviral agents for the treatment and chemoprophylaxis of influenza -recommendations of the advisory committee on immunization practices (acip) who: case management of influenza a(h n ) in air transport influenza: operationeel deeldraaiboek . incidentele introductie nieuw humaan influenzavirus in nederland estimated epidemiologic parameters and morbidity associated with pandemic h n influenza population modeling of influenza a/h n virus kinetics and symptom dynamics household transmission of pandemic influenza a (h n ) virus in osaka contacting passengers after exposure to measles on an international flight: implications for responding to new disease threats and bioterrorism attitudes and behaviors of international air travelers toward pandemic influenza level of concern and precaution taking among australians regarding travel during pandemic (h n ) : results from the queensland social survey perceived risk, anxiety, and behavioural responses of the general public during the early phase of the influenza a (h n ) pandemic in the netherlands: results of three consecutive online surveys eu funded react project: response to emerging infectious disease: assessment and development of core capacities and tools the authors would like to thank josé ferreira for advice on statistics. authors' contributions cs and ra designed the study and collected the data. mk advised on the data management and presentation of the results. js, cs and ra interpreted the data. js critically revised the manuscript. cs wrote the manuscript and all authors commented on drafts and approved the final version. all authors read and approved the final manuscript. the authors declare that they have no competing interests. the pre-publication history for this paper can be accessed here: key: cord- -ug v j authors: madani, tariq a; al-ghamdi, aisha a title: clinical features of culture-proven mycoplasma pneumoniae infections at king abdulaziz university hospital, jeddah, saudi arabia date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ug v j objective: this retrospective chart review describes the epidemiology and clinical features of patients with culture-proven mycoplasma pneumoniae infections at king abdulaziz university hospital, jeddah, saudi arabia. methods: patients with positive m. pneumoniae cultures from respiratory specimens from january through december were identified through the microbiology records. charts of patients were reviewed. results: patients were identified, ( . %) of whom required admission. most infections ( . %) were community-acquired. the infection affected all age groups but was most common in infants ( . %) and pre-school children ( . %). it occurred year-round but was most common in the fall ( %) and spring ( %). more than three-quarters of patients ( . %) had comorbidities. twenty-four isolates ( %) were associated with pneumonia, ( %) with upper respiratory tract infections, and ( %) with bronchiolitis. cough ( . %), fever ( %), and malaise ( . %) were the most common symptoms, and crepitations ( %), and wheezes ( %) were the most common signs. most patients with pneumonia had crepitations ( . %) but only % had bronchial breathing. immunocompromised patients were more likely than non-immunocompromised patients to present with pneumonia ( / versus / , p = . ). of the patients with pneumonia, ( . %) had uneventful recovery, ( . %) recovered following some complications, ( . %) died because of m pneumoniae infection, and ( . %) died due to underlying comorbidities. the patients who died of m pneumoniae pneumonia had other comorbidities. conclusion: our results were similar to published data except for the finding that infections were more common in infants and preschool children and that the mortality rate of pneumonia in patients with comorbidities was high. mycoplasma pneumoniae is a common cause of upper and lower respiratory tract infections. it remains one of the most frequent causes of atypical pneumonia particu-larly among young adults. [ , , , , ] although it is highly transmissible, most infections caused by this organism are relatively minor and include pharyngitis, tracheobronchitis, bronchiolitis, and croup with one fifth of in-fections being asymptomatic. [ , ] only - % of infected subjects develop symptoms consistent with bronchopneumonia and mortality from infection is rare. [ , ] the organism is fastidious and difficult to grow on cultures. therefore, diagnosis of infections caused by this organism is usually confirmed with serological tests or polymerase chain reaction-gene amplification techniques. at king abdulaziz university hospital (kauh), jeddah, saudi arabia, the facility to perform mycoplasma culture has been available since january . as published information concerning m. pneumoniae infections in saudi arabia is scarce, [ , , ] we wished to study the epidemiology and clinical features of cultureproven infections caused by this organism at this hospital. kauh is a tertiary care teaching hospital with a bed capacity of beds and annual admissions of to patients. patients with m. pneumoniae positive cultures from respiratory specimens were identified over a -months" period from january, through december, for this review. during the study period, respiratory specimens (sputum, nasopharyngeal aspiration, endotracheal secretion, and bronchoalveolar lavage) for m. pneumoniae culture were obtained from patients with upper or lower respiratory tract infections seen as inpatients or in the outpatient or emergency departments. respiratory specimens were aslo gram-stained and cultured for bacteria and viruses. m. pneumoniae serological tests for igg or igm were not available at kauh during the study period. all positive culture results were obtained from the microbiology laboratory records. charts of patients were reviewed with standardized data collection. information collected included patients' demographics, comorbidities, clinical manifestations, complications, and outcome. m. pneumoniae was cultured using the classic m. pneumoniae agar medium (m.p. agar) and the pneumofast tray (pneumofast ® , international microbio, signes, france). specimens were processed according to the instructions of the manufacturer. the m.p. agars and pneumofast trays were incubated anaerobically at °c and inspected daily for weeks. the organism was identified based on typical colonial morphology (granular colonies, rarely fried-egg-like, - ∝ in diameter) on the m.p. agar medium and the change in the pneumofast broth color from red to orange then to yellow (glucose fermentation) in the absence of turbidity of the broth. antibiotic sensitivity profile on the pneumofast tray was also used for identification according to the instructions of the manufacturer. bacterial and viral cultures were performed using standard methods. m. pneumoniae isolates were considered community-acquired if they were recovered from unhospitalized patients or within hours of admission to the hospital, and nosocomial if they were recovered beyond that period. pneumonia was diagnosed based on clinical symptoms and signs, along with radiographic evidence of pneumonia when possible. severe pneumonia was defined as pneumonia associated with tachycardia (> /minute), tachypnoea (> /minute), hypotension (systolic blood pressure < mmhg), hypoxemia (arterial oxygen partial pressure < kpa or oxygen saturation < %), and/or more than areas of consolidation. outcome of patients with m. pneumoniae infection was classified into categories; uneventful recovery, recovery following complications, death due to m. pneumoniae infection, or death unrelated to m. pneumoniae infection. the statistical package for social sciences (spss) program was used for data analysis. comparison of categorical data was by chi-square statistic or fisher's exact test for small expected values. a total of respiratory specimens from patients were positive for m. pneumoniae over the -months study period. the demographic and epidemiological characteristics of the patients are summarized in table . of all isolates, ( . %) were community-acquired and ( . %) were nosocomial. thirty-three ( . %) patients required admission to the hospital and the remaining ( . %) were treated as outpatients. twenty-four isolates ( %) were associated with pneumonia, ( %) with upper respiratory tract infections, and ( %) with bronchiolitis. of the cases of pneumonia, were confirmed radiologically and the remaining were diagnosed clinically. the two cases of bronchiolitis occurred in children, one and three years old. thirty-one patients ( . %) had comorbidities. eleven patients ( . %) had cardiopulmonary comorbidities (asthma, , lung fibrosis, , congestive heart failure, , congenial heart disease, ), patients ( . %) were immunocompromised (malignancy, , steroid therapy, , human immunodeficiency virus infection, ), and patients ( . %) had other comorbidities (premature newborns, , and one each of myelodysplastic syndrome, myelopro-liferative disorder, sickle cell anemia, evan's syndrome, down syndrome, sarcoidosis, demyelinating disease, cerebral palsy, and spinal muscle atrophy). organisms concomitantly isolated with m. pneumoniae from the respiratory tract included herpes simplex virus type ( occasions), adenovirus ( occasions), cytomegalo virus ( occasion), respiratory syncytial virus ( occasion), and bacterial isolates ( occasions: acinetobacter species, , and enter obacter cloacae, ). clinical manifestations associated with m. pneumoniae infections are summarized in table . pneumonia was more common than upper respiratory tract infections ( . % versus . %, respectively). immunocompromised patients were more likely to present with pneumonia as opposed to upper respiratory tract infection or bronchiolitis than non-immunocompromised patients ( / versus / , p = . ). similarly, there was a tendency for patients years of age or older to present with pneumonia more frequently than those below ( / versus / , p = . ). of the patients with clinically or radiologically confirmed pneumonia, ( . %) had crepitations and only ( %) had bronchial breath sounds on physical examination. of the patients in whom wheezes were detected, ( . %) were not known to have asthma or other obstructive airway disease. table . of the patients with pneumonia, ( . %) were admitted to the hospital, and ( . %) had comorbidities. all patients with upper respiratory tract infections ( patients) or bronchiolitis ( patients) had uneventful recovery. of the patients with pneumonia, ( . %) had uneventful recovery, ( . %) recovered following some complications (acute respiratory distress syndrome, , respiratory failure, , septic shock, ), ( . %) died because of m pneumoniae infection, and ( . %) died due to underlying comorbidities. the patients who died of m pneumoniae pneumonia had other comorbidities; one had congestive heart failure, the second had congenital heart disease, and the third was a months old infant born prematurely at weeks of gestation who previously had episodes of pneumonia due to other pathogens. mycoplasma pneumoniae is one of the most common causes of atypical pneumonia accounting for - % of community-acquired pneumonia, [ , , , , ] in a study of children with acute respiratory tract infection in riyadh, saudi arabia, mycoplasma pneumoniae was found to be the second most common causative agent after respiratory syncytial virus (rsv) accounting for % of all cases, [ ] in a study of adult patients with community acquired pneumonia admitted to a military hospital in riyadh, saudi arabia, this organism accounted for % of all cases, [ ] in another retrospective study of pneumonic episodes in adult patients from al-qassim area, the organism accounted for % of all episodes, [ ] the organism also causes other relatively minor infections such as pharyngitis, tracheobronchitis, bronchiolitis, and croup. it is transmitted from person-to-person by infected respiratory droplets during close contact. it is most common in school-aged children, military recruits, and college students. [ ] most cases occur singly or as family outbreaks. larger outbreaks can also occur in closed populations such as military recruit camps or boarding schools, [ ] infection occurs most frequently during the fall and winter in temperate climates but may develop year-round, [ ] the average incubation period is weeks following exposure, [ ] although rare, complications are protean and may involve virtually any organ system such as the respiratory system (e.g.: pleurisy, pneumothorax, acute respiratory distress syndrome, lung abscess), the hematologic system (e.g.: hemolytic anemia, intravascular coagulation, thrombocytopenia), the dermatologic system (e.g.: maculopapular or urticarial rashes, erythema multiforme, erythema nodosum), the musculoskeletal system (e.g.: myalgias, arthralgias, arthritis), the cardiovascular system (e.g.: pericarditis, myocarditis), the nervous system (e.g.: meningoencephalitis, guillain-barre syndrome, neuropathies, acute psychosis), or the eye (optic disc edema, optic nerve atrophy, retinal exudation and hemorrhages). [ , , , , , , ] immunity following infection is not long lasting. [ ] in our study, the infection affected all age groups but was most common in infants ( . %) and preschool children ( . %), and least common in adults aged to years ( . %) and elderly above years of age ( %). this contrasts with data from temperate countries where the infection is most common in school-aged children, and young adults. [ ] one possible explanation for this difference is that infants and preschool children perhaps had more severe infections than did school-aged children, and young adults which prompted presentation of the former group to the hospital. the infection occurred year-round but was most common in the fall ( %), and spring ( %), and least common in the summer ( %). most infections were community-acquired ( . %). more than one half of patients ( . %) presented with pneumonia, and about a third ( . %) presented with upper respiratory tract infection, immunocompromised patients and patients years of age or older were more likely to present with pneumonia as opposed to upper respiratory tract infection than non-immunocompromised patients or those below years of age. cough ( . %), fever ( %), and malaise ( . %) were the most common presenting symptoms. cough was usually dry or slightly productive of white sputum and mild to moderate in severity. most febrile patients had mild to mod- erate fever of °c or less; high-grade fever of more than °c was rare. crepitations ( %), and wheezes ( %) were the most common signs. wheezes were as common in patients with no history of obstructive airway disease ( patients) as it was in those with such a history ( patients). bronchial breathing as a sign of consolidation was detected in only one fourth of patients with pneumonia, which is consistent with the known disparity between clinical and radiological signs of m pneumoniae pneumonia. crepitations, however, were detected in the majority ( . %) of patients. pleuritic chest pain and pleural effusion were rare. more than half ( . %) of the patients with pneumonia had uneventful recovery. mortality from m. pneumoniae pneumonia was high ( . %) and occurred only in patients with underlying comorbidities. none of the patients with no underlying comorbidities died of m pneumoniae pneumonia. the relatively high complications rate ( . %) and mortality ( . %) related to m. pneumoniae pneumonia are likely due to selection bias as most patients with pneumonia were sick enough to require admission to the hospital ( / or . %) and most of them had comorbidities ( / or . %). in conclusion, our data shed some light on the epidemiology and clinical features of m pneumoniae infections in one of the saudi tertiary care centers. the data are comparable to those of other countries except for the finding that infections were more common in infants and preschool children than in school children and young adults. additionally, mortality attributable to m. pneumoniae pneumonia was relatively high in patients with comorbidities. it is hoped this information will assist clinicians in their approach and management of respiratory tract infections. community-acquired pneumonia ambulatory patients with community-acquired pneumonia: the frequency of atypical agents and clinical course mycoplasma pneumoniae community-acquired pneumonia: a review of hospitalized adult patients respiration atypical" 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infection mycoplasma pericarditis: evidence of invasive disease clin infect dis cns manifestations associated with mycoplasma pneumoniae infections. summary of cases at the university of helsinki and review clin infect dis we wish to thank mrs. fatin s. gazzaz, microbiologist, and mr. yusri a. al-suruji, microbiology technologist, king abdulaziz university hospital, for performing mycoplasma pneumoniae cultures and for providing the list of patients for this study. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/content/backmatter/ - - - -b .pdf key: cord- -zyaj nh authors: wong, samuel ys; wong, eliza ly; chor, josette; kung, kenny; chan, paul ks; wong, carmen; griffiths, sian m title: willingness to accept h n pandemic influenza vaccine: a cross-sectional study of hong kong community nurses date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: zyaj nh background: the pandemic of influenza a (h n ) infection has alerted many governments to make preparedness plan to control the spread of influenza a (h n ) infection. vaccination for influenza is one of the most important primary preventative measures to reduce the disease burden. our study aims to assess the willingness of nurses who work for the community nursing service (cns) in hong kong on their acceptance of influenza a (h n ) influenza vaccination. methods: questionnaires were posted from june , to june , to community nurses with % response rate. results of the respondents on their willingness to accept influenza a (h n ) vaccine were analyzed. results: twenty-seven percent of respondents were willing to accept influenza vaccination if vaccines were available. having been vaccinated for seasonable influenza in the previous months were significantly independently associated with their willingness to accept influenza a (h n ) vaccination (or = . ; % ci: . - . ). conclusions: similar to previous findings conducted in hospital healthcare workers and nurses, we confirmed that the willingness of community nurses to accept influenza a (h n ) vaccination is low. future studies that evaluate interventions to address nurses' specific concerns or interventions that aim to raise the awareness among nurses on the importance of influenza a (h n ) vaccination to protect vulnerable patient populations is needed. the pandemic of influenza a (h n ) infection has alerted many governments to make preparedness plan to control the spread of influenza a (h n ) infection. with evidence on the effectiveness of vaccination in the control and prevention of seasonal influenza [ , ] , vaccination for pandemic influenza is one of the most important primary preventative measures to reduce the disease burden associated with influenza a (h n ) infection [ ] . several high risk groups have been identified as "the priority group" to receive the influenza a (h n ) vaccination and among these, healthcare workers have been identified "as a first priority" to be vaccinated against influenza a (h n ) by the world health organization [ , ] . although it is considered essential for all healthcare workers to be immunized against influenza a (h n ) to prevent the spread of influenza a (h n ) to patients as the pandemic evolves, previous studies that have examined the acceptability of seasonal influenza vaccination among healthcare workers have generally demonstrated a low acceptance rate of vaccination in this group [ , ] . among all healthcare workers, nurses constitute the largest group with the highest frequency of contacts with patients and staff [ ] . previous findings of the acceptability of seasonal influenza vaccination in nurses showed that their acceptance of vaccination was lowest among all healthcare workers [ , , , ] . acceptability of influenza a (h n ) vaccination in healthcare workers has been shown to be low [ ] [ ] [ ] . a survey conducted in greece found that only % of hospital healthcare were willing to receive influenza a (h n ) vaccination [ ] . of all healthcare workers, nurses were found to have the lowest rate of acceptability of influenza a (h n ) vaccination [ , ] . a study of italian healthcare workers showed % of nurses willing to accept vaccination compared to % of physicians [ ] . in a study conducted of hong kong healthcare workers in hospitals, it was found that only % of nurses were willing to accept influenza a (h n ) vaccination, compared with % of doctors and % of allied professionals [ ] . general practitioners working in the community in france also report a high rate of acceptability of influenza a (h n ) vaccination at % [ ] . it is therefore not surprising that a recent online poll conducted in the uk suggested that nurses may be unwilling to receive pandemic influenza vaccination [ ] . in a cross-sectional survey that was conducted on experienced nurses who were members of the nursing professional organizations in hong kong, the vaccination rate for seasonal influenza vaccination was about % [ ] . in a more recent survey that explored influenza a (h n ) acceptance rate in the same group of nurses [ ] , it was found that only % were willing to accept vaccination for influenza a (h n ) compared to % who plan to receive the seasonal influenza vaccination. however, in the study, there was a low response rate of % of nurses with different clinical settings. there is a lack of studies in hong kong looking at influenza a (h n ) vaccination acceptability particularly in the community setting. nurses who work in the community may be the first group to be in contact with patients who are affected with the influenza a(h n ) infection. a recent study [ ] showed differences in the concerns in using new vaccines during a pandemic than using established vaccine in a non-crisis situation. therefore, we undertook the current study to examine the willingness of frontline registered nurses who work in the community in hong kong to receive vaccination against influenza a (h n ) at the time of a pandemic. all participants in this study were specially trained nurses, who provided nursing care and treatment for patients in their own homes (also known as community nursing service) in hong kong. the responsibility of these community nurses is to provide nursing care and health education to patients through home visits. cns nurses are employed by hospital authority in hong kong and provide continuity of care for patients who have been discharged from hospitals such that patients can recover in their own homes. community nurses were chosen because of their frequent contacts with patients in their homes which is likely to increase their risk for exposure to influenza. we have only included cns nurses who provide medical services in the study. the rest of the cns nurses (around nurses) provide psychiatric services in the community. currently, there are a total of nurses who provide medical related services for the community nursing service (cns) centres that are distributed among the geographical clusters in hong kong (in hong kong, public hospital and primary care services are organized in clusters that covers all of hong kong). in this study, twelve major cns centres were contacted first and all cns nurses were invited to participate in the current study through these major centres. all centres responded to this study and questionnaires were returned with completed questionnaires [ ] . the response rate for this study was % and all questionnaires were received within a week period at a time when there was widespread h n in the community. the survey was sent out from june th to june th , when the who influenza pandemic alert level assigned to h n was phase . phase signifies a widespread human infection, indicating that the virus has caused sustained community level outbreaks in at least one other country in another who region (who pandemic phase description). the pandemic in hong kong started on st may, when a mexican traveller was confirmed with influenza a (h n ). till the end of our data collection, there were confirmed cases and no death were reported. all general managers of the involved community nursing centres were contacted through telephone to obtain approval to send questionnaires to their nursing staff. in total, self administered, anonymous questionnaires were posted to general managers of centres who then passed these questionnaires to the community nurses in their centres. the general managers of centres were then reminded via telephone during the period from nd july and th july one week after the questionnaires were sent out and advised to return the completed questionnaires within the week. once completed, questionnaires were collected and returned by their supervisors, except for one of the (sau mau ping) sub-offices, where nurses mailed back their questionnaires individually. all centres sent their questionnaires back after one telephone reminder. the last pile of completed questionnaires was received on th july, . the questionnaire consisted of six parts with questions and the full questionnaire can be accessed by contacting the authors. the first four parts were based on a conceptual framework developed by patel et al [ ] to guide systematic planning for community primary care service response to pandemic influenza with modifications to make it more relevant for nurses. we added a fifth part on psychological responses to pandemic influenza and a sixth part on demographics of respondents which were based on two studies previously published (one on general practitioners' response to sars and one on general public response to swine flu) [ , ] . in summary, these sections were ) clinical services change as a response to pandemic influenza; ) internal environment changes as a response to pandemic influenza e.g. wearing of mask; ) macro-environmental changes as a response to pandemic influenza e.g. use of guideline etc; ) professional and public health responsibilities with respect to pandemic influenza; ) attitude and psychological responses to pandemic influenza; and ) demographics and year of education of respondents. the willingness to accept influenza a(h n ) vaccination was asked in the professional and public health responsibility sections and the question "will you receive the new influenza a (h n ) vaccine when it is available" was asked with a dichotomous "yes" or "no" response. for those who answered no, they were further asked to give their reasons for refusing to receive the vaccine. only results on willingness of accept influenza a (h n ) vaccination and information related to the analysis on willingness to accept vaccine are reported in this paper. other results from this survey will be presented in a separate report. descriptive results were cross-tabulated. χ test was used to examine characteristics between nurses who were willing to accept influenza a (h n ) vaccination against those who were not willing to accept vaccine. univariate analysis was performed with demographic information (age, post year education and working district), personal protective behaviour (hand washing practice), experience of taking care of sars patients, and influenza vaccination in the previous months as independent variables. dependent variables were the willingness to receive pandemic influenza vaccination. multiple logistic regression analysis was conducted to examine the relationship between pre-defined factors that we think might be associated with the acceptance of the influenza a (h n ) vaccine when constructing the questionnaire and the dependent variable. the level of statistical significance was set at a p-value of ≤ . . among the respondents ( table ) , most of them were females who had worked an average of . years as a community nurse (ranging from months to years) and having been a registered nurse for . years (ranging from year to years). the mean age of respondents was . years and about a third ( %) had had the experience of dealing with sars. one third of them had received vaccination for seasonal influenza in the past months. nurses from each geographical cluster in hong kong participated, with % of respondents working in hong kong island, % working in kowloon and % working in the new territories (hong kong is geographically divided into hong kong island, kowloon peninsula and the new territories). overall, ( %) participants do not want to receive new influenza a (h n ) vaccine when it is available. the reasons for their not intending to receive vaccination when it is available are summarised in table . the characteristics of respondents who were willing to accept influenza a (h n ) vaccination and with those who were not willing to accept influenza a (h n ) influenza vaccination were compared by χ test and were presented table . nurses who were willing to receive influenza a (h n ) vaccine were different from nurses who were not willing to receive influenza a (h n ) vaccine with respect to "being vaccinated against seasonal influenza vaccination in the previous months". there were no statistical significant differences in other characteristics as analyzed by chi-square test. the relationship between demographic and other characteristics of the nursing respondents and their willingness to accept vaccination were analyzed further using forced entry logistic regressions (table ) . having seasonal vaccination in the past months was significantly independently associated with the willingness to accept influenza a (h n ) vaccination (or = . ; % ic: . - . ). washing hands before and between patient contact, however, was negatively independently associated with willingness to accept influenza a (h n ) vaccination (or = . ; % ic: . - . ). to confirm the results, we have also conducted backward logistic regression and the results also indicated that having seasonal vaccination in the past months was significantly associated with the willingness to accept influenza a (h n ) vaccination (or = . , % ci: . - . , p < . ). consistent with findings from previous surveys conducted in hospital healthcare workers and nurses [ , ] , we have shown that the majority of nurses from community nursing services in hong kong were not willing to be vaccinated against h n influenza when the vaccine becomes available. similar to findings from previous studies in healthcare workers [ , , , ] , we showed that the major concerns for vaccination against pandemic influenza was fear of side effects and concern of efficacy of the new vaccine (table ) . moreover, influenza vaccination in the previous months was significantly associated with their willingness to accept the pandemic influenza vaccination. we also showed that in addition to previous vaccination with seasonal influenza, preventive behaviours such as frequent hand washing practice were independently associated with nurses' willingness to accept influenza a (h n ) vaccination. we showed that "have been washing hands between and before patient contact" was negatively associated with willingness to accept vaccination independently although the reason for this is unclear and be a result of our relatively small sample. we can only postulate that the barrier to pandemic influenza vaccination is probably not related to the willingness of nurses to protect themselves against infections or their personal hygiene in general. researchers [ ] have suggested one of the barriers to pandemic influenza vaccination in nurses was misconceptions about the purpose of vaccinations in which nurse might think that the aim of vaccination was for self protection rather than to protect at risk populations in contact with them [ , , ] . specific vaccination policy for health care workers may improve vaccination in this group as nurses have different concerns and priorities when compared to the general public's concerns [ , ] . although some may suggest that more educational programs for healthcare workers may be a solution to the low vaccination uptake [ ] , studies have reported low influenza vaccination rates among healthcare workers even when educational programs were implemented [ ] . other studies including randomized controlled trials also failed to show that better knowledge or educational programmes ( ) other concerns (i.e. pregnancy, poor health status, and the severity of the epidemic of h n ) (< . ) note: the total percentage exceeds % because multiple responses were allowed. were effective in increasing acceptability of vaccination in healthcare workers [ ] . indeed, some suggested that educational campaigns based on the health belief model were unlikely to be enough to change healthcare workers' acceptability of vaccination as evidence showed that perceived seriousness of infection, acknowledgement of increased risk of infection and knowledge of vaccine being safe were unrelated to vaccine uptake in healthcare workers [ ] . others suggested that educational programmes may be counter-productive as many of these healthcare workers do not perceive themselves to be at risk for contracting the infection. recently, ofstead et al [ ] suggested that an ecological model, which included engaging organizations, communities and policy makers to create environments that were more conducive to risk reduction, might be more effective in increasing vaccination rates in healthcare workers. to our knowledge, this is the first study to explore the willingness of nurses who work in the community to be vaccinated for pandemic influenza and our results confirmed that their acceptability of influenza a (h n ) vaccination is low. a strength of our study is our response rate of % which is higher than similar report conducted in hong kong with a response rate of % [ ] . a limitation of our study is that we have only documented nurses' intentions of when a vaccine is available and not the actual uptake of vaccination. furthermore, all data from this study were from self-reports and recall bias, such recalling influenza vaccination in the previous year, might have occurred. a possible contributory factor e.g. recent episode of influenza-like illness which may influence the willingness of vaccination was not enquired. our analysis of results was limited by the relatively small sample size in nurses who are part of the community nursing service in hong kong with no information available on non respondents. however, our results are similar to recent studies conducted in hospital healthcare workers [ ] and members of professional nursing organizations [ ] in hong kong. consistent with previous findings which were conducted in healthcare workers and nurses [ , ] , we confirm that the acceptance rate of pandemic influenza vaccination is low amongst community nurses. since community nurses are at high risk of contracting influenza infection, and play a significant role in caring for community cases, special attention should be paid to this group as successful vaccination strategy has been shown to be beneficial in disease transmission [ ] . future work, including interventional studies evaluating potential interventions based on the ecological model or interventions that aim to increase awareness among nurses on the importance of vaccination in healthcare workers to protect vulnerable populations [ ] is needed. the need to address low influenza vaccination rates in this high-risk group is urgent in the context of pandemic response. vaccines for seasonal and pandemic influenza efficacy and effectiveness of influenza vaccination stockpiling prepandemic influenza vaccines: a new cornerstone of pandemic preparedness plans healthcare workers should get top priority for vaccination against a/h n flu, who says who global influenza preparedness plan: pandemic influenza preparedness and response: a who guidance document. world health organization correlation between healthcare workers' knowledge of influenza vaccine and vaccine receipt factors influencing update of influenza vaccination among hospital-based healthcare workers nurses' contacts and potential for infectious disease transmission influenza immunization: improving compliance of healthcare workers uptake of influenza vaccine by healthcare workers in an acute hospital in ireland low acceptance of vaccination against the pandemic influenza a(h n ) among healthcare workers in greece behviours regarding preventive measures against pandemic h n influenza among italian healthcare workers willingness of hong kong healthcare workers to accept pre-pandemic influenza vaccination at different who alert levels: two questionnaire surveys positive attitudes of french general practitioners towards a/ h n influenza-pandemic vaccination: a missed opportunity to increase vaccination uptake in the general public? vaccine siva n: incidence of swine flu in england continues to fall, but winter surge is predicted impact of severe acute respiratory sundrome and the perceived avian influenza epidemic on the increased rate of influenza vaccination among nurses in hong kong exploring determinants of acceptance of the pandemic influenza a (h n ) vaccination in nurse the public's acceptance of novel vaccines during a pandemic: a focus group study and its application to influence h n . emerg health threats j will the community nurse continue to function during h n influenza pandemic: a cross-sectional study of hong kong community nurses? general practice and pandemic influenza: a framework for planning and comparison of plans in five countries primary care physicians in hong kong and canada-how did their practices differ during the sars epidemic? widespread public misconception in the early phase of the h n influenza epidemic influenza vaccination among primary healthcare workers which determinants should be targeted to increase influenza vaccination uptake among healthcare workers in nursing homes? vaccine influenza vaccination among registered nurses: information receipt, knowledge, and decision-making at an institution with a multifaceted educational program promoting uptake of influenza vaccination among health care workers: a randomized controlled trial health care worker, vaccinate thyself: toward better compliance with influenza vaccination this study was supported by the research fund for the control of infectious diseases (rfcid), food and health bureau, hong kong sar government. we thank ting gao in data entry and her assistance in analysis of data. we also thank louisa lau for coordination with centres* of the community nursing service. we especially thank all nurses and nursing managers of the community nursing service of the hospital authority who participated in this study. all authors were involved in the design of the project. the survey tool was designed by sysw and elyw. the data collection, analysis and the results interpretation were carried by sysw and elyw in consultation with the team. the first draft of this article was composed by sysw and was revised critically by all authors. all authors have approved the final version of the manuscript. the authors declare that they have no competing interests. key: cord- -mnqn t q authors: zhao, xia; wang, lihong; wei, nan; zhang, jingli; ma, wenhui; zhao, huijie; han, xu title: epidemiological and clinical characteristics of healthcare-associated infection in elderly patients in a large chinese tertiary hospital: a -year surveillance study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: mnqn t q background: we analyzed the results of a -year surveillance study on the epidemiological and clinical characteristics of healthcare associated-infections (hais) in elderly inpatients in a large tertiary hospital in china. methods: real-time surveillance was performed from january , to december , . all hais were identified by infection control practitioners and doctors. inpatient data were collected with an automatic surveillance system. results: a total of , inpatients including , ( . %) elderly ≥ years were included. the overall incidence of hai was . %. the incidence of hai in elderly patients was significantly higher than that in non-elderly patients ( . % vs. . %, χ( ) = . , p < . ) and increased with age. the top five sites of hais in the elderly were the lower respiratory tract, urinary tract, blood stream, antibiotic-associated diarrhea, and surgical site. the five most common pathogens detected in elderly hai patients were candida albicans, klebsiella pneumonia, acinetobacter baumannii, escherichia coli, and pseudomonas aeruginosa. the incidence of ventilator-associated pneumonia in the elderly was lower than in the non-elderly, catheter-associated urinary tract infections were more common in elderly patients, and the rate of central line-associated bloodstream infection was similar between groups. the numbers of male patients and patients with comorbidities and special medical procedures (e.g., intensive care unit admission, cerebrovascular disease, brain neoplasms, hypertension, hyperlipidemia, diabetes mellitus, coronary artery disease, chronic obstructive pulmonary disease, malignant tumor, malignant hematonosis, and osteoarthropathy) were significantly higher in the elderly group, but the number of patients who underwent surgery was lower. conclusion: we observed a significantly higher overall incidence of hai in elderly inpatients ≥ compared to non-elderly inpatients < years, but the trend was different for device-associated hais, which was attributed to the higher rates of comorbidities and special medical procedures in the elderly group. the main hai sites in elderly inpatients were the lower respiratory tract, urinary tract, and bloodstream, and the main pathogens were gram-negative bacilli and candida albicans. modern societies have continually growing numbers of elderly inpatients. in , there were an estimated million people aged ≥ years in the world, comprising % of the global population [ ] . the population aged ≥ is growing at an annual rate of~ %. european countries currently have the greatest percentage of population aged ≥ ( %). rapid ageing will also soon occur in other parts of the world, and by all regions except africa will have nearly a quarter or more of their populations ≥ [ ] . population ageing is projected to have a profound effect on societies by increasing demand on fiscal, political, healthcare, and social protection systems of many countries in coming decades. china will be one of the countries affected. healthcare associated-infections (hais) occur in patients under medical care in hospitals or other healthcare facilities. these infections occur during healthcare delivery for other diseases and even after discharge. hais are associated with prolonged hospital stay, poor prognosis, and increased mortality and economic burden [ ] [ ] [ ] . a systematic review and meta-analysis reported that the major risk factors independently associated with hais were diabetes mellitus, immunosuppression, body temperature, surgery time in minutes, reoperation, cephalosporin exposure, days of exposure to central venous catheter, intensive care unit (icu) admission, icu stay in days, and mechanical ventilation [ ] . moreover, invasive devices such as catheters, ventilators, and central lines are associated with hais. the elderly are particularly vulnerable to infections due to reduced immunological competence and a high risk of underlying chronic illness [ ] [ ] [ ] [ ] [ ] . however, there are limited data available regarding hais in elderly inpatients. here we describe and analyze the results of a -year real-time surveillance study on the incidence of hais and the epidemiological and clinical characteristics of elderly inpatients in a large tertiary hospital in china. a cohort study based on real-time surveillance was performed from january , to december , in a large tertiary hospital with beds in beijing, china. we carried out real-time surveillance of hais with an online nosocomial infection surveillance system (niss) that can download inpatients' clinical information and screen potential hais at any time and run automatically at an appointed time every day. pediatric patients ≤ years and patients who had been hospitalized for < days or > days were excluded. all hais that occurred during hospitalization were identified by infection control practitioners and doctors. hais were defined as infections that occurred h after the patients were admitted to hospital according to the definitions published by ministry of health of the people's republic of china [ ] . we implement postdischarge surveillance of surgical site infection (ssi) for patients who undergo surgery. ssi included infection that occurred within days after the procedure if no implant is left in place or within year if an implant is placed. the data were collected using an automatic online real-time niss (rt-niss, version: . . . ). the rt-niss downloads and records clinical information for each participant including demographic characteristics, hospitalization days, diagnoses, operations, specific device days, body temperature, laboratory and auxiliary examinations, microbiologic profile, and bacterial resistance automatically online from other information systems including the hospital information system, electronic medical record, laboratory information system, picture archiving and communication system, mobile nursing information system, and anesthesia operation system. then the rt-niss automatically screens potential hais according to the definitions published by the ministry of public health, which are input into the system by the algorithm of fever history, microbiological reports, serological and molecular testing, radiology information, and antibiotic usage [ ] . the infection control team checked the collected data and removed invalid inputs. then the infection control practitioners and doctors identified potential hais screened by rt-niss according to the definitions published by ministry of health of the people's republic of china. this ensured that the collected data were valid and reliable. previous studies showed that compared with a manual survey of hais (the gold standard) in all inpatients, the sensitivity and specificity of rt-niss were . and . %, respectively [ ] . data analysis spss . (spss inc., chicago, il, usa) and stata . (statacorp, college station, tx, usa) were used for data analysis. rate ratios (rrs), % confidence intervals (cis), and p values were calculated to identify significant differences in incidence density. differences in categorical variables were assessed using χ tests. statistical testing was performed at the conventional -tailed α = . . during the -year study period, a total of , admissions with a total of , , hospital days were involved in the study, including , ( . %) elderly inpatients ≥ years with , hospital days. the median hospital stay length was days in total and days in elderly. the median ages overall and for elderly were and years, respectively. there were hai cases among the , included patients with , , patient-days, and the total incidence and incidence density of hai were . % and . per patient-days, respectively. the incidence density of hai in elderly patients was significantly higher than in non-elderly under years old and increased with age (table ). there were hais in , elderly inpatients and hais in , non-elderly inpatients, with a significantly higher incidence in elderly compared to non-elderly ( . % vs. . %, χ = . , p < . ). the incidence of hai in elderly inpatients significantly increased with age, with rates of . % ( / , ), . % ( / , ), and . % ( / ), respectively in patients aged - years, - years and ≥ years (χ = . , p < . ). the four departments with the highest hai incidence rates were hematology and gerontology in the medicine department and the vascular and general surgical departments: . , . , . , and . % respectively. the departments with the lowest hai incidence were otorhinolaryngology, gynecology and obstetrics, pain, and ophthalmology with . , . , . , and . %, respectively. the ventilator and central line use rates in elderly patients were significantly higher than in non-elderly patients, but the urinary catheter use rate was lower in elderly patients. the incidence of ventilator-associated pneumonia (vap) in elderly patients was lower than in non-elderly patients, catheter-associated urinary tract infections (cautis) were significantly more common in elderly patients, and central line-associated bloodstream infection (clabsi) rates were similar in both groups (tables and ). the top three sites of hais in elderly were the lower respiratory tract, urinary system, and bloodstream. among these three sites, the rates of vap, cauti, and clabsi were . % ( / ), . % ( / ), and . % ( / ), respectively (table ) . the numbers of male patients and patients with comorbidities and special medical procedures (e.g., neurological and chronic noncommunicable diseases and icu admission) were significantly higher in the elderly, but fewer patients in that group underwent surgery ( table ). the top five pathogens detected in elderly hai patients were candida albicans, klebsiella pneumonia, acinetobacter baumannii, escherichia coli, and pseudomonas aeruginosa. however, the main pathogens differed by infection site (table ). analysis of years of data showed that the incidence and incidence density of hais in elderly patients were significantly higher than in non-elderly patients ( . % vs. . %, . / d vs. . / d, p < . ). the incidence of hais in the oldest group (≥ years) was three-fold higher than that in non-elderly ( . % vs. . %, p < . ). although the incidence of hais in this study was lower than most previous publications, the trend was consistent. hais accounts for . - % of all infections in developed and developing countries [ ] . extensive studies in the usa and europe showed that hai incidence density ranged from . to . episodes per patient-days [ ] . incidence rates are higher in icus, affecting~ % of patients [ ] . however, a survey of long-term care facilities for the elderly in japan found that the overall incidence rate of hais was . per resident-days [ ] . a survey of the prevalence of hais in older people in acute care hospitals in scotland found a linear relationship between hai prevalence and increasing age, the incidence of hais in patients younger and older than were . and . %, respectively [ ] . an investigation of the hai incidence in elderly hospitalized patients at a hospital in hunan province, china reported that the hai incidence in patients aged ≥ was significantly higher than in those aged < ( . % vs. . %, χ = . , p < . ) [ ] . we may have underestimated the hai incidence because we did not implement postdischarge surveillance for all inpatients. the lower hai incidence may also be related to the shorter average hospital stay. in this study, the median lengths were only days overall and days for elderly patients. elderly inpatients are at high risk of hai because of their poor immune function, decreased mobility, and comorbid chronic noncommunicable diseases such as cardiovascular disease, cancer, diabetes, and chronic obstructive pulmonary disease (copd). furthermore, elderly inpatients with hais have poor prognosis and increased economic burden [ ] . one study evaluated elderly patients who had an hai in the icu and found that clinical outcomes of the elderly who acquired an infection in the icu were influenced by sociodemographic and clinical variables that increase mortality rates [ ] . another study of hai in elderly patients identified the following risk factors: advanced age; comorbid neurological and chronic noncommunicable diseases such as cerebral hemorrhage, cerebral infarction, brain neoplasms, diabetes mellitus, coronary artery disease, malignant tumor and malignant hematonosis; hospital days before hai; icu admission; and device use [ ] . the participants of that study were elderly ≥ years with or without hais. to control for confounders and identify novel risk factors of hai in this population, we investigated comorbidities and special medical procedures in elderly and non-elderly groups. the percentages of male patients, patients with comorbidities (e.g., cerebrovascular disease, brain neoplasms, hypertension, hyperlipidemia, diabetes mellitus, coronary artery disease, copd, malignant tumor, malignant hematonosis, and osteoarthropathy), and icu admissions were significantly higher in the elderly group. conversely, fewer elderly patients underwent surgery lower. these results suggest that the higher incidence of hai in elderly may be attributable to the higher rates of comorbidities and special medical procedures in elderly inpatients. one of the primary concerns of the current investigation was identifying common hais sites in elderly inpatients. most hais were found in the lower respiratory tract, urinary system, and bloodstream. these findings are consistent with other available studies of both elderly and non-elderly inpatients [ , ] . with recent improvements in implants, it is important to focus on device-associated infections (dais [ ] . for elderly inpatients with lower respiratory tract, urinary system, and bloodstream infections, the percentages of vap, cauti, and clabsi were . % ( / ), . % ( / ), and . % ( / ), respectively. ventilator and central line use rates in the elderly group were significantly higher than in the non-elderly, but the elderly had a lower urinary catheter use rate. the incidence density of vap in elderly was lower than in non-elderly, but cautis were significantly higher, and clabsi rates were similar. these results suggest that the incidence densities of vap, clabsi, and cauti in elderly inpatients did not increase with the ventilator, central line, and urinary catheter use rates. the lower incidence density of vap in the elderly group was probably because the admitting diagnosis often included lower respiratory tract infections, and it was difficult to find evidence of vap in these patients. the high incidence density of cauti in elderly inpatients is consistent with other reports. a study concerned with risk factors for cauti in italian elderly found that increasing age and duration of hospital stay before catheter insertion were associated with cautis [ ] . the high percentage of cauti may be related to specific issues of elderly inpatients, but also due to failures in catheter insertion as we observed here and which may be our next study. the low similar incidence density of clabsi in both the elderly and non-elderly groups may be related to effective interventions to prevent clabsi. hallam and colleagues collected data over a -year period and found a significant and sustained reduction in the clabsi rate from per catheter days to . per catheter days [ ] . the other notable finding of the current investigation was the pathogens detected in elderly hai patients. the five most common were candida albicans, klebsiella pneumonia, acinetobacter baumannii, escherichia coli, and pseudomonas aeruginosa, but they varied by infection site. extensive use of broad-spectrum antibiotics in elderly could account for the high positive detection rate of candida albicans. the main pathogens of lower respiratory tract, urinary system, and bloodstream infections detected in elderly hai patients could serve as reference evidence for empirical use of antibiotics to treat hais. first, this was a single-center study, so our findings cannot be generalized to all elderly patients in china. second, we may have underestimated the hai incidence because we did not implement post-discharge surveillance for all inpatients. finally, the microorganism profile did not include drug sensitivity or antibiotic resistance. we will include those tests in our next study to better prevent hais in elderly inpatients. we observed a significantly higher overall incidence of hais for inpatients ≥ years old compared to those under , which was attributable to higher rates of comorbidities, special medical procedures to treat neurological and chronic noncommunicable diseases, icu admission, and surgery in elderly. the main sites of hais in elderly patients were the lower respiratory tract, urinary system, and bloodstream due to high rates of vap, clabsi and cauti. interestingly, the incidence density of device-associated infections did not increase with the higher device use rate in the elderly group. futures studies to identify risks factors of hais in elderly will help decrease the rate in elderly inpatients. world population prospects: the revision, key findings and advance tables prevalence, incidence burden, and clinical impact of healthcare-associated infections and antimicrobial resistance: a national prevalent cohort study in acute care hospitals in greece effect of healthcare-acquired infection on length of hospital stay and cost hospital-acquired infections in belgian acute-care hospitals: an estimation of their global impact on mortality, length of stay and healthcare costs cañon-montañez w. risk factors for health care-associated infection in hospitalized adults: systematic review and meta-analysis are short-stay units an appropriate resource for hospitalising elderly patients with infection? elderly patients are at high risk from hospital-acquired infection bloodstream infections in older patients risk factors of health care-associated infection in elderly patients: a retrospective cohort study performed at a tertiary hospital in china clinical relevance of age-related immune dysfunction the ministry of public health. the nosocomial infections diagnosis criterion real-time automatic hospitalwide surveillance of nosocomial infections and outbreaks in a large chinese tertiary hospital the prevalence of health care-associated infection in older people in acute care hospitals nosocomial infections: epidemiology, prevention, control and surveillance healthcare-associated infections in the neurological intensive care unit: results of a -year surveillance study at a major tertiary care center current prevention and control of health care-associated infections in long-term care facilities for the elderly in japan incidence of healthcare-associated infection in elderly hospitalized patients at a hospital in hunan province an implementation on the social cost of hospital acquired infections deaths among the elderly with icu infections epidemiology and outcome of ventilator-associated pneumonia in a heterogeneous icu population in qatar international nosocomial infection control consortium report, data summary of countries for - : device-associated module risk factors for catheter-associated urinary tract infection in italian elderly establishing catheter-related bloodstream infection surveillance to drive improvement publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the many healthcare professionals who assisted with the surveillance study in our hospital.authors' contributions xz contributed to implementation of the investigation, data analysis, and manuscript drafting; lhw designed and organized the study. nw and jlz assisted with data analysis; whm, hjz, and xh contributed to data collection. all authors have read and approved the manuscript. the datasets generated and/or analyzed during the current study are not publicly available due the data copyright protection of the author's institute, but are available from the corresponding author on reasonable request. the study design was approved by the ethics review board of xuanwu hospital, capital medical university. the director of the hospital infection management division and the director of the information center of xuanwu hospital at capital medical university grant permission to access the raw data of the study. not applicable. the authors declare that they have no competing interests. key: cord- -pwz rde authors: kalugalage, thilini; rodrigo, chaturaka; vithanage, thamal; somaratne, pranitha; de silva, h janaka; handunnetti, shiroma; rajapakse, senaka title: low serum total nitrite and nitrate levels in severe leptospirosis date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: pwz rde background: the relationship between inducible nitric oxide synthatase activity and disease severity in leptospirosis is unclear. nitric oxide is converted to nitrites and nitrates, thus nitrite and nitrate levels (no(x)) in serum are considered surrogate markers for nitric oxide. no(x) are excreted through the kidneys, and elimination is diminished in renal impairment. we assessed the correlation of no(x) with disease severity in patients with leptospirosis, compared with healthy controls and non-leptospirosis fever patients. methods: all patients admitted over a two-month period to the national hospital, colombo, sri lanka with a clinical picture suggestive of leptospirosis were included. leptospirosis was confirmed by the microscopic agglutination test (titre≥ ). severe leptospirosis was defined by the presence of two or more of the following criteria: jaundice (bilirubin> . μmol/l), oliguria (urine output < ml/day), serum creatinine> μmol/l or blood urea > . mmol/l, or the presence of organ dysfunction. non-leptospirosis fever patients and healthy volunteers were used as control groups. no(x) levels were measured using a modified griess reaction. results: forty patients were confirmed as having leptospirosis and of them had severe disease. no(x) levels were significantly higher in confirmed leptospirosis patients compared to healthy controls, mat equivocal patients and non-leptospirosis fever patients (p< . ). no(x) concentrations were also significantly higher in patients with severe compared to mild leptospirosis (p< . ). once no(x) levels were corrected for renal function, by using the ratio no(x)/creatinine, no(x) levels were actually significantly lower in patients with severe disease compared to other patients, and values were similar to those of healthy controls. conclusions: we postulate that high nox levels may be protective against severe leptospirosis, and that finding low nox levels (when corrected for renal function) in patients with leptospirosis may predict the development of severe disease and organ dysfunction. leptospirosis is a zoonotic illness that has a high morbidity and mortality in the tropics [ ] . it is caused by a spirochaete of the genus leptospira, which is now found to have at least nine pathogenic species and over serovars. the global burden of leptospirosis is difficult to quantify due to under-reporting and difficulties in establishing a serological diagnosis. however, it is estimated that in endemic areas (localized geographical areas in central america, indian subcontinent, oceania, and the caribbean), the incidence of leptospirosis can be as high as clinical infections per , of population per year (in contrast to per , per year in non-endemic areas) [ ] . the majority of infections are asymptomatic or may pass off as a flu like illness. however, severe leptospirosis can be fatal. severe leptospirosis is associated with adult respiratory distress syndrome (ards), pulmonary haemorrhage, acute kidney injury, liver impairment, and multi-organ dysfunction syndrome (mods) [ , ] . the case fatality in severe leptospirosis (weil's disease) can be as high as % [ ] . predictors of disease severity can be useful to the clinician for anticipating complications. factors predicting mortality in severe leptospirosis as reported in various studies worldwide have been reviewed by rajapakse et al. [ ] under the categories of predisposition, insult, response and organ dysfunction (similar to the piro model used to predict mortality in severe sepsis). although there was insufficient data to develop a scoring system for mortality prediction, it was noted in this review that serum markers of acute inflammation (tumour necrosis factor-α, interleukin- , interleukin- ) have not been adequately assessed as prognostic markers. these pro-inflammatory cytokines lead to an increase in the activity of inducible nitric oxide synthatase (inos) to synthesize nitric oxide (no) which is toxic to the bacterium. the role of inos and no production in inflammation has not been clearly determined in leptospirosis; in fact its significance in severe sepsis [ ] and other infections such as malaria [ ] [ ] [ ] [ ] is subject to much debate [ , ] . while its primary role is to combat infection, no levels have been shown to be elevated in severe leptospirosis [ ] , leading to the postulate that high no levels may be involved in the pathogenesis of organ dysfunction in leptospirosis. on the other hand, increased inos activity may actually protect against organ dysfunction. no is an extremely volatile compound that is difficult to measure in serum. it is quickly converted to nitrite (no ˉ) and nitrate (no ˉ) [ ] . it is estimated that more than % of nitrite in whole blood gets converted to nitrate within one hour [ ] . thus, the total blood levels of nitrite and nitrate (no x ) could be considered to be a surrogate marker of serum no levels. no x levels in blood are affected by the amount of ingested nitrates; to control for this, measurements should ideally be made after an overnight fast [ ] . furthermore, no x is excreted renally, thus no x clearance is reduced in the presence of renal impairment [ ] . to correct for this, the use of the ratio of serum no x /creatinine has been suggested to be a more accurate marker of inos activity than crude no x levels, and was used in the study by anstey et al. [ ] to correct no x levels for renal function in patients with malaria. although other factors could influence creatinine levels, and therefore this correction factor too, creatinine levels are the standard index used for assessment of renal dysfunction in acute kidney injury [ ] . therefore the formula no x /creatinine is currently the only practical formula available to correct no x levels for renal function. we previously demonstrated through a preliminary study that serum nitrite levels are elevated in patients with acute leptospirosis compared to healthy controls [ ] . however, the sample size was inadequate to determine a correlation with disease severity. the aim of this study was to determine the relationship between no x (i.e., total nitrite and nitrate) levels in the blood (as a marker of inos activity) and disease severity in leptospirosis. if such a correlation exists, no x could potentially be useful as a prognostic marker. the objectives of our study were to a) determine serum no x (nitrate and nitrite) levels in patients with confirmed leptospirosis, b) compare serum no x levels in leptospirosis patients with mild and severe disease, healthy controls and non-leptospirosis fever patients (nlfs), and c) seek a correlation between serum no x levels and disease severity after correcting for impaired renal clearance. patients suspected to have leptospirosis were selected from the national hospital of sri lanka (nhsl). the nhsl is the premier tertiary care center in sri lanka, with a bed strength of over . it is one of the few state sector centers with facilities for haemodialysis in the country, and most patients with acute kidney injury are transferred to nhsl for further management. it is also the major hospital that covers the heavily populated western province which is an endemic area for leptospirosis [ ] . the annual incidence of leptospirosis in the western province for the year was per , population. all patients with a febrile illness who were clinically suspected of having leptospirosis admitted to medical wards in nhsl during a two-month period from rd june to th august were included in to the study, after obtaining informed consent. the clinical criteria to define a probable case of leptospirosis were adopted from the world health organization (who) surveillance criteria [ ] . microscopic agglutination titre (mat) is the most widely used confirmatory test for leptospirosis, although the duration that mat remains positive after infection is not clearly known [ ] . nonetheless a mat titre of > is generally considered to indicate acute infection even in areas of high endemicity, in the setting of a clinical diagnosis of leptospirosis. based on mat results, patients were retrospectively categorized as confirmed leptospirosis (mat titre≥ , mat equivocal (mat titre and ), and nonleptospirosis fever (mat negative). healthy volunteers (mat negative) were selected as controls. serial haematological and serum biochemical measurements of patients were made during the illness, and included leukocyte and platelet counts, blood culture, erythrocyte sedimentation rate (esr), serum potassium, serum sodium, aspartate aminotransferase, alanine aminotransferase, creatinine, blood urea and indirect, direct and total bilirubin levels. patients with severe leptospirosis were defined as those presenting with acute fever and clinical symptoms compatible with leptospirosis (confirmed serologically by a positive mat result) with two or more of the following criteria: jaundice (bilirubin> . μmol/l), oliguria (urine output < ml/day), serum creatinine> μmol/l or blood urea > . mmol/l [ ] , or the presence of acute organ dysfunction. serum no x levels were determined in all patients who were recruited in to the study. no x levels were measured in blood obtained early morning. total no x levels were used as a surrogate marker for serum nitric oxide levels [ ] . the blood samples collected were centrifuged, and separated sera were stored at − °c. during analysis, the serum samples were first thawed, then deproteinized by adding zinc sulfate. deproteinization is a necessary step in the measurement of serum nitrite concentrations [ ] . ten microlitres of . g/ml zinc sulphate solution was added to ml of serum, vortexed for minute, and centrifuged at , g for minutes at room temperature (rt= °c, i.e., the controlled temperature in the laboratory). the supernatant was pipetted out and centrifuged again at , g for minutes. the clear serum ( μl) was applied in duplicate to a -well elisa plate, μl of vanadium (iii) chloride ( mg/ ml) was added to each well (for reduction of nitrate to nitrite) followed by the addition of μl of griess reagent (equal mixture of % sulphanilamide in % phosphoric acid and . % n-( -naphthyl) ethylenediamine hydrochloride in distilled water). the plates were incubated for minutes at rt and the optical density was measured at nm using the elisa reader (bio-tek instruments inc, usa). a two-fold dilution series ( . - μm) of nano was prepared from μm nano solution using distilled water. each dilution ( μl) was mixed with an equal volume of griess reagent, and the optical density (od) was measured at nm. a standard curve was plotted against optical density and nano concentration. intra-assay coefficient of variability (cv) was . % and inter-assay cv was . %, which indicated good precision and repeatability. ethics approval was obtained from the ethics review committee of the faculty of medicine, university of colombo and the ethics review committee of the nhsl. informed written consent was obtained from all patients and healthy controls prior to recruitment to the study. statistical analysis was performed using spss w version . . results were expressed as mean ± sd. data were analyzed by applying a one-way anova with the bonferonni/dunn post-hoc correction for multiple comparisons. multivariate regression analysis was performed to determine the relationship between no x and other biochemical markers of severity. statistical significance was defined as p < . . on the basis of clinical features, patients were recruited to the study with probable leptospirosis. of these, were confirmed as leptospirosis with mat (titre of ≥ ). there were also patients who had equivocal mat titers and with non-leptospirosis fever (mat negative). twenty three mat negative healthy individuals were also recruited as controls. the mean ± sd of the duration between onset of symptoms and obtaining serum samples in the confirmed leptospirosis patients was . ± . days. the baseline characteristics of these groups are shown in table . of the patients with confirmed leptospirosis, were categorized as having severe disease according to the criteria mentioned above. one patient with severe disease died and all others survived. the laboratory investigations for each category of patients and for nonleptospirosis fever patients are summarized in table . we first compared no x levels of confirmed leptospirosis patients (mild and severe) mat equivocal patients, nlf patients, and healthy controls (table and figure , table ). significantly higher no x levels were observed in confirmed leptospirosis patients when compared against healthy controls, mat equivocal patients and non-leptospirosis fever patients (p< . ). no x concentrations in patients with severe leptospirosis were also significantly higher than in those with mild leptospirosis (p= . ). there was no significant difference in values between mat equivalent patients and non-leptospirosis fever patients. notably, many of these differences were not observed when the serum nitrite levels alone were considered. in order to correct for renal impairment, we calculated the serum no x /creatinine ratio in each of the groups. the comparisons of these groups are shown in table and figure (also table ). corrected no x were lower among patients confirmed to have leptospirosis compared to healthy controls, although the difference was marginal. there was no significant difference seen in corrected no x levels among confirmed leptospirosis patients, mat equivocal patients and nlfs. the most significant finding was that corrected no x levels were markedly lower among patients with severe leptospirosis compared with both mild leptospirosis and mat-equivocal patients. on the other hand, no difference was seen in corrected no x levels in severe leptospirosis patients, healthy controls and nlf patients. corrected no x levels were significantly lower among healthy controls compared with mild leptospirosis patients and mat equivocal patients, but no significant difference was shown between healthy controls and nlfs. elevation of serum no x levels during acute infections such as dengue, malaria and leptospirosis has been shown in previous studies, however the main criticism of these studies has been the lack of correction of no x concentrations for renal function. as mentioned above, no x is excreted predominantly by the kidneys, and no x levels have been shown to be elevated in the presence of renal impairment. we demonstrated that crude no x levels are significantly elevated in leptospirosis, with higher levels correlating with severity of the illness. however, once no x levels were corrected for renal function, they were significantly lower in severe leptospirosis. if no x levels reflect inos activity, this finding suggests that inos activity is diminished in patients developing severe disease. furthermore, the results suggest that inos activity is similar in severe leptospirosis and healthy controls despite the heavy inflammatory response in the former group. we postulate that this indicates that a blunted inos response is seen in severe leptospirosis; whether this is the result of the inflammatory response that occurs, or whether a diminished inos response plays a role in the genesis of severe leptospirosis and organ dysfunction remains to be elucidated. conversely, it is possible that higher levels of nox are protective against organ dysfunction. admittedly, the relationship between inos activity and inflammation is extremely complex. nonetheless, similar patterns have been seen in malaria. al yaman et al. [ ] described an association between high levels of no x levels and coma in children with cerebral malaria. similarly kremsner et al. [ ] showed that higher levels of plasma no were seen in severe malaria; however they also demonstrated that higher levels of no was associated with accelerated recovery. the criticism of both these studies was that crude no levels were considered, and no correction was made for deranged renal function (serum creatinine levels in the patients were not provided), and that the elevated no levels could simply be related to reduced excretion of no due to impairment of renal function [ ] . in fact, anstey et al. [ ] demonstrated that when no x levels were corrected for renal function, using the ratio no x /creatinine, no x levels showed an inverse relationship with the severity of malaria. corrected no x levels were lowest in patients with severe disease, while higher levels were seen in controls as well as those with asymptomatic disease, suggesting that high no x levels may protect against severe malaria. furthermore, in a mouse model, gramaglia et al. [ ] demonstrated that low no bioavailability contributes to the genesis of experimental cerebral malaria. although many confounding factors could be present, the finding that corrected no x levels are low in clinically severe leptospirosis is itself of significance. whether no x levels are low in severe leptospirosis as a result of endothelial dysfunction resulting from severe disease, or whether individuals in whom inos activity does not increase in response to infection are more likely to develop severe disease remains unclear. another possibility is that inos activity is normal, but the no produced is rapidly removed by other molecules such as reactive oxygen species and haemoglobin. the measurement of serial no x levels and correlating these with the onset of organ dysfunction in patients with leptospirosis and in experimental models is likely to provide further insight into this issue. the finding that corrected no x levels in nlfs were similar to those seen in severe leptospirosis is interesting. nlfs presumably represent a heterogenous group, and a significant number in this group had renal dysfunction. whether this suggests a decrease in no x levels in infections which result in organ dysfunction is difficult to determine from this study. clinical features similar to those of severe leptospirosis occur in many other infections, such as dengue, hanta-virus and acute hepatitis; some of the patients in the nlf and mat equivocal groups did present with clinical features similar to severe leptospirosis; however numbers were too small for any realistic comparisons of no levels in these subgroups to be possible. the griess reaction explained in the methodology is specific for nitrite levels in blood. therefore in order to measure the nitrates, they had to be converted to nitrites. this conversion can be achieved with either a chemical or an enzymatic reduction and we opted for the chemical measure by treating the sera with vanadium(iii) chloride [ , ] . the use of vanadium (iii) chloride offers a low cost method compared to the enzymatic reduction and therefore the modified griess assay is an inexpensive, simple, rapid, accurate and a sensitive method for measurement of no x levels, better suited method for resource limited settings in developing countries. one of the limitations of this study is the confounding factors that can affect serum no x levels such as age and diet [ ] . however, the mean ages of the subjects in the different groups were comparable, and dietary influence was minimized by collecting samples in the early mornings prior to the intake of food. it was also possible that patients with severe disease had a lower dietary contribution of nitrites. this is very difficult to quantify in a clinical study, and no standard methods for quantification exist. however in our study, we compared the incidence and severity of symptoms such as nausea, vomiting and loss of appetite, and there was no difference in the incidence of these symptoms in severe disease compared with non-severe disease. all patients, even the sickest, were able to eat and drink. there was a gender bias in the sample with more males than females. however, this is a well established epidemiological fact in leptospirosis in sri lanka, as it is the males who engage more in outdoor activities such as farming which is a major occupational risk factor for leptospirosis [ ] . of the serological tests to diagnose leptospirosis, mat is the preferred method, and it is also the test recommended by the epidemiology unit of the ministry of health in sri lanka [ ] . however, mat serology may be insensitive in early acute-phase specimens [ ] . moreover, patients with fulminant leptospirosis may die before seroconversion occurs. a four-fold rise in mat titer would have helped to differentiate patients with true leptospirosis in the mat equivocal group [ , ] . however, many patients did not return for follow up visits after discharge and we had to restrict the leptospirosis confirmed group to those with high mat titers ≥ . we did not obtain serial no x measurements in patients (due to logistical difficulties and limited resources) which would have enabled us to predict the earliest point at which no x levels would start to differ in those with severe disease, thus establishing the approximate earliest point it would be useful as a predictive marker. based on the results of this initial study a larger multi-centre study was designed by us, and is currently in progress. this study shows that crude serum no x levels were significantly elevated in sri lankan patients with leptospirosis compared to healthy controls and nonleptospirosis fever patients; however once no x levels were corrected for serum creatinine, the relationship between no x levels and disease severity was shown to be strikingly different. corrected no x levels were significantly suppressed in patients with severe leptospirosis. thus, no x levels in patients with leptospirosis may be useful to predict severe disease, i.e., the presence of low no x levels (after correction for renal function) in leptospirosis may predict the development or organ dysfunction. we also demonstrated that chemical conversion of nitrates to nitrite with vanadium (iii) chloride and a global research agenda for leptospirosis epidemic of leptospirosis: an icu experience the kidney in leptospirosis predictors of lethality in severe leptospirosis in urban brazil predictors of mortality in severe leptospirosis; a concept paper on developing a clinically relevant classification is plasma arginine concentration decreased in patients with sepsis? a systematic review and meta-analysis enhanced production of reactive nitrogen intermediates in human and murine malaria high plasma levels of nitrogen oxides are associated with severe disease and correlate with rapid parasitological and clinical cure in plasmodium falciparum malaria association between serum levels of reactive nitrogen intermediates and coma in children with cerebral malaria in papua new guinea nitric oxide in tanzanian children with malaria: inverse relationship between malaria severity and nitric oxide production/nitric oxide synthase type expression reactive nitrogen intermediates and cerebral malaria nitrate levels in malaria high serum nitric oxide levels in patients with severe leptospirosis nitrite and nitrate determinations in plasma: a critical evaluation relation between pro-and anti-inflammatory cytokines and the production of nitric oxide (no) in severe sepsis measuring nitric oxide production in human clinical studies effect of renal function on serum nitrogen oxide concentrations acute kidney injury network: report of an initiative to improve outcomes in acute kidney injury serum nitrite levels in sri lankan patients with leptospirosis an interim analysis of leptospirosis outbreak in sri lanka- . colombo epidemiology unit, ministry of health care and nutrition world health organization: human leptospirosis. guidance for diagnosis surveillance and control. geneva: world health organization assessment of the efficacy of an igm-elisa and microscopic agglutination test (mat) in the diagnosis of acute leptospirosis a rapid, simple spectrophotometric method for simultaneous detection of nitrate and nitrite protien precipitation methods evaluated for determination of serum nitric oxide end products by the griess assay low nitric oxide bioavailability contributes to the genesis of experimental cerebral malaria age-associated changes in nitric oxide metabolites nitrite and nitrate two methods for rapid serological diagnosis of acute leptospirosis low serum total nitrite and nitrate levels in severe leptospirosis we thank ms ratnamali perera and ms thameesha gamage of the department of microbiology, medical research institute, colombo, sri lanka for their help with mat testing, the staff of national hospital sri lanka for helping with patient data and providing patient care, and the institute of biochemistry, molecular biology and biotechnology, university of colombo for facilitating this study. measuring of no x levels (modified griess reaction) is a relatively cheap assay technique that can be employed in resource limited settings.abbreviations no: nitric oxide; no x : total nitrites and nitrates; mat: microscopic agglutination titre; inos: inducible nitric oxide synthatase; nlf: non-leptospirosis fever; who: world health organisation; mods: multi-organ dysfunction syndrome; elisa: enzyme-linked immunosorbent assay; nhsl: national hospital, colombo, sri lanka. the authors declare that they have no competing interests.authors' contributions sr, smh, hjds and tk developed the initial concept for the study. tk and tv collected clinical data and samples. tk and smh performed the laboratory measurements. ps performed leptospirosis diagnostic serology. sr, smh, tk and cr analysed the data. tk, cr, smh and sr wrote the first draft. all authors read and approved the final manuscript. key: cord- -hn o authors: pivette, mathilde; mueller, judith e; crépey, pascal; bar-hen, avner title: drug sales data analysis for outbreak detection of infectious diseases: a systematic literature review date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: hn o background: this systematic literature review aimed to summarize evidence for the added value of drug sales data analysis for the surveillance of infectious diseases. methods: a search for relevant publications was conducted in pubmed, embase, scopus, cochrane library, african index medicus and lilacs databases. retrieved studies were evaluated in terms of objectives, diseases studied, data sources, methodologies and performance for real-time surveillance. most studies compared drug sales data to reference surveillance data using correlation measurements or indicators of outbreak detection performance (sensitivity, specificity, timeliness of the detection). results: we screened articles and included in the review. most studies focused on acute respiratory and gastroenteritis infections. nineteen studies retrospectively compared drug sales data to reference clinical data, and significant correlations were observed in of them. four studies found that over-the-counter drug sales preceded clinical data in terms of incidence increase. five studies developed and evaluated statistical algorithms for selecting drug groups to monitor specific diseases. another three studies developed models to predict incidence increase from drug sales. conclusions: drug sales data analyses appear to be a useful tool for surveillance of gastrointestinal and respiratory disease, and otc drugs have the potential for early outbreak detection. their utility remains to be investigated for other diseases, in particular those poorly surveyed. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. since the mid- s and the raise of concerns about bioterrorism and emerging diseases, non-diagnosis-based data have increasingly been used for routine disease surveillance and outbreak detection [ ] . the cdc defined "syndromic surveillance" as an investigational approach where health department staff, assisted by automated data acquisition and generation of statistical alerts, monitor disease indicators in real-time or near real-time to detect outbreaks of disease earlier than would otherwise be possible with traditional public health methods [ ] . in such efforts, different registries have served as data sources for public health surveillance [ , ] , including data on absenteeism at work or school [ ] , calls to health helplines [ , ] , emergency department consultations [ , ] , ambulance dispatching [ ] , or drug sales. although unspecific, such data sources can have the advantage over diagnosis-based surveillance of providing information within short delays since the event and in readily available electronic form for relatively low-cost, while capturing large parts of the population. drug sales data analysis may overcome the limitation of poor specificity when groups of drugs are exclusively used for the disease or disease syndrome of interest. furthermore, drug sales data may earlier capture changing population health status, as over-the-counter (otc) sales and a dense network of pharmacies in most developed countries make drugs easily accessible to patients at the earliest appearance of their symptoms. despite this potential interest, no state of the art of drug sales based surveillance is available to date. the present systematic literature review therefore summarized the evidence for an added value of drug sales data for infectious disease surveillance. we limited the scope of the review to infectious diseases, as they represent a public health problem for which early and valid signal detection is of particular concern, in light of potentially rapid emergence and opportunity for control interventions. we conducted a literature search from up to june to identify relevant peer-reviewed articles regarding surveillance of infectious diseases based on drug sales data. prisma guidelines were followed in the reporting of the review [ ] . published articles were searched for on electronic databases (pubmed, embase, scopus, lilacs, african index medicus, cochrane library), using combinations of the following key words: ("surveillance" or outbreak detection or warning system) and (overthe-counter or "prescription drugs" or pharmacy or (pharmaceutical or drug or medication) sales). the search was limited to articles in english or french. there were no limitations on study settings. to be included in the review, articles had to describe, test, or review an infectious disease surveillance based on drug sales data; and be original research that presented new data and results. we excluded studies that monitored chronic diseases, as well as prevalence studies whose purpose was not epidemic detection. one reviewer screened and evaluated the titles and abstracts. articles were widely included in a first stage. the full-text review and the final selection of the articles were made by two reviewers. we reviewed and described the articles in terms of objectives, diseases studied, data sources, methodologies, and performance for real time surveillance. to describe methods and results, we separated the articles into three groups based on their main objective: descriptive retrospective studies, drug selection studies, and prediction studies. outcomes selected to compare drug sales data to reference surveillance data of the corresponding disease were correlation measurements (strength and timeliness of the correlation) and indicators of outbreak detection performance (sensitivity, i.e. ability to identify true outbreaks; specificity, i.e. ability to identify true negative and timeliness of the detection). we screened a total of articles, of which were included in the final review. the search and selection process is presented in figure . articles excluded based on fulltext review (no drug sales data, no infectious disease, no outbreak detection) n= figure flow chart of study selection process in a systematic review of drug sales data analysis for syndromic surveillance of infectious diseases. three types of studies were defined: retrospective descriptive studies, drug selection studies and prediction studies. nineteen of the studies were descriptive retrospective studies assessing the strength of the correlation between drug sales and reference surveillance data of the corresponding disease or evaluating outbreak-detection performance [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . five studies used statistical algorithms to select groups of drugs that were closely associated with clinical surveillance data of a given disease and that would be most appropriate for future drugsales-based surveillance [ , [ ] [ ] [ ] [ ] . in a third group of three studies, the authors developed and evaluated statistical models to predict clinical surveillance data based on drug sales data [ ] [ ] [ ] . table summarizes the studies in terms of their general characteristics. most of the studies focused on respiratory illnesses ( studies) [ , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] or gastrointestinal illnesses ( studies) [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , ] . only two other studies evaluated surveillance of pertussis [ ] and syphilis [ ] . most of the studies were set in the united states (n = studies, %), followed by canada (n = ), france (n = ), japan (n = ), the netherlands (n = ) and england (n = ). only one study was conducted in more than one country [ ] . in most retrospective studies, data were collected specifically for the purpose of the study from a sample of pharmacies [ , [ ] [ ] [ ] or from retailers [ , , , ] . for example, in a canadian study [ ] , electronic data were provided by one major retailer for all of their pharmacies in the study area. automatically compiled data sources were used in all the drug selection and prediction studies and in some retrospective studies. drug sales data were routinely collected in samples of a city's or country's pharmacies. such routine data collection systems were mainly implemented by research or public health groups, such as the johns hopkins applied physics laboratory [ , , , ] , the new york city department of health [ ] , the national institute of infectious diseases in japan [ ] , or the real-time outbreak and disease surveillance laboratory at the university of pittsburg [ ] . data are available the day after the day's sales in those systems. in eight other studies, private marketing companies had automatically aggregated and made available drug sales data from a sample ( - %) of pharmacies in a given city or country [ , , , [ ] [ ] [ ] [ ] ] . nineteen studies retrospectively compared drug sales data to gold standard reference data of the disease. details are given in table . reference data of the disease included medical case reports [ ] [ ] [ ] , diagnostic registries of microbiological laboratories [ , , , ] , hospital admission or discharge data [ ] [ ] [ ] [ ] [ ] ] , or clinical emergency department data [ ] [ ] [ ] . the selection of indicator drugs in these studies was based on the literature or expert opinion. for example, edge et al. [ ] selected all anti-nauseant and antidiarrheal otc drugs for gastrointestinal surveillance. in stirling et al. [ ] , pharmacists determined which common antidiarrheal drugs they would report. two methods were commonly used to compare drugsale and diagnostic data time series: correlation analysis and signal detection comparison ( table ). ten studies used cross-correlation function to measure the similarity of two curves and to determine the time lag at which the correlation between the datasets is maximized. cross-correlation is a standard method to determine the time delay between two signals. in three studies, only correlation between the time series was examined without analyzing time-lagged relationship. six studies used aberration detection methods to evaluate whether and by how long the date of signal detection by drug sales precedes the signal based on diagnostic data. the signal definition for aberration detection was based on either a simple threshold to define alerts [ ] or more complex algorithms such as the serfling method [ ] , arima models [ ] , the simple moving average method (ma), the cumulative sum method (cusum) [ , ] , or the exponentially weighted moving average (ewma) [ ] . these studies assessed the performance in terms of sensitivity, specificity and timeliness of disease outbreak detection. five other studies [ , [ ] [ ] [ ] ] only evaluated whether drug sales showed a significant increase during a known epidemic period. twelve of studies evaluating otc sales retrospectively found significant correlations or a significant increase in drug sales [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] . only two studies didn't found any consistent correlation. for example, das et al. [ ] found a poor correlation between otc antidiarrheal drug sales and emergency department visits for diarrhea in new york city, with an r of . . they found however an increase in sales during a known outbreak of norovirus. otc drug sales preceded clinical data in three of eight studies that analyzed timeline correlations [ , , ] . for example, in hogan et al. [ ] , the correlation coefficient between electrolyte sales and hospital diagnoses of respiratory and diarrheal illness was . ( % ci, . - . ) when drug sales were assumed to precede clinical diagnosis data by . weeks. outbreaks were detected with % sensitivity and specificity in of studies that analyzed signal detection [ , , ] . drug sales data provided an earlier outbreak signal in two of them [ , ] . in davies et al. [ ] , the rate of cough/cold sales exceeded a threshold of units per week two weeks before the peak in emergency department admissions during three consecutive winters. in hogan et al. [ ] , detection from electrolytes sales occurred an average , weeks earlier than detection from hospital diagnoses of respiratory and diarrheal diseases. six of the seven studies that focused on prescribed drugs found strong correlations (r = . - . ) with clinical reference data or a significant increase in drug sales, without lead time however. the other study [ ] showed that the cusum signal generated for prescriptions for macrolide antibiotics was linked to a pertussis outbreak in a county of new york state. no association was observed between the type of reference data and the time lags observed. an important challenge for drug-sales-based surveillance is identifying relevant indicator drug groups to monitor diseases. five retrieved articles addressed this question. characteristics of the studies are described in table . two studies [ , ] developed methods to find homogeneous groups of otc products. the authors used unsupervised clustering algorithms for aggregating otc products in groups sharing similar sales histories. for example, magruder et al. [ ] first assigned otc products for respiratory diseases to subgroups qualitatively based on indication, dose form, and age group. a stepwise hierarchical clustering algorithm was then used to form categories sharing a similar sales history, leading to a set of product categories. in two studies [ , ] , the authors developed procedures to identify the drugs correlating with disease incidence. clusters were formed specifically for a particular disease. in pelat et al. [ ] , a hierarchical clustering procedure was applied to the time series of all therapeutic classes and the acute diarrhea incidence rate reported by a network of general practitioners. four therapeutic classes were found to cluster with diarrhea incidence and an algorithm based on the selected drugs allowed the detection of epidemics with a sensibility of %, a specificity of % and a timeliness of . weeks before official alerts. in three studies [ ] [ ] [ ] , the authors developed models to predict clinical data based on drug sales data. vergu et al. [ ] used a poisson regression model on selected otc sales to forecast influenza-like illness (ili) incidence as recorded by a sentinel network of general practitioners. the forecast at the national level - weeks ahead showed a strong correlation with observed ili incidence (r = . - . ). najmi et al. [ ] used least mean square filtering methods to estimate the incidence of emergency room consultations for respiratory conditions from past and present sales of groups of cold-remedy sales. in a later article [ ] , they succeeded in extending the estimation algorithm for predicting increases in clinical data several days in advance. the evidence gathered in this systematic literature review suggests that drug sales data analysis can be a useful tool for surveillance of acute respiratory and gastrointestinal infections. as could be expected, prescribed drug sales data were strongly correlated with clinical case reporting. no lead time was observed, which is consistent with the fact that patients purchase drugs after seeing a healthcare professional. analysis of prescribed drug sales data may nevertheless have an additional utility for epidemic detection, as these data might be available with a shorter delay than clinical surveillance data [ ] . a high correlation between otc drug sales data and reference surveillance data were found in almost all the retrospective studies. several studies also showed that otc drug sales can serve as an early indicator of disease epidemics. patients may buy nonprescription drugs during the early phase of illness when they become symptomatic, before consulting a health practitioner [ ] . a surveillance system based on drug data should ideally detect all the outbreaks, rapidly, with a low false alert rate. however, few studies in the review determined the sensitivity and specificity of the outbreak detection and those aspects should be analyzed in more details in future studies. surveillance based on otc drug sales could be particularly relevant for diseases whose prodromal phase persists for several days before the onset of more severe symptoms. for example, the early stages of dengue fever symptoms are nonspecific (fever, headache, myalgia, arthralgia, etc.) [ ] . the occurrence of grouped cases could trigger an excess of nonspecific drug sales over baseline levels, which in turn could provide an early warning of outbreak in an endemic area. results from drug selection studies showed that it is possible to identify groups of products strongly associated with incidence data, which can then be used to predict future trends in clinical data and help public health authorities to prepare response planning. such product selection procedures, however, depend on the existence of large clinical surveillance databases of the diseases concerned. similarly, the validity of drug sales data analysis has been evaluated mainly for two disease groups, respiratory and gastrointestinal illness, for which clinical reference data, used as the gold standard, are readily available. pertussis and syphilis have been evaluated in only one study each, and still require further confirmation. the concept of drug-based surveillance therefore needs to be validated for other infectious diseases. all the studies were conducted in developed countries or area. surveillance based on drug sales data requires electronic information systems for routine data analysis. besides, its implementation requires that the population has access to the health care system and mainly buy drugs in pharmacies. this could limit the use of drug based surveillance systems in developing countries. by improving the timeliness of epidemic detection compared to clinical data and giving information from a larger part of the population, drug sales data can be an additional source of information for already monitored diseases. besides, drug sales data analysis could have its greatest value in the surveillance of diseases for which clinical surveillance is cumbersome and costly, or where substantial under-reporting is suspected. to confirm the selected drug group as a valid proxy of disease, clinical surveillance may be conducted for a defined period in a representative population. examples of diseases for which this would be useful are typically varicella, urinary infections, allergies/asthma, and parasitic diseases. ideally, the drugs to be monitored should be specific to the disease and widely used to treat it in order to maximize the sensitivity of the signal. for example, benzylpenicillin benzathine . mui is the quasi exclusive treatment for syphilis infection [ ] and is a good candidate. in contrast, the treatment of measles is mostly symptomatic without a specific drug, which makes this disease unattractive for this approach. another limitation applies to diseases that are usually treated in hospitals or specialized centers, such as tuberculosis. surveillance based on drug sales, may not be appropriate to accurately estimate incidence of diseases, as the source population size is not precisely known. moreover, it may be difficult to link the number of drug packages sold to the number of patients with disease. however, the method is very efficient to determine temporal dynamics of a situation and to detect abnormal phenomena. surveillance based on drug sales is therefore well adapted to diseases with seasonal variations such as norovirus gastroenteritis, influenza and other infectious respiratory agents, or community outbreaks (foodborne illnesses, waterborne illnesses, hepatitis a, etc.). drug sales can be influenced by store promotions, sales period (holidays, weekends), and the media. also, we do not know whether people buy medications to treat a disease they currently have or a disease they fear they may have in the near future. for example, during the media coverage of avian influenza a (h n ) in the us, an increase in antiviral medications sales was observed [ ] , which corresponded to stockpiling behavior of the population. health-seeking behaviors also vary by demographic, social, cultural, and economic factors. a survey [ ] in canada analyzed the healthcare-seeking behaviors of patients with acute gastroenteritis. they found significant differences (patient age and sex) between the patients who used otc drugs and those who did not. consequently, factors that prompt self-medication should also be taken into account. the usefulness of drug sales based surveillance is also dependent on the available resources and the organization of the health care system. otc drug sales surveillance is for example less relevant in countries where reimbursement rate are high and patients mainly get prescribed drugs. population mobility, particularly in tourist areas, may lead to an increase in remedy sales, which could wrongly be interpreted as a disease outbreak. inversely, patients with high geographical mobility may not be included in the region of study and lead to an underestimation of the magnitude of an epidemic. despite some limitations, routine collection and analysis of drug sales data are likely to be developed in the coming years. many automated surveillance systems that collect drug data the day after the sales have been implemented in the last decade [ , , , ] . they allow a rapid assessment of the public health situation. early detection of outbreaks allows public health authorities to set up epidemic investigations and control measures sooner. most studies included in this review were published after the year , with their number increasing recently. they illustrate the need for improved surveillance systems, evidenced by recent public health crises (e.g., anthrax in , the sars outbreak in , the a/h n influenza pandemic in , etc.). drug sales data present indeed many advantages in terms of public health surveillance. data can be obtained in a real-time manner and usually cover a large portion of the population. data collection may be exhaustive, without selection of specific sales, and allows the simultaneous monitoring of a large number of diseases, especially new or emerging diseases. although non-specific, drug sales data are directly linked to patients' health conditions. drug sales data are therefore more specific than other syndromic surveillance data, such as tracking search patterns on the web and are likely to reflect more accurately disease activity in the population. moreover, it should be noted that alternative sources of data for disease surveillance are currently under development. healthcare management databases that can provide exhaustive information on drug consumption and diagnosis, as the dossier médical personnel [ ] in france, are promising tools for disease surveillance. our review may be affected by a publication bias since studies unable to show correlations between drug sales and reference data may have been less published. in addition, selections bias may have occurred in the studies. indeed, some studies in the review were based on a limited number of pharmacies and/or a limited study period (e.g. less than one year). language bias may exist as we were not able to identify studies published in languages other than english and french. the review focused on the temporal dynamics of infectious disease; consequently, further analyses are required to determine the capacity of these systems to efficiently monitor other aspects of infectious diseases such as spatial spreading. this review suggests that the analysis of drug sales data is a promising method for surveillance and outbreak detection of infectious diseases. it has the potential to trigger an outbreak alert earlier than most surveillance systems. however, the main challenges consist in the appropriate selection of indicator drug groups and the validation of this approach for diseases for which no or poor quality clinical surveillance data exists. the usefulness of the approach also depends on the available resources and the organization of the health care system. drug sales databases with real-time or near real-time data transmission are available in several countries; future studies should be encouraged to expand their use on other infectious diseases. what is syndromic surveillance? mmwr morb mortal wkly rep cdc: framework for evaluating public health surveillance systems for early detection of outbreaks: recommendations from the cec working group review of syndromic surveillance: implications for waterborne disease detection absenteeism in schools during the influenza a(h n ) pandemic: a useful tool for early detection of influenza activity in the community? using ontario's "telehealth" health telephone helpline as an early-warning system: a study protocol using nurse hot line calls for disease surveillance disease outbreak detection system using syndromic data in the greater washington dc area assessment of a syndromic surveillance system based on morbidity data: results from the oscour network during a heat wave use of ambulance dispatch data as an early warning system for communitywide influenzalike illness preferred reporting items for systematic reviews and meta-analyses: the prisma statement use of medicaid prescription data for syndromic surveillance-new york a practical method for surveillance of novel h n influenza using automated hospital data syphilis surveillance in france monitoring over-the-counter medication sales for early detection of disease outbreaks sales of over-the-counter remedies as an early warning system for winter bed crises syndromic surveillance of gastrointestinal illness using pharmacy over-the-counter sales. a retrospective study of waterborne outbreaks in saskatchewan and ontario syndromic surveillance of norovirus using over-the-counter sales of medications related to gastrointestinal illness detection of pediatric respiratory and diarrheal outbreaks from sales of over-the-counter electrolyte products prediction of gastrointestinal disease with over-the-counter diarrheal remedy sales records in the san francisco bay area evaluation of over-the-counter pharmaceutical sales as a possible early warning indicator of human disease progress in understanding and using over-the-counter pharmaceuticals for syndromic surveillance experimental surveillance using data on sales of over-the-counter medications-japan using oral vancomycin prescriptions as a proxy measure for clostridium difficile infections: a spatial and time series analysis surveillance data for waterborne illness detection: an assessment following a massive waterborne outbreak of cryptosporidium infection pharmaceutical sales; a method for disease surveillance? waterborne cryptosporidiosis outbreak real-time prescription surveillance and its application to monitoring seasonal influenza activity in japan validation of syndromic surveillance for respiratory pathogen activity sales of nonprescription cold remedies: a unique method of influenza surveillance seasonal influenza surveillance using prescription data for anti-influenza medications mining aggregates of over-the-counter products for syndromic surveillance a multivariate procedure for identifying correlations between diagnoses and over-the-counter products from historical datasets a method for selecting and monitoring medication sales for surveillance of gastroenteritis unsupervised clustering of over-the-counter healthcare products into product categories estimation of hospital emergency room data using otc pharmaceutical sales and least mean square filters an adaptive prediction and detection algorithm for multistream syndromic surveillance medication sales and syndromic surveillance implementing syndromic surveillance : a practical guide informed by the early experience value of syndromic surveillance within the armed forces for early warning during a dengue fever outbreak in french guiana in increased antiviral medication sales before the - influenza season factors associated with the use of over-the-counter medications in cases of acute gastroenteritis in hamilton drug sales data analysis for outbreak detection of infectious diseases: a systematic literature review this research was funded by celtipharm (vannes, france) a company specialized in the real time collection and statistical processing of healthcare data (www.celtipharm.orgwww.openhealth.fr), through a doctoral thesis contract for mathilde pivette. mathilde pivette prepares a doctoral thesis under the french framework "cifre" (industrial contract for training through research; www.anrt.asso.fr), in partnership with the company celtipharm (www.celtipharm.org). the other authors declare they have no competing interests. all authors contributed to the study's design. mp and jm carried out the literature search and reviewed articles. mp drafted the manuscript. all authors interpreted the results, revised and approved the final manuscript. key: cord- -vj t hn authors: joffe, michael; wagner, simon d.; tang, julian w. title: case report: a fatal case of disseminated adenovirus infection in a non-transplant adult haematology patient date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: vj t hn background: we report a fatal case of disseminated adenovirus infection in a non-transplant haematology adult patient with chronic lymphocytic leukaemia who had completed combination chemoimmunotherapy a few months before developing respiratory symptoms. in such non-transplant patients, monitoring for adenovirus in the blood is not routine. however, with adenoviruses, when there is a more peripheral (i.e. non-blood) site of infection such as the chest, serial adenovirus monitoring in blood for the duration of that illness may be warranted. case presentation: this case started with an initial bacterial chest infection that responded to treatment, followed by an adenovirus pneumonitis that disseminated to his blood a week later with levels of up to million adenovirus dna copies/ml. despite prompt treatment with cidofovir, his respiratory function continued to deteriorate over the next two weeks and he was moved to intensive care. intravenous immunoglobulin and ribavirin were subsequently added to his treatment. however, he died soon after this with a final adenovirus load of million copies/ml in his blood. conclusions: we recommend that even in non-transplant haematology patients, where such patients present with an acute respiratory adenovirus infection, teams should consider checking the blood for adenovirus to check for signs of disseminated infection. the earlier this can be tested, the earlier treatment can be initiated (if adenovirus positive), which may produce more successful clinical outcomes. human adenoviruses are non-enveloped, doublestranded dna viruses. they exist as seven species (a-g) with greater than types identified so far that can affect different organs and cause a wide spectrum of disease in both immunocompetent [ ] [ ] [ ] [ ] and immunocompromised [ , [ ] [ ] [ ] [ ] individuals, including pneumonitis, hepatitis, gastroenteritis and conjunctivitis [ ] . although adenoviruses are important respiratory pathogens in haematology patients, adenovirus surveillance in blood is not normally performed in non-transplant adult patients. we report here a case of adenovirus pneumonitis which led to a fatal disseminated adenovirus infection in an adult patient with chronic lymphocytic leukaemia (cll) on chemotherapy. the patient was a -year old man with cll, who presented with persistent cough and coryzal symptoms in early . he had a past medical history of epilepsy and rheumatic fever. he had never smoked. cll was first diagnosed in late . the leukaemia progressed and he underwent six cycles of chemotherapy with the fcr combination (fludarabine mg/m , cyclophosphamide mg/m and rituximab mg/m ) over the next months completing the treatment in june . bone marrow and clinical findings showed a complete response to treatment. after his chemoimmunotherapy he continued on acyclovir ( mg bd) and co-trimoxazole ( mg bd, times a week) prophylaxis, both of which he was still taking at the time of presentation. this was in response to a t-cell lymphopenia that persisted up to his hospital admission in july (cd + t helper cells . × [ ]/l). in april he presented with a -week history of a non-productive cough and rhinorrhoea. there were no clinical or laboratory features of cll at that time but serum immunoglobulins were suppressed (igg . g/l (normal range - g/l) and cd + t-cells were low at . × [ ]/l (normal range . to . × [ ]/l). although there were transient improvements, productive cough persisted despite multiple courses of antibiotics (azithromycin mg od, co-amoxiclav mg tds, doxycycline mg od) and a course of oral prednisolone ( mg od). a ct scan of his sinuses showed right maxillary sinus change consistent with chronic sinusitis, and he was subsequently referred to ear nose and throat specialists for further management. however, the radiological sinus changes were not felt to be significant and no specific treatment was initiated. the patient's symptoms worsened and in the june a ct scan of the chest showed centrilobular nodular changes, right-sided patchy consolidation with surrounding ground glass opacity with halo sign. this raised the possibility of atypical infection including fungal pathogens and the patient was subsequently prescribed voriconazole mg bd. he also underwent a bronchoscopy later the same month, from which a broncho-alveolar lavage (bal) sample cultured haemophilus influenzae, but was negative for other pathogens including fungi. no viral screen was carried out on this sample. in response to this, he was given a prolonged course of co-amoxiclav ( mg tds). he showed subsequent clinical improvement on this, together with the voriconazole. one month later (july ), however, he was readmitted with a marked deterioration in the productive cough and shortness of breath on exertion. bilateral crepitations were heard on examination, which was consistent with a chest x-ray showing bilateral, patchy consolidation. c-reactive protein was mg/l. he was started on intravenous (iv) tazocin ( . g tds) and clarithromycin ( mg bd). although there were signs of improvement over the course of the week his symptoms persisted. a repeat ct scan on this readmission showed ground glass changes, tree-in-bud appearance, and nodular changes all of which had progressed from the previous imaging. he underwent another bronchoscopy examination, and was started on a treatment dose of cotrimoxazole ( mg/kg, daily in divided doses) and caspofungin ( mg od). in addition his serum immunoglobulins, which had remained low since his chemotherapy demonstrated pan-hypogammaglobulinaemia, and intravenous immunoglobulin (ivig) . g/kg was administered. this second bal was positive for adenovirus dna by pcr testing, using an in-house respiratory multiplex pcr screening assay, as described elsewhere [ ] . a beta-d-glucan test on the bal was also positive at pg/ml, having been negative in the peripheral blood. fungal, bacterial, pneumocystis and tuberculosis screens were all negative. despite this, a left mid-zone consolidation persisted on chest imaging. at this point he was diagnosed with adenovirus pneumonitis. one week later the patient remained symptomatic with persistent fevers, and peripheral blood was sent for adenovirus pcr, with a result of . million copies/ml. the qualitative and quantitative adenovirus pcr testing on this blood sample were performed at a commercial laboratory (micropathology ltd., coventry, uk). four days later this adenovirus level had risen to . million copies/ml (adenovirus type c , based on viral sequencing and analysis performed by micropathology ltd., coventry, uk). based on these results iv cidofovir ( mg/kg weekly) treatment was given. four days later the blood adenovirus dna levels had increased to million copies/ml. over the course of the following two weeks the patient deteriorated in terms of respiratory function, requiring transfer to intensive care for ventilatory support. his liver function also deteriorated during this time (bilirubin μmol, alkaline phosphatase iu/l. despite additional measures including further dosing with ivig ( . g/kg), and the addition of ribavirin (iv mg/kg), he died as a result of disseminated adenovirus infection and multi-organ failure. the last adenovirus dna level, three days before death was million copies/ml. no post-mortem investigations were performed. thus, the disseminated adenovirus infection was deemed to be the cause of the patient's multi-organ failure and death on the basis of the high levels of viremia, which coincided with the patient's rapid deterioration, as described in other cases [ ] [ ] [ ] [ ] . there are several well-known risk factors for severe adenovirus infections, including: allogeneic stem cell (or solid-organ) transplantation, particularly with t-cell depletion; treatment with anti-cd monoclonal antibody (alemtuzumab or campath) or anti-thymocyte globulin (atg); severe immunosuppression used to treat graft-versus-host disease; and any other cause of severe lymphopaenia that reduces the ability of the host's cell-mediated immunity to defend against adenovirus infection [ ] . this patient's chemotherapy regimen included fludarabine which has severe lymphopaenia as a recognised adverse effect, and which has been present in treatment regimens where various other viral reactivations have occurred, including hepatitis b [ ] [ ] [ ] , bk virus [ ] , herpes simplex and epstein-barr viruses [ ] , cytomegalovirus [ ] , as well as adenovirus [ ] . yet in this case, it was noted that throughout this period during which he acquired and was infected with adenovirus, his total lymphocyte count remained within or even above the normal range of - × [ ]/l, though their specific functionality was not tested. although fatal adenovirus infection has been reported in non-transplant paediatric and adult patients on chemotherapy [ ] [ ] [ ] [ ] , there is still no consensus on how to deal with an isolated adenovirus positive result in a peripheral (non-blood) sample type on a routine basismany such patients also have asymptomatic adenovirus infections. from a virological viewpoint, since all systemic antiviral drugs are only virostatic and not virucidal, earlier adenovirus pcr blood testing allowing earlier treatment to prevent further increases in viral load, may improve clinical outcomes [ ] . however, due to the severe nephrotoxic nature of the mainstay treatment, intravenous cidofovir, most transplant teams are reluctant to prescribe this drug empirically, unless a definitive upward trend is seen in the adenovirus blood levels. this is often the cause of delays in commencing therapy with this drug for disseminated adenovirus infection. thus, in such immunocompromised patients where a peripheral site (e.g. a non-blood sample, such as a respiratory or stool sample) has had an adenovirus pcr positive result, we would recommend that serial (once or twice weekly) monitoring for adenovirus pcr testing be performed for the duration of that specific adv illness, to check for possible disseminated adenovirus infection, earlier. such a test in this context is inexpensive and would allow earlier detection of disseminated adenovirus infection. this in turn then allows treatment to be commenced earlier, which could have a significant, positive clinical impact. finally in such immunocompromised patients, all pathogens: bacteria, fungi and viruses, should be screened for in the initial investigation of an acute infective episode, to allow prompt intervention as required. adenovirus infections in immunocompetent and immunocompromised patients epidemic of adenovirus-induced respiratory illness among us military recruits: epidemiologic and immunologic risk factors in healthy, young adults large epidemic of adenovirus type infection among military trainees: epidemiological, clinical, and laboratory studies outbreak of severe respiratory disease associated with emergent human adenovirus serotype at a us air force training facility in fatal adenovirus hepatitis during standard chemotherapy for childhood acute lymphoblastic leukemia fatal adenovirus hepatitis during maintenance therapy for childhood acute lymphoblastic leukemia disseminated adenovirus infection in cancer patients presenting with focal pulmonary consolidation fatal adenoviral and enteroviral infections and an epstein-barr virus positive large b-cell lymphoma after alemtuzumab treatment in a patient with refractory sézary syndrome resource impact of managing suspected middle east respiratory syndrome patients in a uk teaching hospital hepatitis b virus reactivation after fludarabine-based regimens for indolent non-hodgkin's lymphomas: high prevalence of acquired viral genomic mutations hbv reactivation after fludarabine chemotherapy identified on investigation of suspected transfusion-transmitted hepatitis b virus hepatitis b reactivation in a hbsag-negative, hbcab-positive patient receiving fludarabine for the treatment of chronic lymphocytic leukaemia symptomatic bk virus reactivation following fludarabine, cyclophosphamide and rituximab chemotherapy for chronic lymphocytic leukemia/small lymphocytic lymphoma herpes simplex and epstein-barr virus lymphadenitis in a patient with chronic lymphocytic leukemia treated with fludarabine cytomegalovirus oesophagitis following treatment with fludarabine for refractory lymphoplasmacytic lymphoma fulminant hepatitis due to human adenovirus no funding was required for the writing of this case report availability of data and materials any data (suitably anonymised to maintain patient confidentiality) is available for readers to review if a suitable written request to the corresponding author is made. authors' contributions mj, swparticipated in the clinical care of the patient on the ward. jwtadvised and supervised the laboratory testing, interpretation and reporting. mjwrote the first draft of the paper, which was edited for style and accuracy by jwt and sw. all authors critically reviewed the manuscript for publication. all authors have read and approved the final version of this manuscript.ethics approval and consent to participate not applicable consent for publication written consent to publish this case report and any accompanying images was obtained from the patient's next of kin. jwt is one of the virology section editors for the journal. the other authors declare that they have no competing interests.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- - r dmm authors: seo, jun-won; kim, choon-mee; yun, na ra; kim, dong-min; kim, sung soon; choi, sangho; chu, hyuk title: scalp eschar and neck lymphadenopathy after tick bite (senlat) caused by bartonella henselae in korea: a case report date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: r dmm background: tick-borne lymphadenopathy (tibola) is an infectious disease, mainly caused by species from the spotted fever group rickettsiae and is characterized by enlarged lymph nodes following a tick bite. among cases of tibola, a case of scalp eschar and neck lymphadenopathy after tick bite (senlat) is diagnosed when an eschar is present on the scalp, accompanied by peripheral lymphadenopathy (lap). only a few cases of senlat caused by bartonella henselae have been reported. case presentation: a -year-old male sought medical advice while suffering from high fever and diarrhea. three weeks before the visit, he had been hunting a water deer, and upon bringing the deer home discovered a tick on his scalp area. symptoms occurred one week after hunting, and a lump was palpated on the right neck area days after the onset of symptoms. physical examination upon presentation confirmed an eschar-like lesion on the right scalp area, and cervical palpation revealed that the lymph nodes on the right side were non-painful and enlarged at . × . cm. fine needle aspiration of the enlarged lymph nodes was performed, and results of nested pcr for the bartonella internal transcribed spacer (its) confirmed b. henselae as the causative agent. conclusion: with an isolated case of senlat and a confirmation of b. henselae in korea, it is pertinent to raise awareness to physicians in other asian countries that b. henselae could be a causative agent for senlat. bartonella henselae is a gram-negative, facultative, intracellular bacteria that can cause various diseases, including lymphadenopathy, bacteremia, bacillary angiomatosis, and bacillary peliosis [ ] . one of the typical diseases from b. henselae is cat scratch disease. people usually contract the disease from cats infected by b. henselae, but cases from flea or tick bites have been reported [ ] . the infection is asymptomatic in cats, but for humans, it can result in symptoms or signs such as lymphadenopathy, red papules, fever, headache, malaise, and sometimes, in adults, fever of unknown origin (fuo) [ ] . tick-borne lymphadenopathy (tibola) is an infectious disease, mainly caused by species from the spotted fever group rickettsiae (e.g. rickettsia slovaca, rickettsia raoultii) and is characterized by enlarged lymph nodes following a tick bite. scalp eschar and neck lymphadenopathy after tick bite (senlat) occurs after a bite from a tick and key clinical features occur at the surface of the scalp and cervical lymph nodes. therefore, we consider the tibola case with eschar on the scalp as sen-lat [ ] . there are some cases of senlat caused by b. henselae in other country [ ] , but there are no such case reports in south korea, except for some other clinical syndromes [ , [ ] [ ] [ ] . this study reports a first case of senlat in which b. henselae was confirmed as the etiologic agent in korea. the patient was a -year-old male, who brought home a water deer (hydropotes inermis argyropus) from muangun, jeollanam-do, korea., he had hunted a week prior to his presentation. his symptoms of high fever, diarrhea, and indigestion developed after the hunting incident, and his right cervical lymph nodes suddenly became swollen days following the onset of fever, which prompted him to visit the infectious diseases outpatient clinic at the chosun university hospital. the day after carrying the water deer home, he found a tick on his scalp, but had quickly removed and discarded it. he denied contact with cats or dogs as well as flea. on physical examination, he had a high fever of °c, an eschar-like lesion was found on his right scalp area (fig. a) , and on palpation, non-painful peripheral lymphadenopathy (lap) of . × . cm in size was identified in the right cervical region (fig. b) . blood test and fine needle aspiration were performed on the day of first visit. laboratory investigations revealed a white blood cell count of . × /ul, hemoglobin level of . g/dl, and platelet count of × /ul on routine complete blood count. serum biochemistry revealed the following: total protein concentration, . g/dl; albumin, . g/dl; blood urea nitrogen, . mg/dl; bilirubin, . mg/dl; alkaline phosphatase, u/l; and creatinine, . mg/dl (all were within normal limits). aspartate aminotransferase (ast) of . u/l was within normal limits, but alanine aminotransferase (alt; . u/l) as well as lactate dehydrogenase (ldh; u/l) were mildly elevated. to identify the cause of lap, fine needle aspiration (fna) was performed on the enlarged lymph nodes of the neck. cytology from the fna demonstrated a granuloma with an unclear boundary comprised of epithelioid cells along with giant cells and some lymphocytes. dna was extracted from the buffy coat of the patient's blood and from the lymph node aspirate using a qiaamp blood mini kit (qiagen, germantown, md). the results of the genetic detection were all negative when the -kda gene from o. tsutsugamushi and the ompa gene were targeted by nested pcr for rickettsial detection [ ] . nested pcr on the bartonella internal transcribed spacer (its) [ ] , using blood and lymph node samples, and by using b. elizabethae as a positive control, revealed a positive band by electrophoresis in only the lymph node aspirate. sequencing of the sample was therefore requested at solgent (daejeon, korea). the query output of blastn (ncbi) demonstrated a % identical sequence ( / bp) to the b. henselae strain bm (accession no. hg ) previously identified in human blood (fig. ) . indirect immunofluorescent antibody assay (ifa) against b. henselae were conducted at at the korea centers for disease control and prevention. the sera were examined with commercially available slides for bartonella-ifa igg and igm assay (focus diagnostics, cypress, ca, usa). the kit for detecting igm and igg antibodies utilizing vero cells infected with either b. henselae or b. quintana was used according to the manufacturer's instructions. diagnostic criteria are determined to be bartonella positive when endpoint titer of igg ≥ : or igm ≥ : . the ifa igm antibody titer against b. hensealse was < : at both first visit and fig. a photograph of the eschar on the scalp and right cervical area of a -year-old male patient with a confirmed diagnosis of bartonella henselae, and a cytology report from fine needle aspiration of an enlarged cervical lymph node. a eschar on the scalp at the first visit to the outpatient clinic. b right cervical lymphadenopathy on the first visit to the outpatient clinic. c a photograph showing a marked reduction of size in the right cervical lymphadenopathy days later follow up. the ifa igg antibody titer against b. hensealse was < : at first visit ( . . ), and : after follow up ( . . ). he was treated by doxycycline for first days and then with azithromycin for days. ten days later, the lap resolved (fig. c) . serological tests of orientia and other rickettsia species were performed together and the results were all negative. the incidence of disease caused by b. henselae has been previously reported, especially in association with contact with cats or dogs. the main route of infection is thought to be from scratching the site of a cat bite or a flea bite. the prevalence of bartonella infection in korea, identified by pcr, is estimated to be - . % [ , ] in animals, and - . % [ , , ] tibola commonly occurs in women and young people and has been reported in european countries such as france, spain, and hungary, particularly in cold seasons. rickettsia slovaca is known to be the most commonly confirmed etiologic agent of tibola, and the fig. a phylogenetic tree based on bartonella internal transcribed spacer (its) sequences from genbank most frequently identified vector is dermacentor marginatus [ ] . the scalp area is recognized as the most common site for tick bites. one possible explanation for this is that dermacentor ticks can stick to long hair, which plays a role as a shelter. among tibola entities, disease entity with both the eschar in the scalp and the neck lymphadenopathy are recognized as a new clinical entity named by senlat [ ] , as in our case. senlat has characteristic epidemiological findings that occur frequently in females and young children and are seasonal bimodality (spring and autumn) [ ] . although rickettsia slovaca is the most common pathogen in this syndrome, other several agents like rickettsia raoultii, rickettisa sibirica subsp. mongolitimonae, coxiella burnetii, borrelia burgdorferi, and candidatus rickettsia rioja are also known as etiological pathogens [ ] . the patient in our case had an eschar lesion on his right scalp, and a superficial enlarged right cervical lymph node, consistent with senlat. the result of the nested pcr, using a sample from the enlarged node, was positive for bartonella species in a genus-specific its gene, and the sequencing results confirmed bartonella henselae to be the cause of infection. and then, based on previous several literature showing that water deer living in korea serve as a reservoirs of bartonella species [ , ] , we believed that b. henselae identified in our patient originated from a water deer contacted prior to our hospital visit. in our case, the tick residing on the water deer may have bitten the patient, and hence infected him with b. henselae. as the patient had discarded the tick, we could not investigate the role of the tick as a vector. dermacentor ticks are, however, known to be absent in korea [ ] . cotte et al. showed in their experimental study that potential transmission of b. henselae is possible with ixodes ricinus ticks [ ] . therefore, one cannot exclude a possibility that senlat could have been caused by ixodes nipponensis, which is frequently observed in korea [ ] . in addition, the water deer may be the source of the tick, but it is not clear, and the natural environment may have been the tick source. however, further study is needed to confirm this. angelakis et al. have reported senlat caused by b. henselae following a tick bite [ ] . these three patients who are similar to our patient, but there is one big difference. that is, they are proven to be infected by b.henselae through pcr test with eschar tissue or tick specimen, but we have demonstrated senlat by b.henselae with pcr tests using neck lymph node tissue. all of their examples occurred in the colder months in europe, and the authors suggested that dermacentor ticks are most active during these periods. the occurrence of senlat has mainly been reported in europe. however, no cases of senlat have been reported in asia. difficulties in culturing b. henselae from pus aspirates and lymph node biopsy specimens of patients with cat scratch disease have been reported [ ] , and very low levels of sensitivity in serologic and pcr tests have been found against b. henselae infection. for example, among the patients with a confirmed diagnosis of cat scratch disease, only were noted to have positive pcr results, and the cycle thresholds were reported to be average of (range: . - . ) [ ] . our patient also showed a positive pcr result for b. henselae with lymph node aspirate, but not with a blood sample. ifa antibody test for b. henselae was also negative, presumably due to the low sensitivity of the ifa igg antibody test [ ] . in conclusion, this case report demonstrated a case of senlat in which the patient had ipsilateral lap and a scalp eschar, with a confirmed diagnosis of b. henselae infection from pcr of aspirate from the affected lymph node. this study should raise awareness in clinicians that, in addition to rickettsia species, b. henselae may be a causative agent of tibola or senlat. first case of bartonella henselae bacteremia in korea bartonella henselae and the potential for arthropod vector-borne transmission. vector borne zoonotic dis bartonella henselae as a cause of prolonged fever and fever of unknown origin in children scalp eschar and neck lymphadenopathy after tick bite: an emerging syndrome with multiple causes scalp eschar and neck lymphadenopathy caused by bartonella henselae after 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zoonotic dis prevalence of anaplasma and bartonella spp. in ticks collected from korean water deer (hydropotes inermis argyropus) prevalence of severe fever with thrombocytopenia syndrome virus in ticks collected from national parks in korea. vector borne zoonotic dis transmission of bartonella henselae by ixodes ricinus current status of tickborne diseases in south korea predominance of two bartonella henselae variants among cat-scratch disease patients in the netherlands low sensitivity of bartonella henselae pcr in serum samples of patients with cat-scratch disease lymphadenitis pitfalls and fallacies of cat scratch disease serology: evaluation of bartonella henselae-based indirect fluorescence assay and enzyme-linked immunoassay publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank jeong hee su, lee chae hun, kim sung yun, park sang uk, jeong yu min, kang hye ju, and choe yu seop for helping to review this manuscript. authors' contributions jws, nry, dmk contributed to the management of this patient. dmk was the leader of the clinical team. jws conducted the literature review and wrote the manuscript. dmk revised the article. cmk contributed to molecular identification. ssk and shc, hc contributed to perform isolation and identification of the pathogen and exclusion diagnosis, confirm final diagnosis. all authors read and approved the final manuscript. this work was supported by a grant ( -er - ) from the korea centers for disease control and prevention. funding support for this project had no role in the study design, data collection and analysis, decision to publish, or preparation of this article. all the information supporting our conclusions and relevant references are included in the manuscript. there are no datasets related to this case report.ethics approval and consent to participate not applicable. written informed consent was obtained from the patient for publication of this case report and any accompanying images. the authors declare that they have no competing interests. key: cord- -xc vv x authors: eslahi, aida vafae; badri, milad; khorshidi, ali; majidiani, hamidreza; hooshmand, elham; hosseini, hamid; taghipour, ali; foroutan, masoud; pestehchian, nader; firoozeh, farzaneh; riahi, seyed mohammad; zibaei, mohammad title: prevalence of toxocara and toxascaris infection among human and animals in iran with meta-analysis approach date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: xc vv x background: toxocariasis is a worldwide zoonotic parasitic disease caused by species of toxocara and toxascaris, common in dogs and cats. herein, a meta-analysis was contrived to assess the prevalence of toxocara/toxascaris in carnivore and human hosts in different regions of iran from april to june . methods: the available online articles of english (pubmed, science direct, scopus, and ovid) and persian (sid, iran medex, magiran, and iran doc) databases and also the articles that presented in held parasitology congresses of iran were involved. results: the weighted prevalence of toxocara/toxascaris in dogs (canis familiaris) and cats (felis catus) was . % ( % ci: . – . %) and . % ( % ci: . – . %), respectively. also, pooled prevalence in jackal (canis aureus) and red fox (vulpes vulpes) was . % ( % ci: . – . %) and . % ( % ci: . – . %), correspondingly. weighted mean prevalence of human cases with overall records was . % ( % ci: . – . %). the weighted prevalence of toxocara canis, toxocara cati, and toxascaris leonina was represented as . % ( % ci: . – . %), . % ( % ci: – . %) and . % ( % ci: . – . %), respectively. conclusion: our meta-analysis results illustrate a considerable prevalence rate of toxocara/toxascaris, particularly in cats and dogs of northern parts of iran. the presence of suitable animal hosts, optimum climate and close contact of humans and animals would have been the reason for higher seroprevalence rates of human cases in our region. given the significance clinical outcomes of human toxocara/toxascaris, necessary measures should be taken. zoonoses are those complications which are transmissible between human and animal populations [ ] . in this regard dogs and cats are considered as a public health concern, as they may harbor various pathogens such as zoonotic helminths including toxocara species [ ] . toxocariasis is a worldwide parasitic infection, primarily rendered by t. canis in dogs, t. cati in cats and foxes and t. leonina in a wide range of carnivores [ ] . mature worms lay eggs in the intestinal lumen of their host, which are excreted into the environment via defecation and pass their developmental stages in optimum soil and climate conditions. upon ingestion of embryonated eggs by another host, the larvae would emerge and invade the intestinal mucosa, then migrate through viscera such as lungs, liver, and kidneys. in addition, transplacental and transmammary transmission to puppies and kittens are important routes of infection. in an epidemiological perspective, animal hosts parasitized by adult worms in their gut can disseminate infection by shedding parasite eggs into environment [ ] . in an epidemiological perspective, animal hosts parasitized by adult worms in their gut, can shed parasite eggs, hence considered as a source for dissemination of the infection [ ] . human infection occurs by accidental ingestion of eggs, and, to a lesser extent, via pica and devouring on the paratenic hosts, including chicken, cattle, lamb, pig, and earthworms [ , ] . consequently, developmentally-arrested larvae migrate through body organs, but don't develop into mature worms; hence, they provoke an array of syndromes enclosing vlm, nlm, and olm as well as covert infection and asymptomatic toxocariasis [ ] [ ] [ ] . although rare, cardiac-associated toxocariasis is a serious, life-threatening complication due to vlm which has recently been emphasized [ ] . most of the infected individuals manifest nonspecific symptoms such as a cough, rhonchus, dyspnea and pyrexia along with hepatomegaly and eosinophilic granuloma, which implicates diagnosis of the infection using more sensitive approaches such as immunological assays i.e., elisa for screening and western blot for confirmation, rather than histological or parasitological methods [ , ] . toxocariasis cause by t. cati and t. canis frequently impacts young cats and dogs from birth to year old, entailing respiratory signs (coughing due to pulmonary larval migration), general failure to thrive (retarded growth, emaciation, debilitated body coat and arthralgia) and intestinal disorders (alternating diarrhea and constipation, pot-belly and vomiting). no remarkable trait is seen following toxascaris infection in dogs and/or cats and it is usually well-tolerated [ ] [ ] [ ] . one of the characteristic of helminthic parasites is the stimulation of the immune system that leads to increased th response and high production of il- , il- , il- , il- , il- , eosinophils, and ige. toxocara larvae can causes severe hyper eosinophilia and allergic involvements with effect on ige and il- . consequently, the production of specific antibodies provides the most complete evidence for toxocara infection, which is the base of diagnostic tests such as elisa and western blot for reactivity to larval tes antigen [ ] [ ] [ ] . iran, a middle eastern country, possesses several climatological areas with particular characteristics in each region; this would have a significant bias on the epidemiology of toxocara/toxascaris species. in the previous studies the infection of dogs and cats with toxocara species in different parts of iran has been shown [ ] . despite the prevalence of toxocara canis in the most areas, molecular studies on cat nematodes in shiraz, in south-central iran showed that, the most prevalent one is t. cati [ ] . toxocara vitulorum is frequently found in ruminants. its main hosts are cattle and buffalo in tropical and sub-tropical regions [ ] . it has been reported that % ( % ci: - %; out of samples) of soil samples gathered from public parks of the iran were positive for toxocara spp. eggs [ ] . on the other hand, due to increasing body of work on toxocara prevalence in various human/animal hosts in iran, a comprehensive review would be exceedingly beneficial for appraising progresses about this zoonosis. therefore, this meta-analysis attempts to fill the current gaps and provides insights into parasite prevalence with respect to host type, toxocara and toxascaris species, and geographical region in the country. iran has a population of approximately million (as of ), and is located between ° and ° n and ° and ° e, which covers a wide territory in the middle east area ( , , km ) . the country borders afghanistan and pakistan to the east, iraq and turkey to the west, the persian gulf and oman sea to the south, and azerbaijan, armenia, and turkmenistan to the north. the iranian plateau climate is generally hot and dry, however the caspian sea coast in northern parts, comprising golestan, mazandaran and guilan provinces, is mediterranean-like, demonstrating heavy rainfalls, vegetation-enriched, surrounded by dense forests and a diverse range of carnivorous animals these geo-ecological features would provide a well-established setting for most parasites, e.g. soil-transmitted helminthiases, to localize in the area and parasitize many canid species. also, the country is a mountainous region with several mountain ranges, mostly located at the western and northern parts such as zagros mountain ranges with colder winters and heavy snowfalls. the annual precipitation is mm in the eastern part of the plain and more than mm in the western parts [ ] [ ] [ ] [ ] . the prisma protocol (preferred reporting items for systematic reviews and meta-analysis) was employed to conduct this meta-analysis [ ] . in order to assess the prevalence of t. canis, t. cati and t. leonina in humans and carnivores of different parts of iran, we investigated the available online articles of both persian (sid, iran medex, magiran, iran doc) and english (pubmed, science direct, scopus, ovid) databases. the search include between april and june . also, the articles that presented in held parasitology congresses of iran were involved. a combination of the following search terms were employed in our literature searches as follows: ("toxocariasis" or "toxocara infection" or "toxocara canis" or "toxocara cati" or "toxascaris leonina") and ("carnivora" or "human") and ("prevalence" or "epidemiology") and ("iran"). after hand searching in bibliographic list of obtained full-text records for any related literature as well as removing duplicates, two independent reviewers screened the titles and abstracts for initial inclusion. a third reviewer was also involved for consensus in the case of any disagreements. finally, those records that met the following inclusion criteria were eligible to enter our meta-analysis: (a) peer-reviewed originally-published papers both in english or persian; (b) being available online between april till june ; (c) crosssectional investigations that assessed the prevalence of toxocara spp. in various carnivores and human populations in iran; (d) studies that detected toxocara infection using at least one of the parasitological, serological and molecular methods; (e) exact total sample size, positive samples and the respective prevalence rates were available. empirical studies and any kind of review papers were excluded and failed for further analysis. a detailed variable of each of articles, including: province, year of publication, study design, sample size, detection method, and prevalence rates, in addition to animal species and sampling method for animal-based investigations were gathered. in this study, the jbi critical appraisal checklist for prevalence studies was employed [ ] . the jbi checklist was used for quality assessment of the included articles. this checklist contains eight questions with four options including, yes, no, unclear, and not applicable (additional file : figure s ). briefly, a study can be awarded a maximum of one star for each numbered item. the papers with a total score of ≤ and ≥ points were specified as the moderate and high quality, respectively. based on the obtained score, the authors have decided to include and exclude the papers [ ] . briefly, meta-analysis was yielded as a forest plot representing the prevalence estimates and related confidence intervals of each study along with summary measures. also, the heterogeneity was analyzed using stata statistical software (version . ) to calculate cochran's q and i statistics. i values of , , and % were considered as low, moderate and high heterogeneity, respectively [ ] . furthermore, the funnel plot based on egger's regression test illustrates publication bias and small study effects. in the current study, i was substantial; therefore, we used a random effects model at a % ci, to give a more conservative estimate of the toxocara infection prevalence. following systematic search of eight databases, totally records human studies and animal investigations were found eligible regarding toxocara/toxascaris (fig. ) . during a -years period, , human individuals were examined and the calculated weighted prevalence was . % ( % ci: . - . %) (tables and ). the trend line of human toxocara/toxascaris infection demonstrated that the prevalence has declined in spite of increased bulk of work on human population (additional file : figure s ). most records ( studies) were conducted in both rural and urban circumstances, however seroprevalence was mostly predominant in urban regions with % ( % ci: . - . %) (no showed data). people under years old were mostly examined by serodiagnosis approach, indicating . % ( % ci: . - . %) seroprevalence rate (additional file : figure s ). a number of entries contributed to prevalence of toxocara/toxascaris in dogs (canis familiaris), showing a prevalence of . % ( % ci: . - . %). the weighted prevalence of toxocara/toxascaris was higher in investigations which examined cats (felis catus) [ . % ( % ci: . - . %)] (tables and ) . interestingly, one study also used serodiagnosis in cats indicating a . % ( % ci: . - . %) seroprevalence (additional file : figure s ). four studies (all necropsy-based) dedicated to prevalence of toxocara/toxascaris in jackal (canis aureus), representing a . % ( % ci: . - . %) frequency. a and ). according to the detection method, the highest total prevalence of t. canis in feces was related to the formalin-ether method [ . % ( % ci: . - . %)] (additional file : figure s ). also the most total prevalence of t. cati in feces was related to the formalin-ether method [ . % ( % ci: . - . %)] (additional file : figure s ). among toxocara/toxascaris species examined through included studies in iran, t. cati possessed the highest prevalence rate with . % ( % ci: . - . %) ( (figs. , and ) . there was no statistically significant association between the estimated pooled prevalence of toxocara/toxascaris infection in human population and mean temperature (p = . ), humidity (p = . ), longitude (p = . ), and latitude (p = . ). among three parasite species, only humidity (p = . ) and latitude (p = . ) for t. canis were statistically significant, while others were not remarkably involved (fig. ). the current systematic review and meta-analysis was aimed to elucidate the prevalence of toxocara spp. infection in animal and human hosts in iran. the human infection was highly concentrated in two northern provinces (mazandaran and east azerbaijan) (fig. ) , highlighting optimum geo-ecological milieu in those parts of the country because of high percentage humidity due to vicinity to the caspian sea as well as considerable rainfall during the year; notwithstanding, we didn't found any statistically significant correlation between human toxocara/toxascaris seroprevalence studies and fig. the total prevalence of t. cati infection in carnivores of iran geographical parameters comprising mean temperature, humidity, longitude and latitude (fig. ) . despite of equal records of toxocara/toxascaris infection from rural and urban areas, seroprevalence was partly elevated in urban regions rather than rural territories, resulting from the likely heterogeneity among studies and/or lack of sufficient records; care must be taken in interpreting such result as rural areas are naturally considered as higher risk areas than urban [ , , , ] . toxocariasis due to several species of toxocara and/or toxascaris roundworms is still a seriously notifiable public health issue, particularly due to its intricate transmission routes [ ] . in human this infection is caused by t. canis, in particular, and t. cati renders several issues comprising vlm, olm, nlm and covert disease, each of which is represented by manifestations of the involved organ [ , ] . toxocara/toxascaris infection in human populations is considered as a chronic parasite in nature which is distributed worldwide, particularly in tropical underdeveloped countries [ ] . several risk factors are supposed to play a major role in toxocara/toxascaris distribution among the human population, consisting of habitation in rustic areas, soil contact, consuming the undercooked meat of the infected paratenic host, insufficient and unhygienic water repositories, poor housing and low education as well [ ] [ ] [ ] [ ] . furthermore, owing to the adventurous nature of children, such as tasting any objects, eating soil and/or earthworms and being in the vicinity of dogs and cats, they are considered as a substantial risk group regarding toxocariasis [ , ] . hence, public places in which children may walk around such as parks, playgrounds, beaches and sandboxes are crucial territories for the acquisition of the infection [ , ] . since most individuals do not manifest any pathognomonic symptoms, the actual prevalence rate of the infection remains to be elucidated, even in industrialized nations [ , ] . considering that toxocara parasites do not develop into adult stage in humans, coproscopy is unnecessary; thus, biopsy and direct parasite observation are the gold standard methods [ ] . however, such examination is invasive and relies on the larval load and the infection phase [ ] . therefore, routine diagnosis of infection and/or exposure in human cases can be done by elisa to detect specific antibody against tes antigens, which should be further validated by immunoblotting [ , ] . as previously mentioned, tes-based elisa tests are mostly used for human seroprevalence studies. despite having proper immunogenicity, native tes antigens may cross-react with antibodies elicited against other helminths specifically ascaris lumbricoides which decreases test specificity [ ] . therefore, the results may be regarded as suspicious, particularly when no immunoblotting confirmation is done, specifically in endemic regions where there exists the possibility of helminth co-infections. alternative detection methods in paratenic or accidental hosts are including pathological inspection, larvae morphometry as well as pcr-based experiments [ ] . a great deal of effort has been devoted to revealing the seroprevalence of human toxocara/toxascaris infection worldwide. in africa, elevated seroprevalence rates of infection were detected, encompassing % in egypt to % in gabon and % in réunion island [ , ] . additionally, the seroprevalence ranges in asia and south america included - . % and . - %, respectively [ ] [ ] [ ] . comparable to other territories, rates of seropositive human cases were relatively low in european and north american countries [ ] , implicating improved hygiene practices and public awareness in industrialized nations. in total, seroprevalence data integration in epidemiological investigations is not reasonable for several reasons, comprising sampling disparities, antigen preparation, and quality, different cutoff levels, cross-reactivity especially in the tropics were polyparasitism exist and inability to explicitly distinguish the infection by various toxocara spp. therefore, expanding our evidence based on human toxocara infection would be corroborated by a better understanding of parasite biology, in particular, the immune evasion mechanism of larvae, and utilization of advanced, species-specific diagnostic tools [ ] . the calculated total prevalence of infection in cats (felis catus) was higher [ . % ( % ci: . - . %)] than in dogs (canis familiaris) [ . % ( % ci: - %)] in the country (table ). similar to human seropositive cases, carnivores in northern iran were the most frequent hosts being parasitized by toxocara spp., whereas minimum animals were infected in central parts [ % ( % ci: - %)]. among wild canine species in iran, only jackal (canis aureus) and red fox (vulpes vulpes) were diagnosed with toxocara/toxascaris infection, with . % ( % ci: . - . %) and . % ( % ci: . - . %), respectively (table ) . moreover, it was deduced that the weighted prevalence of t. canis, t. cati, and t. leonina in iran were . % ( % ci: . - . %), . % ( % ci: . - . %), and . % ( % ci: . - . %), respectively. given fig. the total prevalence of t. canis infection in carnivores of iran geographical characteristics, only humidity (p = . ) and latitude (p = . ) were significantly linked to t. canis infection. increasing latitude would likely result in decreased mean temperature and more temperate climates than the equator area. water vaporization and condensation in northern parts of the country due to the vicinity to the caspian sea and high mountain ranges and humid weather substantially implicate in toxocara/ toxascaris larval development, as proved in the laboratory [ , ] . the survey of the infection in carnivores is usually made via traditional parasitological methods (e.g. floatation technique) to detect eggs as well as intestinal necropsy of dead carcasses [ , ] . nevertheless, each detection method may provide a prevalence rate different from other modalities, which this issue would implicate potential biases in reporting and/or interpreting data. as we stated in the results section, necropsy has been shown as a better and efficient detection tool than fecal examination. for instance, more than -fold prevalence of toxocara/toxascaris spp. in dogs was obtained using necropsy [ [ ] [ ] [ ] [ ] . in dogs dwelling in the americas, t. canis infection prevalence varied from . % in canadian provinces to % in cuba. also, t. cati was mostly prevailed in argentina and brazil with and %, respectively [ ] [ ] [ ] [ ] . the highest t. canis and t. cati infection rates in asia were dedicated to russia and china with and . %, respectively [ , ] . additionally, mild toxocara species infections were identified in african domestic carnivores [ ] [ ] [ ] [ ] . globally, the highest t. leonina prevalence (up to %) was observed in domestic dogs from russia [ ] . wildlife probably plays a critical role in the epidemiology of toxocara species, as they may be considered as potent reservoir for these enigmatic roundworms [ ] . patent t. canis infections are generally higher in young foxes (under months of age); although, a relatively high prevalence rate have also been among adult foxes in endemic territories, representing weak immune status against intestinal [ ] . the prevalence of t. canis in european foxes varies between . % (in italy) and . % (in denmark), as well as . and . % prevalence in canada and japan, respectively [ ] . the lowest and highest t. leonina prevalence in red fox was reported from kirghizstan ( . %) and the slovak republic ( . %), respectively [ ] . regarding golden jackal (canis aureus) moderate prevalence rates of toxascaris leonina have been reported around the world, such as in azerbaijan ( . %), bulgaria ( %) and russia ( . %). the prevalence of t. canis in this wildlife species ranges - % in asia and - . % in european countries, whereas t. cati was only detected in jackals dwelling in russia ( - %) [ , ] . considering that there are only golden jackal studies and red fox (vulpes vulpes) studies, there exist paucity of data on toxocara/toxascaris prevalence in wild canine and feline fauna of iran, which highlights more subtle investigations. approximately, since the middle of previous century a periurban rise in european foxes population carrying toxocara/toxascaris worm burdens have posed a great environmental risk of contamination with parasite ova. on the other hand, they act a critical role in maintaining t. canis wildlife cycle with implications in constant transmission to human populations and pet dogs [ ] . the findings of the present study indicated a mild seroprevalence in human population; also, infection in cats was higher than dogs, however unbalanced sampling may have influenced these findings. most of the infected cases were from north of iran, which possess a favorable ecological milieu for appropriate animal hosts and toxocara egg development (i.e., - °c in laboratory-based conditions, during - weeks [ ] . despite the improved hygiene and health surveillance systems as well as a wide-range public awareness in developed countries, still toxocara/toxascaris infection remains a public health concern in those areas and the rest of the world as well. during the time, there have been established a close companionship between dogs and cats with humans, and during past decades it has been even strengthened. however, these associations, particularly in underdeveloped nations, have been accompanied with poor veterinary infrastructures. this, along with free-roaming or community-owned dogs and cats pose a serious threat for zoonoses transmission to human societies [ ] . with respect to the constant infection cycle in carnivores and the life-threatening traits of human toxocariasis, revisiting the epidemiological strategies in companion animals enclosing anti-helminthic medication and screening plans such as the routine fecal examination is of utmost importance. in addition, it is highly emphasized that future human investigations focus on using recombinant tes antigens with high sensitivity and specificity and less cross-reactivity. also, it is better to identify anti-toxocara igg coupled with tes rather than total igg and employ western blot as a complementary diagnostic technique [ ] . moreover, it is recommended to educate laboratory technicians for accurate parasite detection, regularly deworm puppies and kittens to decrease the worm burden, perform proactive chemoprophylaxis approach and cultivate knowledge among the public as well as physicians regarding the clinical consequences of the disease. the interwoven collaboration among blood banks, veterinary diagnostic laboratories and municipalities (control stray dog/cat populations in urban areas) would provide a more completed picture of disease seroprevalence and distribution in people and animals, giving us the opportunity for targeted intervention strategies and better management of this zoonotic enigma. in parallel to above-mentioned recommendations the wsava has recently found a one health committee to highlight the transmission potential of zoonotic infectious agents from dog/cat to human. besides the oie has recently extended the surveillance of wildlife diseases through wahid in the world. all of these expanded fields of epidemilogical data would assist the global community towards better understanding of human-domestic animal-wildlife interplay and control of human zoonotic diseases [ ] . it is noteworthy to mention that some limitations constrained our findings en route performing current systematic review and meta-analysis, including ) lack of risk factor appraisal, ) absence of a standard, easy-touse diagnostic tool in case of human studies to particularly 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jackal parasites reveals high diversity of species changes in the distribution of red foxes (vulpes vulpes) in urban areas in great britain: findings and limitations of a media-driven nationwide survey lehrbuch der parasitologie für die tiermedizin [textbook of parasitology for veterinary medicine surveillance of zoonotic infectious disease transmitted by small companion animals publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank all staff of the department of parasitology of tarbiat modares university and alborz university of medical sciences, iran. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file : figure s . the quality assessment of included studies of human population additional file : figure s . the weighted prevalence of human toxocara/toxascaris by the year in iran additional file : figure s . the weighted prevalence of human toxocara/toxascarisby the age in iran additional file : figure s . the weighted prevalence of toxocara/ toxascaris in iran dogs by study method additional file : figure s . the total prevalence of t. canis in feces of animals according to the different parasitology methodsin carnivore population in iran. mb, ff, and mz extracted the data and wrote the study manuscript. ak and smr contributed to data analysis and interpretation the manuscript. all authors read the manuscript and participated in the preparation of the final version of the manuscript. all authors read and approved the final manuscript. there was no any funding or sponsoring organization for this review. the datasets used and analyzed during the current study are available from the corresponding author on reasonable request. the study design including its ethical aspects was reviewed and approved by the ethics committee of alborz university of medical sciences. not applicable. the authors declare that they have no competing interests.author details key: cord- - rm za z authors: curtale, filippo; perrelli, fabrizio; mantovani, jessica; atti, marta ciofi degli; filia, antonietta; nicoletti, loredana; magurano, fabio; borgia, piero; di lallo, domenico title: description of two measles outbreaks in the lazio region, italy ( - ). importance of pockets of low vaccine coverage in sustaining the infection date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: rm za z background: despite the launch of the national plan for measles elimination, in italy, immunization coverage remains suboptimal and outbreaks continue to occur. two measles outbreaks, occurred in lazio region during - , were investigated to identify sources of infection, transmission routes, and assess operational implications for elimination of the disease. methods: data were obtained from several sources, the routine infectious diseases surveillance system, field epidemiological investigations, and molecular genotyping of virus by the national reference laboratory. results: overall cases were reported, sustained by two different stereotypes overlapping for few months. serotype d was likely imported from romania by a roma/sinti family and subsequently spread to the rest of the population. serotype b was responsible for the second outbreak which started in a secondary school. pockets of low vaccine coverage individuals (roma/sinti communities, high school students) facilitated the reintroduction of serotypes not endemic in italy and facilitated the measles infection to spread. conclusions: communities with low vaccine coverage represent a more serious public health threat than do sporadic susceptible individuals. the successful elimination of measles will require additional efforts to immunize low vaccine coverage population groups, including hard-to-reach individuals, adolescents, and young adults. an enhanced surveillance systems, which includes viral genotyping to document chains of transmission, is an essential tool for evaluating strategy to control and eliminate measles the world health organization regional office for europe (who/euro) has set as the target for elimination of measles in the region [ , ] . this objective has already been achieved in some states through routine immunization programmes which maintain high coverage using a two-dose measles-mumps-rubella (mmr) vaccine schedule [ ] . however, vaccination coverage still remains inadequate in several western european countries, including italy, and although mass vaccination has successfully lowered the incidence of the disease outbreaks continue to occur, often affecting communities with low vaccination coverage [ ] . in italy, measles vaccine (mv) was introduced in and routine administration of one dose of measles vaccine to all children aged ≥ months was recommended since . combined mmr vaccines became available in the early s and the two-dose schedule (first dose at - months and second dose at - or - years) was introduced in [ ] . however, despite the existence of national recommendations, responsibility for implementation of measles vaccination activities lies within each of italy's regions. this has contributed to non uniform coverage across regions, with lower rates observed mainly in central and southern regions (including lazio) with respect to northern regions [ ] . in the years - a large measles outbreak occurred in italy with over , estimated cases among children below years of age [ ] . following the outbreak, in november , all italian regions signed an agreement with the italian ministry of health to implement the "national plan for the elimination of measles and congenital rubella" [ ] . strategies include among others, improving routine mmr coverage among children below years of age, implementing supplementary vaccination activities for older children and adolescents (aged - years) and strengthening disease surveillance. following implementation of the elimination plan, national vaccination coverage for first mmr dose in children at two years of age increased from . % in , to . % in . during the same period of time, incidence of measles in italy decreased from . / , to < / , [ , ] . in the lazio region, the measles elimination plan succeeded in increasing coverage for the first dose of mmr among children at two years of age from . % in to . % in [ ] . during the same five year period, coverage among school-aged children ( - years of age) increased from % to % while coverage for the second dose at years of age increased from % to %. in and a historically low incidence of < case per , was reached in the lazio region, with approximately measles cases reported per year. however, sporadic cases continued to be reported, especially among susceptible adolescent and adults. pockets of low coverage were also present in specific areas and among emarginated and hard-to-reach populations (hrp), such as the roma/sinti population, and illegal immigrants [ ] . between june and august , two measles outbreaks occurred in the lazio region. the first outbreak (june-december ) initially involved the roma/sinti population, and subsequently spread to the rest of the population. the second outbreak overlapped with the first (october -august ) and affected mainly the italian adolescent and adult population. in this article we describe the two outbreaks and highlight the importance of pockets of low vaccine coverage in sustaining such outbreaks. data from the mandatory infectious diseases surveillance system, field epidemiological investigations, and molecular characterization of measles virus by the national reference laboratory are presented. lazio, one of italy's regions, has a population of . million people ( ), . million of which living in the urban area of the capital city of rome. it is divided into provinces (rome, rieti, viterbo, latina and frosinone) and local health units (lhus). the public health agency of the lazio region (agenzia di sanita' pubblica, asp), is responsible for surveillance of infectious diseases and immunisation coverage in the region. the asp monitored the two measles outbreaks and coordinated outbreak control activities in the lhus of the region. in italy, measles is a disease subject to mandatory notification, and according to the routine procedure, physicians must report suspected measles cases to their lhu within hours of diagnosis. the local health authorities then report confirmed measles cases to the asp monthly. at the beginning of the outbreaks this procedure was modified and physicians were asked to report suspected measles cases to both the local health authorities and asp offices within hours of diagnosis. personnel of the lhus performed epidemiological investigation of suspected cases including laboratory investigation and contact tracing. a suspected measles case was defined as a subject with fever (≥ °c), generalised rash and at least one of the following symptoms: cough, coryza, or conjunctivitis. a confirmed case was defined either as a laboratory confirmed case (in which measles-specific igm antibodies were present in serum or saliva samples or measles virus nucleic acid was detected in urine samples by pcr or as a case with an epidemiological link to a confirmed case. for each confirmed case, demographic data characteristics (including whether cases belonged to a roma/sinti community), vaccination history, date of onset of symptoms, and hospitalisation, were collected. information was conveyed to the asp, which discarded non confirmed cases from the database, eliminated redundant records, performed quality check and contacted the local health authorities for any missing information. to assess presence of indigenous transmission or sources of imported virus the data set was integrated with information provided by the national institute of health (istituto superiore di sanità, iss), which conducted viral molecular characterization from urine samples of measles cases, utilising the pcr technique [ ] . data analysis regarding the age distribution and immunization status of cases, as well as the percentage of cases requiring hospital admission was performed on the total number of cases reported in the two outbreaks. phylogenetic analysis based on the available partial nucleoprotein gene sequences of measles virus and tree reconstructions were performed with mega software version . [ ] . virus isolates and genotypes were designated according to the new official who nomenclature [ ] . data from confirmed cases was entered into microsoft excel and converted to sas version (sas institute inc., cary, nc) for analysis. the temporal and geographical distribution of cases, together with the age distribution, was calculated separately for the roma/sinti and rest of the population. the present study did not require approval from an ethics committee. laziosanità -the public health agency of the lazio region is the local government agency responsible for the collection of infectious disease notifications, hospital admission and discharge records and laboratory surveillance data. the management of these data for public health purposes does not require a patient's informed consent nor does it require any authorization regarding privacy laws. from june to the end of august , a total of cases were reported, of which in and in . the two outbreaks overlapped and not all cases were genotyped; therefore, it was not possible to determine the exact number of cases that occurred in each of the two outbreaks. overall, / cases occurred amongst the italian ethnic population and / amongst the roma/sinti population. seventy-eight cases ( %) were laboratory confirmed while the remaining were epi-linked. the virus serotype was identified for fifty-seven cases (serotype d : n = ; serotype b : n = ). the first reported case was an eight year-old roma child of romanian nationality, living in a settlement located in the outskirts of rome. the child was admitted to hospital with rash, fever, diarrhoea, conjunctivitis, rhinitis and otitis on june , reporting onset of symptoms on june . analysis of routine data allowed the identification of an additional case that had been notified one week previously, in rome by a different lhu. this case was an unvaccinated six-year-old roma child, also of romanian nationality, who developed symptoms on june . the child was not hospitalised and the source of his infection was not determined, since his parents refused to answer to questioning by the health officials and then moved away. he transmitted the infection to an eight-year-old child living in the same building, who developed symptoms on july and was hospitalised. in the last week of june two additional roma/sinti children, in two different health authorities, developed measles symptoms on june and june respectively. by the end of july in rome, an additional measles cases had been reported in various settlements of the roma/sinti community and cases among the rest of population. two cases among the italian ethnic population, who developed symptoms on and july, reported contact with roma/sinti patients with measles in a hospital waiting area, on and july in november , a cluster of six measles cases was reported amongst adolescents and young adults attending a professional school in the outskirts of rome (attack rate . %). the infection subsequently spread outside the school and cases continued to be reported amongst the italian ethnic population until the summer . this second outbreak reached a peak in march and was considered over only in august (figure ). during july-august three measles cases were notified per month, bringing the incidence level to that reported in the lazio region before the described outbreaks (≤ cases per month, or < / , population per year). the d genotype, grouped in two different clusters of common origin, was responsible for the first cases reported in the roma/sinti population and detected in several other cases, including the italian ethnic population, up to december . starting in october the b genotype was isolated in a contact of a case from the school outbreak, overlapping for some time with genotype d (figure ). the first outbreak (d ) started in the rm-b lhu (figure rome, where the outbreak in the professional school occurred. (figure ). overall, the median age of cases was years. children aged - years were the most affected age group, with one third ( / ) of cases reported belonging to this age group ( / , incidence for children < years). measles incidence in the total population during the two outbreaks was / , . when analysed separately, the age distribution of measles cases was different among the roma/sinti population (median age two years) with respect to the rest of the population (median age years). seventy percent ( / ) of roma/sinti cases occurred in children aged - years and over % ( / ) were aged below years. the age distribution of cases in the italian ethnic population was more evenly distributed among all age groups. only % of cases ( / ) were aged - years, and less than % ( / ) occurred in children below years of age ( figure ). none of the roma/sinti cases were vaccinated against measles, and only ( . %) cases from the italian ethnic population had received one dose of measles-containing vaccine. overall, over % ( / ) of cases required hospital admission. fifteen cases ( . %), of whom from the first outbreak and three from the second, reported a hospital contact with children affected by measles, either in the waiting area or after admission for a different condition. four of these contacts were healthcare professionals (two nurses and two physicians). out of the reported cases of nosocomial transmission, seven involved contacts with roma/sinti children, including the first two cases in the italian ethnic population. in response to the described clusters of measles cases, active tracing and vaccination of susceptible contacts was performed by local health authorities. a second dose of mmr vaccine was also offered to incompletely vaccinated contacts. in addition, mmr vaccine was offered to all susceptible or incompletely vaccinated children and adolescents attending any of the schools in which cases had been detected and to roma/sinti children up to years of age. vaccination sessions were conducted directly in the involved settlements; in total persons in seven settlements were vaccinated in august . isolation of cases and susceptible contacts (in hospital for roma/sinti patients and at home for other subjects) was recommended, local health authorities were urged to identify possible contacts of all suspected measles cases, and alert general practitioners, family paediatricians, and hospitals about the outbreaks. physicians were asked to report cases within hours of diagnosis. in addition, guidelines regarding respiratory isolation of patients with suspected measles, and vaccination of susceptible hospital staff were forwarded to all hospitals of the region. before the start of the / school year a meeting was organized by the asp with staff in charge of the measles elimination campaign and the public health departments of the health authorities. local media were also informed of the outbreaks. the two described outbreaks, which involved cases (incidence / , ) notified from june to august , represent the most serious episodes after the - measles outbreak [ ] . they confirmed that pockets of low vaccination coverage exist in some areas of the lazio region, particularly among roma/sinti communities and adolescents thanks to relatively high immunisation rates amongst new born children ( , %) [ ] and the work done by the local health authorities, conducting contact investigation of cases', vaccination of susceptible school and household contacts, and implementing isolation measures, the outbreaks did not affect the whole region and, in the city of rome, was mainly limited to a few peripheral districts (figure ). both outbreaks started in populations known to have low coverage, (roma/sinti community, and students of a secondary school). the subsequent spread to the rest of the population, at least for the first outbreak, was facilitated by nosocomial transmission. differences were found in the affected age groups among the roma/sinti and the rest of population. as expected in a susceptible population, the most affected age group in the roma/sinti population was the - year-old age group. conversely, in the italian ethnic population, which had a higher percentage of vaccinated subjects with respect to the roma/sinti population, especially among young children, most cases occurred in the - year-old age-group. molecular characterization of measles virus is an important surveillance tool for monitoring measles elimination [ , ] . in this case it was fundamental in tracing the origin of both outbreaks and showing that two distinct chains of transmission took place in the region. it is highly likely that the first outbreak, due to the d measles serotype, which is not endemic in italy [ ] , was imported from romania. in fact, first cases occurred in families of romanian nationality with family and social links in romania. in addition, the d sequence identified among the first cases in lazio was found to be identical to the d isolated in romania during the - outbreak [ ] in which over cases and deaths occurred [ ] . a d measles strain highly correlated with the one isolated in lazio caused a smaller outbreak in northern italy (south tyrol), between june and august [ ] . the first case was reported during the same week of the beginning of the first outbreak in lazio, and of cases belonged to the roma/sinti population. in south tyrol, a transit camp for roma/sinti travelling between italy and eastern europe appeared to be the entry point for the d measles genotype in italy (figure ) . between august and february other measles outbreaks linked to the lazio outbreak (d ) occurred in sardinia (italy) and barcelona (spain), both affecting mainly roma/sinti communities. in sardinia, nine cases, all aged below years and three of which laboratory confirmed, with genotype d isolated, were reported from a roma/sinti settlement in the town of alghero, between august and september [ ] . four of the children had travelled to rome from to august , to attend a funeral. in the barcelona region (spain) an outbreak occurred from october to february . the first case, a six-year-old girl of eastern european origin had attended a family gathering in italy with her mother where other guests may also have had measles. genotype d was identified [ ] confirming the link with the outbreak in lazio (figure ) . genotype d was no longer isolated after the end of , being replaced by b genotype starting in october . despite an accurate epidemiological investigation, the origin of this genotype was not identified. b is not considered endemic in italy and is most frequently detected in sub-saharan africa, although transmission of this virus within europe has been reported. the isolated b was similar, but not identical, to the strain circulating in uk in [ ] . this serotype was first identified in lazio and subsequently introduced in puglia (figure ) , a region of south italy, where it was responsible for an outbreak from november to january [ ] . the percentage of cases which required hospitalisation during the two outbreaks in lazio was high ( %). this can be partly explained by the well known underreporting of cases by general practitioners and paediatricians, as compared to hospital physicians. the number of cases requiring hospitalisation, especially during july-september , was sufficiently high to create problems to the hospital system. the inadequate number of isolation beds in hospitals may represent a serious problem in case of occurrence of an epidemic due to a more aggressive infective agent, such as sars or pandemic flu. measles nosocomial transmission has been recently documented in several other outbreaks in italy and other european countries [ ] [ ] [ ] [ ] and the public health importance of nosocomial measles transmission has been established in many situations. the cases reported in this paper represent only % of the total number of cases and therefore did not contribute significantly to the measles incidence during the outbreak. however, these cases most likely represent an underestimate of the real number of infections that occurred through hospital contacts and nosocomial transmission. it is likely that isolation measures and separate admission procedures were not always adopted in case of admission of a patient with signs and symptoms compatible with measles. in the first outbreak, nosocomial transmission may have been responsible for the spread of the infection from the roma/sinti to the italian population. also of concern is the fact that four healthcare professionals developed measles. as the incidence of measles declines, nosocomial transmission is likely to become an important source of infection and sustain the occurrence of outbreaks among non-immunised health staff and hospital contacts, representing a serious problem in the elimination of measles [ ] . strategies to minimize nosocomial spread of infection should become a priority for control and effectively implemented in the future. the described outbreaks highlight the threat represented by pockets of susceptible populations, even in the presence of good coverage levels in the overall population [ ] . these groups include hard-to-reach populations (hrp) such as the roma/sinti communities (estimated mmr coverage in italy below %,) [ ] , families who refuse vaccination for ideological or religious reasons, as reported in recent outbreaks amongst students in private religious schools and orthodox communities in europe and israel [ ] [ ] [ ] , and families objecting to having their children vaccinated out of concern for vaccinerelated adverse events [ ] [ ] [ ] . the risk for the community represented by hrp or organized groups of objectors should not be underestimated and represent a more serious treat than sporadic susceptible individuals. susceptible population groups may reintroduce indigenous measles virus transmission even in countries confirmed as disease-free or in a population with high immunization coverage [ ] facilitating the transmission of the disease to susceptible individuals still present in the region. efforts are needed to improve methods to identify areas with low coverage and to develop specific strategies which target susceptible groups. an improvement in health services delivery may be needed to reach roma/sinti communities and new immigrants. at the same time, more effective communication strategies should be defined to address subjects objecting to vaccination either for religious or other reasons, involving these subjects in a wider discussion on their responsibility toward the community. the implementation of catch up campaigns targeting adolescents and young adults should also be considered, with the additional objective of protecting women of childbearing age against rubella. world health organization regional office for europe: strategic plan for measles and congenital rubella infection in the european 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survey on childhood immunization coverage and reasons of non-compliance in lazio region high genetic diversity of measles virus, world health organization european region molecular characterization of measles viruses world health organization: update of the nomenclature for describing the genetic characteristics of wild-type measles virus: new genotypes and reference strains monitoring of measles elimination using molecular epidemiology the benefit of molecular characterization during a measles upsurge in denmark net: measles outbreak in romania measles hits romania: promed-mail cluster of measles cases in the roma/sinti population measles outbreak in the barcelona region of catalonia gentic characterization of measles virus strain isoltated during an epidemic cluster in puglia an outbreak of mealses including nosocomial transmission in abulia current measles outbreak in greece measles outbreak in the region of nosocomial outbreaks -a potential threat to the elimination of measles measles elimination target: the need to meet the specific risk group measles outbreak in a community with very low vaccine coverage, the netherlands. emerging infectious diseases parent du chatelet i, the investigation team: outbreak of measles in two private religious schools in bourgogne and nord-pas-de. calais regionas of france an outbreak of measles in an ultra orthodox jewish community in jerusalem, israel, -an in dept report measles outbreak in germany: over cases now reported in nordrhein westfalen measles in south-west germany imported from switzerland -a preliminary outbreak description implication of a measles outbreak in indiana for sustainable elimination of measles in the united states pre-publication history the pre-publication history for this paper can be accessed here submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank all the health staff in the communicable diseases and immunization units of the lhu of lazio for the time and effort they have devoted to the control of the outbreaks and to assist asp in the data collection and analysis. special thanks to laura alecci (asp), for creating, maintaining, and updating the data-set, claudia fortuna and antonella marchi (mipi) for the laboratory work, and to paolo giorgi rossi (asp) for his help in finalizing the manuscript. authors' contributions fc coordinated the epidemiological data collection and the outbreaks' control measures, supervised the data analysis, and drafted the manuscript. fp supervised the local health units personnel and the implementation of public health measures. jm performed the statistical analysis and produced the graphs. mcda revised the results of data analysis and contributed in drafting the manuscript. af contributed to epidemiological data collection, participated in drafting and revising the manuscript. ln supervised the laboratory work and molecular genetic study. fm carried out the sequence alignment, the molecular genetic studies and contributed in drafting the manuscript. pb formulated the original study hypothesis and participated in the study design. ddl conceived the study, and participated in its design and coordination. all authors read and approved the final manuscript the authors declare that they have no competing interests. key: cord- -k p fr authors: olive, david; georges, hugues; devos, patrick; boussekey, nicolas; chiche, arnaud; meybeck, agnes; alfandari, serge; leroy, olivier title: severe pneumococcal pneumonia: impact of new quinolones on prognosis date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: k p fr background: most guidelines have been proposing, for more than years, a β-lactam combined with either a quinolone or a macrolide as empirical, first-line therapy of severe community acquired pneumonia (cap) requiring icu admission. our goal was to evaluate the outcome of patients with severe cap, focusing on the impact of new rather than old fluoroquinolones combined with β-lactam in the empirical antimicrobial treatments. methods: retrospective study of consecutive patients admitted in a -bed general intensive care unit (icu), between january and january , for severe (pneumonia severity index > or = ) community-acquired pneumonia due to non penicillin-resistant streptococcus pneumoniae and treated with a β-lactam combined with a fluoroquinolone. results: we included patients of whom received a β-lactam combined with ofloxacin or ciprofloxacin and combined with levofloxacin. twenty six patients ( . %) died in the icu. three independent factors associated with decreased survival in icu were identified: septic shock on icu admission (aor = . ; % ci . - . ; p = . ), age > yrs. (aor = . ; % ci . - . ; p = . ) and initial treatment with a β-lactam combined with ofloxacin or ciprofloxacin (aor = . ; % ci . - . ; p = . ). conclusion: our results suggest that, when combined to a β-lactam, levofloxacin is associated with lower mortality than ofloxacin or ciprofloxacin in severe pneumococcal community-acquired pneumonia. streptococcus pneumoniae is the leading causative agent of community-acquired pneumonia (cap). despite new antimicrobial agents and advances in supportive measures, attributable mortality linked to pneumococcal pneumonia remains unchanged and dramatically high when patient are admitted in intensive care units (icu) [ ] . most guidelines have been proposing, for more than years, a combination of a β-lactam with either a quinolone or a macrolide as empirical, first-line therapy of severe cap requiring icu admission [ ] [ ] [ ] [ ] [ ] [ ] [ ] . although a recent study demonstrated combination antibiotic therapy to be associated with a higher survival rate than monotherapy in patients with severe cap and shock [ ] , the rationale for this combination was not to increase efficacy but rather to routinely provide coverage of all common pathogens causing severe cap and particularly, s. pneumoniae and legionella species. in our icu, we followed until the french recommendations [ ] . most patients received an empirical therapy based on a β-lactam-fluoroquinolone combination. before , fluoroquinolones used were ofloxacin and ciprofloxacin. levofloxacin replaced these quinolones since its addition to the hospital formulary. such a replacement was comforted by the ers, french and idsa guidelines published between and [ ] [ ] [ ] . we wished to determine outcomes of patients treated with a combination of β-lactam plus fluoroquinolone for severe pneumococcal pneumonia. this homogenous modification of severe cap antibiotic management in our icu gives us the further opportunity to assess the influence of a fluoroquinolone with enhanced activity against s.pneumoniae. firstly, we retrospectively collected all consecutive patients aged > years who were admitted into our icu ( -bed medical and surgical intensive care unit in a -bed general hospital) between january and january for severe community-acquired pneumonia (cap) and who received a definite diagnosis of pneumococcal pneumonia. secondly, we selected patients who received, as initial antibiotic treatment, a β-lactam plus a fluoroquinolone, used with an appropriate dosage by iv route. thirdly, patients were divided into two groups according to the fluoroquinolone used, group a for ofloxacin or ciprofloxacin, group b for levofloxacin. the study protocol was submitted to the institutional review board for university hospital of lille which gave an approval with waiver of informed consent, in agreement with french regulations concerning such retrospective studies. cap was defined by the following criteria observed at initial presentation or occurring within h following hospitalization: acute onset of signs and symptoms of lower respiratory tract infection and a new pulmonary infiltrate found on the hospital admission chest radiograph. we excluded patients coming from nursing homes or hospitalized within days prior to developing pneumonia or hospitalized > h in general medical wards before icu admission, and those with radiographic abnormalities attributed solely to any other known cause (i.e., pulmonary embolus, lung carcinoma or congestive heart failure). the decision for admission to our icu was made, in all cases, by the attending physicians. however, only patients having a pneumonia severity index (psi) score ≥ were included in this study [ ] . streptococcus pneumoniae was considered as the causative agent of cap when a s. pneumoniae strain was isolated from > blood culture or when validated sputum (< squamous epithelial cells and > polymorphonuclear cells per low-power field) or tracheobronchial aspirates cultures grew with > cfu/ml s. pneumoniae. patients having cap due to a penicillinresistant strain of s. pneumoniae (mic > mg/l) were excluded from our study. appropriate drug dosages were defined in the french recommendations as: amoxicillin > mg/kg/d, cefotaxime > mg/kg/d, ceftriaxone > mg/kg/d, piperacillin > mg/kg/d, ofloxacin = mg/ h, ciprofloxacin = mg/ h, levofloxacin = mg/ h [ , , ] . these drug dosages for β-lactams, ofloxacin and ciprofloxacin were unchanged during the study period. thus, doses used in both groups were similar. within h of admission, all patients underwent clinical, radiological and biological tests. briefly, we recorded age, gender, underlying clinical characteristics and initial vital signs. chronic respiratory insufficiency was assessed combining the usual clinical and radiological criteria and the coexistence of ventilatory impairment assessed either before or after icu stay. immunosuppression was defined as recent use of immunosuppressant or systemic corticosteroids (i.e., prednisolone > . mg/kg/day for more than month), human immunodeficiency virus infection, neutropenia (absolute neutrophil count < . cells/mm ), organ transplantation with ongoing immunosuppressant, cancer chemotherapy within the past months, or asplenia. shock was defined as a sustained (> h) decrease in the systolic blood pressure of at least mm hg from baseline or a resultant systolic blood pressure < mm hg after adequate volume replacement and in the absence of any antihypertensive drug [ ] . severity of illness at admission to icu was assessed using the simplified acute physiology score ii (saps) ii [ ] , the sepsis-related organ failure assessment (sofa) score [ ] and the logistic organ dysfunction (lod) score [ ] . we also calculated the psi at icu admission [ ] . for all patients, information on the following therapeutic topics instituted within hours following icu admission was recorded: supportive measures such as mechanical ventilation or hemodialysis, use of vasopressor drugs, hydrocortisone, drotrecogin alfa (activated), or intensive insulin therapy. the effectiveness of initial antimicrobial therapy was assessed within h after treatment as follows: a lack of clinical improvement days after treatment initiation (worsening or persistent fever or hypothermia, worsening of pulmonary infiltrates or of respiratory function assessed by pao /fio ) defined an ineffective treatment. on day , day and day , body temperature, and sofa score were determined. during the patient's stay in the icu, occurrence of complications was recorded. we distinguished sepsis-related complications (secondary septic shock, acute respiratory distress syndrome or development of multiple organ failure), hospital-acquired lower respiratory tract (ha-lrt) superinfections and icu-related complications (i.e., upper gastrointestinal bleeding, catheter-related infection, deep venous thrombosis and pulmonary embolism). multiple organ failure (mof), acute respiratory distress syndrome (ards) and ha-lrt were defined according to usual criteria [ ] [ ] [ ] . durations of mechanical ventilation, treatment with vasopressor drugs, and icu length of stay were noted. finally, patient mortality was evaluated on d- , and at the time of icu discharge. descriptive analyses were performed in order to check and resume data. characteristics of patients in each group were compared. continuous variables were compared using the student's t test. categorical variables were compared using chi-square test or fisher's exact test when chi-square was not appropriate. differences between groups were considered to be significant for variables yielding a p value < . . a stepwise logistic regression including variables collected within the first hours of icu stay and associated with a p value < . in bivariate analysis was performed. adjusted odd-ratios were computed using a logistic regression analysis including the independent predictors of mortality. the kaplan-meier product limit method and the log-rank test were used to construct and compare survival curves for patients in each group. all statistical analyses were performed using the sas software, v . . during the study period, patients with severe cap were admitted in our unit. among them, ( %) patients exhibited a severe pneumococcal pneumonia and, finally, we identified patients treated with a β-lactam combined with a fluoroquinolone, including men ( . %) and women ( . %). the mean age was . ± . years. s. pneumoniae was identified in blood cultures in patients ( . %). infection was polymicrobial in patients ( . %). causative pathogens associated with s. pneumoniae were haemophilus influenzae (n = ), methicillin susceptible staphylococcus aureus (n = ), enterobacteriaceae (n = ), streptococcus spp. (n = ) and moraxella catarrhalis (n = ). all pathogens were susceptible to at least one drug (β-lactam and/or fluoroquinolone) received by the patients. thirty-eight patients ( . %) were classified as group a. β-lactams used were a third generation cephalosporin (n = ; . %), amoxicillin ± clavulanic acid (n = ; . %) and piperacillin-tazobactam (n = ; . %) combined with ofloxacin (n = ; . %) or ciprofloxacin (n = ; . %). thirty-two patients ( . %) were classified as group b. β-lactams used were a third generation cephalosporin (n = ; . %), amoxicillin ± clavulanic acid (n = ; . %) and piperacillin-tazobactam (n = ; . %) combined with levofloxacin. main patients' characteristics on icu admission are reported table . most characteristics were similar in the two groups. however, underlying chronic respiratory insufficiency and bacteremia were more frequent in group b patients. main therapeutics instituted during icu stay, evolution of severity scores, and occurrence of complications are reported table . the most significant differences between the two groups of patients were the more frequent use of drotrecogin alpha, intensive insulin therapy and hydrocortisone in group b patients. on day , ( %) patients had died, ( . %) in group a and ( . %) in group b (p = . ). overall, patients died in the icu, ( . %) in group a vs. ( . %) in group b (p = . ). so, difference in mortality rates was only significant during the first days of icu stay (figure ). in group a, in-icu mortality was % ( / ) when ofloxacin or ciprofloxacin were combined with a third generation cephalosporin and . % ( / ) when combined with another beta-lactam, respectively (p = . ). in group b, it was . % ( / ) when levofloxacin was combined with a third generation cephalosporin and . % ( / ) when combined with another beta-lactam (p = ). results of icu-discharge survival prognosis bivariate analysis, including factors present on icu admission, are reported table . all underlying diseases (excepted chronic heart failure), mechanical ventilation, use of a third generation cephalosporin combined with a fluoroquinolone, and bacteraemia on icu admission did not appear as significant prognostic variables in this analysis. among the bacteremic patients, mortality was higher in group a patients ( . %) than group b patients ( . %), but the difference was not statistically significant ( / vs. / ; p = . ). among the patients with septic shock on icu admission, mortality was higher in group a patients ( %) than in group b patients ( %), but the difference was not statistically significant ( / vs. / ; p = . ). among variables collected during the icu stay, use of hydrocortisone, intensive insulin therapy, haemodialysis and occurrence of ha-lrt superinfections did not appear as significant prognostic variables. conversely, improvement on d , sofa > on d , d , and d , and occurrence of sepsis-related complications were significantly associated with outcome at icu discharge (table ) . according to the results of the bivariate analysis, the following variables were entered in the stepwise analysis: chronic heart failure, age > yrs, acute respiratory failure requiring mechanical ventilation, septic shock on icu admission, use of hydrocortisone, haemodialysis, psi score = , saps ii > on d , lod > on d , the main finding of this retrospective analysis is that levofloxacin plus a β-lactam appears to be associated with improved survival compared to ofloxacin or ciprofloxacin plus a β-lactam in severe pneumococcal cap. empirical antibiotic regimen for icu-treated severe cap has long been recommended to cover the most common severe cap pathogens (s. pneumoniae, s. aureus and h.influenzae), atypical pathogens and most relevant enterobacteriaceae species. levofloxacin is a fluoroquinolone active against most of these pathogens, especially s. pneumoniae with or without decreased penicillin susceptibility [ , ] . its clinical activity in cap has been well documented in various clinical trials in europe and the usa [ , ] . some studies demonstrated the efficacy of levofloxacin used as monotherapy in severe cap, compared to ceftriaxone plus erythromycin or cefotaxime plus ofloxacin [ , ] . nevertheless, experts continue to propose, for icu-treated severe cap, an empirical antibiotic regimen based on an anti pneumococcal β-lactam combined with either a macrolide or a fluoroquinolone. since respiratory fluoroquinolones with enhanced activity against s. pneumoniae (levofloxacin, moxifloxacin or gemifloxacin) became available, they replaced second generation fluoroquinolones (ofloxacin or ciprofloxacin) in the guidelines [ ] [ ] [ ] . this fluoroquinolone generation shift has never been clearly justified and, to our knowledge, no clinical study has compared these different quinolones combined with a β-lactam in severe cap. our results suggest that, when severe cap causative agent is s. pneumoniae, a combination levofloxacin plus β-lactam is associated with lower mortality than a combination ofloxacin or ciprofloxacin plus β-lactam. these results could be surprising as all patients received an appropriately dosed β-lactam active against s. pneumoniae and as numerous strains of s. pneumoniae remain in vitro susceptible to ofloxacin or ciprofloxacin. however, there might be bacteriological and clinical data explaining our results. a synergy between β-lactams and levofloxacin against s. pneumoniae has been reported [ ] . conversely, synergy was rarely observed between the combination of cefotaxime and ofloxacin [ ] . recent clinical studies suggest that combination therapies could improve the prognosis of pneumococcal pneumonia: waterer et al. retrospectively studying patients with severe bacteremic pneumococcal pneumonia demonstrated that a single effective therapy was an independent predictor of mortality (aor = . ) [ ] . baddour et al. performed a prospective, multicenter, international study including adult patients with s. pneumoniae bacteremia [ ] . although the -day mortality was not significantly different for all patients receiving monotherapy versus combination ( . % vs. . %), a combination of in vitro active agents was associated with a significantly lower mortality than a single active agent ( . % vs. %; p = . ). the present work has numerous limits. the most important is probably major treatment differences among the two groups. patients were recruited during a long period ( - ), during which therapies such as hydrocortisone, drotrecogin alfa (activated), or intensive insulin therapy were introduced. management of septic shock and ards has changed following results of large international studies [ , ] . as most changes in management of patients with multiple organ failures overlap with our antibiotic policy changes, our results might be biased. indeed, hydrocortisone use and intensive insulin therapy were more frequent in group b than in group a. however, these factors were not significantly associated with icu survival in bivariate analysis and hydrocortisone use, in multivariate analysis, was not an independent prognostic factor. moreover, there is no evidence suggesting a survival benefit by most adjunctive therapies in patients with cap [ ] and the benefit of intensive insulin therapy in medical icu and/or low-dose steroids is now highly questionable [ , ] . similarly, the use of cephalosporin is more frequent in group b than in group a. however, the use of a third generation cephalosporin rather than amoxicillin has no impact on prognosis. this is not surprising as, to our knowledge, no clinical study demonstrated a third generation cephalosporin to be superior to amoxicillin for non penicillin-resistant s. pneumoniae cap as far as drug dosage is adequate. finally, some important prognostic parameters such as the time elapsed between admission and the first dose of antibiotic were not taken into account in our study. before , we did not have computerized data charts thus, exact time of admission and antibiotics admission, particularly for patients transferred from other departments/hospitals cannot be obtained. our study suggests that levofloxacin combined with a β-lactam is associated with improved survival in comparison with ofloxacin or ciprofloxacin combined with a β-lactam in severe pneumococcal patients admitted in the icu. this combination, proposed by current guidelines as empirical treatment of severe cap patients admitted in icu could improve their prognosis. obviously, only a prospective, randomized, double-blind trial could confirm this result. list of abbreviations aor: adjusted odd ratio; ards: acute respiratory distress syndrome; cap: community-acquired pneumonia; ci: confidence interval; ha-lrt superinfections: hospital-acquired lower respiratory tract superinfections; icu: intensive care unit; lod score: logistic organ dysfunction score; los: length of stay; mof: multiple organ failure; mv: mechanical ventilation; psi: pneumonia severity index; saps: simplified acute physiology score; sofa: sepsis-related organ failure assessment; sd: standard deviation. epidemiological features and prognosis of severe community-acquired pneumococcal pneumonia quatrième conférence de consensus en thérapeutique anti-infectieuse de la société de pathologie infectieuse de langue française: les infections des voies respiratoires management of community-acquired lower respiratory tract infection in the adult. recommendations by the french language society of pneumology with collaboration of the french language society of infectious pathology, from the recommendations of the practice guidelines for the management of community-acquired pneumonia in olive et al. bmc infectious diseases infectious diseases society of america guidelines for the management of adults with community-acquired pneumonia. diagnosis, assessment of severity, antimicrobial therapy, and prevention european society of clinical microbiology and infectious diseases: guidelines for the management of adult lower respiratory tract infections xve conférence de consensus en thérapeutique anti-infectieuse: prise en charge des infections des voies respiratoires basses de l'adulte immunocompétent consensus 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require vasopressors cefotaxime acts synergistically with levofloxacin in experimental meningitis due to penicillin resistant pneumococci and prevents selection of levofloxacin-resistant mutants in vitro in vitro interaction between ofloxacin and cefotaxime against grampositive and gram negative bacteria involved in serious infections monotherapy may be suboptimal for severe bacteremic pneumococcal pneumonia international pneumococcal study group: combination antibiotic therapy lowers mortality among severely ill patients with pneumococcal bacteremia the acute respiratory distress syndrome network: ventilation with lower tidal volumes as compared with traditional tidal volumes for acute lung injury and the acute respiratory distress syndrome surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock adjunctive therapies for community-acquired pneumonia: a systematic review intensive versus conventional glucose control in critically ill patients edusepsis study group: effectiveness of treatments for severe sepsis pre-publication history the pre-publication history for this paper can be accessed here severe pneumococcal pneumonia: impact of new quinolones on prognosis. bmc infectious diseases the writers thank g. moran for collaboration in the writing of this paper. authors' contributions do collected data and helped to draft the manuscript., hg participated in the design of the study, collected data and helped to draft the manuscript, pd performed the statistical analysis., nb collected data and helped to draft the manuscript, ac collected data and helped to draft the manuscript, am collected data and helped to draft the manuscript, sa collected data and helped to draft the manuscript, and ol contributed to the design of the study and wrote the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -l qcbat authors: rao, huaxiang; shi, xinyu; zhang, xi title: using the kulldorff’s scan statistical analysis to detect spatio-temporal clusters of tuberculosis in qinghai province, china, – date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: l qcbat background: although the incidence of tuberculosis (tb) in most parts of china are well under control now, in less developed areas such as qinghai, tb still remains a major public health problem. this study aims to reveal the spatio-temporal patterns of tb in the qinghai province, which could be helpful in the planning and implementing key preventative measures. methods: we extracted data of reported tb cases in the qinghai province from the china information system for disease control and prevention (cisdcp) during january to december . the kulldorff’s retrospective space-time scan statistics, calculated by using the discrete poisson probability model, was used to identify the temporal, spatial, and spatio-temporal clusters of tb at the county level in qinghai. results: a total of , tb cases were reported from to in qinghai. results of the kulldorff’s scan revealed that the tb cases in qinghai were significantly clustered in spatial, temporal, and spatio-temporal distribution. the most likely spatio-temporal cluster (llr = . , rr = . , p < . ) was mainly concentrated in the southwest of qinghai, covering seven counties and clustered in the time frame from september to december . conclusion: this study identified eight significant space-time clusters of tb in qinghai from to , which could be helpful in prioritizing resource assignment in high-risk areas for tb control and elimination in the future. electronic supplementary material: the online version of this article (doi: . /s - - -y) contains supplementary material, which is available to authorized users. tuberculosis (tb) is an infectious disease caused by mycobacterium tuberculosis. over % of the new tb cases, globally, were reported in developing countries. according to a world health organization report, the tb burden of china is the second largest in the world [ ] . in recent years, although the chinese government has paid an increasing amount of attention to the control of tb, prevention measures are still insufficient, especially in areas with inadequate medical resources, such as qinghai, a province where most of the population suffers a high risk of tb even now [ ] . a large number of studies on the spatial and temporal distribution of tb have demonstrated that tb has a highly complex dynamics and is spatially heterogeneous at the provincial, national, and international levels during certain periods of time; however, the variations in small area are always be ignored by using a relatively large scale [ ] [ ] [ ] [ ] . in our previous study, the moran's i spatial autocorrelation analysis method was used to analyze the tb incidence data from to in qinghai and found that the distribution of tb in this province was not random [ ] . however the global moran's i spatial autocorrelation analysis only evaluates the distribution characteristics of the disease in several specific time points. moreover, this method can not estimate the risk level of high-risk cluster areas [ ] [ ] [ ] [ ] . it is a known fact that time is a critical confounder that might directly bias the determination of the high-risk regions of tb. the kulldorff 's space-time scan statistical method can detect the distribution characteristics in both the temporal and spatial axes, bringing them closer to real-world conditions [ ] [ ] [ ] [ ] . additionally, the relative risk of disease in a cluster area can be estimated by comparing it with the area outside the cluster area. this method has been used wildly in the epidemiology studies of infectious diseases [ ] [ ] [ ] [ ] . analyzing and evaluating the spatio-temporal patterns and trends of tb in qinghai is necessary for tb control and elimination. in this study, our aim was to use the kulldorff's scan statistical analysis to explore the spatial, temporal, and space-time dynamics of tb at the county level in qinghai. qinghai is located in the northwest china and lies to the northeast of the qinghai-tibet plateau. the average altitude is - m. qinghai comprises eight prefectures, including a total of counties (fig. ) . the province is comparatively less developed, with a high annual incidence of tb. the total population is about . million people. we collected data ( - ) of tb cases in qinghai from the china information system for disease control and prevention (cisdcp). we also extracted the demographic data of counties from qinghai's statistical yearbooks ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) . tb is one of the notifiable infectious diseases in china. it is mandated that each case of tb must be reported online within h after diagnosis in a hospital. cases of tb were diagnosed using radiography, pathogen detection, and pathologic diagnosis, based on the diagnostic criteria recommended by the national health and family planning commission of the people's republic of china ( ). a total of , incident cases of tb and tb-related deaths were reported across hospitals and medical institutions in qinghai from january to december . in this study, , cases, aggregated at the county level monthly, were analyzed to detect the spatio-temporal high-risk areas of tb. and cases without detailed information on the residential address were excluded from the analysis. in order to check the missing reports of tb, we randomly selected from all hospitals and medical institutions, and double checked all the medical records of these selected institutions. no missing cases or outbreaks of tb were recorded. we used kulldorff's space-time scan statistical analysis to detect the temporal, spatial, and space-time clusters of tb, and to verify whether the geographic clustering of tb was caused by random variation or not [ ] . since the population in several areas was very small, we used the radius of the population coverage instead of the geographical radius. the discrete poisson probability model was used for scanning since the tb incidence was not very high [ ] . the window with the maximum likelihood is defined as the most likely cluster area, and other clusters with statistically significant log-likelihood ratios (llr) were defined as the secondary potential clusters. the pvalues of llr were estimated through monte carlo simulations [ , ] . a p-value < . indicates a significantly high risk inside of the scan window, which might be a potential cluster of a high risk of tb. the relative risk (rr) of tb in each cluster was calculated to evaluate the risk of tb in the cluster areas [ , , ] . the results of spatio-temporal scan are sensitive to various parameters, like the maximum cluster sizes of spatial and temporal. thus, the selection of the maximum radius of the spatial scanning window and the maximum length of the temporal scanning window were very important [ ] . in order to select optimal parameters, we analyzed the data of using the maximum spatial cluster sizes from % to % of total population at risk by increments of %. the radius was considered as an optimal radius for analysis if there were fewer overlaps between the areas defined by the radius, and the biggest area covered less than seven counties or % of all the counties [ , ] . similarly, we found an optimal temporal cluster size by testing the maximum temporal cluster sizes from % to % of the total study period by increments of % to analyze the data of the preceding years ( - ). based on the the optimal spatio-temporal parameters, retrospective space-time scanning analysis was applied to identify the geographic areas and time periods of potential clusters with significantly higher tb incidents than that of nearby areas. we also used global moran's i spatial autocorrelation analysis to depict the spatial clustering of annual tb incidence at the county level. the moran's i > , = , and < indicate a positive spatial autocorrelation, random distribution, and negative spatial autocorrelation, respectively [ ] . additionally, we conducted time series seasonal decomposition analysis to identify the seasonality of tb incidence in qinghai province [ ] [ ] [ ] . the seasonal index was also calculated to examine the seasonal pattern of tb. the index was calculated as the ratio of the average number of cases for a given month to the average monthly incidents of * months ( - ). an index value close to . indicates no seasonal trend [ ] . the satscan™ software (v . . , kulldorff and information management services, inc.) was used for spatial, temporal, and spatio-temporal analyses. then, we used arcgis (v . . , esri inc., redlands, ca, usa) to visualize the relative risk of tb in high-risk cluster areas. open geoda software (arizona state university, az, usa) was used for global moran's i spatial autocorrelation analysis. p < . indicates a statistical significance. we conducted temporal scanning by using the time window with the length that covers - % of the total study period, by increments of %. the scanning results indicated that the high-risk cluster of tb was predominantly concentrated in the time period between january and may (additional file : table s ). therefore, the maximum temporal cluster size was set as % in this study. for each year, the maximum scan time length was months. determination of the optimal space window for spatial scanning in order to detect an appropriate space window for the spatial scan, we conducted several times of spatial scanning using different maximum circular spatial windows. we started from the radius covering from % to % of the population, increasing by % each time. the results are shown in additional file : fig. s . when the maximum spatial scan size was set to - %, the high-risk clustering areas overlapped, and the most likely cluster covered more than % of all the counties. while for the sizes of - %, selfsame area was detected as the most likely cluster area, but the secondary clusters were slightly different. the cluster areas identified using the windows of - % covered the largest high-incidence areas. according to the venn diagram (fig. ) , we finally set % as the maximum circular spatial window for spatial scanning, covering a population of . million. two counties, huangzhong and datong, were not included in the scanning window. the incidence rates of tb in these two counties were relatively low. therefore, the exclusion would not be influential. the time series seasonal decomposition analysis of tb incidents showed a significant seasonal periodicity, but the seasonal trend was not obvious between and ( fig. a and b) . this was consistent with the result of the seasonal index (fig. e) . the maximum seasonal index value was . in march, and the value appeared to be less than . after july. there was a slowly increase trend for tb incidents from to (fig. c) . the temporal cluster analysis also showed that tb incidents were mainly concentrated in the spring and early summer, annually, ranged from january to may. the high aggregated period for tb was observed in all districts from january to august (llr = . , p < . ). during this period, a total of , tb cases were reported, and the risk of tb related incidents was % (rr = . ) higher than that in other time periods (table ) . spatial clustering analysis of the entire years identified a total of nine statistically significant high-risk areas, covering a total of counties. similarly, the global moran's i values of each year at the county level also indicated a positive spatial autocorrelation in qinghai, ranging from . to . (all p < . ). the high-risk areas with a relative risk greater than three, including the most likely cluster area and two secondary cluster areas, were mainly concentrated in the southwest of qinghai. the center of the most likely cluster area was located in dari county, . °n and . °e (llr = . , p < . ). this circular area covered six counties with a radius of . km, including dari, gande, maqin, banma, jiuzhi, and maduo. the total number of tb cases was , and the risk of tb related incidents was . times (rr = . ) higher than that outside this area (table and additional file : fig. s ). the incidences of tb inside the cluster areas were significantly higher than that in the areas outside every year ( table ) . the results of spatio-temporal cluster analysis suggested a special characteristic in temporal and spatial distribution for tb incidents in qinghai. we detected a most likely cluster area and seven secondary cluster areas by fig. venn diagram of spatial clustering for tb incidents in qinghai, china, . the scan window used in this analysis was set to be % of population using temporal and spatial scanning (fig. ) . the most likely spatio-temporal cluster area was located at the southwest of the province, and the high-risk period was from september to december (llr = . , p < . ). the center of this area was in yushu county, . °n and . °e, which was a circular area with a radius of . km, covering seven counties: yushu, nangqian, chengduo, zaduo, maduo, qumalai, and dari. a total of tb cases were reported in this area during the high-risk period, and the rr was . (table ). a seasonal trend was decomposed from the time-series of tb incidents; c: a long-term trend was decomposed from the time-series of tb incidents; d: the residual data after excluding of seasonality and a long-term trend; e: estimated seasonal index of months ranged from . to . , and the maximum value was recorded in march in this study, spatial patterns and the secular trends of tb in qinghai from to were examined using the kulldorff's scan statistical analysis. to the best of our knowledge, no other similar study has been done in this area. our study demonstrated that there was a significantly space-time clustering in distribution of tb incidents in qinghai province. the high-risk areas were mainly concentrated in the southwest qinghai, and the temporal clusters were mainly concentrated in spring and early summer. kulldorff's retrospective scan statistics take multiple testing problems into account, which is known as the most powerful method for evaluating geographical and temporal distribution by using routinely collected data [ ] . this method has been used worldwide to detect the clusters of diseases [ , , , , ] . as is known, in the temporal and spatial model, selection of a suitable time window and spatial window was very important for model identification. currently, there are two methods for selecting the size of spatial window: one is based on the geographical area, and the other method is based on the population size covered by the scanning area [ ] . in this study, we used the radius of population coverage, because deviation of population in different counties was very large. similarly, size of time scanning window is another important parameter for analysis. generally, the window size of time was set as % of the entire time period of the study. however, there exists some evidence with regard to whether or not this window size is reasonable [ ] . yue ma et al. conducted a simulation study to explore how to choose an appropriate scanning window. they found that the window might be too large to include the low-risk area if the window covered % of the population [ ] . therefore, this situation might lead to a high false positive rate. however, the window which covered a smaller population might be too small to detect the real high-risk area, and the high-risk area would be separated. thus, the high false negative rate would be an issue. tango and takahashi suggested that when using the irregular scan statistic to detect the aggregated region, the coverage area of a single region should not be more than % of the whole study area [ , ] . in addition, several studies also suggested choosing an appropriate window which could identify the cluster areas with less overlap [ ] . based on the tango's criteria, we analyzed the data for many times by using one window value at a time. finally, we selected the temporal window covering % of whole study period and the spatial window covering % of the population at risk. and the overlapping of the identified high-risk clusters was not observed. our temporal scanning results indicated that there was a high-risk period for tb incidents every year and during the entire years, which mainly occurred in spring and early summer, from january to may. as is known, during winter, the reduction in exposure to ultraviolet rays from sunlight and the poor ventilation in indoor settings may increase the incidences of tb infections. additionally, in the case of infectious diseases, time is needed for the symptoms to develop and patients may lack the knowledge on where to seek care for tb. all these factors may delay tb diagnosis and treatment [ ] . some studies reported that the average incubation period of tb infection ranges from to weeks, with a -month interval from the appearance of symptoms to medical diagnosis [ ] . therefore, the high-risk periods in spring and early summer complied with the disease characteristics. such seasonal patterns were consistent with the previous studies done in yunnan province, where is the registration peak of tb cases during spring [ ] . additionally, spatial scanning results displayed statistically significant - cluster areas for tb diseases in qinghai each year, which were similar to the results of our previous study [ ] . compared with the median incidence rate of tb ( / ) out of the cluster areas, tb incidence in our identified tb cluster was higher than / . this indirectly suggested a relatively high sensitivity of this scanning method. the spatiotemporal model used in this study simultaneously considered time and space distributions. compared with the separated spatial scanning model and temporal scanning model, the time-space scanning makes a conclusion more closely to the real-world situation. using this model to detect the spatio-temporal distribution of tb in qinghai, from to , we found that the highrisk counties were concentrated in the southwest qinghai, from september to december . during this period, the risk of tb infection in these areas was obviously higher than in other areas, especially in the zhiduo county. in these areas, the inhabitants have very low income, as well as poorer living conditions and sanitation compared to the eastern region of qinghai. many studies have showed that poverty is one of the most important social factors responsible for the high prevalence of tb, and also the socio-economic status may contribute to the high risk of tb [ ] . our result indicated that further prevention and special tb control strategies should be considered in relation with the economical and sanitary level in the clustered areas. our study also demonstrated the usefulness of spatial and temporal clustering analysis using the arcgis and satscan to identify the significant space-time clusters of tb in qinghai. this could be used to provide strategies for tb prevention at the county level. however, the study had limitations on analysis. first, it is important to note that the data were analyzed at the county level, which is not the smallest unit of administrative regionalization. thus, we may exclude several critical factors. second, the influence of weather and socio-economic factors were not included in this study. our study analyzed the spatial, temporal, and space-time clusters of tb incidents at the county level in qinghai, from to , using the kulldorff's retrospective scan statistic methods. the spatial and temporal clusters were statistically significant every year, and the spacetime scanning result indicated eight high-risk areas for tb incidents which were predominantly located in the southwest qinghai. these results suggested that it is urgent to establish the preventive and controlling strategies to decrease the tb incidence in qinghai by qinghai government and the center for disease control and prevention. additional file : table s . temporal clustering of tb incidents monthly in qinghai, china, - . we set the maximum size for temporal scanning to be months, nearly % of the total study period, by which the scan result was best to fit the raw time-series data of tb incidents. the who global tuberculosis report-further to go space-time clustering characteristics of tuberculosis in china spatial-temporal epidemiology of tuberculosis in mainland china: an analysis based on bayesian theory spatial and temporal analysis of tuberculosis in zhejiang province, china pulmonary tuberculosis space-time clustering and spatial variation in temporal trends in portugal, - : an updated analysis childhood tuberculosis infection and disease: a spatial and temporal transmission analysis in a south african township spatial transmission and meteorological determinants of tuberculosis incidence in qinghai province, china: a spatial clustering panel analysis spatial patterns and secular trends in human leishmaniasis incidence in morocco between area-level global and local clustering of human salmonella enteritidis infection rates in the city of toronto, canada spatial big data analytics of influenza epidemic in vellore analyzing spatial clustering and the spatiotemporal nature and trends of hiv/aids prevalence using gis: the case of malawi spatial, temporal, and spatiotemporal analysis of malaria in hubei province temporal, spatial and spatiotemporal analysis of the occurrence of visceral leishmaniasis in humans in the city of birigui, state of sao paulo detecting spatial-temporal cluster of hand foot and mouth disease in beijing, china spatial disease clusters: detection and inference spatial cluster analysis of human cases of crimean congo hemorrhagic fever reported in pakistan cluster of human infections with avian influenza a (h n ) cases: a temporal and spatial analysis malaria infection has spatial, temporal, and spatiotemporal heterogeneity in unstable malaria transmission areas in northwest ethiopia using the satscan method to detect local malaria clusters for guiding malaria control programmes influence of spatial resolution on space-time disease cluster detection childhood leukaemia in sweden: using gis and a spatial scan statistic for cluster detection a spatial scan statistic for survival data selection of the maximum spatial cluster size of the spatial scan statistic by using the maximum clustering set-proportion statistic a flexibly shaped spatial scan statistic for detecting clusters a flexible spatial scan statistic with a restricted likelihood ratio for detecting disease clusters exploration of diarrhoea seasonality and its drivers in china seasonality of tuberculosis in the united states extreme temperatures and paediatric emergency department admissions the epidemiology of plasmodium vivax and plasmodium falciparum malaria in china serological study of an imported case of middle east respiratory syndrome and his close contacts in china spatiotemporal transmission dynamics of hemorrhagic fever with renal syndrome in china spatiotemporal patterns of japanese encephalitis in china seasonal variations in notification of active tuberculosis cases in china spatial-temporal analysis of pulmonary tuberculosis in the northeast of the yunnan province, people's republic of china the authors have no external support of funding to report. this study was supported by the qinghai center for disease control and prevention (cdc). the authors gratefully acknowledge the staff involved in tb surveillance at all participating levels in qinghai province, china. the authors have no support or funding to report. the dataset analyzed during the current study, while not publicly available, is available from the corresponding author on reasonable request. authors' contributions hr, xs and xz contributed equally to this work. hr conceived and designed the study. hr and xs collected the data and performed the statistical analysis. hr and xz prepared the manuscript. all authors read and approved the final manuscript.ethics approval and consent to participate this retrospective study was consulted to the ethics review board of the qinghai center for disease control and prevention. ethics approval was not available in this study because we did not include any data of patients' personal or health information, including name, identity information, address, telephone number, etc. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -r atew authors: chong, zhuo lin; sekaran, shamala devi; soe, hui jen; peramalah, devi; rampal, sanjay; ng, chiu-wan title: diagnostic accuracy and utility of three dengue diagnostic tests for the diagnosis of acute dengue infection in malaysia date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: r atew background: dengue is an emerging infectious disease that infects up to million people yearly. the growing demand of dengue diagnostics especially in low-resource settings gave rise to many rapid diagnostic tests (rdt). this study evaluated the accuracy and utility of virotrack dengue acute - a new biosensors-based dengue ns rdt, sd bioline dengue duo ns /igm/igg combo - a commercially available rdt, and sd dengue ns ag enzyme-linked immunosorbent assay (elisa), for the diagnosis of acute dengue infection. methods: this prospective cross-sectional study consecutively recruited patients with suspected dengue from a health clinic in malaysia. both rdts were performed onsite. the evaluated elisa and reference tests were performed in a virology laboratory. the reference tests comprised of a reverse transcription-polymerase chain reaction and three elisas for the detection of dengue ns antigen, igm and igg antibodies, respectively. the diagnostic performance of evaluated tests was computed using stata version . results: the sensitivity and specificity of virotrack were . % ( %ci . – . ) and . % ( %ci . – . ), versus . % ( %ci . – . ) and . % ( %ci . – . ) for sd ns elisa, and . % ( %ci . – . ) and . % ( %ci . – . ) for ns component of sd bioline, respectively. the combination of the latter with its igm and igg components were able to increase test sensitivity to . % ( %ci . – . ) with corresponding decrease in specificity to . % ( %ci . – . ). although a positive test on any of the ns assays would increase the probability of dengue to above % in a patient, a negative result would only reduce this probability to . – . %. in contrast, this probability of false negative diagnosis would be further reduced to . % ( %ci . – . ) if sd bioline ns /igm/igg combo was negative. conclusions: the performance of virotrack dengue acute was comparable to sd dengue ns ag elisa. addition of serology components to sd bioline dengue duo significantly improved its sensitivity and reduced its false negative rate such that it missed the fewest dengue patients, making it a better point-of-care diagnostic tool. new rdt like virotrack dengue acute may be a potential alternative to existing rdt if its combination with serology components is proven better in future studies. dengue is an emerging infectious disease endemic to more than tropical countries [ ] . an estimate of up to million dengue infections happened in the year alone, of which only a quarter were detectable [ ] . dengue threatens a pandemic with the spread of aedes mosquito, the vector that carries the pathogen dengue virus that comes in four serotypes (denv - ), to subtropical regions throughout the world [ ] . in malaysia, dengue incidence stood as high as . cases/ , population with case fatality rate ranging from . - . % in recent years. in alone, dengue virus infected up to , people and caused deaths [ ] . despite an absence of medical treatment to date, early disease recognition and timely intervention with proper fluid management and supportive care can prevent mortality due to dengue infection [ ] . the obstacle to early dengue diagnosis lies in its diverse and unspecific clinical symptoms that resemble other diseases, which leads to delay in health-seeking and misdiagnosis of the disease [ , ] . laboratory tests such as virus isolation (vi), reverse transcriptionpolymerase chain reaction (rt-pcr), haemagglutination inhibition (hi), and enzyme-linked immunosorbent assay (elisa) for the detection of dengue non-structural antigen- (ns ) or dengue-specific immunoglobulin (igm/igg) can confirm diagnosis. but they are resourceintensive and not suitable for low-resource settings [ ] . the growing demand for point-of-care diagnostics gave rise to many dengue rapid diagnostic tests (rdt) that flooded the market in the past two decades [ , ] . recent development in biosensors for the rapid diagnosis of acute dengue infection, if proven accurate, may intensify the competition and make rdt more affordable to more patients who need it [ ] . in this evaluation study, we aimed to estimate the diagnostic accuracy and utility of a newly developed biosensors-based dengue ns assay, and one each of commercially available ns elisa and ns /igm/igg rdt. this evaluation study employed a prospective crosssectional design. its reporting followed standards for the reporting of diagnostic accuracy studies (stard) guidelines [ ] . the study adhered to the principles of the revised declaration of helsinki [ ] and obtained ethical approval from the medical research and ethics committee, ministry of health malaysia (nmrr- - - , ) and university malaya medical center (mrecid.no: - ). written informed consent was obtained from each participant and minor assent where appropriate. the study site was shah alam section health clinic, a public clinic with the highest number of dengue patients located in the dengue-endemic district of petaling, selangor state, malaysia. febrile patients aged months and above with symptoms fulfilling world health organisation (who) criteria for suspected dengue that sought treatment at this clinic from th november to th march during normal working hour were recruited consecutively [ ] . patients in need of emergency care or with pre-existing conditions that were prone to complications from blood sampling were excluded. both capillary and venous blood samples were collected from each patient using edta tubes for immediate onsite index tests. another venous sample in plain tube was also collected together, centrifuged, aliquoted, and stored at − °c for laboratory-based diagnostic tests in a virology laboratory in university of malaya, malaysia. no convalescent sample was taken. socio-demographic background and clinical history were captured using structured questionnaire through faceto-face interview at the same setting. the target sample size calculated using single proportion sample size formula based on a % disease prevalence was expected to achieve % absolute precision with % confidence for both sensitivity and specificity estimates [ ] . virotrack dengue acute (blusense diagnostics, denmark), sd bioline dengue duo and sd dengue ns ag elisa (standard diagnostics, korea) were evaluated in this study for comparison. the first two are rdt intended for point-of-care use. they were tested onsite on both capillary and venous blood samples by medically trained research assistants blinded to the clinical pictures of the research participants. additionally, viro-track dengue acute was also tested onsite on the same day with serum samples extracted from the plain tubes. virotrack dengue acute (blusense diagnostics, denmark) is a biosensors-based semi-quantitative immuno-magnetic agglutination assay packed in a polymer centrifugal microfluidic cartridge. its diagnostic mechanism was detailed out previously [ ] . briefly here, for each test, a virotrack micofluidic loaded with mcl of blood sample was inserted into a portable opto-magnetic readerthe blubox. the sample was centrifuged, metered, and mixed with magnetic nanoparticles (mnps) pre-coated with antidengue antibodies located within the cartridge. dengue ns antigen, if present, formed sandwich agglutination with these mnps and were forced to rotate under an oscillating magnetic field, which modulated the intensity of a laser beam passing through them. a photodetector with a blu-ray optical pickup unit would then measure the phase difference between the modulated light transmission and the applied field, which corresponded to the level of dengue ns antigen. this measurement was presented in a relative unit and interpreted by the blubox according to a pre-set threshold value, where positive was defined as > = , negative if < , and equivocal (eq) if - . unit. the whole process after the insertion of the microfluidic was automatic and the result was ready in less than min. the results were recorded by one research assistant and verified by three others independently. for analysis, a patient was considered tested positive for virotrack dengue acute if either capillary or venous sample was positive, eq if both were eq, and negative for all other combinations. no repetition was done for eq results. this is a commercially available rapid immunochromatographic test that comes in a combo of two joint cassettes, one for ns and another for igm/igg. only mcl blood sample was needed for the ns assay, while serology required mcl followed by assay diluent. results were interpreted according to manufacturer's instruction by two independent research assistants - min after the application of specimen, where appearance of a test line was considered positive in the presence of a control line. presence of only control line was considered negative [ ] . discrepancies between first and second interpreters were resolved with the help of a third interpreter. for analysis, a patient was considered tested positive to an assay on sd bioline dengue duo if either capillary or venous sample was found positive, and negative if both were negative. sd dengue ns ag elisa sd dengue ns ag elisa (standard diagnostics, korea), a commercially available direct sandwich elisa, was performed together with reference tests and interpreted according to manufacturer's instruction [ ] . test was considered valid if the negative and positive controls absorbance values were within set ranges. cut-off value was calculated by adding . to the mean absorbance for negative controls. a sample was considered positive if its absorbance was equal to or larger than the cut-off value, and negative if lower. the reference tests comprised of itaq universal sybr green one-step real-time rt-pcr (bio-rad laboratories, hercules, ca), panbio dengue early elisa, and sd dengue igm and igg capture elisa (standard diagnostics, korea). they were performed according to the manufacturers' instructions as described in detail previously [ ] [ ] [ ] [ ] . they were chosen in reference to a previous study [ ] . these tests were conducted from th december to th april , up to around month from sample collection, by trained laboratory personnel blinded to the clinical information and results of the point-of-care index tests. a laboratory-confirmed dengue was defined as ) rt-pcr positive, or ) panbio ns elisa positive; while a presumptive dengue tested negative for both the above, but positive for igm elisa [ ] . both laboratory-confirmed and presumptive dengue were included in the analysis as dengue positive; while patients who did not fall into any category above were taken as dengue negative without further laboratory tests. on top of that, a combination of "recife" method and igm/igg ratio from elisa was used to classify dengue positive patients into primary and secondary dengue, whereby primary was defined as igg negative with positive on either igm, ns or rt-pcr; while secondarypositive igg with negative igm and positive on either rt-pcr or ns . if both igm and igg were present, igm/igg ratio > = . was considered as primary dengue, while < . -as secondary dengue [ , , , ] . descriptive analysis was used to describe the sociodemographic and clinical characteristics of the participants. the interrater agreement between the first and second interpreters for each assay in sd bioline dengue duo was assessed using kappa statistics (k). it was also computed for the agreement of test results between capillary and venous samples for this combo, while virotrack dengue acute also had additional results for capillaryserum and venous-serum. agreement was interpreted as poor if k was < , slight if - . , fair if . - . , moderate if . - . , good if . - . , and excellent if . - . [ ] . the true positive (tp), false negative (fn), false positive (fp), and true negative (tn) of each index test and various combinations of the components of combo test as compared to the reference standard were used to calculate various diagnostic accuracy parameters and their % confidence intervals ( %ci) using standard formulas [ , ] subgroup analyses by exposure (serotype, day of illness or doi, dengue infection status) and outcome (lab-confirmed vs presumptive dengue) were also performed to compare sensitivity estimates. for diagnostic utility, post-test probabilities ( %ci) of dengue for positive and negative test were calculated for each test assay and their combinations. for that, pre-test probability (prevalence) of dengue among participants of this study was first converted to odds: prevalence/( -prevalence). this pre-test odds were then multiplied with corresponding lr to obtain post-test odds that in turn were converted back to probabilities: odds/( + odds) [ ] . data analysis was performed using stata version (statacorp, tx, us). all inconclusive and missing test results, whether of reference standard or index tests, were excluded from the analysis. out of the potentially eligible patients who attended the clinic over the study period, ( . %) agreed to participate in the study. their age ranged from months to . years with a mean of . years (s.d. . ). ( . %) were male. the mean day of fever upon recruitment was . (s.d. . ) days, with a range of to days. all recruited patients had either capillary and/or venous sample tested on both point-of-care rdt. but only were characterised with the laboratory-based index test -the sd dengue ns ag elisa, and all reference tests. absence of results for any assay was due to insufficient test specimens. the flow of participants for the index tests and their results was presented using stard diagrams (figure s , s and s ). out of the patients tested with reference tests, were dengue positive, were negative, and was inconclusive. among the dengue positive patients, were presumptive and were laboratory-confirmed. the latter comprised of positives on both rt-pcr and panbio ns elisa, positives only on rt-pcr, and positives only on ns elisa. among the dengue positive patients, had dengue without warning sign, while reported at least one warning sign according to who classification. one hundred thirty seven of them had primary while had secondary dengue. lastly, there were denv- , denv- , denv- , and only denv- among the patients tested positive on rt-pcr. for sd bioline ns assay, patients had both capillary and venous samples tested and patients had only venous results; while had both results and had only venous result for igm/igg assay. for virotrack dengue acute, patients had both results, while and had either capillary or venous result, respectively. all patients also had serum results for virotrack dengue acute. comparison can only be made between test results from different subgroups belonging to the same patient. the kappa and their %ci for all comparisons were more than . , indicating excellent agreement (table s , s , s ). for sd bioline dengue duo, both interpreters almost completely agreed on ns assay tested on both capillary and venous samples with kappa of . ( %ci . - . ) and . ( %ci . - . ), respectively; while the results were not significantly lower for serology tests with point estimates of k ranging from . - . (table s ). when the final approved test results between capillary and venous were compared, the kappa ranged from . ( %ci . - . ) for igg assay to . ( %ci . - . ) for ns (table s ) . for virotrack dengue acute ns assay, the kappa stood at . ( %ci . - . ) for capillary-venous, . ( %ci . - . ) for capillary-serum, and . ( %ci . - . ) for venous-serum (table s ). these estimates were lower than that of sd bioline ns assay due to the additional eq category, albeit not statistically significant. all the diagnostic accuracy parameters were presented in table . among ns -only assays, the sensitivity of ns elisa from sd was significantly higher than its own rdt, at . % ( %ci . - . ) versus . % ( %ci . - . ), respectively. the sensitivity of viro-track dengue acute ranked second at . % ( %ci . - . ) and did not significantly differ from both the above. the specificities were rather comparable, with sd ns rdt performed better insignificantly at . % ( %ci . - . ). there was no significant difference between all three ns tests for the other parameters ( table ) . the sensitivities of both sd serology rdt were lower when compared to all the ns -only assays, and the difference was significant when compared to elisa and virotrack, with igm at . % ( %ci . - . ) and igg - . % ( %ci . - . ). however, among all individual assays, igm had the highest specificity of . % ( %ci . - . ); while igg had it lowest - . % ( %ci . - . ), which was significantly lower when compared to both sd ns and igm rdt. igg also performed significantly worse in all the other parameters compared to all individual assays except for npv when compared to igm. on the contrary, igm had higher ppv, lr+, and dor; but worse npv, lr-, and auc, when compared to ns -only assays. although significant difference was only found in lr-and auc with elisa, and only lr-with viro-track (table ) . when the results of all three individual assays on sd bioline dengue duo were combined, where positive on either one was considered as dengue positive, the diagnostic accuracy parameters generally improved. for sd ns /igm combination, all the estimates were better compared to elisa and virotrack, but all these differences were insignificant save for virotrack sensitivity, lr-, and auc. when compared to sd ns rdt alone, ns /igm performed better except on specificity, ppv, and lr+, with significant difference only in sensitivity, npv, lr-, and auc. on the contrary, for sd ns /igm/ igg, the insignificant marginal improvement over sd ns /igm in sensitivity to . % ( %ci . - . ), npv to . % ( %ci . - . ), and lr-to . ( %ci . - . ), was compensated with decrease in all other parameters, which were significant for specificity, ppv, and lr+ ( table ) . the sensitivities of the evaluated index tests stratified into different subgroups were presented in table and table . when stratified by serotype, all ns assays whether alone or in combination had higher sensitivity in detecting denv- , followed by denv- and denv- . only the difference between denv- and denv- was significant for virotrack, and sd ns rdt whether individually or in the form of combo. there was no significant difference between the lower sensitivities of sd serology rdt in the detection of various serotypes, although igm appeared to do insignificantly better with denv- followed by denv- . none of the assays was able to detect the sole denv- patient. when compared between index tests, the sensitivities of all assays with ns component did not differ much with each other but were all significantly better than serology-only assays, except between sd ns and igm rdt for denv- ( table ) . when the analyses were stratified by doi that usually coincides with day of fever, all ns -only assays had insignificantly higher sensitivity in the first days compared to later period. in contrast, all serology-only assays performed significantly better in the opposite manner especially for sd igm rdt. the same trend observed for the serology-only assays was carried forward into both the sd rdt combo, although the difference became insignificant. when compared between all ns only assays for the detection of dengue in the first days, elisa performed insignificantly better than viro-track followed by sd rdt. but when the latter was combined with igm, the sensitivity improved such that the italic numbers shown before the parameter estimates are number of correct tests over number of all tests for each corresponding parameter the difference became significant compared to itself alone and virotrack for doi > = days. the improvement was more substantial for sd ns /igm/igg, where sensitivity was significantly higher versus sd ns rdt alone and virotrack at any time, and even against elisa for doi > = days ( table ) . all ns -only assays had significantly better sensitivity in detecting primary dengue versus secondary. sd ns /igm rdt demonstrated similar trend but the difference was insignificant. this pattern was reverse in sd igg rdt, while sd igm rdt and sd ns / igm/igg rdt did not have any within-assay difference between infection status subgroups. when compared between assays with ns component, both sd rdt combo assays had significantly better sensitivity compared to sd ns rdt alone for both primary and secondary dengue, while the sensitivity of sd ns /igm/igg rdt was also significantly better than virotrack and elisa in detecting secondary dengue thanks to the additional igg component. serologyonly assays had significantly lower sensitivity compared to all other assays with ns component in detecting primary dengue, while sd igg rdt was also significantly less sensitive compared to sd igm rdt. however, for secondary dengue, sd igg rdt performed better than all individual assays and significantly better than sd ns rdt (table ) . the italic numbers shown before the sensitivity estimates are true positives over all disease-positives for the respective assay the italic numbers shown before the sensitivity estimates are true positives over all disease-positives for the respective assay similar to the trend observed above, all ns -only assays and sd ns /igm rdt were more sensitive in the detection of laboratory-confirmed over presumptive dengue, albeit without significant difference. on the contrary, both serology-only assays performed significantly better in detecting presumptive over laboratoryconfirmed dengue. again, sd ns /igm/igg rdt performed equally well in both with sensitivity point estimates above %. this combination was an all-rounder with significantly higher sensitivity compared to serologyonly assays in the detection of lab-confirmed dengue owing to its ns component, and significantly higher sensitivity compared to all ns -only assays in the detection of presumptive dengue owing mainly to its igm component (table ) . the pre-test probability of dengue (or proportion of dengue patients among all patients) was . % in this study ( table ) . all index tests if tested positive would increase the probability of dengue diagnosis to above %, except sd igg that registered only . % ( %ci . - . ), which was significantly lower than all other assays. the highest post-test probability of dengue for positive test was achieved by sd igm rdt at . % ( %ci . - . ), followed by sd ns rdt and sd ns /igm rdt at . % ( %ci . - . ) and . % ( %ci . - . ), respectively. these figures achieved by sd rdt ns or/and igm components were significantly higher than the . % ( %ci . - . ) attained by its ns /igm/igg combination. the results for sd elisa and virotrack did not differ significantly between each other nor with other assays, except with sd igg rdt (table ) . when it comes to post-test probability of dengue for negative test, sd ns /igm/igg rdt performed best with . % ( %ci . - . ), significantly lower than all individual assays. sd ns /igm rdt came in second at . % ( %ci . - . ), also significantly better than all individual assays except sd elisa. the latter had the best result among all individual assays followed by viro-track and sd ns rdt, at most published dengue rdt evaluation studies used serum samples. some studies also used whole blood specimen and only one used capillary blood [ ] [ ] [ ] [ ] . the excellent agreement between the results tested on capillary, venous, and serum samples in this study demonstrated that all three of them can be used on rdt, provided that anticoagulant-coated tool is used for the collection of whole blood specimen. the validity of results from capillary blood has practical implication when minimal invasiveness and/or rapidity is required such as in young children or during massive dengue outbreak. no study was published prior to this for the evaluation of virotrack dengue acute, while sd dengue ns ag elisa and sd bioline dengue duo have been extensively evaluated. the individual components of the sd bioline rdt had point estimates of sensitivity and specificity for the diagnosis of acute dengue that ranged within . - . % and . - . % for ns [ ] [ ] [ ] , . - . % and . - . % for igm [ , , ] , and . - . % and . - . % for igg [ , , ] , respectively. on the other hand, the point estimates of sensitivity and specificity of sd dengue ns ag elisa published previously were . - . % and . - . %, respectively [ , , ] . the results in this study fell within the above range except for the igg component of the sd rdt, where both sensitivity and specificity were lower than previously found. this difference may be attributed to the underlying difference in study design, patient population, definition of reference standard, and other study characteristics [ ] [ ] [ ] . since differences in study characteristics would modify the outcomes, it is difficult to directly compare diagnostic accuracy and utility between different studies without proper assessment. in other words, the apparent difference in diagnostic accuracy and utility parameters between two tests evaluated in two different studies may be due to the difference between their study characteristics instead of the actual performance of the tests themselves. however, diagnostic tests evaluated within the same study on the same patient population under the same condition can be directly compared [ ] . in this study, among all the individual assays, sd dengue ns ag elisa had the best performance followed by virotrack, sd bioline dengue duo ns , igm and igg components. this outcome is expected as both elisa and virotrack employed objective result read-out based on physical properties of light transmission in contrast to the subjective interpretation of sd rdt [ , - , , ] . the sensitivities of all the evaluated assays turned out as expected in the subgroup analyses. the ns -only assays performed better in detecting dengue infection in the first days versus days and above as ns antigen is actively produced and secreted in the first week [ , , , , , [ ] [ ] [ ] [ ] . however, the decline in performance was more obvious for sd ns rdt as reducing level of ns towards the end of the week might produce a test line too faint to be detected with naked eyes [ ] . on the contrary, serology-only assays performed better after day when igm and igg started to be secreted to counter the infectious agent [ , , , , , , , [ ] [ ] [ ] [ ] [ ] . the observation that sd igm performed better than igg, especially after day , is most likely due to the use of igm elisa in the definition of the reference standard. this study discovered that ns -only assays had higher sensitivity in detecting primary and laboratoryconfirmed dengue, while serology-only assays were better in secondary and presumptive dengue. again, the reason for this lies predominantly in the definition of reference standard, where primary and laboratoryconfirmed dengue were mostly those tested positive on rt-pcr and/or panbio ns elisa; while secondary and presumptive were more dependent on igm and igg capture elisa for their definitions. this similar trend was observed repeatedly in previous studies with some variations attributable to differences in study characteristics [ , , , - , , , ] . the finding that all ns -only index tests had lower sensitivity while sd igm rdt had it higher for the detection of denv- as compared to other serotypes was unexpected. some previous studies did not demonstrate difference [ , ] , while others did [ , , , , ] . further analysis (data not shown) showed that there were more patients beyond the th day of illness among those with denv- , which may partially explain these results. on the other hand, the failure of all assays to detect the sole denv- was probably due to chance, as this patient had secondary dengue (with low igm level) on th day of illness, when the level of free and detectable igg and ns happened to be too low after their union in vivo [ , , , ] . lastly, the serotype distribution found in this study agreed with previous finding [ ] . notwithstanding the underperformance of sd bioline dengue duo individual components, the diagnostic accuracy drastically improved when they were combined and interpreted as dengue positive if found positive on either one component. combination of sd ns and igm improved sensitivity, npv and lr-(better rule-out test if found negative) without compromising on the other parameters, while the addition of igg component further improved the rule-out parameters at the expense of rule-in parameters. this trend was in line with previous findings [ , , , , , , , , , , , ] . the repercussion of this finding is that dengue combo test is always superior to individual assay rdt as combo has the ability to detect dengue infection regardless of the phase of illness [ , ] . this recommendation is further backed up by the diagnostic utility from this study. as shown in table , tested positive for sd ns /igm rdt was able to double the probability of dengue infection in a patient to be more than %, leaving only less than out of wrongly diagnosed non-dengue patients. although all individual assays except sd igg rdt were able to more or less match this performance, they could not reproduce the same when it came to negative tests. if a patient was tested negative for any of the individual assays, the best post-test probability of dengue was still . %. in other word, at least out of dengue patients were fn that might have been misdiagnosed. in contrast, sd combo tests were able to reduce fn to just to per dengue patients. while dengue fp may not be a big concern due to its small proportion and relatively less harmful supportive treatment unless in the case of misdiagnosis of other more severe diseases, high number of fn might lead to late diagnosis and delayed administration of required life-saving treatment for dengue patients. as such, in a clinical setting, especially in primary care, it is important for a dengue rdt to act as a screening tool that can detect more cases with minimal fn. although this study found that sd bioline dengue duo, a dengue rdt based on immunochromatographic principle, was a more preferred tool when used as a combo test; the new biosensors-based virotrack dengue acute was not without its own advantages. it requires only mcl of blood specimen compared to mcl required for sd ns rdt. this feature is important when blood sampling is difficult and yield is scarce, such as among paediatric patients. its test procedure requires minimal training, and is as simple and user-friendly as that of sd rdt at least in terms of sample loading, but its test duration is slightly shorter. still, after loading blood sample, virotrack must be inserted into a blubox, an electronic device that runs on electricity. this may create a bottleneck if more than one sample must be run concurrently, unless commercialised version of blubox comes with the ability to run multiple tests. yet, the presence of blubox increases test sensitivity and reduces number of false positive from delayed interpretation common for immunochromatographic tests [ ] . furthermore, its ability to connect with local and global network reduces the rate of manual transcription error in reporting [ ] , and makes results available immediately upon diagnosis to treating doctors and public health authority for quick intervention [ ] . nevertheless, virotrack must first be available in the form of a combo test and proven to be more accurate, cost-effective, and beneficial in future studies for this new technology to be popularised. the strength of this study lies in its sound methodology and application. as mentioned above, it is difficult to directly compare diagnostic performance between evaluation studies due to different study characteristics [ ] . in the same way, it is also fundamentally incorrect to directly apply their results into daily practice or for policy making. good performances reported in casecontrol or laboratory-based studies may be due to biases instead of the discriminatory power of the evaluated tests [ , ] . in contrast, the cross-sectional prospective design in an actual primary care clinical setting seen in this study produced a more realistic set of diagnostic performance parameters that is true to other similar clinical settings, making the application of its results easier and more valid. besides, it complied with stardguidelines for quality assurance [ ] . in addition, diagnostic utility presented here is more intelligible to clinical practitioners and policy makers compared to the usually reported diagnostic accuracy. with simple calculation based on the formulas provided here or using rough estimation, the diagnostic utility of the three index tests evaluated in this study can be estimated for any clinical setting [ , ] . however, it should be cautioned that this exercise took into account only dengue diagnostics without consideration of other diseases. practitioners should also use existing clinical reasoning for differential diagnosis. in malaysia, four previously conducted dengue rdt evaluation studies were exclusively laboratory-based and only one employed cross-sectional prospective design [ , , , ] . this study was the first conducted prospectively among consecutively sampled patients in primary care setting. it provided better and more updated insight on the application of dengue rdt in malaysia. furthermore, it was the first that evaluated a biosensors-based rdt in this setting and compared it with extensively used rdt and ns elisa for a more comprehensive understanding of their relative performance, which is absent in most other studies that evaluated only either rdt or elisa. two limitations of this study are, first, only single sample was collected from each patient, making the reference standard based on serology presumptive rather than conclusive [ , , ] ; and second, the diagnostic utility calculated was based on the pre-test probability of disease based on the who guidelines without taking into account of the haematological result. nevertheless, these limitations perfectly reflect the actual situation faced by clinicians in the front line, making the study results more realistic and applicable to the real-world circumstances. in conclusion, for the diagnosis of acute dengue infection, new biosensors-based dengue rdt such as viro-track dengue acute performed almost as well as sd dengue ns ag elisa, while the latter was superior to sd bioline ns assay. the addition of serology component to sd bioline dengue duo, however, greatly enhanced its diagnostic accuracy and utility parameters almost beyond that of sd dengue ns elisa, making it a better point-of-care dengue diagnostic tool as it would miss the fewest dengue patients. as such, virotrack dengue acute can be a potential alternative to existing combo rdts only if its combination with serology components matches or outperforms them in diagnosing dengue. this decision making should be based on the results of properly conducted diagnostic test accuracy and economic evaluation studies in the future that allow within-study comparison of multiple diagnostic tools instead of comparing between heterogeneous studies. world health organization. global strategy for dengue prevention and control - . geneva: world health organization the global distribution and burden of dengue the eye of the tiger, the thrill of the fight: effective larval and adult control measures against the asian tiger mosquito, aedes albopictus (diptera: culicidae), in north america senarai kawasan cluster dengue clinical practice guidelines: management of dengue infection in adults dengue: guidelines for 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nonstructural antigen assays for the diagnosis and surveillance of dengue in singapore commercial dengue rapid diagnostic tests for point-of-care application: recent evaluations and future needs? evaluation of onestep dengue ns rapidip™ instatest and onestep dengue fever igg/igm rapicard™ instatest during the course of a dengue type epidemic evaluation of two new commercial tests for the diagnosis of acute dengue virus infection using ns antigen detection in human serum low specificity of an immunochromatographic serological assay for diagnosis of dengue fever in travelers returning with malaria diagnostic accuracy of ns elisa and lateral flow rapid tests for dengue sensitivity, specificity and relationship to viraemia and antibody responses evaluation of the ns rapid test and the who dengue classification schemes for use as bedside diagnosis of acute dengue fever in adults epidemiology of dengue disease in malaysia ( - ): a systematic literature review comparison of two dengue ns rapid tests for sensitivity, specificity and relationship to viraemia and antibody responses evaluation of a pan-serotype point-of-care rapid diagnostic assay for accurate detection of acute dengue infection measuring the rate of manual transcription error in outpatient point-of-care testing web-based infectious disease surveillance systems and public health perspectives: a systematic review simplifying likelihood ratios a comparative study on the performance of two commercial anti-dengue igm assay kits performance of a commercial rapid dengue ns antigen immunochromatography test with reference to dengue ns antigencapture elisa publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the director general of health malaysia for the approval to publish this article. we sincerely appreciate the good people in petaling district health office and shah alam section health clinic that contributed towards the study. university of malaya, kuala lumpur, malaysia. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file : figure s . stard flow diagram for virotrack dengue acute.additional file : figure s . stard flow diagram for sd dengue ns ag elisa.additional file : figure s . stard flow diagram for sd bioline dengue duo.additional file : table s . interrater agreements and their % ci between two interpreters for capillary and venous specimens tested on different assays of sd bioline dengue duo. table s . agreements and their % ci between the results of capillary and venous specimens tested on different assays of sd bioline dengue duo. table s . agreements and their % ci between the results of different specimens tested on virotrack dengue acute. authors' contributions zlc contributed in design and coordination of the study, data collection, analysis, and led in manuscript writing. sds and cwn conceived of the study, participated in its design and coordination, and helped in drafting the introduction and discussion. hjs contributed in the data collection, analysis, and drafting the methods and results. dp and srlr helped in study coordination and drafting of the discussion. all authors have read and approved the final manuscript. this research was funded by blusense diagnostics, denmark. the funding body has no role in the design of the study; in data collection, analysis, and interpretation; in manuscript writing and decision to submit it for publication. the datasets used and/or analysed during the current study, as well as the study protocol, are available from the corresponding author on reasonable request.ethics approval and consent to participate ethical approval for this study was granted by the medical research and ethics committee, ministry of health malaysia (nmrr- - - ) and university malaya medical center (mrecid.no: - ). written informed consent was obtained from each participant. for participants below years old, minor assent was first sought where appropriate, and if possible, in written form; before parents or guardians provided written informed consent on their behalf. not applicable. the authors declare that they have no competing interests. key: cord- -lq tp z authors: khanafer, nagham; sicot, nicolas; vanhems, philippe; dumitrescu, oana; meyssonier, vanina; tristan, anne; bès, michèle; lina, gérard; vandenesch, françois; gillet, yves; etienne, jérôme title: severe leukopenia in staphylococcus aureus-necrotizing, community-acquired pneumonia: risk factors and impact on survival date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: lq tp z background: necrotizing pneumonia attributed to panton-valentine leukocidin-positive staphylococcus aureus has mainly been reported in otherwise healthy children and young adults, with a high mortality rate. erythroderma, airway bleeding, and leukopenia have been shown to be predictive of mortality. the objectives of this study were to define the characteristics of patients with severe leukopenia at -h hospitalization and to update our data regarding mortality predicting factors in a larger population than we had previously described. methods: it was designed as a case-case study nested in a cohort study. a total of cases of community-acquired, necrotizing pneumonia were included. the following data were collected: basic demographic information, medical history, signs and symptoms, radiological findings and laboratory results during the first h of hospitalization. the study population was divided into groups: ( ) with severe leukopenia (leukocyte count ≤ , leukocytes/ml, n= ) and ( ) without severe leukopenia (> , leukocytes/ml, n= ). results: median age was years, and the male-to-female gender ratio was . . the overall in-hospital mortality rate was . %. death occurred in . % of severe leukopenia cases with median survival time of days, and in . % of cases with leukocyte count > , /ml (p< . ). multivariate analysis indicated that the factors associated with severe leukopenia were influenza-like illness (adjusted odds ratio (aor) . , % ci ( % confidence interval) . - . , p= . ), airway bleeding (aor . , % ci . - . , p= . ) and age over years (aor . , % ci . - . , p= . ). a personal history of furuncles appeared to be protective (or . , % ci . - . , p= . ). conclusion: s. aureus-necrotizing pneumonia is still an extremely severe disease in patients with severe leukopenia. some factors could distinguish these patients, allowing better initial identification to initiate adapted, rapid administration of appropriate therapy. since the description of necrotizing pneumonia due to panton-valentine leukocidin (pvl)-positive staphylococcus aureus by gillet et al. in , numerous cases have been reported worldwide [ ] [ ] [ ] [ ] [ ] [ ] [ ] . they mainly impacted otherwise healthy children and young adults, with a median age of years. they are attributed to either methicillin-sensitive or -resistant s. aureus strains of various genetic backgrounds, but have all pvl genes in common [ , ] . necrotizing pneumonia is characterized by rapid, extensive, bilateral pneumonia frequently evolving towards acute respiratory distress syndrome (ards), despite intensive medical interventions with mechanical ventilation and inotrope support. no specific treatment guidelines have been published so far, but the addition of toxin-suppressing antibiotics, such as clindamycin, linezolid and rifampicin, has been suggested [ ] [ ] [ ] . the overall mortality rate ranges from to % of cases [ , ] . the fatal outcome is rapid, with median survival time between to days from the onset of symptoms [ , , ] . it is associated with classic severity factors, such as the need for mechanical ventilation or inotrope support, and the onset of ards. erythroderma, airway bleeding, and leukopenia have been shown to be predictive of mortality. gillet et al. reported significant differences, by multivariate analysis, in median leukocyte count between patients who survived and those who did not. the survival rate was < % when the leukocyte count was < , leukocytes/ml. the leukopenia observed in patients could reflect pvl cytotoxicity, demonstrated in vitro on human neutrophils [ , ] . as patients with leukopenia face a high risk of mortality, better initial recognition of these severe cases would allow rapid administration of appropriate treatment. in this study, a series of cases of s. aureus-necrotizing pneumonia was analyzed. our aim was to define the characteristics of patients with severe leukopenia at -h hospitalization and to update our data regarding mortality predicting factors in a larger population than we had previously described [ ] . since , the french national reference centre of staphylococci (lyon, france) has collected case reports of documented pneumonia caused by s. aureus strains carrying pvl genes (luks-pv-lukf-pv). informed consent was waived because data were extracted from the surveillance database. according to french law, a study like this one does not require ethics committee approval because it is observational and based on a surveillance database approved under national regulations. the protocol design was approved by the hospital's institutional review board (comité national informatique et liberté). pneumonia was defined by signs and symptoms of lower respiratory tract infection (e.g., cough, expectoration, and chest pain) and pulmonary infiltrates on chest x ray reviewed by a radiologist, that were not attributable to other causes, but coinciding with s. aureus isolation by at least of the following procedures: ( ) pleural effusion or lung abscess; ( ) broncho-alveolar lavage fluid culture ( cfu/ml), wimberley brushing ( cfu/ ml), or protected tracheal aspiration ( cfu/ml); and ( ) blood culture revealing s. aureus as the sole potential pathogen. cases with respiratory symptoms starting at least h after hospitalization were classified as nosocomial and were excluded from the study. leukopenia was defined as severe if median leukocyte count was < , leukocytes/ml within the first h after hospital admission. s. aureus isolates were tested for antimicrobial susceptibility and toxin production. testing of isolates for antimicrobial susceptibility by broth microdilution was undertaken according to the interpretive criteria of the clinical and laboratory standards institute (formerly the national committee for clinical laboratory standards). the following antimicrobial agents were used: penicillin, oxacillin, kanamycin, tobramycin, gentamicin, erythromycin, clindamycin, tetracycline, ofloxacin, fusidic acid, rifampicin, vancomycin, teicoplanin, fosfomycin, trimethoprimsulfamethoxazole, and linezolid. gene sequences encoding superantigens (enterotoxins a-e, g-i, and toxic shock syndrome toxin), pvl and meca gene, which codes for methicillin resistance, were detected by pcr, as described elsewhere [ ] . only cases caused by pvl-positive s. aureus strains were included. designed as a case-case study nested in a cohort study, analyses were restricted to cases where all clinical and biological data were available. the following data were collected by a standardized form and comprised basic demographic information, medical history (including risk factors for infection and history of personal or familial abscesses or furuncles), signs and symptoms, radiological findings and laboratory results during the first h of hospitalization. severity was rated by pediatric risk of mortality (prism) scores for patients < years and the simplified acute physiology score (saps) ii for patients ≥ years, when available. some biological and radiological data were missing because of death shortly after admission to hospital. categorical variables were compared by the chi-square or fisher's exact test, and continuous variables, by student's t-test or mann-whitney tests. survival probability according to median leukocyte count, was estimated by the kaplan-meier method. initially the cases were divided into groups: - , ; , - , and > , leukocytes/ml but we merged the data of the last two groups since no significant difference was found for variables included in the final multivariate regression model (data not shown in this paper). the roc analysis showed a % sensitivity for a cut-off of leukocytes/ml. baseline was the day of admission to hospital because of pneumonia, and patients who survived were censored at hospital discharge. when patients died within h after admission, the observation period was rounded to day. survival distributions were compared by the logrank test. variables independently associated with survival were identified with a cox regression model based on hazard ratios with % confidence interval ( % ci). variables associated with severe leukopenia were tested by the multivariate logistic regression model. when p values of variables described in the first table, were < . in univariate analysis, they were submitted to the multivariate model. variables in multivariate analysis were subjected to the forced entry procedure, with stepwise and backward elimination, using p values of . as criteria for inclusion and elimination of risk variables based on best subset logistic regression with chi-square score fit criteria. the hosmer-lemeshow test assessed the model's goodness-of-fit. adjusted odds ratios (aor) and corresponding % ci were calculated. for all tests performed, -tailed p values < . were regarded as denoting statistical significance. analyses were performed with spss . software (spss inc., chicago, il, usa). from through , cases of communityacquired, necrotizing pneumonia were collected and documented; case reports were excluded because of missing data concerning leukocytes count. in total, cases of community-acquired necrotizing pneumonia were included. median age was years (interquartile range [iqr] . - . ) and the male-to-female gender ratio was . ( males and females). smoking was reported in . % patients. common risk factors for staphylococcal infection, such as diabetes, steroid therapy, and immunosuppressive treatment, were noted for . %, . % and . % of patients, respectively. among the patients for whom data were available, . % had a personal history of furuncles or skin abscess. the median duration of symptoms prior to hospitalization was . days (iqr . - . days), with preceding influenzalike syndrome in . % ( of ), and pre-existing skin and soft tissue infection (ssti) in . % ( of ). the clinical course during the first h after hospital admission was usually severe, with . % of patients requiring mechanical ventilation. the most remarkable clinical feature was airway bleeding, which occurred in . % of patients. s. aureus was recovered from blood culture in . %, in pleural fluid from . %, and in tracheal aspirates from . % of patients. median minimal leukocyte count during the first h of hospitalization was , leukocytes/ml (iqr , - , leukocytes/ml), and . % had leukocyte count ≤ , leukocytes/ml. the minimal platelet count was also low, with a median of , platelets/ml (iqr , - , platelets/ml). the overall in-hospital mortality rate was . %. . % of deaths were attributed to s. aureus and were secondary to refractory shock and/or respiratory failure. the study population was divided into groups: ( ) with severe leukopenia (leukocyte count ≤ , leukocytes/ ml, n= ) and ( ) without severe leukopenia (> , leukocytes/ml, n= ). the group with severe leukopenia was characterized by a significantly higher rate of female patients (p= . ) ( table ) . prior influenza-like illness (ili: . % vs. . %, p< . ) and airway bleeding ( . % vs. . % (p< . ) were also associated with severe leukopenia. conversely, a personal history of furuncles or skin abscess (p= . ) and ssti at admission (p= . ) were more frequent in the group without severe leukopenia. the group with severe leukopenia presented ards onset more frequently and needed artificial ventilation or inotrope support (p< . ). multivariate analysis indicated that ili, airway bleeding and age over years were independent factors associated with severe leukopenia (table ) . a personal history of furuncles appeared to be protective. leukocyte count was negatively correlated with mortality. death occurred in . % of cases ( of ) with severe leukopenia (≤ , leukocytes/ml) with median survival time of days (figure ). only . % of cases ( of ) with leukocyte count > , /ml (p< . ) died. mortality was % in cases with airway bleeding versus . % in those without (p<. ). cox multivariate analysis indicated that the only factors associated with fatal outcome were leukopenia, airway hemorrhage and age (table ). in this article, we compared the characteristics of a group of cases of severe leukopenia (≤ , leukocytes/ml) with a group of patients without severe leukopenia (> , leukocytes/ml) at h of hospitalization. the group with severe leukopenia was exemplified by a significantly higher rate of female patients (p= . ), prior ili (p< . ) and airway bleeding (p< . ). conversely, a personal history of furuncles or skin abscess (p= . ) and ssti at admission (p= . ) were more frequent in the group without severe leukopenia. the mortality rate was significantly different between groups ( . % vs . %, p< . ). necrotizing pneumonia largely occurred in young people with a median age of years, only slightly higher than reported earlier in smaller series [ , , , , ] . the increasing of age of included patients might be attibuable to a delayed exposure to our study confirms the association of leukocyte count with disease severity. by multivariate logistic regression, age over years was associated with severe leukopenia (aor . , p= . ). the reason of this association was not clear. the increasing of age of patients might be attibuable to a delayed exposure to the causative organism. this epidemiological trend should be challenged by similar surveys in other countries. an association between time and lymphocyte count was actually reported in different populations [ , ] . female gender was associated with severe leukopenia (or . , p= . ) by univariate analysis but not by multivariate analysis. females in general mount a more profound immune response after antigenic challenge, and these differences have mainly been attributed to the immunomodulatory effects of sex hormones, despite the lack of human in vivo data. even though we still found some papers reporting an association of neutropenia with female gender. vaneijk et al. demonstrated, in an in vivo study, that females exert a more proinflammatory pattern of cytokine release compared to males during systemic inflammation after the administration of escherichia coli endotoxin [ ] . this difference is associated with more leukocyte sequestration in females [ ] . sterling et al. reported gender-based differences in host immune responses [ ] . in a study analyzing the risk factors for developing neutropenia after mitomycin c administration, multivariate logistic regression showed that female gender was an independent risk factor for neutropenia and the reasons for this association are unknown [ ] . the significant association with a previous ili (aor . , p= . ) might be related to a particular linkage with influenza virus. possible epithelial damage, due to viral infection, could promote the pathogenicity of pvlproducing strains with increasing affinity for collagen (i and iv) and laminin [ ] . a personal history of furuncles appears to be protective (or . , % ci . - . , p= . ). it is conceivable that patients with such a history had previously been exposed to pvl and had developed a degree of protective immunity [ ] . another hypothesis could explain this association: after bacteria reach the lungs through the bloodstream, the necrotizing effect is less serious. the present investigation has some limitations. first, case reporting to the reference center was unsolicited and may not reflect accurate epidemiology and disease severity. severe and dramatic cases among previouslyhealthy young people are more likely to be reported. a bias of notification would not be excluded. this would be caused by some missing data possibly not declared after due to the dispersion of laboratories dealing with pvl gene detection. moreover the median age was slightly higher than had been previously reported which could be related to the introduction of several novelties in the treatment of severely ill patients since the identification of this pathology. another limitation concerns clinical data collection: they were missing or incomplete in % of cases. analysis included only cases for which microbiological and demographic information were available; cases were excluded from further analysis because they lacked data on leukocyte count. comparisons between the excluded cases and the cases analyzed revealed no significant differences. leukopenia may be simply considered as a reflection of disease severity. this is in agreement with in vitro data showing that pvl induces both apoptosis and necrosis in human leukocytes [ ] . the time period between the onset of symptoms and hospitalization could be important to reach a certain level of severity and impaired leukocyte count, but it was not different between both groups. on the other hand, multivariate logistic regression was adjusted to the time factor. furthermore we noticed that factors associated with severe leukopenia were quite different from those associated with mortality. based on our results, we can suggest two syndromes of necrotizing pneumonia: ) those that are mainly related to ili and direct inoculation of staphylococci to damaged respiratory epithelium, airway hemorrhage, severe leukopenia and death; and ) those that are mainly related to hematogenous spread from ssti, less airway hemorrhage, without leukopenia and improved survival. in conclusion, our study emphasizes that s. aureus-necrotizing pneumonia is still an extremely severe disease. we found that some factors could distinguish patients with severe leukopenia from those without leukopenia. leukocytes count is promptly available at admission to the hospital and can be easily used to asses the severity of disease as suggested by this paper. the impact of this marker, on patient management, need to be clarified. empiric therapy should include coverage for s. aureus as soon as possible, without waiting for the bacteriological results. clinical data indicate that neutralizing toxin production improves the outcome [ , ] . the toxin can be blocked by combining a toxin-suppressing agent (e.g., clindamycin, linezolid or rifampin) with bactericidal antibiotics acting on the cell wall [ ] . s. aureus-necrotizing pneumonia is still an extremely severe disease in patients with severe leukopenia. some factors like leukocytes count could distinguish these patients, allowing better initial identification to initiate adapted, rapid administration of appropriate therapy. this paper could be regarded as a preliminary work. variables were adjusted to gender, mechanical ventilation, personal history of furuncles, platelet count, pleural effusion, and previous influenza-like illness. experts are invited to work on a widely accepted score, validate a score including leukocytes count, to predict the severity of this disease. association between staphylococcus aureus strains carrying gene for panton-valentine leukocidin and highly lethal necrotising pneumonia in young immunocompetent patients life-threatening hemoptysis in adults with community-acquired pneumonia due to panton-valentine leukocidinsecreting staphylococcus aureus severe community-onset pneumonia in healthy adults caused by methicillin-resistant staphylococcus aureus carrying the panton-valentine leukocidin genes severe community-acquired pneumonia due to staphylococcus aureus, - influenza season severe community-acquired pneumonia 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polymorphonuclear leukocytes mediate staphylococcus aureus panton-valentine leukocidin-induced lung inflammation and injury staphylococcus aureus panton-valentine leukocidin is a very potent cytotoxic factor for human neutrophils diagnosis of a predisposition of retinoblastoma at the dna level community-associated methicillinresistant staphylococcus aureus in outpatients age-related changes in human hematopoietic stem/progenitor cells htlv outcomes study (host) investigators: long-term increases in lymphocytes and platelets in human t-lymphotropic virus type ii infection gender differences in the innate immune response and vascular reactivity following the administration of endotoxin to human volunteers sex-based differences in t lymphocyte responses in hiv- -seropositive individuals incidence, risk factors, and impact of severe neutropenia after hyperthermic intraperitoneal mitomycin c staphylococcus aureus isolates associated with necrotizing pneumonia bind to basement membrane type i and iv collagens and laminin a history of panton-valentine leukocidin (pvl)-associated infection protects against death in pvl-associated pneumonia staphylococcus aureus panton-valentine leukocidin directly targets mitochondria and induces bax-independent apoptosis of human neutrophils expanded clinical presentation of community-acquired methicillin-resistant staphylococcus aureus pneumonia prompt and successful toxin-targeting treatment of three patients with necrotizing pneumonia due to staphylococcus aureus strains carrying the panton-valentine leukocidin genes submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the clinicians and microbiologists who sent us clinical data and isolates. we acknowledge the contribution of prof. rené ecochard and dr. muriel rabilloud from the department of biostatistics in lyon university hospital. special thanks to mr ovid da silva for editing this manuscript. the authors declare that they have no competing interests.authors' contributions je, pv, fv, yg, od, vm, at, mb and gl conceived of and designed the study; nk, ns, and pv performed the data analysis; and nk, ns, and je wrote the paper. all authors read and approved the final manuscript. key: cord- -ml mgyf authors: huang, linna; zhang, wei; yang, yi; wu, wenjuan; lu, weihua; xue, han; zhao, hongsheng; wu, yunfu; shang, jia; cai, lihua; liu, long; liu, donglin; wang, yeming; cao, bin; zhan, qingyuan; wang, chen title: application of extracorporeal membrane oxygenation in patients with severe acute respiratory distress syndrome induced by avian influenza a (h n ) viral pneumonia: national data from the chinese multicentre collaboration date: - - journal: bmc infect dis doi: . /s - - -x sha: doc_id: cord_uid: ml mgyf background: evidence concerning the efficacy and safety of extracorporeal membrane oxygenation (ecmo) in patients with influenza a (h n ) has been was limited to case reports. our study is aimed to investigate the current application, efficacy and safety of ecmo in for severe h n pneumonia-associated acute respiratory distress syndrome (ards) in the chinese population. methods: a multicentre retrospective cohort study was conducted at hospitals that admitted patients with avian influenza a (h n ) viral pneumonia patients’ admission from provinces in china between october , , and march , . data from the national health and family planning commission of china, including general conditions, outcomes and ecmo management, were analysed. then, successfully weaned and unsuccessfully weaned groups were compared. results: a total of patients, aged ± years, were analysed; . % of patients were male with % mortality. all patients underwent invasive positive pressure ventilation (ippv), and rescue ventilation strategies were implemented for cases ( . %) with an average ippv duration of ± d, pao( )/fio( ) of ± mmhg, tidal volume (vt) of ± ml and plateau pressure (p(plat)) of ± cmh( )o pre-ecmo. after h on ecmo, pao( ) improved from ± mmhg to ± mmhg and paco( ) declined from ± mmhg to ± mmhg. haemorrhage, ventilator-associated pneumonia (vap) and barotrauma occurred in . %, % and . % of patients, respectively. compared with successfully weaned patients (n = ), the unsuccessfully weaned patients had a longer duration of ippv pre-ecmo ( ± d vs. ± d, p < . ) as well as a higher p(plat) ( ± cmh( )o vs. ± cmh( )o, p < . ) and vt ( ± ml vs. ± ml, p < . ) after h on ecmo support. furthermore, the unsuccessfully weaned group had a higher mortality ( % vs. . %, p < . ) with more haemorrhage ( . % vs. . %, p < . ). conclusions: ecmo is effective at improving oxygenation and ventilation of patients with avian influenza a (h n ) induced severe ards. early initiation of ecmo with appropriate ippv settings and anticoagulation strategies are necessary to reduce complications. conclusions: ecmo is effective at improving oxygenation and ventilation of patients with avian influenza a (h n ) induced severe ards. early initiation of ecmo with appropriate ippv settings and anticoagulation strategies are necessary to reduce complications. keywords: extracorporeal membrane oxygenation (ecmo), avian influenza a (h n ), acute respiratory distress syndrome (ards), complications, mortality background avian influenza a (h n ) viral pneumonia can manifest with varying degrees of dyspnea and is associated with a mortality of~ % [ ] . in particular, % of patients develop rapidly progressive pneumonia and % progress to acute respiratory distress syndrome (ards). the mortality of severe ards is as high as % [ ] . timely and effective respiratory support is particularly important to treat severe ards caused by avian influenza a (h n ) pneumonia. however, severe ards induced by avian influenza a (h n ) pneumonia might manifest as refractory hypoxaemia even with appropriate invasive positive pressure ventilation (ippv) support. extracorporeal membrane oxygenation (ecmo) is the ultimate respiratory support method and directly improves the oxygenation and ventilation of patients as well as enables implementation of the "lung protective ventilation strategy" [ ] . ecmo was the breakthrough treatment for the severe avian influenza a (h n ) outbreak of and reduced mortality from this outbreak [ ] [ ] [ ] . therefore, we believe that ecmo could also be effective for other types of severe viral pneumonia. existing studies of ecmo treatment for avian influenza a (h n ) pneumonia are primarily limited to case reports [ ] [ ] [ ] , and no study has systematically reviewed the efficacy or safety of ecmo to treat such diseases. therefore, it is particularly important to understand the current application of ecmo for avian influenza a (h n ) pneumonia-induced severe ards, investigate the application timing and management strategies of ecmo, and explore the possible reasons for treatment failure. based on the current study, we expect to standardize the management of ecmo and provide a description of our experiences using ecmo to treat patients with avian influenza a (h n ) pneumoniainduced severe ards. patients who had laboratory-confirmed avian influenza a (h n ) virus-induced pneumonia were included in this study. patients were admitted to hospitals in provinces of china between october , , and march , , and were reported to the national health and family planning commission of china. we included patients aged > ys who were supported by ecmo. patients who were lacking key detailed records of parameters during ecmo, such as ventilator or laboratory findings, were excluded. the included patients were divided into groups, namely, the "successfully weaned group" and "unsuccessfully weaned group". the former refers to a group of patients whose condition improved and were weaned from ecmo for at least h; the "unsuccessfully weaned group" refers to those who died or voluntarily discontinued treatment due to lack of improvement during ecmo support. the general conditions included age, gender, pregnancy status, underlying disease, time from onset to antiviral drug administration, vasoactive drug administration pre-ecmo, duration of ippv pre-ecmo, whether rescue ventilation strategies (including lung recruitment maneuvre, prone-position ventilation, and high-frequency oscillation ventilation) were implemented pre-ecmo, disease severity score, total duration of ecmo and ippv. we collected the ecmo blood flow at , , , and h on ecmo. improvement in circulatory and respiratory physiological indicators were considered, as well as ippv parameters at h pre-ecmo and , , and h on ecmo. furthermore, anticoagulation indicators during ecmo, including the types of anticoagulant drugs and methods of use; the maximum and minimum values of the activated coagulation time (act) and activated partial thromboplastin time (aptt); and the differences between the maximum and minimum act and aptt at , , and h on ecmo were recorded. finally, data regarding complications during ecmo therapy, including ecmo and ippv-related complications and nosocomial infections, were collected. the primary outcome was in-hospital mortality. the secondary outcomes were the length of stay in the intensive care unit (icu) and total length of hospitalization. three methods were used for a laboratory diagnosis, namely, the real-time reverse transcription-polymerase chain reaction (rt-pcr), viral isolation, and serological testing for the avian influenza a (h n ) virus using a modified haemagglutinin inhibition assay [ ] [ ] [ ] . we defined ards according to the berlin definition in [ , ] . pneumonia was diagnosed as an acute illness with fever, cough, or dyspnea/tachypnea, and at least one new focal chest sign that was supported by a finding of lung shadowing on a chest radiograph and without other noninfectious causes. the primary criteria for severe pneumonia were as follows: < > need for tracheal intubation and mechanical ventilation (mv) and < > need for vasoactive drugs after the active fluid resuscitation due to septic shock. the secondary criteria were as follows: < > respiratory rate ≥ times/ min; < > pao /fio ≤ mmhg; < > multiple lobe infiltration; < > disturbances of consciousness, disorientation, or both; < > blood urea nitrogen ≥ . mmol/l; and < > systolic blood pressure ≤ mmhg that required active fluid resuscitation. patients who met one primary criterion or at least three secondary criteria were diagnosed as having severe pneumonia [ ] . the criteria for the diagnosis of vap are in accordance with the european centre for disease prevention and control [ ] and included the following: < > two or more sequential chest x-rays or ct scans with a suggestive image of pneumonia for patients with underlying cardiac or pulmonary disease, or one definitive chest x-ray or ct scan in patients without underlying cardiac or pulmonary disease; < > a fever greater than °c and/ or leukocytosis greater than or equal to , wbc/ mm or leukopenia less than or equal to wbc/ mm ; and < > at least one of the following: <a > new onset of purulent sputum or change in the characteristics of the sputum; <b > cough, dyspnea, or tachycardia; <c > auscultatory findings, such as rales, bronchial breath sounds, ronchi, or wheezing; or <d > worsening gas exchange (e.g., oxygen desaturation or increased oxygen requirements or increased ventilation demand). for all included patients, we first described the general conditions, ecmo model and parameters, ippv parameters, the changes in circulation and respiratory physiological indicators from pre-ecmo to on ecmo status, anticoagulation on ecmo, and complications during ecmo therapy in all included patients. then, we compared patients who were successfully or unsuccessfully weaned from ecmo with regard to above items. all of the analyses were performed using spss . software. normally distributed continuous variables are expressed as the means ± sd and were compared using the t-test or chi-square test. non-normally distributed continuous variables are expressed as medians and quartiles and were compared using the wilcoxon rank-sum test. categorical variables were compared using the x test. p-values < . were considered significant. a total of patients were diagnosed with avian influenza a (h n ) virus-related pneumonia. patients were admitted to hospitals in provinces of china between october , , and march , , and were reported to the national health and family planning commission of china. the medical records of patients were available, and patients were reported to be supported by ecmo. one of the patients lacked ippv and ecmo parameters pre-ecmo and on ecmo and was eliminated as a participant; therefore, patients were ultimately selected ( fig. ) . data from patients ( . % males), with an average age of ± years, were analysed. there was no patient under the age of . a total of patients had underlying diseases, patients were treated with steroids and immunosuppressive agents within month of admission to the hospital, and patient was pregnant. the sequential organ failure assessment (sofa) score was ± points, and the murray score was . ± . points. the time from onset to antiviral drug administration was approximately ± d, and the time from onset to ecmo support was approximately ± d. high-dose vasoactive drugs [ ] were needed to maintain blood pressure in patients ( . %). the duration of ippv pre-ecmo was approximately ± d. rescue ventilation strategies, including the recruitment manoeuvre (rm), prone-position ventilation (pp) and high frequency oscillatory ventilation (hfov), were needed for patients ( . %) pre-ecmo. the total durations of ippv and ecmo were approximately ± d and d ( - d), respectively. of the patients, ( %) were successfully weaned from ecmo, and the other patients died due to an uncontrolled haemorrhage ( patients), septic shock ( patients due to progressive lung infection, patients due to bloodstream infection), heart failure ( patients) and discontinuation of treatment because of no improvement ( patients). one of patients showed an aggregated lung infection after weaning and eventually died due to septic shock. the inhospital mortality was %. the length of icu stay was ± d, and the total length of hospitalization was ± d (table ) . of the patients, were treated using the veno-venous ecmo (v-v ecmo) model. a total of patients with severe cardiac insufficiency and cardiogenic shock were treated using the venous-arterial ecmo (v-a ecmo) model. the ecmo equipment was mainly provided by maquet (shanghai) medical equipment co., ltd. and sorin (shanghai) medical equipment co., ltd. the pump from sorin was the stockert centrifugal pump system (scp/scpc), and the oxygenator was the d eos ecmo. the pump from maquet was the rota-flow, and the oxygenator was the quadrox pls. changes in ippv parameters and physiological indicators in patients on ecmo the ventilator parameters, including fio , positive end-expiratory pressure (peep), p plat , and vt, were significantly decreased in patients on ecmo. the vital signs, which included the heart rate, respiratory rate, and spo , and the arterial blood gas analysis (abg), which included the ph, paco , and pao levels, were improved in patients after ecmo support ( table ) . monitoring of anticoagulation all patients received a continuous infusion of unfractionated heparin for anticoagulation. however, heparin was discontinued for patients with cerebral haemorrhage and with active gastrointestinal haemorrhage. the act was ± to ± s at h, ± to ± s at h, and ± to ± s at h on ecmo. the aptt was ± to ± s and ± to ± s at and h on ecmo, respectively. complications during ecmo therapy in this study, the rates of gastrointestinal haemorrhage, cerebral haemorrhage, brain death, renal insufficiency, disseminated intravascular coagulation (dic), hyperglycaemia, and ecmo oxygenator thrombosis were higher compared to the relevant data from the ecls registry report [ , ] . new cases of vap developed in patients during ecmo, with an incidence rate of %. new cases of barotrauma occurred in patients, accounting for . % of cases. in addition, patients had a urinary infection, with an incidence rate of . %, and patients had a catheter-related blood stream infection (crbsi), with an incidence rate of . % (table ) . comparison between the patients successfully and unsuccessfully weaned from ecmo group contained patients who were successfully weaned from ecmo, and group included patients who were unsuccessfully weaned from ecmo. compared with patients successfully weaned from ecmo, the unsuccessfully weaned group had a higher mortality ( % vs. . %, respectively, p < . ), and was older ( ± years vs. ± years, respectively, p = . ), and more likely to have diabetes mellitus ( . % vs. . %, respectively, p < . ), had more frequent severe conditions (sofa: ± points vs. ± points, respectively, p < . ) pre-ecmo. meanwhile, they had a longer duration of ippv ( ± d vs. ± d, respectively, p < . ), had lower pao /fio levels ( . ± . mmhg vs. . ± . mmhg, respectively, p < . ), and higher rate of rescue ventilation strategies ( % vs. . %, respectively, p < . ) before ecmo support. no significant differences were found in the total duration of ippv, total duration of ecmo, length of icu stay and length of hospitalization between the two groups (table ) . ecmo blood flow did not significantly differ between the two groups during the initiation of ecmo support. however, in the successfully weaned group vs. the unsuccessfully weaned group, a significant decrease in blood flow correlated with an increase in the duration of support, which was . ± . l/min vs. . ± . l/min, respectively, (p < . ) at h on ecmo and . ± . l/ min vs. . ± . l/min, respectively, (p < . ) at h on ecmo (additional files and ). in the successfully weaned group compared to the unsuccessfully weaned group, fio was ± % vs. ± (fig. , additional file ). the vital signs were improved but did not significantly differ between the two groups pre-ecmo and during ecmo support (additional file ). patients who were unsuccessfully weaned from ecmo compared to patients who were successfully weaned from ecmo had severe acidosis (ph: . ± . vs. . ± . , respectively, (p < . ), a higher paco ( . ± . mmhg vs. . ± . mmhg, respectively, (p < . ), and a higher lactate concentration ( . ± . mmol/l vs. . ± . mmol/l, p < . ) pre-ecmo. ph and paco did not differ significantly between the two groups during ecmo support, while patients who were eventually successfully weaned from ecmo had a gradual ascending tendency of pao at and h on ecmo and a sustained low level of lactate (fig. , additional file ) . during the early stage of ecmo ( and h), the successful weaning group showed smaller differences between the act max and act min than the unsuccessful weaning group, which was ± s vs. ± s at h (p < . ) and ± s vs. ± s at h (p < . ). however, this trend was not found with regard to the difference between the maximum and minimum aptt. there were no differences between the two groups in mechanical complications associated with ecmo, vap and barotrauma. the successfully weaned group compared to the unsuccessfully weaned group had a lower haemorrhage rate ( . % vs. . %, respectively, p < . ), lower rate of renal insufficiency ( . % vs. . %, respectively, p < . ), lower rate of liver failure ( % vs. . %, respectively, p < . ) and lower heart failure rate ( . % vs. . %, respectively, p < . ). this study was the first to systematically and comprehensively discuss as well as elaborate on the current application of the efficacy and safety of ecmo in patients with h n pneumonia-related ards. a few studies [ , , , ] have shown that the mortality of ph n -induced ards was reduced to - % following ecmo treatment. presently, no studies with large samples have investigated the mortality of h n -induced ards, while the in-hospital mortality was as high as % in our study. late initiation of ecmo, inappropriate ippv settings during ecmo, and more ecmo complications might explain the relatively high mortality. moreover, as a multicentre collaboration study, the experiences of ecmo varied among the centres (additional file ), which might be another reason for the high mortality. according to the extracorporeal life support organization (elso) data [ , ] , ecmo is indicated when death risk exceeds %, i.e., when pao /fio < mmhg on fio > % and the murray score is - . our patients met the indications for ecmo support. the duration of mv for more than days pre-ecmo is an important prognostic factor for death [ ] . for patients in the successfully weaned group, the duration of ippv pre-ecmo was ± d; however, the duration was even longer among patients in the unsuccessfully weaned group ( ± d). moreover, rescue ventilation strategies were implemented for most patients before ecmo, which partially delayed the timing of ecmo. in comparison, ecmo was initiated at h ( - h) after ippv among patients with ph n in australia and new zealand in [ ] , which was significantly shorter than that in our cases. therefore, we emphasized early implementation of ecmo in our patients. the principle of ippv during ecmo is the "lung rest strategy" [ ] . the reva registry study examined patients with ph n -induced ards [ ] and showed that the high p plat ( cmh o) on day of ecmo was related to high mortality. in our study, the pre-ecmo p plat level was high ( ± cmh o). high p plat can lead to overdistension of the alveoli and cause lung volutrauma. the shear force between the overdistended and collapsed alveoli further aggregates vili [ ] , which ultimately increases mortality. although the p plat values [ ] showed that a high peep level within d of being on ecmo was related to decreased mortality. although no difference was observed in the peep levels between the two groups, we speculated that the down-regulation of peep during ecmo might have further aggravated the occurrence of collapse-induced injury, which led to atelectasis and sputum discharge obstacles. therefore, the ippv parameters, including high p plat and vt levels and low peep settings, might have been unreasonable in our study; lung rest or the maintenance of open alveoli was not achieved. the incidence of an ecmo oxygenator thrombus, haemorrhage, and organ failure in our study was high, which suggests that some problems existed in the anticoagulation management and organ supportive treatment of ecmo. we found that the unsuccessfully weaned group had larger fluctuations in act (the difference between act max and act min were larger) during the early stage of anticoagulation. this effect might suggest relatively unstable anticoagulation and a higher risk of haemorrhage. moreover, the incidence rate of vap during ecmo was as high as % and was partially attributed to the long course of h n pneumonia and the prolonged duration of ippv. therefore, intensification of airway management was extremely necessary. our study had limitations. the nature of the study required the collection of data at multiple consecutive time points to evaluate the efficacy of ecmo. as a retrospective study with some missing data, we were unable to successfully collect data at h pre-ecmo and , , and h post-ecmo. additionally, the number of subjects was too small to perform a multiple regression analysis to explore the risk factors for unsuccessful weaning from ecmo. ecmo is effective at improving oxygenation and ventilation of patients with avian influenza a (h n )-induced severe ards. early initiation of ecmo with appropriate ippv settings and anticoagulation strategies are necessary to reduce complications. fig. comparison of ippv parameters and abgs between two groups of patients on ecmo. for the successfully weaned group compared to the unsuccessfully weaned group, fio was ± % vs. ± %, respectively, at h (p < . ) and ± % vs. ± %, respectively, at h (p < . ). the monitored p plat was ± cmh o vs. ± cmh o, respectively, at h (p < . ) and ± cmh o vs. ± cmh o, respectively, at h (p < . ). the monitored vt was ± ml vs. ± ml, respectively, at h (p < . ) and ± ml vs. ± ml, respectively, at h (p < . ) after ecmo support. however, there were no differences in peep during ecmo between the two groups. patients who were in the unsuccessfully weaned group compared to patients in the successfully weaned group had severe acidosis (ph: . ± . vs. . ± . , respectively, (p < . ), a higher paco ( . ± . mmhg vs. . ± . mmhg, respectively, (p < . ), and a higher lactate concentration ( . ± . mmol/l vs. . ± . mmol/l, respectively (p < . ), pre-ecmo. the ph and paco did not significantly differ between the two groups during ecmo therapy, while patients who eventually weaned successfully from ecmo had a 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respiratory distress syndrome mechanical ventilation in ards: one size does not fit all mechanical ventilation management during extracorporeal membrane oxygenation for acute respiratory distress syndrome: a retrospective international multicenter study not applicable. dr. zhan was supported by a grant from the national key research and development programme-major chronic non-communicable diseases' prevention and control (qml yfc ). dr. cao was supported by a grant from the national science fund for distinguished young scholars (grant number /h ) and grants from the national natural science foundation of china ( / h and /h ), and the national program for the prevention and control of human infections by avian-origin h n influenza a virus (kjyj- - - ). the funding sources had no role in the design, conduct, or reporting of the study or the decision to submit the manuscript for publication. the remaining authors have disclosed that they do not have any potential conflicts of interest. the datasets used and/or analysed during the current study are available from the corresponding author upon reasonable request. department of infectious diseases, henan provincial people's hospital, zhengzhou, henan province, people's republic of china. additional file : blood flow during ecmo, changes in ippv parameters and physiological indicators pre-ecmo and during ecmo. (docx kb) additional file : blood flow during ecmo between the two groups. in the successfully weaned group vs. the unsuccessfully weaned group, a significant decrease in ecmo blood flow correlated with an increase in the duration of support, which was . ± . l/min vs. . ± . l/ min, respectively, at h (p < . ) and . ± . l/min vs. . ± . l/ min, respectively, at h (p < . ). (tiff kb) additional file : changes in vital signs pre-ecmo and during ecmo between the two groups. vital signs were improved and did not significantly differ between the two groups during ecmo. authors' contributions all authors made substantial contributions to the conception and design of the study, data acquisition, analysis or interpretation of data, and review and approval of the final manuscript. drs. lh, wz, yy, ww and wl contributed equally to the article. drs. bc and qz assumed full responsibility for the integrity of the submission and publication and were involved in the study design. drs. wz, yy, ww, wl, hx, hz, yunfu wu (yw), js, lc, and ll were responsible for caring for the influenza a (h n ) cases and have been involved in gathering data. drs. lh, bc, qz, cw, dl, and yw had full access to all of the data in the study, assume responsibility for the integrity of the data and the accuracy of the data analysis and were responsible for data verification and analysis, as well as the drafting of the manuscript.ethics approval and consent to participate all patients gave written informed consent before ecmo treatment. as a highly pathogenic disease, the chinese national health and family planning commission approved the collection of the data from the patients with h n virus-induced pneumonia. the informed consent was waived to allow for exploration of the characteristics of the emerging infectious disease after rigorous contemplation and discussion by the chinese national health and family planning commission. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord- -x jorbm authors: chughtai, abrar ahmad; stelzer-braid, sacha; rawlinson, william; pontivivo, giulietta; wang, quanyi; pan, yang; zhang, daitao; zhang, yi; li, lili; macintyre, c. raina title: contamination by respiratory viruses on outer surface of medical masks used by hospital healthcare workers date: - - journal: bmc infect dis doi: . /s - - -x sha: doc_id: cord_uid: x jorbm background: medical masks are commonly used in health care settings to protect healthcare workers (hcws) from respiratory and other infections. airborne respiratory pathogens may settle on the surface of used masks layers, resulting in contamination. the main aim of this study was to study the presence of viruses on the surface of medical masks. methods: two pilot studies in laboratory and clinical settings were carried out to determine the areas of masks likely to contain maximum viral particles. a laboratory study using a mannequin and fluorescent spray showed maximum particles concentrated on upper right, middle and left sections of the medical masks. these findings were confirmed through a small clinical study. the main study was then conducted in high-risk wards of three selected hospitals in beijing china. participants (n = ) were asked to wear medical masks for a shift ( – h) or as long as they could tolerate. used samples of medical masks were tested for presence of respiratory viruses in upper sections of the medical masks, in line with the pilot studies. results: overall virus positivity rate was . % ( / ). commonly isolated viruses from masks samples were adenovirus (n = ), bocavirus (n = ), respiratory syncytial virus (n = ) and influenza virus (n = ). virus positivity was significantly higher in masks samples worn for > h ( . %, / versus . %, / , or . , % ci . – . ) and in samples used by participants who examined > patients per day ( . %, / versus . %, / , or . , % ci . – . ). most of the participants ( . %, / ) reported at least one problem associated with mask use. commonly reported problems were pressure on face ( . %, / ), breathing difficulty ( . %, / ), discomfort ( . % / ), trouble communicating with the patient ( . %, / ) and headache ( . %, / ). conclusion: respiratory pathogens on the outer surface of the used medical masks may result in self-contamination. the risk is higher with longer duration of mask use (> h) and with higher rates of clinical contact. protocols on duration of mask use should specify a maximum time of continuous use, and should consider guidance in high contact settings. viruses were isolated from the upper sections of around % samples, but other sections of masks may also be contaminated. hcws should be aware of these risks in order to protect themselves and people around them. infectious diseases are a continuing threat, with constant emergence or re-emergence of serious diseases in various parts of the world and healthcare workers (hcws) are particularly at-risk of exposure to index cases [ ] [ ] [ ] [ ] . various types of personal protective equipment (ppe) are recommended and used by hcws to protect from infections, including medical masks, respirators, gloves, gowns, goggles and face shield [ , ] . in healthcare settings, medical masks are used by hcws to protect from splashes and sprays of blood and body fluids, and by sick individuals to prevent spread of respiratory infections to others [ ] . reuse and extended use of masks are also common in many parts of the world, particularly during outbreaks and pandemics [ , ] . respiratory pathogens may be present on used masks layers and lead to infection of the wearer [ ] . in hospital settings, these pathogens may be generated from breathing, coughing or sneezing patients or during aerosol generating medical procedures [ ] . studies have shown that influenza virus can remain airborne for h after a patient has passed through an emergency department [ ] . while using masks, or during long periods of time of re-using them, these pathogens may cause infection through hand or skin contamination, ingestion, or mucus membrane contact [ ] . currently there are limited data on the presence of respiratory pathogens on surface of ppe and other fomites in hospital settings. previous studies show that influenza and respiratory syncytial virus (rsv) may survive on outer surface of ppe [ ] [ ] [ ] [ ] . a study showed that influenza viruses may survive on hard surfaces for - h, on cloth up to - h and on hands for up to min [ ] . a previous study in an australian neonatal intensive care unit (nicu), respiratory syncytial virus (rsv) rna was identified from % of dress samples and % of environmental samples [ ] . if health departments do not provide clear guidance on the use of masks in these situations, hcws may continue using contaminated masks and may get infection [ ] . the risk of self-contamination of hcws is influenced by the mask itself, its shape and properties, and the virus concentration on its surface. to our knowledge, only one study examined the presence of contamination on mask and various bacteria were isolated from outer surface of medical masks [ ] . the main aim of this study was to study the level of contamination on the surface of medical masks. medical masks were tested as per protocols developed through two pilot studies in sydney australia. the aim of this pilot study was to identify areas of maximum virus concentration on the surface of masks. medical masks were donned on a simple mannequin in a laboratory setting and fluorescent particles (uv glow powder) were sprayed front on and side on from a distance of approximately m using a spray bottle. we performed three experiments from the front and three experiments from the sides of mannequin. uv light was used to quantify the density of particles on mask surface and to identify area of maximum concentration. in all three experiments, most particles were concentrated on upper right, middle and left sections of the masks (figs. and ). the second pilot study was conducted in two tertiary referral hospitals in sydney australia to develop testing methodology. twelve hcws (doctors and nurses) from the infectious diseases, respiratory/ chest wards and intensive care unit (icu) participated in the study. hcws were asked to wear medical masks for a shift (minimum min) used masks were tested in the virology research laboratory, university of new south wales and prince of wales hospital sydney australia. if a respirator was indicated due to airborne inflictions, hcws were excluded from the study and were allowed to use a respirator. medical masks were divided into six sections as shown in fig. . samples were taken from upper three sections of masks i.e. samples were tested in total ( masks x samples). the outer layer of the mask was removed using sterile tweezers. the mask layer was placed into a ml falcon tube containing μl of phosphate buffered saline and vortexed for s. after min incubation the mask was placed in a custom made filter tube inside an eppendorf tube and centrifuged briefly. the filtrate was then transferred to . ml eppendorf tube. total nucleic acid was extracted on the kingfisher flex (thermo scientific) using the magna pure total nucleic acid isolation kit (roche) according to the manufacturer's instructions. presence of respiratory viruses was detected using the seegene allplex™ respiratory panel assays , , (seegene). the main study was conducted in respiratory wards and fever clinics of three selected hospitals in beijing china from december to january . doctors and nurses from selected wards were invited to participate in the study. participants include nursing and medical staff aged > years working full time in the ward who were able to provide written and informed consent. participants with pre-existing respiratory, medical illness or pregnancy were excluded. as we did not test the participants, detail history on respiratory symptoms was taken to rule out contamination of masks by participants themselves. hcws from the participating wards were asked to wear medical masks for a shift ( - h), or as long as they could tolerate the masks with no adverse event. three layered standard medical masks were used. if hcws used more than one mask during their shift, first sample was collected and tested. used medical masks were collected at the end of the day and were stored immediately in zip-lock bags. hcws were advised to store masks in in zip-lock bags while they take off the masks during break time. all masks samples were labelled with participants' id and hospital id. at the end of the study, hcws were asked to complete a short survey to collect information on mask use in routine (type of mask used, number of masks used and situations when masks were normally used) and during the study period (wearing time, number of patients seen, situations when masks were used, aerosol generating procedures performed and hand hygiene during donning and doffing). participants reported "number of masks used" and "number of patients seen" in absolute numbers. "duration of mask use" was recorded in hours as, < h, to h, to h, to h, to h, > h. "situations when masks were used" were categorized into: "used continuously", "used continuously except during breaks", "used only during patients' encounters" and "used only high-risk patient encounters". medical masks were tested in the beijing cdc laboratory. all masks were collected immediately after use in zip-lock bags and kept at − °c until testing. pilot studies showed that upper sections of masks were more contaminated (figs. and ). the outer layers of upper right, middle and left mask were separated with a same size, placed into separated tubes containing μl pbs buffer (gibco, usa), vortexed for min, and finally aliquoted μl for viral testing. we performed three tests on upper right, middle and upper left sections of the masks on around a quarter mask sample ( %) and performed one test on the remaining mask samples ( %). for one testing, outer layers of upper right, middle and left section of mask were separated and placed into the same tube. viral dna/rna was extracted using king-fisher flex viral dna/rna purification kit (thermo fisher, usa) according to the manufacturer's instructions. the reverse-transcription polymerase chain reaction was performed to amplify viral target genes, including influenza a/b virus, influenza a(h n ) and a(h n ), parainfluenza viruses - , rhinoviruses, bocavirus, human metapneumovirus, adenovirus, respiratory syncytial virus, coronaviruses oc , e, nl and hku using a commercial multiplex combined real-time pcr detection kit for respiratory virus, which is developed by "jiangsu uninovo biological technology co. ltd." in china. currently there is very limited data on testing of masks surface for presence of pathogens. in previous studies influenza virus was detected on over % of the fomites tested in community settings during influenza season [ ] . the rate is expected to be higher in the healthcare setting and moreover other viruses will also be tested. assuming % higher positivity rate in the healthcare setting, the required sample size would be masks, with % power and two-sided % significance level for detecting a significant difference. some hcws might not be able to provide mask samples, we aimed to recruited hcws in total for this study. descriptive analysis was conducted, and rates and frequencies were calculated. univariate analysis was performed to identify the factors associated with mask positivity. logistic regression was used to calculate odds ratio (or) and % confidence intervals (ci) data were analyzed in sas (sas institute inc., usa) version . . ethics approval for pilot study was sought from south eastern sydney local health district (seslhd). ethics approval for the main study was sought from human research ethics committee unsw (hc ) and ibr china. written consent as obtained from all participants. of samples in pilot testing, three samples were positive for human enterovirus. two samples were positive from outer sections of mask, while one sample was positive from middle section. no other viruses were detected in mask samples. a total of participants were recruited from three hospitals in the main study. ten participants provided more than one samples for the testing, so we excluded these cases from analysis due to uncertainty around the duration of mask use being tested. most participants were recruited from hospital a ( %, / ), largely from the respiratory ward . %, / ). around half of the participants were doctors ( . %, / ), and majority were female ( . %, / ). in routine clinical practice, almost all participants ( . %, / ) had previously used disposable medical masks. generally, most of the participants had been using or medical masks per day ( . %, / ) and around two third participant ( . %, / ) had been using mask all the time during the clinical work (table ) . during the study period, around / participants used masks for > h -" - h" participants ( . %) and "> h" participants ( . %). the remaining / used masks for ≤ h -" - h" participant ( . %), " - h" participants ( . %) and " - h" participants ( %). most participants ( . %, / ) used masks either continuously or continuously except breaks. the majority of participants ( . %, / ) reported at least one problem adenovirus was most commonly isolated from the masks (n = ), followed by bocavirus (n = ), rsv (n = ) and influenza virus (n = ) ( table ) . compared to the participants working in internal medicine department, virus positivity rates were lower among those working in respiratory (or . , % ci . - . ) and pediatric (or . , % ci . - . ) departments. virus positivity was significantly higher on masks samples worn by participants who used masks for > h, compared to those who used mask for ≤ h that day (or . , % ci . - . ). similarly, virus positivity was significantly higher on masks samples worn by participants who examined > patients per day, compared to who examined ≤ patients (or . , % ci . - . ). virus positivity rates were also higher in mask samples collected from males, participants who used mask during encounters with high risk patients and those who performed aerosol generating procedures (agps), however the difference was not statistically significant (table ). to our knowledge this is the first study examining the presence of respiratory viruses on the outer surface of used medical masks. one in ten masks were positive for any virus which highlights the risk of self-contamination to the wearer, particularly on doffing [ ] . reuse and extended use of masks are very common, particularly in low income countries and during outbreaks and pandemics when supplies are short, and demand is high [ , ] . staff should be aware of the risk associated with the reuse and extended use of masks and respiratory protective devices and high clinical contact. large scale studies should be conducted to determine the contamination on other ppes as well and to quantify the risk of infection among hcws. epidemics of a new infectious disease may be devastating due to global spread, disease burden and high case fatality. ppe are generally considered lowest among infection control hierarchy and recommended to be used with other administrative and environmental control measures [ ] . however, masks, respirators and other ppe are important during initial phase of outbreak and pandemic when drugs and vaccine are not available [ ] . ppe can easily get contaminated during clinical care of sick patients which may result in an increased risk of infection in wearer [ ] . many simulation studies have also shown presence of particles on the potential surface of ppe and associated risk of self-contamination during doffing of ppe [ , [ ] [ ] [ ] . in this study we only tested the presence of viruses on the medical masks. overall virus positivity rate in this was . % ( / ) and adenovirus was isolated from mask samples while bocavirus, rsv and influenza viruses were isolated from samples each. prospero et al. conducted a study in dental settings and estimated the bacterial contamination on surface of masks used by dentist, lamps, areas near spittoons, and mobile trays. sterile nitrocellulose filters were applied on these surfaces to isolate pathogens. highest levels of bacterial contamination (streptococcus species %, staphylococcus species %, and gram-negative bacteria %) were recorded on the external surface of masks wore by dentist [ ] . large scale studies should be conducted to examine presence of various pathogens on the surface of masks and other ppe. in this study, the risk of mask contamination was associated with duration of masks use and number of patients seen. currently there is no standard duration for the time period that facemasks and respirators can safely one study showed that influenza virus may survive on mask surface and maintained infectivity for at least h [ ] . our study showed very low infection among hcws who used masks for ≤ h. high virus positivity on masks samples worn by hcws who examined > patients, may be due to more frequent clinical contact with infective cases and transfer of more pathogens from patients to mask surface. virus positivity rates were also higher in those working in internal medicine department compared to respiratory and pediatric departments. the reason of high virus positivity in internal medicine department is not clear, but this may be due to using varying infection control policies and practices. high risk perception and more infection control measures may result in low virus positivity in in respiratory and pediatric departments. however, the sample sizes and number of positive results were too low to make meaningful comparisons between departments. there is a need for more research to define the exact threshold of safe duration, and to develop a comprehensive policy on the use of masks in hospital settings and protocols should specify a maximum time of continuous use and should consider guidance in high contact settings. we also aimed to identify the area on the mask surface with maximum respiratory virus concentration. laboratory based pilot study showed maximum fluorescent contamination on upper sections of the masks, which is also the likely area to be touched on removal. of the three positive tests in hospital-based pilot study, two samples were positive from outer sections of mask, while one sample was positive from middle section. in the main study we were able to check the location of contamination on a quarter of mask samples. of the mask samples, one or more viruses were isolated from four ( . %) samplestwo from middle section of masks and two from right section of the masks. this presents a large area of potential contamination which place hcw at risk when removing a mask. these data may assist in developing policies on for doffing of masks after encounter with infective cases. as a general rule, hcws should not reuse masks, should restrict use to less than h and avoid touching the outer surface of mask during doffing, and practice hand hygiene after removal. there are limitations of this study. due to funding constraints we tested selected masks samples. we performed three tests on a sub-sample ( %) to identify the area of maximum concentration. moreover, we just tested upper three sections of medical masks based on the first pilot study, while lower three sections should also be tested. then we tested only outer layer of masks and did not check filtering layer and inner layer due to funding constraints. ideally all sections and layers of masks should be tested. we collected detail history from the participants to rule out any existing respiratory illness. although none of the participant had a respiratory or a medical illness, it is not possible to determine whether viruses isolated from the masks surface were from exogenous or endogenous source. for example, adenovirus was most commonly identified in this study and is associated with mild or no respiratory illnesses. ideally participants should also be swabbed to rule out infections, and the inside surface should also be tested. however, given the large variations of infection probability in different types of wards, it is unlikely that all viruses came from the background infection. to overcome this limitation, detailed history on respiratory symptoms was taken to rule out contamination of masks by clinically ill participants themselves. moreover, we only examined viruses on the masks, while bacteria and other pathogens may also be present [ ] . mask use was not monitored, and self-reported compliance was recorded. previous studies show that self-reported compliance is generally reported to be higher compared to the actual compliance [ , ] . we also did not document the method of mask removal, nor the number of times the hcw touched the mask. to maintain the functionality and capacity of the health care workforce during outbreaks or pandemics of emerging infections, hcws need to be protected. this study provides new data, which will help 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vietnam publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations thanks to the staff at the beijing centre for diseases prevention and control and hospitals staff for participating in the study. we would like to thank xin chen (unsw), yimeng liu (beijing center for diseases prevention and control) and jiachen zhao, beijing center for diseases prevention and control, for assisting with sample collection and handling. authors' contributions aac -conception and design of study, data analysis and manuscript writing. ssb, wr, gp -data/ sample collection and lab testing for pilot studies in australia, manuscript review. qw, yp, yz and ll -data/ sample collection for the main study in china, manuscript review. dz -testing for the main study in china, manuscript review. crm -contributed to study design and manuscript writing. all authors approved the study. this study was supported by nhmrc centre for research excellence grant app (integrated systems for epidemic response [iser]). funding body has no role in design of the study and collection, analysis, and interpretation of data and in writing the manuscript". the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate ethics approval for pilot study was sought from eastern sydney local health district (seslhd). ethics approval for the main study was sought from human research ethics committee unsw (hc ) and ibr china. consent was obtained from all participants before recruitment. not applicable . all authors have completed the unified competing interests form (available on request from the corresponding author) and declare that: aac had testing of filtration of masks by m for phd. crm has held an australian research council linkage grant with m as the industry partner, for investigator driven research. m have also contributed supplies of masks and respirators for investigator-driven clinical trials. she has received research grants and laboratory testing as in-kind support from pfizer, gsk and bio-csl for investigator-driven research. the remaining authors declare that they have no competing interests and have no non-financial interests that may be relevant to the submitted work. key: cord- - d yfv authors: porfyridis, ilias; plachouras, diamantis; karagianni, vasiliki; kotanidou, anastasia; papiris, spyridon a; giamarellou, helen; giamarellos-bourboulis, evangelos j title: diagnostic value of triggering receptor expressed on myeloid cells- and c-reactive protein for patients with lung infiltrates: an observational study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: d yfv background: differential diagnosis of patients with lung infiltrates remains a challenge. triggering receptor expressed on myeloid cells (trem)- is a neutrophil and monocyte receptor up-regulated during infection. the aim of this study was to evaluate the diagnostic accuracy of trem- and of c-reactive protein (crp) from patients with lung infiltrates to discern community acquired lung infections. methods: patients admitted to a medical ward with acute respiratory illness were enrolled in the study. neutrophil and monocyte trem- expression were measured by flow cytometry, strem- by an enzyme immunoassay and c-reactive protein by nephelometry. clinical pulmonary infection score was recorded. results: patients were diagnosed with bacterial community acquired pneumonia (group a) and with non-bacterial pulmonary disease (group b). median serum trem- concentration was . pg/ml in group a and lower than . pg/ml (p < . ) in group b. mean±se neutrophil trem- expression was . ± . mfi in group a and . ± . mfi (p = . ) in group b. monocyte trem- expression was . ± . mfi in group a and . ± . mfi (p = . ) in group b and mean±se crp was . ± mg/ml in group a and . ± . mg/ml (p < . ) in group b. a cut-off of . pg/ml of strem- with sensitivity . % and specificity % to discriminate between infectious and non-infectious pulmonary infiltrates was found. strem- at admission greater than pg/ml was accompanied with unfavourable outcome. conclusion: trem- myeloid expression and strem- are reliable markers of bacterial infection among patients with pulmonary infiltrates; strem- is a predictor of final outcome. early diagnosis of lung infections remains a challenge. there is no gold standard for diagnosing microbial infection as clinical and laboratory signs are neither sensitive nor specific enough, and microbiological studies often remain negative. the presence of a new infiltrate on plain chest radiograph is considered indicative for diagnosing pneumonia, especially when is supported by clinical and laboratory findings. however it is difficult to differentiate a chest infiltrate of bacterial origin from a chest infiltrate of non-bacterial origin solely based on radiological criteria [ ] . the diagnosis of infection is not always clear in the acute setting in patients with respiratory tract disease and a surrogate marker of infection would be a major benefit in the diagnostic armamentarium. many inflammatory mediators and acute phase reactants, like c-reactive protein (crp) and procalcitonin, have been described as reliable markers of infection; however none are specific enough, since they are also increased in non-infectious inflammatory conditions [ ] . triggering receptor expressed on myeloid cells (trem)- is a recently described receptor on neutrophils and monocytes. it behaves like a pattern recognition receptor (prr) since its activation leads to the release of pro-inflammatory cytokines, namely of tumour necrosis factor-alpha (tnfα) and of interleukin (il)- . although its ligand is still unknown, activation is mediated by bacteria and fungi [ , ] . a soluble form of trem- , namely strem- , is increased in the bronchoalveolar lavage (bal) of patients with ventilator associated pneumonia (vap) [ , ] , and in the serum of patients with sepsis, with bacterial meningitis and with acute pancreatitis [ ] [ ] [ ] [ ] [ ] [ ] . this same soluble form of trem- seems to be increased in patients bearing noninfectious processes like peptic ulcer, inflammatory bowel disease, viral infections, malignant pleural effusions and chronic obstructive pulmonary disease (copd) but also among patients after cardiac surgery or cardiac arrest. increase of strem- seems particular prominent when the latter non-infectious states are complicated with systemic inflammatory response syndrome (sirs) without infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] . several published studies yielded contradictory results for the diagnostic and prognostic usefulness of trem- and of strem- for infections [ , , [ ] [ ] [ ] . the created impression is that more data are necessary to yield definitive results for its usefulness as a diagnostic and prognostic marker of community acquired pneumonia (cap). the aim of the present study was to define whether expression of trem- on cell membranes of neutrophils (ntrem- ), of monocytes (mtrem- ) and serum strem- may help in the diagnosis of acute bacterial infections for patients admitted with a new pulmonary infiltrate or pleural effusion. in this observation trial, all consecutive admissions to the department of critical care and pulmonary services on predetermined and randomly selected emergency duty days were eligible. inclusion criteria were: i) age above yrs, ii) written informed consent; iii) acute respiratory illness and iii) presence of new pulmonary infiltrates or pleural effusion on chest x-ray or lung computed tomography. exclusion criteria were: i) human immunodeficiency virus (hiv) infection, ii) documented extrapulmonary infection, iii) neutropenia; and iv) oral intake of corticosteroids defined as any more than mg/kg of prednisone for more than month. the study protocol was approved by the ethics committee of the hospital and written informed consent was obtained from all patients within the first hrs after admission. clinical, laboratory, and imaging data were recorded for each patient including: i) clinical presentation; ii) body temperature, iii) arterial blood gas, iv) peripheral blood cell counts, v) gram stains and cultures of all biological fluids obtained (blood, sputum, bronchial secretions, bal, and pleural fluid); vi) imaging findings, vii) antigen serology (legionella spp and streptococcus pneumonia urinary antigen, serological testing for legionella pneumophila, mycoplasma pneumoniae, chlamydia pneumoniae) and viii) in-hospital mortality. the severity of illness was assessed by calculating acute physiology and chronic health evaluation (apache) ii, sequential organ failure assessment (sofa) and clinical pulmonary infection (cpis) scores at admission [ ] . a diagnosis of community-acquired pneumonia (cap) was established in any patient presenting with a combination of fever, cough and purulent sputum, shortness of breath, chest pain, and new consolidation on chest x-ray or computed tomography. the severity of pneumonia was assessed the first hours of admission according to confusion, urea nitrogen, respiratory rate, blood pressure (curb) index. patients having two or more criteria were identified to have severe pneumonia [ ] . sepsis, severe sepsis and septic shock were defined according to current recommendations [ ] . pneumonia was considered to be absent when: i) an alternative cause for pulmonary infiltrate was established (e.g. pulmonary embolus) and ii) full recovery was achieved without antimicrobial therapy. pulmonary embolism was diagnosed according to current recommendations [ ] . lung cancer was ruled out based on histology and/or cytology specimens. congestive heart failure was diagnosed according to american heart association [ ] , and interstitial lung disease according to american thoracic society guidelines [ ] . all cases were evaluated by two clinicians blinded to trem- and strem- results. agreement about the diagnosis was achieved in all cases. patients with cap were classified as having bacterial respiratory infection (group a). all other patients were classified as having non-bacterial respiratory disorders (group b). all patients assigned to group b were subject to chest computed tomography. for the measurement of strem- , mtrem- , ntrem- and crp ml of peripheral venous blood were sampled after venipuncture of the antecubitul vein under sterile conditions on the day of admission and on days and of hospitalization. seven ml were centrifuged and serum was stored in - °c until assayed for strem- . three ml were collected into edta-coated tubes (vacutainer, bd) for estimation of ntrem- and mtrem- expression. briefly, red blood cells were lysed by ammonium chloride. white blood cells were labelled by phycoerythrin-conjugated anti-trem- monoclonal antibodies (r&d inc, minneapolis, usa) for minutes in the dark. ntrem- and mtrem- expression were assessed after passage of labelled cells through a flow cytometer (epics xl/msl, beckman-coulter co, miami florida) and expressed as the mean fluorescence intensity (mfi) with gating for neutrophils and for monocytes by their characteristic fs/ss scattering. determination of strem- was performed in duplicate by a developmental enzyme-linked immunoabsorbent assay according to the instructions of the manufacturer (r&d inc, minneapolis, usa). the lower detection limit and inter-day variation of the assay were . pg/ml and . % respectively. measurement of serum crp was performed by an immunoturbidimetric assay on roche automated clinical chemistry analyzers and was expressed in mg/ml. crp was used as a comparator due to its universal application in all studies of evaluation of biomarkers. asumming that measured parameters between groups a and b differed by %, it was calculated that to patients should be assigned into each group to yield a difference at the % level with % power. values for ntrem- , mtrem- and crp are presented as mean ±se; those of strem- are presented as medians and % confidence intervals (ci) or interquartile range (iqr). comparisons between groups for ntrem- , mtrem- expression and for crp were done by anova, followed by the tukey's test for multiple comparisons. comparisons of strem- between groups were done by mann-whitney u test after bonferroni corrections for multiple comparisons. comparisons of strem- between consecutive days within one group were done by wilcoxon's signed rank test. receiver operator curves (roc) were designed to asses sensitivity, specificity, positive and negative predictive values for the estimated parameters to disclose infectious from non-infectious infiltrates. patients were divided into two categories according to serum levels of strem- upon admission: those with strem- below or equal to pg/ml; and those with serum strem- greater than pg/ml. this concentration has been proposed as a threshold defining final prognosis in septic populations [ , ] . since cap is a common cause of sepsis, this threshold was considered of merit. survival was assessed by kaplan-meier and comparisons were done by log-rank test. correlations between severity scores and measured parameters were done according to spearman. probability values less than . were considered statistically significant. all statistics and graphs were done using the statistical package for the social sciences software version . . (spss inc, chicago, il). the study flow-chart is shown in figure . demographic and clinical data of the patients are summarized in table . patients suffering from tuberculosis and enrolled in group b were presented with pleuritis. group a (n = ) consisted of patients with community acquired pneumonia (cap) likely to be caused by extracellural bacteria. seventeen had microbiological evidence of pulmonary infection, with isolation of the offending pathogens from sputum, blood or bal samples (when bronchoscopy was performed). seventeen patients were diagnosed with cap on the basis of typical clinical and radiological presentation and good response to antibiotic therapy. main radiological group b (n = ) consisted of patients with non-bacterial respiratory disorders. diagnoses were: lung cancer ( patients); pulmonary embolism (six patients); interstitial lung disease (six patients); heart failure (n = ); pulmonary tuberculosis (two patients); rheumatoid pleuritis (one patient); and q-fever (one patient). main radiological findings were: right pulmonary infiltrate (six patients); left pulmonary infiltrate (three patients); bilateral pulmonary infiltrates ( patients); right pleural effusion (four patients); left pleural effusion (one patient); both right lung infiltrate and right pleural effusion (four patients); both left lung infiltrates and left pleural effusion (two patients); bilateral pulmonary infiltrates and left pleural effusion (one patient); and left pulmonary infiltrate and bilateral pleural effusions (one patient). among patients from group a with cap nine (n = ) died; six patients were admitted to the icu and three were not admitted to the icu due to relatives' denial. mean age of patients not admitted to icu was years; the first two patients had a case-history of stroke and chronic heart failure; the third patient had a case-history of lung cancer. all three died from severe sepsis and multiorgan dysfunction syndrome (mods). mean age of patients admitted to icu was years; two patients had a case-history of aortic valve stenosis; two patients were under chronic intake of receiving corticosteroids; the fifth patient suffered from end-stage renal disease; and the sixth patient was suffering from hepatic failure due to alcohol intake. all six patients died from severe sepsis and multiorgan dysfunction syndrome (mods). all patients in the icu accomplished the clinical and radiological criteria for acute respiratory distress syndrome (ards) and were ventilated with the strategy of low tidal volume ventilation, according to current guidelines [ ] , with volume limited mode ventilation, low tidal volumes (about ml/kg ideal body weight), a maximum of - breaths per minute, high positive end-expiratory pressure (peep cmh o) and a goal plateau airway pressure < cmh o. among patients admitted in the icu, two died on the second day post-admission; one died on the third day post-admission; one on the seventh day post-admission; one the eighth day postadmission; and one on the twentieth day post admission. concentrations of strem- and of crp in sera of both groups and expression of ntrem- and mtrem- are given in table . all four parameters were significantly greater in group a than group b. roc of strem- , ntrem- , m-trem- and crp to differentiate whether a chest x-ray infiltrate is due to cap or to a non-infectious process is shown in figure . area under curve (auc) of strem- was . ± . ( %ci: . - . , p = . ). sensitivity and specificity to diagnose between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin were . % and % respectively at concentrations above . pg/ml. auc of ntrem- and mtrem- were . ± . ( %ci: . - . , p = . ) and . ± . ( %ci: . - . , p = . ) respectively. sensitivity and specificity to diagnose between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin were . % and . % for ntrem- above . mfi. sensitivity and • lung cancer ( ) • staphulococcus aureus ( ) • pulmonary embolism ( ) • haemophilus influenzae ( ) • congestive heart failure ( ) • pseudomonas aeruginosa ( ) • interstitial lung disease ( ) • other ( specificity to diagnose between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin were . % and . % respectively for mtrem- above . mfi. auc of crp was . ± . ( %ci: . - . , p < . ). sensitivity and specificity to diagnose between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin were % and % respectively at concentrations above . mg/ml. positive correlations were found between apache ii scores and expression of trem- on monocytes on day (r s : + . , p: . ); and between apache ii scores and strem- on day (r s : + . , p: . ). no significant correlations were found between apache ii scores and expression of trem- on neutrophils on day as well as between sofa scores and any of the measured parameters on day . correlations between serum levels of strem- and crp and expression of trem- on monocytes and neutrophils in relation to the identified causative pathogen of cap are shown in figure . serum levels of strem- were greater among patients with cap caused by gram (+) cocci and haemophilus influenzae than among patients with cap caused by other pathogens. death occurred in three out of patients were no pathogen was defined ( . %); in nil out of three patients infected by atypical pathogens ( %); in three out of seven patients ( . %) infected by gram-negative bacteria; and in three out of nine patients ( . %) infected by gram-positive cocci or h. influenzae (p: . between grouping according to pathogen). survival of patients with strem- on day below or equal to pg/ml was prolonged compared with patients with strem- on day above pg/ml ( figure ) . the results of the present study indicate that trem- can be used as marker of bacterial infection in patients with lung infiltrates. strem- , ntrem- , mtrem- and crp were comparable to their discriminating ability between a pulmonary infiltrate of infectious origin and a pulmonary infiltrate of non-infectious origin. strem- levels were decreased within the first hours in patients with cap with favourable outcome probably after the initiation of appropriate therapy followed by improvement of clinical symptoms. finally, strem- levels above pg/ml were an accurate independent predictor of in-hospital mortality from cap. discrimination of the infectious or non-infectious origin of a pulmonary infiltrate remains an everyday clinical problem. cpis was introduced for that purpose helping considerable in cases of ventilator-associated pneumonia (vap) [ ] . trem- is a surface receptor on cells of the myeloid lineage. activation of trem- leads to the production of pro-inflammatory cytokines [ , , ] . binding of its ligand is possibly linked to the activation of several transcription complexes that synergize with nf-b in order to elicit transcription of genes of pro-inflammatory cytokines [ ] . strem- is the soluble counterpart of trem- and it is probably shed in the systemic circulation from cell membranes of neutrophils and monocytes [ , , ] . the physiologic role of strem- remains under question despite data support a probable anti-inflammatory role [ , ] . trem- has been studied in patients with pneumonia, especially vap [ , , [ ] [ ] [ ] . few data are available on the diagnostic role of trem- and of strem- in patients with lung infiltrates. our data are in agreement with observations from the study by phua [ ] . their proposed strem- cut-off point was ng/ml, which is different than the one we found. this may be result from the different method of assaying strem- the used being western blotting. the results of our study are in contrast to those of another study [ ] that did not disclose any difference in ntrem- expression between patients with and without a bacterial lung infection probably due to the small number of patients included in that former study. el sohl et al [ ] reported elevated alveolar levels of strem- in pulmonary aspiration syndromes, but not in serum. however, serial plasma strem- levels were not obtained and the possibility that plasma levels might rise on subsequent days cannot be excluded. two recent studies [ , ] evaluated the diagnostic role of cpis and of strem- in bal fluid from patients with bilateral lung infiltrates in the intensive care unit (icu). these studies reported controversial results. however authors did not measure strem- in serum on consecutive days. the reported results of the present study are the first to our knowledge that evaluate the diagnostic value of trem- among patients with lung infiltrates to discriminate cap. they also disclose a relationship between levels of circulating strem- and causative pathogens. more precisely, infections caused by streptococcus pneumoniae, sthaphylococcus aureus and haemophilus influenzae were accompanied by greater levels of strem- and by greater expression of trem- on neutrophils than infections caused by other pathogens. although it may be hypothesized that gram-positive cocci and h. influenzae are strong inducers of trem- expression, it should be emphasized that trem- is one prr, the exact agonist of which remains to be found [ , ] . a former study of our group [ ] and another by gibot et al [ ] in heterogeneous populations of patients with severe sepsis of diverse aetiology investigated the role of early assessment of strem- as a determinant of final outcome. results revealed that concentrations greater than pg/ml are accompanied by survival benefit. the exactly opposing finding is reported here. this discrepancy may be explained by the enrolment of more homogeneous populations of patients, compared to these former studies [ , ] , all suffering with cap. our study presents two main limitations: a) no documented cases of cap by legionella pneumophila, mycoplasma pneumoniae, protozoa or parasites were enrolled in group a; b) mortality in the cap patient group was high probably due to the existence of severe co-morbid conditions. in conclusion, the presented results indicate that serum strem- and expression of trem- on neutrophils and monocytes may serve as markers of cap in patients with pulmonary infiltrates. concentrations of strem- in serum are particularly increased in cap caused by gram-positive cocci and haemophilus species. the real clinical value of strem- assay comes when trem- levels are low, allowing the clinician to withhold empiric antibiotics until culture results are available, and thus eliminating unnecessary antibiotic exposure to the patient. and finally, early serum levels of strem- greater than pg/ml in cap are associated with unfavourable prognosis. and evangelos j. giamarellos-bourboulis ass. prof. md have no conflicts of interest to disclose related to this study. evangelos j. giamarellos-bourboulis prof. md has received reimbursement for attending the th international symposium on intensive care and emergency medicine where participated as a speaker and unrestricted educational grants from abbott hellas sa; wyeth hellas sa; sanofi-aventis hellas sa. imaging of pneumonia: trends and algorithms diagnostic and prognostic accuracy of clinical and laboratory parameters in community-acquired pneumonia trem- (triggering receptor expressed on myeloid cells): a new player in acute inflammatory responses trem- amplifies inflammation and is a crucial mediator of septic shock does soluble triggering receptor expressed on myeloid cells- play any role in the pathogenesis of septic shock? soluble triggering receptor expressed on myeloid cells and the diagnosis of pneumonia serum of patients with septic shock stimulates the expression of trem- on u monocytes clinical review: role of triggering receptor expressed on myeloid cells- during sepsis triggering receptor expressed on myeloid cells- (trem- ) is regulated post-transcriptionally and its ligand is present in the sera of some septic patients soluble triggering receptor expressed on myeloid cells- : a biomarker for bacterial meningitis increased levels of soluble triggering receptor expressed on myeloid cells- in patients with acute pancreatitis triggering receptor expressed on myeloid cells- expression on monocytes is associated with inflammation but not with infection in acute pancreatitis soluble triggering receptor expressed on myeloid cells- (strem- ): a new mediator involved in the pathogenesis of peptic ulcer disease role of soluble triggering receptor expressed on myeloid cells- in inflammatory bowel disease trem- expression in tumor-associated macrophages and clinical outcome in lung cancer activation of triggering receptor expressed on myeloid cells- on human neutrophils by marburg and ebola viruses soluble triggering receptor expressed on myeloid cells is released in patients with stable chronic obstructive pulmonary disease increased plasma levels of soluble triggering receptor expressed on myeloid cells and procalcitonin after cardiac surgery and cardiac arrest without infection the increased expression of trem- on monocytes is associated with infectious and non-infectious inflammatory processes soluble trem- is not suitable for distinguishing between systemic inflammatory response syndrome and sepsis survivors and nonsurvivors in the early stage of acute inflammation prognosis of community acquired pneumonia(cap): value of triggering receptor expressed on myeloid cells- (trem- ) and other mediators of the inflammatory response timecourse of strem (soluble triggering receptor expressed on myeloid cells)- , procalcitonin, and c-reactive protein plasma concentrations during sepsis diagnosing pneumonia during mechanical ventilation: the clinical pulmonary infection score revisited infectious diseases society of america guidelines for the management of adult lower respiratory track infections acute pulmonary embolism focused update incorporated into the acc/aha guidelines for the diagnosis and management of chronic heart failure in adults: a report of the american college of cardiology foundation european respiratory society international multidisciplinary consensus classification of idiopathic interstitial pneumonias early changes of cd -positive lymphocytes and nk cells in patients with severe gram-negative sepsis the national heart, lung, and blood institute ards clinical trials network: higher versus lower positive end-expiratory pressures in patients with the acute respiratory distress syndrome trem and trem-like receptors in inflammation and disease the trem receptor family and signal integration monocytes as a site of production of soluble triggering receptor expressed on myeloid cells - (strem- ) in the septic host soluble triggering receptor expressed on myeloid cells- modulates the inflammatory response in murine sepsis soluble triggering receptor expressed on myeloid cells- as an antiinflammatory mediator in sepsis soluble triggering receptor expressed on myeloid cells - in acute respiratory infections triggering receptor expressed on myeloid cells: role in the diagnosis of lung infections triggering receptors expressed on myeloid cells in pulmonary aspiration syndromes diagnostic implications of soluble triggering receptor expressed on myeloid cells- in bal fluid of patients with pulmonary infiltrates in the icu diagnostic utility of the soluble triggering receptor expressed on myeloid cells- in bronchoalveolar lavage fluid from patients with bilateral lung infiltrates funding source: none the present authors would like to thank all patients who participated in the current study, the doctors and nurses of the department of critical care and pulmonary services, national and kapodistrian university of athens, 'evangelismos' hospital, athens, greece for their hand work in the treatment of the patients and the doctors and laboratory personnel of the th department of internal medicine, national and kapodistrian university of athens, 'attikon' hospital, athens, greece for their most helpful support during the study. finally, the current authors thank paris praxitelous authors' contributions ip participated in the study design, the enrolment of patients, the estimation of trem- , strem- , crp, the follow-up of patients and wrote the manuscript. dp participated in the study design and in the estimation of trem- and strem- . vk carried out the estimation of trem- . ak and sap participated in study design, and drafted the manuscript. hg participated in study design and drafted the manuscript ejgb coordinated the lab job, analyzed the data and drafted the manuscript. all authors read and approved the final manuscript. key: cord- - tp i vh authors: hackert, volker h.; dukers-muijrers, nicole h. t. m.; hoebe, christian j. p. a. title: signs and symptoms do not predict, but may help rule out acute q fever in favour of other respiratory tract infections, and reduce antibiotics overuse in primary care date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: tp i vh background: from early , the dutch region of south limburg experienced a massive outbreak of q fever, overlapping with the influenza a(h n )pdm pandemic during the second half of the year and affecting approximately . % of a , population. acute q fever shares clinical features with other respiratory conditions. most symptomatic acute infections are characterized by mild symptoms, or an isolated febrile syndrome. pneumonia was present in a majority of hospitalized patients during the dutch – q fever epidemic. early empiric doxycycline, guided by signs and symptoms and patient history, should not be delayed awaiting laboratory confirmation, as it may shorten disease and prevent progression to focalized persistent q fever. we assessed signs’ and symptoms’ association with acute q fever to guide early empiric treatment in primary care patients. methods: in response to the outbreak, regional primary care physicians and hospital-based medical specialists tested a total of subjects for q fever. testing activity was bimodal, a first “wave” lasting from march to december , followed by a second “wave” which lasted into and coincided with peak pandemic influenza activity. we approached all notified acute q fever cases and a random sample of q fever negative individuals for signs and symptoms of disease. using data from / ( . %) q fever positive and / ( . %) q fever negative respondents from wave , we built symptom-based models predictive of q-fever outcome, validated against subsets of data from wave and wave . results: our models had poor to moderate auc scores ( . to . %), with low positive ( . – . %), but high negative predictive values ( . – . %). male sex, fever, and pneumonia were strong positive predictors, while cough was a strong negative predictor of acute q fever in these models. conclusion: whereas signs and symptoms of disease do not appear to predict acute q fever, they may help rule it out in favour of other respiratory conditions, prompting a delayed or non-prescribing approach instead of early empiric doxycycline in primary care patients with non-severe presentations. signs and symptoms thus may help reduce the overuse of antibiotics in primary care during and following outbreaks of q fever. from march , south limburg, the southernmost region of the netherlands, experienced a massive outbreak of human q fever related to an abortion storm on a local dairy-goat farm. laboratory-confirmed symptomatic human q-fever cases were first reported in april, peaked in may, and then steadily declined over subsequent months. culling of infected goats took place around the turn of the year. by april , no more new cases were reported to the regional public health service (phs), and the number of notified human cases reported to the regional phs had totalled , whereas the number of infections was estimated to run into thousands [ ] . a majority of acute q fever infections are understood to be asymptomatic or only mildly symptomatic. symptomatic patients usually present with a febrile syndrome or flu-like illness frequently said to be associated with myalgia and headache. during the dutch q fever epidemic, which lasted from to , pneumonia was present in as many as % of hospitalized patients. although most cases of acute q fever are self-limiting, early antibiotic treatment with doxycycline within the first days of symptoms may shorten duration of disease, and may prevent progression to persistent focalized infection, commonly referred to as chronic q fever, in cases with underlying risk factors, including vascular and valvular anomalies [ , ] . in patients with known valvular heart disease, combining doxycycline with hydroxychloroquine has been shown to prevent progression to q fever endocarditis [ , ] . definitive diagnosis usually relies on laboratory testing. while polymerase chain reaction (pcr) may provide timely outcomes, serological assays still are the mainstay of laboratory testing, resulting in diagnostic delay and foregone or inappropriate treatment [ ] . during the dutch epidemic of q fever, general practitioners (gp's) with experience in treating q fever patients tended to start empiric antibiotic therapy ahead of laboratory confirmation, which had a median delay of days from onset of illness in [ , ] . while doxycycline was the most commonly prescribed initial antibiotic, a substantial proportion of subjects were treated with a penicillin, which is considered to be ineffective in q fever [ ] . a complicating factor in the diagnostic workup of cases was the influenza a(h n )pdm pandemic which overlapped with the regional outbreak for several months during the second half of the year. several studies have assessed the diagnostic potential of signs and symptoms in respiratory disease, including influenza and q fever [ ] [ ] [ ] [ ] . however, evidence regarding the predictive usefulness of signs and symptoms in patients with suspected q fever is scarce, and has been limited to hospital settings. a dutch study performed during the - q fever epidemic in the netherlands, for example, found that signs and symptoms did not differentiate between acute q fever and other respiratory infections in hospitalized patients [ ] . however, it is the primary care setting where signs and symptoms of disease are essential in the initial diagnostic workup and in guiding early clinical decision-making. our study, which used data from a cohort of subjects a majority of whom were tested by general practitioners, aimed to assess whether signs and symptoms could support decision-making in primary care. specifically, we assessed whether signs and symptoms could accurately identify acute q fever in suspect cases prior to laboratory confirmation, or help rule out the diagnosis in favour of other respiratory infections where, depending on national guidelines, treatment with amoxicillin as a first-line antibiotic or a delayed or non-prescribing approach would be considered more appropriate. the study area was the catchment area of one of the largest dutch general hospitals, located in south limburg, netherlands ( km , municipalities, , inhabitants). in march , the regional food and consumer product safety authority notified the south limburg phs of a large dairy-goat farm where out of pregnant goats had aborted due to laboratory-confirmed q fever. the study period was defined by the time of veterinary notification (march ) and the time when the outbreak source had been eliminated through culling of infected goats and vaccination of remaining goat populations, and new community cases were no longer reported (april ). we performed a retrospective case-control study assessing the association of acute q fever case status with signs and symptoms of disease in a sample of questionnaire respondents from the cohort of all individuals tested for acute q fever by gp's or hospital-based medical specialists in the period from march through april (n = ). medical specialists requesting tests were from a variety of fields, including internal medicine, infectious disease, or respiratory medicine. all notifiable community cases (n = ) were reported to the regional phs by the affiliated regional testing laboratory. disease onset in community cases was physicianreported. the testing laboratory also provided data on all non-notifiable q fever negative individuals tested in the study period, including date of birth, gender, zip code as a proxy for residential address, name and address of gp, testing dates, and testing results. promptly following notification, all notified community cases were approached with a questionnaire assessing the presence or absence of individual presenting signs and symptoms of disease preceding testing, underlying medical conditions, and risk exposure activities, among others. response in this group was . %( / ). among the subjects who had tested negative (non-notifiable controls), a random selection of individuals were approached with the same questionnaire via their gp's (response: . %( / )). the entire cohort of subjects was tested for igg-and igmtype antibodies to phase-i and phase-ii c. burnetii antigen by serion elisa classic, according to manufacturer's instructions (serion elisa classic, institut virion\serion gmbh, würzburg, germany). elisa-positive specimens were subjected to confirmation by indirect immunofluorescent antibody assay (ifa) (c. burnetii ifa igm/igg test kit, fuller laboratories, fullerton, california). pcr was routinely performed on all elisa-negative samples. the presence of phase-ii igm antibodies to c. burnetii (absorbance > % above extinction of the cut-off control) in a single serum sample, confirmed by ifa, or the presence of c. burnetii dna in pcr (cycle threshold ≤ ) was considered diagnostic of acute q fever [ ] [ ] [ ] . overall, . % ( / ) of all patients tested were confirmed with a diagnosis of acute q fever by serology or pcr. testing activity followed a bimodal distribution over time. a larger first testing wave from march to december (wave ) was followed by a second smaller one from december through april (wave ) (fig. ) . the larger first wave, including subjects tested from week (march ) until week (december ), contained % of all tested patients, with a q fever positive rate of %, thus yielding % of all notifiable patients with a laboratory-confirmed diagnosis of acute q fever in the study period. by contrast, the second wave, although it counted more than a quarter of all tested patients, had a positive rate of only % and identified just % of all notified patients. characteristics of tested subjects are summarized in table . statistical analyses were performed using spss statistics . (ibm corporation, new york, usa). for derivation and validation of our symptom-based prediction, we fig. weekly counts of all individuals tested for q-fever by south limburg gp's and hospital-based medical specialists (n = ), along with weekly counts of notified q-fever cases (by gp-reported week of disease onset, n = ) used questionnaire data from all adult questionnaire recipients with a complete questionnaire response tested during wave , i.e., all questionnaire recipients from the age of years who had been tested in the weeks before week and had answered all questions about presenting signs and symptoms of disease which preceded testing. of all patients tested during wave , ( . %) had received the questionnaire, with response from questionnaire recipients (response rate . %), and a complete response from recipients (complete response rate . %). children and adolescents under the age of were excluded since the association of signs and symptoms with q fever in this age group are known to be less clear-cut than in adults [ , ] . a holdout sample of all subjects tested during wave (i.e., the immediate post-outbreak phase) with a complete questionnaire response was used for additional validation of the models derived from our wave data. of all patients tested during wave , ( . %) had received the questionnaire, with response from questionnaire recipients (response rate . %), and complete response from recipients (complete response rate . %). characteristics of questionnaire respondents are summarized in table . we first assessed association of q fever status with sex, age, smoking habits, test ordered by gp versus (hospital-based) medical specialist, and presence or absence of individual presenting signs and symptoms of disease in all complete questionnaire respondents tested during wave , using univariable logistic regression. for a full list of signs and symptoms assessed by our questionnaire refer to table . as a next step, we assessed associations with q fever status, entering the full set of variables into stepwise backward multivariable logistic regression, a procedure that eliminates statistically non-significant variables along the way. variables that were statistically significantly (p < . ) associated with q fever outcome in univariable or multivariable regression were selected for inclusion in our prediction models (refer to next paragraph). sex, age, active smoking habits, and test ordered by gp versus medical specialist were selected as potential predictors regardless of their association with outcome in univariable or multivariable regression in the steps described above. distance of residential address from the outbreak farm was not included as a candidate predictor, since this information would usually be unavailable to physicians at the time when patients present to their office, or may be unknown altogether in situations where no outbreak source has (yet) been identified. the entire dataset of complete questionnaire respondents tested during wave was randomly split into four subsets, each including roughly % of respondents. one subset was set aside for validation (henceforth referred to as the validation subset), while data of the remaining three subsets combined (including roughly % of the respondents, henceforth referred to as the prediction subset) were used for derivation of our prediction model. to build the prediction model, we used the prediction subset, entering all variables selected according to the procedure described in the previous paragraph into backward stepwise logistic regression. coefficients obtained from the variables that were statistically significantly associated with q fever outcome (p < . ) were used to calculate a sum score. predictive performance of the model was then assessed by applying the score to the validation subset to determine area under the curve (auc) of the receiver operator curve (roc), sensitivity and specificity (based on cut-points specific to the model, calculated according to the youden index), and the model's positive (ppv) and negative predictive value (npv) (based on an estimated regional seroprevalence of . %, derived from comparison of two regional population samples, one pre-outbreak dating from , and the second one post-outbreak dating from ) [ , ] . for additional validation, the same score was applied to the immediate post-outbreak holdout sample from wave , again using auc to assess predictive performance of the model. the entire process was repeated for the remaining three subsets, resulting in four prediction models, each applied once to its specific validation subset from wave , and once to the holdout sample from wave . finally, we compared models in terms of their auc's of the roc, assessing statistical differences between auc's using a bivariate approach [ , ] . uni-and multivariable associations of acute q fever outcome with potential predictors univariable associations of q fever status with sex, age, smoking habits, test ordered by gp versus (hospital-based) medical specialist, and presence or absence of individual presenting signs and symptoms of disease in all complete questionnaire respondents (q fever positive n = , q fever negative n = ) tested during wave , based on univariable logistic regression, are summarized in table . statistically significant multivariable associations for the same set of complete questionnaire respondents are summarized in table , eliminating non-significant associations through backward stepwise logistic regression. prediction models derived from the four prediction subsets (model through ) table shows sets of statistically significant predictors, referred to as model through , derived from backward stepwise logistic regression for the four prediction subsets including roughly % of the respondents each. coefficient, p value, standard error (se), and odds ratio (or) are included for each predictor, in addition to relevant model statistics. predictive performance of model through figure summarizes performance characteristics of the four prediction models, based on each model's coefficient score applied to the corresponding validation subset from wave (left column), and to the holdout sample from wave (right column). auc's ranged from . to . from least to best performing model, generally considered to be poor to moderate in terms of predictive accuracy. sensitivity of the models ranged between . and . %, with specificities between . and . %, ppv's between . and . %, and npv's between . and . %. the difference between model performance in terms of auc was statistically significant between the leastand best-performing model applied to their corresponding validation subsets (model versus model , p = . ), but not between the least-and best-performing model applied to the holdout sample (model versus model , p = . ). comparing performance of each model on the validation subset versus the holdout sample (rows in fig. ) showed no statistically significant differences either. given the poor to moderate performance of our prediction models, our study suggests that signs and symptoms of disease do not accurately predict acute human q fever in gp patients, confirming findings from a dutch study in hospitalized patients [ ] . however, signs and symptoms may be useful in ruling out acute q fever in favour of other acute lower respiratory tract infections. this is especially relevant in cases where pneumonia is not suspected and a non-prescribing or delayed prescribing approach would seem more appropriate, helping reduce the overuse of antibiotics. in the cohort of patients tested in our region, this would have been particularly relevant in the immediate post-outbreak phase where the number of tests for acute human q fever remained high but the proportion of seropositive cases was very low ( %), and prevalence of pneumonia was also low ( %). even during the outbreak phase, only . % of tested individuals were q fever positive, and ruling out acute q fever by symptoms would likely have contributed to a reduction in antibiotic overuse. male sex, fever, and pneumonia were positive predictors of acute q fever across all four of our models, in accordance with other studies [ , ] . cough was a negative predictor in three models, suggesting that cough as a symptom may be useful in ruling out q fever in suspected cases. cough is considered a common symptom in upper respiratory tract infections. its presence, according to our findings, may point to respiratory conditions other than q fever [ ] . specifically, cough has been described as a symptom suggestive of influenza, rather than, for example, common cold [ ] . overall, in our sample cough was the most prevalent symptomsecond only to flu-like illnessin questionnaire respondents from the second wave, both in q fever positive and q fever negative subjects. this, combined with the low rate of q fever positive findings during the second wave, may suggest that the rise in q fever testing activity by gp's and medical specialists during the second half of and the early months of mayat least to some degreehave resulted from patients presenting with respiratory symptoms due to increasing pandemic influenza a(h n )pdm activity in that period. moreover, due to long persistence of anti-coxiella phase ii igm following infection, some of the subjects who tested positive during the second wave may have been misclassified as acute q fever. while abdominal pain was a negative predictor of acute q fever across all four models, gastrointestinal symptoms such as abdominal pain and diarrhoea were much less prevalent than cough in both q fever positive and q fever negative subjects from both waves, and the nature of the observed negative association of abdominal pain with q fever remains unclear. studies on the gastrointestinal symptoms in patients with influenza report prevalence rates ranging from . to . % for influenza a(h n ) infections, and . to . % for influenza a(h n )pdm infections, suggesting a possible association of gastrointestinal symptoms in our study with the swine flu [ ] . use of signs and symptoms of disease to rule out acute q fever would be most appropriate in patients with nonsevere lower respiratory tract infections, i.e., in cases where pneumonia is not suspected clinically. in such cases, use of antibiotics has been shown to provide little benefit in primary care, both overall and in patients aged years and above, but may cause slight harms [ , ] . nevertheless, inappropriate use of antibiotics remains common in this population, as a study performed in , outpatients was recently able to show [ ] . in a subgroup of patients with laboratory-confirmed influenza, in whom no pneumonia had been diagnosed, ( %) were prescribed an antibiotic. given the low yield of q fever positives in wave of our study, we assume that q fever testing during wave was in large part instigated by patients presenting with unspecific, but most likely influenza-related, symptoms. although we have no data on rates of antibiotic prescriptions in this group, the percentage of subjects receiving inappropriate empiric doxycycline or other antibiotics may have been even higher than in aforementioned study. therefore, under circumstances where outbreaks of q fever overlap with other respiratory conditions, symptom-based prediction may deliver the greatest gain in terms of reducing antibiotic overuse. in cases with clinical suspicion of pneumonia, however, the benefit of antibiotics would outweigh potential harms. for instance, several national guidelines recommend doxycycline as a second or first line drug for empiric treatment of community-acquired pneumonia (cap), where it is generally considered to be safe and effective [ ] [ ] [ ] [ ] [ ] . in cases of lower respiratory tract infections where acute q fever is included in the differential diagnosis and pneumonia is suspected, use of doxycycline would thus seem an appropriate choice in an outpatient setting. the combination of doxycycline and hydroxychloroquine should be considered in patients with known valvular heart disease to prevent evolution to q fever endocarditis (but is not recommended in patients with increased risk of acute q fever endocarditis as revealed by high igg anticardiolipin levels included in routine testing in some countries) [ , , ] . local antimicrobial resistance patterns are an important consideration in the choice of empirical treatment. while doxycycline is generally considered to be highly effective against atypical pathogens, including c. burnetii, doxycycline resistance is becoming more common in streptococcus pneumoniae, particularly in isolates with reduced penicillin susceptibility. although overall frequency of doxycycline resistance in s. pneumoniae in was %, rates vary widely geographically and over time, ranging from % to more than %, and more than % in penicillin-resistant strains, potentially limiting the use of doxycycline for more severe pneumococcal infections [ ] [ ] [ ] [ ] . in our study, the prevalence of pneumonia in subjects tested during wave was % overall, but % in q fever positive subjects, which is higher than the % rate found in q fever positive patients from a large -year cohort of patients with q fever from the french national reference center for q fever [ ] . nevertheless, a huge majority of patients in our study had no suspicion of pneumonia and would have had potential benefit from symptom-based exclusion of q fever. predictive values are greatly impacted by prevalence of the disease in the base population. positive predictive values (ppv) tend to be low in situations where prevalence in the base population is low, as was the case in our study, where post-outbreak seroprevalence of prior exposure to c. burnetii in the base population was estimated a mere . % [ ] . with ppv ranging between . and . %, mirrored by low areas under the receiver (see figure on previous page.) fig. predictive performance of the four prediction models tested on their corresponding validation subsets from wave (left column) and the holdout sample from wave (right column). (legend). at cut-point calculated according to youden index based on cut-point calculated according to youden index and an estimated regional prevalence of . % operator curves, our models had no use as a diagnostic tool for acute q fever. conversely, negative predictive values (npv) tend to be high under circumstances of low disease prevalence. with npv ranging between . to . %, our models were able to rule out the presence of acute q fever with a relatively high degree of confidence. nevertheless, decisions favouring a delayed or non-prescribing approach should ideally be corroborated by information from patient history, including self-reported exposures to farms, farm animals and farm animal products, and other clinical findings supporting such approach. in other contexts, for example in a well-circumscribed population of patients with high-risk exposure to a known source, prevalence may be (much) higher, with resulting decline in npv. to the best of our knowledge, ours is the first study to use post-outbreak data to validate prediction models for acute q fever derived from outbreak data, thus enhancing the generalisability and robustness of our findings. moreover, our study is first to assess the predictive potential of signs and symptoms for the diagnosis of acute q fever in a large population of subjects most of whom were primary care patients. other studies attempting to predict q fever by signs and symptoms, including a retrospective case-control study from the netherlands, were performed in hospital settings. the dutch study reported that clinical signs and symptoms were not helpful in differentiating adult hospital-referred patients with acute q fever from a hospital-referred control group [ ] . a second study aimed to predict q fever in patients presenting with community-acquired pneumonia to the hospital. the only symptom independently associated with q fever in this study was headache. the prognostic score derived from multivariable logistic regression included male sex, age - years, a low leukocyte count and a high c-reactive protein (crp) level, along with headache, as predictors of q fever pneumonia [ ] . a third study attempted to predict acute q fever in febrile patients from rural kenya, based on parameters including a range of clinical signs and symptoms. the study identified acute lower respiratory infection, abdominal pain, diarrhoea and a history of fever lasting > days as independent significant positive predictors of acute q fever. a prediction score derived from a modelling approach similar to ours was reported to reliably identify acute q fever in febrile patients with undifferentiated illness [ ] . our study had a number of limitations. selection of subjects for inclusion in our study was based on laboratory q fever testing outcomes rather than random sampling, with a potential for selection bias, e.g., due to variations in diagnostic strategies between individual physicians. laboratory confirmed cases of acute q fever and patients who were q fever negative were both selected based on signs and symptoms leading to addition of q fever in the differential diagnosis, possibly resulting in some weakening of the association under study. our laboratory data were strictly limited to outcomes of q fever testing, precluding us from assessing signs and symptoms in relation to possible alternative outcomes. as mentioned above, misclassification of positive laboratory results as acute q fever infection cannot be entirely ruled out, since phase-ii igm antibodies to c. burnetii, which at the time of the outbreak were considered to be reliable markers of acute q fever infection, have been shown to persist for longer periods in individual patients, thus complicating the differentiation between past q fever infections and acute respiratory infections with different aetiologies [ ] . validation and testing of our models were performed on samples from the same base population, potentially compromising generalisability of our findings. the lack of external validation of our models, however, may have partly been offset by the fact that we performed validation against a holdout sample, i.e., data from the second wave of q fever testing. testing during the second wave took place in what may be described as an immediate post-outbreak transition period where q fever was increasingly replaced by other aetiologies of clinical respiratory disease, thus distinguishing the population of individuals tested during the second wave from those included in the first wave. splitting our first-wave dataset for internal validation may have resulted in loss of power, and may have contributed to discrepancies between our four models in terms of predictors included in each model. nevertheless, all four models showed poor to moderate performance in terms of auc, but performed equally well in terms of their negative predictive value, suggesting that signs and symptoms of disease may be useful for symptom-based exclusion of acute q fever. whereas the youden index is a commonly used method for cut-point selection in roc analysis, there are several other approaches, whose application may have led to different results [ ] . our study suggests that signs and symptoms of disease, considered in combination with age, sex and active smoking habits, do not accurately predict q fever. however, presence of cough and gastrointestinal symptoms may point to different, possibly viral respiratory aetiologies, and help rule out acute fever in the absence suspected pneumonia and fever. in these cases, physicians in primary care may favour a delayed or non-prescribing approach if no known risk factors for progression to persistent focalized (or chronic) q fever (e.g., heart valve or vascular anomalies) are present. a history of exposure to farms, single-point source outbreak with high attack rates and massive numbers of undetected infections across an entire region from q fever to coxiella burnetii infection: a paradigm 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what do we know? amoxicillin for acute lower respiratory tract infection in primary care: subgroup analysis by bacterial and viral aetiology amoxicillin for acute lower-respiratory-tract infection in primary care when pneumonia is not suspected: a -country, randomised, placebo-controlled trial outpatient antibiotic prescribing for acute respiratory infections during influenza seasons bts guidelines for the management of community acquired pneumonia in adults: update diagnosis and treatment of adults with community-acquired pneumonia recommendations and guidelines for the treatment of pneumonia in taiwan british thoracic society community acquired pneumonia guideline and the nice pneumonia guideline: how they fit together nice guidelines to family doctors on diagnosis of pneumonia antiphospholipid antibody syndrome with valvular vegetations in acute q fever antimicrobial susceptibility/ resistance of streptococcus pneumoniae distribution of serotypes and patterns of antimicrobial resistance among commensal streptococcus pneumoniae in nine european countries in: mandell, douglas, and bennett's principles and practice of infectious diseases kucers the use of antibiotics: a clinical review of antibacterial, antifungal, antiparasitic, and antiviral drugs clinical features and complications of coxiella burnetii infections from the french national reference center for q fever evaluation of commonly used serological tests for detection of coxiella burnetii antibodies in well-defined acute and follow-up sera defining an optimal cut-point value in roc analysis: an alternative approach lyophilization to improve the sensitivity of qpcr for bacterial dna detection in serum: the q fever paradigm publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank sander van kuijk, clinical epidemiologist at the department of clinical epidemiology and medical technology assessment at maastricht university / mumc+, for statistical advice, review of our manuscript, and valuable suggestions for improvement. we also thank public health nurses rick boesten, elleke leclercq, and hans frantzen, and communicable disease consultant henriëtte ter waarbeek of phs south limburg for their contribution to data logistics. we wish to thank medical microbiologist frans stals, zuyderland medical centre, for providing laboratory data. farm animals or farm animal products may increase the likelihood of acute q fever. it should be noted that pcr testing, whose sensitivity may be enhanced by lyophilisation, may shorten diagnostic delay and support early decision-making [ ] . we recommend further validation of our findings in different larger independent cohorts. authors' contributions vh conceptualised and designed the study, collected, analysed and interpreted the patient data, and wrote the manuscript. nd and ch were major contributors in designing and conceptualising the study, and in writing the manuscript. all authors read and approved the final manuscript. this work was supported by the netherlands organization for health research and development (zonmw) (grant number - - - ). the funder had no role in no role in the design of the study and collection, analysis, or interpretation of data nor in writing the manuscript. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. this study was ethically approved by the medical ethics committee of the maastricht university medical centre (number ). no administrative permissions were required to access the raw data used in this study. all data were de-identified prior to analysis. not applicable. the authors declare that they have no competing interests.received: march accepted: september key: cord- - m vyxq authors: jayathilaka, p. g. n. s.; mendis, a. s. v.; perera, m. h. m. t. s.; damsiri, h. m. t.; gunaratne, a. v. c.; agampodi, suneth buddhika title: an outbreak of leptospirosis with predominant cardiac involvement: a case series date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: m vyxq background: severe leptospirosis is known to cause multi organ dysfunction including cardiac involvement. in the clinical setting with limited resources, high degree of suspicion is needed to diagnose cardiac involvement including myocarditis. although myocarditis is not reported as a common complication due to lack of diagnostic facilities, there are evidence to support myocarditis is more prevalent in post mortem studies of patients died due to leptospirosis. we present a case series of severe leptospirosis with cardiac involvement observed during a period of one month at colombo-north teaching hospital, sri lanka. case presentation: we report here five patients with severe leptospirosis complicated with cardiac involvement, admitted to a single medical ward, colombo-north teaching hospital, sri lanka during a one-month period. out of six suspected leptospirosis patients admitted during that period, five in a raw developed severe leptospirosis with cardiac involvement. in this case series, four patients were confirmed serologically or quantitative pcr and one patient had possible leptospirosis. all patients developed shock during their course of illness. two patients developed rapid atrial fibrillation. one patient had dynamic t wave changes in ecg and the other two had sinus tachycardia. two patients had evidence of myocarditis in d echocardiogram, whereas other two patients had nonspecific findings and one patient had normal d echocardiogram. all five patients had elevated cardiac troponin i titre and it was normalized with the recovery. all five patients developed acute kidney injury. four patients needed inotropic/vasopressor support to maintain mean arterial pressure and one patient recovered from shock with fluid resuscitation. all patients were recovered from their illness and repeat d echocardiograms after recovery did not show residual complications. one patient had serologically proven dengue co-infection with leptospirosis. conclusions: myocarditis and cardiac involvement in leptospirosis may be overlooked due to non-specific clinical findings and co-existing multi-organ dysfunction. atypical presentation of this case series may be due to micro-geographic variation and unusual outbreak of leptospirosis. co-infection of dengue with leptospirosis should be considered in managing patients especially in endemic areas. leptospirosis is a well-known zoonosis which causes outbreaks particularly in tropical countries. the causative organism is a spirochete of the genus leptospira. history of leptospirosis is likely to extend to ancient times which is evident by chinese texts describing "rice field jaundice" [ ] . in , adolph weil describes a syndrome consists of jaundice, splenomegaly, renal dysfunction, conjunctivitis, and skin rash [ ] and few years later, inada described the causative organism of spirochetosis icterohaemorrhegica [ ] now known as leptospirosis. the classical untreated disease is described as a biphasic illness with initial acute leptospireamic phase followed by immune phase. most cases are self-limited, but some patients develop fatal complications with severe disease. jaundice and renal failure ("weil's disease"), pulmonary hemorrhage, acute respiratory distress syndrome (ards), uveitis, optic neuritis, peripheral neuropathy, myocarditis, and rhabdomyolysis are well known complications [ ] . after the resolution of febrile phase with the clearance of leptospiremia, the immune phase can occur in less than % of patients. however, atypical presentations are reported more frequently in the recent history [ ] . in sri lanka, these differences of clinical presentations has been observed and attributed to micro-geographic changes [ ] . there are more than serovars of leptospira which have been classified in to more than serogroups and the different clinical manifestations are partially attributed to specific serovars. understanding and identifying the varying clinical presentations of leptospirosis mimicking other diseases is important in clinical practice for early treatment and management. in this case series, we describe a series of male patients with severe leptospirosis with cardiac involvement, presented to a single medical ward during a period of one month. here we define cardiac involvement as positivity of at least one of following criteria. they are, ) transient echocardiogram abnormalities during the illness ) elevated troponin i titer which came down with the recovery of illness, ) transient electrocardiogram changes during the illness. we present five patients who were treated for leptospirosis with complications. all are male patients admitted to a single medical ward at north colombo teaching hospital, sri lanka during a one month period starting from to - . data were collected by direct interview of patients, during admission and follow up visits, and from hospital records. fifty-eight years old previously healthy mason admitted to the hospital on / / with fever for three days. fever was associated with chills, rigors, headache, body aches, faintishness, mild cough producing whitish sputum for two days, dysuria, two episodes of loose stool on day of illness, and loss of appetite with poor intake. urine output was normal up to the day of admission. he had a history of cleaning a drainage system one week prior to onset of symptoms. on examination, he was febrile ( °f) and dehydrated. he had low volume pulse with a rate of bpm, blood pressure of / mmhg. examination of other systems were unremarkable except, few basal crepitations in the right lung. inward uss abdomen was performed and there was no free fluid indicative of dengue hemorrhagic fever. initial investigations revealed neutrophil leukocytosis with thrombocytopenia (table ) , high c-reactive protein level (table ) , high serum creatinine with marginally elevated liver transaminases (ast > alt). urine analysis showed microscopic hematuria and ecg showed sinus tachycardia. he was resuscitated with intravenous crystalloids. despite adequate resuscitation he remained in shock and oliguric acute renal failure. after five hours of admission he was started on intravenous noradrenalin infusion and later dobutamin also added to the therapy (table ) . clinical diagnosis was made as leptospirosis and intravenous cefotaxime was started in the meanwhile. urine output was improved with the rise of mean arterial pressure. but patient was dependent on ionotrope and vasopressor. d echocardiogram showed mild global hypokinesia with ejection fraction - % and concluded as possible myocarditis. troponin i titre became positive. on day of illness, patient developed rapid atrial fibrillation with shock requiring electrical cardioversion to achieve sinus rhythm. by day five of illness, he became heamodyanemically stable without inotropic/ vasopressor support. during the recovery, he developed asymptomatic hypokalemia and potassium was replaced. by day eleven of illness he was completely recovered clinically and full blood count, liver function tests, renal function tests and ecg were normal. c-reactive protein and troponin i titer were coming down and patient was discharged. after three weeks of illness, d echocardiogram was performed and it was completely normal. leptospira was detected in qpcr (quantitative polymerase chain reaction) performed on day five of illness and leptospirosis antibody test on day seven of illness (mat) was positive. (titre- : ) his urine and blood cultures, dengue antigen were negative. a years old previously healthy male, a retired clerk presented to the medical casualty with a history of fever for three days. it was associated with arthralgia, myalgia, headache and loss of appetite. he did not have respiratory, urinary symptoms and bowel habits were normal. he denied any history of exposure to leptospirosis or contact history of fever. on admission, his general examination was normal with a heart rate of bpm and blood pressure of / mmhg. other system examination was unremarkable. after admission it was noted that his urine output is low while he was on maintenance fluid. initial investigations revealed neutrophilia with normal white blood cell count, thrombocytopenia, elevated blood urea, serum creatinine, c-reactive protein and ast. urine analysis showed - pus cells, - red cells with granular casts. clinical diagnosis of leptospirosis was made on high index of suspicion although there was no significant history of exposure to leptospirosis. patient was started on intravenous cefotaxime. by the day five of illness, he developed confusion (gcs- / ), low blood pressure ( / mmhg) with tachycardia ( bpm), high fever spike ( f), and mild dyspnea with spo % on air. ecg showed sinus tachycardia, non-contrast ct brain was normal, d echocardiogram revealed ejection fraction of > %, chest x ray-pa was normal, and troponin i titer was marginally positive. ultrasound abdomen showed renal parenchymal changes with normal sized kidneys. serum creatinine was rising. patient was started on inotropic and vasopressor support to maintain blood pressure. even after achieving mean arterial pressure > mmhg patient went in to anuric acute renal failure. meanwhile he developed rapid atrial fibrillation which was settled with electrical cardioversion. he was given hemodialysis on day of illness. on day of illness again patient developed rapid atrial fibrillation and it did not respond to electrical cardioversion and started on iv amiodarone infusion and patient regained sinus rhythm and could tail off inotrope and vasopressor. since day , he gradually improved clinically with good urine output, hemodynamic stability and confusion settled. but he did not recover from acute kidney injury and renal functions remained rising again. he was given another hemodialysis on day of illness. then his renal functions slowly improved and discharged on day of illness with a follow up plan at nephrology clinic. on discharge patient had normal platelet count, c-reactive protein, liver transaminases, ecg. serum creatinine was static around micromol/l. repeat d echocardiogram which was done three weeks after recovery was normal. leptospirosis antibody titre (mat) on day of illness was positive. ( : ). a year old male patient presented with fever for two days. fever was associated with chills, rigors, arthralgia, myalgia, frontal headache, faintishness, lower back pain, loss of appetite, vomiting, loose stool - times/day for two days. patient denied a significant exposure to leptospirosis. there was no contact history of fever. he was a manual worker. on admission he was ill looking, febrile ultrasound scan of abdomen showed acute renal parenchymal changes and there was no evidence of free fluid in the abdomen. initial investigations revealed neutrophil leukocytosis with thrombocytopenia, high c-reactive protein ( mg/l), high blood urea ( mg/dl) and serum creatinine ( micromol/l), marginally elevated liver transaminases (ast > alt), microscopic hematuria, ecg showed sinus tachycardia with mild t inversions in v -v . chest x ray was normal. possible diagnosis of leptospirosis was made on clinical grounds and he was started on intravenous cefotaxime. his blood pressure was improved after fluid resuscitation and he had good urine output. his d echocardiogram was normal, but his troponin titer increased and then came down. patient was discharged from the ward on day of illness with complete recovery and normal full blood count, renal and liver function tests. crp and trop i titer was coming down. d echocardiogram which was performed after three weeks of recovery was normal. his dengue antigen test, blood and urine cultures were negative. the leptospirosis qpcr test performed on day three of the illness was reported as not detected though one out of triplicate samples was positive. patient was clinically diagnosed as a "possible" case of leptospirosis. a year old male laborer presented with fever for four days duration. he was previously diagnosed to have diabetes mellitus, but he was not taking treatments. fever was associated with arthralgia, myalgia, headache, lower back pain, dysuria and reduced urine output for two days, cough for one week producing scanty amount of whitish sputum. he had a history of muddy contact within one week prior to symptom onset. on admission, patient was febrile (temp- f), ill looking, mildly dehydrated and had conjunctival suffusion. his pulse rate was bpm with blood pressure of / mmhg. other system examination was unremarkable. initial laboratory work up showed neutrophilia with normal white cell count, thrombocytopenia, high c-reactive protein ( mg/l), high serum creatinine ( micromol/l) and normal liver transaminases. ecg showed sinus tachycardia and chest x ray-pa was normal. depending on clinical grounds, diagnosis was made as leptospirosis and started on intravenous cefotaxime while fluid resuscitation is being carried out. despite adequate fluid resuscitation patient developed shock with low urine output on the same day of admission. (day of illness-pulse rate- bpm, bp- / ) then vasopressor support was given and small dose of frusemide infusion was started after achieving normal blood pressure with noradrenalin. d echocardiogram was performed on d of illness and it showed mild global hypokinesia with ejection fraction - %, dilated left ventricle with concentric left ventricular hypertrophy and concluded as hypertensive heart disease with or without myocarditis. cardiac troponin i titre became positive and had rising titre when repeated and then came down by the time of recovery. us scan of abdomen revealed bilateral renal parenchymal changes with normal sized kidneys. noradrenalin was tailed off within h and urine output was improved with maintenance fluid therapy. patient had rising serum creatinine till day of illness and then started to come down. serum electrolytes were normal throughout and there was no acidosis. patient was improved dramatically and was discharged from the hospital by day of illness. on discharge he had rising platelet count, normal serum creatinine and dropping troponin i titre and crp. d echocardiogram was repeated after weeks of discharge and his ejection fraction was improved to % and there was mild left ventricular hypertrophy with grade i diastolic dysfunction. his diabetes was controlled with soluble insulin during acute illness and changed to oral hypoglycemic treatment with the recovery. leptospirosis antibody titre (mat) done on day of illness was positive ( : ). a years old male patient presented with fever for days. it was high fever associated with arthralgia, myalgia and mild difficulty in breathing. he also complained of reduced urine output and loose stool (two episodes) for one day. there were no other respiratory or urinary symptoms. he denied a significant exposure to leptospirosis. he had a past history of hypertension for which he was not taking treatment and past history of renal calculi for which he has undergone surgery several years back. on admission he was ill looking, febrile (temp- f), and anicteric. pulse rate was bpm and blood pressure / mmhg. other system examination was unremarkable. initial investigations revealed marked thrombocytopenia, neutrophilia with low normal white blood cell count, high c-reactive protein ( mg/l), high serum creatinine ( micromol/l), elevated liver transaminases (ast > alt), urine analysis showed pus cells - , red cells - and albumin + (urine culture became negative). chest x ray-pa was normal. possibility of dengue fever could not be excluded with his full blood count and clinical presentation, but all other initial investigations were supportive towards leptospirosis although there was no history of significant exposure to leptospirosis. on admission ultrasound scan of the abdomen was performed inward and there was no evidence of fluid leakage. therefore, patient was started on intravenous cefotaxime in addition to hydration with maintenance fluid. patient had low urine output and went in to shock (pr- , bp- / mmhg) despite of adequate fluid resuscitation (on day of illness). he was started on iv noradrenalin to maintain blood pressure. ultrasound scan of the abdomen revealed right side scarred kidney with left side renal parenchymal changes with normal size kidney. there was no evidence of leaking by the time of developing shock. d echocardiogram showed severe mitral regurgitation with and there was no evidence of myocarditis. troponin i titer became marginally positive and later came down. ecg showed sinus tachycardia. histological diagnosis or cardiac mri to diagnose cardiac involvement was not accessible due to lack of resources in the hospital. noradrenalin could be tailed off within h. (on day of illness). by day five of illness urine output was gradually improved but serum creatinine remained rising with normal serum electrolytes. dengue ns antigen was negative, but igm and igg antibodies were positive with dropping platelet count and white cell count (neutrophilia persisted). dengue pre-critical monitoring was continued while giving maintenance fluid therapy. daily ultrasound scans were performed to exclude fluid leakage. patient remained hemodynamically stable and platelet and white cell count started to increase by day of illness and serum creatinine started to come down by day of illness. he was discharged from the hospital on day of illness with a plan to be followed up in nephrology clinic for possible chronic kidney disease. d echocardiogram was repeated after three weeks of recovery and it was normal other than trivial mitral regurgitation. leptospirosis antibody titer done on day of illness was positive. ( : ). severe leptospirosis is characterized by multiple organ dysfunction including liver, kidney, lungs and brain. it is also known to cause cardiac involvement as well. cardiac manifestations range from non-specific electrocardiographic changes and arrhythmias to myocarditis, pericarditis, endocarditis and cardiogenic shock [ ] [ ] [ ] . but the pathophysiology behind it is less well understood and the magnitude of the problem is under-reported [ ] . all five patients included in this case series had evidence of acute kidney injury. the most striking feature of these five patients admitted to a single unit within a month was cardiac involvement. all five patients developed shock with low blood pressure during their course of illness. except case number , all other patients needed vasopressor/inotropic support to maintain blood pressure. case number showed evidence of myocarditis in d echocardiogram at the time of shock. case number had possible evidence of myocarditis whereas case number , had normal echo findings. case number had severe mitral regurgitation in his d echocardiogram. all these echocardiogram were performed while the patients were in shock. repeat d echocardiograms performed after three weeks of recovery were completely normal except in case number and . number had mild left ventricular hypertrophy with grade diastolic dysfunction and number had trivial mitral regurgitation. in addition to these various echo findings all of these five patients had more or less positive cardiac troponin i titre which came down with the recovery of illness. case number one and two developed atrial fibrillation which needed intervention for normalization. case number three had mild t wave inversions in anterior leads which was dynamic in serial electrocardiograms. case number and had only sinus tachycardia. all five patients had shock by definition and the most probable explanation is cardiogenic shock due to cardiac involvement of leptospirosis. though not commonly reported, myocarditis in severe leptospirosis may not be a rare complication. the european society of cardiology working group on myocardial and pericardial diseases has developed clinical and diagnostic criteria, when present myocarditis should be suspected. presence of unexplained cardiogenic shock, positive cardiac troponins, variable ecg changes are included for these criteria in addition to several other criteria [ ] . definitive diagnosis of myocarditis ideally should be established by histopathological, immunological and immunohistochemical criteria for which myocardial biopsy is required. this is not practical in most settings as these investigations are not routinely done and not required for patient management. in this case series none of the patients underwent histopathological or cardiac mri diagnosis of cardiac involvement due to lack of resources in the hospital. due to wide variability in presentation and non-specific clinical findings, many cases of myocarditis likely to go undetected. as an example, study conducted examining hearts from patients who had died due to leptospirosis has revealed myocarditis in % of cases histologically. endocardial inflammation had been observed in % of cases [ ] . in sri lanka, myocarditis has been reported previously as a complication of leptospirosis [ , ] and around - % of confirmed cases are being reported as having this complication [ , , ] . however, in most of the previous studies, the details of diagnosis of myocarditis was not clearly given. in our case series, histological diagnosis or cardiac mri to diagnose cardiac involvement was not possible due to lack of resources in the hospital. there is another phenomena coming up in the recent literature to explain the shock in leptospirosis. according to julie cagliero et al. dys-regulation of inflammatory mechanisms in severe leptospirosis can lead to cytokine storm causing sepsis like picture [ ] . systemic inflammatory response syndrome (sirs) is supposed to occur in severe leptospirosis [ ] . sirs itself can cause elevated cardiac troponins [ , ] . therefore, pure cardiac involvement in leptospirosis becomes more difficult to diagnose. all these five patients presented during one-month period in a raw and we had only six total suspected (notified) cases of leptospirosis during that month. observing cardiac involvement in five out of six probable cases of leptospirosis may be due to an outbreak caused by a different strain of a leptospira. as previously observed, outbreaks of leptospirosis with uncommon complications such as pancreatitis [ ] needs more investigations and explanations. however these patients did not have evidence of pulmonary involvement which is a known complication to occur in severe leptospirosis. case number patient had serological evidence of leptospirosis and co-infection with dengue virus. co-infection of leptospirosis and dengue is a known phenomenon in endemic countries with subtropical and tropical climates. a study conducted in malaysia has concluded that there is a considerable prevalence of leptospirosis and dengue co-infection with overlapping demographic, clinical and laboratory presentations [ ] . in sri lanka [ ] as well as in many other places [ ] [ ] [ ] , a co-infection of these two had been reported earlier and possible due to high endemicity of both diseases. it is crucial to consider co-infection with dengue where clinical suspicion arise even in the presence of enough supportive evidence for leptospirosis. because close monitoring and fluid management are the lifesaving principles of management of dengue hemorrhagic fever which must be done timely. developing severe leptospirosis in five out of six cases during same period may be due to outbreak of uncommon strain of leptospirosis. cardiac manifestations of leptospirosis are possibly under-diagnosed due to co-existence with other multi-organ involvement. diagnosis of myocarditis is difficult due to lack of imaging facilities, lack of specificity of available tests as well as unavailability of non-invasive gold standard diagnostic test. to assess the significance of cardiac troponins in diagnosing cardiac involvement in leptospirosis further studies are required. co-infection of dengue in a patient with leptospirosis should be considered especially in endemic areas. Über eine eigenthümliche mit milztumor, icterus und nephritis einhergehende acute infectionskrankheit areport on the discovery of the causative organism (a new spesies of spirochete) of weil's disease. tokyo ijishinshi leptospirosis in humans severe leptospirosis and pancreatitis; a case series from a leptospirosis outbreak in anuradhapura district, sri lanka regional differences of leptospirosis in sri lanka: observations from a flood-associated outbreak in cardiac manifestations in leptospirosis. apropos of cases observed in new caledonia cardiac involvement in severe leptospirosis cardiac and pulmonary involvement in leptospirosis current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases cardiac findings in leptospirosis myocarditis causing severe heart failure -an unusual early manifestation of leptospirosis: a case report co-existent facial palsy and myocarditis in a -year old farmer diagnosed with probable leptospirosis: a case report predictors of the development of myocarditis or acute renal failure in patients with leptospirosis: an observational study leptospirosis outbreak in sri lanka in : lessons for assessing the global burden of disease demographic, clinical and laboratory features of leptospirosis and dengue co-infection in malaysia fatal co-infection with leptospirosis and dengue in a sri lankan male fatal leptospira spp./zika virus coinfection-puerto rico sero-epidemiology study of leptospirosis in febrile patients from terai region of nepal clinical predictors of dengue fever co-infected with leptospirosis among patients admitted for dengue fever -a pilot study we acknowledge the staff of ward of colombo-north teaching hospital, ragama in making this study a success.funding sba is supported through u.s. public health service grants u ai . the funders have played no role in the research.availability of data and materials all data contained within the article.authors' contributions nj perceived the study and prepared the first draft of the manuscript. nj, asvm, mhmtsp, hmtd, avcg provided patient care, followed up the patients, collected and interpreted clinical data. sba involved in design, analysis, interpretation of data and preparing the manuscript. all authors contributed, read and approved the final manuscript.ethics approval and consent to participate not applicable. written informed consent was obtained from all patients for publication of their individual details. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -fvrd w authors: herath, h. m. l. y.; jayasundara, j. m. h. d.; senadhira, s. d. n.; kularatne, s. a. m.; kularatne, w. k. s. title: spotted fever rickettsioses causing myocarditis and ards: a case from sri lanka date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: fvrd w background: spotted fever group of rickettsial infections are emerging in sri lanka. we describe a patient with rapidly progressing ards and myocarditis secondary to spotted fever caused by rickettsia conorii. ards and myocarditis are rare complications of rickettsia conorii infections and only a few cases are reported to date. case presentation: a years old manual worker presented with fever for days and a skin rash. he was in circulatory failure on admission and developed severe hypoxaemia with gross changes in chest radiograph by next day requiring assisted ventilation. he had myocarditis causing left ventricular failure and acute respiratory distress syndrome. he was confirmed to have spotted fever rickettsial infection with rising titre of indirect immunofluorescence antibodies to ricketssia conorii and made a complete recovery with appropriate antibiotic therapy and supportive care. conclusion: rickettsial infections can present with diverse manifestations. even the patients with severe organ involvements such as myocarditis and ards can be completely cured if timely identified and treated. emergence of spotted fever group of rickettsial infections in the hilly central province of sri lanka was first observed in early nineties [ ] . rickettsia conorii, the organism known to cause mediterranean spotted fever (msf) is the most prevalent organism causing spotted fever in sri lanka. few serologically confirmed cases of rickettsia honei and rickettsia japonica has also been reported [ , ] . usual presentation of spotted fever is with a prodrome of high grade fever, headache myalgia, arthralgia and anorexia. less common manifestations include frank arthritis, cough, abdominal pain, conjunctival injection and diarrhea [ ] . various neurological manifestations including confusion, hallucinations, tinnitus, hearing impairment and rarely coma are also seen [ ] . the characteristic skin rash is present only in about % of patients. typically, the rash is maculopapular with predominant involvement of limbs including palms and soles. in severe cases fern leaf type skin necrosis can occur. the typical eschar is rare to be found and often the patients are unaware of of tick bites [ , ] . on rare occasions patients present with fever and multiple organ dysfunction making it difficult for the clinician to find the exact diagnosis since many tropical diseases can cause a similar picture. indirect immunofluorescent antibody assay (ifa) is the reference serology method for the diagnosis. it is available in only a few laboratories in sri lanka. limited availability of ifa has led to underreporting of the cases with rickettsial infections. msf is usually a mild disease with a mortality rate around . %. elderly patients are prone to get more complications [ ] . mortality data regarding sri lankan patients are not available, except few fatal case reports. the following case report highlights myocarditis and acute respiratory distress syndrome (ards) as complications in a severely ill patient with spotted fever group of rickettsioses where timely diagnosis and intervention saved the life. a -year-old male was transferred from peripheral hospital hatharaliyadda (phh) to teaching hospital, kandy (thk) in a state of circulatory failure for specialized care. he was a previously well 'tree cutter' working close to his residence situated in a hilly terrain in the northern slope of central hills of sri lanka where rich lust green vegetations and tropical trees are in abundance. his routine was to cut trees in the tea estates in the area and to carry the logs to the closest motorable road. he developed fever with myalgia and headache days prior to the admission to phh. on the th day of fever he had noticed a rash over his body. as his condition deteriorated on the th day of the illness, he was transferred to thk. on admission, he was febrile and recorded temperature was °f. he had a generalized discrete erythematous macular rash in most areas of the body including palms and soles. also he had swelling of both ankle joints. he denied any tick bite prior to illness. there was no eschar found. he had neither lymphadenopathy nor splenomegaly. but the liver was palpable cm below costal margin. lungs were clear to auscultation. (fig. ) he had a thready pulse of /min with a blood pressure of / mmhg. he was initially resuscitated with intravenous normal saline and managed as septic shock. the presence of skin rash prompted to consider a spotted fever rickettsial infection. therefore, he was started on intravenous ceftriaxone and oral doxycycline. since his blood pressure did not improve with fluid resuscitation a central line was placed and intravenous norepinephrine infusion at a rate of . μg/kg/min was commenced along with septic dose of intravenous hydrocortisone mg/ hourly. his plasma random glucose was normal. initial electrocardiogram did not show st/t wave abnormalities and chest radiograph was normal. his serial investigations during the hospital stay are shown in table . with inotropic support, antibiotics and maintenance fluids he remained stable for the next h. his blood cultures, urine cultures and retroviral studies were negative. during the latter part of the second day of admission to thk he developed progressively worsening shortness of breath with hypoxemia and hypotension. blood gas analysis showed type respiratory failure with po /fio ratio of . . repeat chest radiograph showed bilateral alveolar and interstitial shadowing of both upper and mid zones. ecg revealed sinus tachycardia with no significant st/t wave changes. d echo cardiogram showed a ventricular ejection fraction of - % with global hypokinesia of myocardium suggestive of myocarditis. troponin-i titre was positive at . ng/ml (normal < . ng/ml) and ntprobnp (n-terminal pro b-type natriuretic peptide) value was elevated at pg/ml. at this juncture, elective intubation was done and the patient was transferred to the intensive care unit for assisted ventilation. (fig. ) . the management team identified some issues with regard to diagnosis and choosing the appropriate treatment for the patient. echocardiographic findings, elevated troponin titre and elevated bnp were consistent with myocarditis leading to heart failure causing pulmonary oedema and hypoxemia. the possibility of ards was considered based on clinical, blood gas and radiological evidence. spotted fever was considered to be the most likely diagnosis because of the presence of the typical rash. other differential diagnoses included leptospirosis with myocarditis and pneumonitis. but the rash and lack of liver and renal involvement was not in favor fig. skin rash. skin rash of the patient at the day of presentation. it was erythematous macular rash involving palms and soles with mild pedal edema of leptospirosis. streptococcal and staphylococcal toxic shock syndromes and meningococcal sepsis were also taken into consideration, but inability to fulfill diagnostic criteria and persistently negative cultures were against them. hemophagocytic lymphohistiocytosis (hlh) is also known to complicate many zoonoses including spotter fever infections [ , ] . however, absence of cytopenias and the splenomegaly made it less likely and further screening tests for hlh were not performed. considering the poor response to previous antibiotics, intravenous chloramphenicol mg hourly was added to the treatment regimen as authors personally had seen good response particularly in patients with severe disease. he required inotropes in increasing doses, including norepinephrine . μg/kg/min, dobutamine μg/kg/min and dopamine μg/kg min, to maintain the blood pressure for the next h. in view of myocarditis, iv hydrocortisone dose was increased to mg/ hourly. within h from starting chloramphenicol and increasing the dose of steroids, his clinical parameters started to improve. by next day tailing off of the inotropic support was possible. on the rd day in the intensive care unit, he was extubated and on the th day he was transferred back to the high dependency unit of the medical ward. the skin rash started to fade leaving few necrotic areas over the hands. all three antibiotics were stopped after the completion of days. he did not develop any treatment related complications. he was in two weeks' time, he was reviewed in the out-patient clinic and found to be completely asymptomatic. for the confirmation of diagnosis, st acute blood sample was tested for r. conorii indirect immunofluorescence antibodies (ifa) at the reference laboratory of university of peradeniya which showed moderately high positive titre ( / ). ten days later, nd blood sample was tested with ifa which showed rising titre ( / ) confirming the diagnosis. further species identification was not possible due to unavailability of facilities. unfortunately patient did not turn up for the follow up echocardiogram. we presented a middle aged man, a tree cutter in profession falling ill with fever, then collapsed on th day of illness due to myocarditis and became hypoxic due to ards. timely diagnosis of spotted fever and initiation of appropriate treatment saved his life. even though he denied a tick bite, it was likely that he had an unnoticed tick bite as his occupation carried a high risk of exposure. this case demonstrates a rather rare presentation of spotted fever rickettsial infection where patient deteriorated within short time leading to shock and ards. the development of myocarditis was rapid and severe enough to cause low left ventricular ejection fraction and hypotension. patients with myocarditis and ards are described in the literature in other types of rickettsial infections particularly with scrub typhus. myocarditis has been observed as an autopsy finding in fatal cases of rocky mountain spotted fever. other much rarer forms of tick borne rickettsial infections like sibirica mongolitimonae infections are also known to cause clinically significant myopericarditis [ ] . r. conorii related cardiac involvement is extremely rare and only about cases are described in the literature [ ] . severe forms of spotted fever rickettsioses is also known to be caused by some subspecies such as rickettsia conorii subsp. israelensis [ ] . unfortunately molecular diagnostic methods for identifications of subspecies is not yet available in sri lanka. myocarditis is caused mainly by viruses and also by leptospira spirochetes and toxins. in sri lanka myocarditis is an occasional complication in dengue infection and leptospirosis [ ] . it is not a diagnosis entertained in rickettsial infections despite its high prevalence. in myocarditis, patients usually develop undue tiredness, chest discomfort and dyspnoea which may progress to cardiogenic shock or development of arrhythmias. most often it is diagnosed clinically with ecg abnormalities such as t wave inversions, bundle branch blocks and presence or rhythm abnormalities. echocardiography and elevation of cardiac biomarkers can be used to diagnose myocarditis but these become evident mostly in severe cases. in the background of sepsis, transient cardiac dysfunction can occur due to sepsis induced cardiomyopathy. differentiation between the two diagnoses can only be achieved by endomyocardial biopsy. however, in acutely ill patients it is not justifiable do biopsy as it does not alter the management. further, in the available literature suggests that rickettsial infections related cardiac dysfunction is more likely to be due to myocarditis [ , , ] . newer methods including cardiac mri and segmented inversion recovery gradient-echocardiography pulse sequences have a better sensitivity in diagnosing acute myocarditis [ , ] . our patient had compatible symptoms and signs with global hypokinesia in d echocardiogram and elevated troponin and ntprobnp values to suggest the presence of myocarditis. differentiation between severe pulmonary oedema and ards is difficult both clinically and radiologically at the onset of the illness. but presence of prolonged severe hypoxia and persistent alveolar-interstitial shadows despite treatment with intravenous diuretics was more in favor of ards in our patient than pure pulmonary oedema. management of myocarditis and acute heart failure follow standard guidelines with diuretics, angiotensin converting enzyme inhibitors and beta blockers. place of steroid in acute myocarditis is debatable but it is commonly used by clinicians on empirical evidence and personal experience. european guideline on management of myocarditis recommends immunosuppression only in chronic virus negative myocarditis and inflammatory and autoimmune myocarditis [ ] . none of the reported patients with msf and myocarditis had received steroids. however, we believe that steroids helped in treating our patient. oral doxycycline is the recommended antibiotics for rickettsial infections [ ] . however, chloramphenicol which is a second line agent, is also widely used in many institutions in sri lanka. there are not many studies comparing the efficacy of chloramphenicol with other agents due to risk of major hematological adverse effects. in fact, cdc case report data suggest that patients with rocky mountain spotted fever treated with chloramphenicol are at higher risk for death than persons who received a tetracycline [ ] . in contrast to that our experience suggests that in severe spotted fever rickettsial infections, parenteral chloramphenicol can be used safely with good results like in this patient. out of the reported cases of msf with myocarditis, chloramphenicol was included in treatment regimens of two patients [ ] . limitations of the our report includes the unavailability of histological evidence of myocarditis and not identifying the species due to lack of resources. finally, this case highlights the need of prompt clinical diagnosis and treatment of spotted fever which can present with atypical features. emerging rickettsial infections in sri lanka: the pattern in the hilly central province seroepidemiology of rickettsioses in sri lanka: a patient based study a case series of spotted fever rickettsiosis with neurological manifestations in sri lanka cutaneous manifestations of spotted fever rickettsial infections in the central province of sri lanka: a descriptive study mediterranean spotted fever in spain, - : epidemiological situation based on hospitalization records secondary hemophagocytic lymphohistiocytosis in zoonoses. a systematic review haemophagocytic syndrome and rickettsial diseases acute myopericarditis associated with tickborne rickettsia sibirica mongolitimonae israeli spotted fever in sicily. description of two cases and minireview cardiac complications of a dengue fever outbreak in sri lanka cardiac involvement in a patient with clinical and serological evidence of african tick-bite fever diagnosis and treatment of viral myocarditis current state of knowledge on aetiology, diagnosis, management, and therapy of myocarditis: a position statement of the european society of cardiology working group on myocardial and pericardial diseases diagnosis and management of tickborne rickettsial diseases: rocky mountain spotted fever and other spotted fever group rickettsioses, ehrlichioses, and anaplasmosis -united states analysis of risk factors for fatal rocky mountain spotted fever: evidence for superiority of tetracyclines for therapy not applicable. none.availability of data and materials data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. all data contained within the article. abbreviations ards: acute respiratory distress syndrome; msf: mediterranean spotted fever; ntprobnp: n-terminal pro b-type natriuretic peptide; phh: peripheral hospital hataraliyadda; thk: teaching hospital kandy authors' contributions hmlyh, jmhdj, sdns managed the patient and did the literature review, gathering of data and writing of the initial manuscript. samk, wksk finalized the manuscript and gave expert opinion in management issues. all authors read and approved the final manuscript.ethics approval and consent to participate not applicable. informed written consent for the publication of details and pictures was obtained from the patient. consent form can be made available to the editor on request. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -ztc holk authors: hsieh, ying-hen; cheng, kuang-fu; wu, trong-neng; li, tsai-chung; chen, chiu-ying; chen, jin-hua; lin, mei-hui title: transmissibility and temporal changes of ph n pandemic during summer and fall/winter waves date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ztc holk background: in order to compare the transmissibility of the ph n pandemic during successive waves of infections in summer and fall/winter in the northern hemisphere, and to assess the temporal changes during the course of the outbreak in relation to the intervention measures implemented, we analyze the epidemiological patterns of the epidemic in taiwan during july -march . methods: we utilize the multi-phase richards model to fit the weekly cumulative ph n epidemiological data (numbers of confirmed cases and hospitalizations) as well as the daily number of classes suspended under a unique " " partial school closing policy in taiwan, in order to pinpoint the turning points of the summer and fall/winter waves, and to estimate the reproduction numbers r for each wave. results: our analysis indicates that the summer wave had slowed down by early september when schools reopened for fall. however, a second fall/winter wave began in late september, approximately weeks after the school reopened, peaking at about - weeks after the start of the mass immunization campaign in november. r is estimated to be in the range of . - . for the first wave, and between . - . for the second wave. conclusions: transmissibility of the summer wave in taiwan during july-early september, as measured by r, was lower than that of the earlier spring outbreak in north america and europe, as well as that of the winter outbreak in southern hemisphere. furthermore, transmissibility during fall/winter in taiwan was noticeably lower than that of the summer, which is attributable to population-level immunity acquired from the earlier summer wave and also to the intervention measures that were implemented prior to and during the fall/winter wave. although the first known imported case of pandemic influenza (ph n ) arrived in taiwan on may from the u.s. via hong kong, serological evidence has indicated that the ph n virus had spread to central taiwan by april-june [ ] . local infections and laboratory-confirmed ph n cases in taiwan started to mount in significant numbers in july-august when the schools were in summer recess. by the time the schools reopened in september, multiple intervention measures had been implemented by the government, which include strict border temperature screening starting in may, a " " class suspension policy [ , ] implemented in september, and later a mass immunization program [ ] [ ] [ ] starting in november. the number of cases began to decline by the end of the year, and continued to do so into early next year, until the government announced on february the end of the fall/winter outbreak [ ] with over laboratory-confirmed cases reported, hospitalizations, and deaths [ ] . although school closing was a widely used method of intervention around the world during the ph n outbreak (see, e.g., [ ] [ ] [ ] [ ] [ ] [ ] ), its suitability, timing, and the manner of implementation remains controversial. when k- schools (kindergarten through high schools) reopened on august in taiwan, the government implemented a unique partial school closing policy called the " " class suspension policy aimed toward kindergarten through secondary schools (k- ), cram schools, and after-school institutions. under this policy, if within any three ( ) consecutive school days, two ( ) or more students in the same class are diagnosed with influenza, then that class will be suspended for the next five ( ) days including weekends and holidays [ , ] . the policy was designed to minimize the potential social impact of full-scale school closings in the event of a major influenza outbreak in the community; to detect cluster infections in school settings early and swiftly; and to contain the infections locally without disruption for the other students in the school. at the height of the class suspensions in late november, more than classes with more than , students from almost schools in taiwan were suspended on a single school day (figure ), yet without any visible disruption in the normal functioning of the society. moreover, starting november , a mass immunization program was initiated in taiwan sequentially, according to a priority list of target groups [ ] , with healthcare and public health personnel having the highest priority [ ] . subsequently, preschool children were immunized starting on november ; and followed by pregnant women, k- schoolchildren, and people with major illness/injury being vaccinated starting on november ; - year-olds on november ; and the general population on december . by march , a total of . million doses of adimflu-s (unadjuvanted h n v from adimmune) or focetria ® (mf ® adjuvanted h n v from novartis) were administered, and more than million of the million taiwanese had been immunized [ ] . children and under were advised to receive two doses of vaccine, although many of them eventually received only one dose due to various reasons. a simple mathematical model, the richards model, is utilized to fit publicly accessible cumulative epidemic data in order to obtain estimates for the turning points (the peaks and volleys of the incidence curve) and the reproduction number r of a particular wave of infections. examples of applications of the richards model to infectious diseases include those of sars [ , ] , dengue [ , ] , and the ph n epidemic [ , ] . in this study, we will make use of the richards model to pinpoint the turning points of each wave of the epidemic, in order to ascertain the temporal changes of the epidemic in taiwan in the summer months and during the fall and winter days. the transmissibility of the ph n virus during the outbreak is determined through its reproduction number. the data was accessed from the central epidemic command center website of the taiwan centers for disease control (tcdc). samples were collected from hospitals and clinics participating in the taiwan influenza surveillance system under the taiwan national influenza center (taiwan nic), which was established in to integrate all existing efforts of influenza surveillance and notification with laboratory analysis systems throughout taiwan in order to enhance the epidemic data collection capacity in taiwan [ ] . the weekly laboratory confirmed ph n case data (by the week when the samples were collected and sent to the tcdc-contracted laboratories) and the weekly hospitalization data (by the week the lab-confirmed cases were hospitalized) from june , (epidemiological week or e-week of ) to march , (e-week of ) was accessed from the weekly influenza express made publicly available on the internet by the tcdc [ ] during the epidemic. the surveillance protocols in taiwan remained essentially the same throughout the data period since, by the time the data were collected, clinical characteristics of the ph n infection had already been well understood from the spring outbreaks around the world. we also accessed the daily record of numbers of classes suspended and number of schools with at least one class suspended during the fall school semester (september , to january , ) from the tcdc daily ph n updates [ ] during the epidemic. the time series of class suspension data is given in figure . since this data is for school days only, the days are specified in the horizontal axis of figure in weekly increments of school days, except for weeks with less than school days at the beginning and the end of the school semester as well as the week containing the new year holiday (january ). the richards model [ ] is of the form: where the prime symbol "'" denotes the rate of change over time which is in eweeks. c(t) is the cumulative number of cases at time t (in weeks), k is the cumulative case number over a single wave or phase of outbreak, r is the per capita growth rate of the infected population, and a is the exponent of deviation. the explicit solution of the equation is here the parameter t m is related to the turning point t i of a wave (or the inflection point of the cumulative case curve) by the simple formula t m = t i + lna/(ra), where ln denotes the natural logarithm function. moreover, r = exp(rt) where t is the generation interval of the disease, or the average time interval from the onset of one infected person to the time when the onset of his or her contacts occurs. it has been shown mathematically [ ] that, given the growth rate r, the expression r = exp(rt) provides an upper bound of the basic reproduction number regardless of the distribution of the generation interval that is being used. in this work, we will use the term effective reproduction number r instead, due to the community-level immunity likely achieved by july and the interventions implemented during the two waves. the richards model is a phenomenological model which can be used to describe the phenomenon of a biological growth (of cumulative number in this case) without requiring detailed information on the actual process of disease transmission. the basic premise of the richards model is that the incidence curve of a single wave of infections contains a single peak of high incidence, resulting in an s-shaped cumulative epidemic curve and a single turning point (or peak incidence) of the outbreak. the turning point, defined as the point in time at which the rate of accumulation changes from increasing to decreasing, or vice versa in the event of a multi-wave outbreak, can be easily pinpointed by locating the inflection point of the cumulative case curve, i. e., the moment at which the trajectory begins to decline, as demonstrated in previous applications (see, e.g., [ ] [ ] [ ] [ ] [ ] [ ] . this quantity has important epidemiologic implications, indicating either the valley (i.e., moment of acceleration after deceleration) or peak (i.e., moment of deceleration after acceleration) of a disease incidence curve. multi-wave outbreaks also can be modeled by using the multi-phase richards model [ , ] . simultaneous estimates of the model parameters r, a, t i , and k, based on fitting the explicit solution of the richards model for c(t) to the epidemic data used in the study, can be obtained easily and efficiently using any standard software with a nonlinear least-squares approximation tool, such as sas or matlab. the procedure for locating multiple turning points for multi-wave outbreak, which required the use of the multistage richards model, is detailed in [ ] and hence is omitted here. we first fit the weekly laboratory confirmed ph n case data by sample receiving week in taiwan table with the model fit shown in figure . the turning points for the two waves are estimated at . weeks after e-week and . weeks e-week , respectively. subsequently, the weeks in which the turning points for temporal changes in the weekly confirmed ph n case number took place on e-week ( / - / ) for the first wave with a % ci range of ( . , . ), and on e-week ( / - / ) with a % ci range of ( . , . ) for the second wave. we note that the above results were obtained by rounding off the estimates to the next largest integer, e.g., e-week + . = . and hence e-week is the week during which the turning point for the first wave occurred, and similarly for the second wave. to compute the effective reproduction number r, we use the generation time t = . days ( % ci: . - . ) for the ph n in mexico estimated by fraser et al. [ ] . we note that the given ci's for r reflect the uncertainty in the generation time t as well as in the uncertainty in the least-squared estimates for r, and does not reflect the error due to the model itself, which is always difficult to measure. we also fit the weekly confirmed ph n hospitalization data by hospitalization week in taiwan from eweek ( / - / ) of to e-week ( / - / ) of to the richards model. the results are given in table . the data also fit a two-phase richards model with the first wave spanning e-weeks - ( / / - / / ) of and the second wave from e-week ( / - / ) of to e-week- ( / / - / / ) of ( figure ) . the turning points for the weekly confirmed ph n hospitalizations occurred on e-week ( / - / ) for the first wave with a % ci range of ( . , . ) and on e-week ( / - / ) with a % ci range of ( . , . ) for the second wave, which were the same weeks as the case number data turning points. the estimate for r using an estimated generation time t for ph n in mexico [ ] is again provided. to further analyze and compare our previous results, we also make use of the daily class suspension data in taiwan from september , to january , , which allows us to ascertain the temporal changes in this intervention measure during the time period. since this dataset started near the end of the first wave, according to our previous results, only one wave was modeled via the richards model. the estimation results for model fit using the daily class suspension number data as well as the daily number of schools with at least one class suspended are given in table the actual confirmed case number (approximated by k in our model) is during the first wave and for the two waves. point. a graphical illustration of the temporal timelines of the epidemic, as illustrated by the three model fits, is given in figure . moreover, an illustrative comparison of the estimates for r as obtained by the model fits is also provided in figure . in both figures and , the results from fitting the number of schools with class suspended are omitted for brevity, since they are similar to that of the fitting with class suspension data. figure model fit for the -wave richards model using weekly confirmed ph n case data by sample receiving week in taiwan. the dots are the real cumulative data, the blue curve denotes the first wave, and the red curve denotes the second wave. the arrows indicate the weeks in which turning points had occurred. the actual number of confirmed hospitalizations is for the first wave and for the two waves. the estimates for effective reproduction number r obtained from the confirmed case and hospitalization data are in good agreement, with r in the range of . - . for the first summer wave during july-september, and . - . for the second wave in fall/winter, using the generation time estimated by [ ] for the spring outbreak in mexico. serological evidence has indicated that approximately one in every ten persons was infected with the ph n virus in central taiwan by april- june [ , ] ; hence the estimates using data after july does not yield, and can reasonably be expected to be lower than, the more commonly known basic reproduction number r . a recent modeling study [ ] of the ph n epidemic by geographic region in mexico reveals a threewave pandemic, with an initial wave in april-may (mexico city area), a second wave in june-july (southeastern states), and a geographically widespread third wave in august-december. the estimates for the regional reproduction numbers r were . - . , . - . , and . - . for the spring, summer, and fall waves, respectively. the second and third waves in mexico occurred, respectively, one month earlier than the summer (july-early september) and fall/winter (late september-march ) waves in taiwan under study here and exhibit similar decreasing trend, although with higher r. transmissibility of the fist ph n wave in taiwan during the summer in july-september, as measured by r, was lower than that of the earlier spring outbreak in north america [ , , , ] and europe [ ] , most likely, at least in part, due to decreased social contacts among the population triggered by public awareness of the earlier, well-publicized outbreaks in mexico and north america as well as the subsequent preemptive government campaign to reduce transmissions. it was also lower than that of the winter outbreak in the southern hemisphere around the same time [ , , ] , perhaps attributable to the fact that it was the winter influenza season in the southern hemisphere. moreover, it is lower than the final size estimate of r ( . ; % ci: . - . ) obtained from serological study of a cohort household population in central taiwan during the same period of time [ ] . however, we note that this disparity is reasonable since the serologic data used for this estimate accounts for the asymptomatic cases among the cohort group. the decreased transmissibility (smaller r) during fall/winter can be reasonably attributed to increased community-wide immunity from the first wave, and perhaps to the class suspension policy initiated in early september before the start of the fall/winter wave. significantly higher estimate of r (focused on schoolchildren) in the range of . - . was found for the initial pandemic wave in japan [ ] . using updated epidemic data and an age-structured model, the same authors also estimated r for the subsequent community-wide wave in japan in early summer to be much lower ( . - . ) [ ] , although different population and modeling methodology also may have played a role in the decrease in r in subsequent waves. similar decreases in estimates of reproduction number of h n when more than one pandemic wave had occurred have been reported in many countries, including mexico [ ] , argentina and brazil [ ] , canada [ ] , and japan [ , ] . furthermore, these studies show that it is not uncommon for multiwave outbreaks to be more transmissible in a first wave but less widespread with a smaller number of infections (or perhaps limited to a small subpopulation as was in the case of ph n in japan), when compared to subsequent waves. moreover, the second wave in taiwan started shortly after the school opened in september, when additional infections occurring in school settings (as demonstrated by substantial number of class suspensions) contributed to a large number of cases, but perhaps with relatively less per contact transmissibility when compared to household contacts, as it has been reported that sitting next to a case or being the playmate of a case did not significantly increase the risk of h n infection [ ] . the estimates for r using laboratory-confirmed case data by sample receiving weeks are slightly lower than those obtained by using confirmed hospitalization data. although both the confirmed case and hospitalization datasets identify week as the cutoff week for the two waves, the estimates of turning points for each wave differ by about one week when using the two datasets. since only the more severe confirmed cases were hospitalized, the individuals in the resulting hospitalization time series is a selected subset of those in the confirmed case time series. subsequently, the temporal trends of the two time series might not be closely comparable. however, the cumulative curves in figures , , , indicate some similarity in the temporal trends of the cumulative data, mainly in the form of the turning points. the reproduction numbers of the two datasets, on the other hand, are indeed comparable since they mostly are generated from the initial growth rates and hence less affected by any selection bias. the confirmed case data is generated by sampling week, which could be different from the week of symptom onset and hence pose a potential source of some bias in data. however, samples were typically taken when the physicians diagnosed and reported h n cases. we refer to sars outbreak in taiwan, when it was estimated that the onset-to-diagnosis interval is . days for previously quarantined persons and . days for non-quarantined persons [ ] . given the similarity in symptoms of sars and influenza as well as the heightened public awareness due to the world-wide alarm over the seriousness of the ph n pandemic by september, it is more than likely that the time delay from symptom onset to diagnosis (and sample collection) of ph n cases in taiwan would be no more, if not less, than that of sars. moreover, one would expect that the lesson of sars and the subsequent efforts by the government to educate has taught the general public in taiwan to avoid delays in seeking medical care. subsequently, this delay of one or two days in the weekly data can be expected to be most likely not significant. the use of hospitalization data is mainly for the purpose of estimation of reproduction number and comparison with the resulting estimates using the confirmed case data, which is not affected by this delay that might be present in both data. estimates of r obtained by using other (larger) estimated generation time in literature result in larger values for r, but generally are well within the ranges of the other studies (see, e.g., [ , , , [ ] [ ] [ ] [ ] and table [ ]) and hence is omitted for brevity. note also that the formula for r used here yields an upper bound over all possible distributions for t given the growth rate r, and hence might result in an overestimate of its true value. in taiwan, the fall session for kindergarten to high school started on august , while the universities started the fall semester two weeks later, around mid-september. our analysis using the weekly confirmed case and confirmed hospitalization data shows that the initial summer wave of ph n epidemic in taiwan had peaked by e-week - ( / - / ), around the time schools from kindergarten to grade reopened on august . however, a second fall/winter wave of cases started to emerge near the end of september around eweek ( / - / ), approximately weeks after the schools reopened, which did not reach its peak until mid-november (e-week - or / - / ) and lasted until the turn of the year. it is interesting to note that the state-specific fall pandemic waves in mexico began - weeks after school reopened [ ] , which is consistent with our results on the start of the fall wave in taiwan. note that both turning points of the two waves in taiwan fell on neighboring week using either the lab-confirmed case or hospitalization data. this is reasonable since the hospitalization of confirmed cases and the time that the samples were received by laboratories are closely related, although not necessarily in any particular order. the class suspension data started on september near the end of the first wave when the earliest class suspension occurred, according to our -wave fitting in tables and , hence only one wave was modeled via the richards model (table ) . moreover, november ( % ci: november - ) was determined to be the turning point for the daily class suspension data, while november ( % ci: november - ) is the turning point for the daily number of schools with class suspended. both days fall on e-week , which coincides with the week where the turning point had occurred as pinpointed by using the confirmed case data and one week after the turning point obtained by using the hospitalization data. it is reasonable to expect the class suspension to take place following the occurrence of case reporting and hospitalization. moreover, the use of daily data allows a more precise estimation of the turning point. also of interest is the possible impact of major intervention measures implemented by the taiwan government during this time period, which including the aforementioned " class suspension" policy and the mass immunization program. the daily number of class suspensions started to increase in early september and continued until late november after the implementation of mass immunization campaign (figure ). in particular, the policy, which was designed to minimize the potential social impact of full-scale school closings in the event of a major influenza outbreak in the community; deserve special attention to ascertain its actual effectiveness. in fact, the lower estimates of r for the second wave and for the school closings data might indeed be attributable to the possible effects of school closings after september. however, more detailed class suspension data as well as age-specific epidemic data is needed to further quantify the actual impact or effectiveness of this very unique approach of partial school closure and localized class suspensions on the infections in the school and in the community in a qualitative modeling analysis (see, e.g., [ , , ] ). using routine influenza surveillance data, we modeled the temporal changes of the two waves of ph n epidemic in taiwan in summer and in fall/winter. the mass h n vaccination program was first initiated sequentially on november , where a typical delay of at least two weeks from immunization is needed for protection from the vaccine to take effect in human bodies. our results suggest that the turning point for the second wave of infections in the fall had occurred around mid-november (e-week - or / - / ). moreover, the class suspension data indicate that the number of class suspensions had peaked by november , less than three weeks after the start of mass immunization and most likely before the impact of mass immunizations started to become significant. however, the mass immunization, and perhaps the voluntarily decreased social contacts by the general public in response to the well-publicized mass immunization campaign by the government, could have contributed to the overall mitigation of the disease in the community, as indicated by the early saturation of the winter epidemic by early february. however, this cannot be modeled without detailed vaccination data. the richards model considers only the cumulative infected population size with saturation in growth as the outbreak progresses, which can be caused by other factors such as implementation of control measures. although data by reporting date is often and typically scrambled by artificial factors such as health system alertness, public response, and government responsiveness, the richards model is able to capture the turning points of outbreaks because they are often results of these artificial factors. we note, however, that the skewness in an epidemic curve, as quantified by the exponent of deviation "a" in the richards model which describes the curvature of a given cumulative case data, also could conceivably arise from various other intrinsic factors such as spatial heterogeneity and individual heterogeneity in contact (see [ ] , pp. for example) which is not captured by this simple model. this type of modeling, although somewhat simplistic and subsequently limited in its quantification of complex factors, nevertheless enables us to ascertain the impact of these artificial factors through the temporal changes of an outbreak, especially in the events when detailed epidemic data describing disease transmissions and other relevant data (such as that of intervention measures in this case) are not readily available for the construction of a complete disease transmission model and the reliable estimation of model parameters, as in this study moreover, the use of cumulative numbers could often, or at least partially, smooth out stochastic variations that typically occur in epidemic data, and hence the richards model could be a valuable tool in providing clues to the challenging task of public health policy evaluation and planning. serological evidence of hsieh et al. bmc infectious diseases subclinical transmission of the pandemic h n influenza virus outside of mexico taiwan centers for disease control: national standards for implementing school closure passed at twenty-first meeting held by the central epidemic command center central 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animals authors' contributions yhh conceived and organized the study, carried out the analysis, and wrote the first draft. yhh, kfc, tcl, tnw, cyc, and jwc participated in the study and the interpretation of study findings. kfc participated in the writing of the manuscript. mhl participated in the data collection and analysis. all authors have read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -ylvqxpba authors: ansuini, valentina; rigante, donato; esposito, susanna title: debate around infection-dependent hemophagocytic syndrome in paediatrics date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ylvqxpba background: hemophagocytic syndrome (hps) is clinically defined as a combination of fever, liver dysfunction, coagulation abnormalities, pancytopenia, progressive macrophage proliferation throughout the reticuloendothelial system, and cytokine over-production, and may be primary or secondary to infectious, auto-immune, and tumoral diseases. the most consistent association is with viral infections but, as it is still debated whether any micro-organisms are involved in its pathogenesis, we critically appraised the literature concerning hps and its relationship with infections. discussion: infection-dependent hps has been widely observed, but there are no data concerning its incidence in children. a better understanding of the pathophysiology of hps may clarify the interactions between the immune system and the variously implicated potential infectious agents. epstein-barr virus (ebv) infection has been prominently associated with hps, with clonal proliferation and the hyperactivation of ebv-infected t cells. however, a number of other viral, bacterial, fungal, and parasitic infections have been reported in association with hps. in the case of low-risk hps, corticosteroids and/or intravenous immunoglobulin or cyclosporine a may be sufficient to control the biological process, but etoposide is recommended as a means of reversing infection-dependent lymphohistiocytic dysregulation in high-risk cases. summary: hps is a potential complication of various infections. a polymerase chain reaction search for infectious agents including ebv, cytomegalovirus and leishmania is recommended in clinical settings characterised by non-remitting fever, organomegaly, cytopenia and hyperferritinemia. summary: hps is a potential complication of various infections. a polymerase chain reaction search for infectious agents including ebv, cytomegalovirus and leishmania is recommended in clinical settings characterised by non-remitting fever, organomegaly, cytopenia and hyperferritinemia. hemophagocytic syndrome (hps) is a potentially fatal condition due to dysregulated lymphocyte activation and proliferation, mainly characterised by impaired or inactive natural killer (nk) cells and cytotoxic t cells, which leads to macrophage hyperactivation and over-expression of cytokines [ ] . the result of this process is uncontrolled and ineffective immune activation, multi-organ dysfunction, and hemophagocytosis throughout the reticuloendothelial system [ ] . the pathognomonic characteristic of hps is the activation of well-differentiated macrophages, phagocyting erythrocytes, leukocytes and platelets in bone marrow, lymph nodes, spleen, liver and other organs, which can infiltrate almost anybody district and may account for many of its systemic features [ ] . hsp is still often under-diagnosed and sub-optimally managed in children [ ] , but the epidemiological data are fragmentary. the syndrome was first described in as poorlycontrolled histiocyte proliferation, but has since also been called hemophagocytic histiocytosis and macrophage activation syndrome [ ] [ ] [ ] . it can be divided into a primary genetic form and a secondary reactive form (table ) , a distinction that has historically been used to differentiate cases of often fatal infantile hps from those caused by other etiologies that appear later in life and have a better prognosis. this difference may be artificially scholastic because primary forms can occur at any age (not only during infancy or early childhood) [ ] , and both primary and secondary forms can be precipitated by infections with a substantial risk of mortality [ ] . even secondary hps occurs as an imbalance between insufficient host defense, obstinate hyperinflammation, and a heterogeneous triggering event, which can be of infectious, rheumatic or neoplastic nature: therefore, the clinical disease results as the signature of a dysregulated immune activation, leading to macrophage proliferation and widespread hemophagocytosis in the reticuloendothelial system. the aim of this review is to make a critical appraisal of the literature concerning infectionrelated hps in paediatrics. genetic hps is heterogeneous and arises from defects in the critical regulatory pathways responsible for the natural termination of immune responses that lead to the failure of the homeostatic removal of cells that are superfluous or dangerous to the host. since , various genetic loci related to the activity of perforin and granzyme granules have been associated with genetic autosomal recessive hps, thus explaining the impaired or absent function of nk cells and cytotoxic t cells [ , ] . the familial form, which was first described in [ ] , is an autosomal recessive disorder that is estimated to occur in / , - , births, and usually appears during the first year of life [ , ] . sporadic cases of hps associated with multiple genetic mutations have also been described [ ] . the different primary forms of hps are associated with immune deficiencies, including chediak-higashi syndrome, griscelli syndrome, x-linked lymphoproliferative syndrome, wiskott-aldrich syndrome, severe combined immunodeficiency, lysinuric protein intolerance, and hermansky-pudlak syndrome. acquired hps has also been associated with a variety of viral, bacterial and mycobacterial, fungal, and parasitic infections [ ] , autoimmune diseases [ ] , auto-inflammatory syndromes [ ] , and tumours, particularly t cell malignancies [ ] . the constellation of signs and symptoms of hps is not specific and none of the biochemical abnormalities is distinctive. the dramatic presentation of the syndrome includes unremitting fever, visceromegaly, thrombocytopenia, lethargy, seizures, skin rash, pulmonary failure, and cardiac and/or renal involvement, and the mortality rate is - % [ ] . the most common laboratory findings are due to liver dysfunction, and include low fibrinogen, and high serum triglycerides and ferritin levels [ ] . two highly diagnostic clues are increased plasma concentrations of the alpha chain of interleukin- receptors (also known as scd ) and impaired nk cell activity. as treatment can be life-saving and some of the clinical criteria occur late during the course of the disease, it is not necessary to satisfy all of the criteria before beginning therapy. the hallmark of hps is the phagocytosis of blood cells and their precursors: bone marrow aspiration typically reveals the normal maturity of all cell lineages, and infiltration by activated macrophages "stuffed" with other blood cells [ ] . criteria for a diagnosis of hps on the basis of clinical, laboratory, and histopatological findings are the following [ ] : ( ) genetic diagnosis: genes known to cause the syndrome (prf , unc d, stx , stxbp , rab a) ( ) signs and symptoms (at least five of the following criteria): a) fever b) splenomegaly c) cytopenias (minimum cell lines reduced) d) hypertriglyceridemia (≥ mg/dl) and/or hypofibrinogenemia (≤ g/dl) e) hemophagocytosis in any involved organ f ) very little or no nk cell activity g) increased ferritin ≥ mg/l h) increased soluble cd (serum interleukin- receptor alpha) ≥ . u/ml the main pathophysiological abnormality in hps is cytokine dysfunction, which leads to the uncontrolled accumulation and ectopic migration of activated t lymphocytes, antigen-presenting cells and histiocytes, and multi-system inflammation [ ] . the pathophysiology of acquired hps has not been fully defined, but deficient cytolytic activity leads to the persistent activation of lymphocytes and histiocytes, followed by the hypersecretion of pro-inflamatory cytokines and high soluble interleukin- receptor levels that correlate with the prognosis [ ] . the association between hps and infections has been widely documented and both familial or sporadic cases are often precipitated by acute infections. it must also be pointed out that every form of hps can mimic infectious diseases or overwhelming bacterial sepsis, thus hindering the diagnosis of a precipitating and treatable infectious illness. virus-associated hps was first described in by risdall et al., whose series consisted of patients, most of whom were immunocompromised but without any confirmed genetic or acquired immunodeficiency, and all of whom showed serological signs of viral infection [ , ] . since then, there have been reports of hps associated with a host of infections [ ] . epstein-barr virus (ebv) is the most commonly reported trigger of hps [ ] . the epidemiology of ebv-related hps is not well known, although a higher incidence has been observed in asian countries, where it has been theorised there may be a more pathogenic viral strain that is genetically similar to the strains observed in nasopharyngeal carcinoma cell lines [ ] . two forms of ebv-related hps have been described: the first occurring during primary infection and the second during a reactivation process [ ] . during primary infection, ebv typically infects and replicates in b cells, whereas ebv-specific cytotoxic t cells are required to produce memory cells. in rare cases, ebv may infect t and nk cells and induce persistent ebv infection, which may lead to chronic active ebv infection, lymphoproliferative disorders and fulminant ebv-related hps [ ] [ ] [ ] . serological testing can help determine whether ebv-associated hps has occurred in the setting of acute infection or is the result of a reactivation process. in addition, the real-time polymerase chain reaction (pcr) detection and quantification of ebv nucleic acid is an important laboratory means of adequately reflecting viral replication and assessing ebv load in patients with ebvrelated hps [ ] . the quantitative analysis of cell-free ebv genome copy numbers after four months of treatment can assess therapeutic responses and is prognostically significant [ ] . the clonal expansion of ebv-infected t lymphocytes has been demonstrated in ebv-related hps [ ] and ebvpositive t cell lymphoma [ ] on the basis of the presence of homogeneous viral terminal repeat sequences. the clonality of infected t lymphocytes is further suggested by the finding of monoclonal rearrangements of the t cell receptor-alpha gene in ebv-related hps [ ] . the distinction between the monoclonal proliferation of t lymphocytes seen in ebv-related hps and ebv-positive t cell lymphomas may describe the extremes of a spectrum of disordered t lymphocyte proliferation following ebv infection. the inflammatory cytokine over-production seen in patients with ebv-related hps tends to be much more pronounced than that observed in patients with other forms of hps [ ] . of all of the infections associated with hps, ebv infection has the worst prognosis in the presence of underlying hereditary disorders, diffuse intravascular coagulation, neutropenia, or central nervous system involvement [ ] . treatment strategies vary significantly depending on the clinical features of the infection: mild cases of ebvrelated hps are treated conservatively as spontaneous regression has been reported, and antiviral therapy with acyclovir, ganciclovir or cidofovir has led to disappointing results [ ] . in the case of severe ebv-related hps, the introduction of immuno-chemotherapy and, if necessary, allogenic stem cell transplantation has radically changed the history and prognosis of the disease: in such cases, the optimal treatment strategy can be centred on immunosuppressive medications that inhibit overactive t and nk cell responses (i.e. corticosteroids, cyclosporine a, intravenous immunoglobulin, anti-thymocyte globulins, etoposide, rituximab, and plasma or blood exchange transfusions) [ , ] . hematopoietic stem cell transplantation is the last treatment resort for refractory forms of ebvrelated hps, and in the case of ebv infection occurring in genetic forms of hps [ ] . the most frequent herpes viruses associated with hps other than ebv are cytomegalovirus (cmv) and human herpes virus (hhv ). cmv up-regulates tumour necrosis factor gene expression and has been associated with hps in otherwise healthy patients, patients with inflammatory bowel disease, rheumatological diseases and cancer, and transplant recipients [ ] . hps was observed in seven of a series of patients undergoing hematopoietic stem cell transplantation, and was triggered by cmv in three cases [ ] . younger age may be associated with a worse prognosis [ ] . a recent study has shown that the use of specific anti-cmv therapy, such as cmv immunoglobulin, foscarnet or ganciclovir, may be therapeutic [ ] . hhv has been associated with hps in patients: most of these cases occurred in patients with a lymphoproliferative disorder [ ] or immunocompromised patients [ ] , and rarely in immunocompetent hosts [ ] . treatment based on etoposide, ganciclovir, foscarnet or rituximab has led o successful results [ ] . hps can be associated with human immunodeficiency virus (hiv) infection, alone or with a wide variety of underlying disorders. it is likely that this condition is underestimated as hiv infection and hps have many clinical and laboratory similarities. about % of bone marrow biopsies taken from hiv patients before the start of highly active antiretroviral therapy show active signs of hemophagocytosis [ ] . hiv-related hps can be observed in cases of acute or late hiv infection, and in conjunction with immune reconstitution inflammatory syndrome, opportunistic infections, or malignancies [ ] . hps may even be the initial presentation of hiv infection [ ] , and it has been suggested that hiv itself may play a direct role in triggering the syndrome [ ] . other common viral triggers of hps in hiv patients are ebv, cmv and hhv , and ebv-related hps seems to be more frequent in hiv-infected children [ ] . influenza-related hps has been rarely reported in immunocompromised and otherwise healthy children [ ] [ ] [ ] [ ] . one fatal case of hps was observed among children hospitalised with seasonal influenza in a prospective pediatric study [ ] , but reactive hps has also been associated with avian and swine (non-pandemic) influenza [ , ] . in particular, patients with severe h n (avian) influenza infection have symptoms and laboratory findings that are similar to those observed in patients with hps, mainly encephalitis, organ dysfunction with hemophagocytosis, bone marrow failure, and pro-inflammatory cytokine over-production [ ] . clinical studies have found that mutations in some viral genes (ns , pb , ha and na) are significantly related to cytokine release, and it has been demonstrated that recombinant hemagglutinin (h ) from h n virus can suppress perforin expression and reduce the cytotoxicity of t cells, including their ability to kill h -bearing cells [ ] . some authors have suggested treatment with a shorter course of etoposide and dexamethasone [ ] . hps has been reported in cases of parvovirus b infection, most of whom had hereditary spherocytosis as the underlying disease: fewer than half were children [ ] [ ] [ ] [ ] [ ] . of these patients, did not receive any treatment and survived, thus suggesting that the prognosis of parvovirus-associated hps is better than that of the other viral-mediated forms of hps. fulminant viral hepatitis may mimic and even cause hps, with hepatitis a virus being more frequently associated with hps than the other hepatotrope viruses. fifteen cases (including children) have been described, mainly in asia: three of these patients also had a concurrent rheumatological disease (systemic juvenile idiopathic arthritis or still's disease) and two also had hepatitis c. their treatment consisted of corticosteroids, variously combined with intravenous immunoglobulin, but four patients received no specific treatment and of the experienced a favourable outcome [ ] [ ] [ ] . enterovirus-related hps has been described in pediatric cases: five occurred in infants aged < year, and the oldest patient was years old. an underlying disease was found in four patients who experienced a fatal outcome (lymphoid neoplasms, lymphoblastic leukemia and juvenile idiopathic arthritis). ten patients received intravenous immunoglobulin (six in combination with corticosteroids), but only seven patients survived [ ] . other viruses associated with hps include adenovirus, paramyxovirus (leading to measles and mumps), rubella virus, human parainfluenza viruses, flavivirus (leading to dengue fever) and hantavirus (leading to hemorrhagic fever and severe acute respiratory syndrome), all of which have been treated with varying courses of corticosteroids and intravenous immunoglobulin. reactive hps has frequently been associated with intracellular pathogens. the pathophysiology of hps associated with non-viral agents may be related to the production of high levels of activating cytokines by host lymphocytes and monocytes. although the pathophysiological response of the host immune system to the infectious agent is not fully understood, it is hypothesised that functional deficiencies in nk and cytoxic t cells may occur during the illness [ ] . hps can be associated with disseminated mycobacterium tuberculosis infection. thirty-six cases (including infants and children) have so far been reported, approximately half of which were accompanied by comorbidities: eight patients had end-stage renal disease and were receiving hemodialysis or had undergone renal transplantation, four had a history of a malignancy, two had aids, and one had sarcoidosis. fever was the most frequent clinical feature upon presentation, combined with visceromegaly and pancytopenia, and all of the patients underwent bone marrow aspirations that confirmed hemophagocytosis. evidence of extra-pulmonary tuberculosis was found in % of cases. the concluding remarks of the report stated that tuberculosis-related hps has a poor prognosis, with a mortality rate of approximately %, although anti-tuberculous and immunomodulatory therapy (consisting of high-dose corticosteroids, intravenous immunoglobulin, anti-thymocyte globulin, cyclosporine a, epipodophyllotoxin or plasma exchange) may lead to a better outcome [ ] . early diagnostic confirmation and the timely administration of anti-tuberculous medication seem to be crucial in these patients. one reported case of hps occurred after childhood vaccination with the bacillus calmette-guérin [ ] . hps has also been described in association with brucellosis, with brucella melitensis being the most frequently isolated organism [ ] . leptospirosis can cause life-threatening hps as a result of an insufficient or misdirected immunological response to leptospira itself: antibiotic treatment alone is not sufficient in such cases, and treatment with corticosteroids, intravenous immunoglobulin or etoposide is required [ ] . rickettsial diseases, transmitted to humans by arthropod bites and usually controlled at an intracellular level by nitric oxide synthesis, hydrogen peroxide production, and tryptophan degradation have also been related to hps: overall, cases of rickettsial disease confirmed serologically and complicated by hps have been published in the period - , with only cases occurring in patients less than years and a prognosis influenced by the specific rickettsia species, patient's immunologic equipment, and delay in antibiotic therapy or corticosteroid therapy [ ] . in , sepsis caused by multidrugresistant acinetobacter baumannii following urinary tract infection was reported for the first time in a previously healthy -year-old child, who recovered after multiple doses of granulocyte colony stimulating factor and red blood cell/platelet transfusions without any cytotoxic treatment or immunotherapy [ ] . hps can be associated with leishmania donovani and leishmania infantum infections, but leishmaniasis may also mimic the syndrome, as it is characteristically associated with organomegaly and pancytopenia. this is particularly important in non-endemic areas, where visceral leishmaniasis is unlikely to be included in the differential diagnosis, and repeated bone marrow smears are often required to identify leishmania species by means of pcr with species-specific probes [ ] . specific anti-leishmania treatment with amphotericin b is usually sufficient to control hps. unfortunately, sporadic cases of undiagnosed leishmaniasis have been treated as hps with fatal consequences [ ] . malaria (caused by plasmodium falciparum and plasmodium vivax), toxoplasmosis, babesiosis, and strongyloidiasis have been rarely identified in association with hps: a history of travel from endemic countries may help to identify these triggering agents [ ] . yeast (candida spp., cryptococcus spp. and pneumocystis spp.) and moulds (histoplasma spp., aspergillus spp. and fusarium spp.) have been associated with the occurrence of hps, most commonly during hiv infection, neoplastic diseases, protracted corticosteroid administration, and transplantation [ ] [ ] [ ] . disseminated penicillium marneffei infection is common among hiv-infected patients in many regions in southeast asia: the first case of hps associated with penicilliosis in a thai hiv-infected child was reported in , with complete recovery after antifungal and intravenous immunoglobulin therapy [ ] . many immunological, neoplastic and genetic disorders may underlie hps, but infectious causes are the most prevalent and most frequently reported in association with this syndrome. the specific clinical and laboratory tests for microbiological identification of hps are the following: ( ( ) serum cryptococcal antigen and serum galactomannans as a fatal outcome may occur when infection-related hps is only treated supportively, a multidisciplinary approach by experienced clinicians and infectious disease specialists is required in order to ensure the appropriate management of the syndrome itself, and the precipitating or underlying infection. pediatricians should be alert and aware of the risk of the syndrome, because an early diagnosis can change its natural history and it has been shown that prompt treatment improves the overall prognosis. a combination of high fever unresponsive to broad-spectrum antibiotics, hyperferritinemia, hypertriglyceridemia, hypofibrinogenemia, cytopenia, organomegaly and characteristic histological findings in the setting of an infectious process (particularly ebv infection, but also other viral and bacterial, parasitic and fungal infections) is the key diagnostic clue. a better understanding of the pathophysiology of hps should clarify the interactions between immune system pathways and infections. specific antimicrobial therapy can be beneficial in selected cases, whereas antiviral drugs do not seem to be curative. severe cases of infection-dependent hps require immunosuppressants or chemotherapeutic agents, while bone marrow transplantation is the ultimate choice for persistent refractory cases. hemophagocytic syndrome (hps) can occur as a rare complication of various infections in children. clonal proliferation of t lymphocytes with an excessive activation of macrophages can be triggered by different infectious agents, thus indicating that infection per se is involved in the pathogenetic mechanism of the process. a number of studies have demonstrated that hps is frequently triggered by one of many different viral, bacterial, parasitic or fungal infections, with large differences in terms of treatment responses and overall outcomes. all patients meeting the criteria for hps should undergo initial tests to diagnose the underlying infecting organism, which should be guided by epidemiological data and the patient's medical history. a polymerase chain reaction search for infectious agents, including ebv, cmv and leishmania, is recommended in a clinical scenario characterised by unremitting fever, organomegaly, cytopenia and hyperferritinemia. as hps may be associated with many infectious diseases and immunological, neoplastic or genetic disorders, the close cooperation of pediatricians and infectious disease specialists is crucial in order to define any precipitating or underlying condition. the authors declare that they have no competing interests. authors' contributions va and dr drafted the manuscript and 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acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution key: cord- -uzzs w j authors: ma, xuezheng; liu, fang; liu, lijuan; zhang, liping; lu, mingzhu; abudukadeer, abuduzhayier; wang, lingbing; tian, feng; zhen, wei; yang, pengfei; hu, kongxin title: no mers-cov but positive influenza viruses in returning hajj pilgrims, china, – date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: uzzs w j background: there is global health concern that the mass movement of pilgrims to and from mecca annually could contribute to the international spread of middle east respiratory syndrome coronavirus (mers-cov). in china, about , muslim pilgrims participate in the hajj gathering in mecca annually. this is the first report of mers-cov and respiratory virus molecular screening of returning pilgrims at points of entry in china from to . methods and results: a total of returning hajj pilgrims participated in this study. the test results indicated that of the travelers, tested positive for influenza a virus, for influenza b virus, for metapneumo virus, for respiratory syncytial virus, and for human coronavirus. there was a significant difference in the rates of positive and negative influenza virus tests between hajj pilgrims with symptoms and those without. the detection rates of influenza virus were not significantly different among the three years studied, at . , . and . % for , and , respectively. discussion and conclusion: the mers-cov and respiratory viruses detection results at points of entry in china from to indicated that there were no mers-cov infection but a . % positive influenza viruses in returning chinese pilgrims. as of november , there had been laboratoryconfirmed cases of middle east respiratory syndrome coronavirus (mers-cov) infection reported to the world health organization, and at least cases had died [ , ] . most cases of mers-cov infection were reported from the kingdom of saudi arabia. annually, more than million muslim pilgrims from countries attend the hajj pilgrimage in mecca, saudi arabia [ ] . this mass gathering of pilgrims presents a global health risk due to the potential spread of infectious diseases, and respiratory infections are the most common infections transmitted between hajj pilgrims [ , ] . the viruses most commonly isolated from symptomatic patients during the hajj pilgrimage were influenza virus and coronaviruses [ ] [ ] [ ] [ ] . there is global concern that travelers returning from pilgrimage could contribute to the international spread of mers-cov. the international health regulations (ihr) emergency committee suggested that all countries perform surveillance for mers-cov among pilgrims during and after hajj [ ] . in china, about , muslim pilgrims participate in the hajj gathering in mecca annually [ ] . this is the first report of the molecular screening for mers-cov and respiratory viruses among returning pilgrims at points of entry in china, carried out from to . the participants in this study were adult hajj pilgrims who traveled in groups to mecca, saudi arabia, and stayed there for - days from september to october, - . in china, the government arranged charter flights for hajj pilgrims to visit mecca. infectious disease monitoring and surveillance of foreigners travelers coming from other countries is the responsibility of aqsiq (general administration quality supervision inspection and quarantine of the people's republic of china). aqsiq supervises entry-exit ports in china, and operates on behalf of the national government. for all chinese hajj pilgrims, personal and flight information was recorded and a medical examination was conducted, including vaccination by a local aqsiq office, before the trip to mecca. xinjiang and gansu province have the highest number of hajj pilgrims visiting mecca each year. in this study, our institute, the chinese academy of inspection and quarantine, cooperated with the xinjiang and gansu entry-exit inspection and quarantine bureau. we randomly selected returning pilgrims arriving at xinjiang and gansu airports, and asked for their consent to participate in this study. in china, every entry-exit airport has an infrared radiation thermometer, installed by aqsiq, to screen travelers' body temperatures. among returning pilgrims, returning pilgrims triggered the alarm on passing through the infrared radiation thermometer installed at the airport to monitor travelers' body temperature. sixteen of these travelers were confirmed by using a clinical thermometer to have the onset of fever (> . °c), and also reported a sore throat or cough on the returning flight. the remaining returning pilgrims did not have a fever or other symptoms. the numbers of travelers with fever in each year were ( ), ( ), and ( ). the mean age of all participants was . years old (sd = . ). in this study, were females and were males, and they were all moslem. all pilgrims were asked to undergo a health examination and were vaccinated against influenza a and b in a local travel health center a week prior to departure. all participants included in this study were voluntary and signed consent forms. for the detection of viral infection, samples included lower respiratory tract sputum, washes, and upper respiratory tract oropharyngeal swab specimens. lower respiratory tract sputum samples were used to test for respiratory viruses during this -year period. all pilgrims were tested for influenza and mers, but only those with fever were tested for the other viruses. all specimens were collected immediately when returning pilgrims arrived at each point of entry, and nucleic acid was isolated and immediately screened by real time rt-pcr for the upe and orf a genes of mers-cov provided by the world health organization [ , ] . all real time pcr protocols for influenza a and b followed those used by a previous study [ ] . samples from travelers displaying a fever were also tested by real time rt-pcr for human metapneumo virus (hmpv), human respiratory syncytial virus (hrsv), and human coronaviruses hku , e, and oc [ ] . according to the infection control and health quarantine rules at airports, the time taken between specimen collection and the reporting of results was within h. all real time rt-pcr results for mers-cov were negative. a total of influenza a and influenza b virus positive samples were detected from to (table ) . of these, the test results from participants with a fever indicated that samples were positive for influenza a, were hmpv positive, were hrsv positive, and participant was positive for each of hku , e, and oc . in addition, influenza a and influenza b positive samples were detected from nonsymptomatic travelers. no dual infections were detected. two hypotheses were tested: ( ) there is a significant difference in the positive and negative rates of influenza virus detection between hajj pilgrims with symptoms and those without. pearson's chi-square analysis indicated that there was a significant difference in the influenza virus detection rates between travelers with fever and those without symptoms (χ = . , p = . ). it is of interest, although of unclear significance, that none of the influenza b positive subjects were symptomatic. ( ) there is a significant difference in the rates of influenza (a and b) virus detection among the years , , and . the rates of influenza virus detection for the years , , and were . , . , and . %, respectively, and statistical analysis revealed that there was no significant difference in the rates of influenza virus detection among these three years (χ = . , p = . ). all participants with fever were followed up, and none of these individuals were admitted to hospital after days. in this study, we did not detect any cases of mers-cov infection but respiratory virus infections including influenza a and b, hmpv, hrsv, and human coronavirus were detected among hajj pilgrims returning to china. this result was consistent with the outcomes of similar studies of respiratory virus detection in hajj pilgrims in france, north india, egypt, ghana, saudi arabia, and the uk [ ] [ ] [ ] [ ] [ ] [ ] . regarding the detection of influenza viruses, these studies reported detection rates of . % in france (no vaccination) [ ] , % in north india ( % vaccination rate) [ ] , % in egypt ( % vaccination rate) [ ] , . % in ghana (vaccination rate unknown) [ ] , and % in the uk ( % vaccination rate) [ ] . in our study, all participants had been vaccinated against influenza virus, but . % tested positive for influenza virus infection. we are unable to measure the direct impact of influenza vaccination on the resistance of hajj pilgrims to influenza infection and further studies are required to understand the efficacy of the influenza vaccine among this population. however, increasing the rate of vaccination will help protect individuals, particularly those travelers that are most vulnerable to infection such as older adults and those that may be immunocompromised. a combination of vaccination and rapid antiviral treatment of symptomatic individuals currently offer the best strategy for the prevention and treatment of infections among hajj pilgrims. in this study, mers-cov was not detected in any of the upper respiratory swabs or sputum specimens tested. however, limiting the time taken for sample collection, the type of samples collected and the selection of participants can all affect the rates of positive detection. in previous studies, most samples were nasal swabs collected from strongly suspected symptomatic participants after they were under investigation in hospital [ ] [ ] [ ] [ ] [ ] . however, in our study, swabs were collected from both suspected and asymptomatic returning pilgrims immediately after their arrival at airports. our sampling design would therefore include some healthy pilgrims, thereby decreasing the rate of detection of respiratory virus infections. in addition, upper respiratory samples (nasopharyngeal swabs and sputum) have been demonstrated to have a lower mers-cov genome load than lower respiratory specimens such as tracheal aspirates and bronchoalveolar lavage specimens [ ] . this may also have limited the detection of mers-cov in our study. the findings from our study demonstrate the risk of influenza infection among travelers during mass gatherings, and confirming the need for effective surveillance of imported infectious diseases at entry points into china. the hajj pilgrimage provides a unique opportunity to test the effectiveness of different infectious disease preventive and detective measures that require a large sample size. continued annual monitoring of mers-cov, influenza viruses, and other respiratory viruses (such as human rhinovirus), is needed to increase our understanding of the epidemic patterns of respiratory virus infections among hajj pilgrims in china. world health organization. global alert and response (gar) middle east respiratory syndrome middle east respiratory syndrome hajj-associated viral respiratory infections: a systematic review high prevalence of common respiratory viruses and no evidence of middle east respiratory syndrome coronavirus in hajj pilgrims returning to ghana prevention of influenza at hajj: applications for mass gatherings the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus world health organization statement on the tenth meeting of the ihr emergency committee concerning mers-cov characteristics of traveler with middle east respiratory syndrome assays for laboratory confirmation of noval human coronavirus (hcov-emc) infection detection of a novel human coronavirus by real-time reverse-transcription polymerase chain reaction epidemiology of respiratory rna viruses in a cohort of hospitalized children in riyadh, saudi arabia cross-sectional survey and surveillance for influenza viruses and mers-cov among egyptian pilgrims returning from hajj during - . influenza other respir viruses influenza not mers cov among returning hajj and umrah pilgrims with respiratory illness lack of nasal carriage of novel corona virus (hcov-emc) in french hajj pilgrims returning from the hajj , despite a high rate of respiratory symptoms lack of mers coronavirus but prevalence of influenza virus in french pilgrims after viral respiratory infections at the hajj: comparison between uk and saudi pilgrims respiratory tract samples, viral load, and genome fraction yield in patients with middle east respiratory syndrome we wish to thank dr. dexin li for his extensive support and assistance with the study. all data generated or analyzed during this study are included in this published article.authors' contributions xm and fl carried out sample collection and drafted the manuscript. ll, lz, and lm extracted rna and collected clinical samples. aa, lw, wz, and py recorded the experimental data and collated the results tables. kh designed the study, edited the manuscript, and supervised the experiments. all authors read and approved the final manuscript. the study was conducted according to the protocol approved by the human research ethics committee, chinese academy of inspection and quarantine, in compliance with the provisions for human research in the helsinki declaration (es- / / hq). written informed consent was obtained from all participants. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord- -ow r authors: lokida, dewi; hadi, usman; lau, chuen-yen; kosasih, herman; liang, c. jason; rusli, musofa; sudarmono, pratiwi; lukman, nurhayati; laras, kanti; asdie, rizka humardewayantie; murniati, dewi; utama, i made susila; mubin, risna halim; karyana, muhammad; gasem, muhammad hussein; alisjahbana, bachti title: underdiagnoses of rickettsia in patients hospitalized with acute fever in indonesia: observational study results date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: ow r background: reports of human rickettsial infection in indonesia are limited. this study sought to characterize the epidemiology of human rickettsioses amongst patients hospitalized with fever at tertiary hospitals in indonesia. methods: acute and convalescent blood from hospitalized non-dengue patients was tested for rickettsia igm and igg by elisa. specimens from cases with seroconversion or increasing igm and/or igg titers were tested for rickettsia igm and igg by ifa and rickettsia genomes using primers for rickettsia (r.) sp, r. typhi, and orientia tsutsugamushi. testing was performed retrospectively on stored specimens; results did not inform patient management. results: r. typhi, r. rickettsii, and o. tsutsugamushi igg antibodies were identified in / ( . %), / ( . %), and / ( . %) of samples, respectively. for the / ( . %) non-dengue patients diagnosed with acute rickettsial infection, presenting symptoms included nausea ( %), headache ( %), vomiting ( %), lethargy ( %), anorexia ( %), arthralgia ( %), myalgia ( %), chills ( %), epigastric pain ( %), and rash ( %). no acute rickettsioses cases were suspected during hospitalization. discharge diagnoses included typhoid fever ( ), dengue fever ( ), respiratory infections ( ), leptospirosis ( ), unknown fever ( ), sepsis ( ), hepatobiliary infections ( ), uti ( ), and others ( ). fatalities occurred in ( . %) patients, mostly with co-morbidities. conclusions: rickettsial infections are consistently misdiagnosed, often as leptospirosis, dengue, or salmonella typhi infection. clinicians should include rickettsioses in their differential diagnosis of fever to guide empiric management; laboratories should support evaluation for rickettsial etiologies; and public policy should be implemented to reduce burden of disease. rickettsioses are arthropod-borne zoonoses caused by obligate intracellular bacteria from rickettsia or orientia genera. they include murine typhus, spotted fever, and scrub typhus groups [ ] . small mammals serve as reservoirs and arthropods (ticks, fleas, lice, and mites) as vectors. humans are incidental hosts for many pathogenic rickettsiae [ ] . human rickettsioses in indonesia are not well characterized. limited reports have found murine typhus in travelers returning from indonesia [ ] [ ] [ ] . in , over travel-associated cases were reported worldwide; a significant proportion were r. typhi from tropical and subtropical areas, r. conorii from southern asia and o. tsutsugamushi from the asia-pacific [ , ] . an active surveillance study of children in asia showed that . % of indonesian cases were due to rickettsia [ ] . other fever studies revealed prevalence of murine typhus, spotted fever, and scrub typhus in northeastern papua to be , , and %, respectively [ ] , whereas prevalence of murine typhus in central java was % [ ] . clinically, rickettsioses are difficult to distinguish from other conditions causing acute fever in endemic areas, especially during the early phase. common presentations include fever, abdominal discomfort, headache, myalgia, and rashes. lung, liver, and kidney involvement may complicate the disease [ ] . given the non-specific clinical syndrome and limited access to diagnostics, rickettsioses are likely underdiagnosed in indonesia. underdiagnoses could engender inappropriate management, treatment delays, prolonged hospitalisation, and increased morbidity and mortality [ , ] . therefore, early diagnosis and empirical therapy of rickettsioses are important. to characterize the epidemiology of rickettsioses in indonesia, we performed rickettsia diagnostic panels on blood from subjects in the acute fever requiring hospitalization (afire) study [ ] . presentation of rickettsial infection in subjects that were initially diagnosed with another infection such as dengue, salmonella and leptospirosis were evaluated to identify features that may confound diagnosis of rickettsiosis. patients found to have rickettsial infection by reference laboratory testing were identified from ina-respond's [ ] afire observational cohort study conducted in indonesia from to . it recruited patients presenting to hospital for evaluation of acute fever, at least year old, hospitalized within the past h, and not hospitalized within the past months. study sites were eight tertiary hospitals in seven cities in indonesia: bandung, denpasar, jakarta, makassar, semarang, surabaya and yogyakarta. details of afire have been previously described [ ] . subjects were evaluated at enrollment, between and days post-enrollment and months post-enrollment. demographics, clinical data, blood and other clinically indicated specimens were collected during these visits. blood specimens from the first visit were considered "acute" and specimens from the two follow-up visits were considered "convalescent". buffy coat and plasma from blood were stored at − °c and tested retrospectively for pathogens approximately year after enrollment. specimens from subjects were first screened for dengue infection. non-dengue cases were then tested for other pathogens, including rickettsia. details of the diagnostic procedures are shown in fig. . r. typhi igm, r. rickettsii igm, r. typhi igg, and spotted fever group igg were tested using enzyme-linked immunosorbent assay (elisa) (fuller laboratories, san fransisco, ca). igm and igg for scrub typhus were tested using elisa (inbios, seattle, wa). detailed methods for these assays have been described previously [ , ] . convalescent plasma from patients were tested for igg against r. typhi. due to logistic reasons, only a subset was tested for spotted fever group (n = ) and scrub typhus (n = ). if igg was positive, igm and igg from acute and convalescent plasma were tested simultaneously to assess seroconversion, increase or high optical density and index value in paired samples. in these subjects, indirect immunofluorescence assay (ifa) was performed to detect igm and igg reactivity to r. typhi and r. ricketsii (focus diagnostics, ca) following the manufacturer's procedures as previously described [ ] . a specimen was considered positive when igm or igg fluorescence was observed in the : dilution. to determine four-fold increase, acute specimens were diluted by two-fold until igm or igg fluorescence was not observed. the dilution where igm or igg fluorescence was still detected was the end titer of the specimens. the corresponding convalescent specimens was then diluted four-fold of the end titer dilution of the acute specimens. four-fold increase of igm or igg was confirmed when fluorescence was still detected in these diluted convalescent specimens [ ] . seroconversion of igm or igg antibodies was confirmed when no fluorescence was detected in : dilution in acute specimens but was detected in : dilution in convalescent specimens. acute plasma and buffy coat from subjects with seroconversion or increased igm/igg and from subjects that only had acute specimens were tested using pcr. bacterial dna was extracted using qiaamp dna mini kit (qiagen, hilden, germany). rickettsia sp. were detected by the -kd outer membrane protein ( -kd omp) gene of rickettsia sp., while r. typhi was identified by the ompb gene of r. typhi following previously published methods [ , ] and o. tsutsugamushi by its -kd omp gene [ ] . specimens positive for -kd omp gene of rickettsia sp but not the ompb gene of r. typhi underwent pcr and nested pcr amplification using primer set rompb f, rompb r, rompb r targeting -bp sequence of rickettsia sp. ompb gene and followed by dna sequencing to determine rickettsial species [ ] . randomly selected samples with positive r. typhi based on qpcr, were confirmed with the same amplification and sequencing method. sequence chromatograms were edited using bioedit . . . software [ ] ; edited sequences were searched for similarity using blastn. phylogenetic analysis was conducted in mega and inferred using the neighbor-joining method. the analysis involved -bp (nucleotide - of r. typhi ompb gene). ) when both -kd omp gene of rickettsia and ompb gene of r. typhi were detected and/or seroconversion or four-fold increase of r. typhi igm and/or igg by ifa was observed. ) when the detection of -kd omp gene of rickettsia or ompb gene of r. typhi was supported by dna sequencing of the -bp sequence of rickettsia sp. ompb gene or sero-conversion or four-fold increase of r typhi igm and/or igg by ifa. spotted fever group was confirmed by the detection of -kd omp gene of rickettsia and dna sequencing of the -bp sequence of rickettsia sp.ompb gene and /or sero-conversion or four-fold increase titers of spotted fever group igm and igg by ifa. scrub typhus was confirmed by the detection of -kd omp gene of o. tsutsugamushi and/or sero-conversion or four-fold increase o. tsutsugamushi igm and/or igg by elisa. data were collected in openclinica v. . (openclinica, llc) and analyzed using stata v. . (statacorp llc). clinical and laboratory profiles of confirmed cases were characterized by descriptive statistics and compared according to the three most commonly attributed diagnoses. proportions were compared between groups using the chi-squared test. the t-test was used to compare means between groups. specimens from of subjects were evaluated using the rickettsia diagnostic panels. rickettsia was identified as the etiology of febrile illness in / ( . %) cases ( fig. ) . none of these patients were diagnosed with rickettsia upon clinical presentation. one case was clinically diagnosed as rickettsia during hospitalization but was not laboratory confirmed. characteristics of patients with acute rickettsial infection at enrollment are shown in table ( ). (fig. ) . subjects averaged days (range - days) of fever before hospital admission (table ) . other reported symptoms included nausea ( %), headache ( %), vomiting ( %), lethargy ( %), anorexia ( %), arthralgia ( %), myalgia ( %), chills ( %), and epigastric pain ( %). the clinical triad of r. typhi infection (fever, headache and rash) was found in %. the three most frequent confirmed diagnoses in the study cohort, dengue, typhoid and leptospirosis demonstrated overlap with rickettsial infection. details are shown in table . most subjects presented with normal hematocrit (median . %) and leukocyte count (median /mm ). the majority had low lymphocyte proportion (median . %) and platelets (median , /mm ). mildly increased liver enzymes were found in %, with bilirubin increases primarily attributable to direct bilirubin. during hospitalization, no clinically relevant changes were observed. the hematologic profile of r. typhi cases was similar to typhoid, but distinguishable from dengue and leptospirosis. dengue showed lower leukocyte and platelet counts. leptospirosis showed higher leukocyte and neutrophil counts, but lower absolute lymphocyte counts. increased total bilirubin and direct bilirubin were more prevalent than in dengue or typhoid, while increased total and indirect bilirubin were more frequent in leptospirosis. ast above iu was more common in r. typhi cases compared to the three diseases, whereas creatinine > . mg/dl was more common in leptospirosis. for the rickettsioses patients, discharge diagnoses were: typhoid fever ( ), dengue fever ( ), leptospirosis ( ), respiratory infections ( upper, and lower), unidentified fever ( ), sepsis ( ), hepatobiliary infections ( ), unidentified viral infections ( ), uti ( ) and others (one each: hiv, chikungunya, enteritis, meningoencephalitis, and diabetic neuropathy). in all cases of rickettsia initially suspected to be leptospirosis, typhoid fever, chikungunya, or dengue fever, diagnostic assays for those pathogens at the reference laboratory were negative, except in one r. felis case where leptospira pcr was positive and leptospira igm and igg sero-converted, suggesting co-infection. clinicians diagnosed typhoid despite negative or weak positive s. typhi igm rapid tests in presumed typhoid cases; in other cases rapid tests were not performed. in the remaining cases [ ] , positive results from the rapid test were not supported as blood culture, pcr and elisa igm tests for salmonella were negative. in these cases, r. typhi was confirmed by pcr and/or serological assays. fifteen dengue diagnoses were not supported by rapid dengue antigen or antibody tests. in contrast, presumed leptospirosis cases had positive rapid tests, but pcr and elisa at the reference laboratory were negative except in the r. felis case above. details of the diagnostic tests to confirm rickettsia infection and to exclude s. typhi, dengue, and leptospira infections are shown in additional file (see additional file: table s ). antibiotics were taken prior to hospitalization in subjects, including amoxicillin ( ), cephadroxil ( ), cotrimoxazole ( ), chloramphenicol ( ), cefixime ( ), spiramycin ( ), and a combination of antibiotics ( ). antibiotics were given at hospitals in of ( %) subjects with documented treatment data as shown in table . the majority received ceftriaxone ( ) , ciprofloxacin ( ) and levofloxacin ( ), or a combination of antibiotics ( ) . the drug of choice for rickettsia infection, doxycycline was given to patients, one in combination with ceftriaxone and one with amoxicillin. ( %) subjects with suspected viral infections received antibiotics at hospital several days after no clinical improvement with symptomatic treatment. the median hospital stay was days (range - ). twenty-four subjects ( . %) recovered with sequelae and ( . %) recovered without sequelae. seven ( . %) patients (median . years, range . - years) died. of these, had underlying disease (stroke, hiv and chronic liver disease, hiv and tb, dm, and copd). six deaths were attributed to sepsis; in one hiv positive patient with meningoencephalitis, death was attributed to cardiogenic shock. all patients who died received antibiotics, however none received doxycycline. contribution of rickettsial infection to these deaths could not be ascertained. note: significant (p < . ) a between r. typhi and dengue, b between r. typhi and s. typhi, c between r. typhi and leptospira. †one rickettsia felis case is not included our results confirm that rickettsial infections are an important, and often overlooked, cause of fever in hospitalized patients in indonesia. the prevalence of subjects with r. typhi igg was unexpectedly high ( . %), suggesting significant population exposure. furthermore, acute rickettsial infection was the etiology of acute febrile illness in . % of hospitalized subjects, none of whom were clinically diagnosed or managed as having rickettsial infection during hospitalization. the most common clinical manifestations in our subjects (fever, headache, and nausea/vomiting) have been reported in other studies [ , ] . however, rash, the hallmark of ricketsial disease diagnosis that usually occurs late (around days of illness in patients with murine typhus) [ ] was less common than other reports ( % vs. to %, respectively) [ , ] . this may be attributable to the design of the afire study, which only recorded clinical signs and symptoms of subjects during admission, while other studies monitored them throughout illness. lack of longitudinal monitoring may explain why prevalence of the r. typhi infection clinical triad was lower in our study ( %) than in other studies ( - %) [ , ] . several factors may contribute to the misdiagnosis of rickettsioses during hospitalization. first, presentation of rickettsia infection overlaps with that of other infectious etiologies, particularly typhoid fever, as demonstrated in our study and by data from the us cdc [ ] . second, clinicians may not include rickettsioses in their differential diagnosis. literature demonstrating the importance of rickettsia infection in the hospital setting is lacking. previous reports were from a few serologically-confirmed patients from three cities in , [ ] , - [ ] , [ ] and may not have reached clinicians. third, access to diagnostic tests for rickettsioses is poor and specificity is low for available rapid diagnostics for other pathogens such as s. typhi, dengue virus and leptospira spp. reports from laos also describe difficulties in differentiating these pathogens [ ] . prior reports also suggest that rickettsioses are an important etiology of fever in indonesia. a hospital-based study by groen et al. in semarang, central java found rickettsioses in of ( %) suspected dengue cases during - [ ] . a fever study by gasem et al. in the same city years later reported rickettsia cases amongst children and adults visiting primary health centers and hospitals. it is unclear if these cases were due to r. typhi [ ] . as part of a dengue vaccine study, copeding et al. found that % of childhood fever was caused by rickettsia, based on elisa igm [ ] . previous studies have typically confirmed rickettsioses by serologic assays only. our study applied a panel of diagnostic assays including molecular assays, elisa, and ifa, and therefore provides additional information about rickettsia subgroups, as well as the acutely infecting species. furthermore, this study demonstrated the occurrence of human rickettsiosis in geographical areas excluded from previous studies (yogyakarta, surabaya, and makassar) and reconfirmed that rickettsioses continue to circulate in semarang, bandung, jakarta, and bali, both in children and adults. although the prevalence of rickettsioses in indonesia was as high as in thailand or malaysia, predominant species differed. in our study, r. typhi was most common, whereas in thailand and malaysia o. tsutsugamushi was more frequent [ , ] . as o. tsutsugamushi has been found in hosts and vectors from several areas in indonesia and evidence of previous infection was detected in our samples, we likely underdiagnosed scrub typhus. this may be because our study did not include primary health centers, where cases of o. tsutsugamushi may be managed [ ] . it is also possible that there are factors, such as exposure or genetic constitution, in our population that predispose to murine typhus [ ] . in % of subjects initially diagnosed as typhoid fever or dengue fever, diagnoses were unchanged despite subsequent negative or doubtful rapid test results, suggesting that in the absence of comprehensive diagnostic tests, clinicians had no choice but to judge based on the clinical presentations. in cases with positive (≥ ) rapid test, the detection of igm may be associated with persistent igm that can be detected more than a year after infection [ ] and/or multiple exposure for people living in endemic area [ ] . in contrast, the six initially suspected leptospirosis cases had positive leptospira igm, although reference laboratory confirmatory antibody testing and pcr were negative, except in one case with possible co-infection. this suggests that severe r. typhi may clinically resemble leptospirosis (myalgia, abdominal pain, icterus) [ ] and clinicians should interpret the results of leptospira antibody tests cautiously. poor specificity of the leptospira igm test, both with rapid testing and elisa, has been reported [ ] . leukocyte count may also help differentiate between rickettsiosis and leptospirosis as it is more likely to be leukocytosis in leptospirosis. the case fatality rate of r. typhi infection in our study ( . %) is higher than previously reported ( . to %) [ ] [ ] [ ] . our study's higher mortality may be related to presence of comorbidities in of the fatal cases. however, we cannot exclude the possibility of severe r. typhi. complications including meningitis and encephalitis, acute respiratory distress syndrome, acute liver failure, acute renal failure, endocarditis, and multi-organ failure have been reported [ ] . diagnostic inaccuracy can result in inappropriate management of patients. ideally, rickettsioses should be quickly identified and doxycycline, the antibiotic of choice, administered. amongst patients with rickettsial infection in the afire study, two subjects initially diagnosed as leptospirosis received doxycycline in combination with other antibiotics. in % of suspected viral infection subjects, antibiotics were later given, suggesting that clinicians considered bacterial infections but had difficulty making definitive diagnoses. broad spectrum antibiotics such as ceftriaxone or ciprofloxacin are effective against rickettsioses and are reasonable empiric choices while awaiting laboratory confirmation given the overlap of clinical presentations of rickettsia and other infections. however, unnecessary administration of broad spectrum antibiotics should be discouraged. appropriately targeted treatment could hasten recovery, reduce healthcare utilization, and minimize development of antibiotic resistance [ , ] . on the other end of the antimicrobial stewardship spectrum, misdiagnosis of rickettsial infection as dengue could result in witholding necessary antibiotics. this is of particular concern in environments where dengue, which is managed supportively, is diagnosed empirically without laboratory confirmation and rickettsia is not considered. policy makers and clinicians should prioritize diagnosis, treatment and prevention of rickettsioses in indonesia. improved detection with subsequent appropriate management could decrease patient morbidity and reduce healthcare costs. ideally, laboratories should be equipped with valid diagnostic assays (pcr for molecular detection during acute illness and ifa as the gold standard for serology). however, pcr and ifa have several disadvantages, including the need for an expensive thermal cycler or a fluorescence microscope, which are often unavailable in endemic resource-limited settings, and experienced technicians [ ] . therefore, elisa can be an alternative when both are unavailable [ ] . proper empiric management, including administration of appropriate antibiotics, and early diagnostic strategies will minimize disease sequelae. finally, development and implementation of prevention guidelines may also reduce disease burden. our study had several limitations. first, we only enrolled hospitalized patients with fever, and therefore the results cannot be generalized to cases not requiring hospitalization. second, this study was conducted in large indonesian cities, so may not reflect what is seen in more rural areas or other cities. finally, as the parent afire study was not designed as a rickettsia study, we did not specifically collect clinical data or request laboratory tests targeting rickettsial infections. to address this limitation, we retrospectively reviewed medical records for additional data not recorded in case report forms. lastly, we do not know that outcomes would be different if cases had been diagnosed and appropriate targeted treatment provided. in conclusion, our study demonstrates the importance of including rickettsioses in the differential diagnosis for fever in hospitalized patients, developing laboratory capacity and point of care test to rapidly and accurately diagnose rickettsioses, and implementing public policy to reduce disease burden. further studies should be conducted to better characterize the epidemiology of rickettsioses in indonesia and evaluate 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lipopolysaccharide enzyme-linked immunosorbent assay kinetics of the natural, humoral immune response to salmonella enterica serovar typhi in kathmandu limited diagnostic capacities of two commercial assays for the detection of leptospira immunoglobulin m antibodies in laos clinical and laboratory characteristics, epidemiology, and outcomes of murine typhus: a systematic review diagnosis and management of tickborne rickettsial diseases: rocky mountain spotted fever, ehrlichioses, and anaplasmosis. united states: a practical guide for physicians and other health-care and public health profesionals: cdc diagnosis and management of tickborne rickettsial diseases: rocky mountain spotted fever and other spotted fever group rickettsioses, ehrlichioses, and anaplasmosis -united states state of the art of diagnosis of rickettsial diseases: the use of blood specimens for diagnosis of scrub typhus, spotted fever group rickettsiosis, and murine typhus comparison of commercial enzyme-linked immunosorbent assay and immunofluorescence assay for diagnosis of acute rickettsia typhi infections. vector borne zoonotic dis publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank our patients and their families for support of this study and nurses and laboratory technicians for collecting and testing the specimens at sites. we thank dr. richards and dr. widjaja for advice on appropriate rickettsial testing and reference laboratory team (gustiani, ungke anton jaya, deni pepi butarbutar, wahyu nawang wulan, yuanita djajadi, rizki amalia sari) for testing the specimens. we also would like to thank antonius arditya pradana and aly diana for technical assistance with the manuscript. received: february accepted: april supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file : table s . diagnostic tests to confirm rickettsia infection and to exclude s. typhi, dengue, leptospira, and chikungunya infections and diagnostic tests to confirm rickettsia infection in an hiv patient and patients with non-rickettsial clinical diagnoses. this study was conducted by ina-respond, a collaborative research network of nihrd, ministry of health, indonesia, and us-niaid, nih. this project has been funded in whole or in part with federal funds from the niaid, nih, under contract nos. hhsn e and hhsn i. niaid collaborators contributed to design of the study; collection, analysis, and interpretation of data; and writing of the manuscript. the content of this publication does not necessarily reflect the views or policies of the department of health and human services, nor does mention of trade names, commercial products, or organizations imply endorsement by the u.s. government. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate all subjects provided written informed consent before study participation. the study was conducted in accordance with the declaration of helsinki, and the protocol was approved by the not applicable. the authors declare that they have no competing interests.author details key: cord- - x euj authors: nickol, michaela e.; kindrachuk, jason title: a year of terror and a century of reflection: perspectives on the great influenza pandemic of – date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: x euj background: in the spring of , the “war to end all wars”, which would ultimately claim more than million lives, had entered into its final year and would change the global political and economic landscape forever. at the same time, a new global threat was emerging and would become one of the most devastating global health crises in recorded history. main text: the h n pandemic virus spread across europe, north america, and asia over a -month period resulting in an estimated million infections and – million deaths worldwide, of which ~ % of these occurred within the fall of (emerg infect dis : - , , bull hist med : - , ). however, the molecular factors that contributed to the emergence of, and subsequent public health catastrophe associated with, the pandemic virus remained largely unknown until , when the characterization of the reconstructed pandemic virus was announced heralding a new era of advanced molecular investigations (science : - , ). in the century following the emergence of the pandemic virus we have landed on the moon, developed the electronic computer (and a global internet), and have eradicated smallpox. in contrast, we have a largely remedial knowledge and understanding of one of the greatest scourges in recorded history. conclusion: here, we reflect on the influenza pandemic, including its emergence and subsequent rapid global spread. in addition, we discuss the pathophysiology associated with the virus and its predilection for the young and healthy, the rise of influenza therapeutic research following the pandemic, and, finally, our level of preparedness for future pandemics. influenza viruses have posed a continual threat to global public health since at least as early as the middle ages, resulting in an estimated - million cases of severe illness and , - , deaths annually worldwide, according to a recent estimate [ ] . regional influenza epidemics occur on an annual basis, resulting in millions of illnesses and hospitalizations despite intensive vaccination and awareness programs [ , ] . moreover, influenza pandemics arise sporadically due to the introduction of an antigenically-distinct influenza a virus within a population, which can result in devastating effects on global public health and healthcare networks. the emergence of influenza subtype h n in , which ultimately resulted in an estimated - million deaths worldwide, would forever change the course of human history and will be discussed in detail in the following sections [ ] [ ] [ ] . the aims of this short review are to discuss: i) the emergence and spread of the virus; ii) the unique severity of disease in young, healthy individuals; and iii) the subsequent influence of the pandemic on influenza virus therapeutic and future preparedness. it is postulated that % of the worldwide population is infected by an influenza virus each year, resulting in a total economic burden of $ . billion usd [ , ] . as a testament to the significant toll posed by influenza on public health and healthcare systems, the us centers for disease control and prevention (cdc) estimated that from to , influenza infections resulted in . - . million illnesses and , - , hospitalizations annually in the us alone [ ] . it has been suggested that children are likely the primary transmitters of influenza [ ] . lethal influenza infections are primarily associated with high risk populations, including infants (< year), the elderly (> years), and individuals with pre-existing comorbidities, including chronic respiratory abnormalities, cardiac disease, immunodeficiency, and pregnancy [ , ] . mortality in children and young adults is generally low [ ] . symptoms manifest as a sudden high fever, headache, pharyngitis, cough, myalgia, nausea, vomiting, and fatigue, which generally resolve within days in healthy adults [ , ] . severe and/or lethal disease is typically associated with viral pneumonia or secondary bacterial infections in the lower respiratory tract [ ] . to be considered a pandemic, an influenza virus must: i) spread globally from a distinct location with high rates of infectivity resulting in increased mortality; and ii) the hemagglutinin (ha) cannot be related to influenza strains circulating prior to the outbreak nor have resulted from mutation [ , ] . it should also be appreciated that prior to the first isolation of a human influenza virus in , the cause of influenza outbreaks and pandemics could only be inferred based on physiological symptoms of disease, along with the speed and breadth at which illness was spread [ ] . as early as bc, hippocrates, the father of modern medicine, described the first known account of an influenza-like illness in his sixth "book of epidemics" [ , ] . here, he recounted an annual recurring upper respiratory tract infection characterized by pharyngitis, coryza, and myalgia which peaked around the winter solstice [ ] . this seasonal epidemic occurred in perinthus, a northern port town located in what is now turkey, and is referred to as the "cough of perinthus" [ ] . it has been suggested that potential pandemics may have occurred in and ; however, it is unanimously agreed that the first documented influenza pandemic occurred in , resulting in high morbidity [ , ] . the virus originated in asia, before spreading to africa, and then simultaneously spreading from both continents to europe. it reportedly spread across the entire european continent within months, before eventually reaching the americas [ , ] . two pandemics were recorded in the eighteenth century. the first began in russia in , eventually moving across the entirety of europe within months and, ultimately, across the known world over the following years [ ] [ ] [ ] [ ] . the second pandemic began in china in , before spreading to russia and, subsequently, across all of europe. interestingly, this second pandemic had a high proclivity for young adults [ ] . two major pandemics also occurred throughout the nineteenth century. the first began in in china, with subsequent spread to southeast asia, russia, europe, and north america and had a low overall mortality rate [ , , , ] . a second pandemic emerged in russia in and spread rapidly to europe and north america, circumnavigating the globe in just months [ , ] . the virus, suggested to be of subtype h n , reappeared at least more times in successive years resulting in an estimated million global fatalities [ , , , ] . four influenza pandemics have occurred over the past century ( fig. ) . the - spanish flu pandemic, subtype h n , resulted in an estimated - million deaths worldwide and will be discussed in detail in the following sections. the - asian flu pandemic, subtype h n , originated in china in february and spread throughout asia and then globally by the summer. case fatality rates were approximately . % with - million deaths worldwide [ , [ ] [ ] [ ] [ ] . just a decade later, the - hong kong flu pandemic, subtype h n , emerged in china in july and spread throughout europe, north america, and australia by early [ ] . although mortality rates were low, the pandemic would ultimately claim between , and million lives [ ] . in april , the - swine flu pandemic, subtype h n , began with nearly simultaneous outbreaks in mexico and the us, before spreading globally over the next weeks. while the - pandemic had a low associated case fatality rate, resulting in , deaths worldwide, it had devastating effects on global economies and healthcare networks [ , ] . conventionally, influenza pandemics result in the extinction of previously circulating virus strains; however, this view was complicated by events in . although h n was replaced by h n as the circulating strain following the - asian flu pandemic, a descendant of the virus "re-emerged" suspiciously in , likely as a result of a man-made event, and established itself as a co-circulating strain, along with the reassortant h n virus (following the - hong kong flu pandemic) [ , ] . the suspicious "re-emergence" of a descendant of the virus in has been postulated to have been the result of a man-made event. this hypothesis has gained traction, as both the ha and na of the re-emerged virus show incredible similarity to a reference virus, and it is unlikely that this strain was maintained in an animal reservoir for almost two decades without having undergone detectable mutation [ ] . in , a triple reassortment (made up of avian, swine, and human influenza genes) pandemic h n jumped from pigs to humans, resulting in the co-circulation of three influenza strains [ ] . the first wave of the pandemic one hundred years following its emergence, the origin of the pandemic influenza virus remains shrouded in mystery. the pandemic began early in the final year of the first world war. whereas prior pandemics had spread largely along trade routes, the global context of the war enabled greater viral spread facilitated by the mass mobilisation of military personnel and civilians [ , ] . this was further augmented by the poor health and sanitation conditions found within trenches along the frontlines of the war, facilitating disease transmission [ ] . public knowledge regarding the severity of the pandemic was hindered, as many news agencies were barred from writing about the global health threat, instead reporting solely on morale boosting subjects [ ] . however, as spain was a neutral party in the war, newspapers were able to report on the devastating effects that the pandemic virus was exhibiting in spain. thus, it was generally perceived that this devastating illness originated in spain, resulting in the pandemic being incorrectly labeled as "the spanish flu" [ ] . a century following its emergence, there remains a relative paucity of knowledge regarding the ancestry and regional origin of the virus. sequence analysis suggests that the virus was derived from an avian-like influenza virus and that all eight gene segments likely evolved in parallel [ , ] . analyses of influenza virus genome sequences also suggest that the initial entry of the precursor virus into human circulation began in and did not appear to have jumped directly from an avian source [ , , ] . however, improved understanding regarding the emergence of the virus, as well as factors (biological, social, environmental) that contributed to viral transmission and pathogenesis, have been vital to the development of current epidemic and pandemic influenza outbreak response efforts. descendants of the pandemic influenza virus strain have been the cause of almost every seasonal influenza a infection worldwide over the past century [ ] . additionally, each of the pandemics occurring in , , and were caused by descendants of the pandemic ( , , and ). circulation of h n was reinitiated in and has therefore been added to this timeline. grey arrows designate the circulating or co-circulating strains during the interpandemic periods influenza virus strain, earning the viral strain the nickname "the mother of all pandemics" [ ] . investigations concerning the origins of the first wave of the pandemic, beginning in march , have primarily focused on the us and china, though recently it has been suggested that the origin may have been an outbreak of a respiratory disease misidentified as pneumonic plague in china [ , , ] . humphries suggests that the dissemination of labourers from china to assist allied war efforts during this outbreak resulted in the inadvertent spread of the virus to europe [ ] . from to , the route of travel to europe for the labourers included checkpoints in singapore, durban, cape town, north africa, and canada. additional reports of the first wave of the virus in the spring of suggest that the pandemic originated with chinese workers at camp funston, kansas, where the workers began suffering from to day fevers, gastrointestinal symptoms, and general weakness [ , ] . within weeks soldiers had been hospitalized, and thousands more had received out-patient treatment [ ] . the illness was able to spread to other military camps within the us, before traversing the atlantic ocean via soldiers supporting allied operations in europe. the us army reported that from march-may , . % of us soldiers were hospitalized due to this unidentified respiratory illness [ ] . while illness rates were high during this initial wave, mortality rates were largely similar to seasonal outbreaks of influenza. spain reported that the mortality rates for pneumonia and influenza was only . % [ ] . although there was some acceptance that this new illness was indeed influenza, this was not generally accepted [ ] . radusin reported that although the physiological symptoms were similar to influenza, the illness was too mild and short-lasting with minimal complications for it to be influenza [ ] . infections began to subside in many regions by the early summer [ ] . the generally accepted lines of spread of the first and second waves of the virus are provided in fig. . in mid-august of , reports suggesting a second wave of this severe illness began to surface [ ] . in some regions, primarily northern europe, the period between the end of the first wave and the beginning of the second wave was incredibly short, making the two waves almost indistinguishable [ , ] . this second wave, occurring from september-november , was responsible for the majority of illnesses and fatalities associated with the pandemic. although the origins of the first wave [ ] continue to be debated, the origin of the second wave is generally agreed to be the harbour town of plymouth in southern england, which allowed the pandemic influenza virus strain to easily spread to the rest of the world [ ] . ships from plymouth were dispatched to freetown, sierra leone in august , which allowed the virus to spread across the african continent [ ] . new zealand soldiers, who stopped in freetown on their way to and from the war front in europe, facilitated transfer of the pandemic virus to new zealand [ ] . from plymouth, the virus also spread to boston, from which it was able to disseminate across the rest of north america resulting in > million fatalities over the ensuing four months [ , ] . this second wave spread globally throughout the fall of with illness seen first amongst military personnel and, subsequently, within the general population [ , ] . the second wave of the pandemic differed from the first in that much higher morbidity and mortality rates were reported, with the majority of all fatalities associated with the pandemic occurring during this wave [ ] . ultimately, the pandemic would result in an estimated million infections worldwide (~ / of the world's population at the time) and a case fatality rate > . %, more than times higher than any other pandemic [ , ] . as a testament to the severity of this second wave, during the fall of , the first - pages of spanish newspapers were filled with obituaries of those who had succumbed to the pandemic virus [ ] . further, reports from philadelphia, pennsylvania stated that across hospitals in the city, every hospital bed was occupied by patients with influenza [ ] . the pandemic was especially problematic in highly isolated communities where many individuals had limited contact with prior influenza strains, thus lacking any pre-existing immunity. for example, some inuit settlements reported case mortality rates as high as %, while certain communities in africa were completely decimated [ ] . interestingly, individuals who had been infected throughout the first wave seemed to be protected against this secondary wave, and recent analyses have suggested that these individuals had up to % protection throughout the fall wave [ , ] . a third and final wave of the pandemic appeared in most of the world in the early months of [ , , ] . this final wave generally overlapped the first wave in terms of regional distribution; however, it seemed to spare areas where the second wave had been especially severe. overall, morbidity rates were lower throughout this final influenza wave; however, mortality rates are believed to have been just as severe as the second wave [ , ] . three successive annual winter post-pandemic recurrences occurred following the third wave of the pandemic with continually decreasing mortality rates, in particular within those - years of age [ ] . classically, fatal influenza infections are primarily associated with the very young (< years) and the elderly (> years) resulting in a characteristic "u"-shaped mortality curve (fig. ) . interestingly, however, the - h n influenza pandemic mortality curve exhibits a "w"-shape due to excess mortality in young adults - years of age due to influenza-related illness. it has been postulated that the increased disease severity in young adults was likely associated with immune status due to the lack of pre-existing immunity in this population [ ] . further, more than % of fatal infections occurred in those < years of age and nearly % of all influenza-related deaths during the pandemic were in those aged - years [ ] . influenza and pneumonia fatality rates in those aged - years were more than times higher than in previous years and absolute risk of influenza-related death was higher in those < years of age than those > years old [ ] . it is still not fully understood why this occurred, but it is possible that an antigenically similar influenza strain circulated prior to , providing a level of protection against the novel h n pandemic strain to those born prior to [ ] . additionally, archaeserological and epidemiological evidence have shown that an h subtype influenza virus may have been responsible for the influenza pandemic, which circulated until the emergence of the pandemic virus, leaving those individuals who had not been exposed to an h subtype virus highly susceptible to the pandemic virus [ ] . it has also been suggested that the generation of an excessive inflammatory response ("cytokine storm") in healthy, young adults infected with the virus may have contributed to the excess mortality seen within this age group [ ] . recent in vivo studies with the virus have shown a marked upregulation of inflammatory cytokines, along with the suppression of important antiviral immune responses [ , ] . in addition, other influenza strains, such as fatal h n infections in humans, have also been associated with the deleterious consequences of an excessive inflammatory response [ ] . ultimately, the case fatality rate was so severe in young adults during the - pandemic that the average life expectancy rate in the us dropped by~ years [ ] . physiological symptoms of the pandemic virus generally lasted for days and were described as feeling cold, shivering, high fever, weakness, nausea, loss of appetite, pharyngitis, cough, and bloodshot eyes [ ] . in some patients, a short "rebound" to normal health would occur that was followed by an aggressive recrudescence of disease and, ultimately, death [ ] . similar to the pandemic, the majority of fatal infections resulted from respiratory complications. however, it has also been demonstrated that excess influenza fatalities during the - pandemic were associated with an acute aggressive bronchopneumonia (including epithelial and vascular necrosis, hemorrhage, edema, and bacterial-associated variant pathology within the lungs) and a severe acute respiratory distress-like syndrome associated with severe facial cyanosis [ ] . autopsies performed on preserved lung tissues in the modern era have revealed acute pulmonary hemorrhage and secondary bacterial infections associated with pulmonary lesions in nearly all the fatal cases examined [ , , ] . streptococcus pneumoniae was present in many cases; however, staphylococcus aureus, haemophilus influenzae, and streptococcus pyogenes also appeared to complicate fatal cases [ , ] . neutrophilic pulmonary infiltration was seen in cases of pneumococcal pneumonia, while cases of staphylococcal pneumonia were marked by multiple microabscesses infiltrated by neutrophils [ ] . however, alveolar cell damage was seen in each case along with pulmonary repair and remodelling [ ] . tissues from each of the fatal cases examined had similar pathologic presentation, independent of which pandemic wave they were associated with. despite the difference in mortality rates, each wave showed similar cellular tropism, infecting both type i and type ii pneumocytes, as well as the bronchiolar respiratory epithelium [ ] . a multitude of scientific and technological advances have occurred over the past century, allowing for a greater understanding of the dynamic relationship between the host and influenza viruses during infection. these advances, along with access to autopsy samples and the reconstitution of the pandemic virus, have facilitated a greater understanding of how the pandemic virus differs from other seasonal and pandemic influenza virus strains. moreover, technological advancements following the - influenza pandemic virus have facilitated the development of preventative measures, including vaccines and antivirals, to limit widespread illness due to influenza infections. the determination of the genomic sequence of the pandemic virus, and the subsequent reconstruction of the virus, has provided us with the opportunity to decipher the viral-and host-specific properties that contributed to the severity of the - pandemic. it has been demonstrated that in contrast to other influenza viruses, the pandemic virus is highly virulent and pathogenic in multiple animal species without prior adaptation [ , ] . while obvious knowledge gaps remain, in particular with respect to the origin of the virus and the molecular mechanisms (host and/or viral) underlying differential pathogenesis as compared to other influenza viruses, there have been considerable advances in our understanding of the pandemic virus. [ , ] . means with standard deviations are presented for the prepandemic mortality curve. adapted from taubenberger and morens [ ] since the isolation of the first human influenza virus in , researchers have worked to develop an effective influenza vaccine [ ] . current influenza vaccines are reformulated seasonally and provide protection against circulating influenza a and b viruses [ ] . the world health organization conducts worldwide surveillance studies throughout the year on currently circulating influenza strains, and thus recommends which strains should be included in each influenza vaccine [ ] . while the seasonal influenza vaccine is approximately % effective, this protection is dependent on the characteristics of the individual being vaccinated, including age and overall health, as well as the match between the strains included in the vaccine formulation and currently circulating strains [ ] . individuals who have been vaccinated are generally protected from illness and provide a measure of protection for those who are not able to be vaccinated due to their age or other health issues through herd immunity [ ] . there has also been increasing interest in the development of "universal" influenza vaccines designed to provide protection against a wide range of antigenically-distinct influenza viruses, including those currently in circulation and those that may emerge in the future [ ] . these will not be discussed in detail as recent reviews have provided excellent discussions of this topic [ ] [ ] [ ] [ ] [ ] [ ] [ ] . two major classes of antivirals have emerged for therapeutic treatment of severe influenza virus infections. adamantane antivirals target the matrix- (m ) surface protein, while neuraminidase (na) inhibitors target the na viral surface protein. adamantane compounds were the first licensed influenza antivirals and block the m ion channel protein from properly functioning, thus effectively blocking membrane fusion [ , ] . unfortunately, adamantane antivirals are only able to target influenza a viruses limiting their application for influenza b virus infections [ ] . further, more than % of influenza a viruses are resistant to this class of drugs due to the high mutation rate of the virus [ , ] . thus, the use of na inhibitors is recommended [ ] . na inhibitors block the na surface protein and prevent the release of progeny virus and infection of additional cells [ ] . while resistance to na inhibitors has been observed in some influenza virus strains, they are still highly effective in the majority of patients [ ] . studies have shown that both adamantane antivirals and na inhibitors provide protection against the virus [ ] . although outside the auspice of this commentary, it should be mentioned that advances in mechanical ventilation modalities, including non-invasive positive pressure ventilation, from the s onwards, have provided an additional support mechanism for treatment of severely ill patients [ ] . the routine clinical use of antibiotics in the early twentieth century also heralded a new era for combating influenza viruses. as a testament to this, excess influenza mortality declined significantly from to onwards [ ] [ ] [ ] . however, the widespread general administration of antibiotics has resulted in an escalating public health crisis due to multi-drug resistance. this has impacted the treatment of severe influenza infections, as methicillin-resistant s. aureus (mrsa) is the most frequently isolated bacteria from patients with severe influenza-bacterial co-infections in the us [ , ] and complicated up to % of fatalities during the pandemic [ ] [ ] [ ] [ ] . influenza preparedness and lessons for the future although it has now been a century since the start of the spanish flu pandemic, lessons from this global health catastrophe continue to inform modern-day pandemic preparedness. investigations of the pandemic, including those with the reconstructed virus, have allowed researchers, as well as the global public, to understand the mechanisms that underlie pandemic emergence and escalation to public health crisis. it also allows researchers to predict the potential public health risks which may be caused by new pandemic viruses. for example, sequencing of the pandemic virus revealed similarities in the h protein of the pandemic virus, allowing researchers to predict that a lack of protection, and thus a high mortality rate may be seen in healthy, young adults throughout the h n pandemic [ ] . thus, when vaccines were limited during the early stages of the pandemic, young adults were prioritized over the elderly, who demonstrated some degree of protection to this influenza strain, resulting in a lower mortality rate in young, healthy adults [ ] . the average age for laboratory-confirmed fatalities during the pandemic was years in the us, supporting this vaccine prioritization initiative [ ] . additionally, the awareness of the complications caused by secondary bacterial co-infections from the pandemic ensured that the medical community was aware of this threat throughout the pandemic, likely resulting in a reduced mortality rate due to severe influenza infections with complications [ ] . however, the pandemic, albeit milder than previous pandemics in terms of overall mortality, resulted in significant strains on global healthcare networks and economies [ ] . in canada, direct healthcare costs (including hospitalizations, outpatient visits, and therapeutics) related to the pandemic have been estimated at $ billion cad, with $ million cad related directly to hospital care [ ] . a computational modeling study by smith and colleagues suggested that direct costs related to illness would be between . - . % of gdp in the uk for pandemics ranging from low to extreme [ ] . further, the - severe acute respiratory syndrome outbreak resulted in~$ billion total gdp loss in toronto alone [ ] . this highlights the importance of pandemic preparedness beyond a healthcare-centric approach to one that also includes downstream economic effects. the - pandemic resulted in incredible improvements to public health as well as scientific advances. however, our current understanding of influenza viruses, and their ability to cause illness in humans is still in its infancy in many aspects, and further underlines our inherent need for continued influenza research. the identification of key molecular determinants involved in the pathophysiology of severe influenza infections will also assist drug discovery and development strategies, including insights on appropriate timing for administration of antivirals and/or antibiotics. the development of efficacious broader-spectrum or "universal" influenza vaccines is also of incredible importance. the emergence of novel highly pathogenic avian influenza (hpai) viruses, including h and h subtypes, are of particular concern due to their pandemic potential. circulating hpai viruses are of potential concern to global public health [ ] . asian lineage avian influenza a (h n ), which circulates in fowl, is rarely found in humans but has resulted in life-threatening cases when able to establish stable lineages [ ] and h n has resulted in sporadic human infections in china resulting in > infections with an estimated % case fatality rate since [ ] . because hpai viruses can arise from previously known low-pathogenicity viruses with only minor mutations, it is important to be vigilant concerning these potential pandemic viruses [ , ] . in spite of the public health advancements in the years following the - pandemic, including widespread access in the developed world to an efficacious influenza vaccine, influenza viruses remain a global public health threat. this pas year, there were > , reported influenza infections, influenza-associated hospitalizations, and deaths across canada [ ] . further, during the - influenza season, vaccination rates in those - years of age was only and % in those ≥ , 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authors' contributions men and jk conceived of the ideas presented herein and made substantial contributions to the drafting and revising of the manuscript. both authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors have declared that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -ji qq q authors: lagare, adamou; maïnassara, halima boubacar; issaka, bassira; sidiki, ali; tempia, stefano title: viral and bacterial etiology of severe acute respiratory illness among children < years of age without influenza in niger date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: ji qq q background: globally, pneumonia is the leading cause of morbidity and mortality in children, with the highest burden experienced in sub-saharan africa and asia. however, there is a dearth of information on the etiology of severe acute respiratory illness (sari) in africa, including niger. methods: we implemented a retrospective study as part of national influenza sentinel surveillance in niger. we randomly selected a sample of nasopharyngeal specimens collected from children < years of age hospitalized with sari from january through december in niger. the samples were selected from individuals that tested negative by real-time reverse transcription polymerase chain reaction (rrt-pcr) for influenza a and b virus. the samples were analyzed using the fast track diagnostic respiratory pathogens plus kit (biomérieux, luxemburg), which detects respiratory pathogens including viral and bacterial agents. results: among the samples tested, ( %) tested positive for at least one viral or bacterial pathogen; in ( %) sample, only one pathogen was detected. we detected at least one respiratory virus in ( %) samples and at least one bacterium in ( %) samples. respiratory syncytial virus ( / ; %), rhinovirus ( / ; %) and parainfluenza virus ( / ; %) were the most common viral pathogens detected. among bacterial pathogens, streptococcus pneumoniae ( / ; %) and haemophilus influenzae type b ( / ; %) predominated. conclusions: the high prevalence of certain viral and bacterial pathogens among children < years of age with sari highlights the need for continued and expanded surveillance in niger. acute respiratory infections (aris) are responsible for substantial morbidity and mortality globally, especially in children < years of age, and the highest burden is observed in developing nations [ ] . in , approximately . million ari-associated deaths occurred among children < years of age of which % were in africa and southeast asia [ ] . both bacteria and viruses have been identified as the etiological agents of ari. the viruses most frequently detected in children with aris include respiratory syncytial virus (rsv), influenza virus (inf) types a and b, adenovirus (av), parainfluenza virus (piv), human metapneumovirus (hmpv) and rhinovirus (rv) [ ] [ ] [ ] ; however, the clinical presentations of respiratory tract infections are similar, making it difficult to distinguish between etiologic agents without a laboratory diagnosis [ ] . in addition, the interpretation of a viral detection is complicated by the fact that infections with multiple viruses are common in children with ari and many viruses are frequently found in asymptomatic children [ ] . streptococcus pneumoniae and haemophilus influenzae type b (hib) are the most commonly isolated bacteria from ari cases, while, other atypical pathogens such as mycoplasma pneumoniae and chlamydophila pneumoniae are less frequently reported [ ] [ ] [ ] . s. pneumoniae and hib are commonly identified in nasopharyngeal samples from asymptomatic children due to high rates of carriage; however, their identification from the nasopharynx is rarely indicative of invasive disease [ ] . the viral and bacterial etiology of ari has been well documented in countries from the northern hemisphere [ ] [ ] [ ] [ ] ; however, few studies are available from africa [ , ] . in niger, a sentinel surveillance system for influenza viruses was instituted in april ; however, no studies on the etiology of ari have been conducted in the country. we aimed to document the prevalence of selected viral and bacterial infections among children < years of age hospitalized with severe acute respiratory illness (sari) at selected hospitals in niger from january through december . niger is a west african country with a saharan climate characterized by four distinct seasons: the cold season from mid-december to mid-february, the dry and hot season from mid-february to may, the rainy season from june to september, and the hot season from october to mid-december [ , ] . since april , influenza surveillance has been conducted among patients hospitalized with severe acute respiratory illness (sari) at sentinel sites located in of the regions of the country by the centre de recherche médicale et sanitaire (cermes), the national reference laboratory for influenza. the influenza surveillance program in niger has previously been described [ ] . briefly, all patients hospitalized at the participating sentinel sites that met the sari case definition were eligible for enrollment. verbal informed consent was obtained from all cases who were years of age and older. proxy informed consent was obtained from parents or legal guardians of minors. patients who did not meet the case definition or did not provide verbal consent were not included. a sari case was defined as a hospitalized child < years of age with onset of cough or difficulty breathing within days prior to admission, and at least one of the following danger signs: inability to drink or breastfeed, lethargy, vomiting everything, convulsions, nasal flaring, chest indrawing, stridor in a calm child or tachypnea [ ] . a standardized questionnaire was administrated by clinical personnel, to record patients' demographic characteristics and medical history. the questions included information on date of enrollment and symptom onset, sex, age and clinical symptoms. nasopharyngeal (np) swabs were collected from all enrolled cases and placed in cryovials containing virus transport medium (copan kit, italy). the specimens were kept refrigerated at °c at the sentinel site and then transported twice weekly to cermes for testing. samples were aliquoted, screened for influenza a and b viruses by real-time reverse transcription polymerase chain reaction (rrt-pcr), and then stored at − °c. we conducted a retrospective study on the etiology of influenza-negative sari cases among children < years of age enrolled in influenza surveillance during january through december in niger. we randomly selected (using random selection procedures available in stata) a sample of stored np specimens from sari cases which had tested influenza a and b negative. this sample represented % of the total cases aged < years enrolled during the study period. only . % of sari cases in the < year old age group were tested positive for influenza. influenza-positive cases were mainly detected during the cold season [ ] . nucleic acid was extracted using the qiaamp mini kit (qiagen, germany) in accordance with the manufacturer's protocol. an internal control (ic) was added to each extraction tube in order to assess the quality of the extraction at the end of the amplification. the Χ and the fisher's exact tests were used to assess the difference between categorical variables by comparing expected and observed frequencies across evaluated groups. in addition, we compared the characteristics of selected and non-selected children (including influenza-positive cases) using the x statistics. the statistical analysis was implemented using stata version . (statacorp®, texas, usa). in , the national ethics committee of niger approved the national influenza surveillance program (reference no / /ccne of april ). in , prior to the investigation of other respiratory pathogens, the ministry of health provided an extended approval. access to the study samples was granted by the director of cermes. from january through december we enrolled children < years of age hospitalized with sari into the influenza surveillance program, of which ( %) tested negative for influenza virus. of these, ( %) were randomly selected for our study. the age, sex and symptom duration distribution did not differ significantly among selected and non-selected children (including those that tested positive for influenza). however, among the selected group there was a significantly lower proportion of specimens collected during the cold season, when the majority of influenza-positive cases were detected and excluded from randomization (table ) . among the selected children, % ( / ) were < year of age (median age among children age < years: months), % ( / ) were female and % ( / ) had a duration of symptoms ≤ days. most patients presented with a recorded temperature > °c ( %; / ), cough ( %; / ), dyspnea ( %; / ) and chest indrawing ( %; / ). few patients presented with tachypnea or had difficulty in breastfeeding ( %; / ). overall, / ( %) of children included in the study tested positive for at least one pathogen (viral or bacterial). at least one respiratory virus was detected in / ( %) samples and at least one bacterium was detected in / ( %) samples. among the samples positive for any pathogen, ( %) were positive for a single pathogen. of these ( %) were positive for s. pneumoniae, ( %) for rsv and ( %) for rv, while hmpv, cv, piv and hib each accounted individually for < % of the single organisms detected (fig. ) . among the children in whom ≥ organisms were detected, both viral and bacterial pathogens were detected in samples ( %). among the samples tested, rsv (n = ; %) was the most frequently detected virus, followed by rv (n = ; %) and piv types - (n = ; %) ( table ) . cv, hmpv, hbv, ev and av were detected individually in < % of the specimens. no pv was detected in our study. of the samples that tested positive for cv, ( %) were type oc , ( %) were type e, ( %) were type nl and ( %) were type hku . of the samples that tested positive for piv, ( %) were type , ( %) were type , ( %) were type and ( %) were type . even though we selected co-detections with different subtypes were detected in ( %) and ( %) of the cv and piv positive cases, respectively. rsv, piv and hmpv were detected more frequently in infants < year of age compared to children - years of age (table ) . rsv, piv and hmpv were detected more frequently during the hot season (october to mid-december); while rv and hbv were detected more frequently during the rainy season (june to september). the other viruses were detected with similar frequencies across seasons (table ) . among the samples tested, s. pneumoniae (n = ; %) was the most frequently detected bacteria, followed by hib (n = ; %), s. aureus (n = ; %) and c. pneumoniae (n = ; . %) ( table ) . m. pneumoniae was not detected in our study. we report the detection rate of selected viral and bacterial pathogens among children < years of age hospitalized with sari in niger. we detected respiratory viruses in % of our study sample. the high detection rate of viruses found in our study is consistent with results from similar studies conducted in burkina faso ( %) [ ] , kenya ( %) [ ] and brazil ( %) [ ] . however, lower rates of viral detection were reported from other studies from countries such as ghana ( %) [ ] , china ( %) [ ] and egypt ( %) [ ] . these differences can be attributed to different climatic conditions, enrollment criteria, case definitions and testing platforms. in our study, rsv was the predominant virus detected and was most commonly found in children < year of age. rsv has been reported to be an important pathogen in children and especially in young infants in several studies [ , [ ] [ ] [ ] [ ] [ ] . in addition, rsv detection has been reported to be strongly associated with illness from studies comparing symptomatic cases to controls [ ] . rhinovirus was the second most commonly detected virus ( %) with similar rates among infants < year of age and children aged - years, which has been reported in previous studies [ , ] . however, other studies reported rv as the most prevalent virus among children < years of age [ , , , ] . in addition rv has been commonly detected among asymptomatic persons in several studies indicating that rv can act as both pathogen and by-stander, consequently hindering the ability to infer an association between detection and illness [ ] [ ] [ ] . among the piv and cv detected in this study, piv type and cv type oc were the most common virus types, which has been reported in other studies [ , ] . we also found a high detection rate of bacterial pathogens. s. pneumoniae ( %) and hib ( %) were the most common bacteria detected in nasopharyngeal specimens. elevated colonization rates of these bacteria have been reported in children, but only a proportion of colonizations result in invasive disease [ , , ] . the high detection rate of s. pneumoniae in our study is likely due to the fact that s. pneumoniae is a commensal of the nasopharynx [ ] . it has been shown that the prevalence of s. pneumoniae carriage in healthy children < years of age ranges from % to % in low income countries [ ] . the detection of s. pneumoniae from sterile sites like blood or cerebrospinal fluid, reflecting invasive pneumococcal disease, has been shown to be lower ( - %) [ , ] . nonetheless, s. pneumoniae has been reported to be responsible for % of meningitis cases in niger based on cerebrospinal fluid testing; different serotypes were detected among cases of meningitis prior to the introduction of the pneumococcal conjugate vaccine in [ ] . hib and s. aureus, the nd and rd most prevalent bacterial pathogens in our study, have also been shown to be commensal organisms with high nasopharyngeal carriage rates especially in young children [ ] . the substantial hib nasopharyngeal colonization density found in this study should be investigated further as hib vaccine has been available in the niger expanded immunization program since . nasopharyngeal specimens may be used to aid in the diagnosis of certain bacterial respiratory pathogens that do not tend to colonize the nasopharynx, such as m. pneumoniae and c. pneumoniae [ , ] . c. pneumoniae was detected at low rates ( . %), and m. pneumoniae was not detected in our study. using serological methods, prevalence rates as high as % have been reported for c. pneumoniae [ ] ; in contrast, other studies report significant detection of m. pneumoniae (> %) and low detection of c. pneumoniae (< %) [ , , ] . in our study we found an elevated prevalence ( %) of viral-bacterial co-detections, which has been reported in other studies [ , ] . it has been shown that viral infections may predispose to bacterial super-infection by favoring bacterial attachment sites on nasopharyngeal epithelial cells and through increased mucous production that promotes bacterial growth [ , ] . our study has limitations that warrant discussion. first, the small sample size of our study hindered our ability to accurately assess the seasonality of the pathogens included in our study. nonetheless, our results suggest that rsv, piv and hmpv are more commonly detected during the hot season (october to december), while rv and hbv are detected more frequently during the rainy season (june to september). no difference in the detection rate of the other viruses and bacteria was noted across seasons in our study. the small sample size of our study also hindered our ability to detect patterns of co-detection and the association between specific viral and bacterial co-detections. second, we did not keep formal records of the proportion of patients consenting to participate in the sari surveillance. however, a review of the performance of the surveillance system implemented through hospital record review at sentinel sites revealed that only a few patients that met the study case definition were missed by the surveillance program. third, the lack of controls in our study limited our ability to assess the association of pathogen detection with disease. while most of the viral and bacterial pathogens identified in this study have been described by previous studies as causative agents of ari, the assignation of causality remains challenging [ , ] . fourth, influenzapositive samples were excluded from our study. codetection of other viral and bacterial pathogens with influenza is expected and this may have resulted in an underestimation of the prevalence of the pathogens included in this study, especially during the cold season when the majority of influenza-positive cases were detected. last, we did not systematically collect information on progression of illness (including in-hospital outcome) or risk factors for severe disease, which hindered our ability to evaluate pathogen contribution to the more severe spectrum of illness or to identify groups at high risk for severe disease. this study reports the detection rate of viral and bacterial pathogens among children < years of age hospitalized with sari in niger. the high prevalence of certain viral and bacterial pathogens highlights the need for expanded surveillance in niger so as to inform policies and interventions. given the high rsv detection rate observed in this study and the reported association of rsv detection with illness [ ] , rsv should be included in routine surveillance programs in niger. other selected pathogens could be considered for routine surveillance in the country following further assessment to determine association with illness. in addition, information 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molecular epidemiology of human rhinovirus infections in kilifi, coastal kenya genetic diversity and molecular epidemiology of rhinoviruses in south africa the role of multiplex pcr test in identification of bacterial pathogens in lower respiratory tract infections high nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya determination of pneumococcal serotypes in meningitis cases in niger surveillance for hospitalized acute respiratory infection in guatemala bacterial and viral etiology in hospitalized community acquired pneumonia with molecular methods and clinical evaluation huamn coronaviruses associated with upper respuratory tract infections in three rural areas of ghana we are thankful to the national ministry of public health for financing this study and our partners: the pasteur institute of paris, the world health organization, and the us centers for disease control and prevention for providing reagents, the rrt-pcr machine and technical supports for influenza surveillance in niger. key: cord- - h wvv authors: li, fengqin; wang, yonglan; sun, linlin; wang, xiaoxia title: vancomycin-resistant enterococcus faecium pneumonia in a uremic patient on hemodialysis: a case report and review of the literature date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: h wvv background: even though enterococci can cause serious infections in multiple sites, they are a rare cause of pneumonia. we reported a uremic patient with vancomycin-resistant e. faecium (vre-fm) pneumonia, possibly related to epileptic seizures. case presentation: a -year old man with uremia on hemodialysis was admitted to the hospital with complaint of recurrent epileptic seizures, followed by a two-week history of recurrent fever and cough with purulent sputum. chest ct demonstrated multiple exudation of both lungs. he was diagnosed as community acquired pneumonia. despite antibiotic combination therapy, abnormal chest shadows aggravated. sputum and blood cultures were initially negative, but later blood culture grew vre-fm. we suspected aspiration of gastrointestinal content induced by epilepsy as the most likely mechanism. the patient was successfully treated with a four-week course of linezolid according to the antibiotic susceptibility testing. conclusions: physicians should consider multi-drug resistant organisms such as vre in uremic patients with pneumonia that fails to resolve with broad-spectrum antibiotics, especially in the cases with aspiration induced by epilepsy, immunocompromised conditions, and repeated or prolonged hospitalizations. enterococci are gram-positive cocci which normally inhabit the intestinal tract of humans. e. faecalis and e. faecium are the most common strains. they started appearing as common pathogens in nosocomial infections in the s. at the same time, antibiotic resistance among them started increasing [ ] . vancomycinresistant e. faecium (vre-fm) are multi-drug resistant micro-organisms, and the treatment options and infection control measures are limited. additionally, there is a low clinical awareness. therefore, infections caused by vre-fm are a unique challenge to the clinician. even though the most commonly reported enterococcus infections are intra-abdominal infections, urinary tract infections, bacteremia and endocarditis, pneumonia is rarely described [ ] . in this case report, we aimed to present a uremic patient with vre-fm pneumonia, possibly related to epileptic seizures, being treated successfully with linezolid. a -year old man with uremia on hemodialysis complicated by severe renal anemia, hypertension and heart failure, presented with a two-week history of recurrent fever and cough with purulent sputum. chest pain, nausea, vomiting, abdominal pain, diarrhea and night sweat was denied. he had been diagnosed with end-stage renal disease (esrd) nearly months ago, and the etiology was primary glomerulonephritis. hemodialysis with the central venous catheter started at that time. two weeks before admission, he suffered from recurrent epileptic seizures, characterized by convulsions of the whole body, unconsciousness and fecal incontinence in other hospital. shortly afterwards, fever and cough with yellow phlegm gradually appeared. his labs were notable for white blood cell count (wbc) of . × /l with % neutrophils, hemoglobin (hb) g/l, and c-reactive protein (crp) . mg/l. blood and sputum cultures were negative. computed tomography (ct) scan of the chest revealed pneumonia. intravenous vancomycin ( . g three times a week) and meropenem ( . g q h) was administered empirically for suspected aspiration (given his lethargy after epileptic seizure). then the symptoms improved gradually and indicators of infection dropped to normal during week. unexpectedly, the patient had fever again on the day of admission, with a temperature of . °c, accompanied by deteriorating general status. therefore, he was transferred to our hospital for further treatment. physical examination was significant for appearance of severe anemia, a temperature of . °c, decreased breath sounds at the lung bases bilaterally, a diffuse moist rale on respiratory exam and a slight exudation around the right jugular hemodialysis catheter. initial laboratory investigations revealed wbc of . × /l with . % neutrophils, hb g/l, crp . mg/l, procalcitonin (pct) . ng/ml, scr . umol/l and btype natriuretic peptide (bnp) greater than pg/ml. chest ct demonstrated multiple exudation of both lungs, bilateral pleural effusion and atelectasis of both lower lobes (fig. a) . no valvular vegetation was found in echocardiography, and left ventricular ejection fraction (lvef) was %. initial diagnosis of admission was esrd with hemodialysis, sever renal anemia, community acquired pneumonia, heart failure and suspected catheter-related infection. treatment with intravenous piperacillin / tazobactam ( . g q h) for pneumonia and correction of heart failure and anemia were commenced. the dialysis catheter and urinary catheter were removed and peripheral blood cultures were collected. but his fever with a maximum temperature of °c still persisted. three consecutive blood, sputum culture and catheter cultures were negative. additionally, thoracentesis and drainage were performed and hydrothorax culture was also negative. laboratory detection of tuberculosis and fungi was all negative. antibiotics were switched successively to cefoperazone sodium / sulbactam sodium ( . g q h) + moxifloxacin ( . g/day) + fluconazole ( . g/day), and vancomycin ( . g three times a week) + meropenem ( . g q h), according to consultation results of respiratory department. however, no apparent improvement was noted, and his general condition deteriorated progressively. response of temperature and indicators of infection to antibiotic therapy was shown in fig. and fig. . a repeated chest ct showed increased multiple exudation of both lungs (fig. b) . finally, fourth blood culture became positive for e. faecium at> cfu/ml (vana genotype) on hospital day . at that point, antibiotic therapy was switched to intravenous linezolid ( mg q h) based on the sensitivity pattern of isolates that were vancomycin, moxifloxacin, gentamicin, penicillin, rythromycin and ampicillin resistant, and linezolid, teicoplaninand and tegafycline sensitive. in the following days, the fever subsided gradually (fig. ). and the crp and pct levels decreased steadily (fig. ) . the patient completed a four-week course of linezolid with complete resolution of chest ct abnormalities (fig. c ). e. faecalis is more pathogenic than e. faecium, but the latter exhibits more resistance, taking up the majority of vre infections [ ] . vre is an important nosocomial pathogen spreading in hospitals worldwide. it was reported by the national healthcare safety network (nhsn) that . % of enterococcal hospital-associated infections were resistant to vancomycin from to , which ranked as the second most common cause of nosocomial infections in the us [ ] . additionally, markwart et al. reported the proportion of vre-fm was increasing from . % in to . % in in german hospitals, particularly in southern regions in germany [ ] . in china, there was a rapid increase in vancomycin resistance from . % in to . % in among nosocomial enterococcal isolates in icu, according to the taiwan nosocomial infection surveillance system [ ] . however, pulmonary infections due to enterococcus are distinctly unusual. a prospective and observational study of patients with serious infections due to enterococcus across six hospitals found that there was % of those infections located in the respiratory tract over the course of year [ ] . the best evidence that enterococcal pneumonia is rare has been reported by richards et al. they found only two cases of enterococcal pneumonia among , patients evaluated for a total of , patient-days in american medical intensive care units [ ] . recently, according to the national healthcare safety network, only % of ventilator-associated pneumonias were caused by enterococcus spp. [ ] . we conducted a literature search based on pubmed in an attempt to identify all published cases of pleuropulmonary infection due to enterococcus, without time limits. twenty-four cases of enterococcus-associated pleuropulmonary infection previously published in the literature were eventually summarized in table [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the enterococcal pneumonia cases previously described in the literature mainly occurred in elderly patients with immunosuppression, organ transplantation, hematological malignancies, solid cancer, renal failure, cardiocerebrovascular disease and chronic obstructive pulmonary disease. ten of the cases of enterococcus pneumonia were complicated by lung abscess and empyema requiring drainage. there were cases of pneumonia due to e. faecium of which were vancomycin-resistant. to our knowledge, ours is the first vre-fm pneumonia after epileptic seizure in a patient with esrd on hemodialysis. interestingly, the patient had recurrent epilepsy before he developed pneumonia, which raised the likelihood that aspiration of gastrointestinal content induced by epilepsy (given his altered mental status and lethargy) occurred at some point. and his pulmonary infiltrates involved the middle and lower parts of two lungs, which were typical for aspiration mechanism. according to previous reports, enterococcal-associated aspiration pneumonia also occurred in patients fed by dobb-hofftube and experiencing near drowning [ , ] . since our patient was admitted with a hemodialysis catheter accompanied by purulent exudation at the outlet of the catheter, recurrent fever, heart failure and positive blood culture, another possible mechanism that pneumonia secondary to a septic embolus originated from the dialysis catheter or the heart was suggested. however, imaging of vessels at the catheter and an echocardiography showed no embolism and the catheter culture was negative in the current case. from the cases summarized in table , it is notable that only cases of enterococcal pneumonia were associated with endocarditis [ , ] . furthermore, previous literature data reported cases of vre-fm pneumonia were treated with linezolid, of which survived and died. in contrast, the isolate recovered from our patient was sensitive to linezolid according to the drug sensitivity test, and his infection indicators and body temperature showed good response to the treatment of linezolid, and the chest ct abnormalities completely resolved after a four-week course of linezolid. previous case reports of enterococcal pneumonia were based on clinical findings and infiltrate on chest x ray or ct in combination with the isolation of enterococci in cultures from a transtracheal aspirate, protected brush (ps), bronchoalveolar lavage (bal), sputum or lung tissue. since isolation of enterococcus from respiratory secretions usually represents colonization, lung tissue, ps or bal culture may identify true infection of the lower respiratory tract more accurately than the sputum or endotracheal aspirate cultures. however, in our case, bronchoscopy and lung needle biopsy was not performed to obtain lung tissue, ps or bal specimens, because the patient was seriously ill at that time. evidence supporting a diagnosis of vre-fm pneumonia in our patient included persistent respiratory symptoms, multiple infiltration on chest ct, bacteremia with vre-fm and good response to linezolid based on susceptibility testing. currently, nine phenotypes of vancomycin resistance described are van a, van b, van c, van d, van e, van g, van l, van m and van n [ ] . van a contributes to most of the human cases of vre around the world, and is mostly carried by e. faecium. moreover, a study by bocanegra-ibarias et al., which involved phenotypic and genotypic characterization of vre-fm clinical isolates from two hospitals in mexico, first detected vanb phenotype-vana genotype [ ] . hypermutability, increased mobile genetic elements, metabolic alterations and hypermutability confer drug resistance to e. faecium. our patient was found to be infected by e. faecium with van a gene, so he was clinically unresponsive to vancomycin. the main causes for emergence of vrefm in our patient may be his immunocompromised conditions due to esrd, repeated hospitalizations, mechanical instrumentation (invasive hemodialysis catheter) and exposure to multiple antibiotics (specifically vancomycin). therefore, clinicians should minimize unnecessary invasive procedures and vancomycin abuse. deescalation from initial broad-spectrum antibiotics to narrow spectrum antibiotics immediately after receiving the antibiotic sensitivity report is necessary. additionally, strict adherence to infection control practices can prevent further emergence and spread of drug resistance. in conclusion, vre pneumonia is rarely reported. physicians should consider multi-drug resistant organisms such as vre in uremic patients with pneumonia that fails to resolve with broad-spectrum antibiotics, especially in the cases with aspiration induced by epilepsy, immunocompromised conditions, and repeated or prolonged hospitalizations. better clinical outcomes can be expected if the empirical antibiotic treatment covers vre and early adjustment of sensitive antibiotics based on susceptibility testing. the datasets used and/or analyzed during the case report are available from the corresponding author on reasonable request. ethics approval and consent to participate not applicable. written informed consent for publication of his clinical details and/or clinical images was obtained from the patient. a copy of the consent form is available for review by the editor of this journal. vancomycinresistant enterococci vancomycin-resistant enterococcal infections: epidemiology, clinical manifestations, and optimal management antimicrobial-resistant pathogens associated with healthcare-associated infections summary of data reported to the national healthcare safety network at the centers for disease control and prevention the rise in vancomycin-resistant enterococcus faecium in germany: data from the german antimicrobial resistance surveillance clinical and microbiological characteristics of vancomycin-resistant enterococcus faecium bloodstream infection in central taiwan an analysis of serious enterococcal infections. epidemiology, antibiotic susceptibility, and outcome nosocomial infections in medical intensive care units in the united states antimicrobial-resistant pathogens associated with healthcare-associated infections: annual summary of data reported to the national healthcare safety network at the centers for disease control and prevention the disease picture in enterococcal pneumonia enterococcal lung abscess: medical and surgical therapy empyema and splenic abscess in infective endocarditis enterococcal bacteremia: clinical features, the risk of endocarditis, and management. medicine (baltimore) a rare case of pleuropulmonary infection and septic shock associated with enterococcus faecium endocarditis aerosol polymyxin and pneumonia in seriously ill patients community-acquired pneumonia caused by enterococcus faecium enterococcal empyema community acquired pneumonia due to enterococcus. an entity for consideration? a critical pneumonia by multidrug-resistant enterococcus faecium in a chronic hemodialysis patient. a case report enterococcus faecium as a cause of pulmonary abscesses in kidney transplant recipient pleural enterococcus faecalis empyema: an unusual case enterococcal-associated lower respiratory tract infections: a case report and literature review enterococcus pneumonia complicated with empyema and lung abscess in an hiv-positive patient. case report and review of the literature a case of pneumonia due to enterococcus faecium after near drowing strongyloides hyperinfection syndrome and vre pneumonia vancomycin-resistant enterococcus faecium empyema in an asplenic patient enterococcal pneumonia. occurrence in patients receiving broad-spectrum antibiotic regimens and enteral feeding high prevalence of vanm in vancomycin-resistant enterococcus faecium isolates from shanghai phenotypic and genotypic characterization of vancomycin-resistant enterococcus faecium clinical isolates from two hospitals in mexico: first detection of vanb phenotype-vana genotype the authors express great gratitude to all members of the department of radiology and all members of the department of nephrology at tongren hospital, shanghai jiao tong university school of medicine for their contributions to this case. the authors declare that they have no competing interests.received: august accepted: february authors' contributions fl analyzed the patient data and draft the manuscript. yw and ls performed laboratory analysis and supervised the antibiotic therapy. xw conducted the writing and revision of the paper. all authors read and approved the final manuscript. key: cord- -pw coi v authors: ballus, josep; lopez-delgado, juan c.; sabater-riera, joan; perez-fernandez, xose l.; betbese, a. j.; roncal, j. a. title: surgical site infection in critically ill patients with secondary and tertiary peritonitis: epidemiology, microbiology and influence in outcomes date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: pw coi v background: surgical site infection (ssi) remains a significant problem in the postoperative period that can negatively affect clinical outcomes. microbiology findings are typically similar to other nosocomial infections, with differences dependent on microbiology selection due to antibiotic pressure or the resident flora. however, this is poorly understood in the critical care setting. we therefore aimed to assess the incidence, epidemiology and microbiology of ssi and its association with outcomes in patients with severe peritonitis in the intensive care unit (icu). methods: we prospectively studied consecutive patients admitted to our surgical icu from to with a diagnosis of secondary or tertiary peritonitis. we collected the following data: ssi diagnosis, demographics, acute physiology and chronic health evaluation (apache) ii score, simplified acute physiology score (saps) ii score, type of surgery, microbiology, antibiotic treatment and outcomes. microbiological sampling was done by means of swabs. results: we identified episodes of ssi in patients ( . %) aged . ± . years, of which episodes occurred in men ( . %). the mean apache ii and saps ii scores were . ± . and . ± . respectively. the mean icu and hospital stays were . ± . and . ± days respectively. pseudomonas spp. (n = , . %), escherichia coli (n = , . %) and candida spp. (n = , . %) were the most frequently isolated microorganisms, but gram-positive cocci (n = , . %) were also frequent. microorganisms isolated from ssis were associated with a higher incidence of antibiotic resistance ( . %) in icu patients, but not with higher in-hospital mortality. however, patients who suffered from ssi had longer icu admissions (odds ratio = . , % confidence interval . – . , p = . ). conclusions: the incidence of ssi in secondary or tertiary peritonitis requiring icu admission is very high. physicians may consider antibiotic-resistant pathogens, gram-positive cocci and fungi when choosing empiric antibiotic treatment for ssi, although more studies are needed to confirm our results due to the inherent limitations of the microbiological sampling with swabs performed in our research. the presence of ssi may be associated with prolonged icu stays, but without any influence on overall mortality. the skin is the main barrier against bacterial infection of internal tissues, and surgical wounds create a physical disruption to that barrier. the movement of bacteria across the skin barrier can lead to surgical site infections (ssi), one of the most frequent infectious complications of surgical procedures, with the potential risk for adverse outcomes [ ] . ssi involves different inflammatory responses that range from low to high clinical significance [ ] , with that following abdominal surgery being a typical example associated with increased morbidity and mortality [ ] [ ] [ ] [ ] . worse still, ssi can spread to surrounding areas and vital deep structures, often requiring debridement or drainage [ , ] . consequently, the treatment of ssi leads to increased costs, especially when we consider the high number of surgical procedures and their complexity in a typical referral hospital [ ] [ ] [ ] [ ] . peritonitis, which is defined as inflammation of the serous membrane that covers the abdominal cavity and their organs, is classified into primary (spontaneous), secondary (process-related pathology in the visceral organs) and tertiary (persistent or recurrent after initial adequate surgical treatment). secondary and tertiary peritonitis are associated with higher morbidity with mortality rates of - % [ , ] . tertiary peritonitis usually occurs in icu settings at least - h following adequate treatment of secondary peritonitis, and has a mortality rate of - % [ ] . centers for disease control and prevention (cdc) estimates that the risk of ssi associated with abdominal surgery ranges from approximately to %, depending on the type of surgery [ , , , ] . ssi is classified into several categories: clean ( %), clean-contaminated ( %), contaminated ( %) and dirty ( %) [ , ] . stratification before surgery could help identify at-risk patients suitable for surveillance [ ] . despite the marked influence of ssi associated with severe peritonitis on public health and clinical practice, it has been poorly addressed in the literature, even in the icu setting. this study therefore aimed to describe the incidence, epidemiology, microbiology and outcomes of ssi in patients admitted with secondary or tertiary peritonitis to the icu of a tertiary referral hospital. this prospective, observational study was carried out at from january to december . at the time this study was performed, the hospital universitari de bellvitge (hub) was a tertiary hospital with general care beds and icu beds. we included all consecutive patients from any type of abdominal surgery who required icu admission beyond h for secondary or tertiary peritonitis. all patients received standard preoperative hygiene care and antibiotic prophylaxis at anaesthetic induction consistent with our institutional protocols for elective and emergency surgery [ ] . ssi was defined using the cdc definition [ ] and diagnosis was by the responsible physicians, based on clinical criteria. any purulent drainage from a surgical incision with signs of inflammation of the surrounding tissue was considered an ssi, whether microorganisms were isolated in cultures or not. the infection had to present at the surgical site within days of surgery [ ] [ ] [ ] . the study was approved by the institutional ethics committee of our hospital (comité d' Ètica i assajos clínics de hub (ceic); ethics and clinical assays committee of hub), and informed consent was waived due to the observational design. in all patients, the decisions regarding icu admission and treatment were made by the treating physician. data were recorded from the medical registry of each patient in real time, using a standardised questionnaire, and collected in a database. the following information was recorded on admission: demographic data, medical history and comorbidities, surgical indication and type of surgery (elective or emergency), surgical technique, intraoperative variables (number of drains inserted), microbiologic findings, arterial lactate on admission and treatment characteristics. during their icu stay, we also recorded the following: need for vasopressor drugs, mechanical ventilation and renal replacement therapy (rrt) and; any new microbiological findings, including the appropriateness, changes and resistance to antibiotic treatment. illness severity was quantified with the acute physiology and chronic health evaluation (apache) and simplified acute physiology score (saps) scoring systems during the first h of icu admission for all patients. after icu discharge, follow-up was planned to collected data on in-hospital mortality and patients were followed until discharge from icu or until resolution. the surgical teams collaborated with icu physicians to control the ssi, using simple washouts or serial debridements when appropriate. we obtained tissue samples and exudate samples, and direct needle aspiration was used when needed, in collaboration with the surgical team. the microbiological samples were obtained under conditions as sterile as possible in order to avoid colonizers of the superficial wound. the deepness of tissue sample was evaluated based on ssi characteristics. if necessary, drainage was performed, and any necrotic tissue was debrided and foreign material removed. intensive irrigation with saline solution was employed when necessary to facilitate mechanical debridement [ ] . we provided rational antibiotic therapy based on local guidelines and after consultation with an infectious disease physician. for diagnosis purposes, microbiological samples were sent to the laboratory as swabs in culture media for semiquantitative aerobic and anaerobic cultures. to isolate anaerobes, specimens were inoculated into columbia blood agar plates enriched with hemin and menadione, incubated in an anaerobic chamber at °c, and specimens were gram stained at and h for direct examination. statistical analysis was conducted using pasw statistics . (spss inc., chicago, illinois, usa). continuous data are expressed as mean ± standard deviation and categorical data are expressed as percentages. comparisons between groups with non-normal distributions were by two-sample t-tests or mann-whitney u tests after applying the one-sample kolmogorov-smirnov test. the χ -test was used to evaluate categorical variables. multivariable analysis was done to assess the influence of ssi and other ssi-related factors, such as the microbiology results, on mortality and outcomes. odds ratios (ors) and % confidence intervals (cis) are quoted as appropriate. a p-value of . was considered statistically significant in all cases. of the patients hospitalised for secondary or tertiary peritonitis in our icu, we identified episodes of ssi in patients. the ssi rate of . % was higher in icu compared with the rest of hospitalized patients who underwent major abdominal surgery (n = / ; . %) during the study period (p < . ). patient characteristics, inflammatory response, type of surgery and outcomes are shown in table . the mean duration of hospitalisation prior to surgery was . ± . days. urgent abdominal surgery comprised %- % of all abdominal surgeries performed at our hospital, showing a difference in the type of surgery in comparison with icu patients (p = . ). the types of surgery (based on the anatomical location) of the different identified ssi episodes are shown in fig. . all patients were monitored with a central venous catheter, arterial catheter and urinary catheter, and all patients were on vasoactive drugs or inotropic support. in the studied population, we identified positive culture isolates: ( . %) were from ssis, ( . %) were from intra-abdominal abscesses, ( . %) were from positive blood cultures (blood cultures) and ( . %) were from other cultures. the microbiology results for isolates from ssi are shown in table , with a preponderance of escherichia coli (n = , . %) and pseudomonas aeruginosa (n = , . %), while gram-positive cocci and candida spp. were also frequent at rates of . % (n = ) and . % (n = ) respectively. antibiotic resistance to two or more antibiotics occurred in . % (n = ), with rates of extended spectrum beta-lactamase-producing enterobacteriaceae (n = ) and pseudomonas aeruginosa carbapenem-resistant (n = ) of . % and . % respectively, but with low rates of typical multi-resistant microorganisms such as acinetobacter baumanii ( . %, n = ) and methicillin resistant staphylococcus aureus (mrsa) ( . %, n = ). the microorganisms isolated from intra-abdominal abscesses were the same of those isolated in ssi samples in the . % of the patients (n = ) with similar rates of of multi-resistant bacteria. no relationship was established between the intraabdominal abscesses and the occurrence of ssi based of clinical and surgical evaluation. based on the culture antibiograms, . % of patients received appropriate antibiotic treatment. the most commonly used antibiotics are shown in table , with the use of multiple, simultaneous or sequential antibiotics being used in . % of the cases (n = ). a mean of . antibiotics was used per ssi, with treatment lasting ± days. total mortality was . % (n = ), from which . % (n = ) correspond to icu deaths. although ssi was not associated with higher mortality in our population when confounders, such as variables that reflected disease severity, were included in the multivariate analysis, it was associated with a longer icu stay (or = . , % ci: . - . ; p = . ). indeed, mortality was lower in the group with ssi (or = . , % ci . - . ; p = . ). the need for rrt (or = . , % ci: . - . ; p = . ) and prolonged icu stay (or = . , % ci: . - . ; p < . ) were associated with higher in-hospital mortality. this study provides data on the incidence and microbiology of ssis for a large cohort of critically ill patients admitted with secondary or tertiary peritonitis to a surgical icu. it confirms that there was a high incidence of ssi in those patients. the main findings of our study were that ssi was associated with a prolonged icu stay, but that it had little impact on the overall in-hospital mortality in our population. the development of postoperative ssi is known to have been multifactorial, arising from a complex relationship between host and environmental factors [ , ] . host risk factors for ssi include morbid obesity, disease severity, advanced age, low blood-protein levels and malnutrition, diabetes, malignancy and sepsis, while other risk factors that include susceptibility include immunosuppression, smoking and having a distant infection site [ ] . pre-existing morbidity, the time of surgery and the type of ssi may also play key roles in the development of ssis [ ] . thus, an increasingly elderly population with a greater number of comorbidities significantly increases the risk of developing an ssi [ , ] . critically ill patients represent an increasing proportion of the inpatient population that will undoubtedly lead to greater diagnostic and management challenges, especially given that most ssis in the icus are nosocomial [ ] . ssis are most common in high-risk patients, with an incidence of about . % [ , ] . antibiotic prophylaxis reduces postoperative morbidity and length of hospital stay, which positively affects ssirelated costs [ ] . wounds with a risk of infection below % do not generally require antibiotic prophylaxis, but notable exceptions include the placement of a prosthesis, cardiovascular surgery and neuro-surgery [ ] . up to % of all elective surgical patients may develop an ssi, with rates as high as % being common in contaminated or dirty surgical procedures [ ] . in our study, the majority of procedures were considered dirty or contaminated, and many of the critically ill patients had markedly decreased serum protein concentrations. together, these may ultimately explain our higher ssi rate. our higher rates may also reflect the inherent risks of tertiary care institutions and the severity of our cohort. therefore, our results may not be applicable to secondary and non-referral hospitals. an ssi can increase hospital stay by about six days and can add - % to hospital costs, even leading to death; therefore, prevention and control should be an [ , ] . ssis may occur following any surgical incision, even after the use of minimally invasive techniques, so ssis need to be reported through systematic monitoring programmes for nosocomial infection [ ] . we showed that patients suffering from ssi in our cohort had longer icu stays. however, we do not think this was simply a surrogate of higher illness severity in the ssi group because of the comparable severity scores between groups. the dominant causative microorganisms and treatment options have changed over time. today, most common pathogens are resistant to common antibiotics [ ] with the need for a high index of suspicion, prompt operative intervention, appropriate antibiotic treatment and proper resuscitation [ ] . hypovolemia and hypothermia create peripheral vasoconstriction that leads to poor tissue perfusion, which facilitates the development of ssi in the presence of necrotic tissue, foreign bodies, hematomas and seromas [ ] . the microbiology of intra-abdominal infections also varies depending on the source of infection, prior use of antibiotics, the site of infection and if it is community acquired or nosocomial. besides the host and wound factors, physicians should be aware of the increase in high-virulence species, such as staphylococcus aureus and streptococcus pyogenes. in addition, escherichia coli, bacteroides fragilis and other gram-negative, anaerobic pathogens are common in large bowel perforations [ ] . nosocomial intra-abdominal infections often involve microorganisms such as pseudomonas spp., enterococcus spp. and fungi [ ] . in our population, there was an increased presence of multidrugresistant pathogens and fungal ssi rates when compared with other series [ , ] . this could be explained by the higher antibiotic pressure, use of broad-spectrum antibiotics, illness severity and prolonged treatment periods. indeed, concomitant treatment for peritonitis compounded matters. we also showed higher reliance on total parenteral nutrition (tpn) because enteral nutrition was poorly tolerated and we opted to initiate it early to avoid hypoalbuminemia, which is a risk factor for fungal infection and ssi in critically ill patients [ ] . however, we concede that tpn is a risk factor for all types of fungal infection in icu, especially among surgical patients [ ] . the increasing trend to reduce hospital stays by implementing innovative surgical techniques (particularly minimally invasive and endoscopic procedures) makes it necessary to ensure that accurate measurement and monitoring of adverse events can take place after discharge. without doing so, we cannot establish the real impact of ssi on morbidity and mortality [ , , ] . control measures with an emphasis on the education of healthcare professionals, such as frequent hand washing and the need to isolate patients with multi-resistant bacteria in cluster units, are necessary to reduce ssi rates [ ] . although the total elimination of ssi is not possible, a reduction in the rate of infection to a minimum should be achievable, even in critically ill patients [ ] . our study presents certain limitations. the most important is that we used for microbiological sampling a skin swabs instead of the "gold standard" for culture of skin, which are tissue biopsy or aspiration sampling of infected tissue. we could have obtained colonizer microorganisms that are not responsible for the infection and cultures may be misleading organisms in the deep tissue infection. thus, our results should be considered cautiously and more studies are needed to confirm them. secondly, this was a single-centre observational study and our results cannot be extrapolated to other less severely ill populations. among the strengths of this study are the large sample size, the prospective entry of all data and the use of postoperative scores, which are not used in contemporary studies, even though their importance in risk stratification has been emphasised over recent decades. furthermore, this investigation was conducted at a large tertiary referral hospital with a high level of complexity over a four-year period. in summary, the incidence of ssi was very high in patients with secondary or tertiary peritonitis requiring icu admission. when they prescribe antibiotic therapy, physicians should consider that microorganisms isolated from patients with ssi are more likely to include multidrugresistant pathogens, including pseudomonas spp., grampositive cocci and fungi, although more studies are needed to confirm our results due to the inherent limitations of the microbiological sampling with skin swabs performed in our research. despite the presence of an ssi may be associated with prolonged icu stays, we did not find any effect on the in-hospital mortality in our population. abbreviations ssi: surgical site infection; icu: intensive care unit; apache: acute physiology and chronic health evaluation; saps: simplified acute physiology score; rrt: renal replacement therapy; tpn: total parenteral nutrition; ards: acute respiratory distress syndrome in adults. there is no funding support or conflicts of interest for the present paper. authors' contributions jb was involved in the conception and design of the research, acquisition of data and wrote the paper. jcld performed partial statistical analysis, acquisition of data and wrote the paper. jsr contributed to the design of the research and acquisition of data. xlpf supervised and performed statistical analysis. ajb was involved in the conception, design of the research and interpretation of data. jar was involved in the design of the research and supervised the writing of the present manuscript. all authors read and approved the final version of this manuscript. epidemiology and microbiology of surgical wound infections risk factors for and epidemiology of surgical site infections surgical wound infection: a general overview the epidemiology of chest and leg wound infections following cardiothoracic surgery surgical site infections: epidemiology and prevention the epidemiology of intra-abdominal flora in critically ill patients with secondary and tertiary abdominal sepsis intensive care unit management of intra-abdominal infection risk factors for severe sepsis in secondary peritonitis surgical infections in the critically ill risk factors for wound infection after surgery for colorectal cancer cdc definitions of nosocomial surgical site infections, : a modification of cdc definitions of surgical wound infections surgical wound infection: epidemiology, pathogenesis, diagnosis and management apache ii: a severity of disease classification system implications of , consecutive surgical infections entering year adverse impact of surgical site infections in english hospitals national nosocomial infections surveillance system (nnis): description of surveillance methods microbiological diagnosis of intra-abdominal infections overview of the etiology of wound infections with particular emphasis on community-acquired illnesses a risk factor analysis of healthcare-associated fungal infections in an intensive care unit: a retrospective cohort study fungal infections in icu patients: epidemiology and the role of diagnostics continuous, -year wound infection surveillance: results, advantages, and unanswered questions the authors wish to thank the icu nurses and all members of the general surgery department for their contribution to the study and for their care of the patients reported in this paper. key: cord- - ldtrjf authors: chuang, pei-hung; chuang, jen-hsiang; lin, i-feng title: a dynamic estimation of the daily cumulative cases during infectious disease surveillance: application to dengue fever date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ldtrjf background: in infectious disease surveillance, when the laboratory confirmation of the cases is time-consuming, there is often a time lag between the number of suspect cases and the number of confirmed cases. this study proposes a dynamic statistical model to estimate the daily number of new cases and the daily cumulative number of infected cases, which was then applied to historic dengue fever data. methods: the duration between the date of disease onset and date of laboratory confirmation was assumed to follow a gamma distribution or a nonparametric distribution. a conditional probability of a case being a real case among the unconfirmed cases on a given date was then calculated. this probability along with the observed confirmed cases was integrated to estimate the daily number of new cases and the cumulative number of infected cases. results: the distribution of the onset-to-confirmation time for the positive cases was different from that of the negative cases. the daily new cases and cumulative epidemic curves estimated by the proposed method have a lower absolute relative bias than the values estimated solely based on the available daily-confirmed cases. conclusion: the proposed method provides a more accurate real-time estimation of the daily new cases and daily cumulative number of infected cases. the model makes use of the most recent "moving window" of information relative to suspect cases and dynamically updates the parameters. the proposed method will be useful for the real-time evaluation of a disease outbreak when case classification requires a time-consuming laboratory process to identify a confirmed case. timeliness and accuracy of case reporting are two of the most important performance measures when evaluating an infectious disease surveillance system [ ] [ ] [ ] [ ] [ ] . laboratory confirmation is usually needed for case diagnosis in many infectious diseases. when laboratory confirmation of the diagnosis is time-consuming, however, there is often a time-lag between the onset date of symptoms and the diagnosis date [ ] . for example, the median time for confirmation of the meningococcal disease is about days based on the national notifiable diseases surveillance system (nndss) dataset for the united states from to [ ] . time from disease onset to diagnosis has been also reported to account for most of the delay in case reporting in korea [ ] . correct estimation of daily cases or daily-cumulative infected cases helps the implement of immediate disease control and allows prevention activities for infectious diseases to be instituted [ ] . using a disease surveillance system, one is able to apply statistical methods, such as cumulative sum (cusum) [ , ] or autoregressive integrated moving average (arima) [ ] [ ] [ ] [ ] [ ] , in order to forecast an epidemic curve or to detect aberrations in disease spread. these estimations are based on having a correct daily number of cases or a daily-cumulative number of cases. an epidemic of dengue fever occurs every year in southern taiwan. in the year in particular, there was a large-scale epidemic with , confirmed cases out of totally , suspect cases [ ] . this epidemic continued until march . surveillance and the control of dengue fever have become one of the most important routine areas of work at the taiwan centers for disease control (taiwan cdc) in recent years. by , taiwan cdc had defined a confirmed case of dengue fever as an acute febrile illness together with one of the following criteria: ( ) isolation of dengue virus; ( ) demonstration of positive results by real-time reverse transcription--polymerase chain reaction (real-time rt-pcr); ( ) demonstration of positive seroconversion or a fourfold increase in dengue-specific igm or igg antibody titers in paired serum samples; or ( ) demonstration of high-titer dengue-specific igm and igg antibodies in a single serum specimen [ ] [ ] [ ] . when the dengue fever case classification only included confirmed cases by this protocol, the time needed for isolating the agent or measuring the titers for the acute-and convalescent-phase serum specimens was significant. the result was that there was a gap between the available daily cases or the daily-cumulative cases for given a day and the actual final confirmed cases for the same day given that all diagnosis had been completed on that given day. assuming that a time cost for laboratory confirmation of diagnosis is sometimes inevitable, daily numbers of infected cases and daily-cumulative number of infected cases may be underestimated during an epidemic. the objective of this study was to develop a new method to estimate the number of daily cumulative cases and that this method will be applied to dengue fever in taiwan, as an example. since there are almost no dengue fever cases occurred during the winter in taiwan, we chose may as the beginning of the dengue epidemic season when estimating the cumulative epidemic curve. the data come from the dengue notification dataset containing suspect cases in taiwan whose date of onset was from may , to april , . all serum samples from suspect cases were sent to the two reference laboratories at the taiwan cdc in order to further identify if they were positive (dengue fever infected) or negative cases. the reason we retrieved data based on the date of onset rather than the report date was to avoid the influence of lag reporting on the course of the disease. all imported cases of the disease were removed. the variables we used were the date of onset, the date of laboratory confirmation (diagnosis date), and the final confirmed status of each suspect case (a binary variable that is either positive or negative). in this article, we use confirmed dengue cases and positive cases interchangeably. no personal identification information was contained in the dataset. our proposed method estimates the real-time daily new number of cases and the daily cumulative number of dengue cases; specifically, these numbers of dengue cases are updated daily. let c be the "current" date when the number of dengue cases is to be estimated. in this study, the date c runs from may , to april , . for the i th reported suspect case counting from the st day of the epidemic season, that is may in this study, we define the suspect case's onset date as o i and the laboratory confirmation date as d i if d i >c on date c, the case i does not have a confirmation result as of date c; on the other hand, if d i ≤ c, this case i is either confirmed to be a positive dengue case or has a negative result as of date c. let the final confirmation status for the i th suspect case be, where as a positive dengue case, and as a negative case. in the situation where there are unconfirmed suspect cases as of date c, we assigned a probability of being a dengue case, p(i), to those unconfirmed cases (d ≤ c). then for each suspect case i, the expected final confirmation status on date c, e i (c), can be written as the values of p i (c), and e i (c)are updated for each case i every day. without applying the proposed method, one would be only able to observe the case status from the upper part of e i (c)in equation ( ) . after e i (c)is calculated for each suspect case, daily new cases are easily estimated by summing the e i (c)over all new suspect cases on date c, and cumulative cases can be obtained by summing e i (c)over all cases from i = to the newest suspect cases on date c. is estimated for unconfirmed cases using information from the confirmed cases before date c within one year. let t i be the onset-to-confirmation time (oc-time), the time interval between the onset date and the lab-confirmation date. the oc-time for the i th suspect case as of date c, t i (c), is calculated as follows, the t i (c) is the oc-time for confirmed cases and the censored oc-time for unconfirmed cases on date c. by applying several steps of bayes' rules, the probability p i (c) is given by: to estimate p i (c) using the information as of date c, we applied the following steps. we first estimated p(y i = ) by calculating the proportion of confirmed positive dengue cases out of the suspect cases using the data with onset date within year before the date c. based on a parametric approach, we assumed the oc-time for a given case status, p(t|y i ), follow a gamma distribution. gamma distributions are frequently used to fit time-delay distributions or time event distributions when carrying out disease surveillance analysis [ , ] . the probability density function of the gamma distribution is , where . the gamma distribution is denoted by with two parameters, the shape parameter α and the scale parameter β, and the mean and variance are αβ and αβ , respectively. the values of α and β were estimated and solved by setting up the sample mean and the sample variance of the oc-time equal to αβ and αβ , respectively. as mentioned in the previous section, the mean and standard deviation of the oc-time were different between positive and negative cases, we estimated different sets of α and β for the positive dengue cases (y = ) and negative cases (y = ) separately. a nonparametric approach was also performed in which the probability p(t >t i (c)|y i ) was simply replaced with the cumulative proportion of confirmed data given their final status. both the parametric and nonparametric models were based on the data within a -year "moving window" before date c. the p i (c) and e i (c) were also updated everyday. to evaluate the performance of the proposed method, we estimated the daily new cases and daily cumulative cases for each calendar date c from may , to april , . four epidemic curves are presented. there are: ( ) the final status curve, which is the number of dengue cases based on their final confirmation status ("real data", "gold standard"). ( ) the daily confirmed curve, which is the number of dengue cases based on the confirmed cases as of date c. ( ) the gamma-model curve, which is the number of dengue cases, estimated using the gamma distribution. ( ) the nonparametric-model curve, which is the number of dengue cases, estimated using the nonparametric distribution. to summarize the magnitude of the bias, we defined the absolute relative bias (arb) at date c as: where are the cumulative cases estimated by the proposed methods or by the confirmed cases observed on date c without using the proposed methods, n c and are the cumulative confirmed cases based on the final status ("real data", "gold standard"). an arb closer to zero is a more accurate estimate. all analyses were performed using sas . . software (sas institute, inc., cary, nc). special sas macros for estimating the cumulative cases and daily new cases, based on our proposed model, were developed. figure compares the daily new cases estimated by the proposed models, the confirmed curve (confirmed new cases observed on date c without using the proposed models), and the final status curve (confirmed new cases based on final status; the "gold standard"). since the daily new cases estimated by the proposed methods or the daily new cases observed on date c were different when viewed on different dates, arbitrary view dates of august , (beginning of the epidemic), september , (rising stage, before the peak), october , (rising stage, before but closer the peak) and november , (around the peak), december , (downward stage, after the peak), and january , (end of the epidemic) were chosen to illustrate the results of the estimated daily new cases. each graph in figure shows the epidemic curves three weeks before the view dates. when viewed on august , most of suspect cases had been lab-confirmed before july thus all four curves were close to each others before that date. from july to august , the estimated curves by the proposed methods (red dashed lines with triangle symbols by gamma distribution and blue dashed lines with cross symbols by the nonparametric method) were much closer to the final status curve (shaded area) than that by simply observing the dailyconfirmed new cases (purple dashed line). similar patterns were observed when the results are viewed on september , october , november , and december . the observed daily-confirmed cases usually underestimated the true daily new cases as would be expected, especially within the two weeks before the view date. the curves estimated by gamma distribution or the nonparametric approach were quite similar. however, the daily new cases, as estimated by the proposed method, did not give an accurate estimate towards the end stage of the epidemic, namely when viewed on january , . the epidemic curves in terms of daily cumulative cases are shown on figure . in this figure, the cumulative number of positive cases was updated every day. the two estimated daily-cumulative curves by the proposed methods are quite similar to the final status curve before january but again the proposed method does not work well during the end stage of the epidemic. table compares the arb of the daily cumulative number of positive cases between the different methods. after the first confirmed positive case appeared on july , , the estimates based on the gamma model results in an estimate closer to the real data than the other methods. for other two curves, the nonparametric method performs worst at the end of the epidemic after january and there was about cases higher than the final status curve. the daily confirmed curve was about cases lower than the final status curve during the peak of epidemic. figure showed the daily parameter estimates, α and β, of the gamma distributions used to dynamically calculate the daily number of positive cases. the parameter estimates varied from day to day and thus the probability of being a positive case changes. for the negative cases, the parameters had a jump during late september. as noted previously, timeliness and accuracy are the two of most important characteristics when we evaluate an infectious disease surveillance system. our results show that when an infectious disease required a time-consuming process for diagnosis, such as the dengue fever using the previously mentioned protocol, the actual daily number of infected cases and cumulative positive cases are potentially underestimated. the proposed method dynamically updates the parameters daily by making use of the most recently available information on suspect cases, and then performed estimates with a lower absolute relative bias than when using observed daily lab-confirmed cases only. as shown in table , the proposed method performed a lower median absolute relative bias (abs range . % ~ . %) than those solely based on daily confirmed cases (abs range . % ~ . %) between july and december . these dates covered the rising stage and around the peak stage which were of public health interest. the proposed method provides a more accurate estimate of the epidemic curves when applied to the dengue fever dataset for taiwan during the - season. based on these results, this approach can be used for the real-time evaluation of the severity of a disease outbreak when case classification requires that a confirmed case involves a time-consuming process. in this study, we first established the different distributions for the onset-to-confirmation time of the positive cases and negative cases. next, either a gamma distribution was assumed in order to estimate the probability of being a confirmed case given cases status in equation ( ), or, alternatively, a nonparametric approach was used. we actually experimented with several types of distribution. the estimates using a log-normal distribution were numerically very similar to the results for the gamma distribution. the estimates using a weibull distribution did not perform as well as the gamma distribution applied in our dengue fever data. from figure , we learn that the shape parameter α changed from . to and therefore an exponential distribution may not be appropriate. for simplification, we have chosen to present only the results from the gamma distribution as one example of a parametric approach and compare this with a nonparametric approach. as shown in figure for daily new cases, the differences in the estimates based on parametric approach with gamma distribution and those with nonparametric approaches were minor. the figure and table for cumulative cases showed that a gamma distribution is a more appropriate assumption for the onset-todiagnosis time when estimating the probability of being a positive case using the dengue fever example; nonetheless, the difference between the gamma and the nonparametric method is again only slight except towards the end stage of the epidemic after january . the reason that the nonparametric method did not work well after january is because p(y i = ), p(y i = ), and p(t|y i ) had not changed substantially, resulting in a near constant estimate of the daily positive cases. in practice, any form of the probability of being a positive case can be assumed. it is also not restricted to certain distributions when the models are adapted to different types of infectious disease. when applying this approach to other diseases, researchers should investigate several distributions according to the shape of their data and choose an appropriate one based on some appropriate measures, for instance, those shown in table . our method estimated the probability of being a positive case based on the data within a -year "moving window" before date c and updated p i (c) and e i (c) everyday. the epidemic profiles of dengue fever are different from one year to another in taiwan. choosing the data from most recent one year was done in order to insure that there was enough information to cover a whole epidemic season. the study shows that before the first positive case appeared on july , the proposed method did not work well and are not that useful (table ). our method worked well after the first positive case appeared during the - season. indeed, it needed only four days to be able to consistently estimate the final status curve. in the - season, taiwan cdc activated a central command center for intensively dengue epidemic control on october . the task of this command center included expanded blood sample collection and it is likely that this resulted in more suspect cases for laboratory confirmation, which might have led to a lower proportion of positive cases. this would influence the estimation of probability of being a positive case over the following few days. as we can see on figure , it also influenced the estimation of the parameters for negative cases. while our manuscript was being prepared, the taiwan cdc changed their laboratory protocol for dengue fever to one that requires only a single laboratory test for dengue surveillance and control. the result is a substantial reduction in the waiting time for laboratory confirmation. however, confirmation time can never be completely avoided with dengue fever. a situation where a large number of serum specimens are sent for diagnosis at the same time will result in overloading at the laboratory, which might increase the confirmation waiting time. as described previously, the estimation used information based on a "moving window" time period before the estimated date and the parameters of the model are updated everyday. since the observed confirmed cases counts on date c are always underestimated as long as there is a time lag, our method potentially can be applied while waiting for further investigation of the status of cases. there are some limitations to our method. firstly, the approach needs sufficient historical data to be available in order estimate the parameters of the model; therefore our model cannot be applied effectively to an emerging disease, such as sars or avian flu. secondly, we used confirmed cases, the dates of onset of which were within year before the date estimated and if a case needs more than -year for diagnosis such a case might never provide any information to the parameter estimation; in such a circumstance a different "moving window" needs to be chosen. thirdly, when missing diagnosis dates exist, the estimated curve using the nonparametric method cannot converge with the final status curve. there were and cases missing confirmation results for the - season and the - season, respectively, at the time that the manuscript was prepared. the nonparametric method estimates by plugging in the cumulative proportion of confirmed data given the final status. as we mention before, at the end stage of epidemic, the probabilities in equation ( ) almost remained unchanged. in this study, we could only assume that the proportion of positive cases out of all suspect cases among the missing observations were similar to those having results, which basically assumes that the missing data were missing at random thus ignorable. when diagnosis of infectious diseases required laboratory confirmation, the time lag between onset and confirmation of a positive cases often exists and case counts are usually underestimated. this study has proposed a statistical method that more accurately estimates the real-time daily new cases and daily cumulative number of infected cases using a dengue fever epidemic as an example. the model makes use of the most recent "moving window" of information on suspect cases and dynamically updated the parameters of the assumed probability distributions. different parametric or nonparametric distributions of the onset-to-confirmation times can be specified for different infectious diseases. the results show that, after the first confirmed case occurred, the estimated daily new cases or the cumulative case count fit the real data well compared to the daily counts based only on the available confirmed cases; this was done by assuming a gamma distribution for the onset to confirmation times and involved the use of a dynamic one-year "moving window" of suspected cases when applied to dengue fever outbreaks in taiwan. this method can be used for the realtime evaluation of a disease outbreak when case diagnosis requires time-consuming laboratory process. how complete and accurate is meningococcal disease notification? public health surveillance of aids and hiv infections updated guidelines for evaluating public health surveillance systems: recommendations from the guidelines working group cdc's national violent death reporting system: background and methodology an outbreak of leptospirosis, thailand--the importance of the laboratory evaluation of reporting timeliness of public health surveillance systems for infectious diseases syndromic surveillance systems: public health and biodefense timeliness of national notifiable diseases surveillance system in korea: a crosssectional study methods for monitoring influenza surveillance data the relative efficiency of the sets and the cusum techniques in monitoring the occurrence of a rare event time series analysis forecasting and control on the application of integer-valued time series models for the analysis of disease incidence use of time-series analysis in infectious disease surveillance dynamic linear model and sarima: a comparison of their forecasting performance in epidemiology a monitoring system for detecting aberrations in public health surveillance reports number of reported and confirmed cases -by month world health organization: dengue haemorrhagic fever: diagnosis, treatment and control laboratory-based dengue surveillance in taiwan, : a molecular epidemiologic study current status of dengue diagnosis at the center for disease control epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong sars incubation and quarantine times: when is an exposed individual known to be disease free? this study was supported in part by the centers for disease control, department of health, taiwan, republic of china (doh -dc- and doh -dc- ) and was also supported by the aim for the top university plan of national yang-ming university. the authors would like to thank the reviewers' excellent comments for improving this manuscript. the authors declare that they have no competing interests. authors' contributions ifl designed and conducted the study, and finalized the manuscript. phc participated in the design of the study, performed the statistical analyses, and drafted the manuscript. jhc helped conceive the study, participated in the data collection, gave input to the manuscript, and provided medical advice from the public health perspective. all authors have read and approved the final manuscript. key: cord- -h ch x authors: ebuy, hiluf; bekele, alemayehu; redae, getachew title: hiv testing, test results and factors influencing among infants born to hiv positive mothers in public hospitals of mekelle city, north ethiopia: a cross-sectional study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: h ch x background: timely infant testing for hiv is critical to ensure optimal treatment outcomes among exposed infants. while world health organization recommends hiv exposed infants to be tested between to weeks of age, in developing countries like ethiopia, access to timely infant testing is still very limited. the study is intended to assess timely infant testing, testing for hiv at the th month, test results and factors influencing hiv positivity among infants born to hiv positive mothers in public hospitals of mekelle, ethiopia. methods: a cross-sectional study design was employed on hiv exposed infants, using consecutive sampling technique. a checklist was used to extract years (january –december ) secondary data, collected from january–april . data were analyzed using spss version , and binary logistic regression model was used to examine the association of independent variables with the outcome variables. results: timely infant testing for hiv accounted for ( . %). mothers who attended antenatal care (aor: . ; % ci: . , . ) and who were counselled on feeding options (aor: . ; % ci: . , . ) were strongly associated with timely infant testing. poor maternal adherence status was associated with infants’ hiv positivity at the th month of antibody test (aor: . ; % ci: . , . ). being rural resident (aor: . ; % ci: . , . ), being low birth weight (aor: . ; % ci: . , . ) and not receiving arv prophylaxis (aor: . ; % ci: . , . ) were positively associated with the overall hiv positivity. conclusions: a considerable proportion of exposed infants did not undergo timely testing for hiv. antenatal care follow-up and counselling on feeding options were associated with timely infant testing. mother’s poor adherence status was associated with infant’s hiv positivity at the th month of antibody testing. being rural resident, being low birth weight, and not receiving arv prophylaxis were the factors that enhance the overall hiv positivity. timely infant testing, counselling on feeding options and adherence should be intensified, and prevention of mother-to-child transmission program in rural settings need to be strengthened. there are many ways of hiv transmission. one way of transmission is from an hiv positive woman to her child during pregnancy, labor, and breastfeeding. it is also called vertical transmission which accounts for % of all new hiv infections worldwide. in developing countries, despite the availability of proven interventions for the prevention of mother-to-child transmission (pmtct), pediatric hiv is still a largely uncontrolled epidemic [ ] [ ] [ ] . timely infant testing for hiv infection is critical to ensure optimal treatment outcomes among exposed infants. without testing and effective treatment, one-third of hiv-positive infants will die before the age of one year, and almost half by their second year of life. while world health organization (who) recommends hiv exposed infants to be tested between four to six weeks of age, in developing countries access to timely infant testing is still very limited [ , ] . globally, in , only % of infants exposed to hiv during pregnancy were tested within the recommended period of time. majority of the non-timely tested infants were from developing countries, particular sub-saharan africa. in sub-saharan africa, delayed infant testing is emerging as one of the challenging complex issues facing children infected with and affected by hiv. similar to the other sub-saharan african coutries, studies from ethiopia also showed that timely infant testing for hiv is very low [ ] [ ] [ ] [ ] . the prevalence of hiv among exposed infants can reach up to % if left without pmtct interventions. nearly two-third of pregnant women living with hiv in the middle east and north africa passed the virus onto their infants in the year alone , ] . in ethiopia, vertical transmission, which accounted for more than % of pediatric hiv, is a very critical issue. accordingly, hiv related estimates and projections showed that the national estimate of mtct rate was % in and % in . this high magnitude makes hiv/aids one of the top priorities of the health sector transformation plan (hstp) of ethiopia [ ] [ ] [ ] [ ] . although timely hiv testing of infants is not yet optimal, some strategies and solutions have proven successful, including community-based interventions and support and education of mothers. the joint united nations program on hiv/aids (unaids) - strategy set about ten targets to end the aids epidemic by ; one of the targets is to make new hiv infections among children zero and improve the health and wellbeing of mothers. the global plan to eliminate new hiv infections among children and improve the health of mothers targets to reduce the mtct rate to less than % among breast feeding population and to less than % among non-breast feeding population [ , , ] . pertaining to the factors for hiv positivity of exposed infants, different studies reported various determinants. accordingly, mothers with high viral load, symptomatic disease, failure to use arv drugs during pregnancy, vaginal delivery, rupture of membrane > h, low birth weight babies, premature births, and infants' failure to use antiretroviral (arv) prophylaxis were found to be associated with hiv positivity among infants [ ] [ ] [ ] . in most resource-limited countries, deoxyribonucleic acid polymerase chain reaction (dna/pcr) test is not available for the timely testing of exposed infants, which plays a big role in infants' late initiation of treatment which then leads to hiv related pediatric mortality. in ethiopia, previous studies tried to demonstrate exposed infants' hiv testing and determinants of pmtct. however, most of the studies were confined to a single health facility and only tried to find out factors associated with hiv positivity at weeks of testing. there is paucity of information regarding the factors affecting timely infant testing and hiv positivity at the th month of antibody test, until the infant completes the program. thus, the present study is intended to assess timely infant testing, testing for hiv at the th month, test results and factors influencing overall hiv positivity and hiv positivity at months among infants born to hiv positive mothers in public hospitals of mekelle city, north ethiopia. this study was conducted in mekelle city. mekelle is the capital city of the tigray region and is located around km north of addis ababa, the capital city of ethiopia. there are three public hospitals in the city namely; ayder comprehensive specialized, quiha and mekelle general hospitals. the college of health sciences (chs), ayder comprehensive specialized hospital (acsh) is a public hospital with a capacity of inpatient beds and has the responsibility of rendering patient care, training for medical & health science students and also carrying out research. the institution has a clinic dedicated for maternal, neonatal and child health services (mnch) including pmtct services. quiha general hospital, which was established in by italian cooperation, served as a health center until and then become a general hospital. currently, the hospital has a capacity of beds and the hiv care and treatment clinic is one of the core parts of the hospital's activities. mekelle general hospital, which was established in , is the oldest general hospital in the region. it has its own separate fully functional art clinic and pmtct services. since there was no access to the dna/pcr test in all the public hospitals, the test was done in the regional laboratory after dbs was collected in the public hospitals. on the other hand, antibody test was conducted in the study setting [ ] [ ] [ ] . a cross-sectional study design was employed in the public hospitals of mekelle city from january -december . study population, sample size and sampling procedure the study participants consisted of all mother-infant pairs who were eligible to be enrolled to the pmtct program in the public hospitals of mekelle city during the study period. four years data were extracted retrospectively, from january -december . the reason why we started extracting data from was due to the fact that in ethiopia option b plus strategy for pmtct was adopted in [ ] . however, in the study area consistent and full documentation of option b plus services were available starting . in the four years period, a total of mother-infant pairs who fulfilled the inclusion criteria were enrolled to the study, using consecutive sampling technique. we used consecutive sampling technique because it was a secondary data, % of the participants were excluded from the study due to either incomplete information, loss to follow up or transfer out. all mother-infant pairs who sought and completed the pmtct program were included in the study. we excluded participants who had an incomplete medical records, lost to follow up subjects and participants who were transferred into other health facilities. loss to follow up, which makes identifying and managing hiv-infected children very difficult, could be a reflection of failure in pmtct. after reviewing different literatures [ , , , ] , a checklist developed by the authors was used to collect data regarding socio-demographic and health-related factors. data were extracted from secondary data sources retrospectively by reviewing patient charts (both paper and electronic medical recording systems), pmtct, antenatal, delivery, and postnatal care registration books and partographs from january-april, . there is a special hiv detection test done for hiv exposed infants after dried blood spot (dbs) is collected called dna/pcr test. this hiv testing is recommended to be done within - weeks of age and is referred to as timely infant testing. this is the aggregate of mtct of hiv at the th week of dna/pcr test and the th month of antibody test, until weaning. ratios of art medication adherence equal to or greater than % were defined as good dosage adherence. adherence status of the participants was measured from a self-report. the checklist was prepared in english and reviewed by senior researchers and feedback was incorporated accordingly. the secondary data were extracted by three midwives, one for each hospital and training was given concerning the checklist, data collection technique, purpose of the study, and keeping confidentiality. the research team had day to day on-site supervision to make sure data extraction from secondary sources was going smoothly. the research team discussed how to take corrective measures like checking and discarding data if an error, such as illegibly documented checklists, occurred during the data collection. the collected data were checked for completeness, consistency, and clarity. the collected data were coded and entered into an excel spreadsheet; it was then exported to statistical package for social sciences (spss) version statistical software for analysis. tables and figures were used for descriptive statistics. frequency and percentage were used for categorical variables. odds ratio (or) with % confidence interval was used to measure strength of the association. binary logistic regression model, with bivariate and multivariable analysis, was used to examine the association of independent variables with the outcome variables and calculate their crude as well as adjusted odds ratios. those variables which happened to have a p-value < . with crude analysis of logistic regression model were fitted into the final multivariable logistic regression model and their adjusted odds ratios were calculated. statistical significance was declared using an odds ratio and % confidence interval. absence of multicollinearity was checked and found to be satisfied with the maximum variance inflation factor (vif) value of . . goodness of fit was checked by hosmer-lemeshow test which yielded a chi-square value of . with df and p-value of . for the final model of overall hiv positivity. ethical clearance was obtained from the institutional review board of the ethiopian public health association (epha). the collected data was kept anonymous; mothers and infants' names were not recorded but instead were written down in code and additional measures like keeping the completed checklist in a safe place were taken. this study did not impose any harm to the community or to the research team, and it was conducted in line with national and international ethical guidelines. a letter of collaboration was also obtained from the institutional review board of the college of health sciences, ayder comprehensive specialized, quiha and mekelle general hospitals. socio-demographic characteristics of the study participants among the total mother-infant pairs who were enrolled in the pmtct cohort over the past four years, ( . %) of them fulfilled the inclusion criteria and were included in the final analysis. two hundred thirtynine ( . %) participants were excluded from the study, of which ( . %) had an incomplete medical record, ( . %) were lost to follow up and the remaining ( . %) were transferred into other health facilities. the mothers' ages ranges from to years; with a mean age of . years and a standard deviation of . years. regarding residence, the majority of mothers, ( . %), were urban dwellers. there were ( . %) female hiv exposed infants (table ) pmtct service and clinical characteristics among mothers the majority ( . %) of the mothers had attended anc and ( . %) mothers were on art on entry to the pmtct cohort. of the ( . %) mothers whose adherence status was known, ( . %) had good adherence status. who clinical staging was documented in the majority ( . %) of mothers and ( . %) of them were in clinical stage i or ii (table ) . the mean weight of the infants was . kg, with a standard deviation of . kg. eighty-four ( . %) infants' were low-birth-weights. the majority ( . %) of the infants were given arv prophylaxis. regarding the feeding of exposed infants, no mixed feeding was practiced. timely infant testing for hiv accounted for ( . %) ( table ) . overall study participants were tested for dna/pcr. of these, ( . %) ( % ci: . , . %) tested positive for hiv. among the hiv exposed infants for whom the rapid antibody test was done, ( . %) ( % ci: . , . %) tested positive for hiv. therefore, the overall mtct rate was . % ( % ci: . , . %). in the multivariable logistic regression analysis, factors found to be significantly associated with timely infant testing were anc follow up and feeding option counselling. the odds of practicing timely infant testing of mothers who attended anc follow up were . times higher than those who didn't have anc follow up (aor: . ; % ci: . , . ). the odds of practicing timely infant testing of infants whose mothers were counselled on feeding options were -fold higher than their counterparts (aor: . ; % ci: . , . )( table ) . factors associated with hiv positivity at the th month at the th month of antibody test the mother's adherence status was the only variable that had a significant association with hiv positivity. the odds of hiv positivity of infants whose mother's adherence status was poor were . times higher than those with good adherence status (aor: . ; % ci: . , . ) ( table ) . in the multivariable logistic regression, the variables; place of residence, infants' birth weight, and infants' arv prophylaxis had a significant association with overall hiv positivity among exposed infants. accordingly, the likelihood of hiv positivity among rural residents was times higher than urban dwellers (aor: . ; % ci: . , . ). the odds of hiv positivity of low birthweight infants were . times higher than normal birthweight infants (aor: . ; % ci: . , . ). the likelihood of hiv positivity among infants who did not receive arv prophylaxis was . -fold higher than their counterparts (aor: . ; % ci: . , . ) ( table ). the present study was intended to assess timely infant testing, testing for hiv at the th month, test results and factors influencing overall hiv positivity and hiv positivity at months among infants born to hiv positive mothers in public hospitals of mekelle, tigray, ethiopia. the percentage of hiv exposed infants tested for hiv timely ( . %) in our study were comparatively higher than studies conducted in south gondar, ethiopia in ( . %) and asella teaching and referral hospital, ethiopia from to ( . %) [ , ] . this high percentage of timely infant testing in our study could be due to the fact that the regional laboratory, where the dna/pcr test is done, is located in the same city where the study is conducted, which makes it easier for the dna/pcr test to be done in a timely manner. in the first six weeks the mtct rate of hiv, as determined by a dna/pcr test, was . %( % ci: . , . %). this was one of the lowest mtct rates of reports compared to a cross-sectional household survey conducted [ , , [ ] [ ] [ ] . this could be attributed to the cor crude odds ratio, aor adjusted odds ratio *significant association ci confidence interval reason that almost all of our study participants gave birth in a health institution, which allowed them good access to the first six weeks of a pmtct program. in our study, the overall mtct rate of hiv infection among exposed infants was . % ( % ci: . , . %), which was much less than a study conducted in brazil. a possible explanation for this might be that the study conducted in brazil was a years study, which started when pmtct program wasn't as efficient and modernized as recently. our finding was also less than ethiopia's national estimate of mtct in and and from studies done in arsi add dire dawa, ethiopia [ , , , , ] . this may be explained by the reason that no mixed feeding was practiced in our study, which enhances the odds of hiv positivity. in addition, the majority of our participants were from urban areas where they can easily access pmtct services, while the national estimates account for the overall mtct of the country, including rural residents and home deliveries. mothers who attended anc follow up through the course of the pregnancy were . times more likely to practice timely infant testing of hiv compared to the mothers who didn't have anc follow up. the conceivable reason could be that one of the components of pmtct services during anc is health education and awareness creation about the advantages of timely infant testing. this was given to the mothers who had anc follow up when they visited health institutions. counselling on feeding options was another factor associated with timely infant testing. the odds of practicing timely infant testing of infants whose mothers were counselled on feeding options were -fold higher than mothers of infants who weren't counselled on feeding options. this could be attributed to the reason that when mothers were counselled on feeding options, at the same time, they were also being counselled on other pmtct cascades, such as advantages of timely infant testing. mother's adherence status had a significant association with hiv positivity among exposed infants at the th month rapid antibody test. infants born to hiv positive mothers whose adherence status was poor were . times at higher risk of acquiring hiv infection than infants born to hiv positive mothers whose adherence status was good. the possible risk difference could be due to the fact that poor maternal adherence status causes drug resistance, which then leads to the elevation of maternal viral load, putting exposed infants at high risk of hiv positivity. the odds of hiv positivity of infants born to hiv positive mothers from rural residences were times higher than infants born to mothers of urban residents. this is consistent with the finding of studies conducted in dil chora hospital in dire dawa, ethiopia and gondar university hospital in northwest ethiopia [ , ] . a likely explanation is that mothers from rural settings might have limited access to anc clinics and therefore poor access to pmtct services compared to mothers who are urban dwellers. the odds of overall hiv positivity of low birth-weight infants were . times higher than the normal birthweight infants. this is consistent with researches done in arsi and addis ababa, ethiopia [ , ] . the attributed risk difference could be due to the reason that the immature gastrointestinal tract in low birth weight infants might facilitate mtct of hiv through mother's breast milk. arv prophylaxis for infants was another factor associated with exposed infants' overall hiv positivity. the odds of acquiring hiv among infants who did not receive arv prophylaxis was . -fold higher than those infants who received arv prophylaxis. the observed risk difference could be due to the reason that arv prophylaxis for exposed infants decreases vertical transmission of the virus from hiv positive mothers. this finding is in tune with studies conducted in brazil and ethiopia [ , , , ] . this study gives a clue about the factors associated with timely infant testing and potential factors associated with hiv positivity among exposed infants. furthermore, the findings of the study are meant to strengthen and enhance the implementation of the pmtct program at the hospital level in the public health system. this study has some limitations. the fact that it is a crosssectional study design is one of the study's weakness. since the study used secondary data, the information gathered was incomplete. as a result, some sociodemographic variables such as household income and educational status were not documented. additionally, this study didn't specify the time when infants' arv prophylaxis was initiated. in conclusion, considerable proportion of exposed infants did not undergo timely infant testing for hiv. there was relatively low mtct rate of hiv at six weeks, eighteen months and for overall hiv testing. antenatal care follow-up and counselling on feeding options were associated with timely infant testing. mother's poor adherence status was associated with infant's hiv positivity at the th month of antibody test. infants born to hiv positive mothers from rural residences, low birth-weights and who didn't receive arv prophylaxis after birth were the factors that enhance the risk of overall hiv positivity. health care providers should work on strengthening timely testing of hiv among exposed infants. counseling on feeding options, anc and adherence needs to be provided in a sustainable way. continuous follow up of exposed infants has to be intensified. in addition, policymakers and program managers should focus on scaling up pmtct program in rural settings. a prospective study need to be conducted to assess the effectiveness of pmtct programs and to address additional factors associated with hiv positivity among exposed infants. deoxyribonucleic acid polymerase chain reaction; ebf: exclusive breast feeding; eff: exclusive formula feeding; epha: ethiopian public health association; hiv: human immunodeficiency virus; hstp: health sector transformation plan lsi: leadership in strategic information; mnch: maternal, neonatal and child health; mtct: mother -to-child transmission; or: odds ratio pmtct: prevention of mother-to-child transmission; spss: statistical package for social science; unaids: joint united nations programme on hiv/ aids; who: world health organization references . unaids. unaids report on the global aids epidemic mother-to-child transmission of hiv in the era of highly active antiretroviral therapy early infant diagnosis of hiv: review of current and innovative practices towards universal access: scaling up priority hiv/aids interventions in the health sector. aids epidemic update pediatric hiv/aids in sub-saharan africa: emerging issues and way forward risk of hiv and associated factors among infants born to hiv positive women in amhara region assessment of effectiveness of prevention of mother to child transmission of human immunodeficiency virus in asella hospital, ethiopia prevention of mother-to-child transmission ( pmtct) of hiv joint united nations programme on hiv/aids. aids by the numbers fdre; federal ministry of health. country progress report on the hiv response hiv related estimates and projections for ethiopia- analysis of the prevention of mother-to-child transmission (pmtct) service utilization in ethiopia federal minstry of health. hiv related estimates and projections for ethiopia . health sector transformation plan (hstp) joint united nations programme on hiv/ aids the global plan towards the elimination of new hiv infections among children by and keeping their mothers alive mode of delivery in hiv-infected pregnant women and prevention of mother-to-child transmission: changing practices in western europe factors associated with viral load suppression in hiv-infected pregnant woman in rio de janeiro, brazil maternal risk factors for hiv infection in infants in northeastern brazil mekelle general hospital public relations office quiha general hospital public realtions office, quiha general hospital profile report federal ministry of health. national comprehensive pmtct/mnch/rh training package guideline reference manual mother-to-child transmission of hiv infection and its determinants among exposed infants on care and follow-up in dire dawa city review of prevention of mother to child transmission of hiv in addis ababa, ethiopia. univ s afr pretoria reconstructing the pmtct cascade using cross-sectional household survey data: the pearl study determinant and outcome of early diagnosis of hiv infection among hiv-exposed infants in southwest ethiopia mother-to-child transmission of hiv and its predictors among hiv-exposed infants at a pmtct clinic in northwest ethiopia an assessment of the outcomes of prevention of mother-to-child transmission of hiv services in addis ababa publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the ethiopian public health association. our gratitude also goes to ayder comprehensive specialized, quiha and mekelle general hospitals for providing us the necessary information. last but not least we would like to acknowledge the data collectors.ethical approval and consent to participate this study was approved by the institutional review board of the ethiopian public health association. collected secondary data were kept anonymous; mothers and infants' names were not being recorded instead was written down in code. ethiopian public health association, addis ababa, ethiopia. authors' contributions he had taken a principal role in the conception of ideas, writing the protocol, developing methodologies, analyses and write up of the article and drafted the manuscript. ab contributed to the proposal writing and design, developing methodologies and made a critical revision to the paper and manuscript for intellectual content. gr contributed to the write up of the study protocol and made revision to the paper. ab and gr supervised the study. all authors read and approved the final manuscript. this work was supported by the ethiopian public health association. the funding agency had no involvement in the design of the study, data collection and analysis, interpretation of data and writing the manuscript. the datasets generated and/or analyzed during the current study are not publicly available due to ethical and confidentiality reasons but are available from the corresponding author on reasonable request under the ethics committee's approval. not applicable. there is no competing interest. key: cord- -ld eieik authors: ng, man wai; zhou, gangqiao; chong, wai po; lee, loretta wing yan; law, helen ka wai; zhang, hongxing; wong, wilfred hing sang; fok, susanna fung shan; zhai, yun; yung, raymond wh; chow, eudora y; au, ka leung; chan, eric yt; lim, wilina; peiris, js malik; he, fuchu; lau, yu lung title: the association of rantes polymorphism with severe acute respiratory syndrome in hong kong and beijing chinese date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ld eieik background: chemokines play important roles in inflammation and antiviral action. we examined whether polymorphisms of rantes, ip- and mig affect the susceptibility to and outcome of severe acute respiratory syndrome (sars). methods: we tested the polymorphisms of rantes, ip- and mig for their associations with sars in hong kong chinese sars patients and controls. then we tried to confirm the results in beijing chinese sars patients and controls. results: rantes - g allele was associated with sars susceptibility in hong kong chinese (p < . , or = . , %ci: . – . ). individuals with rantes - cg and gg genotypes had a . -fold ( %ci: . – . ) and . -fold ( %ci: . – . ) increased risk of developing sars respectively (p < . ). this - g allele conferred risk of death in a gene-dosage dependent manner (p = . ) with cg and gg individuals having a . -fold ( % ci: . – . ) and . -fold ( % ci: . – . ) increased risk. for the replication of rantes data in beijing chinese, the - g allele was not associated with susceptibility to sars. however, - cg (or = . , %ci: . – . ) and gg (or = . , %ci: . – . ) were associated with admission to intensive care units or death due to sars (p = . ). conclusion: rantes - g allele plays a role in the pathogenesis of sars. severe acute respiratory syndrome (sars) is an infectious disease caused by sars coronavirus [ ] with > cases and deaths reported in [ ] . pathogenesis of sars is complex and host genetic background is one of the factors in determining susceptibility and outcome [ ] . we have demonstrated that genetic haplotypes associated with low serum mannose-binding lectin (mbl) are associated with sars [ ] and our findings were confirmed in another independent study [ ] . we furthermore showed that the interferon gamma gene polymorphism, + a/ t, is associated with sars [ ] . other susceptibility genes, such as liver/lymph node-specific icam- -grabbing nonintegrin (l-sign) which is encoded by clec m, ', '-oligoadenylate synthetase gene (oas- ) and myxovirus resistance (mxa) were also identified [ , ] . chemokines play important role in cells trafficking during immune responses. acute respiratory viruses commonly induce inflammatory chemokines in local tissue [ ] . in the case of sars, our previous study confirmed that sars coronavirus induces upregulation of a number of inflammatoemokines, i.e. regulated upon activation, normal t cell-expressed and secreted (rantes), interferon-gamma inducible protein (ip- ) and monocyte chemoattractant protein- (mcp- ) [ ] . the upregulation of these chemokines recruit inflammatory cells and leukocytes into the tissue [ ] . therefore, we hypothesized that the polymorphisms of inflammatory chemokine genes might be associated with sars. in this study, we investigated the single nucleotide polymorphisms (snps) of inflammatory chemokine genes, i.e. rantes, ip- and monokine induced by gamma interferon gene (mig) in two chinese cohorts from hong kong and beijing and found that the rantes - g allele was associated with disease susceptibility and severity of sars. the study included hong kong chinese patients with sars. the mean ± sd age was . ± . years with males and females (table ) . at least % of the patients were documented with sars-cov antibody seroconversion and/or detectable sars-cov rna in respiratory secretions by rt-pcr as described previously in our studies [ , ] . approval for the study was granted by the clinical research ethics committee of the institutional review board of the university of hong kong/hospital authority hong kong west cluster. the patients were further divided into two groups, the death group and the survival group. the death group consisted of patients who died from sars and their mean ± sd age was . ± . years, with males and females. the survival group consisted of patients and their mean ± sd age was . ± . years, with males and females. a population of hong kong chinese comprising healthy red cross blood donors served as the control subjects. their mean ± sd age was . ± . years, with males and females (table ) . three hundred and fifty six beijing chinese patients with sars were recruited as described previously (mean ± sd age = . ± . , male and female) (table ) [ ] . among them, patients were classified as severe group, which were identified by their admissions to intensive care units or deaths from sars (mean ± sd age = . ± . , male and female). the remaining patients were classified as mild group (mean ± sd age = . ± . , male and female). a total of ethnically matched healthy individuals (mean age ± sd = . ± . , male and female) served as controls (table ) . rantes - c/g (rs ) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) as described previously [ ] . rantes - a/g (rs ), rantes in . t/c (rs ), ip- nt a/g (rs ), ip- nt c/ a (rs ) and mig nt a/g (rs ) were genotyped by the massarray system (sequenom, san diego, ca). in brief, the samples were amplified in a μl reaction mixture, containing ng genomic dna, . pmol each of specific forward and reverse primers, μl of each dntp, . mm mgcl and . units of hotstartaq polymerase (qiagen, valencia, ca). pcr conditions included initial hot-start for min at °c, cycles of amplification ( sec at °c, sec at °c and min at °c) and final extension for min at °c. the pcr products were treated with alkaline phosphatase to dephosphorylate residual amplification nucleotides. a mixture of . μl of hme buffer, . μl of shrimp alkaline phosphatase ( unit/μl, sequenom) and . μl of ddh o was added to the pcr products. the reaction solutions were incubated for min at °c, followed by min at °c to inactivate the enzyme. mass-extend reactions to determine genotypes were performed in four groups of different terminations according to the design rationale (ddacg, ddact, ddagt and ddcgt, respectively). the reaction volume was μl including unit of thermosequenase (sequenom), μm of the respective termination mix, and . pmol of each assay specific extension primer ( table ). all assays were run with the same thermal cycle conditions: initial denaturation for min at °c followed by cycles of extension ( sec at °c, sec at °c and sec at °c). products of the massextend reactions were desalted and transferred onto a spectrochip by a nanoliter dispenser according to the manufacturer's instructions (sequenom). genotype determination was performed on a maldi-tof mass spectrometer (sequenom). mass spectrometric data were automatically imported into the spectrotyper (sequenom) database for data analysis including noise normalization and peak area analysis. the expected molecular weights of all relevant peaks were calculated by the massarray assaydesign software (sequenom) before the analysis and identified from the mass spectrum. in every -well plate for assay, there is one well for blank control and five wells for duplicate check on five samples for internal quality control. a two-step analysis was used to determine the association of polymorphisms with sars. the genotype frequencies and allele frequencies of all the genes were compared between sars patients and controls by a × chi-square test and a × chi-square test respectively, then logistic regression was used for calculating odds ratios ( % confidence interval) and corresponding p-values of different genotype frequencies among sars patients and controls by adjusting for age, sex and all significant single nucleotide polymorphisms (snps). association with the outcomes of sars infection (death vs survival) was then tested by comparing the genotype frequencies and allele frequencies of all the genes between the death group and the survival group of sars patients by a × chi-square test and a × chi-square test respectively. the genotype frequencies of all the snps were tested for hardy-weinberg equilibrium (hwe) separately in sars patients and controls by chi-square test. significant p-value for multiple testing was adjusted with bonferroni's correction and all statistical analysis was performed by sas, version . and sas/genetics (sas institute inc., nc, usa). rantes - c/g,rantes - a/g, rantes in . t/c, ip- nt a/g, ip- nt c/a and mig nt a/g were genotyped in all sars patients and controls from hong kong. their genotype and allele frequencies were shown in table and respectively. rantes - cg and gg genotypes were significantly associated with sars susceptibility with or of . ( % ci: . - . ) and . ( % ci: . - . ) respectively (p < . ) ( table ) . rantes - g allele was also significantly increased in the patients (p < . , or = . , %ci: . - . ) ( table ). after correction by bonferroni method, the significant p value should be less than . , the association of rantes - c/g to sars susceptibility remained to have significance. we then compared the genotype and allele frequencies of the rantes - c/g between the death group and survival group of the sars patients. rantes - g allele associated with death from sars in a gene-dosage dependent manner (p = . ), with - cg and gg individuals having a . -fold ( % ci: . - . ) and . fold ( % ci: . : . ) increased risk of death from sars respectively ( table ) . to further confirm the association of rantes with sars, we studied the three rantes snps in a beijing chinese cohort [ ] . three hundred and fifty six sars patients and healthy controls were genotyped and their genotype and allele frequencies were shown in (table ). all genotype distributions of the two groups were in hwe. no significant difference was observed between the frequencies in the snps between patients and controls. next, we investigated the association of rantes - c/g with sars severity. twenty patients were classified as severe group as defined by their admissions to intensive care units or deaths due to sars [ ] . the genotype and allele frequencies of this snp in severe and mild patients were shown in table . the genotype distribution among the two groups were significantly different (p = . ). the frequencies of cg and gg genotypes were over-represented in the severe group (cg: or = . , %ci: . - . ; gg: or = . , %ci: . - . ). the frequency of g allele was also significantly increased in the severe group (or = . , %ci: . - . , p = . ). we described here that hong kong chinese with rantes - cg and gg genotypes had a . -fold ( % ci: . - . ) and . -fold ( % ci: . - . ) increased risk of developing sars respectively (p < . ) ( table ) . this - g allele also increased the risk of death of hong kong chinese patients with sars in a gene-dosage dependent (table ). more importantly, we further confirmed the association of rantes - c/g with the severity of sars by studying the beijing chinese cohort and found that beijing chinese patients with cg (or = . , %ci: . - . ) and gg (or = . , %ci: . - . ) genotype were more severe, as defined by admission to intensive care units or deaths due to sars. to further investigate the association of rantes with sars, we have also performed haplotype analysis using the studied snps of rantes, i.e. - a/g, - c/ g and in . t/c, for constructing the haplotypes. however, the major effect of the haplotypes was due to the snp rantes - only (data not shown). rantes is responsible for the recruitment of eosinophils, lymphocytes, monocytes and basophils at the site of inflammation and is involved in many viral infections [ , ] . we found that - g allele of rantes associated with the susceptibility to and death from sars. indeed, rantes - c/g is located at the nf-κb binding site, which is confirmed by gel-mobility shift assays [ ] , meaning that this snp may be involved in the regulation of rantes expression. further in vitro studies show that rantes - g allele enhances nf-κb binding that leads to elevation of promoter activity and increases rantes expression in cd + t cells, cd + t cells and monocytes/ macrophages [ , ] . therefore, together with our observation that - g allele associated with sars, we conclude ns = not significant. *p-value and or ( % ci) were calculated with the use of logistic regression models, adjusted with sex and age. after correction by bonferroni method, the significant p value should be less than . this study showed that rantes - g allele was a risk factor that associated with severe clinical outcomes in both hong kong and beijing chinese sars patients. it has to be noted that many cytokines/chemokines released from activated immune cells not only take part in the process of anti-viral immune response, but are also involved in cell damage and organ dysfunction [ ] . apart from the chemokine receptor signaling pathway, rantes could activate t cells through herbimycin a-sensitive protein tyrosine kinase (ptk)-mediated pathway at high concentration [ ] . this triggers the release of inflammatory cytokines and chemokines such as il- , il- , ifn-γ and mip -β [ ] . we have recently demonstrated sars coronavirus can induce high level of expression of chemokines from human dendritic cells [ ] . too high a level of rantes may intensify lung inflammation and lead to lymphopenia, increasing the chance of secondary infection and hence death rate among sars patients [ , ] . therefore, we speculate that the - g allele that associates with the higher level of rantes may enhance the inflammation and lead to severe clinical outcomes of sars. ns = not significant. p-value and or ( % ci) were calculated with the use of logistic regression models, adjusted with sex and age. indeed, rantes - g allele did show a strong association with death in hong kong chinese patients with sars (table ) and this observation was confirmed in beijing chinese that the rantes - g allele was associated with admission to intensive care units or deaths due to sars (table ). in the case of sars susceptibility, the rantes - g allele was associated with hong kong chinese patients only but not in beijing chinese patients. it has been suggested that chinese in southern and northern china may be genetically distinct [ , ] , accounting for the different observations with regard to sars susceptibility. we demonstrated that the rantes - g allele, which correlates with high rantes production, was associated with sars susceptibility in hong kong chinese. it was also associated with adverse outcomes from sars in both hong kong and beijing chinese. these suggest that a high rantes level may play a role in the pathogenesis of sars. sars study group: coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome pathogenesis of severe acute respiratory syndrome mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection the interferon gamma gene polymorphism + a/t is associated with severe acute respiratory syndrome homozygous l-sign (clec m) plays a protective role in sars coronavirus infection association of sars susceptibility with single nucleic acid polymorphisms of oas and mxa genes : a case-control study chemokine regulation of inflammation during acute viral infection chemokine upregulation in sars coronavirus infected human monocyte derived dendritic cells defense against influenza a virus infection : essential role of the chemokine system polymorphisms in the promoter region of rantes and the regulatory region of monocyte chemoattractant protein- among chinese children with systemic lupus erythematosus selective attraction of monocytes and t lymphocytes of the memory phenotype by cytokine rantes cytokine rantes released by thrombin-stimulated platelets is a potent attractant for human eosinophils nuclear factor-kappa b potently up-regulates the promoter activity of rantes, a chemokine that blocks hiv infection polymorphism in rantes chemokine promoter affects hiv- disease progression chemokines -chemotactic cytokines that mediate inflammation rantes: a versatile and controversial chemokine characterization of cytokine/chemokine profiles of severe acute respiratory syndrome genetic relationship of populations in china towards a genetic history of china the authors do not have any commercial or other association that might pose a conflict of interest. mwn the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -eyigl wz authors: ionidis, georgios; hübscher, judith; jack, thomas; becker, britta; bischoff, birte; todt, daniel; hodasa, veronika; brill, florian h. h.; steinmann, eike; steinmann, jochen title: development and virucidal activity of a novel alcohol-based hand disinfectant supplemented with urea and citric acid date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: eyigl wz background: hand disinfectants are important for the prevention of virus transmission in the health care system and environment. the development of broad antiviral spectrum hand disinfectants with activity against enveloped and non-enveloped viruses is limited due to a small number of permissible active ingredients able to inactivate viruses. methods: a new hand disinfectant was developed based upon . % w/w ethanol and . % w/w -propanol. different amounts of citric acid and urea were added in order to create a virucidal claim against poliovirus (pv), adenovirus type (adv) and polyomavirus sv (sv ) as non-enveloped test viruses in the presence of fetal calf serum (fcs) as soil load. the exposure time was fixed to s. results: with the addition of . % citric acid and . % urea an activity against the three test viruses was achieved demonstrating a four log( ) reduction of viral titers. furthermore, this formulation was able to inactivate pv, adv, sv and murine norovirus (mnv) in quantitative suspension assays according to german and european guidelines within s creating a virucidal claim. for inactivation of vaccinia virus and bovine viral diarrhea virus s exposure time were needed to demonstrate a log( ) reduction resulting in a claim against enveloped viruses. additionally, it is the first hand disinfectant passing a carrier test with adv and mnv. conclusions: in conclusion, this new formulation with a low alcohol content, citric acid and urea is capable of inactivating all enveloped and non-enveloped viruses as indicated in current guidelines and thereby contributing as valuable addition to the hand disinfection portfolio. virus transfer via human hands is a major route of infection inside and outside medical settings [ ] . the efficiency of virus transfer from hands to hands (direct transmission) or via contaminated surfaces (indirect transmission) is closely connected with virus persistence [ ] . detailed studies regarding virus persistence are still lacking however non-enveloped viruses generally are persisting much longer than enveloped-viruses [ ] [ ] [ ] . viruses of the respiratory tract like the non-enveloped rhinovirus and the enveloped viruses like influenza virus and coronavirus can persist on surfaces for several days [ ] . when dried, enveloped blood-borne viruses as hepatitis c virus can be infectious for more than a week [ ] . non-enveloped viruses like hepatitis a virus (hav), adenovirus (adv) and human norovirus may even persist for several weeks [ , ] . the actual level of the viral contamination in the environment is likely underestimated due to limited detection of known viruses and presence of unknown viruses [ , ] . often the detection of viral genomes by nucleic acid assays is the only way to get insights: carducci et al. detected viral nucleic acid in . % of all surface samples in healthcare settings [ ] . a disinfectant for hand hygiene therefore would ideally possess broad antiviral spectrum covering nonenveloped in addition to enveloped viruses offering increased protection against persistent, unexpected or unknown viruses [ , ] . the recommendation of broad spectrum alcohol-based hand rubs (abhrs) for preventing virus transmission by hands is the most important feature of current guidelines [ ] . worldwide there are differences regarding the number of test viruses needed to be inactivated in standardized tests before a broad spectrum claim including nonenveloped viruses can be given by manufacturers for a hand disinfectant [ ] . regulatory authorities in usa admit activity against certain stable reference viruses using methods of american society for testing and materials (astm) ( table ). the choice of reference test virus is left to the manufacturer [ ] . hand antiseptics can be tested on artificially contaminated hands or fingerpads with test viruses such as adv, rhinovirus type or , human rotavirus, surrogates of human norovirus like mnv or feline calicivirus (fcv) and hav [ ] [ ] [ ] . yet cases are well documented in which a disinfectant active against a reference virus was not active against an non-enveloped virus like parvovirus [ ] . regulatory authorities in germany have established a minimum set of test viruses [ ] which are not only difficult to inactivate, but also vary in their susceptibility to disinfectants and thus are thought to be representative of the whole known virus families. under the guideline of deutsche vereinigung zur bekämpfung der viruskrankeiten e.v. and the robert koch-institute (dvv/rki guideline) [ ] , disinfectants achieving at least log titer reduction factor (rf of ) against vaccinia virus and bovine viral diarrhea virus (bvdv) are active against all enveloped viruses (limited spectrum virucidal) [ , ] . disinfectants also inactivating poliovirus (pv), adv and polyomavirus sv (sv ) and since also mnv can claim activity against all viruses (virucidal) according dvv/rki guideline [ ] . the discrimination between enveloped only / all viruses was proved successful and has been taken up in the european en for hand rubs being valid in whole europe (table ) [ ]. in , an additional dvv guideline for testing the antiviral activity of disinfectants on stainless steel disks carriers simulating practical situations was established [ ] and the discrimination limited spectrum virucidal / virucidal claim exists there as well (table ) . at present, work is in progress to develop a en norm for the virucidal carrier test testing mnv and adv as test viruses [ ] . most biocidal active substances used in hand hygiene have no difficulty inactivating enveloped viruses [ ] , which are sensitive to alcohol-based hand rubs even in the presence of interfering substances [ ] . achieving the virucidal claim of the dvv/rki guideline is considerably more difficult and products that can claim inactivation of all non-enveloped viruses according to the german regulatory model are rare in hand hygiene [ ] . the fulfilment of dvv/rki guideline and en in hand hygiene area is even complicated by the practical requirement of a short exposure time. a user will normally not wait more than about - s for a abhrs to act and will not reapply the product if it has dried up before the target exposure time [ ] . to date all of the products that claim virucidal activity for hand hygiene under german dvv/rki guideline are alcohol-based formulations containing either high amounts of ethanol or an ethanol/ -propanol mixture supplemented with phosphoric acid [ ] . yet high alcohol content hand disinfectants are problematic for reasons of fire safety and toxicity [ ] and it is also desirable to produce abhrs with reduced acidity. finally, alcohols were reported to inactivate adv on carriers [ ] , but little is known regarding the ability of abhrs to fulfil the dvv carrier test. therefore, it was the aim to develop a hand disinfectant with a virucidal claim in suspension and carrier tests. we now present a novel abhr based on ethanol (ca %), supplemented with variable amounts of citric acid and urea additives that fulfils dvv/rki guideline and en for virucidal activity in quantitative suspension tests. the formulation with the optimum ratio of additives was further characterized in detail showing to possess virucidal activity (without enteroviruses and parvoviruses) on carriers according to dvv guideline [ ] . the formulations were supplied by oro clean chemie ag, p.b. , ch- fehraltorf, switzerland containing . % weight/weight (w/w) ethanol, . % w/w propanol, different amounts of citric acid ranging from . to . % and of urea between % and . % plus polyethylengylcols as skin care compound. the formulations are manufactured following strict quality criteria. purified water, prepared by a combination of ion exchange and reverse osmosis from municipal water, was used in preparation of all formulations. the microbial count of purified water was under the cfu/ml acceptance criterion specified in european pharmacopoeia (ph. eur.) . . all other components were of ph. eur. quality. tests were carried out in accordance with the dvv/rki guideline at °c [ ] . one part by volume of test virus suspension and one part by volume of aqua bidest. or fcs were mixed with eight parts by volume of the formulations. infectivity was stopped by immediate serial dilution with ice-cold medium and later determined by means of end point dilution titration in microtiter plates. μl of each dilution were placed in eight wells of a sterile polystyrene flat bottomed -well microtiter plate containing μl suspension of permissive cells. cultures were observed for cytopathic effects (cpe) after - days of inoculation depending on the cell culture system. all tests without the initial screening step were conducted in two independent test runs on different days. virus controls were incorporated after the longest exposure time. the different formulations of the new hand rub based on ethanol, citric acid and urea were screened undiluted ( . % due to the addition of test virus suspension and interfering substance) against pv, adv and polyomavirus sv as non-enveloped test viruses of the guideline of dvv/rki in the presence of fcs with a fixed exposure time of s. the ethanol and -propanol amounts were constant ( . % w/w and . % w/w, respectively) in these assays while citric acid and urea were used in a dosedependent manner. the concentration of urea varied between % and . %, whereas the concentration of citric acid ranged from . to . %. for determination of cytotoxicity the formulations were serially diluted -fold in mem up to a dilution of − . one part by volume of water of standardised hardness (instead of test virus suspension) was mixed with one part by volume of interfering substance and eight parts by volume of the disinfectant. aliquots of μl of each test concentration and each dilution were then inoculated into eight wells of a -well microtiter plate containing μl suspension of permissive cells. a control studying the suppression of activity was included. the cell cultures were observed for cytotoxic effects for the same incubation time as afterwards used for the quantitative suspension tests. virus titers were determined using the methods of spearman [ ] and kaerber [ ] and expressed as log tcid /ml including standard deviation. titer reduction is presented as the difference between the virus titer after the exposure time with the disinfectant and the control virus titer (water). according to the guideline of the dvv/rki, a formulation under test conditions must give at least a . log reduction in infectivity titer of test virus (inactivation ≥ . %) at the recommended concentration and exposure time to be considered active [ , ] . tests according to en were run in parallel to the guideline of dvv/rki with pv, adv and mnv as test viruses of the en and the corresponding permissive cells [ ] . the main difference to the german guideline is the change from aqua bidest. and fcs as interfering substances to clean conditions ( . % bovine serum albumin, final concentration in the test procedure . g/l) and the use of water for dilutions of ready-to-use products like hand rubs. a control of efficacy for suppression of disinfectant's activity was included. the quantitative carrier test according to the guideline of dvv was performed with clean conditions [ ] . the cleaning of the stainless steel disks ( mm diameter, gk formblech gmbh, d- berlin, germany) was performed as described in the guideline [ ] . a total of μl of the virus inoculum was deposited on each pretreated carrier and dried. then, inoculum was covered with μl new formulation (for the control μl of hard water was applied) and incubated for and min, respectively. immediately at the end of the exposure time, the disks were transferred into plastic vial holders (sarstedt ag & co. kg, d- nümbrecht) with μl of ice-cold culture medium to stop the activity of the formulation. vials were vortexed for min to recover the residual viruses and the eluate was immediately diluted -fold (quantal test method) for determining viral infectivity. cytotoxicity was measured as described in the guideline [ ] . in addition, a control of efficacy for suppression of disinfectant's activity was included. to estimate differences in the dose-dependency of the virucidal effect of the tested compounds, we calculated the slope of a linear regression model fitted line for urea titration at each citric acid concentration and vice versa. steeper slopes indicate greater dose-dependency. development and virucidal screening of novel formulations containing constant alcohol and different urea and citric acid concentrations as shown in table increasing amounts of citric acid and urea with a constant concentration of ethanol and -propanol resulted in a higher virucidal activity ( table ) . for pv the addition of . % urea and . % citric acid to the alcohols compounds were sufficient to reach a log reduction (table ). in case of adv either the combination of . % urea and . % citric acid or . % urea with . % citric acid were needed to achieve sufficient reduction in viral titers (table ) . for the polyomavirus sv greater virucidal activity as for adv and a lower activity as for pv with a combination of . % urea and . % citric acid could be observed (table ) . to compare the dose-dependency virucidal effects of urea and citric acid, we calculated the slope of a linear regression model fitted line for urea titration at each citric acid concentration and vice versa. as depicted in fig. for poliovirus, when the urea concentration was kept constant with increasing citric acid concentrations a clear dose-dependent increase of the virucidal effect could be observed (fig. a) . titration of urea with constant citric acid concentrations did not results in such combinatory effect (fig. a) . similar findings could be observed for adv and sv , although here the dosedependent effect of citric acid was not as pronounced as for pv (fig. b and c) . in our system, urea without the addition of acid was not achieving virucidal activity: at % urea the reduction of poliovirus titer was . log steps (data not shown). in conclusion, the screening experiments of a novel ethanol-based formulation supplemented with urea and citric acid showed a strong virucidal activity against the three non-enveloped test viruses of the guideline of dvv/rki in the presence of fcs. consequently, the formulation with the sufficient virucidal activity containing . % w/w ethanol, . % w/w -propanol, . % urea and . % citric acid was tested against several non-enveloped (mnv, adv, pv, polyomavirus sv ) and enveloped viruses (bvdv, vaccinia virus strain elstree) in the presence or absence of fcs according to guideline of dvv/rk or in clean conditions according to en . the results are presented in table and show that a virucidal activity against enveloped viruses was achieved in already s independent of the soil load. also the nonenveloped mnv, pv and adv were inactivated within such a short exposure time of s in clean conditions, whereas s were needed for pv, adv and polyomavirus sv in the presence of fcs (table ). these results show that the final formulation supplemented with . % urea and . % citric acid exerts a strong virucidal activity against a broad panel of viruses. in general, the non-porous surface test method is designed to evaluate the ability of chemical biocides to inactivate vegetative bacteria, viruses, fungi, mycobacteria and bacterial spores on inanimate surfaces. here, we evaluated the final formulation as described above to inactivate dried vaccinia virus strain elstree, adv and mnv as test viruses of the dvv guideline [ ] within and min exposure time on stainless steel disks enabling a virucidal claim (fig. ) . all test viruses proved to be very stable during the drying process and finally the new formulation achieved the following reduction factors: . (mnv), . (vaccinia virus) and . (adv) (fig. ) . longer exposure times resulted in higher reduction factors for all viruses tested. in conclusion, the alcohol-based formulation containing the development of broad antiviral spectrum hand disinfectants with activity against all enveloped and nonenveloped viruses is limited by the small number of permissible active ingredientsbroad spectrum hand rubs are generally based on alcohol. with ethanol alone in concentrations above % v/v stable non-enveloped viruses like pv and adv can be inactivated but not polyomavirus sv [ , ] . in contrast, > % v/v -and -propanol being often used in abhrs can easily inactivate adv and sv but not pv [ , ] . human enterovirus was only inactivated by % ethanol and not by % and % ethanol or any concentration of isopropanol [ ] . the reasons for this differential sensitivity of viruses to alcohols are presumed to result from the hydrophobic / hydrophilic nature of the viral particles. the hydrophilic pv is more susceptible to ethanol and the more hydrophobic adv and polyomavirus sv are fig. virucidal efficacy of the final formulation with % urea and % citric acid against dried viruses. a carrier assay was performed with murine norovirus (mnv), vaccinia virus strain elstree and adenovirus (adv) as test viruses at two different exposure times. the reduction factor was determined and is displayed with standard deviations more susceptible to both propanols [ , ] . an improvement in activity can be achieved by re-formulation of alcohol solutions with additional ingredients that may enhance the activity. it was therefore a subject of recent developments to look for such additives that would produce a virucidal alcohol-based hand disinfectant. yet products on the market, which are able to inactivate pv, adv and mnv as required in the en and also polyomavirus sv as required by the dvv/rki guideline are few. they are based either on - % ethanol, achieving an activity time of min or about % ethanol with the addition of . % phosphoric acid, achieving a sufficient activity time of min [ ] . we now report that urea in combination with citric acid can enhance the virucidal activity of ethanol solution (ca %) and inactivate all reference viruses of the dvv/rki guideline within min exposure time. tests with bacteria and fungi are still under investigation for this antiseptic formulation which requires a broad spectrum as shown earlier for a product based on chlorine and alcohol [ ] . the virucidal activity of concentrated aqueous urea solutions against pv is well known from early experiments with monkeys [ ] . organic acids used as a diluted aqueous solution are active against enveloped but not against non-enveloped viruses on their own [ ] . the activity of % ethanol solution against non-enveloped fcv increases from . log to > . log reduction when the ph of the solution is lowered from . to . [ ] . citric acid has shown a virucidal efficacy against rhinovirus at artificially contaminated hands [ ] and has already been incorporated in an alcohol-based hand rub [ ] . inorganic acids achieve the highest increase in virucidal activity of alcohol formulations [ ] . a synergistic virucidal effect of urea and citric acid additives with ethanol was evaluated by measuring the activity against pv, adv and polyomavirus sv according to guideline of dvv/rki in the presence of fcs. keeping urea concentration stable and increasing acid and vice versa, as well as increasing the concentration of both compounds generally increased the antiviral activity of the mixture against all three non-enveloped test viruses. in the presence of fcs, the optimal concentrations of citric acid and urea for the new formulation were identified which resulted in a log reduction against all three test viruses ( table ) within to s. interestingly, we found a better activity in the quantitative suspension test against pv and polyomavirus sv compared to adv with lower concentrations of urea and citric acid. a urea amount of . % and citric acid amount of . % was sufficient to inactivate pv and an urea amount of . % in combination with . % citric acid was sufficient to inactivate polyomavirus sv, but neither of these formulations was sufficiently active on adv. these results are consistent with the data from the study of kramer et al., who tested a virucidal alcoholic hand rub containing a low amount of ethanol and phosphoric acid and found adv type to be more stable than pv and sv [ ] . these results strengthen the idea to test viruses from different virus families with various susceptibilities as found in the en and dvv/rki guideline although it is known that important virus like hepatitis a virus and parvoviruses might be more stable than the test viruses used [ ] . in table it can be seen that the formulation containing % citric acid and % urea possesses the needed activity for all required test viruses at the lowest citric acid and urea contents, which was then adopted for further analysis. the virus testing of this formulation confirmed the activity against a broad spectrum of human pathogenic viruses in the quantitative suspension assay. enveloped viruses like bvdv and vaccinia virus were inactivated within s exposure time. pv, adv and polyomavirus sv were inactivated with a. bidest. and fcs as interfering substances within one minute exposure time. under clean conditions according to the en an exposure time of s was achieved with adv, pv and mnv. the results also shows that with en higher rfs were achieved compared to the dvv/ rki guideline (table ) . tests on carriers confirmed the activity of the formulation found in the suspension assays also against viruses dried on the surface. the disinfectant inactivated adv, mnv and vaccinia virus within one minute, making it active against non-enveloped viruses at low level (without enteroviruses and parvoviruses) on carriers, as defined according to dvv guideline. the activity of the formulation is sufficient for virucidal activity on carriers according to the current version of pren . stainless steel carrier methods have shown a good overall reproducibility between different labs [ ] , but the results on carriers may be not directly transferable to in-vivo situation. fingerpad methods may be a better alternative for abhrs testing under practical conditions, however fingerpad methods seem to lack reproducibility, which may be in part due to the inability of the method to properly distinguish the washing out of virus by mechanical means from virus inactivation by disinfectant. when comparing a mixture of propan- -ol and propan- -ol (rf = . ) and a hand wash product (rf = . ) in the fingerpad test, tuladhar et al. concluded that washing hands with soap and water is better than using hand rubs based on alcohol for removal of norovirus from hands [ ] . own data with the astm e - [ ] including modifications derived from the en [ ] and mnv as test virus demonstrated rfs of . and . after s for ethanol-based disinfectants with addition of an acid, whereas even water was able to achieve a rf of . [ ] . other works report that abhr in fingerpad tests achieve rfs of - against nonenveloped viruses, with hard-water rinse achieving rf of [ ] . ethanol-based hand rub fortified with phosphoric acid achieved in the fingerpad test with pv a rf of . after s [ ] , whereas % ethanol alone was not active against pv on contaminated hands in earlier tests [ ] . further research should provide clear discrimination between mechanical removal (addition of water) and the additional inactivation by chemical biocides for fingerpad tests. a direct comparison of results from european stainless steel carrier method and artificially contaminated fingerpad or whole hand methods should be carried out. hygienic hand disinfection can only be done with intact skin. for dermal tolerance no data for the product developed are available. in another study with a formulation containing % ethanol and % citric acid % of the panelists were not included due to skin irritation [ ] . it can be expected that % citric acid will lower the described rate of adverse effect. the amount of urea on human hands after application of the new formulation is unknown. but urea has a positive effect on transepidermal water loss and on skin barrier function [ , ] . in conclusion, this new formulation with a low alcohol content, citric acid and urea is capable of inactivating all enveloped and non-enveloped viruses as indicated in en and dvv/rki guideline in quantitative suspension tests and inactivates mnv, adv and vaccinia virus on stainless disk carriers. the formulation contributes a valuable addition to the hand disinfection portfolio. it is of course not possible to test the activity of a hand disinfectant against each virus. the test viruses as mentioned in european norms or german guidelines analogous to bactericide testing are representatives for the whole spectrum of relevant viruses. therefore, the new formulation will not only inactivate the test viruses from the european norm or german guideline but is also covering the whole spectrum of all enveloped and all nonenveloped viruses being directly or indirectly transferred by human hands. financial support e. s. was supported by the dfg (ste / - ) and intramural young investigator award of the helmholtz centre for infection research. author details epidemiologic background of hand hygiene and evaluation of the most important agents for scrubs and rubs nosocomial spread of viral disease virus resistance in a hospital environment: overview of the virucide activity of disinfectants used in liquid form different virucidal activities of hyperbranched quaternary ammonium coatings on poliovirus and influenza virus survival of enveloped and non-enveloped viruses on surfaces compared with other micro-organisms and impact of suboptimal disinfectant exposure how long do nosocomial pathogens persist on inanimate surfaces? a systematic review inactivation and survival of hepatitis c virus on inanimate surfaces significance of fomites in the spread of respiratory and enteric viral disease enteric viruses of humans and animals in aquatic environments: health risks, detection, and potential water quality assessment tools computational tools for viral metagenomics and their application in clinical research environmental survey to assess viral contamination of air and surfaces in hospital settings microbicides and the environmental control of nosocomial viral infections viruswirksame desinfektion: die prophylaxe nosokomialer virusinfektionen guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force some principles of virucidal testing hygienic hand antiseptics: should they not have activity and label claims against viruses? standard test method for determining the virus-eliminating effectiveness of liquid hygienic handwash and handrub agents using the fingerpads of adult volunteers standard test method for evaluation of handwashing formulations for virus-eliminating activity using the entire hand the fingerpad protocol to assess hygienic hand antiseptics against viruses disinfection efficacy against parvoviruses compared with reference viruses testing virucidal activity in germany: an update deutsche vereinigung zur bekämpfung der viruskrankheiten e.v. "(dvv; german association for the control of virus diseases) and robert koch institute (rki; german federal health authority) for testing the virucidal efficacy of chemical disinfectants in the human medical area rki) sowie des fachausschusses "virusdesinfektion" der deutschen vereinigung zur bekämpfung der viruskrankheiten (dvv) und der desinfektionsmittelkommission der deutschen gesellschaft für hygiene und mikrobiologie (dghm suitability of vaccinia virus and bovine viral diarrhea virus (bvdv) for determining activities of three commonlyused alcohol-based hand rubs against enveloped viruses leitline der deutschen vereinigung zur bekämpfung der viruskrankheiten (dvv) e.v. zur quantitativen prüfung der viruziden wirksamkeit chemischer desinfektionsmittel auf nicht-porösen oberflächen. anwendung im bereich humanmedizin standard in development. chemical disinfectants and antiseptics -quantitative non-porous surface test without mechanical action for the evaluation of virucidal activity of chemical disinfectants used in the medical area -test method and requirements (phase /step ) virucidal efficacy of a combination of . % peracetic acid and % (v/v) ethanol (paa-ethanol) as a potential hand disinfectant update on hand hygiene virucidal activity of a new hand disinfectant with reduced ethanol content: comparison with other alcohol-based formulations evaluation of a virucidal quantitative carrier test for surface disinfectants the method of right and wrong cases (constant stimuli) without gauss's formulae beitrag zur kollektiven behandlung pharmakologischer reihenversuche leitlinie der deutschen vereinigung zur bekämpfung der viruskrankheiten (dvv) e. v. und des robert koch-instituts (rki) zur prüfung von chemischen desinfektionsmitteln auf wirksamkeit gegen viren in der humanmedizin inactivation of murine norovirus by chemical biocides on stainless steel handbuch der viruswirksamen desinfektion virucidal composition. organization wip, wo virucidal activity of disinfectants: studies with the poliovirus efficacy of alcohols and alcohol-based hand disinfectants against human enterovirus virucidal activity of alcohols. virucidal efficiency of alcohols against viruses in liquid phase antimicrobial activity of a new intact skin antisepsis formulation virucidal (rabies and poliomyelitis) activity of aqueous urea soltutions virucidal activity of organic acids comparative efficacy of seven hand sanitizers against murine norovirus, feline calicivirus, and gii. norovirus effectiveness of hand sanitizers with and without organic acids for removal of rhinovirus from hands improved inactivation of nonenveloped enteric viruses and their surrogates by a novel alcohol-based hand sanitizer reducing viral contamination from finger pads: handwashing is more effective than alcohol-based hand disinfectants chemical disinfectants and antiseptics. hygiene hand disinfection. test method and requirements comparison of virucidal activity of alcohol-based hand sanitizers versus antimicrobial hand soaps in vitro and in vivo activity of an alcohol-based hand gel against human adeno-, rhino-, and rotaviruses using the fingerpad method experiments on antiviral activity of hand disinfectants. some theoretical and practical considerations a randomized trial of the efficacy of hand disinfection for prevention of rhinovirus infection urea-containing moisturizers influence barrier properties of normal skin urea uptake enhances barrier function and antimicrobial defense in humans by regulating epidermal gene expression • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -f xc uu authors: milinovich, gabriel j; avril, simon m r; clements, archie c a; brownstein, john s; tong, shilu; hu, wenbiao title: using internet search queries for infectious disease surveillance: screening diseases for suitability date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: f xc uu background: internet-based surveillance systems provide a novel approach to monitoring infectious diseases. surveillance systems built on internet data are economically, logistically and epidemiologically appealing and have shown significant promise. the potential for these systems has increased with increased internet availability and shifts in health-related information seeking behaviour. this approach to monitoring infectious diseases has, however, only been applied to single or small groups of select diseases. this study aims to systematically investigate the potential for developing surveillance and early warning systems using internet search data, for a wide range of infectious diseases. methods: official notifications for infectious diseases in australia were downloaded and correlated with frequencies for internet search terms for the period – using spearman’s rank correlations. time series cross correlations were performed to assess the potential for search terms to be used in construction of early warning systems. results: notifications for infectious diseases ( . %) were found to be significantly correlated with a selected search term. the use of internet metrics as a means of surveillance has not previously been described for ( . %) of these diseases. the majority of diseases identified were vaccine-preventable, vector-borne or sexually transmissible; cross correlations, however, indicated that vector-borne and vaccine preventable diseases are best suited for development of early warning systems. conclusions: the findings of this study suggest that internet-based surveillance systems have broader applicability to monitoring infectious diseases than has previously been recognised. furthermore, internet-based surveillance systems have a potential role in forecasting emerging infectious disease events, especially for vaccine-preventable and vector-borne diseases. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. prudent detection is a cornerstone in the control and prevention of infectious diseases. traditional infectious disease surveillance systems are typically characterised by a bottom-up process of data collection and information flow; these systems require a patient to recognise illness and seek treatment and a physician or laboratory to diagnose the infection and notify the relevant authority [ , ] . for emerging infectious disease events, this process is reported to take, on average, days from onset to detection and a further - hours for the world health organization to be notified [ ] . the development and implementation of more efficient systems for gathering intelligence on infectious diseases has the potential to reduce the impact of disease events. internet-based surveillance systems are one such system [ ] . internet-based surveillance systems produce estimates of disease incidence through analysis of various digital data-sources. targeted sources include internet-search metrics, online news stories, social network data and blog/ microblog data [ ] . currently, the most promising approach appears to be those based upon monitoring of internet search behaviour. this approach works on the premise that people will actively seek information on diseases they develop and that estimates of disease activity with the community may be developed by monitoring the frequency of related internet searches. through targeting people earlier in the disease process, internet-based systems are able to access a larger fraction of the community and produce more timely information. furthermore, internet-based surveillance systems are intuitive and adaptable, cheap to run and maintain (once established), do not require a formal public health network and have the capacity to be automated and operate in near-real time. despite these advantages, internet-based surveillance systems have a number of significant shortcomings and must not be considered an alternative to traditional surveillance approaches [ ] . firstly, as these systems crowd-source data, resolution will be contingent on the size of the population serviced and may be further limited by national communications infrastructure availability and distribution [ ] . secondly, as internetbased surveillance systems are limited to people who use the internet to source health information, there is the potential that estimates produced by these systems may not accurately reflect the entire community [ ] . finally, as internet-based surveillance systems essentially rely upon self-reporting, bias may be introduced through differences in internet usage between sectors of the community (the elderly, for example, may not use the internet as a source of health information, despite being a high-risk group for many infectious diseases) and/or through media driven interest in emerging disease events [ ] . infectious diseases surveillance systems have been developed using internet search metrics to estimate incidence of influenza (google flu trends) [ ] and dengue (google dengue trends) [ ] . currently, operational systems that utilise this approach are limited, however, studies of the potential for internet-based surveillance have been conducted for a range of other infectious diseases, including: acute respiratory illness [ ] , aids [ ] , chickenpox [ , ] , cryptosporidiosis [ ] , dysentery [ ] , gastroenteritis [ ] , hepatitis [ ] , listeriosis [ ] , lyme disease [ ] , methicillin-resistant staphylococcus aureus [ ] , norovirus [ ] , respiratory syncytial virus [ ] , rotavirus [ ] , scarlet fever (streptococcus pyogenes) [ , ] , salmonella [ ] , tuberculosis [ , ] and west nile virus [ ] . previous studies have focused on single diseases, or a small number of diseases, and the justification of the focus on a particular disease has been specific to each study. the published results have largely been promising; however, to date there has been no systematic, generalizable analysis to identifying diseases that are suited to monitoring through the analysis of internet-search metrics. the underpinning goal of this study was to provide direction for future approaches to developing digital surveillance systems; such as the development of predictive models and/or integrative surveillance models that draw upon multiple traditional and digital data source to create estimates of disease within the community. this study, however, did not aim to develop actionable surveillance systems, produce predictive models of infectious disease based on internet-based data or to identify the best search terms for use in these models. rather, this study aimed to determine which diseases have most promise for monitoring by surveillance systems built on internet search metrics; this was achieved by assessing the level of correlation between a wide range of infectious diseases and internet search term metrics. finally, this study aims to identify diseases for which internet-based data could be used to create early warning systems. surveillance data on notifiable infectious diseases were collected from the national notifiable disease surveillance system (nndss) which is maintained by the australia government department of health (doh) [ ] . monthly notifications (case numbers) aggregated at state/territory and national level, were downloaded for the period of january to september . a full list of notifiable diseases in australia and case definitions can be accessed through the doh webpage [ ] . sixty-four diseases are monitored and these are categorised in the nndss as belonging to one of eight groups: bloodborne diseases; gastrointestinal diseases; other bacterial diseases; quarantinable diseases; sexually transmissible infections; vector-borne diseases; vaccine preventable diseases; and zoonoses. for the purpose of consistency, we have reported diseases according to these groupings. whilst notifiable, data were not downloaded for human immunodeficiency virus infection/acquired immunodeficiency syndrome, creutzfeldt-jakob disease or variant creutzfeldt-jakob disease because surveillance for these diseases is not performed by doh or for severe acute respiratory syndrome, because reporting to the doh is informal; as such, these diseases are not listed on the nndss. in the construction of google flu trends model, the authors identified search terms by performing correlations between influenza-like illness data from the us cdc and the top million google search queries performed in the us over the corresponding period [ ] . such data is not available to the public and an alternative approach to identification of search terms was required; two approaches were used. firstly terms related to diseases, the aetiological agents and colloquialisms (such as "hep" for hepatitis or "flu" for influenza) were manually identified. secondly, google correlate (www.google.com/trends/correlate) was queried using monthly surveillance data (described above). google correlate provides a list of up to search terms that correlate most highly with the query data. to account for potential language shifts that may have affected search behaviour [ ] , this was performed three times using surveillance data covering the periods - , - and - . up to search terms were downloaded from google correlate for each notifiable disease ( search terms per period analysed) and manually sorted; any term related to the queried notifiable disease was included, regardless of the nature of the potential association suitable terms were combined with the manually identified search terms to create a list of search terms (see additional file ). no attempt was made to filter search terms based upon biological plausibility; any term that may be perceived to have any association with the disease of interest was included. search frequencies for terms of interest were collected through google trends (www.google.com/trends/). all data extractions were performed on the nd of october, . google trends was queried using each of the identified terms at a national and state/territory level using the entire time range available ( -present). google trends presents search frequency as a normalised data series with values ranging from to (with representing the point with the highest search frequency and other points scaled accordingly); functionality for exporting search frequency data as a .csv file is provided. for the purpose of privacy, data are aggregated at a daily, weekly or monthly level (or are restricted if there is insufficient search volume). the level of aggregation applied is determined by the period analysed and the search frequency; the level of aggregation is not able to be specified by the user. as the notifiable disease surveillance data used was in monthly format, monthly indices of query search frequencies were required. monthly indices are displayed graphically by google trends when querying periods greater than months; rather than downloading. csv files, a script was developed to scrape data from the google trends webpage, allowing the problems associated with the level of data aggregation to be overcome. analyses were performed at both national and state levels for the period - . as state-level search frequency data were not always available, particularly for less common diseases (due to low search frequency at this level of disaggregation), correlations between state-level notification data and national search frequency data were also performed. owing to the large number of correlations performed in this study, bonferroni adjustments [ ] were applied to significance levels by the equation -( -α) /n ; all p-values reported in this document correspond to onetailed tests. spearman's rank correlation coefficients were used to rank performance. time-series cross correlations were performed to assess linear associations between disease notifications and google trend search indices. cross correlations were calculated using lag values for google trends data ranging from − to . this range allowed for assessment of biologically plausible associations that were relevant to the development of early warning systems. cross correlations were performed on national data using ibm spss version (spss inc; chicago, il, usa). seasonal differencing was applied (value ) to all analyses to remove cyclic trends. whilst all available data ( - ) were downloaded, analyses for this study were focused on the most recent five years ( - ) as preliminary data analyses indicated that google trends data were not available prior to for numerous search terms ( figure ; panels , , , , and ). additionally, shifts in language are known to affect surveillance systems built upon textual data [ ] . the shortened period ( - ) was selected to minimise the effects of language shifts. however, this period still provides the requisite pairs of observations for performing cross correlations [ ] . in this section we discuss analyses of time series data. briefly, the time series analysed were monthly case numbers for the infectious diseases monitored by the australian government's national notifiable disease surveillance system (nndss) and google trends monthly search metrics for related internet search terms. in total, search terms were analysed in this study; this ranged from a single term for some diseases, up to search terms for influenza and search terms for pneumococcal disease. the majority of terms could be categorised as diseases or aetiological agents ("brucellosis" or "brucella"), colloquialisms ("flu", "hep" or "tb"), symptoms ("cough", "white discharge" or "cervical mucus") or medication or general health/treatment related queries ("whooping cough treatment", "symptoms of dengue" or "flu and pregnancy"). a few terms that may have environmental ("flash floods" for leptospirosis) or behavioural ("african tours" for malaria) meanings were also included. a full list of the search terms analysed is presented in the supplementary material. evaluation of the bivariate associations between surveillance and corresponding search frequency data was performed using the spearman's rank correlation. spearman's rank correlations for the top ranked notifiable diseases and terms are presented in figure and raw data for the corresponding diseases and search terms are presented in figure . results of spearman's correlations indicated diseases to be significantly correlated (p < . ; bonferroni corrected: p < . e − ) with at least one search term; p-values for of these were < . (bonferroni corrected: p < . e − ). marked differences were observed in correlations between the various disease groups. correlations for vaccine-preventable diseases were generally highest with six of fourteen exhibiting strong (rho = . - . ) or very strong (rho = . - . ) correlations, followed by sexually transmitted infections ( / ), the vector-borne diseases ( / ), blood-borne diseases ( / ), other diseases ( / ), zoonoses ( / ), gastrointestinal infections ( / ) and, finally, quarantinable diseases ( / ). state level correlations are also reported in figure . consistency between state correlations were variable with some diseases exhibiting reasonable consistency (pertussis; rank ), whilst others were inconsistent (hepatitis c; rank ). results of cross correlations are demonstrated in figure . cross correlation results should be interpreted as product-moment correlations between the two time series; they allow dependence between two time series to be identified over a series of temporal offsets, referred to as lags. lag values indicate the degree and direction of associations. a lag value of − indicates that correlations were performed using time series data for which the first series (google trends' data) has been shifted backwards one unit (a month). conversely, a lag value of indicates that the primary series had been shifted forward one unit. significant positive correlations for lag vales of ≥ or above are of most interest in the context of this study as they indicate a positive relationship between the two time series with google trends data leading the notifications (a pre-requisite for google trends data to be a suitable early warning tool). it should also be noted that seasonal differencing was applied to cross correlations to remove cyclic seasonal trends. disease notifications positively correlated at a lag of one month (lag ) with search term frequency for of the diseases that exhibited significant spearman's rank correlations. overall, of the notifiable diseases exhibited significant, positive correlations at lag of one month. significant positive associations were observed for four of the nine vector-borne diseases (barmah forest virus infection, dengue virus infection, murray valley encephalitis virus infection and ross river virus infection), six of the vaccine preventable diseases (haemophilus influenzae type b, influenza, pertussis, pneumococcal disease and varicella zoster (chickenpox and shingles)), two of the six blood-borne diseases (hepatitis b (unspecified) and c (unspecified)), two of gastrointestinal diseases (campylobacteriosis and cryptosporidiosis) and one zoonosis (leptospirosis). positive significant correlations were not observed at a lag of one month for any of the quarantinable diseases (n = ), sexually transmissible infections (n = ) or other bacterial infections (n = ). it should be noted that positive significant correlations were observed at lags of over one month (but not at lag ) for two of the top ranked diseases (gonococcal infection and meningococcal disease) and diseases overall (see additional file ). additionally, the terms "haemolytic uraemic syndrome" and "leprosy" exhibited significant negative correlations with the respective disease notifications at a lag of one month. the development and application of internet-based infectious disease surveillance systems has the potential to enhance infectious disease control and prevention. whilst this is widely recognised [ , , , , , , , ] the investigation and application of internet-based surveillance has not been systematically applied across infectious diseases; the lack of systemic knowledge regarding the potential breadth of internet-based surveillance appears to have restricted the development of systems to a small number of diseases. to our knowledge, assessments of the use of internet-based surveillance have only been performed for five of the diseases that were demonstrated to have a significant association with internet search terms (influenza [ ] , dengue [ , ] , chickenpox [ , ] , hepatitis b [ ] and cryptosporidiosis [ ] the authors of the final study were, however, not able to detect signals from internet search queries). our study suggests that internetbased surveillance systems have potential application to a wider range of diseases than is currently recognised. however, correlations alone should not be viewed as definitive evidence that such systems are viable; some discretion must be applied, particularly as the analyses performed were univariate. correlations between internet metrics and both gonococcal infection and chlamydia (figure , boxes and ) were high; this appears to be due to a general upward trend in both and internet metrics appears to have little value in detecting perturbations in cases beyond this. this is supported by the cross correlation results (which are seasonally differenced); despite being ranked nd and th by spearman rho (figure ), no positive correlations were observed for these disease/search term cross correlations, even at lag ( figure ) . further research needs to be performed; however, this study suggests surveillance systems build on internet search data to have significant promise for a number of diseases beyond those previously described, most notably pneumococcal disease, ross river virus infection, pertussis, barmah forest virus and invasive meningococcal disease. the application of internet-based data to monitoring systems of interest has been termed "nowcasting"; this approach does not predict the occurrence of future events, but rather seeks to produce more timely information on the systems of interest [ ] . for infectious disease surveillance, this is typically achieved through the ability of internet-based surveillance systems to collect data at an earlier time point than is possible for traditional systems or by circumventing bureaucratic structures inherent to traditional systems that impede information flow [ ] . search terms that exhibit a high level of correlation with disease notifications are of value as they may be used to provide faster intelligence on emerging disease events. results of cross correlations (figure ) , however, indicated that forecasting of infectious disease events may also be possible using internet-based data. of the diseases that exhibited significant spearman's correlations, also had significant positive cross correlations at a lag of one month. overall, cross correlations indicated that forecasting of notification rates using internet-based metrics would be most realistic for the vaccine-preventable and vector-borne diseases. despite search terms offering strong or very strong correlations for two of the sexually transmissible diseases, neither exhibited significant correlations at a lag of one month. whilst internet metrics may provide valuable information regarding disease status, it is important to view these within context. the term "dengue mosquito" (figure , panel ) leads notifications by up to one month. the data imply dependence of dengue notifications on searches for the term "dengue mosquito". the mechanism of this dependence is more likely that environmental conditions that increase the abundance of mosquitos in dengue risk areas correlate with both an increase in dengue notifications and increased search interest for "dengue mosquito", allowing the search term to be used as an indicator for notifications. in this context the internet metrics also provide information that is of potential significance with respect to control of dengue fever; there is increased interest regarding mosquitos in the community and this may be driven by an increase in mosquito numbers. conversely the incidence of disease in the community may also affect search habits. the search term "chikungunya" lags notifications for chikungunya virus infection (figure , panel ). searches for "chikungunya" are probably driven by media exposure. media bias has previously been reported to adversely affect internet-based surveillance systems [ , [ ] [ ] [ ] [ ] [ ] and an increase in cases of a disease in the community will likely result in the publication of stories about the disease in the media; in turn, media exposure will drive internet searches on the topic. these processes, however, are not necessarily mutually exclusive. searches for a disease may lead notifications, however, increased notifications and reporting of an emerging disease event in the media may also drive internet searches. the complexity of this relationship may make interpretation of google trends' data more difficult. for pertussis (figure , (see figure on previous page.) figure cross correlation results for the diseases with the highest spearman's rho values . cross correlations for two search terms are displayed for each disease. coloured bars correspond to the search term with the highest spearman's rho value for each disease (red bars indicate values that exceed the % confidence interval, whereas blue bars do not). unfilled bars indicate cross correlation results for alternative search terms with highest cross correlation values at a lag value of . confidence intervals ( %) are indicated by the grey lines. panel ), the term "whooping" exhibits a significant positive correlation with disease notifications from lag − through to lag . it appears that both mechanisms occur for the same term, demonstrating a potential difficulty in interpreting these data. it is imperative that any terms used in the development of forecasting models are heavily screened to address the complexities of the driving forces behind health-information seeking and routinely re-evaluated to account for any shifts in search behaviour which may occur [ ] . there were a number of obvious limitations to this study. the temporal resolution of the data used was monthly. internet-based surveillance systems built upon monthly data are unlikely to provide better intelligence than existing traditional surveillance systems; these commonly rely upon weekly or daily reporting. this was a function of the availability of the notification data. secondly, the analyses were performed for a specific setting: australia. the nuances of language will create differences in the applicability, not just for different countries, but also within a country and between different settings (such as during an influenza pandemic) [ ] . australia was selected as the study area because internet penetration in australia is very high (> %) [ ] and use is largely restricted to a single search engine; google maintains a market share of over % in australia [ ] . these features reduce biases associated with unequal patterns of use and/or access. additionally, owing to its extensive size, australia exhibits a range of climates and varying environmental conditions, making it susceptible to a wide range of infectious diseases, including endemic and nonendemic vector-borne diseases. additionally, australia has a strong public health network and comprehensive infectious disease surveillance systems which compile high quality data on a range of diseases. combined, these features of internet usage and availability, infectious disease surveillance systems and diseases susceptibility patterns make australia an ideal system in which to study the potential application of internet-based surveillance systems. it is hoped that this work will stimulate further research into internet-based infectious disease surveillance systems beyond australia. even within our own study, however, we observed variation in correlations between internet search metrics and disease notifications for the various states ( figure ). it is imperative to develop models specific to the region of interest and to assess the performance of any internet-based system against traditional surveillance data specific to the region being monitored. thirdly, this study analysed the performance of only single search terms in estimating infectious disease notifications. whilst google has not revealed the terms utilised, or the weightings applied, google flu trends is reported to incorporate around search terms [ ] . despite using only a single search term for each analysis, notifications for diseases were identified as having a strong or very strong correlation with the selected search terms. compounding this is the fact that bonferroni adjustments were applied in assessing significance. bonferroni adjustments have previously been criticised for being overly conservative and for increasing the occurrence of type ii errors (false negatives) [ ] . as such, whilst this study provides a base for future research, it would be remiss to limit future investigations to just these diseases. this study identified numerous infectious diseases of public health significance that had not previously been investigated to have potential for monitoring using internetbased surveillance systems however, this study did not seek to produce robust, accurate, internet-based surveillance systems or early warning systems that are able to produce actionable and timely data for public health units. the aim of this study was to identify the diseases for which this is possible and to focus future research efforts into these. to achieve this aim, this study used univariate analyses to determine the usefulness of internet search metrics for monitoring a wide range of infectious diseases. whilst this simplistic approach was useful for screening diseases, it will not suffice in monitoring or forecasting incidence. future studies should focus on developing composite indexes incorporate multiple search terms, or data sources (such as weather data). models built in such a manner are more resilient to media-driven behaviour, fear-based searching and evolutions in language [ ] . internet-based surveillance systems have the potential to be applied to more than just enumerating disease cases within the community or predicting the onset, peak and magnitude of outbreaks. internet-based systems also have value as tools for planning emergency department staffing and surge capacity [ , ] or for healthcare utilisation [ ] . future research needs to also investigate to application of internet-based data; the greatest challenge in this field may not actually be creating models for forecasting or monitoring disease within the community, but rather applying and articulating the significance in a manner that is beneficial. internet-based surveillance systems have broader applicability for the monitoring of infectious diseases than is currently recognised. furthermore, internet-based surveillance systems have a potential role in forecasting of emerging infectious disease events. additional file : complete tables of results for google correlate searches, google trends data, spearman correlations and cross correlations. trends and directions of global public health surveillance modeling the effects of epidemics on routinely 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the salary for gjm was provided through the australian national health the authors declare that they have no competing interests.authors' contributions gjm and wh developed the original idea for this study. development of the script for data collection was performed by smra. data analysis was performed by gjm with the assistance of wh, jsb, st and acac. the manuscript was primarily written by gjm with editorial advice from wh, smra, jsb, st and acac. all authors read and approved the final manuscript. key: cord- -woref g authors: fragoso-saavedra, sergio; iruegas-nunez, david a.; quintero-villegas, alejandro; garcía-gonzález, h. benjamín; nuñez, isaac; carbajal-morelos, sergio l.; audelo-cruz, belem m.; arias-martínez, sarahi; caro-vega, yanink; calva, juan josé; luqueño-martínez, verónica; gonzález-duarte, alejandra; crabtree-ramírez, brenda; crispín, josé c.; sierra-madero, juan; belaunzarán-zamudio, pablo f.; valdés-ferrer, sergio i. title: a parallel-group, multicenter randomized, double-blinded, placebo-controlled, phase / , clinical trial to test the efficacy of pyridostigmine bromide at low doses to reduce mortality or invasive mechanical ventilation in adults with severe sars-cov- infection: the pyridostigmine in severe covid- (pisco) trial protocol date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: woref g background: severe acute respiratory syndrome coronavirus (sars-cov- ) infection, the causative agent of coronavirus disease (covid- ), may lead to severe systemic inflammatory response, pulmonary damage, and even acute respiratory distress syndrome (ards). this in turn may result in respiratory failure and in death. experimentally, acetylcholine (ach) modulates the acute inflammatory response, a neuro-immune mechanism known as the inflammatory reflex. recent clinical evidence suggest that electrical and chemical stimulation of the inflammatory reflex may reduce the burden of inflammation in chronic inflammatory diseases. pyridostigmine (pdg), an ach-esterase inhibitor (i-ach-e), increases the half-life of endogenous ach, therefore mimicking the inflammatory reflex. this clinical trial is aimed at evaluating if add-on of pdg leads to a decrease of invasive mechanical ventilation and death among patients with severe covid- . methods: a parallel-group, multicenter, randomized, double-blinded, placebo-controlled, phase / clinical trial to test the efficacy of pyridostigmine bromide mg/day p.o. to reduce the need for invasive mechanical ventilation and mortality in hospitalized patients with severe covid- . discussion: this study will provide preliminary evidence of whether or not -by decreasing systemic inflammation- add-on pdg can improve clinical outcomes in patients with severe covid- . trial registration: clinicaltrials.gov nct (registered on april , ). severe acute respiratory syndrome coronavirus (sars-cov- ) infection, the causative agent covid- , may result in severe systemic inflammatory response. about one third of hospitalized patients with covid- develop acute respiratory distress syndrome (ards) [ ] , while % require invasive mechanical ventilation associated to a high mortality rate [ ] . the two main causes of death in patients with severe covid- are respiratory and multiple-organ failure as a result of overwhelming inflammatory response [ , ] . therefore, patients with severe covid- will theoretically benefit from therapeutic interventions that modulate the inflammatory response [ ] . pyridostigmine, an acetylcholinesterase inhibitor (i-ach-e), increases acetylcholine (ach) half-life by inhibiting its peripheral degradation. pyridostigmine has been used for decades in the symptomatic treatment of myasthenia gravis [ ] and as pre-exposure prophylaxis against nerve gas (chemical) warfare [ ] . pyridostigmine has well-characterized pharmacokinetic and safety profiles. recently, pyridostigmine has been shown to reduce persistent inflammation in people living with hiv- infection [ ] [ ] [ ] . the proposed anti-inflammatory effect occurs after the ach binds to nicotinic receptors on the surface of immune cells and this interaction causes a decrease in the production of pro-inflammatory cytokines. this so-called inflammatory reflex, originally described in response to vagus nerve stimulation [ ] , leads to the release of ach with a resulting reduction in acute [ ] and chronic inflammation [ ] . our primary objective is to evaluate whether or not add-on pyridostigmine to best medical management of hospitalized covid- patients will result in reduced need for invasive mechanical ventilation and death. our aim is to tests the efficacy of pyridostigmine use as an immunomodulator to reduce the incidence of complications leading to critical illness or death in hospitalized adults with severe covid- . in order to test this, we propose a randomized, double-blinded, placebocontrolled trial. participants will be randomly allocated in a : ratio to receive either oral pyridostigmine at a dose of mg/day or a matching placebo for a maximum of days in parallel groups. we will compare the need of invasive mechanical ventilation and fatality rates during the days following randomization (fig. ). unblinding will be permissible in case of severe adverse events at the request of the treating group of physicians, or at the request of the external data and safety monitoring board (dsmb). the study is planned in two parts: a phase aimed at determining security, followed by a phase part aimed at evaluating the effect -or lack thereof-of pyridostigmine in patients with severe covid- . phase started recruiting on may . during the security (phase ) part, we aimed at evaluating the safety and feasibility of the study and explore in a preliminary way the magnitude of the effect of the intervention. safety was evaluated according to the frequency of outcomes as well as of reported adverse events. enrollment for the security phase was limited to patients hospitalized at instituto nacional de ciencias médicas y nutrición salvador zubirán (incmnsz) in mexico city. on july , a pre-appointed dsmb performed an ad interim analysis after the first participants ( % of the calculated sample) had been recruited and, as results derived from this security part indicated that pyridostigmine was not associated with an increased frequency of outcomes or adverse events (safety outcome), the dsmb recommended to proceed to a multi-center, phase trial. during this ongoing phase component of the rct, the primary outcome to be evaluated will be a composite outcome including ) the requirement of invasive mechanical ventilation, ) an increase in the sofa scale ≥ points, or ) death. the dsmb also suggested to repeat an ad interim analysis every time a %-recruiting milestone is reached. we are including adult (≥ -year-old), hospitalized patients with confirmed sars-cov- infection based on a positive rt-pcr test for sars-cov- rna in a respiratory specimen (nasopharyngeal or nasal swab) and an imaging study compatible with pneumonia, and at least one high-risk criteria of death (see table ). exclusion criteria include one or more of the following: allergy to pyridostigmine; pregnancy or breastfeeding status; concomitant autoimmune disease; diagnosed immunodeficiencies (including hiv infection); need for mechanical ventilation, admission to the icu, or meeting criteria for septic shock before providing signed, informed consent; inability to receive orally or enterally administered drugs; use of immunosuppressants or immune-modulators (including chemotherapy and corticosteroids) in the preceding -day period unless recommended by the treatment medical team as part of the therapeutic approach for sars-cov- infection; and participation in clinical trials of any kind in the previous days. participants will be randomized in a : ratio, with parallel assignment to receive either placebo or pyridostigmine as an add-on medical treatment to the best medical care available for severe covid- in participating centers. the block-randomization process will be performed using the publicly available online resource (www.randomizer.org). patients in the treatment group will receive pyridostigmine bromide, mg/day per os. participants randomized to the control group will receive matching placebo (identical in appearance) made of pharmaceutical grade starch. participants will be receiving the assigned intervention until the occurrence of either ) any of the prespecified outcomes; ) hospital discharge; or ) a maximum period of in-hospital days (fig. ) . the primary outcomes are a composite of requirement of invasive mechanical ventilation, an increase of ≥ points in the sofa scale, or all-cause mortality, during the -day period following enrollment; and, ) safety of the study drug. the secondary outcome is the change in interleukin (il)- levels (Δ il- ) between baseline samples and those taken on days , , , and (for an outline of the protocol, please refer to fig. ; for the timeline of interventions and measurements, please refer to table ). we will collect demographic information from participants at baseline, including age, sex assigned at birth, presence of comorbidities which will include diabetes mellitus, systemic arterial hypertension, obesity, cardiovascular disease, and lung disease, and other chronic medical conditions from the clinical charts. safety of the intervention will be actively evaluated by daily interrogation of the following common adverse effects of pyridostigmine [ ] : abdominal pain or cramps; diarrhea; nausea, vomiting, or both; hypersalivation/drooling; urinary incontinence; muscle weakness or fasciculations; and, blurred vision. on day , patients will be contacted by telephone to assess their vital and functional status ( fig. ; table ). all collected data will be safeguarded on a coded database with access limited to project investigators. only the principal investigators and the dsmb will have access to the final trial dataset. the final results will be published for generalized access, regardless of the outcome. • abg x • il- measurement x x x x x • mass cytometry x x x x x follow-up phone call x a blood sampling will be performed only while participants are hospitalized. protocol does not require participants to return for further blood sampling after hospital discharge. abbreviations: cbc complete blood count, abg arterial blood gases, il- interleukin currently, recruiting for this study is undergoing at, instituto nacional de ciencias médicas y nutrición salvador zubirán (incmnsz), and instituto nacional de cardiología ignacio chávez, two covid- -designated hospitals in mexico city, mexico. we estimate that a sample size of participants ( in each group) would produce a one-sided % confidence limit that would exclude us finding a %-point difference that would be statistically significant in the complete trial [ ] . however, calculating a % loss, we will recruit participants for this part of the study. we estimate that a sample size of participants ( per group) estimating a % reduction in the occurrence of the primary outcome in the intervention group to be clinically significant. based on recent evidence from china, we estimate that % of patients hospitalized with severe sars-cov- infection will develop complications leading to the need of invasive mechanical ventilation or death [ ] accordingly, we estimate that this sample size will allow us to identify with an % power a reduction in the need of invasive mechanical ventilation or death of % in the group receiving pyridostigmine in comparison with the group on placebo, using a two-sided ttest at the . significance level. primary analysis will be performed by intention-to-treat analysis comparing the proportion of outcome events between groups using x test. we will also compare point estimates and its corresponding confidence intervals between groups. in a secondary analysis, we will use multivariate logistic regression models to explore variables associated with the primary outcomes. this is an ongoing study. recruiting started on may ; at submission, we have recruited participants. no results have been made available, and the therapeutic arms remain double blinded. therefore, no results have been submitted for publication or published. here, we propose to evaluate the potential usefulness of pyridostigmine as add-on therapy to best medical care of patients admitted to a hospital due to severe covid- . recent evidence indicates that between and % of patients hospitalized for covid- required care in intensive care units (icu) for severe hypoxemia. the reported mortality in those first cases that required hospital management is %, but in those with severe disease, the reported mortality is between and % [ , ] , and we assume that it will be similar elsewhere. severity and mortality of covid- appear to be mediated not by infection, but by the disproportionate inflammatory response of the host. hence, finding novel immunomodulatory strategies is a promising strategy to reduce severity and mortality of covid- . furthermore, the repurposing of drugs with well characterized safety profiles and readily available production lines, might lead to faster development of anti-covid- therapies if proven efficacious in well-designed, randomized clinical trials. in mammals, the central nervous system has mechanisms to control the inflammatory response. during inflammatory states, the vagus nerve can inhibit the synthesis and release of inflammatory cytokines [ ] , thereby reducing both local damage and mortality secondary to severe systemic inflammation in murine models as diverse as sepsis, ischemia and re-perfusion damage, or obesity [ ] [ ] [ ] [ ] . the vagus nerve can be stimulated electrically and chemically. chemical stimulation using cholinergic agonists has shown promising effects in murine and cellular models of inflammation [ , ] . acetylcholine esterase inhibitors (i-ach-e) are a family of drugs used regularly by millions of patients, including older adults with alzheimer disease and other dementias, as well as in patients with myasthenia gravis and dysautonomia [ , [ ] [ ] [ ] [ ] . these drugs inhibit the enzymatic degradation of endogenous ach, resulting in greater bioavailability and, therefore, an increase in the possibility of binding to both nicotinic and muscarinic receptors. in addition to the approved uses of i-ach-e in human pathology, there is evidence in various murine models of their efficacy in experimental sepsis and severe inflammatory response [ , , ] , suggesting that i-ach-e drugs have a potential immunomodulatory effect in patients with severe systemic inflammatory response syndrome. pyridostigmine, an acetylcholinesterase inhibitor, has been previously shown to decrease inflammation in people living with human immunodeficiency virus (hiv) infection [ ] [ ] [ ] ; therefore, it is possible that pyridostigmine may lead to a decrease in the production of pro-inflammatory cytokines in patients with covid- at high risk of severe disease. regarding safety concerns, at the proposed dose of pyridostigmine, the rate of adverse events is less than - % with no reported serious adverse effects [ ] . from this perspective, we consider that pyridostigmine can function as an immunomodulator and reduce morbidity and mortality in these patients. the reduction in the frequency of the need for mechanical ventilation would contribute to reducing mortality and the demand for these services. clinical features of patients infected with novel coronavirus in wuhan clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study clinical predictors of mortality due to covid- based on an analysis of data of patients from wuhan, china covid- : consider cytokine storm syndromes and immunosuppression the challenges of long-term sepsis survivors: when surviving is just the beginning myasthenia gravis: subgroup classification and therapeutic strategies pyridostigmine used as a nerve agent pretreatment under wartime conditions acetylcholine-esterase inhibitor pyridostigmine decreases t cell overactivation in patients infected by hiv add-on pyridostigmine enhances cd + t-cell recovery in hiv- -infected immunological non-responders: a proof-of-concept study the effect of pyridostigmine on small intestinal bacterial overgrowth (sibo) and plasma inflammatory biomarkers in hiv-associated autonomic neuropathies vagus nerve stimulation attenuates the systemic inflammatory response to endotoxin xanomeline suppresses excessive pro-inflammatory cytokine responses through neural signal-mediated pathways and improves survival in lethal inflammation vagus nerve stimulation inhibits cytokine production and attenuates disease severity in rheumatoid arthritis acute pyridostigmine overdose: a report of nine cases sample size calculations for pilot randomized trials: a confidence interval approach characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention essential neuroscience in immunology cholinergic agonists attenuate renal ischemia-reperfusion injury in rats galantamine alleviates inflammation and other obesity-associated complications in high-fat diet-fed mice forebrain cholinergic dysfunction and systemic and brain inflammation in murine sepsis survivors forebrain cholinergic signaling regulates innate immune responses and inflammation alzheimer's disease alzheimer's disease vascular dementia pyridostigmine treatment trial in neurogenic orthostatic hypotension publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. this study is funded by peer-reviewed, competitive grants from consejo nacional de ciencia y tecnología (conacyt): grants and , both to sivf. the final datasets will be made publicly available in the final manuscripts, supplemental materials, or public repositories. all protocol documents are available in spanish upon reasonable requests. this study is being carried out in accordance with the recommendations of the institutional ethics in human research committees. all participants will have given written informed consent to one of the study investigators in accordance with the declaration of helsinki. the protocol was approved by the ethics in human research committees of instituto nacional de ciencias médicas y nutrición salvador zubirán (incm nsz), and instituto nacional de cardiología ignacio chávez, both in mexico city, mexico; and comisión federal para la protección contra riesgos sanitarios (cofepris), the federal mexican pharmacological regulatory commission. not applicable. pablo f belaunzarán-zamudio is an associated editor for the hiv and coinfections section of bmc infectious diseases. key: cord- -ykisq nz authors: kallel, hatem; matheus, séverine; mayence, claire; houcke, stéphanie; mathien, cyrille; lavergne, anne; hommel, didier title: capillary leak-syndrome triggered by maripa virus in french guiana: case report and implication for pathogenesis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: ykisq nz background: we report hereby a severe case of hantavirus pulmonary syndrome” (hps) induced by maripa virus in french guiana and describe the mechanism of severity of the human disease. case presentation: a -year- old patient started presenting a prodromic period with fever, dyspnea, cough and head ache. this clinical presentation was followed by a rapid respiratory, hemodynamic and renal failure leading to admission in the icu. biological exams revealed an increased haematocrit level with a paradoxical low protein level. echocardiographic and hemodynamic monitoring showed a normal left ventricular function with low filling pressures, an elevated extravascular lung water index and pulmonary vascular permeability index. these findings were compatible with a capillary leak-syndrome (cls). conclusions: the severity of hps caused by the virus maripa in french guiana can be explained by the tropism of hantavirus for the microvascular endothelial cell leading to a cls. hantavirus generally stands as a rodent-borne virus infection. rodent shed the virus in their droppings, saliva and urine. human infection occurs after breathing air contaminated by the virus. this can result on a severe respiratory syndrome named 'hantavirus pulmonary syndrome' (hps) which can be associated to cardiac failure leading to 'hanta virus cardio-pulmonary syndrome' (hcps) [ ] . in french guiana, cases of hps due to a hantavirus named maripa virus were diagnosed between and [ , ] . we report hereby the th human case of hps diagnosed in french guiana and describe the mechanism of severity of the human disease. our -year-old patient with a history of tobacco, alcohol, and illicit-drug consumption was admitted to the icu for fever ( °c), tachycardia ( beat/min), hemodynamic shock (blood arterial pressure was / mmhg), acute respiratory distress syndrome (ards; pao /fio ratio was ) with signs of intra-alveolar haemorrhage and, acute renal failure. symptoms including headache, fever, cough and dyspnea leading to respiratory failure started days before admission. the treatment consisted of crystalloids and norepinephrine infusion as well as mechanical ventilation support and continuous renal replacement therapy (rrt). initial laboratory testing showed renal impairment (urea nitrogen at . mmol/l, serum creatinine at μmol/l), a rise in inflammatory parameters (leucocytes count at . g/l and c-reactive protein at mg/l), an increased haematocrit level ( . %), thrombocytopenia ( g/l), a low protein level ( g/l) and cellular dysoxia (lactates dosage at . mmol/l). all other biological tests including the dosage of hepatic, muscular and cardiac enzymes were normal on admission. chest x-ray showed bilateral alveolar infiltrates and bilateral pleural effusion. transthoracic echocardiography showed normal and homogeneous left ventricular contractility with low filling pressures, with an aortic velocity time integral (vti) of cm (normal: - cm), a normal right heart function with tricuspid annular plane systolic excursion (tapse) at mm (normal: - mm), and a small pericardial effusion. hemodynamic monitoring using transpulmonary thermodilution (picco system; pulsion medical systems se, feldkirchen, germany) showed a cardiac output (co) of . l/min (normal: - l/min), a global end diastolic volume index (gedvi) of ml/m (normal: - ml/m ), an extravascular lung water index (evlwi) of . ml/kg (normal: - ml/kg), and a pulmonary vascular permeability index (pvpi) of . (normal: . - . ). chest computed tomography showed vessels enlargement, peribronchial cuffing, bilateral kerley lines, alveolar edema, and abundant right pleural effusion needing pleural drainage. the protein level in the pleural fluid was g/l and the microbiological culture was sterile. the viral investigations (igm and rt-pcr in the blood) confirmed an acute infection by hantavirus. the complete rna coding sequence of the s rna segment (genbank accession no. mg ) was also generated and compared with those of the other previous hantavirus cases from french guiana using a bayesian approach. this s rna sequences showed a nucleotide identity of . to . % with the five other previously described sequences of the maripa virus belonging to the rio mamoré clade. phylogenetic relationships were inferred from alignment with nt of the s segment [ ] . laboratory testing concerning other infectious agents (bacteria, fungal or parasite) were negative. overall, the patient's management included mechanical ventilation, norepinephrine, fluid infusion, sedation (midazolam and sufentanil), curarisation (cisatracrium), broad spectrum antibiotics, corticosteroids, and renal replacement therapy. concerning the outcome, the patient recovered gradually with a concomitant rise in serum protein level and a decrease in haematocrit concentration (fig. ) . he was weaned from mechanical ventilation at day , norepinephrine was stopped at day and rrt at day . he fully recovered and left hospital on the th day. the patient was examined in the outpatient clinic three weeks after discharge and his clinical examination was normal. we report here a human case of acute maripa virus related pulmonary syndrome managed in the icu of french guiana with a clear evidence of associated capillary leak syndrome responsible for the severity of the disease. in case of hantavirus infection, initial symptoms include fever, myalgia and headache followed by gastrointestinal symptoms such as abdominal pain, vomiting and diarrhea [ ] . this stage lasts around to days before the onset of respiratory failure, hypotension and cardiovascular shock. thrombocytopenia with haemorrhagic symptoms and renal injury are also frequently reported [ ] . in our case, symptoms recorded at admission were compatible with acute infection by hantavirus and required admission to the icu. the mechanism which may explain the severity of the disease is the tropism of hantavirus for the microvascular endothelial cell [ ] . this tropism causes microvascular hyperpermeability with fluid and proteins leakage leading to hypovolemia and to a non cardiogenic pulmonary oedema. biologically, we observe an increased haematocrit level due to hemoconcentration, and a paradoxical reduced serum fig. evolution of the serum protein and hematocrit levels during the first days (norepinephrine was stopped at day ) proteins level secondary to the transfer of proteins from the vessel to the interstitial space. under normal conditions, the endothelium plays the role of a selective permeable barrier to regulate plasma fluid exchange, as well as molecules and cells trafficking. disruption of cell junctions, with combination of cell retraction process, lead to the loss of the vascular endothelium barrier function. in such conditions, fluids and proteins infusion are ineffective because of the immediate leakage to the interstitial space with a worsening of the respiratory failure without any efficacy on the hemodynamic state. this mechanism is similar to that reported by clarkson in and is explained by a plasma leakage [ ] which was also described in arbovirus infections where the diagnosis was based on thoraco-abdominal sonography and scanography [ ] [ ] [ ] . in our patient, hemodynamic investigations using echocardiography and the picco system showed hypovolemia with low filling pressures and without any ventricular dysfunction. an elevated amount of extravascular lung water as well as an increased vascular permeability were also observed. this result is confirmed by the chest ct scan findings, showing a large amount of water in the alveoli, in the perivascular and in the pleural space. the pleural effusion was exudative and contained a high quantity of protein which can be explained by a protein leakage rather than by an inflammatory origin. the pathogenesis of capillary leakage remains undefined. some evidence suggest that hantavirus disease pathogenesis is immunologically mediated by cytotoxic t lymphocytes and other immune cells in target organs producing inflammatory cytokines. overall, three hypotheses have been reported to explain the mechanism of increased capillary permeability involved in hantavirus infection: a) the attack of infected endothelial cells by virus-specific cytotoxic t lymphocytes (ctls), b) tnf-α production by infected monocyte/macrophages and finally c) the direct effect of the virus on the endothelial cell functions [ , ] . bradykinin, a potent inflammatory and vasoactive nonapeptide generated by kallikrein at the sites of tissue injury is supposed to be the key mediator of the vascular leakage resulting from hantavirus infection. it acts by disrupting inter-endothelial junctions and causes changes in vascular tone. two patients with severe capillary leak syndrome caused by a puumala hantavirus infection were successfully treated with a bradykinin receptor antagonist [ , ] . experimental data demonstrating the plasma kallikrein-kinin system activation during hantavirus infection were also reported [ ] . in the same way, the intensity of the inflammatory syndrome was correlated to the importance of the capillary leakage and to the level of thrombocytopenia [ ] . despite abundant literature on hantavirus, few reports have focused on the aetiology of shock in severe hantavirus infected patients. many studies assume that the shock associated to hantavirus pulmonary syndrome is cardiogenic and hantavirus induces a typical myocarditis. these data were based on the examination of postmortem tissue from human hps cases [ ] . however, in our case, myocardial dysfunction was neither observed during echocardiography and nor during picco investigation. any inotropic agent support has been needed. in addition, troponine levels were normal despite severe shock. consequently, we think that maripa virus is more responsible for hps rather than hcps. such a finding is important as it raises the question about the effectiveness of extracorporal membrane oxygenation (ecmo) in patients presenting maripa virus infection with severe shock. we conclude that hps secondary to maripa virus infection in french guiana can cause severe damages leading to multi organ failure. the severity of the disease may be explained by a dysregulated inflammatory and immune reaction causing a severe capillary leakage without cardiac involvement. physicians should be aware of hps occurring in french guiana and any immediate management in the icu should be considered. none. clinical data will not be made available according to the french cnil recommandations (commission nationale informatique et libertés) that require specific authorizations to transfer data from one center to another. however, data from the medical chart of the patient can be obtained contacting the corresponding author since, the patient has given consent to share his medical informations. authors are cited in the same order that they are cited in the title page. ethics approval and consent to participate the patient has given consent to participate. hantavirus pulmonary syndrome hantavirus pulmonary syndrome caused by maripa virus in french guiana hantavirus pulmonary syndrome, french guiana maripa virus rna load and antibody response in hantavirus pulmonary syndrome immunopathogenesis of hantavirus pulmonary syndrome and hemorrhagic fever with renal syndrome: do cd + t cells trigger capillary leakage in viral hemorrhagic fevers? cyclical edema and shock due to increased capillary permeability capillary leakage in travelers with dengue infection : implications for pathogenesis zika virus in the americas: a review for clinicians zika and chikungunya: emerging arboviruses in the new world increased permeability of human endothelial cell line ea.hy induced by hantavirus-specific cytotoxic t lymphocytes a severe case of puumala hantavirus infection successfully treated with bradykinin receptor antagonist icatibant severe puumala virus infection in a patient with a lymphoproliferative disease treated with icatibant endothelial cell permeability during hantavirus infection involves factor xiidependent increased activation of the kallikrein-kinin system thrombocytopenia associates with the severity of inflammation and variables reflecting capillary leakage in puumala hantavirus infection, an analysis of finnish patients mechanisms of shock in hantavirus pulmonary syndrome written informed consent was obtained from the patient for publication of this case report and accompanying images. a copy of the written consent is available for review by the editor of this journal. the authors declare that they have no competing interest. key: cord- -ef d cg authors: han, seung beom; bae, e young; lee, jae wook; lee, dong-gun; chung, nack-gyun; jeong, dae-chul; cho, bin; kang, jin han; kim, hack-ki title: clinical characteristics and antimicrobial susceptibilities of viridans streptococcal bacteremia during febrile neutropenia in patients with hematologic malignancies: a comparison between adults and children date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ef d cg background: this study was performed to compare the clinical characteristics and antibiotic susceptibilities of viridans streptococcal bacteremia (vsb) between febrile neutropenic adults and children with hematologic malignancies. methods: the consecutive medical records of neutropenic patients with hematologic malignancies who were admitted to the catholic blood and marrow transplantation center between april and july , and who were subsequently diagnosed with vsb were reviewed retrospectively. a comparison was made between the clinical and laboratory characteristics of adults and pediatric patients and also between patients with cefepime susceptible or not susceptible vsb. results: a total of episodes ( in adults, in children) of vsb were identified. among them, ( . %) cases had severe complications including four ( . %) cases of death attributable to vsb. for antibacterial prophylaxis, most adults received ciprofloxacin ( . %), but children more frequently received trimethoprim/sulfamethoxazole ( . %). oral mucositis (p = . ) and abdominal pain (p = . ) were found more frequently in adults, and cough was found more frequently in children (p = . ). the occurrence rates of severe complications and death attributable to vsb were not significantly different between adults and children. susceptibility rate to cefepime was significantly higher in adults than children ( . % vs. . %, p = . ). however, in multivariate analysis, cefepime susceptibility had no impact on clinical outcome. conclusions: there was no significant difference in clinical outcome between adults and children with vsb despite a difference in cefepime susceptibility. hence, different antibiotic treatment strategies may not be necessary. bacteremia is identified in - % of febrile neutropenic patients with hematologic malignancies [ ] [ ] [ ] , and - % of the bacteremia is caused by viridans streptococci [ , , ] . although gram negative bacteria were the most common isolates to cause bacteremia in febrile neutropenic patients in the past [ ] , viridans streptococci are currently one of the most common isolates in both adults and children [ , , , ] . viridans streptococcal bacteremia (vsb) has been reported to cause severe complications such as shock and acute respiratory distress syndrome (ards) in - % of infected neutropenic patients and death in up to % [ ] [ ] [ ] . a higher occurrence rate of these severe complications was reported in children compared to adults [ ] . although the infectious diseases society of america (idsa) and korean guidelines state that β-lactam antibiotics are adequate for viridans streptococcal infections [ , ] , it is uncertain whether the same practice guidelines can be applied to treat infections in adults and children because of the different complication frequencies [ ] and the potentially different antibiotic susceptibilities to viridans streptococci in febrile neutropenic adults and children with vsb. we performed this retrospective study to compare clinical characteristics including the occurrence of severe complications and antibiotic susceptibilities of viridans streptococci between febrile neutropenic adults and children with hematologic malignancies, and to propose appropriate antibacterial treatment strategies for adults and children. the consecutive medical records of patients diagnosed with vsb during febrile neutropenia were reviewed retrospectively. the patients were admitted to the catholic blood and marrow transplantation (bmt) center between april and july , and received conventional chemotherapy or hematopoietic cell transplantation (hct) for their hematologic malignancies. the catholic bmt center is affiliated with seoul st. mary's hospital in seoul, republic of korea and is a university-affiliated tertiary center with about , beds. there are separate hematology wards for adults and children, and the adult hematology ward consists of separate wards for intensive conventional chemotherapy and hct patients. the institutional review board (irb) of seoul st. mary's hospital approved this research protocol with a waiver of informed consent (kc risi , approved on september , ). patients who were younger than years were categorized as children, and the rest as adults according to the irb guideline, and clinical and laboratory characteristics and antibiotic susceptibilities were compared between the adults and children. the same clinical and laboratory characteristics were also compared between patients with vsb susceptible to cefepime, one of the empirical antibiotics used for febrile neutropenic patients, and those with vsb not susceptible to cefepime. data gathered on patients' demographics and clinical characteristics consisted of gender, underlying disease with remission status, type of therapy preceding febrile neutropenia, number of days from the beginning of respective therapies to the diagnosis of vsb, use of antibacterial prophylaxis, and occurrence of oral mucositis, respiratory symptoms, gastrointestinal symptoms, severe complications and polymicrobial infection by other bacteria or fungi. laboratory characteristics consisted of white blood cell (wbc) count and absolute neutrophil count (anc) upon the diagnosis of vsb, the number of neutropenic days before the diagnosis of vsb, total number of neutropenic days during the febrile neutropenic episode, and the peak c-reactive protein (crp) level within a week after the diagnosis of vsb. ceftazidime or cefepime with aminoglycoside, and piperacillin/tazobactam with aminoglycoside were administered as initial empirical antibacterial therapy for febrile neutropenia in adults and children, respectively. after three to five days of initial antibacterial therapy, an adjustment, if needed, was made according to the korean guideline for febrile neutropenia [ ] . glycopeptides were given based on the indications recommended by the korean guideline [ ] . blood for culture was sampled using sterile technique with one set from a peripheral vein and another set from a central catheter. in adults, - ml of blood was inoculated into each aerobic and anaerobic culture bottle (bd bactec™ plus aerobic/f, lytic/ anaerobic/f culture vials, becton dickinson, sparks, md, usa), and in children, - ml of blood was inoculated into a culture bottle (bd bactec™ peds plus culture vial, becton dickinson, sparks, md, usa). the bottles were immediately transported to the clinical microbiology laboratory. automated culture systems were used to detect bacterial growth (bactec™ fx, becton dickinson, sparks, md, usa) and to identify the exact bacterial type (vitek® , biomériux, hazelwood, mo, usa). antibiotic susceptibility tests were performed on a muller-hinton agar plate with % sheep blood, according to the clinical and laboratory standards institute (clsi) recommendations [ ] . the susceptibilities were determined by using an e-test for penicillin and cefotaxime, and using a disk diffusion method for cefepime, erythromycin, clindamycin, vancomycin, and linezolid. a result of 's' was considered susceptible, and results of 'i' and 'r' were considered not susceptible. susceptibility rates to each antibiotic drug were calculated and compared between adults and children. because antibiotic susceptibilities and clinical characteristics were not significantly different among viridans streptococcal species [ , ] , we did not identify the different species of viridans streptococci. vsb was defined as growth of viridans streptococci from at least one peripheral or central blood sample. neutropenia was defined as having an anc lower than /μl or an anc lower than , /μl that was predicted to be lower than /μl within two to three days, and fever was defined as a body temperature higher than . °c with a tympanic thermometer or . °c with an axillary thermometer [ ] . severe complications included shock, any kind of mechanical ventilator care, ards, and death. shock was defined as hypotension (mean arterial pressure less than mmhg in adults, and systolic blood pressure less than the th percentile to age in children) requiring an intravenous fluid bolus or inotropic agents to maintain normal blood pressure [ , , ] , and ards was defined as pao /fio < in arterial blood gas analysis of a patient with hypoxia of spo < % and bilateral pulmonary infiltrates on the chest x-ray [ ] . the severe complications were considered to be attributable to vsb if there was no clinical improvement after the diagnosis of vsb with severe complications, no other infectious isolates were detected, no deterioration in underlying malignancy was observed, and no other clinical diagnoses were made. death attributable to vsb was defined as death accompanied by severe complications attributable to vsb within days after the diagnosis of vsb, and overall death included death from all causes within a month after the diagnosis of vsb [ ] . statistical analysis was performed with spss statistics . (spss inc., chicago, il, usa), and statistical significance was defined as a two-sided p < . . in comparisons between adults and children and patients with vsb susceptible and not susceptible to cefepime, a student's t-test was used for numerical variables, and a χ test was used for categorical variables. multivariate analysis using multiple logistic regression tests was performed for statistically significant factors derived from univariate analysis to determine factors related to the susceptibility of viridans streptococci to cefepime. the peak crp level within a week after the diagnosis of vsb, predicting the development of severe complications attributable to vsb, was determined by a receiver operating characteristic (roc) curve. during the study period, there were , admissions in , adults and , admissions in children for conventional chemotherapy, hct, or febrile neutropenia following chemotherapy. in adults, episodes of bacteremia in patients and episodes of vsb in patients were identified, and the incidence of bacteremia and vsb were . and . episodes per , person-days, respectively. in children, episodes of bacteremia in patients and episodes of vsb in patients were identified, and the incidence of bacteremia and vsb were . and . episodes per , person-days, respectively. among the total episodes of vsb in adults and children, ( . %) cases with severe complications including ( . %) deaths were identified, and ( . %) of them, including four ( . %) deaths were attributable to vsb. the other cases leading to mortality were due to uncontrolled underlying hematologic malignancies. multiple episodes of vsb were diagnosed in patients. eight patients (five adults and three children) each experienced two episodes, and three patients (one adult and two children) each experienced three episodes of vsb. each episode was diagnosed during separate admissions. none of the patients experienced multiple episodes of severe complications. among the total cases of vsb, ( . %) cases were male, and ( . %) cases suffered from acute myeloid leukemia (aml). vsb occurred a median of days (inter quartile range, iqr: - ) after the preceding therapy. conventional chemotherapy and hct accounted for . % and . % of the preceding therapy, respectively. diarrhea ( / , . %) was the most common symptom accompanying fever, and was followed by oral mucositis ( / , . %) and abdominal pain ( / , . %). more children were male compared to the adult group (p = . , table ). aml accounted for about % of the underlying diseases in both adults and children, and the distribution of underlying diseases was not significantly different between the two groups ( table ) . all children with vsb had been treated with conventional chemotherapy, whilst . % of adults had been treated with conventional chemotherapy, and . % with hct. the type of preceding therapy was not significantly different between the two groups ( table ). the median number of days from the beginning of the preceding therapy to the diagnosis of vsb was days (iqr: - ) in adults and days (iqr: [ ] [ ] [ ] in children, and this difference was statistically significant (p < . , table ). this difference in antibacterial prophylaxis occurred because fluoroquinolones are not recommended to children aged less than years in korea due to the risk of skeletal abnormalities. the seven patients in the pediatric group who received ciprofloxacin prophylaxis were older than years. among the symptoms accompanying vsb, oral mucositis (p = . ) and abdominal pain (p = . ) were more common in adults, and cough was more common in children (p = . , table ). the occurrence rates of severe complications attributable to vsb, overall mortality, and mortality attributable to vsb were not significantly different between adults and children (table ) . crp levels were measured a median of three times (iqr: - ) within the first week after the diagnosis of vsb, and the peak crp levels within a week were detected a median of four days (iqr: - ) after the diagnosis of vsb. the frequency of measuring crp levels and the time of the peak crp levels were not significantly different between adults and children. there was no significant difference in laboratory results between adults and children ( table ) . antibiotic susceptibility was assessed in / ( . %) of bacterial isolates, that is, all the isolates except for one from an adult patient ( ). the susceptibility rates to cefotaxime, cefepime, and erythromycin were significantly higher in adults than in children (table ) . comparison between patients with severe complications attributable to viridans streptococcal bacteremia and those without severe complications the median of peak crp levels within a week after the diagnosis of vsb was . mg/dl (iqr: . - . ) in patients with severe complications attributable to vsb and . mg/dl (iqr: . - . ) in those without severe complications. these were significantly different (p < . ). peak crp levels were detected a median of four days (iqr: - ) after the diagnosis of vsb in the two groups without a significant difference. the cut-off value of the peak crp level predicting the development of severe complications attributable to vsb was determined using an roc curve as . mg/dl (area under the curve = . ) with sensitivity, specificity, positive predictive value, and negative predictive value of %, %, %, and %, respectively. there were no other significant differences in clinical and laboratory characteristics between the two groups. the antibiotic susceptibility rate of each antibiotic drug was not significantly different between the two patient groups (table ) . comparison between patients with viridans streptococcal bacteremia susceptible to cefepime and not susceptible to cefepime susceptibility tests to cefepime were conducted in isolates, and ( . %) isolates were susceptible to cefepime (table ). in univariate analysis, patients with vsb susceptible to cefepime were older (p = . ), more likely to be in complete remission status (p = . ), more likely to have received ciprofloxacin prophylaxis (p < . ), and had a longer duration of neutropenia before the diagnosis of vsb (p = . ) than patients with vsb not susceptible to cefepime (table ) . however, there was no significant factor related to cefepime susceptibility in multivariate analysis (table ) . medical records on the complete course of chemotherapy and antibacterial therapy for febrile neutropenia with antibiotics which have anti-pseudomonal effect since the diagnosis of hematologic malignancies were reviewed in cases ( adults, children). of the remaining cases, medical records of patients who had been referred from other hospitals were not completely reviewed, and patients who had been newly diagnosed with hematologic malignancies were excluded because they had no previous history of antibacterial therapy for febrile neutropenia. the interval from the diagnosis of hematologic malignancy to the diagnosis of vsb was a median of three months (iqr: - ). among the patients, patients with vsb susceptible to cefepime and patients with vsb not susceptible to cefepime received a median of one course (iqr: - ) and a median of two courses (iqr: - ) of antibacterial therapy for febrile neutropenia, respectively. the number of preceding antibacterial therapies for febrile neutropenia was not significantly different between patients with vsb susceptible and not susceptible to cefepime (table ). we investigated the clinical and laboratory characteristics of vsb in febrile neutropenic patients with hematologic malignancies and the antibiotic susceptibilities of the viridans streptococci. the data were compared between adults and children and also in patients with vsb susceptible and not susceptible to cefepime. vsb occurred most commonly in aml patients ( . %), days (iqr: [ ] [ ] [ ] [ ] [ ] after the beginning of consolidation chemotherapy ( . %), and six days (iqr: - ) after the onset of neutropenia. this pattern of vsb occurrence was consistent with previous reports [ ] . while oral mucositis, a risk factor for vsb, occurred in roughly % of patients with vsb in previous reports [ , ] , it occurred at a lower rate of . % in this study. on the other hand, gastrointestinal symptoms were common in all patient groups, and cough was common in children. considering that viridans streptococci are normal flora of the gastrointestinal and upper respiratory tracts as well as oral mucosa [ , ] , and that mucosal damage can occur at these sites following chemotherapy or hct, this was a predictable result. since young children often cannot adequately complain of their oral and abdominal pain, and their parents or medical personnel might easily recognize objective symptoms, such as diarrhea and cough, the reported incidence of oral mucositis and abdominal pain might be lower in children than in adults. other clinical and laboratory characteristics were not significantly different between adults and children, and the aforementioned symptoms occurred in less than one-third of patients. therefore, we concluded that there were no distinctive characteristics to distinguish between vsb in adults and children. the . % occurrence rate of severe complications attributable to vsb was lower than that of previous reports, which showed an occurrence rate of - %, and the . % mortality attributable to vsb in this study was also lower than that of previous reports, which showed mortality up to % [ ] [ ] [ ] . although martino et al. [ ] reported a higher occurrence rate of severe complications and death due to vsb in children than in adults, the occurrence rate of severe complications and death attributable to vsb and overall mortality were not significantly different between adults and children in the present study. previous researchers did not find significant factors related to a worse prognosis in children, and there have been few studies comparing the clinical characteristics and prognoses between adults and children. comparisons between adults and pediatric patients with severe complications attributable to vsb showed that children more commonly complained of cough and had a longer duration between the beginning of the preceding therapy and the diagnosis of vsb, similar to the comparison between all adults and children with vsb. when comparing patients with severe complications attributable to vsb and those without severe complications in this study, there was no significant difference except for the peak crp level within a week after the diagnosis of vsb. this had a low positive predictive value of % for the occurrence of severe complications. thus, we were also unable to identify a definite factor that could help anticipate severe complications in vsb. antibiotic susceptibility rates to cefotaxime, cefepime, and erythromycin were lower in children than in adults. although we performed both univariate and multivariate analyses to determine risk factors for decreased susceptibility to cefepime, no significant factors were found. recurrent antibiotic use may be related to the increase in antibiotic resistance [ , ] ; however, there was no difference between patients with vsb susceptible and not susceptible to cefepime in the number of antibacterial therapies for febrile neutropenia after previous conventional chemotherapy or hct. the fact that the first-line antibiotic agent for patients with hematologic malignancies was cefepime or ceftazidime in most adults and piperacillin/tazobactam for almost all children in our hospital also supports the finding that previous antibiotic use is not related to decreased susceptibility to cefepime. we also analyzed the effect of prophylactic antibiotics on susceptibility to cefepime since ciprofloxacin, principally given to adults, has a limited effect on gram positive bacteria [ , ] , while tmp/smx, principally given to children, has a satisfactory effect [ , ] . the effect of prophylactic antibiotics on decreased susceptibility to cefepime may be small since antibacterial prophylaxis has been reported to be unrelated to increased antibiotic resistance in a meta-analysis [ ] , and since patients in the present study received ciprofloxacin or tmp/smx rather than β-lactam antibiotics and antibacterial prophylaxis with these antibiotics is not known to trigger antibiotic resistance in viridans streptococci [ , , ] . nevertheless, prophylactic antibiotic effects on decreased susceptibility to cefepime should not be ignored. viridans streptococci can acquire β-lactam resistance through transfer of the mutated penicillin binding protein gene from streptococcus pneumoniae [ , ] , and it has been reported that the resistance of s. pneumoniae to β-lactam antibiotics after tmp/smx prophylaxis in human immunodeficiency virus-infected patients can increase by a factor of . [ ] . however, resistance to penicillin of s. pneumoniae was . % in nonmeningeal isolates and . % in meningeal isolates, and ceftriaxone resistance was . % in nonmeningeal isolates and % in meningeal isolates from to in the republic of korea [ ] . the exact effect of prophylactic antibiotics on the development of antibiotic resistance remains controversial [ ] , and the type of antibiotics, duration of prophylaxis, bacterial species, and host factors may influence the development of antibiotic resistance [ , ] . in this study, there were no definite differences in clinical and laboratory characteristics, mortality, or occurrence of severe complications between febrile neutropenic adults and children with vsb, despite a significant difference in antibiotic susceptibility to cefepime between the two groups. antibiotic susceptibilities were not significantly related to the development of severe complications. thus, our study results show that different antibiotic treatment strategies for adults and children with vsb are not necessary. the lower susceptibility rate of . % to cefepime in children may indicate the need for initial glycopeptide therapy in febrile neutropenic children. however, bacteremia is diagnosed in - % of febrile neutropenic children [ ] [ ] [ ] , and - % of the bacteremia is caused by viridans streptococci [ , , ] . in addition, since severe complications occurred in . % of the patients with vsb according to our results, we estimate that the incidence of severe complications of vsb in febrile neutropenic children is . %. therefore, considering that antibiotic susceptibility is not significantly related to the prognosis of vsb in febrile neutropenia [ , ] , universal initial glycopeptide therapy targeting only . % of febrile neutropenic children with hematologic malignancies should not be considered. instead, we should consider glycopeptide therapy if antibiotic susceptibility tests revealed that the isolated viridans streptococci were not susceptible to antibiotics being administered to the patient and susceptible to glycopeptides. this study has several limitations including its retrospective nature. we tried to eliminate selection bias by including all consecutive hematologic malignancy patients with vsb who were treated in the same hospital environment. also, there were some limitations in our tests for antibiotic susceptibility. the results of the e-test and disk diffusion method for antibiotic susceptibility in this study may be different from results of broth microdilution methods. additionally the clinical laboratory of our hospital did not perform piperacillin/ tazobactam susceptibility test for viridans streptococci; thus, we assumed that cefepime susceptibility was similar to piperacillin/tazobactam susceptibility. this assumption may not be applicable to clinical settings. lastly, past histories of antibacterial therapy for febrile neutropenia were reviewed to evaluate its effect on the differences in antibiotic susceptibility; however, information from patients was missing. although we assumed that previous antibacterial therapies should not influence β-lactam susceptibilities, the relationship should be further investigated. in this study, no definite differences in clinical and laboratory characteristics or prognosis were found between febrile neutropenic adults and children with vsb. while susceptibility to cefepime was lower in children, there were no differences in clinical characteristics or prognosis between patients with vsb susceptible and not susceptible to cefepime. therefore, this study showed that different antibiotic treatment strategies for adults and children with vsb are not necessary, and also confirmed that current idsa and korean guidelines for febrile neutropenic patients may be applied to both febrile neutropenic children and adults with vsb. further studies on the cause and clinical significance of the difference in antibiotic susceptibility rates between adults and children are needed. a prospective study on the epidemiology of febrile episodes during chemotherapy-induced neutropenia in children with cancer or after hemopoietic stem cell transplantation clinical practice guideline for the use of antimicrobial agents in neutropenic patients with cancer: update by the infectious diseases society of america etiology and clinical course of febrile neutropenia in children with cancer clinical investigation of bacteremia in children with hemato-oncologic diseases serious complications of bacteremia caused by viridans streptococci in neutropenic patients with cancer changing epidemiology of infections in patients with neutropenia and cancer: emphasis on gram-positive and resistant bacteria epidemiology and clinical features of bloodstream infections in hematology wards: one year experience at the catholic blood and marrow transplantation center predictors of viridans streptococcal shock syndrome in bacteremic children with cancer and stem-cell transplant recipients viridans streptococci bacteremia in children with malignancy: relevance of species identification and penicillin susceptibility viridans streptococcal bacteraemia due to penicillin-resistant and penicillin-sensitive streptococci: analysis of risk factors and outcome in patients from a single cancer centre before and after penicillin is used for prophylaxis viridans streptococcal bacteremia and viridans streptococcal shock syndrome in neutropenic patients: comparison between children and adults receiving chemotherapy or undergoing bone marrow transplantation evidencebased guidelines for empirical therapy of neutropenic fever in korea performance standards for antimicrobial susceptibility testing; twentieth informational supplement. clsi document m -s approach to the patient with shock. in harrison' s principles of internal medicine nelson textbook of pediatrics acute lung injury and the acute respiratory distress syndrome: a clinical review bacterial and fungal bloodstream isolates from hematopoietic stem cell transplant recipients between infections with viridans group streptococci in children with cancer. pediatr blood cancer the impact of mucositis on alpha-hemolytic streptococcal infection in patients undergoing autologous bone marrow transplantation for hematologic malignancies infections caused by viridans streptococci in patients with neutropenia viridans group streptococcal infections among children with cancer and the importance of emerging antibiotic resistance reduced susceptibility to penicillin of viridans group streptococci in the oral cavity of patients with haematological disease increased carriage of resistant non-pneumococcal alpha-hemolytic streptococci after antibiotic therapy in mandell, douglas, and bennett's principles and practice of infectious diseases efficacy of oral prophylactic antibiotics in neutropenic afebrile oncology patients: a systematic review of randomised controlled trials infections during induction therapy for children with acute lymphoblastic leukemia. the role of sulfamethoxazole-trimethoprim (smx-tmp) prophylaxis. pediatr blood cancer meta-analysis: antibiotic prophylaxis reduces mortality in neutropenic patients antimicrobial susceptibility of viridans group streptococci isolated from patients with acute leukemia receiving ofloxacin for antibacterial prophylaxis emergence of quinolone resistance among viridans group streptococci isolated from the oropharynx of neutropenic peripheral blood stem cell transplant patients receiving quinolone antimicrobial prophylaxis penicillin-resistant viridans streptococci have obtained altered penicillinbinding protein genes from penicillin-resistant strains of streptococcus pneumoniae erythromycin and penicillin resistance mechanisms among viridans group streptococci isolated from blood cultures of adult patients with underlying diseases trimethoprimsulfamethoxazole prophylaxis and antibiotic nonsusceptibility in invasive pneumococcal disease ansorp study group: changing trends in antimicrobial resistance and serotype of streptococcus pneumoniae isolates in asian countries: an asian network for surveillance of resistant pathogens (ansorp) study fluoroquinolone consumption and resistance in haematology-oncology patients: ecological analysis in two university hospitals - submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution there is no competing interest for any authors.authors' contributions sbh, dgl, bc, and jhk designed this study. sbh, eyb and jwl collected data, and ngc and dcj analysed the data. sbh, jwl and dgl wrote the manuscript, and bc, jhk and hkk critically reviewed the manuscript. all authors read and approved the final draft. key: cord- - dus u m authors: civaner, murat; arda, berna title: can "presumed consent" justify the duty to treat infectious diseases? an analysis date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: dus u m background: aids, sars, and the recent epidemics of the avian-flu have all served to remind us the debate over the limits of the moral duty to care. it is important to first consider the question of whether or not the "duty to treat" might be subject to contextual constraints. the purpose of this study was to investigate the opinions and beliefs held by both physicians and dentists regarding the occupational risks of infectious diseases, and to analyze the argument that the notion of "presumed consent" on the part of professionals may be grounds for supporting the duty to treat. methods: for this cross-sectional survey, the study population was selected from among physicians and dentists in ankara. all of the participants were given a self-administered questionnaire. results: in total, . % of the participants said that they either had some degree of knowledge about the risks when they chose their profession or that they learned of the risks later during their education and training. of the participants, . % said that they would not have chosen this profession if they had been informed of the risks. it was found that % of the participants believed that there is a standard level of risk, and % of the participants stated that certain diseases would exceed the level of acceptable risk unless specific protective measures were implemented. conclusion: if we use the presumed consent argument to establish the duty of the hcw to provide care, we are confronted with problems ranging over the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the near-impossibility of being able to evaluate retrospectively whether every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. our findings suggest that another problem can be added to the list: one-fifth of the participants in this study either lacked adequate knowledge of the occupational risks when they chose the medical profession or were not sufficiently informed of these risks during their faculty education and training. furthermore, in terms of the moral duty to provide care, it seems that most hcws are more concerned about the availability of protective measures than about whether they had been informed of a particular risk beforehand. for all these reasons, the presumed consent argument is not persuasive enough, and cannot be used to justify the duty to provide care. it is therefore more useful to emphasize justifications other than presumed consent when defining the duty of hcws to provide care, such as the social contract between society and the medical profession and the fact that hcws have a greater ability to provide medical aid. in the course of providing health care service, health care workers (hcws) are continually exposed to many workrelated health risks. one of these risks is the exposure to infectious diseases. these diseases can include the flu, aids, tuberculosis, and hepatitis, and can be transmitted through physical contact, exposure to contaminated blood, or via the respiratory system. and, needless to say, such risks do indeed at times prove fatal. the consequences of occupational exposure to pathogens are not limited solely to bodily infections. each year, thousands of hcws are adversely affected by psychological trauma stemming from months of anxiously awaiting the results of serological tests, tests made necessary due to potential infection incidents. the anxiety experienced by hcws is related to the perception of risk from the incident and the resulting infection that may occur, and by the worry of what the reactions of others might be, such as colleagues, family, and friends, all who have to be informed. during this uncertain waiting period hcws will frequently experience intrusive thoughts, problems concentrating, difficulty sleeping, frequent loss of temper, and a decrease in sexual desire, which can act as a catalyst to exacerbate any pre-existing and unresolved emotional issues [ ] . and if it turns out that the health care worker has indeed been infected by one of these contagions, the serious personal consequences to that health care worker can include the postponement of childbearing, damaged personal relationships, having to alter sexual practices, experiencing the side effects of prophylactic drugs, chronic disabilities, loss of employment, denial of worker compensation claims, possible need for a liver transplant, and premature death [ ] . aids, sars, and the recent epidemics of the avian-flu have all served to remind us of the occupational risks faced everyday by hcws; the result being that the recent appearance of these diseases has forced this issue onto the common agenda and helped to spark renewed interest in the debate over the limits of the moral duty to treat. in seeking an answer to this question it is useful to have an understanding of the occupational risks faced by hcws as well as an understanding of the attitudes of hcws to these risks. for example, studies conducted in various countries have shown that, especially when there was a risk of being infected with aids, hcws may refuse to treat a patient on the grounds that there is a risk of being infected by this patient [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . and despite the fact that the hepatitis viruses are transmitted more easily than hiv, it is the fear of being infected with hiv that causes many hcws to experience the greatest amount of stress and anxiety [ ] . in a study which compared the relative risks of transmission of both hbv and hiv, the reasons for physicians' underlying fears of particular contagions were also inves-tigated and described. [ ] . according to the study, people initially percieve the risk to be greater when there is a high likelihood of death involved with infection (as with hiv) even though there may be less risk of infection, as opposed to when there is a higher risk of infection but a lower risk of death involved with that infection (as with hbv). additionally, since the likelihood of sexually transmitting hbv between heterosexual partners is less than that of transmitting hiv, the consequences of hbv infection are again percieved to be less severe than the consequences of hiv infection. in this way, the hazards posed by hbv infection conflict less with the obligation to protect family members from harm. it was also found to be important that there is less of a stigma attached to having hbv than there is to having hiv. and, finally, the fact that there is a vaccine for hbv infection, which is more than percent effective (for vaccinated hcws the risk of death from infection is reduced by a factor of nearly twenty), also was found to greatly influence the perceptions of the physicians. additionally, factors other than a fear of the contagion can contribute to the reluctance to treat a particular patient. some physicians and dentists express concern that if it is discovered that they treat patients with aids, then those patients who don't have hiv may shun their practice. still, other physicians insist they do not know enough about hiv infection and are too busy to learn [ ] . another reason for which a physician may refuse to treat hiv-positive patients is that the physician feels they have a duty to protect their other patients, basing their reasoning on the principle of "first do not harm". by treating hiv-positive patients they claim that they may potentially be putting their other patients at risk for infection [ ] . furthermore, as has been reported, there is always the possibility that when a hcw is able to reject the patient based on a more benign excuse, for example if the patient does not have enough money, it is even easier, and all the more likely, for treatment to be refused, even though this refusal was done in the interest of protecting the physical health of the individual health care provider [ ] . in the literature, most studies have concentrated primarily on the attitudes and rationale behind the refusal to treat. before one can set out to effectively explore the attitudes of hcws however, it is important to first consider the question of whether or not the "duty to treat" might be subject to contextual constraints, such as providing health care to a patient suffering from an infectious disease which may be particularly contagious or for which adequate treatment measures may not yet be available. clark, in his article about physicians' duty to treat, claimed that there are three reasons in which such a duty is grounded [ ] : "... since the ability to render aid is greater, the obligation to assist is (...) elevated. second, by consideration of daniels' argument that by freely joining a profession designed to combat disease, one consents to some standard of risk, and third, by realizing that the profession has flourished due to socially negotiated promises to be available in such times of duress." in his article "duty to treat or right to refuse", daniels argues that when a person chooses a career in a particular profession, it must be understood by all parties that this individual has both accepted and is willing to take the risks that are inherent to that profession [ ] : "consent is crucial where obligations to take risks exist in various occupations or professions. for example, we assume that in choosing their careers, undergoing the training involved, and agreeing to follow the codes and practices regulating their work, firefighters and police have given consent to facing the significant risks they are obliged to take. there are strong parallels to medicine. people who enter medical fields clearly had alternatives. there is a general understanding that physicians face an increased risk of contagion from disease, an understanding refined during schooling and training." daniels proposes, however, that some situations can exceed the standard level of risk (slr) [ ] : "for example, it is common to screen new house staff and nurses in medical centers to determine whether any individuals face special risks of contagion, such as immunosuppression or pregnancy. those at high risk may then be asked to avoid certain treatment situations, materials, or hospital areas.(...) protecting immunosuppressed providers is reasonable "risk management", a measure taken to reduce bad outcomes. but such special protection supports the claim that only standard risks are included in the duty to treat.(...) some nosocomial risks clearly take us beyond what duty requires." it is perhaps more illustrative if this argument (from this point on, this statement will simply be referred to as the "presumed consent") is written in classic form: health care services should be provided to patients who have a contagious disease. contracting an infectious disease while providing health care services to a patient with a contagious disease is an occupational risk. it is generally assumed that by joining the health care profession physicians have given their consent to be exposed to an increased risk of disease contagion. this assumption is based on the following facts: a. there is a general understanding that physicians face an increased risk of contagion from disease, an understanding refined during schooling and training. b. people who enter medical fields clearly had alternatives. some nosocomial risks clearly take us beyond what duty requires. there is a moral duty to treat patients who have a contagious disease so long as the risk to the hcw is below the slr. if we are to accept this argument, then the pressing question becomes how to determine and define the risks which are deemed to be standard and acceptable versus those which are believed to exceed and, indeed, outweigh the duty of the health care provider to treat. in order to begin to answer this question, it will be useful to investigate the nature of the choice (and all that goes along with making it) that an individual makes when they decide to enter a particular profession. for instance, how wise is it to assume that at the time of choosing their future profession the hcw was fully aware of the risks involved with such work? perhaps they were not made aware of the risks until their education and training. furthermore, if they were aware of, and fully appreciated, the risks prior to deciding on a particular profession, would they have even chosen that profession in the first place? and, finally, how is the slr to be determined, and which of the infectious diseases would then exceed this slr? in order to effectively analyze the presumed consent argument it is necessary to have an awareness of the diverse opinions and beliefs of hcws and, also, to understand their different motives and backgrounds. additionally, knowledge of what hcws feel about the risk concept and of how they feel about their duty to treat patients with contagious diseases can also be of great value to educators as they plan their curricula and it can be used by the authorities in charge of health care systems in order to better organize their services. the purpose of this study was to analyze whether or not the third premise grounds the duty to treat, namely, "it is generally assumed that by joining the health care profession physicians have given their consent to be exposed to an increased risk of disease contagion". in order to carry out this analysis, the opinions and beliefs of physicians and dentists regarding the occupational risks of infectious diseases were investigated; and, by extension, the argument that the notion of "presumed consent" may be grounds for supporting the hcws' duty to treat was also analyzed. for this cross-sectional survey, the study population was selected from among physicians and dentists in ankara, the capital of turkey. a self-administered questionnaire designed to assess the beliefs and opinions of the participants regarding the occupational risks of infectious diseases was used. this questionnaire was also used to obtain the socio-demographic information of the participants. the items on the questionnaire were developed by reviewing previous studies in the literature [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a draft of the questionnaire was distributed to experienced health care professionals and later revised based on their criticism and suggestions. in both of the universities in which this study was conducted there are ethics committees which had been established for the purpose of determining the ethical appropriateness of pharmaceutical trials using humans; since our study involved only the use of a questionnaire and not an experimental drug, we did not apply for approval from either of these ethics committees. instead, written permission to carry out the study was granted by the dean of the faculty of medicine and by the chief manager of university hospitals. in addition, all of the potential participants were fully informed about the aim and structure of the study. furthermore, potential volunteers were all made aware that participation was strictly voluntary and that all of the answers they provide would be done so anonymously. the questionnaire was administered to a total of health care workers: all of the physicians who work in surgical specialties at the ankara university ibn-i sina hospital and to all of the dentists in the gazi university faculty of dentistry. dentists were included in this study because, aside from being hcws themselves, there are a number of studies in the literature which show that dentists, citing various reasons, may also refuse to treat patients with contagious diseases. and, in order to better assess the fact on which the third premise of presumed consent is based, we decided to include only professional health care workers, instead of students and others who might still be in the process of deciding whether or not to currently enter the field. in total there were participants, physicians and dentists, who completed the questionnaire, for an overall response rate of . %. the questionnaire was later sent back to the non-respondents one month after the first survey, and of these were completed and returned to us. the mean age of the participants was . ± . years, while . % were male and . % were female. additionally, the average amount of time that they had been working in the medical profession was found to be . years (min. , max. ). all of the data was collected anonymously. the difference between the two groups, physicians and dentists, was compared using the chi-square test, with a p-value of < . accepted as statistically significant. all analyses were carried out using spss . . of the hcws surveyed in this study, roughly half stated that they understood that by choosing their profession they would be exposing themselves to an increased risk of contracting contagious disease ( . %). and at the time of entering the faculty, . % of the participants expressed that they were unaware of any increased risks; however, they later learned of these risks during their education and training. in other words, . % of the participants stated that they had known about the risks either at the time they chose their profession or that they had later learned of the risks during their training and education. additionally, . % of the participants answered that they had only come to realize the kinds of risks they would face after starting to work. the percentage of participants who claimed that if they had been aware of the risks earlier they would not have chosen to enter or continue in the medical profession was . %. table are statements which physicians and dentists chose as best reflecting their personal opinions regarding the occupational risks of infectious disease. in general, the physicians, prior to their education and training, were significantly more aware of the potential risks associated with their profession than were the dentists (p < . ). a significantly higher percentage of the dentists however, stated that they only learned of the occupational risks of dentistry during their education and training (p < . ). there was no statistically significant difference between the two groups in terms of the other opinions questioned. the participants were also asked whether or not they agree with the argument "when people choose, and continue to practice, the medical or dentistry profession, they are then required to accept all of the occupational risks resulting from the infectious diseases they might confront". the aim of this question was to determine whether or not the hcws each have their own individual working-definition for the slr. of the participants, . % believed that there is such a level. . % felt that certain diseases would exceed the level of acceptable risk unless specific protective measures were implemented, and . % said that some diseases were always beyond the slr, no matter what precautions might be taken. no statistically significant difference was found between the physicians and the dentists. listed in table are the diseases which, under certain circumstances, were cited as potentially exceeding the slr. among the participants who stated that there would be a slr for providing health care to the patients of specific diseases unless protective measures were implemented, aids and hepatitis c and b were the most frequently cited of these diseases ( . %, . %, and . %, respectively). the participants who felt that some diseases would always exceed a slr expressed, that hepatitis b, tuberculosis, and bacterial meningitis always would go beyond the slr ( . %, same for all). according to these participants, the occupational risk of potentially being infected with hiv is paramount to all other risks. percentage-wise, aids was the most frequently mentioned disease that would exceed the slr, more so than sars. all of the participants who answered that some diseases would be beyond the slr were then asked what criteria they used to make their determination. the most com-monly expressed criteria, in order, regarding the diseases, were the likelihood of transmission, whether or not protective measures are available, and whether or not immunization is possible ( . %, . %, and . %, respectively). the distribution of these criteria among the physicians and dentists can be seen in table . physicians expressed significantly more often than dentists that if there was no immunization or treatment available for a particular disease, then that disease would exceed the slr (p < . ). in terms of other criteria, there were no significant differences observed between the two groups. the primary aim of this paper is to evaluate the claim that presumed consent may constitute grounds for the moral duty to treat. the presumed consent argument is valid, because its conclusion should logically be accepted if its premises are taken into account. to analyze the soundness of the argument we carried out a survey investigating the opinions of hcws about the occupational risks of infectious diseases. in total, . % of the participants said that they either had some degree of knowledge about the risks when they chose their profession or that they learned of the risks later during their education and training. in other words, one fifth of the participants either lacked adequate knowledge about the occupational risks when they chose their profession or were not sufficiently informed of these risks during their faculty education and training. this means that the assumption stated in premise may be wrong for an important proportion of health care workers. it seems reasonable to suggest that the words "there is a general understanding" would be misleading if used to characterize a social concept of which the applicability and, indeed, the very existence, are yet to be established by sociological studies. it is also useful to discuss the other problems associated with presumed consent; in particular, the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the nearimpossibility of being able to evaluate, in retrospect, whether or not every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. if we are to use the presumed consent argument, then the findings of this study indicate that when a new epidemic of a contagious disease occurs, or when the medical profession is confronted with a disease for which no immunization or treatment options are available, some hcws are not bound by the duty to treat according to the presumed consent argument. this seems potentially problematic and demands serious consideration. how appropriate is it to describe the healthcare provider's responsibility and duty as stemming from their 'consent'? to address this question, it is helpful to reflect on the conditions required for an individual to be able to give consent that is well-informed. for an hcw's consent to be informed, the following should first be explained to them: (a) the risk posed by each of the contagious diseases known at that given time, (b) commonly agreed criteria and definitions of situations that would surpass the slr, and (c) the fact that there will always be a degree of uncertainty involved with working in the medical profession, as new risks may emerge at any point during one's professional life. if not necessarily when they choose their profession, then at least after being given the relevant knowledge during education and training, the person's choice should be regarded as informed. it should therefore be ensured that hcws are acquainted with each new and emerging risk, and with any methods of prevention developed during or after their education and training. if a person's choice is to be confidently regarded as informed, it is imperative that these conditions be met. of course, the question now becomes: how possible is it to satisfy all these conditions? it is quite easy to imagine more than one answer to this question, but one thing is for sure: any thoughtful answer would acknowledge that choice is determined both by factors that are under the control of the individual and by factors that are not. personal factors such as educational status, perception of the world and ambitions all influence an individual's choice of profession strongly. nevertheless, factors outside the individual's control also play a large role in determining that choice. the environment in which the person grew up -their family life, the jobs of their parents, their community, social class and cultureall contribute to forming that individual's background, which (needless to say) has a very large influence on the opportunities and choices available to them. even though a person may not have been sufficiently informed when they chose their profession, it can be argued that during their education and training they will learn all relevant knowledge about the occupational risks associated with working in the medical profession. if so, it is fair to assume that when this individual begins to work after graduation they will be willing to confront any of those risks. in theory at least, it can be presumed that every student who passes their exams and goes on to graduate from the faculty is informed of the risks; so it can be argued that all hcws who are currently active in their profession have consented to accept the risks posed by all the contagious diseases known at the time of their graduation. of course, the diverse factors that determine the quality of education, such as the particular educational methods used, the course content, the abilities and knowledge of the instructors, role-models, and the personal features and motivations of students, are all potential sources of variation. but for the sake of argument, let us assume that a standardized education program is implemented throughout all medical schools. if that were the situation, then the argument that the individual has been made aware of the occupational risks during education would be true to the extent that the education program addressed those risks sufficiently. nevertheless, it would be hard to claim that presumed consent is valid for every individual. for many people, a degree in medicine is very costly, both financially and in terms of time and energy. under such circumstances, it is difficult for a person to quit their schooling despite the awareness they gain of the occupational risks involved. individuals might feel pressured and confused by the two options confronting them: on the one hand, dropping out of medicine and forfeiting all the time, effort and money spent on schooling towards that aim; and on the other, reluctantly accepting the occupational risks, which may look frightening to the individual at that moment. of course, it is important to remember that the person chose the medical profession in the first place, and numerous positive and beneficial elements are associated with working within it, which may ultimately serve to temper and override the individual's fear of the risks. an additional source of pressure may be that, for whatever reasons, switching educational tracks is too difficult; it may appear too daunting or be financially untenable. it seems likely that in the end the individual will choose to continue with their education and embark on a career in medicine, despite hesitation and fear of the risks. it is difficult to describe a decision made under conditions of such uncertainty and stress as 'informed'. as can be seen, we do not choose our profession from among a wide array of possibilities spread out in front of us by thoroughly researching each one so that we are fully informed of its nature; everybody's options are different and it is a large and difficult task to make oneself sufficiently informed of them. moreover, as described above, the decision to quit medical school can be quite difficult: on one side of the dilemma there are occupational risks that must be accepted regardless of misgivings on the part of the individual; on the other side, very influential factors pressure the individual to continue their medical education. thus, the claim that "people who enter medical fields clearly had alternatives" is debatable and sometimes even doubtful. in theory it sounds right; nobody has to be a physician. but in practice, having alternatives does not mean that all our decisions are made freely or autonomously. theoretically, the fundamental problem with the presumed consent argument is that it cannot explain why there is always some degree of uncertainty about the occupational risks of working in the medical profession, particularly stemming from new and emerging diseases. quite simply, if there was little or no knowledge of a risk at the time the individual became informed and gave their (tacit) consent, then this individual never accepted the risk, implicitly or otherwise, because it was unknown at the time the individual was informed. from a historical perspective, it is possible to see that while the medical profession was once concerned only with treating diseases, its vocational responsibility came in time to include preventative, promotive and rehabilitative healthcare services. as the notions of human rights and patient rights have developed and become widespread, perceptions about the health profession have changed at the community level. diseases that once killed millions of people can now be treated with a simple medicament, but today we are faced with new and challenging diseases unheard of in the past. as a result, the continuous cycle of changespurred on by greater knowledge and technological advances and confrontations with new and untreatable diseases -serves to alter the identity and nature of the medical profession, and out of all this arises a constant degree of uncertainty. this characteristic uncertainty is present both when the individual chooses the profession and throughout their education and training periodand, indeed, for the entirety of their professional career. it is therefore not possible for someone to be fully informed when they choose their profession, nor is it possible for them to become fully informed during their education and training; nevertheless, the person should be informed about the uncertainty involved with working in the medical profession. in the light of this uncertainty, answers such as "if i had known i would not have chosen it" are not very meaningful, because there is no way to anticipate all the potential risks one might encounter in the course of a professional life. the only sure thing amid the uncertainty is that diseases such as sars and avian 'flu will always continue to emerge. so far in the discussion, the difficulties of satisfying the conditions needed to validate the presumed consent argument have been described. it seems virtually impossible to fulfill all these conditions satisfactorily. at this point it is important to discuss two particular problems regarding the argument itself. first, it seems nearly impossible to evaluate individually whether the choice made by every hcw to enter the medical profession was informed. the only way to do that would be laboriously to ask each hcw whether they were informed when they decided on the health care profession. even then, irrespective of whether the person's answer to this question reflects the truth, the only thing that could be learned from such a broad and extensive interrogation would be the individual's perception, not their actual knowledge. by extension, we could not question the participants' level of knowledge in this study, but rather how they perceive their level of knowledge. because it is futile to seek objectivity in people's perceptions, it would not be sound to use those perceptions to determine whether the hcw's choice was informed, and thus whether they have taken on the duty to treat. and if these perceptions are regarded as subjective, as they should be, then it would be very difficult to develop a set of standard criteria that could be used to establish whether a hcw has been informed. this would also complicate efforts to reach a consensus on forming criteria by which various levels of risk could be defined universally. unfortunately, such difficulties can only hamper efforts to protect the right of every patient to receive the best treatment available. the second problem is that there is very likely to be more discrimination against patients with certain diseases, as hcws use this argument to justify their refusal to treat those diseases. the world medical association's declaration of geneva, which states the basic moral values of the medical profession, specifies that there should be no discrimination, regardless of the circumstances: "i will not permit considerations of age, disease or disability, creed, ethnic origin, gender, nationality, political affiliation, race, sexual orientation, or social standing to intervene between my duty and my patient" [ ] . however, the physicians' right to choose their patients is described by the same organization in another policy called "twelve principles of provision of health care in any national health care system" [ ] : "any health care system should allow the patient to consult the physician of his choice, and the physician to treat only patients of his choice, without the rights of either being affected in any way." for regulations at the national level, it is generally held that if there is an urgent need for medical care [ , ] , or if no other physician is available to whom the patient can apply [ , ] , this right would be negated and the available physician must treat the patient. if we look at these obligations in reverse, we can see that the physician might refuse to provide health services to the patient if there is no urgent need for medical care and/or if another physician is around to whom the patient can be referred. besides, the physician might also refuse the patient on the grounds that their prejudices may adversely affect the advice or treatment that they provide [ ] . in actuality, this flexibility was written into the regulations in order to ensure that the patient receives the highest quality care possible; but as can be seen, it could also be abused. and if the presence or absence of consent is used as a criterion to define the duty to treat, in addition the existing flexibilities, then a mechanism would be created by which hcws may freely, and perhaps excessively, discriminate among patients. to summarize, considering all the points discussed above, the soundness of the presumed consent argument can be doubted. therefore, it should not be claimed that there is a duty to treat on the basis of the presumed consent argument, as the argument itself is not persuasive. it is also important to note that . % of our participants said that they would not have chosen this profession if they had been informed of the risks. in other words, most of those hcws who claimed that they were uninformed of the occupational risks when they entered the faculty, or were not fully informed of them during their education period, stated that they still would have chosen the medical profession even if they had been more aware of the risks. this finding tells us that, generally speaking, hcws place relatively little importance on being informed beforehand. further support for these findings comes from the answers given by the participants to the other questions. nearly half said that there is no slr, and the other half felt that the diseases they evaluate would not surpass slr if the appropriate protective measures are available. also, table indicates that at least . % of the participants thought that none of the diseases listed in that table would exceed the slr regardless of circumstances. it can therefore be concluded that a large majority of the hcws place more emphasis on their working conditions than on being informed beforehand. in addition, the criteria most commonly stated by the participants for determining the slr were the likelihood of transmission of a disease, whether protective measures are available and whether immunization is possible. each of these criteria is related to protecting the hcw from infection, not to the treatment or the effects of a particular disease. this means that as long as protective measures are available, the hcws would regard a given disease as below the slr, so it has nothing to do with being informed beforehand. besides, the only disease used as an example in this study that could be claimed to exceed the slr was sars; aids, hepatitis c, hepatitis b, tuberculosis and bacterial meningitis all fell below the slr according to these criteria. nevertheless, only . % of the participants suggested that sars would surpass the slr. to put this into perspective, sars was not even observed until , and the research in the present study was conducted in and . therefore, most participants in this study were not aware of sars when they chose the medical profession, nor were they ever informed of it during education or training. they nonetheless felt that the duty to treat pertained even to patients with sars. all of this suggests that factors more useful and relevant than presumed consent influence the decision of hcws to choose and continue in the medical profession; these factors may include the social contract between society and the medical profession, and the greater ability of hcws to provide medical care [ ] . it is these factors that should be investigated and emphasized when defining a moral duty to treat. this study could be limited by several factors. the first limitation could be due to a socially desirable response bias; some participants might have given what they perceived as the 'right' answers to the questions rather than the answers that reflect their opinion or belief. in order to address this concern, future studies could benefit by using qualitative methods, which provide more reliable results about the motives and opinions of participants. also, this study was not prospective, so recall bias might have affected the responses of the participants. furthermore, the extent to which the results of this study are applicable to hcws such as nurses or physicians who work in internal specialties is uncertain. future studies that include other hcws as participants may broaden our understanding of the beliefs and opinions of hcws, thereby allowing us to state our claims and shape our arguments more precisely. finally, it should be mentioned that the response rate for this study ( . %) was slightly lower than is generally expected for a survey. nevertheless, despite all these methodological limitations, we believe that our findings support our conclusion about the persuasiveness of the presumed consent argument. if we use the presumed consent argument to establish the duty of the hcw to provide care, we are confronted with problems ranging over the difficulty of choosing a profession autonomously, the constant level of uncertainty present in the medical profession, the near-impossibility of being able to evaluate retrospectively whether every individual was informed, and the seemingly inescapable problem that this practice would legitimize, and perhaps even foster, discrimination against patients with certain diseases. our findings suggest that another problem can be added to the list: one-fifth of the participants in this study either lacked adequate knowledge of the occupational risks when they chose the medical profession or were not sufficiently informed of these risks during their faculty education and training. as we stated above, in order for a candidate hcw to be informed literally, three items should be explained to them: (a) the risk posed by each of the contagious diseases known at that given time, (b) commonly agreed criteria and definitions of situations that would surpass the slr, and (c) the fact that there will always be a degree of uncertainty involved with working in the medical profession, as new risks may emerge at any point during one's professional life. in this study it has been shown that at least some hcws may not be informed of (a). also, it is not currently possible to inform hcws of (b) since there are no widely-agreed criteria and definitions to allow for a universally accepted slr; and there is currently no standard education for all hcws to ensure that (c) is satisfied. considering this in addition to the problems mentioned above, the third premise of the presumed consent argument appears implausible and, consequently, the duty to treat cannot be grounded persuasively on the consent assumption. it is therefore more useful to emphasize justifications other than presumed consent when defining the duty of hcws to provide care, such as the social contract between society and the medical profession and the fact that hcws have a greater ability to provide medical aid. furthermore, in terms of the moral duty to provide care, it seems that most hcws are more concerned about the availability of protective measures than about whether they had been informed of a particular risk beforehand. it seems important that further research be carried out to improve understanding of the opinions and perceptions of hcws and the basis of their definitions, as this information could prove very helpful in defining a duty to treat that can be effectively put into practice. it is also important that a well-organized ongoing educational program that is needs-based and easily accessible be provided to hcws at both the graduate and postgraduate levels. in particular, this program must be continuously updated regarding aids and other diseases that may cause the hcws to behave discriminatively towards patients, even though these diseases are below the slr. such continuing medical education is the best answer to the justification "when i chose the profession/when i graduated, this disease did not exist!" for refusing treatment. emphasizing the social role of hcws, and educating them about the professional obligations derived from the social contract betweeen the profession and the wider social order, would further reduce that kind of reasoning. in addition, stricter standards for the duty to provide care should established by determining the criteria for a slr and identifying the situations and conditions that would exceed this slr. each of these measures could serve to remind hcws that they have a moral responsibility, as individual hcws, to be aware of professional obligations and to act as responsible members of the profession. moreover, the working environment of hcws should be provided with preventative measures that can be applied both generally and specifically and should emphasize their use. for a circumstance in which a preventative measure has been developed for a disease but is not available for treating a particular case, it would not be easy to justify the claim that there is an undeniable duty to provide care at that moment. international health care worker safety center: annual number of occupational percutaneous injuries and mucocutaneous exposures to blood or potentially infective biological substance knowledge, practices and attitudes towards hiv positive and aids patients among dental auxiliaries. east african medical journal do p: knowledge, attitudes, and practices among physicians on hiv/aids in quang ninh, vietnam. aids patient care and stds knowledge, attitudes, and practices regarding sexually transmitted infections among general practitioners and medical specialists in karachi survey of hiv/aids knowledge and attitudes of kuwaiti family physicians. family practice an investigation of dentists knowledge, attitudes and practices towards hiv+ and patients with other bloodborne viruses in south cheshire knowledge and attitudes of japanese dental health care workers towards hivrelated disease a knowledge, attitudes, beliefs and practices (kabp) survey on hiv infection and aids among doctors and dental surgeons in singapore oral care of hiv infected patients: the knowledge and attitudes of irish dentists discriminatory attitudes and practices by health workers toward patients with hiv/aids in nigeria the role of psychosocial assessment and support in occupational exposure management guidelines for prevention of transmission of human immunodeficiency virus and hepatitis b virus to health-care and public-safety workers duty to treat or right to refuse? hastings cent rep semmelweis revisited: the ethics of infection prevention among health care workers in harm's way: ama physicians and the duty to treat the world medical association: declaration of geneva the world medical association: twelve principles of provision of health care in any national health care system general medical council: good medical practice college of physicians and surgeons of alberta: physician/patient relationships -cpsa guideline turkish medical association: turkish medical association professional code of ethics right to refuse work becomes another sars issue we would like to thank to prof. huma Ömürlü, vice dean, for her helps to the survey's administration in gazi university faculty of dentistry, assoc. prof. alp ergör for his comments on the first draft of the field study, and mr. anthony clark for his kind effort to polish the language of the text. as dr. singer says: "there is a threshold beyond which health care workers aren't obliged to take personal risks. we don't expect firefighters to jump into a burning pit, or police officers to throw themselves in front of a bullet. how health care workers define this threshold is an intensely personal decision. ... but obviously, it has serious implications for our collective response to a problem like sars." [ ] . it is clear that to rely upon the presumed consent argument to define the duty to treat will not make our collective response to potential epidemics such as sars or avian 'flu any more effective or robust. hcws: health care workers; slr: standard level of risk the author(s) declare that they have no competing interests. mc and ba have contributed equally to the conception, design and writing of the manuscript. all authors read and approved the final manuscript. the questions referred to:• whether the participant knew when they chose their profession that they would have an increased risk of being infected by a contagious disease?• if the participant did not know of this occupational risk, whether or not they later learned of it during their training?• if they still have not been formally made aware of the risks, do they think that they have enough knowledge about the current risks that they face?• if they had known about the risks earlier, would they still have chosen this particular profession?• when somebody chooses to be a physician or a dentist, are they obligated to accept all of the occupational risks regarding infectious diseases?• if not, what criteria must we use to determine that a particular disease is below the slr?• which diseases are below the slr?publish with bio med central and every scientist can read your work free of charge the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -jc k fki authors: gardner, emma g.; kelton, david; poljak, zvonimir; van kerkhove, maria; von dobschuetz, sophie; greer, amy l. title: a case-crossover analysis of the impact of weather on primary cases of middle east respiratory syndrome date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: jc k fki background: middle east respiratory syndrome coronavirus (mers-cov) is endemic in dromedary camels in the arabian peninsula, and zoonotic transmission to people is a sporadic event. in the absence of epidemiological data on the reservoir species, patterns of zoonotic transmission have largely been approximated from primary human cases. this study aimed to identify meteorological factors that may increase the risk of primary mers infections in humans. methods: a case-crossover design was used to identify associations between primary mers cases and preceding weather conditions within the -week incubation period in saudi arabia using univariable conditional logistic regression. cases with symptom onset between january – december were obtained from a publicly available line list of human mers cases maintained by the world health organization. the complete case dataset (n = ) was reduced to approximate the cases most likely to represent spillover transmission from camels (n = ). data from meteorological stations closest to the largest city in each province were used to calculate the daily mean, minimum, and maximum temperature ((ο)c), relative humidity (%), wind speed (m/s), and visibility (m). weather variables were categorized according to strata; temperature and humidity into tertiles, and visibility and wind speed into halves. results: lowest temperature (odds ratio = . ; % confidence interval = . – . ) and humidity (or = . ; % ci = . – . ) were associated with increased cases – days later. high visibility was associated with an increased number of cases days later (or = . ; % ci = . – . ), while wind speed also showed statistically significant associations with cases – days later. conclusions: results suggest that primary mers human cases in saudi arabia are more likely to occur when conditions are relatively cold and dry. this is similar to seasonal patterns that have been described for other respiratory diseases in temperate climates. it was hypothesized that low visibility would be positively associated with primary cases of mers, however the opposite relationship was seen. this may reflect behavioural changes in different weather conditions. this analysis provides key initial evidence of an environmental component contributing to the development of primary mers-cov infections. middle east respiratory syndrome coronavirus (mers--cov) is an emerging zoonotic agent that was first isolated in from a patient hospitalized in saudi arabia [ ] , and has since infected over people with a % case fatality ratio [ ] . after an incubation period of - days [ ] , the virus causes a disease (middle east respiratory syndrome, or mers) characterized by fever, cough, and shortness of breath, which commonly leads to pneumonia and respiratory failure [ ] . the virus circulates silently in dromedary camels, the only known reservoir species and zoonotic source of spillover to humans [ ] . however, not all primary human cases have documented exposure to dromedaries or their products, such as milk and meat. although human-to-human community-acquired infections have not been documented, there is evidence that asymptomatic infections of mers-cov exist and could be a source of community transmission [ ] . zoonotic spillover from dromedary camels to humans has been documented in the arabian peninsula [ ] . subsequent secondary cases can occur after unprotected contact with family members and within healthcare facilities once the primary case seeks medical assistance [ ] . while the sizes of mers-cov outbreaks have decreased thanks to improved infection control in healthcare settings in affected countries, cases continue to be reported regularly, especially in saudi arabia, where surveillance is strong [ ] . in order to further reduce cases and prevent human outbreaks, a better understanding of zoonotic transmission of mers-cov is needed. a deeper understanding of the epidemiology of primary human cases can inform evidence-based interventions at the level of the community at the animal-human interface. zoonotic modes of mers-cov transmission have not yet been definitively determined. mers-cov in dromedary camels causes a mild upper respiratory infection with no documented viremia [ ] , and therefore droplet or aerosol transmission by close camel contact is most likely. however, transmission through contaminated milk, meat, and urine is possible, although the contribution of camel products cannot currently be estimated due to a lack of scientific evidence. the effects of weather and environmental conditions on respiratory diseases with similar modes of transmission (direct contact or droplet), such as influenza and respiratory syncytial virus, have been documented. temperature and humidity are associated with transmissibility of influenza virus [ ] , and the seasonality of both influenza and respiratory syncytial virus is linked to these two factors [ ] . air quality is also associated with respiratory infections. air pollution has been linked to pneumonia and acute lower respiratory infections [ , ] , while dust storms are associated with infectious respiratory disease by acting both as a carrier of pathogens and increasing airway susceptibility to infection [ ] . the risk of acquiring primary mers may be influenced by changes in weather conditions in two ways. first, weather conditions may affect the viability and persistence of the virus in the environment and therefore its transmissibility [ , ] . secondly, weather influences behaviour, and it is plausible that the likelihood of people contacting camels depends on environmental conditions. seasonal or meteorological patterns of primary mers-cov infections have yet to be explored. this study examined whether meteorological conditions were associated with the development of known primary mers-cov infections using a case-crossover study design. case-crossover studies are designed so that exposures during a period of interest before a case are compared to exposures during control periods before or after the case. in this regard, case-crossover studies answer the question "why now?" as opposed to "why these subjects?" [ ] . the design is well suited for rare diseases with short incubation periods such as mers-cov. the effect period, that is, the period of time after the proposed "trigger", typically has a degree of uncertainty [ ] , leading to exposure windows with intervals of biological relevance to the outcome of interest. for infectious diseases, this would equate to the incubation period [ ] . furthermore, with appropriate selection of referent windows, the case-crossover design controls for confounding effects of temporal fluctuations such as climatic and livestock-associated seasons (e.g. the dromedary breeding cycle) [ ] . by comparing weather conditions immediately before mers cases to weather conditions at other times, this study aimed to identify environmental factors that are associated with primary human mers in saudi arabia. the world health organization (who) maintains a list of all human laboratory confirmed cases of mers-cov. publicly available case data from january -december were obtained. case data prior to were excluded due to a lack of standardized data collection prior to [ ] . a mers case was defined throughout the study period as "a person with laboratory confirmation of mers-cov infection irrespective of clinical signs and symptoms" [ ] . of the confirmed cases with onset dates between january -december , cases were removed where exposure to camels and camel products were known not to have occurred. geographically, cases were restricted to those reported from saudi arabia, where the province of exposure was provided (n = ). cases that were likely primary cases were retained by excluding healthcare workers and cases with documented contact with known mers cases (n = ). cases were further removed where symptom onset date was after hospitalization date (n = ). of the remaining cases (n = ), ( . %) had missing symptom onset dates. to retain these ten cases, the median time between symptom onset date and lab confirmation date was calculated ( days) and subtracted from the lab confirmation date to obtain an estimate of the symptom onset date. visual inspection of the timeline of retained cases identified a spike from riyadh province around august , which corresponds to a documented mers-cov outbreak in the city of riyadh from july-september . data from a published report of the outbreak contained weekly counts of primary and secondary cases [ ] . these weekly counts were compared with the case list for this analysis and thirty-two secondary cases associated with the riyadh outbreak were removed. the final number of retained cases fitting the primary case definition was ( fig. ). for the purposes of the descriptive results, age groups were chosen for ease of reading while still providing a visualization of the distribution, and according to age categories provided by the statistical yearbook of the general authority for statistics of the kingdom of saudi arabia, which was used for standardization. meteorological stations closest to the largest city in each province were identified by a numeric identifier and location using google earth [ ] (fig. ) . meteorological data were obtained from the noaa global hourly index [ ] . the daily mean, minimum, and maximum temperature, wind speed, and visibility were calculated. relative humidity was calculated using temperature and dew point data [ ] . a case-crossover design was used to explore the associations between primary mers cases and preceding meteorological conditions [ , ] . each case's exposure status on individual days before disease onset (the exposure window) was compared to the exposure status on different days during a control period. under the assumption that weather effects on virus transmission were immediate, the exposure window, that is, the time lag between weather events and disease onset, was set to be equal to the mers incubation period of - days [ ] . univariable conditional logistic regression was used to assess statistical associations between cases and weather variables on each day within the case and control exposure windows. associations with p < . were considered statistically significant. a time-stratified design was used, with a -day strata length with random bi-directional controls matched by day of the week. using a -day time window provides at least three control days for each case exposure day while minimizing bias introduced from seasonal changes [ ] . temperature and humidity variables were categorized into tertiles calculated within each time stratum. wind speed and horizontal ground visibility were categorized into two groups within each stratum with the median as cutoff. therefore, there is no single threshold for each weather variable, but rather "low", "medium" and "high" are determined according to the measurements in each stratum. statistical analyses were conducted using stata . (stata corporation, college station, tx). four hundred and forty-six cases of mers-cov in saudi arabia with symptom onset dates between january -december were included in the analysis. table presents the case counts as well as crude and age-and sex-standardized rates by province, sex, and age group. all provinces in saudi arabia reported cases during this -year period. riyadh province had the highest count of reported cases with cases ( %), although qasseem had the highest cumulative incidence ( . cases per , people), followed by riyadh ( . cases per , people). the median age of cases was years (range, - ), and % of cases were male. age and sex proportions are similar to figures reported for primary cases in previously published literature [ ] . figure presents the case count by month from to for the entire country. cases were reported in every month of the year, although no clear seasonality is apparent. temperature and humidity conditions were associated with case occurrence - days later. the odds of a mers case days after low minimum temperatures was . ( % confidence interval [ci], . - . ) higher than after control days, while low mean daily temperature was similarly associated with cases at (or, . ; % ci, . - . ) and day lags (or, . ; % ci, . - . ) (fig. ) . conversely, high minimum, maximum, and mean temperatures were protective at similar lag days. for example, the odds ratios of mers cases for the high mean daily temperature was . ( % ci, . - . ) with a -day lag, and . ( % ci, . - . ) with a -day lag. humidity followed a similar pattern to temperature. when maximum daily humidity was low days earlier, the odds ratio for a mers cases was . ( % ci, . - . ). high humidity was associated with fewer cases across all three daily measurements (fig. ) . for example, the odds ratios of cases for high maximum daily humidity was . ( % ci, . - . ) and . ( % ci, . - . ) at -and -day lags, respectively. high visibility was positively associated with occurrence of a mers case days later, whereas low visibility demonstrated protective effects for risk of mers (fig. ) . the odds of a mers case days after both minimum and mean daily visibility were high was . and . times higher than after control days ( % ci, . - . and . - . ). conversely, when minimum and mean wind speed results were conflicting, with low minimum daily wind speed and high maximum wind speed both positively associated with cases at similar time lags (fig. ) . the odds of a mers case was . times higher days after low minimum wind speed ( % ci, . - . ), while the odds ratio of cases for when minimum wind speed was relatively high was . ( % ci, . - . ). conversely, the odds of a case when maximum wind speed was relatively high was . ( % ci, . - . ) (not shown in figure) . mers is a global public health threat that causes severe respiratory disease with a high case fatality ratio, identified by who as a priority pathogen for research and development in public health emergency contexts [ ] . it is primarily characterized by healthcare-associated outbreaks triggered by index cases who acquire infection from dromedary camels and possibly from unidentified asymptomatic human carriers. improving our understanding of the epidemiology and risks of primary cases of mers is vital for designing effective interventions that aim to reduce these index cases and prevent subsequent outbreaks in humans. the list of cases maintained by the who was restricted to a subset of primary cases based on explicit inclusion and exclusion criteria and was used to analyze the effect of weather on case occurrence using a case-crossover design. all four weather variables demonstrated statistically significant correlations within the incubation period for mers in humans. the statistically significant time lags for each variable do not match up perfectly, which is to be expected and could be due to a number of reasons including natural variability in incubation periods, variable impact of weather on transmission, the interaction of unmeasured cofactors on weather variables as well the direct effect of unmeasured factors on transmission, and stochasticity in general. acute weather events as well as general seasonal patterns may affect disease transmission rates by altering pathogen viability and persistence in the environment as well as by influencing human behaviour and contact patterns. this study found that mers-cov, although a zoonotic disease, follows similar environmental transmission patterns to other non-zoonotic respiratory diseases with analogous modes of transmission such as influenza and respiratory syncytial virus. tamerius et al. [ ] have shown that global trends of influenza broadly follow either a "cold-dry" or "humid-rainy" pattern, corresponding to temperate and tropical climates. additionally, temperate climates tend to have a single annual peak and tropical climates have semi-annual peaks. they further demonstrate that for countries with an annual influenza peak such as saudi arabia, temperature and humidity can be predictive of those peaks, even at latitudes close to the equator. respiratory syncytial virus also follows similar environmental conditions, with peak timing in the arabian peninsula from december to february, following the distribution of cases in the temperate northern hemisphere [ ] . the influence of weather is further supported by experimental evidence, which has demonstrated that lower temperatures and lower relative humidity each favour influenza transmission [ ] . furthermore, coronaviruses have been shown to exhibit strong seasonal variation in natural hosts, and the theory that these fluctuations may increase risk of zoonotic transmission at certain times of the year has been discussed [ ] . the results here demonstrate that colder, drier conditions may increase the risk of zoonotic transmission of mers from dromedaries to humans. sandstorms, dust storms, and air pollution in saudi arabia and elsewhere have been associated with increased morbidity and mortality, including from respiratory disease [ , ] . a case-crossover study in the united states demonstrated the short-term effects of air pollution on acute lower respiratory infections [ ] , while another study demonstrated increased numbers of pneumonia admissions following acute dust storm events in taiwan [ ] . dust storms can act as a pathogen carrier and also induce inflammatory reactions, potentially increasing both exposure and susceptibility to disease agents [ ] . horizontal ground visibility and wind speed were used as proxies for the occurrence of sandstorms and acute air pollution events. visibility can be reduced to m for an average of . h during a sandstorm [ ] . summarizing the weather data used in this study, the mean daily visibility by province ranged from m to over , m, although the median value was over m in all but one province. the distribution of visibility indicates that fig. daily mean and minimum temperature and risk of primary mers by province in saudi arabia. odds ratio (solid line) and % confidence limits (dashed lines) are plotted on the y-axis, while time lags preceding case occurrence are plotted on the x-axis. the odds of primary mers is increased with low temperature at and day lags (a &b), while the odds of primary mers are decreased with high temperatures at and day lags (c & d). asterisks indicate statistically significant odds ratios on corresponding days anything less than full clarity was categorized as low visibility, and that according to the measurements in [ ] , could indicate the presence of a sandstorm. it was hypothesized that primary mers infections are more likely to increase following sandstorms or other severe events of air pollution that affect visibility. however, results indicate that the risk of primary mers infection increased following high visibility days, and decreased following low visibility days. this may be due to behaviour, if people are more likely to stay inside during acute weather events, and less likely to engage in activities such as interacting with camels. it was further hypothesized that higher wind speeds would be associated with more cases of mers. while a positive association was found between cases and high maximum wind speeds days prior, there were also similar results to those of visibility. low minimum wind speed was positively correlated with cases, and conversely, when minimum wind speed was relatively high there were statistically fewer cases of mers. results suggest that further investigation of wind speed as a factor for primary mers is warranted. there are several limitations and potential sources of bias in this study. the major cities in saudi arabia are severely polluted and exceed who guidelines, as measured by particulate matter (pm) [ , ] . sand and dust storms as well as other sources of air pollution such as industrial activities, fuel combustion, and traffic emissions contribute to elevated levels of pm in the country [ ] , all of which contribute to reduced visibility [ ] [ ] [ ] . this study did not differentiate between sandstorms and other acute events that reduce visibility, and discerning between different forms of air pollution may provide insights about the risk of mers-cov transmission under different environmental conditions. fifty-two cases ( . %) in the subset of primary cases had no known exposure history (no information on camel exposure, contact with a known case, nor healthcare worker status). the subset of primary cases investigated likely also include secondary cases, and is a source of selection bias. furthermore, given that mers is an emerging disease, case reporting and data collection standardization may have improved over the -year period included here. geographical case data were available only at the provincial level, while exposure data from the weather station closest to the largest metropolitan city in each province were used. while camel raising in the middle east is moving from extensive to intensive production systems and concentrating around cities [ ] , human spillover cases would be scattered throughout the provinces to an unknown degree. therefore, if environmental conditions differ significantly within a province, this could be a source of misclassification bias. the risk of primary human cases of mers was associated with a decrease in temperature and humidity, and an increase in ground visibility. the temperature and humidity findings are consistent with associations between the environment and other respiratory diseases. further study of weather and seasonal risk factors may strengthen the evidence for an environmental component of mers-cov transmission. a better understanding of virus viability in different environmental conditions is also a key research need. evidence of environmental risk factors for mers could be utilized by public or one fig. daily visibility and wind speed variables and risk of primary mers by province in saudi arabia. odds ratio (solid line) and % confidence limits (dashed lines) are plotted on the y-axis, while time lags preceding case occurrence are plotted on the x-axis. the odds of primary mers is increased with high visibility and decreased with low visibility after days (a & c), while the odds of primary mers are increased with low wind speed and decreased when wind speed is high at -day lags (b & d). when maximum wind speed was high, the odds of a mers case were increased with a -day lag (not shown). asterisks indicate statistically significant odds ratios on corresponding days health practitioners for targeted interventions during higher-risk periods. the risk of mers acquired from zoonotic transmission, or from asymptomatic carriers in the community, appears to be sensitive to weather conditions, providing key initial evidence of an environmental component for the development of primary mers-cov infections. isolation of a novel coronavirus from a man with pneumonia in saudi arabia world health organization. who | middle east respiratory syndrome coronavirus (mers-cov). who world health organization. investigation of cases of human infection with middle east respiratory syndrome coronavirus (mers-cov) interim guidance middle east respiratory syndrome coronavirus (mers-cov) fact sheet mers-cov technical working group. mers-cov: progress in global response to epidemic threat, remaining challenges and way forward: report from the fao-oie-who global technical meeting on mers-cov presence of middle east respiratory syndrome coronavirus antibodies in saudi arabia: a nationwide, cross-sectional, serological study evidence for camel-to-human transmission of mers coronavirus middle east respiratory syndrome world health organization. mers situation update replication and shedding of mers-cov in upper respiratory tract of inoculated dromedary camels influenza virus transmission is dependent on relative humidity and temperature latitudinal variations in seasonal activity of influenza and respiratory syncytial virus (rsv): a global comparative review international approach to environmental and lung health a perspective from the fogarty international center short-term elevation of fine particulate matter air pollution and acute lower respiratory infection lung health in era of climate change and dust storms seasonality of infectious diseases and severe acute respiratory syndrome-what we don't know can hurt us should we use a case-crossover design? a comparison of case-crossover and case-control designs in a study of risk factors for hemorrhagic fever with renal syndrome bias in the case-crossover design:implications for studies of air pollution middle east respiratory syndrome coronavirus (mers-cov) disease outbreak news world health organization. who | middle east respiratory syndrome coronavirus: case definition for reporting to notes from the field: nosocomial outbreak of middle east respiratory syndrome in a large tertiary care hospital national centers for environmental information weathermetrics: functions to convert between weather metrics the case-crossover design: a method for studying transient effects on the risk of acute events referent selection in case-crossover analyses of acute health effects of air pollution reported direct and indirect contact with dromedary camels among laboratory-confirmed mers-cov cases world health organization. who | list of blueprint priority pathogens environmental predictors of seasonal influenza epidemics across temperate and tropical climates the pulmonary consequences of sandstorms in saudi arabia: a comprehensive review and update the effect of sandstorms and air pollution on causespecific hospital admissions in taipei asian dust storm events are associated with an acute increase in pneumonia hospitalization predicting the development of weather phenomena that influence aviation at abu dhabi international airport. pretoria: university of pretoria outdoor particulate matter ( pm ) and associated cardiovascular diseases in the middle east world health organization. who air quality guidelines for particulate matter, ozone, nitrogen dioxide and sulfur dioxide: global update : summary of risk assessment the influence of meteorological conditions and atmospheric circulation types on pm and visibility in tel aviv estimation of particulate matter from visibility in bangkok fine particulate matter characteristics and its impact on visibility impairment at two urban sites in korea: seoul and incheon human-dromedary camel interactions and the risk of acquiring zoonotic middle east respiratory syndrome coronavirus infection the authors would like to thank all of the many individuals who investigated and collected information from mers patients in saudi arabia. availability of data and materials all laboratory confirmed human cases of mers included this publication can be found on the who disease outbreak news website, at the following website: http://www.who.int/csr/don/archive/disease/mers-cov/en/ authors' contributions eg designed the study, analyzed and interpreted the data and wrote the manuscript. ag and dk provided significant guidance in all aspects of the research. ag, dk, svd, mvk and zp substantially contributed to the conception of the study and interpretation of the results, critically reviewed the manuscript and provided final approval for publication.ethics approval and consent to participate not applicable: all data used were publicly available. not applicable. publicly available, non-individually identifying data were used in this publication. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -ajafw authors: bozza, fernando a; cruz, oswaldo g; zagne, sonia mo; azeredo, elzinandes l; nogueira, rita mr; assis, edson f; bozza, patricia t; kubelka, claire f title: multiplex cytokine profile from dengue patients: mip- beta and ifn-gamma as predictive factors for severity date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ajafw background: dengue virus pathogenesis is not yet fully understood and the identification of patients at high risk for developing severe disease forms is still a great challenge in dengue patient care. during the present study, we evaluated prospectively the potential of cytokines present in plasma from patients with dengue in stratifying disease severity. methods: seventeen-cytokine multiplex fluorescent microbead immunoassay was used for the simultaneous detection in dengue patients. glm models using bimodal or gaussian family were determined in order to associate cytokines with clinical manifestations and laboratory diagnosis. results: il- β, ifn-γ, il- , il- , il- , il- and gm-csf were significantly increased in patients with severe clinical manifestations (severe dengue) when compared to mild disease forms (mild dengue). in contrast, increased mip- β levels were observed in patients with mild dengue. mip- β was also associated with cd +nk cell circulating rates. il- β, il- , tnf-α and mcp- were associated with marked thrombocytopenia. increased mcp- and gm-csf levels correlated with hypotension. moreover, mip- β and ifn-γ were independently associated with both dengue severity and disease outcome. conclusion: our data demonstrated that the use of a multiple cytokine assay platform was suitable for identifying distinct cytokine profiles associated with the dengue clinical manifestations and severity. mip-β is indicated for the first time as a good prognostic marker in contrast to ifn-γ that was associated with disease severity. during the last decades dengue became the most important arthropod-borne emerging viral disease in tropical countries [ ] . it is estimated that about . % notified cases are classified as dengue haemorrhagic fever (dhf) and about . - % of dhf cases are lethal [ ] [ ] [ ] . in the last two decades, latin america saw a dramatic increase in frequency and in geographic extension of dengue fever. specifically, the situation in brazil has worsened during the last decade since the introduction of the dengue- serotype. in the past years brazil had dengue outbreaks with at least million cases ( ) ( ) and within the last months thousand cases were reported [ ] . in addition, severe disease forms are occurring with increased frequency and mortality rates. dengue pathogenesis is not completely understood, and the main determinants of the development of severe forms are not yet well established. increase in capillary permeability associated with endothelial activation and haemorrhagic phenomena are landmarks of severe clinical manifestations, strongly suggesting an alteration in immunoregulation [ ] . cytokines are proteins secreted during innate and adaptive immunological responses, acting as inflammatory mediators or modulatory molecules during several haemorrhagic fevers [ ] . clinical studies support a key role for cytokines in the dhf pathogenesis [ ] [ ] [ ] [ ] [ ] [ ] . during dengue virus infections, cytokines are involved in the disease onset and homeostatic regulation. specifically, tnf-α, il- β and il- have been associated with both coagulation (f + and tatc) and fibrinolysis (t-pa, papc, and d-dimmer) activation markers [ ] . this activation is more striking in patients with severe clinical manifestations, although it can be found at lower degrees in patients with mild disease [ , ] . despite the fact that cytokine network and their multiple regulatory pathways are highly complex and not fully elucidated during dengue fever, these molecules seem to represent interesting markers for patient stratification or prognosis. an emerging interest has appeared in order to define biomarkers that may have pathophysiological roles during disease and that may be used as future therapeutic targets. new technologies have been developed in order to detect multiple biomarkers within a single and small blood sample. such approaches may lead to the development of specific marker panels for dengue fever. accordingly, cytokine patterns have been indicated as serum biomarkers during infectious diseases such as hepatitis c [ ] , ards [ ] and sepsis [ ] . in this study, we prospectively evaluated the potential use of plasma cytokine concentrations for severity stratifica-tion of patients with dengue, using a cytokine-multiplex assay. among tested cytokines, we were able to recognize ten significantly altered circulating factors and to characterise cytokine patterns related to determined clinical manifestations and disease severity. the ethics committee of the oswaldo cruz foundation approved this study protocol and written informed consent was obtained from all patients or their guardians prior to blood collection. we included prospectively dengue-infected patients ( a detailed history and physical examination was performed to detect hemorrhagic manifestations (positive tourniquet test for capillary fragility, skin haemorrhages, epistaxis, gingival, gastrointestinal, or urinary tract haemorrhage), signs of plasma leakage (pleural or pericardial effusion, ascites), signs of circulatory failure (cold extremities, cyanosis, hypotension, tachycardia, shock), and hepatomegaly. in addition to the suggestive clinical diagnosis, all patients had the dengue virus infection confirmed either by antidengue enzyme-linked immunoabsorbent assay (elisa)-igm, serotype specific reverse transcription-polymerase chain reaction (rt-pcr) or by virus isolation [ ] [ ] [ ] . dengue immune response was considered as primary or secondary by igg elisa according to previously established criteria [ ] . as previously reported [ ] [ ] [ ] , we also were often unable to characterize the severe disease forms based on who criteria [ ]. in nicaragua, harris et al. [ , ] described four key severe clinical manifestations associated with dengue -shock, plasma leakage, marked thrombocytopenia or internal haemorrhage -that do not fit dhf/dss classification as single parameters. according to these criteria, we considered: • severe dengue -dengue confirmed cases plus severe thrombocytopenia (< , platelets/mm ) and/or hypotension (postural hypotension with decrease in systolic arterial pressure in mm hg in supine position or systolic arterial pressure < mm hg) and/or plasma leakage (either haemoconcentration fluctuation of packed cell volume ≥ % during illness course and recovery or clinical signs of plasma leakage, such as pleural effusion) and/or severe haemorrhagic manifestations. • mild dengue -dengue confirmed cases in absence of severe thrombocytopenia, hypotension, plasma leakage signs or haemorrhagic manifestations. blood samples were collected from a peripheral vein and kept on ice. plasma was collected by centrifugation at g for min at °c, aliquoted, and stored at - °c until the analysis day. a multiplex biometric immunoassay, containing fluorescent dyed microspheres conjugated with a monoclonal antibody specific for a target protein, was used for cytokine measurement according to the manufacturer's instructions (bio-plex human cytokine assay; bio-rad inc., hercules, ca, usa). cytokines measured were: il- β, il- , il- , il- , il- , il- , cxcl (il- ), il- , il- (p ), il- , il- , granulocyte colony stimulating factor (g-csf), granulocyte-monocyte colony stimulating factor (gm-csf), monocyte chemoattractive protein (mcp- /ccl ), macrophage inflammatory protein (mip- β/ccl ), and tnf-α. briefly, μl plasma samples were diluted : and incubated with antibodycoupled beads. complexes were washed, then incubated with biotinylated detection antibody and, finally, with streptavidin-phycoerythrin prior to assessing cytokine concentration titres. concentrated human recombinant cytokine was provided by the vendor (bio-rad laboratories). a range of . - , pg/ml recombinant cytokines was used to establish standard curves and to maximize the sensitivity and the assay dynamic range. cytokine levels were determined using a multiplex array reader from luminex™ instrumentation system (bio-plex workstation from bio-rad laboratories). the analyte concentration was calculated using software provided by the manufacturer (bio-plex manager software). liquid nitrogen cryopreserved peripheral blood mononuclear leukocytes were isolated by histopaque- (sigma chemical co., saint louis, mo, usa) from out of dengue patients. cells were stained for cd surface marker using anti-cd -cy (igg , clone b ) from pharmingen (san diego, ca, usa) and positive cells were detected by flow cytometry as described before [ ] using facscalibur (becton-dickinson). events ( , - , ) were acquired and analyses were carried out with flowjo (treestar, version . ) software. the nonparametric mann-whitney u test was used to evaluate differences between cytokine ratios from severe and mild dengue patients. glm models were used to evaluate factors independently associated with quantitative variables. analysis of factors independently associated with dengue severity and other clinical manifestations was performed with glm with logistic regression or gaussian family. results from the logistic regressions are given as odds ratio (or). the confidence interval (ci) was established at %. alternatively, for a glm gaussian family t values were recorded. a probability value of p< . was considered to be significant. the statistical programs r [ ] and prism (graph-pad software, san diego, ca, usa) were employed. the fisher's exact test was applied to determine the significance of positive samples from patients when comparing different virus serotypes or sequential infections. correlation between platelet counts and cytokine production in blood samples was estimated by spearman's correlation. from the patients included, were classified as severe dengue and as mild dengue. detailed demographic, clinical, and laboratorial data from dengue patients are summarized in table . blood collection was performed between and days after disease onset. in order to avoid effects due to differences in the blood collection time, we compared mild and severe dengue patient groups using mann-whitney u test, which showed no differences in the disease onset time at the moment of sample collection [see additional file ]. the original data used to perform this analysis is shown at figure . patients with mild and severe dengue were investigated for prior incidence of infection, detected by serologic immune response (igg antibodies for denv). patients with severe dengue ( %; out ) were more likely to be experiencing a secondary dengue virus infection than patients with mild dengue ( %; out ), although no statistical significance was found in fisher's exact test (p = . ). among patients with denv- , were classified as secondary infection, whereas among patients with denv- , were classified as secondary infection (p = . ). dengue fever is characterised by a high fever phase and an abrupt drop in body temperature that has been called defervescence phase. characteristically the disease outcome is defined during this phase, when patients can either recover rapidly or progress to a severe life-threatening stage. cytokines and immunoactivation markers such as ifn-γ, il- , soluble cd and receptors for tnf-α [ , ] are associated with the defervescence phase and with disease severity. ifn-α levels are higher in dhf than in df during defervescence [ ] . during the febrile phase significant increase in cytokine circulating levels was detected including il- , il- , il- , mcp- and mip- β levels, which were maintained also elevated in defervescence (data not shown, analysed by non parametric kruskal-wallis test and dunn's multiple comparison test when compared with controls, p < . ); no significantly altered febrile levels were found when compared to defervescence. during the febrile cytokine levels in plasma from patients with mild and severe dengue figure cytokine levels in plasma from patients with mild and severe dengue. box-and-whiskers graph. the box extends from the th to the th percentile and the line at the middle is the median. the error bars, or whiskers extend down to the lowest value and up to the highest. factors were sorted according to their functional groups. mann-whitney u test was used to evaluate differences between cytokine concentration from severe and mild dengue patients. * p < . , ** p < . and ** p < . . phase il- was significantly higher than in defervescence. il- β, il- , ifn-γ were significantly increased during defervescence as compared to control samples. significant levels of il- , il- , and il- were not detected during dengue disease in our patients. il- was detected both in healthy individuals and in dengue patients but no difference between these two groups was detected [see additional file ]. we studied the cytokine profile from brazilian patients in order to compare severe and mild dengue cases during the acute phase of the disease. figure shows data from patients with regard to their plasma cytokine contents, which were sorted in four groups according to their reported function. we observed that cytokine concentrations of il- β, ifn-γ, il- , il- , il- , il- and gm-csf were significantly increased during severe dengue as compared to mild dengue, while mip- β levels are higher in mild dengue. mip- β and ifn-γ were independent variables associated disease outcome as determined by a logistic regression model (table and figure ). while mip- β was increased during mild dengue with odds ratio (or) of . and confidence interval (ci) . - . , ifn-γ was associated with severity with or of . (ci, . - . ). to assess relationships between cytokine levels and several clinical manifestations, the patient cohort with severe dengue was divided into distinct subgroups: those with hypotension, thrombocytopenia (≤ . counts/mm ) and/or haemorrhagic manifestations. a logistic regression model was used for binomial response subgroups and glm models using gaussian family were employed for subgroups with continuous response in order to determine cytokine profiles. il- β was associated with marked thrombocytopenia with or = . (ci, . - . ) in dengue patients. tnf-α was inversely related to thrombocytopenia with or = . (ci, . - . ) (table , figure ). considering platelet counts as a continuous variable for statistical analysis with a gaussian family, it was possible to determine that il- (p = . ) and mcp- (p = . ) levels are inversely related to their counts, displaying therefore an association with thrombocytopenia, while mip- β (p = . ) confirms its association with higher counts -normal or tending to normal (table ) . gm-csf (or = . ; ci, . - . ) was related with hypotension, whereas il- β had a negative predictive table and figure . natural killer (nk) cells have been earlier related to mild cases of dengue [ ] . forty-eight pbmc samples from thirty-five patients had their cd + rates determined by flow cytometry and a good correlation was observed with cytokines detected in plasma as independent factors in predicting severity table . their respective mip- β plasmatic levels (r = . ; p = . ). considering that different cytokines act in the immunological network as stimulating/up regulating factors and also in a feedback loop as down regulators, the cytokine balance might play a role in the immune response outcome. therefore we calculated mip- β/ifn-γ ratios for every patient and compared those from mild dengue with those from severe dengue. ratio averages ± sem were respectively ± and ± (p = . ; mann whitney u test), confirming our earlier data that these cytokines are acting as opposing factors. the different models built here using clinical manifestations as independent variables each exhibit specific cytokine profiles. the cytokine profile identified in patients with dengue may represent a valuable tool for the characterisation of immunological response patterns and may assist the identification of patient groups at risk for developing severe disease. in the present study, the use of a multiplex analysis for cytokine plasma detection in patients with dengue could identify cytokine profiles associated with the disease severity. early identification and management of severe dengue disease are essential to prevent death. it has been increasingly recognized that the inflammatory response and deregulated cytokine production play key roles in the development of severe clinical manifestations [ ] . however, cytokine profiles associated with dengue evolution and prognosis are not well established. new technologies for cytokine quantification were developed including the multiplex immunofluorescent bead array analysis system, allowing multiple biomarkers to be tested simultaneously in a small volume from one single plasma aliquot. recently, this methodology has been used for cytokine profile evaluation during several infectious diseases including viral infections [ , , ] and sepsis [ ] , among others. we were able to identify models for cytokine circulation during dengue acute phase that may vary with clinical manifestations. mip- β was for the first time associated with a good prognostic and was identified in the different disease models presented here. mip- β has been earlier detected after dengue virus cell stimuli in vitro [ , ] but preliminary studies in vivo did not report their role in severity. in accordance with a protective role for mip- β, changes in mip- β levels were significantly correlated with decreases in viral titre after hepatitis c treatment [ ] . in addition, mip- β was up regulated in acute infection in chimpanzees only when viral clearance took place, but not in those animals which failed to eradicate the virus [ ] . mip- β is produced by human monocytes and dendritic cells upon different stimuli [ ] as well as by activated nk cells [ ] and lymphocytes [ , ] . activated nk cells release granzyme a, which displays cytolytic functions and mip- β is chemoattractant for nk cells, recruiting them to inflammatory sites. nk cells have been associated with mild dengue [ ] . here a good correlation between mip- β plasma levels and nk cells was observed, reinforcing the relevance of these pathways and strongly suggest- ing their role in dengue protective mechanisms. an early and efficient virus clearance by direct or indirect nk functions is likely controlling virus replication, restricting intense immunological activation and the dengue immunopathology and therefore favouring a mild dengue disease. in previous studies, tnf-α has been reported to be associated with severity, mainly during dhf in brazilian patients [ , , ] . in the present study, however, this cytokine was not strongly associated with severity. indeed, other authors also found inconsistency or no difference in tnf-α levels in severe vs. mild disease forms [ , ] . we may hypothesize that differences in dengue virus serotypes or in host immune response such as different tnf-α genetic polymorphisms may explain the disease outcome. in our study from (braga et al., ) , patients were dengue- infected, while in the present study, patients were dengue- and - infected. a recent report [ ] describes non-significant tnf-α serum levels in adult patients and suggests that the discrepancy may have been caused by a transient tnf-α peak which was not detected at a later time point. in the present study we observed an association of ifn-γ with disease severity. indeed, increased ifn-γ concentrations have been detected in dengue patients in a variety of studies [ , [ ] [ ] [ ] [ ] . dhf induced by dengue- was associated with higher viremia early in illness and earlier peak plasma ifn-γ levels; maximum plasma viremia levels correlated with the degree of plasma leakage and thrombocytopenia [ ] . however, in a previous study from our group we failed to observe association of ifn-γ with disease severity [ ] , probably due to the small number of severe patients analyzed or to the dengue- incidence. ifn-γ is produced during a t-lymphocyte helper response type and may reflect cd + t cell activation with production of inflammatory cytokines. high levels of ifn-γ were observed in patients with dengue from asian and latin america and were associated with severity [ ] . ifn-γ produced by t cells may also activate mononuclear phagocytes (monocytes and dendritic cells), which would produce factors such as tnf-α, tissue factor, and plateletactivating factor, among other mediators. these factors may all participate in platelet and endothelial cell activation, leading to platelet consumption, increase in endothelial permeability, hypotension and ultimately to shock. ifn-γ has also been associated with secondary heterologous dengue virus infections [ , ] inducing a strong antigenically cross-reactive inflammatory response, probably inefficient in terms of antibody and t-cell specific response. indeed, we observed earlier in several patients a cd +t cell activation with hla-dr+ subset increase that was associated with severity [ ] . gm-csf acts at early differentiation processes at myeloid progenitors or resting monocytes [ ] . an additional stimulus may be required to activate monocytes or dendritic cells in order to produce proinflammatory cytokines [ ] . gm-csf was associated with hypotension as well as mcp- , likely acting both in concert, contrasting with mip- β, once more associated with good prognostics. mcp- was earlier detected in dhf patients [ ] but for the first time we reported clinical and laboratory findings associated with severity. il- and mcp- , here associated with thrombocytopenia, are chemokines and may contribute to platelet activation, either by their chemoattractant properties or by their effect on endothelial permeability. both factors were detected in patients with dhf [ , ] . these cytokines are produced by monocytes after various activation stimuli, such as virus infection, and increase the endothelial permeability by disrupting tight junctions among cells [ ] . despite the fact that our study could identify cytokines with good accuracy for the stratification and/or prognosis of dengue, it has potential limitations. here we identified cytokines related to dengue severity, but the small sample size represents a shortcoming regarding the generalization of our results. in addition, only one time point was used for the measurement of cytokines, not allowing further insights provided by sequential measurements. moreover, classification of disease severity has been a matter of debate, especially for adult patients' management and classification. indeed, the who criteria for dhf has failed to identify severe disease, including fatal cases, in adult latin america population [ , ] (s.m.o. zagne, r.m.o. nogueira, unpublished) and clearly do not satisfy the stratification of our studied population. accordingly, in the present study severe disease forms were classified following other proposed criteria [ ] . while a direct correlation of cytokine concentrations and the pathophysiology of severe dengue is tempting, we believe that the full burden of disease severity cannot be attributed to a single cytokine. cytokines may be increased simply as one of the several steps in the network loops without necessarily playing a direct harmful role and most likely more than one factor may be involved, including others not tested here such as il- , tgf-β, and mif among others [ ] . we can suggest a mechanism explaining our cytokine models for dengue fever (figure ) . mip- β would be associated with a protective pathway for its chemoattractive and activating effect on nk cells, which in turn are efficient cells in early virus clearance, by their antiviral cytokine production and cytotoxic activity against infected target cells. ifn-γ has a deleterious effect for the host regarding its action in activating t cells for virus anti-genic cross-reactive response and monocyte/dendritic cell activation. mononuclear monocytes are activated by ifnγ and gm-csf among other cytokines and in turn produce several factors such as il- β and mcp- that may act on vascular permeability leading to plasma leakage and haemoconcentration. as suggested by other authors, it is likely that viral replication in antigen presenting cells, cytokine liberation and circulation, and t cell activation may not be a linear process [ ] , but in fact a complex interaction network, with positive and negative feedbacks, where viral clearance and pathologic events take place, such as increased vascular permeability and circulatory collapse, and their balance may determine the disease outcome. our study demonstrated the plasma cytokine profile in dengue fever from a brazilian population detected by a multiplex bead immunoassay. mip-β is indicated for the first time as a good prognostic marker and in contrast to ifn-γ that was associated with the disease severity. both cytokines can discriminate mild from severe cases. moreover, we show here for the first time that during the dengue course different cytokine profiles may be present and vary according to determined clinical manifestations. the cytokine profiles identified herein by bead array multiplex system may favour an early identification of patients with the worst prognosis and may contribute to the establishment of more directed therapeutic procedures than the present ones. hypothetic mechanism to explain cytokine models during dengue fever figure hypothetic mechanism to explain cytokine models during dengue fever. mip- β is associated with a good prognostic and ifn-γ has a predictive value for severity. gm-csf, mcp- , il- β, il- , il- , il- , il- are also playing important roles during dengue pathogenesis (see text for detailed description). epidemic dengue/dengue hemorrhagic fever as a public health, social and economic problem in the st century world health organization: dengue hemorrahagic fever diagnosis treatment and control secretaria de vigilância em saúde-dengue -informe epidemiológico da dengue immunopathological mechanisms in dengue and dengue hemorrhagic fever viral hemorrhagic fevers role of t cells, cytokines and antibody in dengue fever and dengue haemorrhagic fever activated peripheral 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cytokine responses of dengue-specific cd + t cells to heterologous serotypes dengue-specific t cell responses in peripheral blood mononuclear cells obtained prior to secondary dengue virus infections in thai schoolchildren alternative activation of macrophages effects of granulocyte-monocyte colony-stimulating factor (gm-csf) on expression of adhesion molecules and production of cytokines in blood monocytes and ovarian cancer-associated macrophages mcp- , a highly expressed chemokine in dengue haemorrhagic fever/dengue shock syndrome patients, may cause permeability change, possibly through reduced tight junctions of vascular endothelium cells il release, tight junction and cytoskeleton dynamic reorganization conducive to permeability increase are induced by dengue virus infection of microvascular endothelial monolayers predictors of spontaneous bleeding in dengue dengue and dengue hemorrhagic fever, brazil of cascades and perfect storms: the immunopathogenesis of dengue haemorrhagic fever-dengue shock syndrome (dhf/dss) this work was supported by fundação oswaldo cruz (pdtsp-dengue), decict/conselho de desenvolvimento científico e tecnológico (cnpq), fundação de amparo à pesquisa do estado do rio de janeiro (faperj). the authors thank the program for technological development in tools for health-pdtis-fiocruz for use of its luminex facilities. we acknowledge in memoriam dr. jussara p. nascimento for her constant encouragement, dr. marcelo a. pinto for suggestions and dr. andrea schwager for the manuscript revision. the authors declare that they have no competing interests. fab and ogc contributed equally to the study. fab contributed to the study conception and design, carried out clinical studies, helped in data analysis and in drafting the manuscript. ogc performed data and statistical analysis. smoz carried out the clinical studies. efa and carried out the luminex immunoassays. ela collected and stored samples and patient data and helped in the luminex immunoassays. rmrn was responsible for the confirmatory diagnostics. ptb conceived the study and design and helped to draft the manuscript. cfk conceived the study and design, participated in data and statistical analysis and drafted the manuscript. all authors read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -te jsd authors: liu, qiyong; liu, xiaodong; jiang, baofa; yang, weizhong title: forecasting incidence of hemorrhagic fever with renal syndrome in china using arima model date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: te jsd background: china is a country that is most seriously affected by hemorrhagic fever with renal syndrome (hfrs) with % of hfrs cases reported globally. at present, hfrs is getting worse with increasing cases and natural foci in china. therefore, there is an urgent need for monitoring and predicting hfrs incidence to make the control of hfrs more effective. in this study, we applied a stochastic autoregressive integrated moving average (arima) model with the objective of monitoring and short-term forecasting hfrs incidence in china. methods: chinese hfrs data from to were used to fit arima model. akaike information criterion (aic) and ljung-box test were used to evaluate the constructed models. subsequently, the fitted arima model was applied to obtain the fitted hfrs incidence from to and contrast with corresponding observed values. to assess the validity of the proposed model, the mean absolute percentage error (mape) between the observed and fitted hfrs incidence ( - ) was calculated. finally, the fitted arima model was used to forecast the incidence of hfrs of the years to . all analyses were performed using sas . with a significant level of p < . . results: the goodness-of-fit test of the optimum arima ( , , ) model showed non-significant autocorrelations in the residuals of the model (ljung-box q statistic = . ,p = . ). the fitted values made by arima ( , , ) model for years - closely followed the observed values for the same years, with a mean absolute percentage error (mape) of . %. the forecast values from to were . , . , and . per , population, respectively. conclusion: arima models applied to historical hfrs incidence data are an important tool for hfrs surveillance in china. this study shows that accurate forecasting of the hfrs incidence is possible using an arima model. if predicted values from this study are accurate, china can expect a rise in hfrs incidence. hemorrhagic fever with renal syndrome (hfrs), or epidemic hemorrhagic fever (ehf) is an acute viral syndrome caused by infection with one of hantaviruses. hfrs is an important infectious disease in developing countries. in china, hfrs is caused mainly by types of hantaviruses, hantaan virus (htnv) and seoul virus (seov), each of which has coevolved with a distinct rodent host. htnv is associated with apodemus agrarius, whereas seov, which causes a less severe form of hfrs, is associated with rattus norvegicus [ ] . in hantavirus -endemic areas, hfrs is most common among farmers and others who may have close contact with excreta of infected rodents [ , ] . in mainland china, hfrs remains a serious public health problem with approximately , - , human cases reported annually, approximately % of the total cases worldwide [ ] [ ] [ ] . currently, hfrs is endemic in of provinces in mainland china [ , ] . in response to the spread of hfrs in china, the chinese center for disease control and prevention designed a surveillance system for hfrs and created educational programs for the general public. however, the impact of control efforts remains difficult to measure due to the inherent complexities of hfrs as a disease: multiple viral strains with identified genetic polymorphisms, complex disease manifestation, diverse animal reservoirs, and multiple routes of transmission [ ] . infectious diseases have certain characteristic features that lead themselves to modeling, such as: speed of pathogen variation, accumulation of susceptible hosts, and environmental indices [ ] . thus, epidemic modeling and forecasting can be essential tools to prevent and control hfrs. recently, statistical methods including linear regression [ ] [ ] [ ] , correlation coefficients [ ] , grey swing model [ ] , back propagation artificial neural network model [ ] have been used for prediction of hfrs incidence. the variation of hfrs incidence, which is influenced and constrained by diversified factors, is characterized by tendency and randomicity. these statistical tools are inappropriate for analyzing the randomicity of hfrs. autoregressive integrated moving average (arima) models, which take into account changing trends, periodic changes, and random disturbances in time series, are very useful in modeling the temporal dependence structure of a time series. in epidemiology, arima models have been successfully applied to predict the incidence of infectious diseases, such as influenza mortality [ ] , malaria incidence [ ] , as well as other infectious diseases [ , ] . this study aimed to develop a univariate time series model for the hfrs incidence; specifically, a stochastic arima model, for short-term forecasting of hfrs incidence (per , population) in china. chinese hfrs incidence data from to was obtained from the chinese center for disease control and prevention. all hfrs cases were initially diagnosed by clinical symptoms. patient blood samples were also collected and sent to local centers for disease control and prevention (cdc) laboratories for serological confirmation. finally, data were collected by case number according to the sampling results. there might be admission rate bias in the disease report, but this has been reduced as much as possible. in china, hfrs is a nationally notifiable disease and hospital physicians must report every case of hfrs to the local health authority within hours. local health authorities later report monthly hfrs case totals to higher the national level cdc for surveillance purposes. due to mandatory reporting, it is believed that the degree of compliance in disease notification over the study period was consistent. we used the box-jenkins approach to arima (p, d, q) modeling of time series [ ] . this model-building process is designed to take advantage of associations in the sequentially lagged relationships that usually exist in periodically collected data [ ] . the following were the parameters selected when fitting the arima model: p, the order of autoregression; d, the degree of difference; q, the order of moving average. the annual data used in this study did not show seasonal pattern, so the series was differenced at the nonseasonal level to induce stationarity. autocorrelation function (acf) graph and partial autocorrelation function (pacf) graph were used to identify the order of moving average (ma) and autoregressive (ar) terms included in the arima model. estimates of the model's parameters were obtained by the conditional least squares method. diagnostic checking including residual analysis and the akaike information criterion (aic) was used to compare the goodness-of-fit among arima models. the ljung-box test was used to measure the acf of the residuals. in addition, we used the mean absolute percentage error (mape) and fitting effect diagram to assess forecast accuracy. the present study was reviewed by the research institutional review board of shandong university and the china cdc, and found that utilization of disease surveillance and meteorological data did not require oversight by an ethics committee. from to , the hfrs incidence in china rose regularly with a peak in of . cases per one hundred thousand population. after , the incidence descended sharply with a dramatic fluctuation until ( figure ). the lowest incidence could be seen in , . per one hundred thousand. according figure , the series showed a non-stationary mean, so we stabilized the mean of hfrs incidence by taking both second and third order differences. all further statistical procedures were performed on the transformed hfrs incidence. based on the distribution characteristics, we conducted five models, arima( , , ), arima( , , ), arima( , , ), arima( , , ), and arima ( , , ) . of all the models tested, the arima ( , , ) model was the best fit for the data ( table ). the transformation series by taking third-order differences is shown in figure . the plots of acf and pacf ( figure ) described the temporal dependence structure in hfrs incidence. the slow decay in the pacf, associated with a acf cutoff at lag suggested a ma(q = ). the parameter estimates for the optimum arima( , , ) model are shown in table time series analysis of surveillance data on incidence of various infections is very helpful in developing hypotheses to explain and anticipate the dynamics of the observed phenomena and subsequently in the establishment of a quality control system and reallocation of resources [ ] . arima model is one of the most widely used time-series forecasting techniques because of its structured modeling basis and acceptable forecasting performance [ ] . in this paper, we applied an arima (p, d, q) model to analyze the surveillance data of hfrs in china. disease monitoring by public health department entails ongoing data collecting, processing, and updating. however, the national level china cdc is the appropriate level of organization for the implementation of an arima predictive model, because reported data is continually received and updated. we found that model predictions are further improved by the assured availability of the health department data. in this study, we have obtained an arima model that closely fits hfrs incidence in china. the autoregression and moving average parameters of our model imply the incidence of hfrs in a month can be estimated by the residual occurring one month prior. according to the results above, the conducted model is reliable with a high validity. once a satisfactory model has been obtained, it can be used to forecast expected numbers of cases for a given number of future time intervals [ ] . thus, the fitted arima( , , ) model can be used to predict the next three years' hfrs incidence in china. the forecast results suggest that the hfrs incidence in china will experience a slight growth in the next three years ( ) ( ) ( ) . a rise in the number of hfrs incidence may also result from an increase in the number and size of natural foci [ ] , climate change, especially the increase of mean temperature [ , ] . therefore, knowledge of hfrs forecasts is necessary to prompt health departments to strengthen surveillance systems and reallocate resources in anticipation of increasing hfrs incidence. several studies have used arima model to fit and predict changing trends in infectious disease. luz et al applied an arima( , , )×( , , ) model to predict dengue incidence in rio de janeiro [ ] and found that arima models were useful tools for monitoring dengue incidence. earnest et al indicated that arima models provided useful tools for administrators and clinicians in planning for real-time bed capacity during infectious diseases outbreaks such as sars [ ] . li et al have applied an arima model to monthly incidence of hfrs in linyi city, china to predict hfrs incidence, and found that the arima model could be used to predict hfrs incidence with high predictive precision in the short-term [ ] . in the present study, we further confirmed the consensus that arima model is a useful tool in monitoring and predicting changing trends in infectious diseases. to the best of our knowledge, this is the first study to apply arima model to fit the hfrs incidence in china with as many as observations at year level. some previous studies [ , ] in china also used arima model to fit and forecast hfrs incidence of some regions, but they had the same problem that the number of observations was not enough, which led to the instability of their forecast results. in order to conduct a stable and effective arima model, we have to collect at least observations [ ] . thus, parameter estimates of the fitted model would be more robust. the longer the series, the better; however, the series should not extend so far into the past as to include periods during which a different case definition was applied or in which any other reporting artifact resulted in a mean number of cases per interval that differs from the mean of recent intervals. as mentioned above, for adequate arima modeling, a time series should be stationary with respect to mean and variance. if the mean increases or decreases over time, or if the variance does, the series may need to be transformed to make it stationary, before being modeled. otherwise, the prediction effect of the model will be poor. in order to improve the model, updating the forecasts is very important. a model without seasonal terms will need to be updated frequently. confidence intervals that widen rapidly as time increase from the starting point of the forecasts also indicate a model that needs frequent updating. generally speaking, there are two ways to implement the updating. the model can be reapplied to the original series with extra observations added at the end to give forecasts based on a later starting point. alternatively, a new model can be fitted to the longer series. this is probably preferable, since fitting a model is quick, especially when the old model is used as a guide, and it makes better use of the additional observations. some limitations of this study also need to be taken into account when interpreting the results. in this study, the interval of hfrs incidence is one year, so we could not analyze its seasonal characteristic. in further study, we would use monthly data to predict hfrs incidence in order to get seasonal pattern and higher predictive precision. in addition, the data are from a passive surveillance system, the possible biases in disease reporting and potential underreporting of hfrs cases might influence the precision of our analysis. there is an urgent need for monitoring and predicting hfrs incidence to reduce the substantial morbidity and mortality caused by this disease [ ] . arima models applied to historical hfrs incidence data are an important tool for hfrs surveillance. accurate forecasting of the incidence of hfrs is possible. our modeling approach can be used to monitor and predict hfrs incidence in china. the arima model could be used to optimize hfrs prevention by providing estimates on hfrs incidence trends in china. spatiotemporal dynamics of hemorrhagic fever with renal syndrome puumala virus infections in finland:increased occupational risk for farmers association of intraspecific wounding with hantaviral infection in wild rats the epidemic characteristics and preventive measures of hemorrhagic fever with renal syndrome in china emerging viruses: the case 'hantavirus spatial analysis of hemorrhagic fever with renal syndrome in china landscape elements and hantaan virus-related hemorrhagic fever with renal syndrome, people's republic of china the epidemiological research status and problems and prospects of hemorrhagic fever with renal syndrome in china forecasting model for the incidence of hepatitis a based on artificial neural network applying linear regression statistical method to predict the epidemic of hemorrhagic fever with renal syndrome predicting high risk for human hantavirus infections seasonal rainfall variability, the incidence of hemorrhagic fever with renal syndrome, and prediction of the disease in low-lying areas of china relating increasing hantavirus incidences to the changing climate: the mast connection appling grey swing model to predict the incidence trend of hemorrhagic fever with renal syndrome in shenyang prediction for incidence of hemorrhagic fever with renal syndrome with back propagation artificial neural network model influenza and the winter increase in mortality in the united states, - modelling malaria incidence with environmental dependency in a locality of sudanese savannah area time series analysis of dengue incidence in rio de janeiro, brazil applications of multiple seasonal autoregressive integrated moving average(arima) model on predictive incidence of tuberculosis time series analysis: forecasting and control an autoregressive integrated moving average model for short-term prediction of hepatitis c virus seropositivity among male volunteer blood donors in karachi use of poisson regression and time series analysis for detecting changes over time in rates of child injury following a prevention program time series forecasts of the construction labour market in hong kong: the box-jenkins approach. construction management and economics use of time-series analysis in infectious disease surveillance epidemiological analysis of hemorrhagic fever with renal syndrome in china from climatic, reservoir and occupational variables and the transmission of hemorrhagic fever with renal syndrome in china relating increasing hantavirus incidences to the changing climate: the mast connection using autoregressive integrated moving average(arima) models to predict and monitor the number of beds occupied during a sars outbreak in a tertiary hospital in singapore a time series model in incidence forecasting of hemorrhagic fever with renal syndrome comparison of gm( , ) gray model and arima model in forecasting the incidence of hemorrhagic fever with renal syndrome fitting research on arma model in the prediction of incidence trend of hemorrhagic fever with renal syndrome gao hx compilation: sas system·sas/ets software manual surveillance of hemorrhagic fever with renal syndrome in china pre-publication history the pre-publication history for this paper can be accessed here forecasting incidence of hemorrhagic fever with renal syndrome in china using arima model authors' contributions ql, xl, bj and wy conceived the study, undertook statistical analysis and drafted the manuscript. xl and bj assisted with data collection and statistical analysis. all authors contributed to the writing of the manuscript and approved the submitted version of the manuscript. the authors declare that they have no competing interests. key: cord- -i aq b authors: chung, grace ty; chiu, rossa wk; cheung, jo lk; jin, yongjie; chim, stephen sc; chan, paul ks; lo, ym dennis title: a simple and rapid approach for screening of sars-coronavirus genotypes: an evaluation study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: i aq b background: the severe acute respiratory syndrome (sars) was a newly emerged infectious disease which caused a global epidemic in – . sequence analysis of sars-coronavirus isolates revealed that specific genotypes predominated at different periods of the epidemic. this information can be used as a footprint for tracing the epidemiology of infections and monitor viral evolution. however, direct sequencing analysis of a large number of clinical samples is cumbersome and time consuming. we present here a simple and rapid assay for the screening of sars-coronavirus genotypes based on the use of fluorogenic oligonucleotide probes for allelic discrimination. methods: thirty sars patients were recruited. allelic discrimination assays were developed based on the use of fluorogenic oligonucleotide probes (taqman). genotyping of the sars-coronavirus isolates obtained from these patients were carried out by the allelic discrimination assays and confirmed by direct sequencing. results: genotyping based on the allelic discrimination assays were fully concordant with direct sequencing. all of the sars-coronavirus genotypes studied were characteristic of genotypes previously documented to be associated with the latter part of the epidemic. seven of the isolates contained a previously reported major deletion but in patients not epidemiologically related to the previously studied cohort. conclusion: we have developed a simple and accurate method for the characterization and screening of sars-coronavirus genotypes. it is a promising tool for the study of epidemiological relationships between documented cases during an outbreak. the severe acute respiratory syndrome (sars) is a recently emerged infectious disease which led to a global epidemic between and . a novel coronavirus (sars-cov) was identified as the causative agent [ ] . the genomic sequence of the sars-cov was promptly characterized [ , ] . thereafter, studies had focused on the early detection of sars-cov and the development of diagnostic tools [ ] [ ] [ ] . systematic analysis of the sars-cov sequence information have demonstrated that characteristic viral genotypes predominated at certain periods during the course of the outbreak [ ] [ ] [ ] [ ] . furthermore, characterization of the viral sequences have been shown to be a useful tool for confirming epidemiological associations between infected individuals as suspected from conventional epidemiological investigations [ ] [ ] [ ] . in-depth analysis of the available sequence data on sars-cov also revealed that the viral isolates could be readily subclassified into several major genotypes based on nucleotide variations at specific genomic positions [ , ] . in a large-scale phylogenetic analysis of sars-cov sequences [ ] , a -nucleotide motif at the gz [genbank :ay ] reference nucleotide residues , , , , , , , , and , was identified to be most useful for distinguishing the major sars-cov genotypes. these major viral genotypes predominated at different periods of the epidemic [ ] . thus, it is evident that viral sequence and molecular epidemiological data provide valuable information and tools for our combat against infectious diseases. however, direct sequencing of viral isolates from a large number of clinical samples is cumbersome and time consuming. therefore, a rapid system for the characterization and screening of viral genotypes, such as for sars-cov, would potentially be useful. in this study, we demonstrate the feasibility of the adoption of allelic discrimination assays based on the use of fluorogenic oligonucleotide probes for the genotyping of sars-cov isolates. viral culture isolates from sars patients who were admitted to the hospitals of the new territories east cluster of hong kong during the sars epidemic were retrieved. the study was approved by the institutional review board. sars was confirmed in all cases either by positive reverse transcription-polymerase chain reaction (rt-pcr) detection of sars-cov rna in clinical specimens or documented seroconversion. we focused on the development of allelic discrimination assays for the five previously described characteristic single nucleotide variations (snv) [ ] . rna was extracted from viral isolates cultured from sars patients' clinical specimens using the qiaamp viral rna mini kit (qiagen, valencia, ca, usa), according to manufacturer's instructions. eleven microliters of the extracted viral rna was reverse transcribed by superscript iii (invitrogen, carlsbad, ca, usa) with random hexamer according to manufacturer's instructions. genotyping of the five snvs was determined using taqman (applied biosystems, foster city, ca, usa) allelic discrimination assays on an abi prism ht sequence detection system (applied biosystems). each assay consisted of two allele-specific minor groove binding probes associated with either, -carboxyfluorescein (fam) or vic™ as the fluorescent label, for the discrimination of the two respective alleles at each snv locus. one assay was designed for each of the snvs. the primer and probe sequences, designed using the primer express . software (applied biosystems) are listed in table . the probes were designed such that the discriminatory nucleotide is placed close to the middle portion of the oligonucleotide. the assays were set up according to the manufacturer's instructions (taqman core pcr kit; applied biosystems) in a reaction volume of µl. each reaction consists of x buffer a, mm mgcl , . µm datp, . µm dctp, . µm dgtp and . µm dutp, nm forward and reverse primers, nm of each fluorescent probe, . u ung, . u taq polymerase and . µl of cdna as template. the thermal profile consists of an initial incubation at °c for min, and then a denaturation period at °c for min, followed by cycles of denaturation at °c for s, and min of combined annealing and extension at °c. the genotypes were scored with the sds . software. all viral sequences were confirmed by direct sequencing. rt-pcr was performed to specifically amplify genomic segments of sars-cov encompassing each of the snvs using primers and protocols previously described [ ] . the dna of each amplicon was sequenced by the dideoxy terminator method on an automated dna sequencer ( genetic analyzer, applied biosystems) based on capillary electrophoresis. taqman allelic discrimination assays for the snvs were first tested on synthetic templates (sigma genosys, australia) ( table ) and verified using viral isolates, cuhk-w [genbank :ay ] and cuhk-su [genbank :ay ]. cuhk-w is a sars-cov isolate with a g:a:c:t:c motif at the gz reference nucleotide residues , , , , , , , , and , , characteristic of sars-cov strains isolated before worldwide dissemination of sars [ , ] . on the other hand, cuhk-su demonstrates a t:g:t:t:t motif which is characteristic of sars-cov strains isolated after global spread was evident. as evident from figure , the newly developed allelic discrimination assays were able to differentiate the viral isolates and genotype each snv correctly (table ) . following initial development and optimization, the allelic discrimination assays were used to genotype sars-cov in clinical samples. we were able to successfully determine the sars-cov genotypes in all samples. genotypes of virus isolates at the snv positions are shown in table . sars-cov from all but seven cases showed the t:g:t:t:t motif resembling that of the cuhk- the sars-cov genotypes isolated from the patients were also confirmed by direct sequencing. the sequencing results were fully concordant with that based on the allelic discrimination assays at all the snvs. the seven samples which gave no allelic signal by the allelic discrimination assay at snv , showed a shortened amplicon encompassing the region. direct sequencing of this short amplicon revealed a deletion of nt identical to a sars-cov deletion variant previously reported by our group [ , ] . this deletion variant was first isolated from a discrete cohort of epidemiologically related sars patients [ ] . in the previous cohort of patients, the origin of the deletion variant was traceable to mid-april in two patients residing in an estate, t, in hong kong with subsequent spread predominantly at the north district hospital, hong kong [ ] . to further determine if the newly identified cases were epidemiologically related to the original patient cohort, the case histories were reviewed. the seven patients had fever onset between april to , which predated the disease onset dates of all cases in allelic discrimination plot of cuhk-su and cuhk-w figure allelic discrimination plot of cuhk-su and cuhk-w . allelic discrimination at each of the studied snvs described in the text as demonstrated using the synthetic templates and cdna from cuhk-su and cuhk-w vero cell culture isolates is presented in the successive plots. ( ) synthetic template for the fam-labeled allele, ( ) synthetic template for the vic-labeled allele, ( ) cuhk-w , ( ) cuhk-su , ( ) no template control. it is interesting to note that this study provided additional anecdotal evidence pointing to estate t as a propagation site for the deletion variant. in addition, we were able to trace the emergence of this deletion variant to early april , weeks before the first appearance reported previously [ ] . our study has clearly demonstrated the feasibility of using allelic discrimination assays as a method for genetic characterization of sars-cov genotypes in patients. it is particularly useful when there is already extensive sequence information. direct sequencing is still the gold standard for identifying new sequence variations when new agents of infectious disease continue to emerge and old ones reemerge. once the variations have been identified, allelic discrimination assay is more efficient and suitable for large-scale population investigations. a recent study illustrated the use of mass spectrometry-based technology in characterizing sars sequence variations [ ] . however, this method requires post-pcr manipulations and the availability of specialized equipment. on the other hand, allelic discrimination assays have been widely used in the study of associations between single nucleotide polymorphisms and diseases such as cancers [ ] and rheumatoid arthritis [ ] . the validity of the approach for single nucleotide polymorphism genotyping has been previously demonstrated [ ] [ ] [ ] . thus, this study further extended the usefulness of allelic discrimination approach based on fluorogenic oligonucleotide probes. the approach provides a rapid and simple means to accurate genotype screening, making it ideal for epidemiological investigations. we have evaluated a rapid approach for characterizing sars-cov genotypes. the assay is simple, easy to perform and reproducible. it can therefore be used as an efficient means to screen for virus genotypes and track the transmission of a particular viral strain in times of epidemics. incidentally, we identified a previously reported deletion variant of the sars-cov in a new cohort of patients and traced the emergence of this variant to an earlier date than previously reported. allele t allele c aetiology: koch's postulates fulfilled for sars virus genotype of sars-cov culture isolates from patients determined by taqman allelic discrimination assays petric m, skowronski dm characterization of a novel coronavirus associated with severe acute respiratory syndrome quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome rapid and sensitive detection of severe acute respiratory syndrome coronavirus by rolling circle amplification early diagnosis of sars coronavirus infection by real time rt-pcr genomic sequencing of a sars coronavirus isolate that predated the metropole hotel case cluster in hong kong molecular evolution of the sars coronavirus during the course of the sars epidemic in china tracing sars-coronavirus variant with large genomic deletion molecular epidemiology of the novel coronavirus that causes severe acute respiratory syndrome molecular epidemiology of sars--from amoy gardens to taiwan comparative full-length genome sequence analysis of sars coronavirus isolates and common mutations associated with putative origins of infection coronavirus genomic-sequence variations and the epidemiology of the severe acute respiratory syndrome sars transmission pattern in singapore reassessed by viral sequence variation analysis common brca variants and modification of breast and ovarian cancer risk in brca mutation carriers a biologically important single nucleotide polymorphism within the toll-like receptor- gene is not associated with rheumatoid arthritis real-time pcr for simultaneous detection and genotyping of bovine viral diarrhea virus detection and genotyping of varicella-zoster virus by taq-man allelic discrimination real-time pcr detection and typing of herpes simplex virus (hsv) in mucocutaneous samples by taqman pcr targeting a gb segment homologous for hsv types and the work is supported by the research fund for the control of infectious disease (rfcid) from the health, welfare and food bureau of the hong kong government sar. ymdl, rwkc and sscc have filed patent applications on aspects concerning the genomics and detection of the sars-coronavirus. gtyc, rwkc and ymdl have contributed in the preparation of the manuscript and the overall study design. gtyc, rwkc and yj have contributed in the assay designs, data analysis and conducting the genotyping experiments. rwkc, sscc, jlkc and pksc have contributed in the collection and analysis of clinical data from the patients. jlkc and pksc provided the viral samples. t t t tc t g t t nd t c t g t t t tc t g t t nd t c t g t t t t c t g t t t t c t g t t t t c t g t t t t c t g t t t t c t g t t t t c t g t t t t c t g t t t tc t g t t nd tc t g t t nd t c t g t t t tc t g t t nd t c t g t t t tc t g t t nd tc t g t t key: cord- -zfpg dgj authors: zhang, xu-sheng; de angelis, daniela title: construction of the influenza a virus transmission tree in a college-based population: co-transmission and interactions between influenza a viruses date: - - journal: bmc infect dis doi: . /s - - -x sha: doc_id: cord_uid: zfpg dgj background: co-infection of different influenza a viruses is known to occur but how viruses interact within co-infection remains unknown. an outbreak in a college campus during the pandemic involved two subtypes of influenza a: persons infected with pandemic a/h n ; persons infected with seasonal a/h n viruses; and persons infected with both at the same time (co-infection). this provides data to analyse the possible interaction between influenza a viruses within co-infection. methods: we extend a statistical inference method designed for outbreaks caused by one virus to that caused by two viruses. the method uses knowledge of which subtype each case is infected with (and whether they were co-infected), contact information and symptom onset date of each case in the influenza outbreak. we then apply it to construct the most likely transmission tree during the outbreak in the college campus. results: analysis of the constructed transmission tree shows that the simultaneous presence of the two influenza viruses increases the infectivity and the transmissibility of a/h n virus but whether it changes the infectivity of a/h n is unclear. the estimation also shows that co-transmission of both subtypes from co-infection is low and therefore co-infection cannot be sustained on its own. conclusions: this study suggests that influenza a viruses within co-infected patients can interact in some ways rather than transmit independently, and this can enhance the spread of influenza a virus infection. electronic supplementary material: the online version of this article (doi: . /s - - -x) contains supplementary material, which is available to authorized users. co-circulation of multiple types and subtypes of influenza virus has been commonly observed in human populations [ , ] . with advanced molecular techniques [ , ] , it is now becoming possible to rapidly detect different subtypes or strains of a disease agent within infected patients. there is accumulating evidence to show that the phenomenon that multiple subtypes of influenza a virus infect an individual simultaneously (i.e. co-infection) is not as rare as we previously thought [ ] [ ] [ ] [ ] [ ] [ ] . hence it is interesting to know how the simultaneous presence of two strains alters, compared to singly infected individuals, the transmissibility of each subtype and of both subtypes together. some observations of transmission involved with co-infected individuals have been reported [ , ] . one important observation reported is individuals co-infected with pandemic a/h n and seasonal a/h n influenza viruses within one outbreak at a college campus in beijing, china during the pandemic [ ] . liu et al. [ ] provides direct evidence that individuals co-infected with different subtypes can transmit viruses separately or simultaneously and provides detailed data for us to quantify the interactions between virus strains. the transmission characteristics can be directly estimated from the transmission tree; however, the difficulty for constructing the transmission tree is that some contacts were missed. fortunately, recent development in statistical inference [ , ] allows us to construct the transmission tree of a single influenza a virus from such a partial contact network. in this short report we extend this inference method to construct the transmission tree that includes two influenza a viruses and their co-infection. from the constructed tree we estimate the impact of co-infection on transmission. we use two parameters to characterise the impact: the co-transmission rate and an interaction parameter. here we define the former as the rate at which two strains simultaneously transmit from doubly infected individuals to susceptible individuals; and the latter as the ratio of transmissibility of each single virus from coinfected individuals to the transmissibility of each single virus from singly infected individuals. here we briefly summarize the data collection method and data of liu et al. [ ] . investigations were conducted on all influenza like illness (ili) cases identified during the outbreak. epidemiological, clinical and contact tracing data were collected by interviewing patients and retrieving medical records. viruses were identified by reverse-transcription polymerase chain reaction assays followed by sequence analysis. the heamagglutination inhibition tests were used to detect antibodies to both viruses. the outbreak is reported to have taken place within three buildings (two dormitories and one college clinic). no other cases at the college were reported. buildings and (with a total membership of and persons, respectively) are next to each other and there is restricted access between the two and to the wider community. forty five ili cases were reported from august to september and forty (n = ) had laboratory-confirmed influenza a infection. three different types of infection were reported: infected with pandemic a/h n virus, infected with seasonal a/ h n virus and six co-infected with both influenza a viruses. in their sequences no substantial differences were observed between patients with mixed and single infections in either pandemic a/h n or seasonal a/h n virus. the clinical features were similar for patients with different infections and the six co-infected patients showed no more severe symptoms than the singly infected patients. contacts between infected people are shown in fig. of liu et al. [ ] but this only extends to the contact network within one dormitory. the 'index' case with pandemic influenza a/h n infection was a college student whose symptoms first occurred on august, days after his returning to college. except for the index case, all patients with a/h n infection had not left the campus during the previous week. in contrast, the source of seasonal a/h n virus infection cannot be determined exactly although available data indicate that a/h n virus might have been prevailing in the college when the pandemic h n virus was introduced. before the isolation of cases and the initiation of prophylaxis among the campus population ( september ), several patients visited the college clinic and the mixing between students of different dormitories was not frequent in comparison to the mixing between students within each dormitory. for a fully traced transmission tree (i.e., the information of the infector v and time of symptom onset t are collected), the infector v(i) for each case i (except the index case) and the duration between symptom onset of case i and symptom onset of its infector v(i): t i -t v(i) should be known. from these it is straightforward to estimate the generation interval distribution and transmissibility of infection. in reality, however, it is rare and difficult to record all the information. based on the partially known contact tracing data and dates of symptom onset, hens et al. [ ] illustrated an inference method to reconstruct the most likely transmission tree that involved with only one virus. here we further develop it to a transmission tree during an outbreak that involves with two viruses of similar epidemiological characteristics. in general, a possible transmission tree can be described by p ij (v,w,φ;θ), the probability that case j is the infector of case i, given the duration between symptom onset of case i and case j, given the information on the possible infector v and the known contacts w, and given the types φ of infection of both cases. following hens et al [ ] , its total log-likelihood is given by: the sum runs through all the non-zero p ij . here g(Δt |θ) denotes the probability density of the generation interval distribution of influenza infection, with θ representing the set of parameters that characterise the probability density distribution. different distributions such as gamma, lognormal and weibull can be used to describe the distribution of generation intervals [ ] . here we assume it follows a weibull distribution: the distribution has two parameters (i.e., θ = {a, b}): scale parameter a and shape parameter b, such that the mean is compared with outbreaks that involved one virus and one transmission process [ ] , this outbreak [ ] involved two viruses and five possible transmission processes: from a/h n to a/h n ; from a/h n to a/h n ; from coinfection to a/h n ; from co-infection to a/h n and from co-infection to co-infection. however, the data given in liu et al. [ ] only provide the relevant information for three transmission processes: from a/h n to a/h n ; from co-infection to a/h n ; and from co-infection to co-infection. the simple calculations show that the mean generation intervals (and their standard deviations) for the three transmission processes are . ( . ), . ( . ), . ( . ) days, respectively. in this data there is no evidence that they differ. as no data is available for the transmission processes that were involved with a/h n [ ] , it is difficult to estimate their generation intervals and judge how they differ from those generation intervals involved with pandemic a/h n . nevertheless, a recent systematic review [ ] indicates that the generation interval of pandemic a/h n virus was similar to that of the seasonal flu. further, as observed by liu et al. [ ] , the three different types of infections have similar epidemiological characteristics. in view of these, the same generation interval distribution is assumed for the three types of infection. the probability that case i is infected by case j, p ij , can be calculated as the probability of observing the duration between the symptom onsets in cases i and j, g(t i -t j |θ), times the probability of a potentially infectious contact between i and j, π ij , normalized by the probability of i being infected by any other case k: the probability of a potentially infectious contact between i and j, π ij , is based on the contact information (v,w) collected during the outbreak and the types φ of infection of both cases i and j. to distinguish different types of infection and to reflect the fact that there is only limited mixing between students in building and building , we define the following, ψ ij ¼ if case i and case j reside in the same dormitory and are the same type of infection or if case j is a co−infection; ψ ij ¼ w if case i and case j reside in different dormitories and are the same type of infection or if case j is a co−infection; the variable w defines the extent of contact rates between students in two dormitories in relation to contact rates within dormitories: w = implies that contacts between dormitories are forbidden and w = suggests that contacts between dormitories are the same as that within each dormitory. student access to the college clinic was not restricted. the original numberings of patients in fig. of liu et al. [ ] are given in accordance with the order of symptom onset within each building. for the convenience of our analysis, the forty patients have been re-indexed in the order of symptom onset as , , …, (see fig. ). if cases i and j form a likely transmission pair (i.e., v(i) = j) and there is only one possible infector, π ij (v,w,φ) = ψ ij and p ij (v,w,φ;θ) = . if the outbreak investigation reveals that case i is not the index case and does not contact any of the i- cases that developed symptoms before case i, the probability of a potentially infectious contact is π ij (v,w,φ) = ψ ij /η i- here η i- is the 'effective' number of infections that developed symptoms before case i and are of the same type of infection as case i or co-infection, and is calculated as given the contact information (v,w), the most likely transmission tree can be obtained by finding the values of parameters θ = {a, b} that maximize the total loglikelihood ( ). the downhill simplex method [ ] was used to locate the maximum-likelihood estimate (mle), θ , of the parameters. we also used the mcmc method to sample the values of parameters θ and find their medians, . percentile and . percentile. from the probabilities that case i is infected by case j: {p i , …, p ni }, we can sample a transmission tree in which all cases are connected and case is the index case ( fig. ) and further estimate transmissibility. the case reproduction number is the average number of secondary cases generated by primary cases, which measures the transmissibility of infection. the overall casereproduction number on day t, r t , can be estimated by summing over all these infectious contacts: we can further estimate the infection type-specific reproductive numbers when both case i and case j are of the same type of infection: here the symbol δ ij,type is defined as: δ ij,type = if both cases i and j are of the infection type; δ ij,type = otherwise. to characterise the interaction between two types of virus within co-infected individuals, we further estimate the reproductive numbers of a/h n and a/ h n due to co-infected individuals as: here Δ ij,type is defined as: Δ ij,type = if infector case j is co-infected while the infectee case i is singly infected with type type; otherwise Δ ij,type = . the strain interactions within co-infections are estimated as: here ϕ measures the effect on infectivity of a/h n within co-infections and ϕ measures the effect on infectivity of a/h n within co-infections. fig. one plausible transmission tree. the tree was constructed under the assumption of limited mixing rate between students in two dormitories in relation to within building mixing rates (w = %). in the construction, the other possible sources of seasonal a/h n virus (e.g., the two question marked boxes above building and clinic in fig. of liu et al. [ ] ) have been ignored. the thick arrows represent transmissions from liu et al. [ ] while the thin arrows display one of the most likely transmissions data that were used in the analysis of this study were extracted from a previous study [ ] and hence did not require human resource ethics committee approval. the data shown in fig. of liu et al. [ ] indicates that the index case is infected with pandemic a/h n virus. among the six cases that the index case infects, three patients (i.e., case , , and ) are co-infected with both a/h n and a/h n . it was assumed that seasonal a/ h n virus was endemic [ ] , however, the infectious contacts with a/h n of these three co-infected cases were unknown. several possible scenarios are possible: cases , and were exposed to pandemic a/h n virus when they were still infectious with a/h n ; cases , and were further exposed to seasonal a/h n virus soon after they had acquired pandemic a/h n virus from the index case; the three cases were infected simultaneously with both a/h n and a/h n viruses from the index case who was actually co-infected before contact with cases , and but was incorrectly typed as a single infection with pandemic a/h n virus. for simplicity, we use all the contact information for cases within building , especially the pathways from the index case to the three co-infections, ignoring the possible pathways for transmitting background endemic seasonal a/h n virus. the maximum likelihood estimates of the infective contact probabilities p ij (v,w,φ;θ) are listed in table from which one sample transmission tree is shown in fig. . the mles of the generation interval distribution g(Δt|θ) and the time course of the mean case reproductive number r t are shown in fig. . the generation interval has a mean of . days and a standard deviation (sd) of . days. the mean case reproductive number over the whole outbreak is . with sd = . . before the isolation of cases and the initiation of prophylaxis among the campus population occurred ( september ), the reproductive number is estimated to be . with sd = . ; after this it declines to . with sd = . . this clearly shows the effectiveness of isolation and prophylaxis. to measure the interactions between two influenza a viruses, we first estimated the values of infection typespecific reproductive numbers before the initiation of isolation and prophylaxis occurred. because most transmissions that involve a/h n and co-infections are known, the estimates of the infection type-specific reproductive numbers are stable at r e = . and r e c = . , and the reproductive number of a/h n due to co-infection is r e,c = . . in contrast, transmissions that involve a/h n were missing and so estimates of their reproductive numbers show some uncertainty: r e has a mean . and % confidence interval of [ . , . ], and the reproductive number of a/h n due to coinfection is r e,c = . [ . , . ]. although the estimate of r e for a/h n is in agreement with the usual estimates [ , ] , that of r e for a/h n is much smaller than other estimates which range from . to . [ ] . the likely reason for this difference lies in the fact that pandemic a/h n is a novel virus while a/ h n is an endemic seasonal virus in the study region [ ] so some pre-existing immunity against a/h n fig. constructed transmission tree of influenza a virus: a the relative frequency of the generation intervals; b the average case reproduction number r t as it varies with time. bars represent % nonparametric bootstrap percentile confidence intervals generated from one million possible transmission trees sampled from the contact probabilities listed in table due to the previous infections and/or vaccination reduces its susceptibility and hence its spread. in the presence of co-infection, infectivity of a/h n increases moderately with ϕ = . . however, the increased infectivity of a/h n in the presence of co-infection is large: ϕ = . [ % ci: . . ] and so co-infection is estimated to contribute greatly to the outbreak. this is not inconsistent with liu et al. [ ] 's observation that no patients had detectable hemagglutination inhibiting antibodies against pandemic h n virus in their acutephase samples . although the presence of co-infection can increase the infectivity of each component influenza virus, co-infection itself cannot spread successfully because the co-transmissibility measured by r e c is estimated to be . , which is less than one. the above results are obtained under the assumption of w = %, which is used to reflect the observation although there were activities outside the dormitory, the members of the buildings did not interact with each other to any significant extent (p , second column, liu et al. [ ] ). if different values of w are chosen, the estimation of transmissibility involved with a/h n virus changes although the results of the overall transmissibility and those involved with a/h n remain nearly the same. with a low value of w (i.e. the mixing between two dormitories become more restrictive), the reproductive number of a/h n due to co-infection (r e,c ) decreases while r e increases; this consequently reduces the value of ϕ . for example, the estimate of ϕ becomes . [ . , . ] when w = . . this is the outcome because without the index case of a/h n infection, the only possible infectors of a/h n cases , , , in building are coinfection cases and , which is independent of the limited mixing rate between two dormitories (w). in the situation without limitation in mixing rates between two dormitories (i.e., w = . ), the estimate of ϕ is . [ . , . ]. this result implies that in the absence of any unidentified index cases of a/h n infection, co-infection can enhance the transmissibility of each component virus. however, all the cases reported in building were infections with a/h n virus alone. in view of the restrictive mixing between two dormitories, it is likely that there is an unknown index case of a/h n infection within building , although it was not reported. an analysis listed in the additional file shows that such a hidden index case could change the above conclusion: the reproductive number of a/h n due to co-infection (r e,c ) might not exceed the reproductive number by its own (r e ) and therefore co-infection could not enhance the infectivity of a/h n . jombart et al. [ ] have developed a bayesian method to reconstruct disease outbreaks by combining epidemiological and genomic data. this may allow for the tests of whether co-infection cases in building are the infectors of a/h n cases in building or whether there is a hidden index case of a/ h n infection in building . unfortunately, the representative sequences deposited in genbank by liu et al. [ ] were not complete genome sequences and were not marked with the relevant symptom onset information. hence they cannot help to distinguish and/or find the potential sources of a/h n infection in the transmission tree. conditional on the available information, the evidence about how co-infection alters the infectivity of a/h n virus is lacking. nevertheless, the conclusion about enhanced infectivity of a/h n within coinfection is not affected. the original method of reconstructing the transmission tree by hens et al. [ ] relies on three assumptions: all cases are observed; all of them except the index case are infected by another observed case; and the generation interval distribution remains unchanged. to apply to such outbreaks as studied here involving two viruses, some assumptions have been strengthened. there must be two index cases that were singly infected with different viruses or one index case that was co-infected with the two viruses. there are more than one transmission processes. in our study example, there are five transmission processes: a/h n to a/h n ; a/h n to a/ h n ; co-infection to a/h n ; co-infection to a/h n and co-infection to co-infection. unfortunately the available data does not provide direct information for all the transmission processes, and it is not directly possible to assess the heterogeneity in generation intervals among transmission processes. in view of the similar estimates for generation intervals of different transmission processes [ ] , the generation interval distribution has been assumed to be unchanged over different types of infection as well as over the course of the outbreak. another aspect is how co-infection is generated. it could be due to an infection with one virus becoming a co-infection, or it could be a consequence of a cotransmission. and it is possible that going from a/h n to co-infection is easier than going from a/h n to coinfection, or vice versa. though this is an interesting issue [ ] the limited information that we can collect from liu et al. [ ] cannot allow us to detect the order in which the two viruses are acquired by an co-infected individual and therefore no way to investigate the effect of the order in which the two viruses are acquired. reconstructing transmission trees provides useful information about generation interval and transmission rate of infectious diseases, which are important for designing containment strategies. in this study, the method of reconstructing the plausible transmission tree from the incomplete data of an outbreak caused by one virus (hens et al. [ ] ) has been extended to the outbreak caused by two influenza a viruses. our estimates of epidemiological characteristics such as the generation interval and the transmission rate of influenza a virus are well within the ranges estimated by others [ , ] . our estimation shows that although co-infection with a/h n and a/h n viruses cannot be sustained by co-transmission, it enhances the single transmission of both viruses. however, the concluded enhancement of a/h n virus infectivity within predicting pneumonia and influenza mortality from morbidity data global patterns in seasonal activity of influenza a/h n , a/h n , and b from to : viral coexistence and latitudinal gradients rapid differentiation of mixed influenza a/h n virus infections with seasonal and pandemic variants by multitemperature single-stranded conformational polymorphism analysis evaluation of alere i influenza a & b for rapid detection of influenza a and b cross-subtype protection in humans during sequential, overlapping, and or concurrent epidemics caused by h n and h n influenza-viruses mixed infection and the genesis of influenza virus diversity fitness of pandemic h n and seasonal influenza a viruses during co-infection pandemic (h n ) and seasonal influenza a(h n ) co-infection co-infection with pandemic h n and seasonal h n influenza viruses dual infection of novel influenza viruses a/h n and a/h n in a cluster of cambodian patients isolation of influenza a/h and b viruses from an influenza patient: confirmation of co-infection by two influenza viruses mixed infections of pandemic h n and seasonal h n viruses in outbreak different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures robust reconstruction and analysis of outbreak data: influenza a(h n )v transmission in a schoolbased population transmission parameters of the a/ h n ( ) influenza virus pandemic: a review a simplex method for function minimization estimates of the reproduction number for seasonal, pandemic, and zoonotic influenza: a systematic review of the literature bayesian reconstruction of disease outbreaks by combining epidemiologic and genomic data influenza and community-acquired pneumonia interactions: the impact of order and time of infection on population patterns the impact of coinfections and their simultaneous transmission on antigenic diversity and epidemic cycling of infectious diseases strain interactions as a mechanism for dominant strain alternation and incidence oscillation in infectious diseases: seasonal influenza as a case study this research was funded by the public health england. the authors gratefully acknowledge the assistance of tom nichols and also express gratitude to drs niel hens and michael worobey for their constructive and helpful comments. co-infection should be taken with caution owing to the unknown infection source of seasonal h n virus.cross-immunity, which characterises the interaction between different viruses when one virus re-infects individuals recovered from previous infection with another virus, has been well recognized and measured. due to their relative rareness, co-infection and interactions between viruses within co-infections have not yet attracted the attention they deserve. to our knowledge this is the first analysis that estimates the interactions between influenza a viruses within co-infection. theoretical modelling illustrates their potential role in generating the recurrent epidemics and alternation of the dominant virus in seasonal influenza [ , ] . surely this urges more empirical studies to investigate this important issue of influenza and other infectious diseases caused by multiple strains. abbreviations ili: influenza like illness; mle: maximum likely estimate; sd: standard deviation. the authors declare that they have no competing interests. conceived and designed the study: xsz and dd; performed the study: xsz; wrote the paper: xsz and dd. both authors read and approved the final manuscript.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -drw nqge authors: weerasooriya, nilusha; fernando, tharanga; serasinghe, pasan; alahakoon, buddhika; madurapperuma, chirath; jayanaga, ananda title: staphylococcal endocarditis in a quadricuspid aortic valve following uncomplicated dengue infection: a case report date: - - journal: bmc infect dis doi: . /s - - -w sha: doc_id: cord_uid: drw nqge background: dengue fever is endemic and a leading health problem in sri lanka. increased incidence of concurrent bacteremia in patients with dengue infection is a recognized complication. however, staphylococcal endocarditis following dengue fever is uncommon. quadricuspid aortic valve (qav) is a rare congenital anomaly and few cases of infective endocarditis have been reported in qav. case presentation: a -year-old sri lankan male presented to the national hospital of sri lanka with recurrence of fever and acute left hemiplegia following an uncomplicated recovery of dengue fever. he was diagnosed to have staphylococcal infective endocarditis of quadricuspid aortic valve, with septic emboli to brain and spleen. he was managed with intravenous vancomycin initially, however, due to inadequate response, intravenous linezolid was added. he developed rhabdomyolysis with very high creatine phosphokinase leading to acute kidney injury, which settled with the cessation of linezolid. the patient succumbed to his illness despite aggressive antimicrobial therapy and maximum supportive care while being assessed for aortic valve replacement. conclusions: this case illustrates three clinical issues that a clinician should be aware of. firstly, the possibility of a serious secondary bacterial infection as a cause for recurrence of fever following dengue infection. secondly, this case highlights the importance of identifying qav as a cause for complicated infective endocarditis of increased severity. the report also denotes the value of being vigilant of linezolid induced rhabdomyolysis which had a causal relationship with the commencement of the drug and its cessation. dengue fever is endemic and a leading health problem in sri lanka. a few case series have recognized an increased incidence of concurrent bacteremia in patients with dengue infection [ , ] . the quadricuspid aortic valve (qav) is a rare congenital anomaly with an incidence of . to . % [ ] . few cases of endocarditis in qav have been reported [ ] . we report a patient presenting with endocarditis of previously undiagnosed qav, soon after recovering from dengue fever. the clinical course of illness was complicated by an embolic stroke and treatment-related rhabdomyolysis. a -year-old sri lankan male presented to the national hospital of sri lanka with acute left hemiplegia. two weeks before the presentation, he was treated for uncomplicated dengue fever at a local hospital. the next day of discharge from the hospital, his fever recurred. on admission to our unit, he was febrile and confused. he had a dense paralysis of the left arm and leg with a gcs of / (e- , v- , m- ). his pulse rate was bpm with a blood pressure of / mmhg and an early diastolic murmur in the lower left sternal edge compatible with aortic regurgitation (ar) was audible. the investigations revealed the following: white cell count- , /mm (neutrophils %) haemoglobin- . g/dl, platelets- , /mm , c reactive protein- mg/l. a non-contrast computed tomography of head revealed multiple hypodensities in the right cerebral hemisphere with cerebral oedema. the second day, intravenous vancomycin g twice daily was started since his blood culture grew methicillin-resistant staphylococcus aureus (mrsa), which was sensitive to vancomycin and teicoplanin. transoesophageal echocardiogram (toe) showed two oscillating masses ( × mm and × mm) separately attached to the inferior side of the aortic valve with moderate ar (fig. ) . a diagnosis of definite infective endocarditis was established according to the modified duke criteria. the left hemiplegia was presumed to be due to an embolic stroke and subsequently, a splenic abscess was detected ultrasonically. despite being on intravenous vancomycin for days, his clinical status deteriorated as his consciousness further declined. the mri brain which was done on day five revealed haemorrhagic transformation of ischaemic foci. on day six, his gcs was declining to / (e- , v- , m- ). he had a continuous fever and persistent sinus tachycardia of - /min with the blood pressure of / mmhg maintained with . μg/kg/min intravenous noradrenaline infusion. the investigations done on the sixth day revealed no improvement: white cell count- , /mm (neutrophils %) haemoglobin- g/ dl, platelets- , /mm , c reactive protein- mg/l. therefore, on day six, the patient required mechanical ventilation and the addition of rifampicin and intravenous linezolid mg twice daily. before commencing linezolid, on day six, his serum creatinine was . mg/dl and creatine phosphokinase (cpk) was u/l. on the fourth day of linezolid therapy, his fever started to settle, however, his gcs and inflammatory markers did not improve. on the same day, his cpk rose to , u/l with a serum creatinine of . mg/dl. on the seventh day of linezolid therapy, he developed acute rhabdomyolysis with raised cpk of , u/l and acute kidney injury (aki) (serum creatinine- . mg/dl). furthermore, the patient's fever recurred and he became more haemodynamically unstable, requiring escalated inotropic support. as there were reported cases of linezolid induced rhabdomyolysis, we considered linezolid as the offending agent [ ] . withdrawal of linezolid resulted in the recovery of aki and rhabdomyolysis. subsequently, cpk normalized. even though he recovered from aki, over the next days, his heart failure worsened. he had persistently low gcs with recurring fever spikes complicated by ventilator-associated pneumonia and candidaemia. the patient succumbed to his illness despite aggressive antimicrobial, antifungal therapy, and maximum supportive care while being assessed for aortic valve replacement. this patient's recovery from dengue fever was complicated with mrsa endocarditis of aortic valve with septic embolization to the brain and spleen. he was found to have qav. we could not find any reports of increased incidence of infective endocarditis in qav. however, qav endocarditis has increased risk for complications such as progressive ar, decompensated heart failure, and valve perforation [ ] . hypotheses on the pathogenesis of concurrent bacteremia in patients with dengue include anti-ns antibody-induced endothelial cell apoptosis, which leads to endothelial dysfunction allowing bacteria to invade tissues [ ] . defective t cell activation by dengue virusinfected dendritic cells and simultaneous increase of il- and il- , which are immunosuppressants, also contribute to the poor host response [ ] . there are two reported cases of linezolid induced rhabdomyolysis [ , ] . rhabdomyolysis has been associated with drugs that inhibit mitochondrial function [ ] . the inhibition of mitochondrial protein synthesis by linezolid may be the mechanistic cause for rhabdomyolysis [ ] . this case illustrates three clinical issues that a clinician should be aware of. firstly, the possibility of a serious secondary bacterial infection as a cause for recurrence of fever following dengue infection. secondly, this case highlights the importance of identifying qav as a cause for complicated infective endocarditis of increased severity. the report also denotes the value of being vigilant of linezolid induced rhabdomyolysis which had a causal relationship with the commencement of the drug and its cessation. risk factors for concurrent bacteremia in adult patients with dengue secondary bacteraemia in adult patients with prolonged dengue fever incidence, description, and functional assessment of isolated quadricuspid aortic valves quadricuspid aortic valve infective endocarditis endothelial cell apoptosis induced by antibodies against dengue virus nonstructural protein via production of nitric oxide il- and socs are predictive biomarkers of dengue hemorrhagic fever rhabdomyolysis in a patient treated with linezolid for extensively drugresistant tuberculosis mechanisms of zidovudine-induced mitochondrial toxicity and myopathy we acknowledge the support given by dr. geethika patabendige and dr. dammika vidanagama, consultant microbiologists, dr. manoj edirisuriya, and dr. dilshan priyankara, consultant intensivists, all the staff in ward and medical intensive care unit and the laboratory staff of national hospital of sri lanka, colombo. all authors were involved in the management of the patient. nw wrote the first draft of the manuscript. aj revised it. tf performed the transthoracic and transoesophageal echocardiography on the patient. cm clerked the patient on admission and cm, ba and ps helped in treating the patient in the ward. all authors have read and approved the final manuscript. not applicable.availability of data and materials not applicable.ethics approval and consent to participate not applicable. written informed consent was obtained from the patient's wife (next-of-kin) for publication of patient's clinical details along with images in this case report. a copy of the written consent is available for review by the editor-in-chief of this journal. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - mki dp authors: fawaz, sarah; barton, stephen; nabhani-gebara, shereen title: comparing clinical outcomes of piperacillin-tazobactam administration and dosage strategies in critically ill adult patients: a systematic review and meta-analysis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: mki dp background: recently, continuous administration of piperacillin-tazobactam has been proposed as a valuable alternative to traditional intermittent administration especially in critically ill patients. however, antibiotic dosing remains a challenge for clinicians as antibiotic dosing regimens are usually determined in non-critically ill hospitalized adult patients. the aim was to conduct a systematic review to identify and highlight studies comparing clinical outcomes of piperacillin tazobactam dosing regimens, continuous/prolonged infusion vs intermittent infusion in critically ill patients. meta-analyses were performed to assess the overall effect of dosing regimen on clinical efficacy. methods: studies were identified systematically through searches of pubmed and science direct, in compliance with prisma guidelines. following the systematic literature review, meta-analyses were performed using review manager. results: twenty-three studies were included in the analysis involving critically ill adult participants in total (continuous/prolonged infusion = and intermittent infusion = ) from geographically diverse regions. continuous/prolonged resulted in significantly: higher clinical cure rates (odds ratio . , % confidence interval . – . , p = . ), lower mortality rates (odds ratio . , % confidence interval . – . , p = . ), higher microbiological success rates (odds ratio . , % confidence interval . – . , p = . ) and decreasing the length of hospital stay (mean difference − . , % confidence interval − . — . , p = . ) in critically ill patients. conclusion: results from this study show that there is a significant level of evidence that clinical outcome in critically ill patients is improved in patients receiving piperacillin-tazobactam via continuous/prolonged infusion. however, more rigorous scientific studies in critically ill patients are warranted to reach a sufficient level of evidence and promote further implementation of c/pi as a dosing strategy. recently, continuous administration of piperacillintazobactam has been proposed as a valuable alternative to traditional intermittent administration especially in critically ill patients. however, correct antibiotic dosing remains a challenge for clinicians as antibiotic dosing regimens are usually determined in non-critically ill hospitalized adult patients. patient that are in intensive care units (icu) differ from other hospitalized patients in terms of pathophysiology and disease severity; these factors not only affect metabolism but also drug pharmacokinetics/ pharmacodynamics (pk/pd) behaviour. critically ill patients also have an increased risk ( - times more likely) of having or developing infections and infectious complications than those in general wards [ ] . dosing strategies that have been validated in patient populations that are non-critically ill fail to consider the substantial changes in organ function that occur with critical illness [ ] . augmented renal clearance of antibiotics is increasingly reported in critically ill patients. antibiotic dosing concentrations will vary greatly within intensive care patients with normal kidney function or renal failure as the pharmacokinetic target attainment is dependent on kidney function [ ] . given the enhanced renal elimination reported in critically ill patients, antimicrobial dosing requires extensive consideration due to important clinical consequences as accurate and timely drug exposure is essential for clinical success. the augmented renal clearance is possibly associated with the ( ) immune response to infection, ( ) inflammation to fluid loading and, ( ) use of vasoactive medications. an increase in both cardiac output and blood flow is therefore observed, leading to enhanced glomerular filtration that results in sub-therapeutic piperacillin-tazobactam concentrations due to substantial drug elimination [ ] . the optimisation of antimicrobial agents is a relatively unexplored area where further research is needed. continuous infusions (ci) and prolonged infusions (pi) of piperacillin-tazobactam has been directly linked to improved clinical outcome displaying capabilities such as lowering the possibility of resistance and decreasing mortality [ , , ] . the aim here is to systematically review the literature comparing the clinical outcome of piperacillin tazobactam dosing regimens, continuous/ prolonged infusion c/pi and ii. a systematic review of the literature was conducted [ ] [ ] [ ] [ ] ; references published between and were acknowledged through searches on pubmed and science direct, in compliance with prisma guidelines. search terms used were: (penicillin or penicillins or piperacillin or tazobactam or piperacillin-tazobactam or piperacillin/ tazobactam) and (intermittent or bolus or short or prolonged or extended or continuous) and (infusion or duration or administration or interval or dosing) and (intensive care or icu or critically ill or critical care or septic shock or sepsis or severe sepsis). however, like any database, their coverage is not complete, therefore the authors retrieved additional articles using supplementary approaches such as manual searching of journals, google scholar and checking reference lists of articles to identify additional text. a full review of published studies was implemented addressing and comparing clinical outcome of iv piperacillintazobactam dosing regimens administered to infected critically ill patients. the last search was on the st of august [prospero registration number: crd ]. initially, all articles reporting comparative outcomes of critically ill patients treated with c/pi versus ii piperacillin-tazobactam were considered eligible. the eligibility criteria were separated into two components: study characteristics and report characteristics. study eligibility criteria included the types of a) studies, b) participants, c) interventions and d) outcome measures; these measures are presented in table . report eligibility criteria included: publications written in english language, study status is "published" and inclusion of both old and new data. exclusion criteria included: pharmacoeconomic studies, non-human subjects, non-adult subjects, non-critically ill subjects, non-english language studies and pilot studies. systematic reviews, meta-analysis and editorials were also excluded. a data extraction form was developed based on cochrane data extraction template. the information extracted from each of the included studies consisted of: . characteristics of participants (didn't necessarily comprise characteristics such as age and sex however, includes characteristics such as the disease patient is diagnosed with and the method of diagnosis) and the eligibility criteria (inclusion and exclusion measures); . the type of interventionmode of administration, continuous vs intermittent dosing (including the drug, dose, duration of infusion and frequency); . type of outcome measure (including clinical outcome and clinical efficacy in terms of clinical cure). one reviewer extracted the following data from included studies (s.f); the second and third reviewers verified the relevance of the extracted information (s.n-g and s.b). variances in opinions were resolved by discussion between the three reviewers. methodological assessment of included rcts was undertaken using the cochrane risk of bias tool. two reviewers individually assessed the risk of bias (s.f and s.n-g) with disagreements resolved by a third reviewer (s.b). six domains of bias were assessed including: ( ) random sequence generation, ( ) allocation concealment, ( ) blinding of participants and personnel, ( ) incomplete outcome data, ( ) selective reporting and ( ) other biases. publication bias was evaluated using funnel plots. the methodological quality of included rct's was assessed with the jadad scale [ ] that evaluated the trial's randomisation, double blinding and reports of withdrawals and dropouts. an overall score of - points was assigned, where an overall score of three and above was regarded as adequate trial quality. the newcastle-ottawa scale is a quality assessment tool for selection, comparability and outcome assessment used to assess the quality of included observational studies (retrospective and prospective) [ ] . studies scoring more than six stars are considered as being good quality. no studies were excluded on the basis of quality assessment however their quality scores were taken into account when describing results. meta-analysis was performed using review manager for windows version . to compare the clinical efficacy of c/pi vs ii in terms of clinical cure, mortality, microbiological cure rates, adverse events and length of hospital stay. pooled odds ratio (or) and % confidence intervals (c.i) were calculated for dichotomous data, taking into account all outcomes from included studies. pooled mean difference (md) and % c.i were calculated for continuous data. statistical heterogeneity was assessed by employing χ test and i statistic. the presence of heterogeneity between studies was assessed by χ test (p < . indicates significant heterogeneity) and the extent of the inconsistencies was considered using i statistic (i > % indicates considerable heterogeneity). the pooled outcomes were calculated using mantel-haenszel fixed effect model when there was no significant heterogeneity otherwise the random effects model was chosen. 'emergence of resistance' was narratively reviewed instead of statistical analysis considering the few sample sizes included. the search of pubmed and science direct provided citations. of these, studies were excluded following review of the abstracts, as they did not meet the inclusion criteria. twenty articles were discarded after reviewing the full article due to the following reasons: non-human (n = ), on non-critically ill (n = ) and children (n = ) subjects. a further four studies were eliminated due to the focus being on pharmacoeconomics and renal replacement therapy. an additional two studies that met the inclusion criteria were acknowledged through checking references of relevant studies. twenty-three studies met the described inclusion criteria and were included in the systematic review . the article selection process is illustrated in fig. and selected studies comparing clinical outcome between ci and ii of piperacillin are listed in table . characteristics of included studies comprising of demographic characteristics, c/pi and ii dosage, drug regimen treatment results as well as study outcomes and suggestions were extracted from all studies and summarised ( table ). out of the twenty-three studies included, only an abstract (and no full article) could be obtained for four of the studies [ , , , ] . 'clinical cure' was defined as 'the complete resolution of clinical signs and symptoms of infection, with no new signs or symptoms associated with the original infection' [ , ] . 'microbiological cure' was defined as 'the eradication and presumed eradication of organisms at the infection site' [ ] . 'adverse events' were defined as 'any unexpected medical occurrences in patients administered piperacillintazobactam caused by either the drug or dosing regimen being received' [ ] . the type of studies included in the systematic review and meta-analysis were rct's (n = ), observational cohort studies (n = ; retrospective n = , prospective n = ) and a quasi-experimental study (non-randomised trial) (n = ). the quality of the majority of rct's included was moderate to high (table ) . according to the jadad scale, seven out of ten rct's ( %) obtained a score of three and above. the studies by ye [ ] and lu [ ] had a score of one and two respectively due to retrieval of only the abstract (full text unavailable). rafati [ ] received a score of two as the article did not describe randomisation method and study was not blinded. all observational studies assessed using the newcastle ottawa scale scored eight or nine stars and recognised as being of high quality (table ) . seventeen of the included studies reported clinical cure rates (table ) [ , - , - , , , - , - ] . patients that received c/pi had a statistically significantly higher clinical cure rate compared to those who received treatment via ii ( patients; or . , % c.i . - . , p = . ; fig. ). no significant heterogeneity was found among the studies (i = %, p = . ). the symmetrical funnel plot obtained indicates the absence of publication bias (fig. ) . despite methodological differences among selected studies, patients receiving c/pi displayed higher clinical cure rates compared with patients receiving ii; overall, clinical cure rate was . and . % for c/pi and ii respectively. pooling results from the studies that reported clinical cure showed that the odds of clinical cure was higher in patients receiving c/pi. the pooled or shows that c/pi piperacillin-tazobactam was . ( % c.i . - . , p = . ), indicating clinical cure rates are % higher than in ii with the true population effect between and %. eighteen of the included studies reported patient mortality rates (table ) [ - , , , , - , ] . statistically significantly fewer mortality rates were found among patients receiving c/pi compared with patients receiving conventional ii ( patients; or . , % c.i . - . , p = . ; fig. ). no significant heterogeneity was found among the studies (i = %, p = . ). the symmetrical funnel plot obtained indicates the low possibility of publication bias (fig. ) . results obtained from meta-analysis suggested that c/ pi piperacillin-tazobactam resulted in significantly lower mortality rates. overall, icu mortality rate was . and . % for c/pi and ii respectively. combining results from studies that reported mortality, the pooled or shows that c/pi piperacillin-tazobactam was . ( % c.i . - . ), indicating lower mortality rates compared with conventional ii. this was statistically significant (p = . ) with the true population effect between and %. seven of the included studies reported microbiological cure rates [ , , , , , , ] . lau et al. [ ] found no statistically significant difference between the dosing regimens however, higher microbiological success was seen in patients receiving ii. in contrast, abdul-aziz et al. [ ] found c/pi piperacillin-tazobactam had significantly higher microbiological cure rates compared with ii. pooling of the outcomes of seven studies that reported microbiological cure rates showed that patients receiving c/pi had significantly higher microbiological success rates ( patients; or . , % c.i . - . , p = . ; fig. ). no significant heterogeneity was found among studies (i = %, p = . ). the symmetrical funnel plot obtained demonstrates the absence of publication bias (fig. ) . the pooled or shows that c/pi piperacillintazobactam was . ( % c.i . - . ), indicating c/ pi piperacillin-tazobactam achieved higher microbiological cure rates compared to conventional ii. overall, microbiological cure rates were . and . % for c/ pi and ii respectively. this was statistically significant (p = . ). six of the included studies reported adverse events [ , , [ ] [ ] [ ] [ ] . participants enrolled in three of these studies table quality assessment of randomised control trials in meta-analysis based on the jadad scale quality assessment of rct's lau [ ] rafati [ ] robert [ ] li [ ] ye [ ] lu [ ] jamal [ ] abdul [ ] cotrina [ ] bao [ ] up to two points are given ( ) : described as randomised (yes = ) (no = ) and ( ) randomisation method described (yes = ) (no = ) double blinding: up to two points are given ( ) : described as double blind (yes = ) (no = ) and ( ) double blinding method described (yes = ) (no = ) reports of withdrawals and dropouts: up to one point is given ( ) : description of withdrawals (yes = ) (no = ) experienced adverse event [ , , ] . lau et al's [ ] , bao et al. [ ] and schmees et al. [ ] observed treatment-related adverse events in patients receiving both c/pi and ii; ci: . % vs ii: . %, ci: . % vs ii: . %, ci: % vs ii: %, respectively. boa [ ] reported serious adverse events in patients (pi: vs ii: ), including renal failure, tachycardia and confusion. the average occurrence of adverse events was . % for c/pi and . % for ii, respectively. participants in the other three studies did not experience adverse events [ , , ] . data obtained from studies showed no significant difference between the two infusion strategies ( patients; or . , % c.i . - . , p = . ; fig. ). no significant heterogeneity was found among studies (i = %, p = . ). although adverse events were not observed in the study by grants et al. [ ] , dosing and administrative errors arose where one patient was administered . g piperacillin-tazobactam dose over a min ii rather than a -h ci. cortina et al. [ ] reported that the most common side effects experienced by patients were gastrointestinal and allergic reactions but the number of patients that experienced these was not reported. the meta-analysis demonstrated that no adverse events that are directly associated to the dosing regimens occurred. c/pi resulted in a lower percentage of adverse events however, the difference between the two groups did not reach statistical significance ( patients; or . , % c.i . - . , p = . ; fig. ). fifteen of the included studies reported length of hospital stay [ - , , , , , - , - , ] . pooling of studies showed that patients receiving c/pi had a significantly shorter length of hospital stay ( patients; mean difference − . , % c.i - . - . , p = . ; fig. ) the meta-analysis suggests there is a significant reduction in the length of hospital stay in patients receiving c/pi compared to those receiving ii. moderate heterogeneity among studies evaluating 'length of hospital stay' (i = %, p = . ) was observed. this is likely due to clinical heterogeneity in the design and outcomes of the included studies. the length of hospital stay was an independent risk factor for mortality, however the influence of mortality on the length of hospital stay could not be evaluated. data regarding the emergence of resistance was reported in four of the included studies [ , , , ] . two resistant pathogens were isolated in one study [ ] however, resistant strains were not isolated in three studies [ , , ] following the initiation of piperacillintazobactam treatment. three studies reported that no resistant pathogen was isolated following the initiation of piperacillin-tazobactam treatment. in the study conducted by grant et al. [ ] , two resistant strains were isolated from patients receiving ci piperacillintazobactam. the majority of rct's and prospective studies assessed were judged to have a low risk of bias for random sequence generation, allocation concealment, incomplete outcome data, selective reporting and other biases. however, evaluations of blinding of participants and personnel parameter was judged to have a high or unclear risk of bias (fig. ). to the best of our knowledge, this systematic review and meta-analysis is the largest study describing clinical outcomes of severely ill patients treated with either c/pi or ii piperacillin-tazobactam. the selected studies involved critically ill adult participants in total (c/pi = and ii = ) from geographically diverse regions. it is the first meta-analysis that shows c/pi resulted in significantly: ( ) higher clinical cure rates ( ) lower mortality rates ( ) higher microbiological success rates and ( ) decreasing the length of hospital stay specifically in critically ill patients. in all the studies, the primary outcome assessed was clinical efficacy. the current study differs from previously published systematic reviews and meta-analyses [ , , [ ] [ ] [ ] [ ] [ ] [ ] as it specifically focuses on use of piperacillin-tazobactam in critically ill icu patients. the present systematic review and meta-analysis identified a significant clinical cure, mortality, microbiological cure and length of hospital stay benefit for c/pi across all included studies. in theory, c/pi of piperacillin-tazobactam is a broadly recognised strategy to optimize antibiotic therapy, where concentrations remain above the mic for a higher percentage of time. studies have demonstrated that the amount of time in which the free or non-protein bound antibiotic concentration exceeds the mic (ft > mic) of the organism is the best predictor of clinical and microbiologic response for β-lactams [ , ] . however, data to backup this developing practice have been sparse [ ] . twenty-three published studies comparing c/pi and ii of piperacillin-tazobactam fit the inclusion criteria ( table ) . outcomes of the current study correlate and expand upon previously published reviews including several analyses comparing clinical efficacy of dosing regimens for beta-lactams generally [ ] [ ] [ ] [ ] . these studies pointed towards a more favourable outcome of c/pi for improved clinical cure and resolution of illness. falagas et al. [ ] and vardakas el al reviewed outcomes of c/pi and ii beta-lactams. there was a significant reduction in mortality rates among patients receiving c/pi in both studies. roberts et al. [ ] observed higher clinical rates and reduced mortality in c/pi patients and lal et al. [ ] found c/pi to reduce clinical failure rates. finding in this study are consistent with published reviews focused specifically on piperacillin-tazobactam [ , , , ] . yusuf el at [ ] reviewed literature comparing the effectiveness of c/pi and ii administration of piperacillin-tazobactam. they documented c/pi improved clinical cure, mortality and length of hospital stay in comparison to ii. yang et al. / [ , ] observed similar beneficial effects of c/pi in their systematic reviews. recently, rhodes et al. [ ] evaluated a wide range of severely ill patients, from hospitalised patients to critically ill patients admitted to icu. c/pi piperacillin-tazobactam is associated with improved clinical outcome and significantly reduced mortality rates. several observations were encountered from reviewing this data which led to reduced comparability among studies. first, clinical heterogeneity was present as selected studies comparing c/pi and ii in terms of clinical outcomes have confounding factors including patient sample size, study settings, study design, quality, intervention and outcomes. second, information regarding monotherapy and combination antibiotic therapy were not reported in the included studies. this reduces the validity of conclusions on c/pi, as agents used possess different antimicrobial spectrum, and drug-drug interactions were unknown hence not considered. third, assessing safety was challenging due to under-reporting of adverse events. higher serum concentrations in c/pi patients over a longer period could potentially result in an increased number of adverse events. fourth, a large number of included studies were rct's ( / ; . %) with small sample size. small sample size may result in bias and the probability of small study effects contributing to the favourable outcome for c/pi. however, metaanalyses including small and large studies did not indicate significant discrepancies and similar outcomes were observed with fixed and random effect models. fifth, duration of piperacillin-tazobactam administration and dosing is not homogenised between studies. ci was administered over the entire dosing interval and the duration of a pi between studies ranged between and h which is in line with proposed guidelines ( - h). traditional ii durations between studies ranged between and min (usually - min) [ ] . heterogeneity of dosing was also noted. in / studies piperacillintazobactam treatment was initiated with a loading dose to ensure rapid achievement of therapeutic concentrations. also, the total daily dose administered differed between ci, pi and ii, providing an additional confounding factor as to whether the duration of infusion or total daily dose attributed to clinical outcome ( table ) . finally, it wasn't apparent how critically ill the patients within studies were as only four studies reported sofa scores. findings of this meta-analysis should be interpreted in view of certain limitations. first, throughout this review, pi and ci were combined and referred to as c/pi, thus, it is unclear which of the two dosing strategies is most effective for critically ill patients. additionally, all studies were evaluated for quality and risk of bias and based on the overall assessment of these two factors no studies were excluded (tables , and fig. ). also, a medical librarian was not involved in this study. in conclusion, c/pi of piperacillin-tazobactam in critically ill patients was associated with ( ) higher clinical cure rates ( ) lower mortality rates, ( ) higher microbiological success rates and, ( ) decreasing the length of hospital stay in critically ill icu patients. no reduction in 'adverse events' and 'emergence of resistance' has been demonstrated. results obtained in this study show that clinical outcome in critically ill patients is significantly better in those receiving c/pi. however, the superiority of the benefits and outcome gains achieved with c/pi administration in comparison to ii is difficult to deduce as studies selected show considerable heterogeneity in terms of: ( ) type of isolated bacteria, ( ) piperacillin-tazobactam dose, ( ) mic of pathogen, ( ) patient renal function, ( ) duration of hospital stay and ( ) outcome definitions. more rigorous scientific studies in critically ill patients are warranted to reach a sufficient level of evidence to promote the widespread adoption and further implementation of c/pi piperacillintazobactam. nosocomial bacterial infections in intensive care 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prolonged-infusion dosing of beta-lactam antibiotics publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. this research did not receive any specific grant from funding agencies in the public, commercial, or for not-for-profit sectors. data generated or analysed during this study are either included in this published article or are available from the corresponding author on reasonable request. not applicable. not applicable. the authors declare that they have no competing interests.received: february accepted: june key: cord- -qhul z authors: chen, jie; zheng, xu-dong; dai, qi-he; hong, wei-li; li, you-peng; chen, rui; ye, bing-bing; mo, xiao-jie; cui, peng; ruan, zhan-wei title: diagnosis of severe scrub typhus infection by next-generation sequencing:a case report date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: qhul z background: scrub typhus is an acute febrile illness, which was caused by orientia tsutsugamushi and transmitted through the bite of chiggers. the diagnosis of scrub typhus could be missed diagnosis due to the absence of the pathognomonic eschar. case presentation: a -year-old man was hospitalized with fever and kidney injury and was diagnosed of hemorrhagic fever with renal syndrome first. however, the situation of the illness deteriorated into refractory septic shock and multiple organ dysfunction rapidly,although the treatment of anti-sepsis was used in rd- th day. orientia tsutsugamushi was determined to be the causative pathogen by next-generation sequencing of his plasma sample in th day. then, the patient was treated with doxycycline and azithromycin and recovered quickly. conclusions: next-generation sequencing was a new diagnostic technology and could identify scrub typhus in accurately and fast without the pathognomonic eschar. scrub typhus is an acute febrile illness caused by orientia tsutsugamushi (a gram-negative coccobacillus) and transmitted through the bite of chiggers [ ] . themortality of scrub typhus is reported to be as high as % in severe cases with multiple organ dysfunction (mods) [ ] . the diagnosis of scrub typhus is difficult due to the absence of the pathognomonic eschar, which is the characteristic clinical manifestations and varies widely ( - %) [ ] . serology, biopsy, culture, and polymerase chain(pcr) reaction were routine diagnostic methods and had many defects in the diagnosis [ ] . for example, the indirect fluorescent antibody test needs a four-fold rise in titers over a -day period [ ] . polymerase chain reaction testing is only used as confirmatory test, but not as a screening test, because of multiple pathogenic bacterium in the clinic [ ] . the culture of orientia tsutsugamushi was very difficult and dangerous, and should be accomplished at a special research institution [ ] . lymphohistocytic vasculitis was the pathological hallmark of scrub typhus on skin biopsy, but not definitive [ ] . next-generation sequencing (ngs) technologies have been used in the diagnosis of other pathogens such as leptospira santarosai [ ] , mycobacterium tuberculosis [ ] , human immunodeficiency virus [ ] etc. however, the use of ngs has not been reported in the case of scrub typhus. here, orientia tsutsugamushi was determined to be the causative pathogen by ngs technologies in a case of mods, and the results contributed directly to the patient's dramatic diagnosis and treatment, resulting in a favourable outcome. a -year-old man, rural mountain inhabitant who frequently encountered mice, had a history of benign prostatic hyperplasia (bph), was admitted to the department of nephrology due to the difficulty in urination and fever for days on th july (fig. a) . his body temperature was . °c, accompanied by fatigue, anorexia, chest tightness, coughing with a small amount of haemoptysis. the vitals read as, bp: / mmhg, rr: bpm, hr: bpm, while slight conjunctival hyperaemia, mild scleral yellow stain, wet voice and wheezing of the lungs, right ear anabrosis (fig. d) , no obvious abnormalities in the abdomen and other systems, no bites and eschars were found. blood test showed white blood cell(wbc) count: . × /l, neutrophil: . %, atypical lymphocytes: %, platelet: × /l, hypersensitive c- were decreased with the treatment of doxycycline (black arrows) in th day, th day and th day respectively. although platelet have been infused by iv in in th day, the blood platelet (plt, green line) was still declined in th day. however, the blood platelet was increased obviously in th day own to the treatment of doxycycline and azithromycin in th day. c shows systemic vascular resistance index (svri) was increased with the treatment of doxycycline (black arrows) in th day, whereas it was reduced in the first days. d shows the skin anabrosis on the right ear. panel e shows the bone marrow cells are hyperplastic obviously, poisoning alteration was existed in the granulocytic cell. the megakaryocytes were obviously proliferated with maturating disorderly. hemophagocytic phenomenon was pointed by the arrow. panel f shows blurred shadows around the pancreas, indicated the pancreatic leakage. besides that, the pancreatic head forms a cystic space with dense shadows, indicated the pancreatic head hematoma by ct reactive protein (crp): . mg/l, procalcitonin (pct): . ng/ml, serum creatinine (cr): umol/ l, alanine aminotransferase (alt): u / l, bilirubin: . umol / l. the urine volume was ml/h, urine routine urine protein +, red blood cells +++/hp. ct scan showed a little exudation in the lungs, bph and no other abnormalities. this patient was diagnosed as "haemorrhagic fever with renal syndrome" caused by hantavirus first (fig. a) . unfortunately, the patient developed rapid atrial fibrillation and delirium, and was transferred to icu, because sequential organ failure assessment score increased from to . continuous renal replacement therapy (crrt) was used due to high serum creatinine (> μmol/l). in the next h, the patient developed a high fever ( . °c), and went into coma. a large amount of haemoptysis and growth of gram-positive bacteria by blood culture smears were found. we intubated him to protect the airway. however, in the rd- th day after hospitalization ( th july to th july), the condition worsened and the patient went into refractory shock (mean arterial pressure: mmhg) although norepinephrine ( . mcg/kg.min) and adrenaline ( . mcg/kg.min) were used, low blood vessels tension (minimum of systemic vascular resistance index: dsm /cm ), hyperlactemia (maximum: . mmol/l), further reduction of platelets (minimum: /ml), hyper-procalcitonin (maximum: . ng/ml), hyperbilirubinemia (maximum: . μmol/l) (fig. b, c) , epidemic haemorrhagic fever antibody was negative, hemophagocytic phenomenon in the bone marrow (fig. e) in spite of organ support therapy, yet antisepsis treatment was given. (fig. a) . fortunately, on the th day after hospitalization ( th july), orientia tsutsugamushi was determined to be the causative pathogen by ngs of the patient's plasma sample. high-quality sequencing data were generated by removing low-quality, and short (length < bp) reads, followed by computational subtraction of human host sequences mapped to the human reference genome (hg ) using burrows-wheeler alignment. the remaining data by removal of low-complexity reads were classified by simultaneously aligning to four microbial genome databases, consisting of virus, bacteria, fungi, and parasites. a total of sequences of orientia tsutsugamushi were detected in plasma sample with a total coverage of . % (fig. a) . orientia tsutsugamushi specific amplification was detected from plasma sample by pcr (fig. b) . the distribution of bacterial sequences (n = reads) was identified in the patient's plasma including orientia tsutsugamushi (n = , . %), propionibacterium, staphylococcus, acinetobacter, sphingomonas, pseudomonas (fig. c) . then, the antiinfection regime was changed to doxycycline ( . g oral bid) and azithromycin ( . g intravenous drops qd). after days of treatment ( th july), the circulation collapse was recovered and the vasoactive drug was stopped. after days of the treatment ( th july), respiratory failure was corrected and tracheal intubation was removed. the patient was removed haemodialysis and returned to the nephrology ward successfully on th july. then, the patient had abdominal distension, elevated blood amylase, pancreatic exudation and haemorrhage by ct in th aug (fig. f, g) and anti-pancreatitis treatment was timely. after the patient recovered, he was then discharged on th aug and returned to normal serum creatinine in th sep by follow-up (fig. a ). scrub typhus is a natural epidemic caused by orientia tsutsugamushi, spread globally, mainly in the asia-pacific region [ , , ] and is considered as the world's leading rickettsial infection, threatening the health of one billion people every year and causing more than one million deaths [ ] . the hosts of orientia tsutsugamushi are larvae and rats, while it could also infect humans and spread through the sputum in grass or soil on the floor [ ] . indeed, this patient lived on the mountain side where mice often appeared in eastern part of china. scrub typhus is characterised mainly by ascended vascular permeability directly co-related with the bacteria count, damaged endothelial cell junctions in the small and medium blood vessels due to the escalated tnf-α [ ] . the typical clinical manifestation of scrub typhus are the fever and eschar. severe scrub typhus infection could affect a variety of systems and lead to multiple complications, including meningitis, acute lung injury, myocarditis, hepatitis, acute renal failure, pancreatitis, disseminated intravascular coagulation, septic shock, and mods [ , ] . unfortunately, because this patient had no specific eschar, he was misdiagnosed as "haemorrhagic fever with renal syndrome" previously. moreover, condition still deteriorated into refractory septic shock and mods rapidly, although crrt, tracheal intubation and antibiotics were used during rd - th days. scrub typhus might be easily misdiagnosed as other febrile diseases due to non-specific symptoms except the hidden eschar. laboratory diagnosis is mainly done with serology, molecular assays, pathogen characterization and tissue culture,which is only supported by large scale laboratories. serology would be positive after - days of illness and has poor sensitivity and specificity due to the lack of species identification techniques in the market. besides that, cross-reactions among closely related members should be noticed [ ] . molecular assays (such as pcr tests) have the advantages of rapid and sensitive diagnosis when an eschar is still present. however, it is difficult to apply on time when the eschar is negative and the initial diagnosis is missed. tissue biopsy, culture, and pathogen characterization require special laboratories to avoid biosafety risks [ ] . ngs technologies have been used in the diagnosis of other pathogenesis widely, which contained whole genome sequence of viral taxa, bacteral genomes or scaffolds, fungi related to human infection, and parasites associated with human diseases [ ] [ ] [ ] . however, the use of ngs has not been reported in the case of scrub typhus. indeed, in the th day after hospitalization ( th july), orientia tsutsugamushi was determined to be the causative pathogen by ngs. scrub typhus could also be treated effectively if it could be diagnosed in time [ , ] . doxycycline has been proved to the preferred drug in the treatment of scrub typhus, and intravenous doxycycline isolation or enteral doxycycline combined with intravenous azithromycin is the better choice in severe cases with shock or intestinal absorption difficulties [ ] . our patient got the combination therapy and recovered quickly. in summary, it was difficult to diagnose of scrub typhus timely owing to the lack of specific eschar and many limitations in conventional diagnostic methods. ngs was a new diagnostic technology which could identify scrub typhus in accurately and fast and wouldbe a promising critical tool to find the aetiology of multiple organs failure or septic shock. supplementary information accompanies this paper at https://doi.org/ . /s - - -y. additional file . tsutsugamushi derived from ngs data. a total of reads mapped to o. tsutsugamushi in the reference database which contains about pathogen genomes, and got a total coverage of . % respectively. b shows the confirmation of o. tsutsugamushi specific amplification by pcr. the primer was ′-aactgattttattcaactaatgctgct- ′ and ′-tatgcctgagtaagatacrtgaatrgaatt- ′. the bp pcr products was detected in case sample. lane : sample case, lane : negative control, lane : the dna ladder was dl from takara. c shows the the distribution of bacterial sequences (n = reads) identified in the patient's plasma included orientia tsutsugamushi(n = ; . %), propionibacterium, staphylococcus, acinetobacter, sphingomonas, pseudomonas endemic scrub typhus in south america severe scrub typhus infection: clinical features, diagnostic challenges and management a concise review of the epidemiology and diagnostics of rickettsioses: rickettsia and orientia spp optimal cutoff titers for indirect immunofluorescence assay for diagnosis of scrub typhus use of eschar swabbing for the molecular diagnosis and genotyping of orientia tsutsugamushi causing scrub typhus in quang nam province laboratory-acquired scrub typhus and murine typhus infections: the argument for a risk-based approach to biosafety requirements for orientia tsutsugamushi and rickettsia typhi laboratory activities actionable diagnosis of neuroleptospirosis by next-generation sequencing multiplexed identification, quantification and genotyping of infectious agents using a semiconductor biochip affordable hiv drug-resistance testing for monitoring of antiretroviral therapy in sub-saharan africa imported scrub typhus: first case in south america and review of the literature the clinical significance of upper gastrointestinal endoscopy in gastrointestinal vasculitis related to scrub typhus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors wish to thank the patient for participating in this study. we also thank dr. yu hao and dr. suwas bhandari for their help in the case discussion. zwrand xdz and jc were responsible for the study design, literature searchand manuscript drafting. ypl and rc and bby and pc were responsible for the data collection and statistical analysis. zwr and jc and wlh and xjm were mainly responsible for the data interpretation. rzw and xdz and qhd were responsible for the study concept and critical revision. all authors contributed to the discussion, writing and reviewing the manuscript and all authors have approved the final manuscript. no funding.availability of data and materials all data generated or analyzed during this study are included in this published article. the study protocol was approved by the institutional review board of third affiliated hospital, wenzhou medical university. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor of this journal. none of the authors have conflicts of interest to disclose or a financial relationship with a commercial entity that has interest in the subject of the manuscript.author details key: cord- - lybl r authors: dubert, marie; visseaux, benoit; birgy, andré; mordant, pierre; metivier, anne-cécile; dauriat, gaelle; fidouh, nadhira; yazdanpanah, yazdan; grall, nathalie; castier, yves; mal, hervé; thabut, gabriel; lescure, françois-xavier title: late viral or bacterial respiratory infections in lung transplanted patients: impact on respiratory function date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: lybl r background: respiratory infections are a major threat for lung recipients. we aimed to compare with a monocentric study the impact of late viral and bacterial respiratory infections on the graft function. methods: patients, who survived months or more following lung transplantation that took place between and , were classified into three groups: a viral infection group (vig) (without any respiratory bacteria), a bacterial infection group (big) (with or without any respiratory viruses), and a control group (cg) (no documented infection). chronic lung allograft dysfunction (clad) and acute rejection were analysed months after the inclusion in the study. results: among included lung recipients, ( %) had at least one positive virological respiratory sample during the study period. patients were classified as follows: in the vig, in the big (among which co-infections with a virus) and in the cg. the big presented a higher initial deterioration in lung function (p = . ) than the vig. but months after the infection, only the vig presented a median decrease of forced expiratory volume in s; − ml (iqr; − ; + ) in the vig, + ml (+ ;+ ) in the big and + (− ;+ ) in the cg, p < . . acute rejection was more frequent in the vig (n = ( %)), than the big (n = ( %)) and cg (n = ( %)), p < . , despite presenting no more clad (p = . ). conclusions: despite a less severe initial presentation, single viral respiratory infections seem to lead to a greater deterioration in lung function, and to more acute rejection, than bacterial infections. thanks to a better selection of recipients and donors, to an improvement in surgical / anaesthetic procedures and to better management of immunosuppressive therapies, early post-operative survival of lung transplant recipients (ltrs) has improved since the advent of lung transplantation. whereas most early deaths are related to primary graft dysfunction or acute rejection (ar), the long-term prognosis is threatened by chronic lung allograft dysfunction (clad), usually in the form of a bronchiolitis obliterans syndrome (bos), that affects to % of lung recipients within years after transplantation [ ] [ ] [ ] [ ] . clad represents the leading cause of death year after lung transplantation [ , , ] . infectious respiratory complications are also a major cause of morbidity and mortality for ltrs and are responsible for a third of deaths occurring in the first year post transplant, and half of all deaths during longterm follow-up [ , ] . the severity of these infections results from several factors including induced immunosuppression, direct exposure of the graft to microorganisms, and finally less effective mucociliary function and lymphatic drainage and cough reflex following denervation of the graft [ ] [ ] [ ] . moreover, stenosis and ischemic processes occurring at the surgical anastomosis decrease the clearance of secretions, and promote their colonization and invasion by microorganisms [ ] . bacterial infections are the leading cause of respiratory infections in ltrs [ , , ] . their association with the occurrence of clad is well established [ , ] . many authors admit that viral respiratory tract infections (vrti) may be associated with clad [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but this remains controversial, depending on the definition of respiratory infection, the virus panel studied, the time limit between vrti and spirometric analysis, and the consideration of intercurrent events possibly influencing the respiratory function [ ] . similarly, the association between vrti and ar continues to be debated in the literature. while some studies have identified an association between these two events [ , , ] some others, including a recent meta-analysis, have not found any link between them [ , , ] . however, to our knowledge, the impact of viralbacterial co-infections on graft survival has not been specifically studied, and was not compared to single vrti or patients without any respiratory infections. the development of rapid antigenic tests and molecular biology techniques has facilitated the detection and diagnosis of several viruses. the new multiplex pcr methods (polymerase chain reaction) are fast, sensitive andable to detect an enlarged number of viruses not easily detected before (e.g. metapneumovirus, coronavirus nl and hku , bocavirus, rhinovirus c) [ , ] . thus, we hypothesis that the impact of the viral infection is at least as severe as bacterial respiratory infections on lung graft function among ltx. the objectives of our study were to assess, in a cohort study, the occurrence of late viral and bacterial respiratory infections in ltrs and to compare their respective impact on graft function with those without any respiratory infections. we retrospectively screened all individuals who underwent lung transplantation between september and september at the bichat-claude bernard teaching hospital, paris, france. in this cohort we evaluated the occurrence of late viral and bacterial respiratory infections and graft function overtime. exclusion criteria were: ( ) death during the first months after transplantation, ( ) no available pulmonary function assessment at the time of documented infection or months after, and ( ) herpetic or cytomegalovirus pneumonia. in order to include patients at a steady state, we studied only the late respiratory infections, i.e. to censure the first months after the lung transplantation, a period during which postoperative complications are frequent and usually intertwined. these patients were regularly followed up. at each routine follow-up visits with patients, symptoms were recorded. spirometric measurements, blood tests, radiological explorations, bronchoscopic procedures with bronchoalveolar fluid and transbronchial biopsies were systematically performed and tested for both bacterial and viral pathogens according to local guidelines. all respiratory samples, bronchial aspirates, bronchoalveolar fluids and nasopharyngeal samples were reviewed. several [ ] [ ] [ ] , as confirmed by our internal method validations and the similar viral diversity observed over time [ ] . their reliability was also assessed throughout the study period by regular qcmd controls (glasgow, uk). a sample was defined as bacteriologically positive if the bacteria were present at cfu/ml or more for sputum, cfu/ml or more for bronchial aspirates and cfu/ml or more for bronchoalveolar fluid. according to virological and bacteriological results from respiratory samples, patients were divided into three groups, as follows: -patients presenting a viral respiratory infection: viral infection group (vig), i.e. -patients with at least one positive virological respiratory sampling, symptomatic or not. nasopharyngeal swabs that tested positive only for rhinovirus were not considered to be positive for a significant virus, and were ignored. -patients without any respiratory infection: control group (cg), i.e. patients with no virological or bacteriological positive respiratory samples during the study, or those with nasopharyngeal swabs positive for rhinovirus only; or with a positive bacteriological respiratory sample not followed by antibiotic treatment (i.e. considered as a simple bacterial carriage). the date of inclusion was defined as the date of the respiratory sample of interest for the two infected groups (vig and big), and the date of the first outpatient consultation for the cg. the primary endpoint was the occurrence of bos months after inclusion. secondary endpoints were the occurrence of ar and the quantitative change of forced expiratory volume in s (fev- ) at months after inclusion, as well as death during the whole study period. the following definitions were used to assess the outcomes: biopsy that demonstrated at least a grade a of acute rejection as defined by the ishlt for cellular rejection. in patients in whom a biopsy could not be performed, acute rejection was defined by deterioration in lung function with no other identifiable aetiology and that positively responded to a high-dose corticosteroid therapy. . fev- delta was defined as -month-fev- last fev- before inclusion. quantitative variables were presented using the median (quartile - ) and were compared by t-test or variance analysis. qualitative variables were compared using a chi test or a fisher-exact test. outcomes were compared using a logistic regression multivariable model, with adjustment for time since transplantation, age and sex. a sensitivity analysis was carried out excluding patients who had had a bos before inclusion. all of the statistical analysis was done using r software, version ( . . ). this study was approved by the cepro (comité d'evaluation des protocoles de recherche observationnelle) ethical committee, number cepro - . between september and september , patients received lung transplants at bichat hospital. fiftyfive patients were excluded from the analysis: ( %) because of death within months following transplantation, six ( %) due to a lack of spirometry assessment at inclusion or months afterwards, and two because of pathological, proven herpetic pneumonia. among the remaining ltrs, ( %) had at least one positive virological respiratory sample. thus, patients were divided into three groups according to the criteria described above: ( %) in the vig, ( %) in the big ( patients mono-infected by bacteria and co-infected patients) and ( %) in the cg. the corresponding flow chart is presented in fig. . nineteen % of the respiratory samples were broncho-alveolar lavages or bronchial aspirations. general characteristics, type of immunosuppression and prior viral infections were similar across the three groups with the exception of renal dysfunction (egfr < ml/min/ . m ), more prevalent in the vig (table ) . previous ar requiring treatment in the months prior to inclusion occurred for ( %) patients in the vig, ( %) in the big, and none of the cg (p < . ). among patients presenting a bos at inclusion, were classified in vig, compared to in the big, and in the cg (p = . ). clinical presentation (table ) patients were included at a median time of (iqr, - ) days after transplantation. both infected groups displayed similar respiratory symptoms. likewise, the biological presentations of both infected groups were close, and inflammatory surrogates (leucocytes, creactive protein, platelets) did not differ statistically. big patients were more prone to be hospitalized ( vs. %, p = . ), and to present a more severe decrease of fev- at inclusion (− ml (− - ) vs. - ml (− - )), compared to the vig. among patients examined with a thoracic tomodensitometry, seven patients ( %) presented a new infiltrate. picornavirus was the most frequently detected virus in the vig (n = ), followed by parainfluenzae virus (n = ), and syncytial respiratory virus (n = ) ( table ) . pseudomonas aeruginosa was the main detected bacteria; found in ( %) patients in the big. in the cg, the neglected bacteria were pseudomonas aeruginosa (n = ), corynebacteriae striatum (n = ) and others (n = ). three patients had more than one detected virus and eight patients had more than one detected bacteria. the overall rate of worsening -month-bos was % with no significant differences between the three groups (table ). sixteen patients ( %) died during the study period ( bos, strokes, neoplasia, severe infections, from unknown cause), with no significant differences between the three groups ( in the two infected groups, these outcomes were not different between the symptomatic patients (n = ) and the asymptomatic patients (n = ). thirteen patients had a prior bos at inclusion: ( . %) in the cg, ( . %) in the vig and ( . %) in the big. there was a trend toward more viral respiratory investigation among patients with a bos at inclusion (median of . ( . - . ) samples/patients) than for patients without bos at inclusion ( . ( . - . ), p = . ). however, the sensitivity analysis performed on the remaining patients without bos at inclusion found similar results in terms of significance and association effect with the development of a new bos or with ar (table s ). in the same way, infected patients were significantly more investigated than those in the cg: this study shows that, after the first months following transplantation, more than half of ltrs were affected by vrti. clinical presentations for late viral and/or bacterial infections at baseline were very similar, albeit with additional signs of severity for the bacterial infections. single late vrti strongly impacted the patients' prognosis by leading to an increased risk of ar, a trend to an increased risk of bos (without significant association), and a more severe secondary decline in respiratory function compared to the late bacterial respiratory infection. the consequences of these different infections were similar whether or not the infection was symptomatic at the time of viral or bacterial detection. in this study, patients ( %) exhibited at least one positive viral respiratory sampling during follow-up. this rate varies among studies as do screening techniques and reasons for withdrawal. while studies using cell cultures in their screening method report virus detection rates in respiratory samples at around % [ , ] , the use of molecular biology tests significantly increases this prevalence from to % [ , , ] . we decided to exclude infections with cmv, especially because cmv diseases respond to different triggers than those infections, were prevented by different protocol evolutions during the study period, and can induce the death or graft rejection in both of our patient groups. with regard to microbiological aetiology, we confirmed that the most frequent viruses detected with the pcr test were those corresponding to the picornavirus group, followed by parainfluenzae viruses and coronaviruses, as already described [ , , , , , ] . it is worthy of note that, in the big, picornavirus were the most frequently detected viruses in co-infections ( %). picornavirus are often considered as a contaminant with a controversial clinical impact. indeed, a recent prospective study demonstrated that rhinoviruses were frequent in ltrs, even in those patients who were asymptomatic [ ] . influenza were rarely identified among lung graft patients, especially when compared to the non-lung graft patients in our hospital using the same mpcr assays ( vs %) [ ] . this is explained by the high vaccination rates and specific prevention measures compliance among lung graft patients and their relatives. among bacterial infections, pseudomonas aeruginosa and corynebacteriae striatum were more often detected. both bacteria are known to be responsible for serious infections in ltrs. as already shown, the symptomatic feature of the initial infection did not impact on graft survival [ , , ] , suggesting that, for these patients, the symptomatic nature of the infection should not be taken into account. concerning ar, the impact of late respiratory viral and/ or bacterial infections on the graft function was significantly different with three times more ar within months for both the vig and big compared to the cg. while some studies supported this association [ , ] , other studies, including a meta-analysis, did not find any significant link [ , , , ] . this difference could be explained by the variety of criteria used to define ar. we chose to identify ar when the histological pattern showed a stage of at least a . indeed, previous studies demonstrated that minimal rejection (≥a ) was associated with an increased risk for bos development and progression that was comparable to a rejection [ ] . on top on that, we noticed a significantly longer delay in ar in the vig than in the big, suggesting that the impact of viral infection on lung graft function must be screen even after several weeks. especially in asymptomatic ltrs and the lack of specific management, the morbidity of viral infection could be attributed to a trivialization of viral colonization, leading to a neglected and chronic cause of inflammation and, thus, to potential rejection. therefore, it seems important to assess the impact of respiratory viral infections on the graft function: to emphasize the prevention of viral infections for immunocompromised with more frequent sampling of patients including wide respiratory virus detection by molecular techniques and to strengthen spirometric controls after viral infections. the all-cause mortality was evaluated to % at months in this study. this rate in consistent with other studies [ , ] . it is explained by the high rate of comorbidity among our patients (more than half with arterial hypertension and diabetes mellitus), the deep immunosuppression required and the numerous complications of lung grafting. to our knowledge, this study is the first to allow a direct comparison of the impact of late viral and bacterial respiratory infections in ltrs. we were able to analyze all the spirometry data both at inclusion and months after, and to present results of an extensive panel of viral pcr tests. this study has several limitations. firstly, the small number of patients could restrict the power of the conclusions, especially for patients classified only on nasopharyngeal swabs (n = ). aors present wide ranges and must be interpreted accordingly. however, this study remains one of the largest available cohort on the topic to date. despite this small sample size we were able to illustrate that late vrti strongly impacted the patients' prognosis by leading to an increased risk of ar. in addition, although not significant there was a trend to higher risk of occurrence of other outcomes such as bos and death with vrti. we believe that these findings are important and should lead to the design of larger studies. because of the small number of patients among the group with bacterial infections and the group with bacterial and viral infections, we decided to merge these two groups and this could be also debated. however, this was done first because of the similar presentation of patients in these groups and second on the basis of the hypothesis that: (i) bacterial infections were responsible of the major acute part of the lung graft malfunction, and (ii) these groups were both subject to an intervention with the use of antibiotics. secondly, the retrospective design leads to several biases: an indication bias leading to higher infection detection in patients presenting ar months prior to inclusion or a bos prior to infection because of more frequent followup visits and a trend to more respiratory sampling streptocoque, n (%) ( ) ( ) other bacteria, n (%) ( ) ( ) abbreviations: big bacterial infections group; vig viral infections group a among patients with bacterial infections, had no bacteria identification, but samples were examined after antibiotic therapy (n = ) and/or presented a localized chest x-ray condensation (n = ) and/or patients had purulent sputum (n = ) for these patients. although treatment for ar months prior to inclusion could be considered as a biais, this rate was not significantly different between the infected groups. because of the small number of patients, we could not perform a sensitivity analysis among these patients who had ar months prior to inclusion. nevertheless, we performed a sensitivity analysis to address the hypothesis of indication bias. we repeated the multivariable model without considering patients with a bos at inclusion and found similar results, suggesting that this bias, if it existed, did not alter our conclusions. we also assume a survival bias that may explain why despite higher lung function deterioration at months after inclusion in the viral infections group, the rate of bos was not significantly different. the relatively short months follow-up period used in our study may not be sufficient to identify a potential difference in the progression of bos between groups. indeed, the incidence of bos development months after the first infection episode was at % in our study, while previous studies described an incidence rate of % over a five-year period [ ] . to conclude, viral respiratory infections and virusbacteria co-infections were frequent in lung graft recipients and led to similar clinical and biological presentations. bacterial infections strongly diminished initial lung function and led to more hospitalizations whereas single viral respiratory infections seem to lead to a greater deterioration in lung function, and to more acute rejection, than bacterial infections. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : table s . multivariate analysis of association with development of bos. without patients with bos at inclusion service de maladies infectieuses et tropicales, rue henri huchard, f- 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determinants symptomatic respiratory virus infection and chronic lung allograft dysfunction viral respiratory tract infection during the first postoperative year is a risk factor for chronic rejection after lung transplantation respiratory viruses in lung transplant recipients: a critical review and pooled analysis of clinical studies the epidemiology of parainfluenza virus infection in lung transplant recipients human metapneumovirus infection in lung transplant recipients: clinical presentation and epidemiology upper and lower respiratory tract viral infections and acute graft rejection in lung transplant recipients development of a respiratory virus panel test for detection of twenty human respiratory viruses by use of multiplex pcr and a fluid microbeadbased assay incidence and outcomes of respiratory viral infections in lung transplant recipients: a prospective study comparison of the luminex xtag rvp fast assay and the idaho technology filmarray rp assay for detection of respiratory viruses in pediatric patients at a cancer hospital comparison of anyplex ii rv with the xtag respiratory viral panel and seeplex rv for detection of respiratory viruses comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder assay and respifinder smart assay prevalence of respiratory viruses among adults, by season, age, respiratory tract region and type of medical unit role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients respiratory metapneumoviral infection without co-infection in association with acute and chronic lung allograft dysfunction association of minimal rejection in lung transplant recipients with obliterative bronchiolitis springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions md: designed the research, monitored the database, analyzed and interpreted the patient data, contributed to the writting of the manuscript. bv: performed the virological study, interpreted the patient data and contributed to the writting of the manuscript. ab: monitored the database, analyzed and verified the patient data, contributed to the writting of the manuscript. pm: took care of the lung transplants reciepients, contributed to the writting of the manuscript. acm: took care of the lung transplants reciepients, contributed to the writting of the manuscript. gd: took care of the lung transplants reciepients, contributed to the writting of the manuscript. nf: performed the virological study, interpreted the patient data and contributed to the writting of the manuscript. yy: designed the research, monitored the database, interpreted the patient data, wrotte the manuscript. ng: performed the bacteriological study, interpreted the patient data and contributed to the writting of the manuscript. yc: took care of the lung transplants reciepients, contributed to the writting of the manuscript. hv: took care of the lung transplants reciepients, contributed to the writting of the manuscript. gt: took care of the lung transplants reciepients, contributed to the writting of the manuscript. fxl: designed the research, analyzed and interpreted the patient data, contributed to the writting of the manuscript. the author(s) read and approved the final manuscript. no funding was obtained for this study. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. data used in this study was anonymised before its use. the authors declare that they have no competing interests. key: cord- -lwiulri authors: fragnoud, romain; flamand, marie; reynier, frederic; buchy, philippe; duong, vasna; pachot, alexandre; paranhos-baccala, glaucia; bedin, frederic title: differential proteomic analysis of virus-enriched fractions obtained from plasma pools of patients with dengue fever or severe dengue date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: lwiulri background: dengue is the most widespread mosquito-borne viral disease of public health concern. in some patients, endothelial cell and platelet dysfunction lead to life-threatening hemorrhagic dengue fever or dengue shock syndrome. prognostication of disease severity is urgently required to improve patient management. the pathogenesis of severe dengue has not been fully elucidated, and the role of host proteins associated with viral particles has received little exploration. methods: the proteomes of virion-enriched fractions purified from plasma pools of patients with dengue fever or severe dengue were compared. virions were purified by ultracentrifugation combined with a water-insoluble polyelectrolyte-based technique. following in-gel hydrolysis, peptides were analyzed by nano-liquid chromatography coupled to ion trap mass spectrometry and identified using data libraries. results: both dengue fever and severe dengue viral-enriched fractions contained identifiable viral envelope proteins and host cellular proteins. canonical pathway analysis revealed the identified host proteins are mainly involved in the coagulation cascade, complement pathway or acute phase response signaling pathway. some host proteins were over- or under-represented in plasma from patients with severe dengue compared to patients with dengue fever. elisas were used to validate differential expression of a selection of identified host proteins in individual plasma samples of patients with dengue fever compared to patients with severe dengue. among host proteins tested, two could differentiate between dengue fever and severe dengue in two independent cohorts (olfactomedin- : area under the curve (auc), . ; and platelet factor- : auc, . ). conclusion: a novel technique of virion-enrichment from plasma has allowed to identify two host proteins that have prognostic value for classifying patients with acute dengue who are more likely to develop a severe dengue. the impact of these host proteins on pathogenicity and disease outcome are discussed. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. infection by one of the four serotypes of the dengue virus (dv), a member of the flaviviridae family, can cause a wide spectrum of clinical manifestations. although the majority of symptomatic patients develop a febrile illness known as dengue fever (df) with nonspecific symptoms such as headache, fever or myalgia, around % of patients develop a more severe form of disease, severe dengue (sd), that may include plasma leakage, severe hemorrhage and organ failure [ ] . the dv genome is a positive single-stranded rna molecule encoding a polyprotein that is processed into three structural proteins (the capsid protein c, the membrane protein m, and the envelope proteins e) and seven non-structural (ns) proteins involved in replication and pathogenicity [ ] . dv enters target cells via receptor-mediated endocytosis and traffics via the endosome, where the acidic environment triggers fusion of viral and host cell membranes. once within the target cell, the virus manipulates the host cell membrane to create an optimal environment for the assembly of its replication complex and subsequent rna amplification [ ] . virion assembly occurs on the surface of the endoplasmic reticulum (er), followed by budding of an immature particle into the er lumen. the immature virion is then transported to the trans-golgi network, matured via proteolytic cleavage, and finally released by exocytosis into the extracellular medium. no specific antiviral treatment against dv currently exists. the available therapies are symptomatic and are administered to control the clinical manifestations. the ability to diagnose dv infection at an early stage and successful prognosis of the resulting complications of dengue are urgently required to improve the management of patients. it is possible that the identification of proteins specifically present in plasma before the onset of severe symptoms may ultimately lead to the discovery of new prognostic biomarkers. despite an incomplete understanding of the mechanisms of pathogenicity, several hypotheses have been formulated to explain the disease process in patients infected with dv. however, the lack of animal models capable of reproducing the features of human disease has hampered the identification of reliable parameters and indicators to explain or predict the development of sd. a number of host immune components, especially antibodies, are associated with the pathogenicity of viral infections. such mechanisms include antibody-dependent enhancement of a secondary infection or cross-reactivity with proteins such as endothelial cell or coagulation proteins [ ] [ ] [ ] [ ] [ ] . other immune components, including memory t-cells, innate immunity effectors and complement factors have been shown to modulate the outcomes [ , ] . the dv non-structural protein (ns ) may also play a major pathogenic role, as it interacts with host complement proteins [ , ] . numerous studies have investigated associations between altered levels of circulating cytokines/chemokines and dengue severity [ , ] . alternatively, as there is a strong biological rationale for investigating markers implicated in vascular pathologies, many markers such as the soluble intercellular adhesion molecule- (sicam- ), the soluble vascular cell adhesion molecule- (vcam- ), the e-selectin or the thrombomodulin have been identified and seemed to correlate with disease severity [ ] . unfortunately, no clear consensus has emerged from these studies. predicting outcome in dengue remains challenging, and the search for robust markers remains crucial. previous studies have demonstrated patients with sd have higher viremia than patients with df [ , , ] . additionally, reports from cuba and australia have suggested that circulating dv may become more virulent through passage in successive hosts during an epidemic [ ] [ ] [ ] [ ] . as the virus cycle and the virus pathogenicity are strongly linked with the host metabolism, it is assumed that host proteins interacting with virions are a reflection of pathological status of the patient. therefore, in the present study, a dv-enrichment procedure combining sucrose gradient ultracentrifugation and a polymer-based technique was developed for differential proteomic analysis of plasma pools from patients with df and sd by liquid chromatography coupled with tandem mass spectrometry (lc-ms/ms). the objective was to identify the viral proteins and the host proteins incorporated into virions, and also the host proteins that interact/are associated with virions. the identified markers were validated by quantitative elisas using samples from dengue patients from two different geographical regions. the possible role of the co-identified host proteins in disease pathogenicity and the potential of these proteins as fingerprints of disease severity in patients infected with dv are discussed. plasma samples were provided by the universidad industrial de santander, bucaramanga, colombia and the institut pasteur, phnom-penh, cambodia. samples were collected from dengue patients as part of retrospective (colombia) or prospective study (cambodia). both studies were reviewed and approved by the local medical ethics committees (universidad industrial de santander, colombia; national ethic committee, cambodia) and performed in compliance with the ethical standards set out by the declaration of helsinki. all patient plasma samples were anonymized after a physical examination and obtaining informed consent. dr villar-centeno (universidad industrial de santander, bucaramanga, colombia) and dr philippe buchy (institut pasteur, phnom-penh, cambodia) granted the authors permission to use the samples. all samples were collected between the onset of symptoms and defervescence. the cases were classified as primary or secondary infections by the physician based on hemagglutination assays performed on different dv serotypes and on japanese encephalitis virus. the serotype and the copy number were determined by real-time quantitative rt-pcr (qrt-pcr). dv-negative plasma specimens from healthy donors were obtained from the french national blood bank (etablissement français du sang, lyon, france). dengue samples were tested for the presence of the viral ns protein using the platelia™ elisa (biorad, marnes-la-coquette, france) following the manufacturer's instructions. rna was extracted from the plasma using the qiamp viral rna kit (qiagen, hilden, germany). viremia was measured using a qrtpcr kit (primerdesign southampton, uk) according to the manufacturer's instructions. qrt-pcr was performed in a final volume of μl, containing μl of extracted rna, μl of x precision onesteptm qrt-pcr mastermix, and μl of dengue primer/probe mix. assays were carried out using a lightcycler® . (roche applied science, bâle, switzerland) using the onestep amplification protocol recommended by the manufacturer. hepg cells (atcc hb- ) were cultivated at °c in % co in dmem supplemented with % decomplemented fetal calf serum, × iu penicillin, mg streptomycin and mm l-glutamine (invitrogen, paisley, uk). the cells were infected as previously described [ ] with a serotype dv (dv , strain d - thailand) graciously provided by dr v. barban (sanofi-pasteur, lyon, france). sub-confluent hepg cell cultures (approx. cells/ cm flask) were incubated with virus diluted in serum-free culture medium at various multiplicities of infection (mois) for min, the supernatant was removed, the cells were washed once with pbs (invitrogen) and ml of fresh complete medium was added to the cells. after days of culture, the supernatant was harvested and clarified by centrifugation at , g for min at °c. denaturing polyacrylamide gel electrophoresis, western blotting and silver-staining following denaturation in sds sample-buffer (novex invitrogen, paisley, uk) at °c and denaturing polyacrylamide gel electrophoresis (page) on - % polyacrylamide gels in sds-mops buffer (nupage invitrogen), samples were electro-transferred to pvdf membranes (millipore, billerica, mt, usa) in % caps- % methanol buffer, blocked in tbs- . % tween - % skimmed dried milk (régilait, macon, france), incubated with anti-e monoclonal antibody (diluted to μg/ml; biomerieux, marcy-l'etoile, france) followed by horseradish peroxidase-labeled conjugate (diluted at . μg/ml; p.a.r.i.s., compiegne, france) for h each at room temperature (rt). after washing with tbs- . % tween , the proteins were revealed using the supersignal west dura kit (thermo scientific, rockford, il, usa) and imaged using the versadoc™ imaging system (biorad, hercules, ca, usa). alternatively, the gels were silver-stained after electrophoresis using the silverxpress kit (life technologies, paisley, uk). densitometry analysis was carried out using quantity-one software (biorad). five milliliters of pre-clarified plasma pools ( g/ min/ °c; cf. table ) or ml of pre-clarified cell culture supernatant were centrifuged for h at , g using a beckman sw rotor in an optima l ultracentrifuge (beckman, fullerton, ca, usa). after centrifugation, the pellet was dissolved in a small volume of cold pbs (euromedex, souffelweyersheim, france), loaded on a discontinuous sucrose gradient constituted of ml of % sucrose in pbs (w/w) and ml of % sucrose in pbs (w/w), and centrifuged for h at , g. the fraction containing virions, located at the interface of the two sucrose solutions, was collected, diluted ten-fold in cold pbs, centrifuged for h at , g and the pellet was resuspended in μl of cold pbs. all centrifugation steps were performed at °c. ultracentrifugation was complemented by additional purification steps using viraffinty™ (biotechsupportgroup, monmouth, usa), a water insoluble elastomeric polyelectrolyte developed for the capture and recovery of viruses and μl of viraffinity™ were added to the resuspended pellet obtained after ultracentrifugation. the mixture was incubated for min at rt and centrifuged at g for min. the supernatant was discarded and the pellet was rinsed three times with mn buffer. finally, the viral fraction was separated from the polymer by heating for min at °c in sds-buffer (novex invitrogen). after a final centrifugation step at g, the supernatant was harvested and stored at − °c for immunoblotting and lc-ms/ms analysis. the following experiments were conducted by the edyp laboratory (edyp-service, cea grenoble, france). the virion-enriched preparation was loaded on a % polyacrylamide gel and electrophoresed until all proteins entered the gel. the band containing the proteins was manually excised, washed three times in buffer containing % acetonitrile and dried using % acetonitrile. proteins with a mascot score higher than (p < . ) were selected for further analysis [ ] . the mascot score is a measure of the reliability of identification: the higher the score, the better the identification. in order to eliminate false matches and incorrect protein identification, consecutive searches against the concatenated swiss-prot and trembl_decoy databases (versions . and . decoy database, respectively; homo sapiens taxonomy, , entries) were performed for each sample using mascot . software (matrix science). irma software [ ] was used to filter the results to achieve a false positive rate lower than %. in-gel digestion and lc-ms/ms analysis was performed twice for each sample. ingenuity pathways analysis (ipa) software (ingenuity systems, redwood city, ca, usa) was used to investigate the interactions among all of the host proteins identified. interactive pathways were generated to observe the potential direct and indirect relationships among the proteins that were differentially expressed in the df and sd samples. commercial elisas (uscn, wuhan, china) targeting potential severity markers were used to measure in duplicate the levels of the proteins in individual df and sd plasma specimen, using the protocols recommended by the manufacturer. for each test, a standard curve was established by serial dilution of the calibrator provided in the kit to determine the protein concentration. the optical density values were determined at nm using a microplate reader (eon; biotek, vinooski, vt, usa). statistical analysis (mann-whitney u test, chi-square test) and receiver operating curve analysis were performed using graphpad prism v. . software (graphpad software, san diego, ca, usa). the mann-whitney and the chi-square tests were used to examine differences in demographic and clinical characteristics between patients and to assess potential confounding variables. comparisons of continuous variables were performed using mann-whitney u test that can be applied on unknown distributions for two small sets of observation (n < ). comparisons of proportion were performed using chisquare test. p < . was considered significant [ ] . plasma obtained from patients with df or sd were pooled to create two samples ( table ) ; all of the plasma samples used for this step came from colombian patients who had secondary dv serotype or infections, and were collected between the onset of symptoms and the development of severe symptoms [ ] . classification of the infections as secondary infections and the degree of severity were based on review of the patients' medical records. in these medical records, the estimation of the severity was based on the who criteria of [ ]. df and dhf grade i (dengue haemorrhagic fever, minor haemorrhages) were considered as classicdengues (not severe). dhf grade iii/iv (dss, dengue shock syndrome) were considered as severe dengues. each medical records compiled details on platelet and blood cell counts, transaminase levels, the presence/absence of warning signs (persistent vomiting, abdominal pain…), haemorrhagic signs (petechiae, ecchymosis, epistaxis…) or shock signs (cold extremities, cyanosis…) that help to the patients classification by the physician. the proportion of samples from male patients was higher for the df pool ( %) than the sd pool ( %). the age of the patients (mean, years) and the number of days the samples were collected after the onset of symptoms (mean, approximately days) were similar between the two groups. no comorbidity was reported for any patient in either group. each of the individual plasma samples was confirmed as ns -positive. the viremia of each pool was estimated using a commercial qrt-pcr kit. all patients were positive for dv, indicating all samples were collected during the acute phase of disease. the average number of rna copies per ml was . × and . × for the samples in the df and the sd pools, respectively. a technique based on ultracentifugation (step ) followed by concentration using a commercial water-insoluble elastomeric polyelectrolyte specially engineered for the capture and recovery of viruses (viraffinity™; step ), was created to obtain a fraction of plasma enriched with dv particles. initially, this technique was developed using the cell culture supernatant of hepg cells harvested five days after infection with dv at a moi of or . the samples obtained after each step of the purification process (step and step + ) were analyzed by western blotting using an anti-e monoclonal antibody (fig. a) . a positive signal corresponding to monomeric ( kda) and dimeric e protein ( kda) was observed in the dv-infected samples, and became more intense after the viraffinity™ step (fig. a, step + ). the purity of the step + sample was assessed by electrophoresis using denaturing page and silver-staining (fig. b) . bovine serum albumin (bsa), an intense protein band of approximately kda was observed in the sample before purification ("not purified" lanes); however, this band was almost absent from the final purified sample ("step + " lane). only a small number of bands were detected in the purified sample. densitometry indicated that the protein complexity of the purified samples was reduced by roughly -fold compared to the unpurified samples. fig. characterization of the viral-enriched fraction of dv-infected hepg supernatant or plasma obtained from patients with dengue. a after infection with dv at a moi of or , hepg cell culture supernatants were purified by ultracentrifugation (step ) followed by the use of viraffinity™ (step + ). b silver-staining assessment of the protein complexity of dv-infected hepg cell culture supernatant before and after purification; not diluted (lane "not purified"), / diluted (lane "not purified / ") dv-infected cell supernatant before purification or the same sample as shown in lane after purification (lane "step + ") were separated by denaturing polyacrylamide gel electrophoresis and silver-stained. c electron microscopy of dv-infected hepg culture supernatant after ultracentrifugation. grids were negatively stained using uranyl acetate. bars: nm, nm. d representative western blotting analysis of plasma pools obtained from patients with dengue after purification by ultracentrifugation and viraffinity™ using an anti-e monoclonal antibody. mw: molecular mass the presence of intact viral particles was also confirmed by electron microscopy, which was performed by the ezus laboratory (université claude bernard, villeurbanne, france). after negative staining, virions could be visualized in the purified samples obtained from the supernatant of cells infected at a moi of (fig. c) . the viral membrane, including the e spikes, was visible. the spikes observed on the virion surface are probably due to the slightly basic ph (ph . ) of the pbs-buffered solutions used during centrifugation and electron microscopy. virions tended to aggregate in clusters of - viral particles as seen in images taken at a lower magnification. little background signal was observed around the virion clusters. following the two-step purification process, the moi sample was also analyzed by nano-lc ms/ms. all structural viral proteins were identified ( table ). the e protein peptides were the most largely represented ( peptides; single peptides). the sequence coverage for the e protein represented % of the entire protein. the protein m, which is part of the external viral layer, along with the e protein, was identified three times ( single peptides) with coverage of more than % of the protein sequence. the pr peptide and capsid were also identified (one peptide each). in conclusion, combining ultracentrifugation with viraffinity polymer yielded a fraction with a considerably reduced protein complexity that contained a large quantity of enveloped virions. this technique was then applied to the pooled plasma samples obtained from the colombian patients (table ) . after purification, the preparations were analyzed by western blotting using a monoclonal antibody directed against the e viral protein (fig. d , sd pool as an example). strong signals corresponding to monomeric ( kda) and dimeric e protein ( kda) were observed. the e protein was not detected in purified plasma obtained from healthy individuals, which was tested as a mock-control. to identify the proteins present in the purified samples and to compare their composition, nano-lc-ms/ms was conducted on the purified df and sd plasma pools. this experiment was also performed on the purified mock-control sample to determine the host background. the same quantity of total protein was analyzed for each sample. these experiments were performed independently twice. both viral and host peptides were identified in the purified pooled plasma samples from the patients with df and sd. one peptide corresponding to the viral envelope protein was detected twice in the sd sample; this peptide (gwgngcgllfgk) was also identified in the purified dv-infected hepg supernatant (table ) . after removal of the mock-control background, the remaining peptides identified in the df and sd samples were analyzed. in order to consolidate the results obtained by nano-lc ms/ms, only proteins for which the variance of the average number of peptides was lower than % were selected [ ] ; host proteins met this criterion. the sd/df peptide ratio was calculated for these proteins (see additional file ). the highest sd/df ratio was obtained for c s esterase (sd/df peptide ratio = . ) and the vitamin k-dependent protein s (ratio = ). the lowest ratio was obtained for beta- spectrin (ratio = . ). six proteins were only identified in the sd sample; consequently, the sd/df ratio could not be calculated for these proteins. ingenuity pathway analysis (ipa) was conducted to further elucidate the specific pathways associated with the identified host proteins. the specific location and function of each pathway was attributed by ipa for of the proteins; the most prominently represented pathways are illustrated in fig. . the ipa diagram shows that the highest p-values were attributed to acute phase response signaling [−log(p-value), lp = ], the lxr/rxr activation pathway (lp = . ), the complement system (lp = . ), the coagulation system (lp = . ) and the extrinsic prothrombin activation pathway (lp = . ). the p-values indicate the likelihood that the focus genes in a network are found in these pathways by random chance alone. although represented to a lesser degree, three pathways related to host proteins involved in the dv cell cycle, clathrin-mediated endocytosis signaling (lp = . ), caveolar-mediated endocytosis signaling (lp = . ) and the virus entry via endocytic pathway (lp = . ), were also identified. other identified pathways, including integrin (lp = . ) and paxillin signaling (lp = . ), correspond to proteins involved in cell-matrix interactions and cell-to-cell communication. figure also illustrates the ratio of identified proteins for each canonical pathway. higher proportions of proteins mapped to the complement system (ratio = . ), coagulation system (ratio = . ) and extrinsic prothrombin activation pathway (ratio = . ) than the other pathways (ratios between . and . ). ipa software was also used to investigate possible interactions among all of the identified proteins and to assess the representation of the identified proteins in acute phase response signaling, the complement system and the coagulation system with prothrombin activation (see additional file ). twenty of the ( %) secreted proteins identified by ipa as part of the acute phase response signaling pathway, which is activated in macrophages and endothelial cells upon infection and inflammation, were over-represented in the pooled sd plasma sample compared to the pooled df plasma sample. thirteen complement component proteins were also over-represented in sd plasma, with high numbers of peptides identified for c r and c s in particular. complement factor c and complement factor b (cfb), which are involved in the complement alternate pathway, were only identified in the pooled sd plasma sample. the c protein was enriched in the pooled df plasma sample. fig. ingenuity pathway analysis for host proteins identified in the viral-enriched plasma fraction of patients with dengue. pathway classification according to canonical pathways was performed using ipa software. the x-axis represents the pathways identified. the y-axis (left) shows the − log of the p-value calculated using fisher's exact test. the ratio (y-axis, right) represented by the line is calculated as follows: number of proteins in a given pathway that meet the cutoff criteria divided by total number of proteins that make up that pathway the extrinsic and intrinsic prothrombin activation pathways are part of the coagulation system. the majority of the proteins involved in the fibrinogen/fibrin cascade (i.e. proteins out of ) were over-represented in the sd sample compared to the df sample. overall, the network analysis also provided evidence of strong links between these three pathways. the coagulation factor (f ) and the c and c proteins are located at the interface of the coagulation system and the complement system (see additional file ). to validate the mass spectrometry data, the levels of selected host proteins were assessed by quantitative elisa both in the virus-enriched fraction and in individual plasma samples from df or sd patients. for the elisa, proteins with a sd/df ratio higher than . or lower than . were selected (see additional file ); other criteria, such as the number of peptides identified in the sd sample and the availability of a commercial elisa kit were also considered. some proteins, such as ribosomal protein p (accession number p ; ratio = . ) and histone h (accession number p ; ratio = . ) were not deemed relevant enough for the study and were consequently not tested. the proteins readily found at high concentrations in plasma and known to be frequent contaminants in proteomic experiments, such as the immunoglobulin heavy chain (accession number p ; ratio = . ), were not tested. finally, a non-exhaustive list of proteins to assay was established. these proteins were ceruloplasmin (cp), vitamin k-dependent protein s, complement factor properdin, antithrombin iii, secretory component p , complement factor c r (c r), complement factor c s, angiotensin, factor , anti-factor viii, serum amyloide p-component, olfactomedin- (olfm ), thrombospondin, gelsolin, platelet factor (pf ), complement factor c q, complement factor b, moesin and complement factor c . multimerin- , apolipoprotein b- and von willebrand factor were also tested. these proteins were first quantified in the virusenriched fraction of the individual plasma samples obtained from df or sd patients of the colombian cohort. the elisa signal levels for these samples were close to background levels (purified mock-control sample); the results were not interpretable. to investigate the potential interaction of these proteins with the virion, fresh plasma was incubated for various times with purified viral particles. the change of protein concentration before and after the incubation was assessed by elisa. this experiment was performed for proteins out of (olfm , cp, c r, pf ) and showed that no significant signal change was observed for these proteins, whatever the condition tested (data not shown). the virus preparation is probably not concentrated enough to induce a significant variation in host protein concentration. furthermore, the plasmatic concentration of the four proteins is too high to be significantly affected by the interactions with the virus coated in the microplates. the elisas were then performed on the whole plasma samples obtained from colombian patients ( table , colombian patients). all patients had secondary infections associated with various dv serotypes (dv , dv and dv ); all samples were collected between onset and defervescence (between days and ). the male/female comorbidity ( %) ( %) - ( %) ( %) -iu international units, ns not significant, na not applicable chi-square or mann-whitney tests were used to analyze the differences between groups sex ratio and age were similar between the groups of patients with df or sd. the samples were confirmed positive for both dv by qrt-pcr and the viral ns protein using a commercial elisa (ns platelia™). no comorbidity was recorded in any patient. among the proteins tested, only cp, c r, olfm and pf , had different plasma concentrations between df and sd patients. the average concentration of c r was higher for patients with sd than patients with df (p = . ). moreover, the variance between the two groups was significantly different (p = . ). the protein concentration of olfm was considered higher for sd patients (p = . ). for cp, the difference between the two groups was significant (p = . ). the largest difference was observed for pf with a higher concentration for patients with df (p < . ; fig. ). to further confirm the relevance of c r, cp, olfm and pf as potential markers to differenciate df and sd, plasma samples from patients with acute dengue from cambodia, another dengue-endemic area, were tested. as for colombian patients, the cambodian patients were classified by the local physician using the who classification of . for the present study, patients classified df or dhf grade i were considered as classic dengue. patients classified dhf grade iii and iv were considered as severe dengue. clinical data were collected and included details on the platelet count, transaminases level, blood cells count, biochemistry (cholesterol, triglycerides, bilirubin…), ultrasound imagery, hemorrhagic signs (petechiae, ecchymosis, epistaxis…), the presence/or absence of warning signs (persistent vomiting, abdominal pain…), plasma leakage and shock signs (cold extremities, cyanosis…). the main characteristics of the cambodian patients are detailed in table (cambodian patients). the mean age of patients from cambodia (about years-old) was lower than that of the colombian patients. the male/female ratios of the df and sd groups were similar. all cambodian patients were infected with serotype viruses. the plasma samples were collected approximately days after the onset of symptoms (=acute samples) and also just before the discharge from the hospital (=discharge samples). in contrast to the samples obtained from the colombian patients, elisas showed that the levels of c r and cp were not significantly different between df and sd patients in the cambodian cohort (see additional file ); however, concentrations of olfm (p < . ) and pf (p < . ) during the acute phase were significantly different between df and sd plasma (fig. ) . the olmf concentration in df patients was lower than in sd patients. in contrast, pf concentrations were higher in df patients than in sd patients and sd patients showed no significant difference with healthy controls (n = ). for these two markers, at the time of discharge, concentrations tended to decline back to the levels observed in controls. receiver operating characteristic (roc) curves compare sensitivity versus specificity across a range of values. roc curves are used to assess the discriminatory ability of each markers. area under the roc curve (auc) is another measure of test performance. the auc quantifies the fig. assessment of the protein concentrations of c r, cp, olfm and pf in the individual plasma samples of colombian patients using specific quantitative elisas. each value corresponds to the mean of two independent experiments, each performed in duplicate. *: . < p < . ; **: . < p < . ; ***: p < . overall ability of the test to discriminate between individuals with df and those with sd. a perfect test (zero false positives and zero false negatives) has an auc of . . roc curve analysis was used to determine aucs and specificity/sensitivity values for olfm and pf as prognostic biomarkers of disease severity in cambodian acute patients with dengue. the roc curves indicated good discrimination between df and sd as the auc were . for olfm and . for pf . the highest specificity/sensitivity values obtained using this model are summarized in table . for a sensitivity of %, the specificity exceeded % (pf ) or %(olmf ). when the sensitivity reached %, the specificity dropped to . % (pf ) or . % (olfm ). dengue viral infections are prevalent in tropical and subtropical areas, and are associated with substantial morbidity and mortality. the pathogenesis of dengue remains unclear. various proteomic approaches have been used to characterize host-protein changes during dv infection and identify prognostic biomarkers of disease severity; several studies have been conducted on cells infected in vitro [ ] [ ] [ ] . studies on plasma specimens have identified a number of candidate biomarkers [ ] [ ] [ ] . as the dynamic range for plasma proteins is large (over orders of magnitude) and the most abundant proteins ( . % of the total number) constitute up to % of the plasmatic protein mass, identification of biomarkers present at low concentrations is a major challenge in proteomic studies dealing with plasma. however, proteomic techniques are constantly being improved to assess low abundance plasma proteins [ ] . viruses facilitate their replication and propagation by subverting host cellular pathways and processes, and constantly adapt to and modulate their host's environment. enveloped viruses are able to incorporate numerous host proteins, both into virus particles as well as the host-derived viral envelope. as the genomes of rna viruses only encode a small number of proteins, they must rely on host proteins during an infection. interactions between the virus and host may have unforeseen consequences, depending on viremia level, the host's genetic background and other relevant factors [ ] . host proteins can be incorporated into virions either randomly, by being present at the site of budding, or specifically, as a result of interacting with viral proteins. the functional significance of the host proteins that associate with viral particles remains to be thoroughly investigated; however, it is very likely that such interactions contribute to pathogenicity if they disturb the metabolism of the host cell [ ] . for instance, the incorporation of cellular proteins such as integrins or hla class ii proteins can affect the ability of hiv- to infect host cells and contributes to immunopathogenesis [ , ] . viroproteomic analyses have already been conducted on dna viruses [ ] and rna viruses, such as retroviruses [ , ] , paramyxoviruses [ ] , coronaviruses [ ] and flaviviruses [ ] ; these studies have proven that many cellular proteins are incorporated into the virion or associate with the viral membrane. usually, prior to ms analysis, protease hydrolysis is combined with ultracentrifugation to remove host proteins that may co-purify with virions. in the present study, as we aimed to characterize the host proteins that interact with viral membrane proteins and particles, enzymatic hydrolysis of the proteins outside of the virion could not be performed. therefore, a new technique combining ultracentrifugation with water-insoluble polyelectrolyte-based enrichment was developed to enable proteomic characterization of a fraction of human plasma enriched with virus particles and depleted of the host proteins predominantly expressed in plasma. previous studies measuring viremia have demonstrated that patients with sd have higher viremia than patients with df [ , , ] . higher viremia may lead to biosynthesis of virions with an altered host protein composition, which may reflect an increased level of cellular stress, or subtle changes in the assembly pathway. these host proteins may be packaged into the virus particle along with the viral components or incorporated into the viral membrane. alternatively, they can be simply co-purified along with virions. such proteins may potentially be fingerprints of the virus assembly pathway and may also play a role in viral pathogenicity. nano lc-ms/ms was used in this study to analyze the protein composition of the virion-enriched samples purified from the plasma pools. some proteins had a higher average number of peptides in the sd sample than the df and control samples, suggesting the presence of greater amounts of these proteins associated to viruses purified from the plasma of patients with sd; conversely, other proteins were mainly identified in the samples purified from the df plasma pool. interestingly, the identified host proteins belong to three main pathways: the complement system, the coagulation system and the acute response signaling pathway. the complement system plays an important role in both innate and adaptive immune responses, is first line of defense against infectious agents, and modulates b-and t-cell responses. in flavivirus infections, excessively-activated complement proteins have been reported to induce a deleterious, exacerbated inflammatory response [ ] [ ] [ ] . recently, it has been shown that the level of complement proteins positively correlates with the severity of dengue in indonesian patients [ ] . coagulation is the process by which the blood changes from a liquid to a gel. coagulation is required to stop blood loss and enable the subsequent repair of damaged vessels. hemorrhage is one of the major symptoms of sd, and is probably caused by vasculopathy and a deficiency in coagulation and fibrinolysis. the normal vascular endothelium produces inhibitors of coagulation and fibrinolysis. hemostasis can be impaired if the endothelium is stimulated by excessive levels of cytokines or by a pathogen, which may in turn result in thrombosis and bleeding [ , ] . the acute phase response corresponds to the inflammatory response observed in response to an infection, tissue injury or an immunological disorder [ ] , and is mainly characterized by increased levels of inflammatory factors and a change in the protein composition of plasma. interestingly, these changes can inhibit complement activation [ ] . interconnections between the coagulation process and complement cascade have been reported by many authors [ , ] . deregulation of one or both systems can result in the clinical manifestations of diseases with inflammatory complications. in a previous study, differentiallyexpressed genes associated with the immune response were identified by microarray analysis of peripheral blood mononuclear cells isolated from colombian children with dengue fever or dengue hemorrhagic fever. these results indicated that the complement and numerous cytokines are deregulated in patients with dengue-hemorrhagic fever. such changes may enhance disease severity by disturbing coagulation and inducing endothelial cell damage [ ] . in another recent study, sera from indian patients with df were compared with that of healthy controls using d-dige associated with maldi tof/tof ms. the authors reported that dv infection led to altered expression of multiple serum proteins involved in complement cascades, blood coagulation and acute phase response signaling, providing further clues regarding the pathogenesis and host immune response to dv infection [ ] . the complement system, coagulation system and acute response signaling pathway are strongly interconnected and share proteins whose expression is modulated during an infection. they are all related to the inflammatory process and the innate immune response, and act upstream of activation of the adaptive immune response. expression deregulation of these proteins could induce deleterious effects in endothelial cells [ ] . in our hands, none of the seven complement proteins tested by elisa showed a difference in concentration levels between sd and df acute samples, minimizing the potential role of these proteins in dengue pathogenesis, at least in the early phase of the disease. several authors have reported proteomic analysis of samples from patients with dengue using different approaches [ , , ] . to our knowledge, this study is the first assessment of the proteins that are differentially present in a virus-enriched fraction purified from plasma specimens obtained from patients during the acute stage of dv infection. we were unable to confirm that proteins identified by lc-ms/ms were directly associated with the viral particles. neither elisas conducted on virions purified from plasma, nor electron microscopy using specific goldlabeled antibodies succeed to yield reliable results. consequently, we cannot discard the possibility that the host proteins identified in this study may be contaminants that co-purify reproducibly with viral particles. elisas carried out on individual plasma specimens from the colombian cohort indicated trends towards higher levels of cp, c r and olfm in patients with sd compared to patients with df. cp, an acute phase protein, is a ferroxidase involved in iron metabolism. elevated levels of cp are generally observed during inflammation. c r is a complement protein that interacts with c s at the beginning of the complement cascade. olfm is an anti-apoptotic factor that promotes tumor growth and facilitates cell adhesion, probably via interacting with cell surface lectins and cadherin [ ] . the associations between these proteins and disease severity in dengue remain to be explored. interestingly, a recent paper mentioned the interest of olfm as a marker of disease severity in respiratory syncytial virus infection in children [ ] . elisas also showed that the concentration of pf was higher in the plasma samples of colombian patients with df than patients with sd. pf is a small cytokine released by platelets that promotes blood coagulation by moderating the effects of heparin-like molecules. pf also plays a role in wound repair and inflammation. alterations in platelet function have been associated with plasma leakage, which is one of the major features of severe disease in patients with dengue [ ] . recently, it has been demonstrated that dv replicates and produces infectious virus in platelets [ ] . in , srichaikul t. et al. demonstrated that the level of pf increases during acute phase in both shock or non-shock dhf children. it is difficult to compare these results with the results presented here because there is no strict correspondence between the patient classification used by srichaikul t. et al. and the classification of used in the present work [ ] . interestingly, among the six proteins involved in coagulation and having a sd/df peptide ratio higher than . or lower than . (see additional file ), pf is the only one that was confirmed by elisa. pf is significantly less concentrated in sd acute patient plasma. the trends observed for olfm and pf by elisas for the colombian patients were also confirmed in the cambodian patients. by testing different time points during the course of the disease, we showed that these markers evolved through time and tended, during the process of healing, to get close to the protein concentration found in healthy patients. pf was overabundant in df acute patients, but remained surprisingly low in sd patients, with levels remaining similar to control individuals. it has previously been proposed that pf interacts with the vasculature and is involved in thrombus formation at sites of vascular injury [ ] . murine studies suggest that pf may have an overall salutary effect in sepsis [ ] . therefore, a basal level of pf expression during the acute phase of dv infection could be an interesting marker of a future severe dengue. the severity of dengue is modulated by multiple risk factors such as the age, genetic background and nutritional status of the host, as well as the genotype and serotype of the virus. these factors could explain the differences in the concentrations of specific markers observed between the plasma samples from the colombian and cambodian patients. for example, sd in south-east asia is mainly observed in children, whereas adults are predominantly affected in south america [ ] . as a consequence, results should be extrapolated with caution to different geographic areas or different demographic groups. in conclusion, we describe the development of a novel technique of dv-enrichment from complex biological fluids based on centrifugation combined with a waterinsoluble polyelectrolyte-based technique, for subsequent proteomic analyses. we found no evidence that the identified host proteins are specifically associated with virions. however, this purification technique enables the analysis of a plasma fraction enriched in virions and from which the most abundant plasma proteins were removed. the host proteins characterized in this study may potentially reflect how dv infection disturbs the function of the cellular proteome. in this regard, further studies are required to assess the prognostic value of host proteins associated with inflammation, complement cascade and coagulation for disease severity by analysis of additional biological samples from patients infected with dv. analysis of a selection of the identified proteins using elisas identified two host proteins, olfm and pf , which had significant prognostic value for classifying patients with dengue who were likely to develop sd. further prospective studies are warranted to confirm and validate the prognostic value of olfm and pf as potential biomarkers of disease severity in larger cohorts of patients from a variety of dengue-endemic areas around the globe. complement factor r; ci: confidence interval; cp: ceruloplasmin; df: dengue fever; dige: difference gel electrophoresis; dv: dengue virus; elisa: enzyme linked immunosorbent assay; er: endoplasmic reticulum; iu: international unit; lc-ms/ms: liquid chromatography coupled to mass spectrometry chain; mes: -(n-morpholino)ethanesulfonic acid; moi: multiplicity of infection; na: not applicable; ns: non-structural references . world health organization. dengue: guidelines for diagnosis, treatment, prevention and control: new edition. geneva: world health organization recent advances in deciphering viral and host determinants of dengue virus replication and pathogenesis modification of intracellular membrane structures for virus replication the dengue virus nonstructural- protein (ns ) generates antibodies to common epitopes on human blood clotting, integrin/adhesin proteins and binds to human endothelial cells: potential implications in haemorrhagic fever pathogenesis molecular mimicry between virus and host and its implications for dengue 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syndrome the host complement system and arbovirus pathogenesis correlation between complement component levels and disease severity in dengue patients in indonesia hemostatic defects in dengue hemorrhagic fever is clinical outcome of dengue-virus infections influenced by coagulation and fibrinolysis? a critical review of the evidence cytokines and the hepatic acute phase response complement factor b gene regulation: synergistic effects of tnf-alpha and ifn-gamma in macrophages complement and coagulation: strangers or partners in crime? interactions between coagulation and complement-their role in inflammation comparison of the transcriptional profiles of patients with dengue fever and dengue hemorrhagic fever reveals differences in the immune response and clues in immunopathogenesis serum proteome changes in dengue virus-infected patients from a dengueendemic area of india: towards new molecular targets? a physical interaction network of dengue virus and human proteins olfactomedin is a novel target gene of retinoic acids and -aza- ′-deoxycytidine involved in human myeloid leukemia cell growth, differentiation, and apoptosis olfactomedin serves as a marker for disease severity in pediatric respiratory syncytial virus (rsv) infection platelet function alterations in dengue are associated with plasma leakage dengue virus binding and replication by platelets platelet function during the acute phase of dengue hemorrhagic fever interactions of platelet factor with the vessel wall role of the platelet chemokine platelet factor (pf ) in hemostasis and thrombosis dengue viruses -an overview we would like to thank dr. y. coute data supporting the findings and materials are available upon request to frederic bedin, biomerieux sa, chemin de l'orme, marcy l'etoile (france). additional file : virus-enriched fraction proteome change in purified plasma pools obtained from acute dengue patients. authors' contributions rf, mf, fr and fb carried out all the experiments and performed the statistical analysis. rf and fb drafted the manuscript. ap, fr and gb participated in the design of the study. rf, fb, gb participated to draft the manuscript. dv and pb collected cambodian samples and managed the patient data and ethics. all authors read and approved the final manuscript. key: cord- - u rlwzp authors: biribawa, claire; atuhairwe, joselyn annet; bulage, lilian; okethwangu, denis othuba; kwesiga, benon; ario, alex riolexus; zhu, bao-ping title: measles outbreak amplified in a pediatric ward: lyantonde district, uganda, august date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: u rlwzp background: measles is a highly infectious viral disease. in august , lyantonde district, uganda reported a measles outbreak to uganda ministry of health. we investigated the outbreak to assess the scope, factors facilitating transmission, and recommend control measures. methods: we defined a probable case as sudden onset of fever and generalized rash in a resident of lyantonde, lwengo, or rakai districts from june- september , plus ≥ of the following: coryza, conjunctivitis, or cough. a confirmed case was a probable case with serum positivity of measles-specific igm. we conducted a neighborhood- and age-matched case-control study to identified exposure factors, and used conditional logistic regression to analyze the data. we estimated vaccine effectiveness and vaccination coverage. results: we identified cases ( probable, confirmed); patients ( . %) died. in the case-control study, % of case-patients and . % of controls were hospitalized at lyantonde hospital pediatric department for non-measles conditions – days before case-patient’s onset (or(adj) = , %ci: . – ). estimated vaccine effectiveness was % ( %ci: – %) and vaccination coverage was % ( %ci: – %). during the outbreak, an “isolation” ward was established inside the general pediatric ward where there was mixing of both measles and non-measles patients. conclusions: this outbreak was amplified by nosocomial transmission and facilitated by low vaccination coverage. we recommended moving the isolation ward outside of the building, supplemental vaccination, and vaccinating pediatric patients during measles outbreaks. starting approximately four days before to four days after rash onset [ ] . treatment for measles virus infection is only supportive and most patients recover within to weeks. however, measles can cause serious complications, including blindness, encephalitis, severe diarrhea, ear infection, and pneumonia, particularly in malnourished children and immune-compromised patients [ ] [ ] [ ] . the casefatality rate in developing countries is usually - %; however, in some localities it may be as high as - % [ , ] . measles vaccination is the best strategy for preventing measles outbreaks and achieving the goal of measles elimination. to effectively prevent measles outbreaks and achieve the goal of measles elimination, who recommends a -dose vaccination administered at and - months of age, and a vaccination coverage (vc) at ≥ % for the -dose vaccination schedule [ ] . the uganda national expanded programme on immunization, as in most countries in the who-afro region, currently implements a one-dose vaccination at months of age. however supplemental immunization activities are organized periodically to interrupt transmission and spread. on august , the uganda ministry of health (umoh) received a notification of a measles outbreak in lyantonde district. serum samples from six suspected measles patients tested positive for measles-specific igm at the uganda virus research institute. to support the district in controlling this outbreak, we conducted an epidemiological investigation to determine the scope of the outbreak, assess risk factors for transmission, assess vaccine effectiveness and recommended evidence-based measures to prevent future outbreaks. the outbreak occurred in a tri-district area in central uganda comprising lyantonde, lwengo, and rakai districts. the estimated total population was approximately , ( , in lyantonde, , in lwengo, and , in rakai), based on the projected populations from the census [ ] . the three districts border each other and share several public hospitals in lyantonde district. we defined a probable case as sudden onset of fever and generalized rash in a resident of the tri-district area from june to september , plus ≥ of the following: coryza, conjunctivitis, or cough. a confirmed case was a probable case with measles-specific igm positivity. for case-finding, we reviewed outpatient and inpatient records at health facilities in the tri-district area, and actively searched for cases with the help of members of village health teams and community leaders. we collected data on patients' symptoms, onset dates of symptoms, treatment outcomes, demographic characteristics, place of residence, receipt of care, and vaccination status. we analyzed the line-listed cases by onset of symptoms, age, sex, and place of residence. to calculate attack rates (ar) by age and sex, we used the estimated population in the tri-district area, provided by uganda bureau of statistics [ ] . we drew a choropleth map using qgis software to describe the ars by sub-county for lyantonde district, where most ( %) of the cases came from. using a semi-structured questionnaire, we interviewed a convenience sample of caretakers for case-patients regarding their potential exposures during their likely exposure period (i.e., - days before their rash onset, or between minimum and maximum incubation periods). the exposures of interest included attending social gatherings, attending worship places, visiting health facilities, visiting communal gathering points, and immunization status. we generated hypotheses about exposures based on findings from the descriptive epidemiology analysis and hypothesis-generation interviews. we conducted a case-control study to test the hypothesis on potential exposures. at the time of the casecontrol study, case-patients were line-listed. we recruited of those case-patients aged ≥ year to participate in the case-control study. for each case, we selected controls in the same immediate neighborhood (i.e., within three homes of the case-patient's) who had no measles symptoms from july to september . we individually matched controls to the case by age (± years). we assessed the exposure risk factors for both the case-patient and the matched controls during the case-patient's likely exposure period, using a structured questionnaire. vaccination status was determined by either reviewing the vaccination records or, if unavailable, by asking whether the child had received an injection on the upper arm at months of age (which is the standard practice for measles vaccine in uganda). cases and controls were considered vaccinated only if they were vaccinated prior to the onset of the outbreak. we also collected data on demographic characteristics (e.g., age and sex) of both case-patients and controls. to account for individual matching in the study design, we used conditional logistic regression to analyze the data, using the matched set as the matching variable. we first assessed the association between each individual risk factor and measles. risk factors that were statistically significant at the p < . level during the univariate analysis were included in the multivariate conditional logistic regression model to calculate the adjusted odds ratios (or adj ) and their associated % confidence intervals (ci). non-significant variables in the multivariable model (p ≥ . ) were backward-eliminated until all were significant. multivariate logistic regression analysis was used to control for potential confounding variables that could be included in the model by holding all the other variables constant. we estimated measles ve using the following formula [ ] : where or adj is the odds ratio associated with having been vaccinated for at least one dose of measles vaccine, adjusted for risk factors that were significant during the univariate analysis, using conditional logistic regression. we estimated the vc using the percentage of controls with a history of measles vaccination in the case-control study, assuming the controls to be representative of the general population. we also obtained the administrative data from the district surveillance officer on vc in lyantonde district. we observed the layout of the pediatric department at lyantonde hospital, especially the pediatric wards in question, and examined the ventilation system. we also reviewed the patient log to assess the type of illnesses admitted in the pediatric department. between june and september , we found cases ( probable and confirmed) in the tri-district area, with four deaths (case fatality rate = . %). of these cases, were from lyantonde, from rakai, and from lwengo district. common symptoms included fever ( %), rash ( %), coryza ( %), cough ( %), and conjunctivitis ( %). among those that died, there was one female and males, aged - months. only one case that died had history of one dose measles vaccination. the major cause of death was respiratory complications ( / ) and the cause of death could not be ascertained for one of the cases. all the cases that died were managed at home for the measles virus infection. in addition of those cases had other underlying disease conditions. the initial cases had rash onset on june. cases started to increase in july and august, and the last case occurred on september. the epidemic curve was indicative of a propagated outbreak. an emergency mass vaccination was rolled out in the tridistrict area. this involved health facility based mass vaccination campaign for all children below years of age (fig. ) . of all age groups, children aged months- years (ar = / , ) and those - months (ar = / , ) were the most affected. females (ar = . / , ) and males (ar = . / , ) were similarly affected ( table ) . the attack rate in the tri-district area was . / , . the initial cases in june occurred in rakai district. the outbreak started to affect lyantonde district in july, and later spread to villages in lwengo district bordering lyantonde district. lyantonde district had the highest ar ( / , ) of all districts (with % of all cases). within lyantonde district, lyantonde rural sub-county was the most affected (ar = / , ) (fig. ) . of the probable measles case-patients interviewed, % reported having visited lyantonde hospital during the weeks before onset of symptoms; % of the patients reported having gone to school, % had visitors with measles at home, % went to a church, and % went to communal water-collection points; % of the case-patients had no history of measles vaccination. in the case-control study, case-patients and controls were comparable in mean age ( . years among casepatients vs. . years among controls) and sex distribution ( % of case-patients and % of controls were males). during the bivariate analysis, % of casepatients and . % of controls had been hospitalized in the pediatric department of lyantonde hospital for nonmeasles conditions - days before case-patients' rash onset (or = , % ci: . - ). visiting any health facility - days before case-patient's rash onset was a significant risk factor. going to church and going to communal water collection points were inversely associated with illness. in the final conditional logistic regression model, hospitalization at the pediatric department (or adj = , %ci: . - ), going to communal water collection points (or adj = . , %ci: . - . ) and measles vaccination history (or adj = . , %ci: . - . ) remained significant. all vaccinated cases and controls reported received at least one dose of measles vaccine. the associations of measles with other risk factors became non-significant ( table ) . the estimated ve was % ( % ci: - %). the estimated vc, based on the percent of controls that had a history of measles vaccination, was % overall, and did not differ greatly between age groups ( table ). the estimated vc based on administrative data for lyantonde district was %. the pediatric department at lyantonde hospital had wards. initially, measles patients and other patients were mixed in the same wards because the measles diagnoses had not been made. after the measles outbreak was confirmed based on results from uganda virus research institute, the hospital attempted to put non-measles patients into ward and suspected measles patients into ward . however, the wards were adjacent to each other and only separated by a half-constructed wall; air moved freely between the wards. moreover, when ward exceeded its capacity, some non-measles patients were transferred into ward . windows of both wards were kept closed. later during the outbreak, a windowless "isolation room" was set up to hold critical measles patients. the "isolation room" was at the extreme end of the pediatric department; patients had to pass through the whole department to access this room, allowing measles and non-measles patients to mix (fig. ) . during our environmental assessment, we observed free mixing of measles and non-measles patients in the reception area. this measles outbreak was facilitated by mixing of measles and non-measles patients when they were hospitalized in the pediatric department of lyantonde hospital. as in previous studies, the role of uncontrolled nosocomial transmission of measles in the propagation of community outbreaks cannot be refuted [ ] [ ] [ ] . in this study on-site assessment of the pediatric department revealed infection-control lapses, this likely facilitated measles transmission. as non-measles patients were infected from the measles patients and developed symptoms, they returned to seek care at the pediatric department and transmitted the disease to other nonmeasles patients, creating vicious cycles of transmission. the subsequent public health interventions, e.g., moving the "isolation" room outside of the pediatric department and the emergency mass vaccination campaign might have helped to break the vicious cycles. measles viruses become airborne after patients expel the droplets by coughing or sneezing. these droplets may remain suspended in the air and remain infectious for up to h after the infectious patient has departed the area [ ] . transmission occurs when susceptible individuals share the same confined spaces with, or within h a some records had missing values for exposure variables, including for "hospitalized at pediatric department, lyantonde hospital", for "went to communal water point", for "went to church", and for "went to school". these records were excluded from the respective analysis b or = crude odds ratios from univariate conditional logistic regression analysis, in which the matching variable was the case-control set c or adj = odds ratios from multivariable conditional logistic regression d case-patient's likely exposure period = - days (minimum-to-maximum incubation periods) before case-patient's rash onset of the departure of infectious patients [ ] . however since patients can become infectious as early as four days before the classic measles rash appears, infection can spread for days before the need for isolation becomes apparent, making healthcare facilities a fertile ground for measles transmission [ ] [ ] [ ] [ ] [ ] [ ] [ ] . preventing nosocomial transmission should be an important part of the overall measles control strategies [ ] . when a non-immune person is exposed to measles virus, a prophylactic measles vaccination could provide protection if administered within h of exposure [ , ] . therefore, researchers have advocated for pediatric departments to offer measles vaccination to pediatric patients during active measles outbreaks to prevent nosocomial transmission, especially in pediatric healthcare settings [ ] . in addition who recommends that a supplementary dose of measles vaccine is given to infants from months of age during a measles outbreak as part of intensified service delivery [ ] . however, these approaches were not used during this outbreak. measles is endemic in uganda. outbreaks have been recently reported in various parts of the country, and this outbreak is likely linked to one of more of those recent outbreaks, several of which have also been linked to exposure in healthcare settings [ ] . in another outbreak, measles was associated with congregation of children at water-collection points [ ] . in this outbreak, visiting water-collection points was protective. this might be because going to the communal water-collection point was a sign of being healthy and healthy children would have less chance of going to and being exposed at the pediatric department of lyantonde hospital. in this study history of measles vaccination was protective of measles virus infection. measles vaccination is the best strategy for preventing measles outbreaks and achieving l measles elimination [ ] . who recommends a -dose vaccination administered at and - months of age, and a vc at ≥ % for the -dose vaccination schedule [ ] . the estimated ve for the one-dose vaccination during this investigation ( %) was higher than previous estimates of % [ ] % [ ] and % based on a review of published literature on the field effectiveness of live attenuated measles containing vaccines [ ] ; however, the confidence interval for the current estimate ( - %) overlaps with those of the previous estimates [ - % [ ] and - % [ ] , respectively]. observed ve in the field varies and is influenced by many factors, such as number of doses administered, vaccine quality, cold-chain failure, and host factors [ ] . in addition to ensuring a high ve, maintaining a high vc through routine vaccination activities is crucial for measles control. who-afro has set a target of ≥ % in vc to achieve herd immunity for preventing measles outbreaks [ ] . in this investigation, the overall estimated vc for all persons was %, the age-specific vc estimates were ranging from to % and the administrative vc estimate was %. all these were well below the who-afro target of ≥ %. the low vc likely facilitated the current outbreak. recent investigations in uganda [ , ] and in other countries [ , , ] have also attributed measles outbreaks to low vc. these low vc estimates showed weaknesses in the routine vaccination system. this investigation had multiple limitations. due to the need of providing a quick answer for outbreak control, we only included early cases in the case-control study, which might have severely limited the power of the study to find exposure risk factors other than the most overwhelming ones. moreover, the exposure risk factors of the early cases might have been different from the ones during the later stage of the outbreak. we used controls in the case-control study to estimate vc. by using this method, we assumed that the controls were representative of the general population. this assumption might not be valid, thereby introducing bias in the vc estimate. however, the vc estimated from the casecontrol study ( %) was close to the administrative vc ( %); the latter is known to often overestimate the true vc. these data suggested that the bias, if any, might have not been substantial. the vaccination status of some of the children was based on their parents' recall, which might have been inaccurate. we conclude that this measles outbreak was amplified through nosocomial transmission in the pediatric department in lyantonde hospital. at our recommendation, lyantonde hospital moved the isolation ward to a room outside of the building, which did not share air with the other buildings of the hospital. the hospital also triaged suspected measles patients (with fever and rash) and isolated them in this properly-constructed new separated room. subsequent to the outbreak, the district health authorities conducted training of the village health teams on signs and symptoms of measles, and on the appropriate process to promptly report suspected measles cases. in addition, the district health office changed their policy from conducting vaccinations only on certain fixed days each month to providing vaccination on any day a child was brought to the health facilities. after these intervention measures were implemented, the outbreak declined and eventually stopped. assessment of the global measles mortality reduction goal: results from a model of surveillance data assessment of the epidemiology and burden of measles in southern mozambique causes of childhood deaths in bangladesh: results of a nationwide verbal autopsy study measles complications in a nigerian hospital setting risk factors for measles death: kyegegwa district, western uganda evaluation of measles surveillance systems in afghanistan- control of communicable diseases manual world health organisation. who guidelines for epidemic preparedness and response to measles outbreaks the national population and housing census -main report vaccine epidemiology: efficacy, effectiveness, and the translational research roadmap strategies for minimizing nosocomial measles transmission nosocomial transmission of measles: an updated review factors contributing to measles transmission during an outbreak in kamwenge district an ongoing large outbreak of measles in merseyside nosocomial and community transmission of measles virus genotype d imported by a returning traveller from nepal nosocomial outbreaks-a potential threat to the elimination of measles? measles transmission in healthcare settings in australia measles in a dutch hospital introduced by an immunocompromised infant from indonesia infected with a new virus genotype nosocomial measles: a proposal for its control in hospitals immunoglobulin (ig) as postexposure prophylaxis measles vaccination in pediatric emergency departments during a measles outbreak world health organization. measles vaccines: who position paper measles outbreak propagated by children congregating at water collection points in mayuge district, eastern uganda field effectiveness of live attenuated measlescontaining vaccines: a review of published literature a review of measles vaccine failure in developing countries measles outbreak in an orphanage spotlight on measles : measles outbreak in ireland publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank the lyantonde health team and health workers in lyantonde, rakai and lwengo districts for their efforts during the measles outbreak response. we also acknowledge the makerere university school of public health, uganda public health fellowship program secretariat, us cdc, and who-uganda office for the technical support during the response to this outbreak and during development of this manuscript. authors' contributions cb: led the outbreak investigation, data collection and data analysis. cb wrote the drafts of the manuscript and revised the paper for substantial intellectual content. ja participated in the outbreak investigation, data collection, analysis and manuscript writing under the supervision of lb and aa. doo participated in analysis, manuscript writing and revision of the paper for substantial intellectual content. the manuscript was reviewed for intellectual content and scientific integrity under the technical guidance and supervision of lb, bk, aa, and bz. all the co-authors have read and approved the final version of this manuscript. this project was supported by the president's emergency plan for aids relief (pepfar), cooperative agreement number gh - , awarded to makerere university school of public health though the us centers for disease control and prevention to support the uganda public health fellowship program at umoh. its contents are solely the responsibility of the authors and do not necessarily represent the official views of the us centers for disease control and prevention, makerere university school of public health, or umoh. the datasets used and analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate uganda ministry of health through the office of the director general health services gave the directive and approval to conduct this investigation. the office of the associate director for science, center for global health, us centers for disease control and prevention, atlanta, determined that this investigation was in response to a public health emergency and not human subjects' research. we obtained verbal informed consent from case-patients and controls ≥ years. for participants < years of age, we sought verbal consent from their parents or guardians. we informed the participants that their participation was voluntary and their refusal to participate would not have any negative consequences to them. before sharing and analyzing the data collaboratively among members of the investigation team, we deleted all identifiable information (i.e., name, address, phone number, etc.). not applicable. all authors declare that they have no competing interests. key: cord- - u t u authors: mao, ying; zhang, ning; zhu, bin; liu, jinlin; he, rongxin title: a descriptive analysis of the spatio-temporal distribution of intestinal infectious diseases in china date: - - journal: bmc infect dis doi: . /s - - -x sha: doc_id: cord_uid: u t u background: intestinal infectious diseases (iids) have caused numerous deaths worldwide, particularly among children. in china, eight iids are listed as notifiable infectious diseases, including cholera, poliomyelitis, dysentery, typhoid and paratyphoid (tap), viral hepatitis a, viral hepatitis e, hand-foot-mouth disease (hfmd) and other infectious diarrhoeal diseases (oidds). the aim of the study is to analyse the spatio-temporal distribution of iids from to . methods: data on the incidence of iids from to were collected from the public health science data centre issued by the chinese center for disease control and prevention. this study applied seasonal decomposition analysis, spatial autocorrelation analysis and space-time scan analysis. plots and maps were constructed to visualize the spatio-temporal distribution of iids. results: regarding temporal analysis, the incidence of hfmd and hepatitis e showed a distinct increasing trend, while the incidence of tap, dysentery, and hepatitis a presented decreasing trends over the last decade. the incidence of oiid remained steady. summer is the season with the greatest number of cases of different iids. regarding the spatial distribution, approximately all p values for the global moran’s i from to were less than . , indicating that the incidences of the epidemics were unevenly distributed throughout the country. the high-risk areas for hfmd and oidd were located in the beijing-tianjin-tangshan (btt) region and south china. the high-risk areas for tap were located in some parts of southwest china. a higher incidence rates for dysentery and hepatitis a were observed in the btt region and some west provincial units. the high-risk areas for hepatitis e were the btt region and the yangtze river delta area. conclusions: based on our temporal and spatial analysis of iids, we identified the high-risk periods and clusters of regions for the diseases. hfmd and oidd exhibited high incidence rates, which reflected the negligence of class c diseases by the government. at the same time, the incidence rate of hepatitis e gradually surpassed hepatitis a. the authorities should pay more attention to class c diseases and hepatitis e. regardless of the various distribution patterns of iids, disease-specific, location-specific, and disease-combined interventions should be established. electronic supplementary material: the online version of this article ( . /s - - -x) contains supplementary material, which is available to authorized users. intestinal infectious diseases (iids), also known as infectious enteric diseases, are transmitted via the faecal-oral route through contaminated food, water, or fomites. the main symptoms of intestinal infectious diseases include nausea, vomiting, fever, headache, limb pain, abdominal pain, loss of appetite, systemic poisoning, diarrhoea, and other gastrointestinal symptoms, which may lead to death if not treated promptly. iids have posed public health threats and resulted in severe social and economic burdens due to the high incidence and morbidity rates, particularly in young children [ ] [ ] [ ] . several large, severe global disease outbreaks have been associated with iids, such as the outbreaks of hfmd in east and southeast asia in the early twenty-first century [ ] , the constantly high burden of cholera that persists in many african countries, and the cholera outbreaks with active cholera transmission in many sub-saharan african countries [ ] . according to the sustainable development goals (sdg), we should combat hepatitis, water-borne diseases and other communicable diseases. iids represent a significant obstacle to achieving sdg [ ] . the chinese government enacted the law of the people's republic of china on the prevention and treatment of infectious diseases and regularly reports cases of notifiable diseases to effectively monitor and control infectious diseases. all reported infectious diseases are divided into classes a, b and c in terms of severity, which is shown in additional file . in china, iids are one of the main types of infectious diseases, presenting the characters of a high morbidity rate and low mortality rate [ ] . eight iids are listed in the law, including cholera, poliomyelitis, dysentery, typhoid and paratyphoid (tap), viral hepatitis a, viral hepatitis e, hand-foot-mouth disease (hfmd) and other infectious intestinal diseases (oiids). among these diseases, cholera and poliomyelitis have been almost completely eradicated. in contrast, according to the data reported by the chinese cdc in , viral hepatitis a, viral hepatitis e, and dysentery are among the top five most severe diseases in class b. at the same time, hfmd and oidds were the most severe diseases in class c. the status of intestinal infectious diseases requires further attention. previous studies have investigated the epidemiology of different intestinal infectious diseases. first, temporal analysis of different intestinal infectious diseases was conducted. the initial efforts in conducting a temporal analysis investigated the temporal variations in the incidence rates of different diseases in different age groups and areas. for example, tian et al. analysed the temporal characteristics of hfmd and the relationship between meteorological factors and the incidence of hfmd in beijing, china. may to july is the period with peak hfmd incidence each year in beijing [ ] . xiao ( , ) ] to predict the trend in the incidence of typhoid using data collected from wuhan city in china from to [ ] . liu et al. forecasted the incidence of bacillary dysentery diseases by applying the seasonal trend model based on the moving average method, which forecast the incidence of hepatitis a in [ ] . ming et al. investigated the seasonal signals and long-term trends in a series of igs sites in china [ ] . xing et al. conducted a study examining the epidemiological characteristics in china from to , emphasizing seasonal patterns among people at different ages [ ] . temporal analyses of infectious diseases have allowed researchers to develop a wide range of research methods, namely, time series analysis and seasonal analysis. however, temporal analysis failed to reflect the overall circumstances of infectious intestinal diseases. on the other hand, the academic world gradually began to realize the importance of performing spatial analysis of infectious diseases. zhang et al. conducted a space-time scan analysis of hfmd in liaocheng city in china from to at the town level [ ] . the distribution of the identified cluster was described in the article. zheng et al. mapped the incidence rate and coefficient of determinants of hfmd to present and identify the most severely affected areas and the most influential determinants [ ] . as shown in the study by wang et al., the typhoid prevalence is spatially clustered and exhibits a gradually decreasing trend [ ] . according to ma et al., bacillary dysentery is not equally distributed across sichuan province in china [ ] . nie et al. used a spatial correlation analysis to explore the associations between selected factors and bacillary dysentery in guangxi province in china [ ] . the spatial analysis visually displayed the discrepancies among different regions, which would be able to present additional results if combined with the temporal analysis. in conclusion, temporal and spatial analyses are useful. previous studies have focused on the epidemiological characteristics of different iids. however, a comprehensive study analysing the spatial and temporal distributions of all reported iids in china is lacking. second, an overall introduction to iids in china to researchers in other countries is unavailable. therefore, this study will collect data on the basic iids and populations, use seasonal decomposition analyses to explore the temporal epidemiology, and perform spatial autocorrelation analysis and space-time scan analysis to explore the spatial epidemiology. among the eight iids, cholera and poliomyelitis have been almost completely eradicated. the number of deaths related to cholera and poliomyelitis since were and , respectively, indicating that the two diseases were less emergent. at the same time, according to the chinese classification of viral hepatitis, the hepatitis a and e should be analysed separately [ ] . overall, this study analyses the spatiotemporal distribution of six intestinal infectious diseases, namely, dysentery, typhoid and paratyphoid (tap), viral hepatitis a, viral hepatitis e, hand-foot-mouth disease (hfmd) and other infectious intestinal diseases (oiid). to better demonstrate the trends of incidence of iids, we analysed these diseases from to , which represents two equal periods: - and - . the data were collected from public health science data centre issued by chinese center for diseases control and prevention (china cdc) [ ] . the sorted data are presented in additional file and additional file . additional file displays the number of cases of iids. additional file displays the number of cases of iids by month and the morbidity of iids by month. intestinal infectious diseases are strictly monitored by the chinese cdc. the centre aims to integrate the scattered data distributed by governments, universities, research institutes and scientists. the seasonal decomposition analysis was based on the seasonal trend decomposition using loess (stl), which incorporates three components: trend, seasonal, and remainder or residual [ ] . advantages of the method include its simplicity and speed of computation, the robustness of results, and flexibility in the period of the seasonal component [ ] . in the present study, iids were analysed by performing a seasonal decomposition of the time series. this assay employed holt-winters filtering and ljung-box test to define the structure (additive or multiplicative) and seasonality (stationary or non-stationary). using an additive model, the iid results were compiled by summing three components: where z t represents the monthly incidence rates of the diseases, m t represents the trend, s t symbolizes the seasonal variation, and r t denotes the remainder. the result of seasonal decomposition analysis is displayed in a figure with the time from to on the horizontal axis and the incidence rate on the vertical axis. the concept of spatial autocorrelation was proposed by tobler in the first geography law [ ] : "everything is related to everything else, but nearest things are more related than distant things." moran's i was the tool used to measure spatial autocorrelation and consists of two types: global moran's i and local moran's i. global moran's i measures the general spatial autocorrelation and the spatial distribution of research object. local moran's i reflects the local spatial autocorrelation and the cluster regions. in the present study, spatial autocorrelation was applied to analyse the iids. global moran's i shows the overall cluster level and distribution of iids; local moran's i reveals the specific cluster regions and cluster categories and the hotspots of iids [ ] . global moran's i is an index ranging from − to . when the index was distributed around − , the overall spatial distribution displayed the dissimilarity, indicating that high cluster regions bordered on low cluster regions. when the index was approximately , a distinct spatial cluster was not observed in the studied regions. when the index was close to , the overall spatial distribution revealed the similarity, indicating that the same cluster category was bounded on another cluster category. the following equation was used to calculate the global autocorrelation: where n denotes the number of observed values, x i represents the incidence rate in province i, x j represents the incidence rate in province j, x indicates the mean value, and w ij represents a spatial weight matrix of systematic binomial distribution, which represents neighbouring relations between geographical units with n representing the total number of those units . in the present study, the data were based on regions. the value for w ij is if province i and province j are adjacent. otherwise, the value is . local moran's i avoids the weaknesses of global spatial autocorrelation by analysing the spatial autocorrelation of certain characters in local regions. the range and explanation for the local moran's i was same as the global index. the cluster results obtained from local moran's i were classified into four types: high-high cluster (hh, which indicated that the high cluster areas were surrounded by other high cluster areas), high-low cluster (hl, which indicated that the high cluster areas were surrounded by low cluster areas), low-high cluster (lh), and low-low (ll) cluster. the clusters were visualized using lisa cluster maps. the following equation was used to calculate local moran's i: where y i represents the incidence rate in province i, y j represents the incidence rate in province j, y indicates the mean value. [ ] in the present study, we analysed a long period to investigate the incidence rates of different iids. the years , , were selected to show the long-term changes. space-time scan statistics were introduced by kulldorff [ ] . the space-time scan statistic based on the discrete poisson model was applied to detect the space-time cluster of iid cases in high-risk or low-risk regions in china [ ] . the shape of space-time scanning windows is cylindrical with the geographic units and the height associated with time [ ] . the null hypothesis presumed that the window area and outside areas have the same relative risk (rr) of incidence. the difference in the incidence inside and outside the windows was evaluated by calculating the log likelihood ratio (llr): where c denotes the total number of cases, c represents the number of observed cases inside the window, and n represents the number of expected cases inside the window [ ] . the space-time analysis was applied to identify the clusters according to the llr value, including the most likely cluster, secondary cluster , secondary cluster , secondary cluster , and secondary cluster , as well as the cluster time. statistical significance was evaluated using a monte carlo simulation with , replicates and a significance level of . . for the other parameters, the maximum radius of the circular base was set to % of the total population at risk and the maximum height of the cylinder was set to % of the total study period. the method is sensitive to user-controlled parameter choices. a reliability analysis was conducted to determine the sensitivity and consistency of the results. multiple scans with different maximum sizes ranging from to % of the population were conducted. reliability was measured using the following equation: where r i denotes the reliability of different provincial units, s indicates the number of scans, and c i represents the number of high-risk areas in these scans. the range of reliability is from to points, where indicates that all scans report a place as high risk and indicates no scan reports [ ] . the reliability results were visualized by constructing a map (additional file and additional file ). the seasonal decomposition analysis were visualized using microsoft excel (version , microsoft corp, redmond, wa, usa). the seasonal decomposition analysis was performed using ibm spss statistics (version , ibm, armonk, ny, usa). geoda (version . . , github, san francisco, ca, usa) was employed to attain the global moran's i and local moran's i hierarchical maps, and lisa cluster maps, and space-time scan maps were obtained using arcgis (version . , esri inc., redlands, ca, usa). the space-time scan was analysed using satscan (version . , kulldorff and information management services, inc., boston, ma, usa). we listed all iids reported in each provincial unit in china from to . table presents a descriptive analysis of selected iids, including the average, maximum and minimum incidence from to . all provincial units were divided into east, central and west china. according to the descriptive summary, hfmd was the most severe disease, with an average incidence of . . oidd ranked second with an average incidence of . from to . the third most common disease was dysentery, with an average incidence of . . regarding the region, east china had the highest incidence rates for hfmd, oidd and hepatitis e. the west area was the area with the greatest number of tap, dysentery and hepatitis a cases. the seasonal decomposition plots contain four parts, the raw data, remainder, seasonal variation, and trend. the raw data represent the original incidence rate of certain diseases. the remainder represents the irregularity of data. seasonal variation exists when the rangeability is greater than . . the trend symbolizes the long-term variation in the data. the seasonality and variation in the trends in monthly data were distinguished from the remainder or residual using a decomposition analysis. figure shows the results of the decomposition analysis for all iids. regarding the seasonal variation, the incidence rates of hfmd and dysentery were high in summer. higher incidence rates for oidd and hepatitis a were observed in summer and autumn. hepatitis e showed a high rate in spring. a distinct seasonal variation in the incidence of tap was not observed. regarding the trend, the hfmd, oidd and hepatitis e displayed increasing trends, and the other diseases showed decreasing trends. based on the remainder, a -month stochastic variance was displayed. the spatial autocorrelation analysis was divided into global spatial autocorrelation and local spatial autocorrelation. the former was considered to represent geographical -difference in whole areas, while the latter was regarded as the difference at the regional cluster level. global spatial autocorrelation table shows the results from the global spatial autocorrelation analysis and significance test. in terms of the significance, all moran's i values for dysentery and hepatitis e reached the significance threshold, while of the values for oidd, tap, and hepatitis a achieved results, with one insignificant index each. hfmd had five significant indexes. in the global spatial autocorrelation analysis, moran's i for hfmd ranged from . to . throughout the years without an obvious increasing or decreasing trend, suggesting that the disease local spatial autocorrelation analysis beijing and shanghai were the areas with the highest incidence rates, while the areas with the lowest rates included xizang, qinghai, yunnan, and guangxi, among others. figure displays the spatial clusters of all iids, reflecting the regional variation from to . the hot spot for hfmd is located in guangdong, while cold spots were detected in some areas of the central and west provinces, such as sichuan, chongqing, hubei, guizhou, and jiangxi. in , the hot spots shifted to adjacent provinces, guizhou and jiangxi; new cold spots shifted to areas in the north province, such as gansu, jilin, xinjiang, and inner mongolia. for oidd, the hh cluster feature was observed in btt areas, while heilongjiang and jilin in northwest china showed the ll cluster feature in . throughout the ensuing years, jilin and hebei were no longer present in the original hot spots and cold spots. jiangxi showed the hh cluster character as well. for tap, hh cluster character was witnessed in yunnan, jiangxi, and guizhou throughout the -year period, while xizang, sichuan, neimenggu and xinjiang showed the character of a cold spot. in terms of dysentery, inner mongolia, beijing, and tianjin were located in hh cluster areas, while the lh cluster was located in hebei. guangxi and guangdong contained the ll cluster. in and , the cluster characters presented the same trends. beijing, tianjin, and hebei were the hh cluster areas, while the ll cluster was located in guangdong. hebei experienced a change from a cold spot to a hot spot. for hepatitis a, the hh cluster character was present in west china, including xinjiang, gansu, qinghai, xizang, sichuan, and chongqing, throughout the -year period, while the ll cluster character was located in the btt area, jiangsu and guangdong. xizang was a hot spot in and , but was a cold spot in . the hot spots for hepatitis e were mainly concentrated in the btt area and yangtze river delta area, while cold spots were located in areas of west china, jiangxi and hubei. space-time scan analysis was used to explore the cluster likelihood, the level of which was classified as the most likely cluster, secondary cluster, nd secondary cluster, rd secondary cluster and th secondary cluster. this study analysed iids in china from to . fig. and table studies of the epidemiology of different intestinal infectious diseases, particularly the temporal and spatial distributions, have played an important role in preventing infections prevention. however, previous studies have neglected to analyse the correlation between the temporal and spatial analyses. a systematic spatio-temporal analysis of iids has not been conducted in china. this study supplements data from other related research in the area of infectious intestinal diseases by performing a temporal analysis and spatial analysis and determining the correlation between them. the evidence provided insights into potential solutions to diminish the diseases. on one hand, we would like to compare the temporal analyses of the six intestinal infectious diseases. according to the incidence rates recorded from to , the temporal trends differed. regarding the absolute incidence of cases, the incidence of hfmd was higher than the other iids, and it gradually became a wide-spread disease, which is consistent with the results from the study by zhang [ ] . then, dysentery and oidd were less severe diseases. hepatitis a, hepatitis e and tap were the least severe diseases, according to the incidence rates. regarding the trend, the incidence rates of hfmd and hepatitis e showed a distinct increasing trend, consistent with the results from the study by zhu. as the epidemic of hepatitis a was controlled, the percentage of hepatitis e cases among patients with viral hepatitis and among patients with iids has increased. the mortality rate of hepatitis e has increased among infectious diseases [ ] . the trend in the incidence of oidd was almost unchangeable. dysentery and tap displayed obvious decreasing trends. the analysis of dysentery filled the research gap in the study by xie et al., which showed a decreasing trend in the incidence of dysentery from to [ ] . the results for tap were similar to the findings reported by liu [ ] , which showed a decreasing trend. the improvement in sanitation facilities and the reduction in food and water pollution likely contributed to the decreasing trend in the incidence rates of these diseases. hepatitis a exhibited a slight increasing trend in the first years, followed by a decreasing trend. this result was similar to the findings reported by zhu [ ] . in conclusion, class b diseases were prevented with high efficiency. class c diseases experienced a higher incidence rate and gradually increasing trend. regarding the seasonal changes, the incidence of hfmd and dysentery peaked in the summer, hepatitis e exhibited a high-incidence season in spring, oidd peaked in summer and a smaller peak in autumn, the peak incidence of hepatitis a occurred in summer and spring, and a distinct peak for tap was not observed. previous studies have revealed an association between the iids and seasons [ , [ ] [ ] [ ] [ ] . the occurrence of intestinal infectious diseases is related to climatic factors, such as the sunshine duration, temperature and humidity, as well as the quality of food and drinking water [ ] . due to the high temperature and humidity in summer, which are conducive to bacterial reproduction, food and water are easily contaminated. at the same time, the human immune system is relatively weak due to higher bodily exertion in summer. in conclusion, summer is the season with the highest incidence rates for iids and should receive closer attention. on the other hand, the high-risk areas for different iids were mainly located in the beijing-tianjin-tangshan (btt) region, yangtze river delta, south and west china. the former two sites are developed areas with extensive urbanization, which attract larger mobile populations characterized by low immune systems, poor living environments and living conditions, and poor health and knowledge of epidemic prevention measures. the hot spots for hfmd, oidd, dysentery and hepatitis e are located in the btt region and yangtze river delta [ ] . south china is located closer to the equator with a subtropical monsoon and tropical monsoon climate, which are characterized by high humidity, temperature, rainfall, and wind speed. iids are strongly correlated with the climatic character of south china [ ] , which is a high-risk area for hfmd, oidd, and tap. the high-risk areas for tap were located in southwest china (yunnan, guizhou, and guangxi), which are also the most likely cluster areas and hh cluster areas. the main reasons are that southwest china borders southeast asia (vietnam, laos, and myanmar), which has a high-risk population due to low sanitation. moreover, the population of southwest china has a medical history of tap and very poor climatic, geographical (karst landform) and sanitation conditions. who estimates of the global burden of foodborne diseases from farm to plate, make food safe food safety: what you should know from farm to plate, make food safe. world heal organ reg off south-east asia, indraprastha estate, mahatma gandhi marg integrated management of childhood illness: distance learning course: who epidemiologic and clinical features of non-polio enteroviral infections in northern taiwan in cholera outbreaks in africa transforming our world: the agenda for 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variables on hand, foot, and mouth disease in mainland china the emergence and outbreak of multidrug-resistant typhoid fever in china springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the national social science fund of china for its support and the chinese center for disease control and prevention for sharing the valuable data. finally, personal eating habits contribute to the incidence of tap [ ] . the west area experiences poor sanitation and a lower economy and is a high-incidence area for hepatitis a and dysentery.the strengths of this study are listed below. first, the study performed a comprehensive analysis of iids, including the temporal and spatial analyses of all reported intestinal diseases in china, to provide a complete picture of iids to other countries. second, the visualization of diseases represented a convenient method to show the distribution of diseases. however, this study had some limitations. we were unable to obtain more sophisticated results for the provincial units in china than were obtained for the county unit. regional discrepancies were also observed among provinces. furthermore, additional studies that explore the temporal and spatial distributions in smaller region units are needed. in conclusion, seasonal patterns and trends in different provincial geographical units were determined. higher incidence rates of iids were observed from may to october, which is the season with a climate characterized by heavy rain, high temperature, and high humidity. the climate zone is associated with the incidence. the highrisk areas for iids were detected in part of the border region, the south region and the region with better economic development.based on the results of our temporal and spatial analysis of iids, we identified the high-risk periods and cluster regions for the studied diseases. hfmd and oidd exhibited high incidence rates, reflecting the negligence of the government in monitoring class c diseases. at the same time, the incidence rate of hepatitis e gradually surpassed that of hepatitis a. the authorities should closely monitor class c diseases and hepatitis e. notable epidemiological trends were observed among different provinces. an effective response requires the implementation of a series of coherent and coordinated measures, which should be specific for the diseases that are endemic to a particular area. authors' contributions nz, ym conceptualized and designed the study. nz put forward the outline of the article with ym. bz, nz made data analysis, draw pictures and drafted the manuscript. jll and rxh revised the article. all authors read and approved the final manuscript. the study was funded by the major project of national social science fund of china: research on big health putting prevention first and construction of healthy china (grant number zda ). the funder had no role in the study design, data collection and analysis, interpretation of data, and writing the manuscript. all data generated or analyzed during this study are included in this published article and its supplementary information files.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.author details key: cord- -legakasx authors: qiu, junke; wang, caihong; pan, xiaohong; pan, lei; huang, xiaoqing; xu, jiekun; ji, xiaobo; mao, minjie title: apache-ii score for anti-tuberculosis tolerance in critically ill patients: a retrospective study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: legakasx background: to investigate the status of anti-tuberculosis treatment in critically ill patients, and to explore the value of apache-ii score in guiding anti-tuberculosis treatment. methods: this analysis included critically ill patients with tuberculosis. the utility of apache-ii score for predicting drug withdrawal was evaluated using receiver operating characteristic (roc) curve analysis. results: among patients enrolled ( ± years; males), ( . %) had drugs withdrawn. the drug withdrawal group had higher apache-ii score (median [interquartile range]: [ – ] vs. [ – ] points), higher cd %, lower hemoglobin level, higher rates of chronic obstructive pulmonary disease (copd) and chronic renal failure, and lower rate of extrapulmonary tuberculosis (p < . ). logistic regression identified apache-ii score > (odds ratio [ % confidence interval]: . [ . – . ], p < . ), copd ( . [ . – . ], p < . ) and hemoglobin level ( . [ . – . ], p < . ) as independent factors associated with drug withdrawal. at an optimal cutoff of . , the sensitivity, specificity, positive predictive value and negative predictive value of apache-ii score for predicting drug withdrawal was . , . , . and . %, respectively. conclusions: apache-ii score > points might predict patient tolerance of anti-tuberculosis treatment. tuberculosis is a global public health problem and the leading cause of death due to infection [ ] . in , there were . million incident cases of tuberculosis and . million deaths due to the disease [ ] . although the prevalence of smear-positive tuberculosis in china fell from per , people in to per , people in [ ] , the burden remains high. indeed, china has the third-highest incidence of tuberculosis after india and indonesia [ ] . medical advances, lifestyle changes and improvements in socioeconomic factors have resulted in people in china living longer than before [ ] , but with poorer physical and mental health than their counterparts years ago [ ] . furthermore, the prolongation of lifespan has led to an increase in the number of elderly people with tuberculosis. tuberculosis can potentially be cured with appropriate medication in those with a timely diagnosis [ ] . however, elderly patients with tuberculosis often have comorbidities, organ dysfunction or organ failure, which complicates the successful management of the infection. in particular, elderly patients with tuberculosis tend to have more extensive pulmonary lesions, higher sputum positivity, a higher susceptibility to the adverse effects of anti-tuberculosis drugs (including hepatic dysfunction) and higher mortality [ ] [ ] [ ] [ ] . elderly patients with tuberculosis have a lower completion rate than younger patients [ , ] and are considered by some healthcare workers to be an "obstacle" to the control of tuberculosis in china [ ] . thus, the treatment of elderly patients who are critically ill with tuberculosis is highly challenging. non-adherence to anti-tuberculosis therapy remains a major issue, and knowledge of the factors that affect treatment completion would enable healthcare providers to implement appropriate strategies to improve adherence. a wide range of factors have been suggested to be associated with non-adherence to treatment for tuberculosis, including male gender, age > years, drug adverse effects, economic factors (including transportation costs), inadequate knowledge about tuberculosis and its therapy, lack of social support, poor relationship/communication with healthcare providers, feeling unwell, tobacco smoking and co-infection with human immunodeficiency virus (hiv) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, to the best of our knowledge, no studies have investigated the factors influencing the tolerability of anti-tuberculosis therapy in patients who are critically ill with tuberculosis. we hypothesized that comorbidities and apache-ii score, a disease severity score widely used in the intensive care unit (icu) [ ] , might be predictors of the tolerability of anti-tuberculosis therapy in critically ill patients with tuberculosis. therefore, the aim of this retrospective analysis was to investigate the factors associated with the tolerability of anti-tuberculosis medications in patients with tuberculosis being treated at an icu in china. it was envisaged that the findings might provide guidance regarding the selection of the anti-tuberculosis regimen in critically ill patients with tuberculosis. critically ill patients with tuberculosis at the department of tuberculosis intensive care unit, hang zhou red cross hospital between march and may were retrospectively analyzed. the inclusion criteria were: ) diagnosis of tuberculosis [ ] ; ) failure of one or more organs, such as respiratory, heart, renal and/or liver failure; ) admitted to the icu ≥ days; and ) anti-tuberculosis treatment ≥ days. the exclusion criteria were: ) non-tuberculous mycobacterial disease; and ) incomplete clinical data. the clinical ethics committee of hangzhou red cross hospital approved the study. informed consent was waived due to the retrospective study design. data including age, gender, clinical characteristics of the tuberculosis episode, comorbidities and other relevant clinical factors, blood tests, sequential organ failure assessment (sofa) score [ ] , apache-ii score [ ] , anti-tuberculosis treatment regimen used, drug therapy, drug withdrawal, and adverse reactions (based on the toxicity and allergy reporting system implemented in chinese hospitals) were recorded. patients who had discontinued at least one anti-tuberculosis drug during therapy were included in the drug withdrawal group. data were analyzed using spss . (ibm, usa). normally distributed data are presented as the mean ± standard deviation and compared using student's t-test. non-normally distributed data are expressed as median (interquartile range) and compared using the wilcoxon rank sum test. a two-sided p-value < . was indicated statistical significance. differences in the underlying diseases were assessed with the χ test. multivariable logistic regression analysis with forward selection was used to identify factors associated with anti-tuberculosis tolerance; factors with p values < . in univariable analyses were considered for inclusion in the multivariable analysis. receiver operating characteristic (roc) curve analysis was used to evaluate apache-ii score for predicting the discontinuation of drug therapy. a total of patients (age, ± years; males, . %) were included in the analysis, with ( . %) in the drug withdrawal group and ( . %) in the therapy completion group. the demographic and clinical characteristics of the patients in the two groups are presented in table . compared with the therapy completion group, the drug withdrawal group had a significantly higher apache-ii score (within h after admission to the icu) and cd % (i.e. percentage of lymphocytes that are cd -positive), a significantly lower hemoglobin level, significantly higher frequencies of chronic obstructive pulmonary disease (copd) and chronic renal failure as comorbidities, and a significantly lower rate of extrapulmonary tuberculosis (all p < . ; table ). a total of patients were using vasoactive drugs. however, there were no significant differences between groups in any of the other parameters assessed (see table for details). a total of patients received anti-tuberculosis treatment. information regarding the anti-tuberculosis drug regimens used in patients from the two groups is shown in table [e] ) was used in the majority of patients ( . % in the drug discontinuation group and . % in the therapy completion group), and over % of patients received at least anti-tuberculosis drugs ( . % in the drug discontinuation group and . % in the therapy completion group). there were no significant differences between groups in the treatment regimen used ( table ) . among those patients, there were six patients with streptomycin (s) resistance, two with r resistance, four with hrs resistance, one with hres resistance, five with hr resistance, and nine with h resistance. there were patients without susceptibility results. the rates of withdrawal of each anti-tuberculosis drug are presented in table . the most frequently withdrawn drugs were rifapentine ( . %) and rifampicin ( . %) followed by pyrazinamide ( . %), isoniazid ( . %) and ethambutol ( . %). as detailed in table , the most common reasons for drug withdrawal were liver dysfunction ( . %), drug allergy ( . %), thrombocytopenia ( . %), disease deterioration ( . %) and renal dysfunction ( . %). in the therapy completion group, patients were transferred to a hospital ward and patients died. in the drug withdrawal group, patients were transferred to a hospital ward and patients died. the overall icu mortality rate was . %, and the icu mortality rate was significantly higher in the drug withdrawal group than in the therapy completion group ( . % vs. . %, p < . ). based on the results of the univariable analysis ( were independently associated with the withdrawal of anti-tuberculosis treatment (table ) . the area under the roc curve was . (fig. ) , and the youden index was . . using an optimal cutoff value of . points, the sensitivity, specificity, positive predictive value and negative predictive value of apache-ii score in the prediction of anti-tuberculosis drug withdrawal was . , . , . and . %, respectively. a notable finding of the present study was that patients in the drug withdrawal group had higher apache-ii score, lower hemoglobin level and higher rates of copd. furthermore, logistic regression identified an apache-ii score > points, comorbid copd and hemoglobin level as independent factors associated with anti-tuberculosis drug withdrawal. in addition, when an optimal cutoff of . was used, apache-ii score had reasonable sensitivity, specificity, positive predictive value and negative predictive value in the prediction of drug discontinuation. taken together, our findings suggest that an apache-ii score > points might be a useful indicator for predicting patient tolerance of anti-tuberculosis treatment. adverse reactions caused by anti-tuberculosis drugs are important factors affecting the success of standard therapy for tuberculosis. previous studies have reported that the total incidence of adverse reactions caused by anti-tuberculosis drugs is . % in china [ ] , . % in portugal [ ] , . % in korea [ ] , and . % in iran [ ] . the apparent variation between studies may be related to differences in the incidence of tuberculosis, treatment strategies used or definitions of adverse reactions, as well as other factors. the incidence of adverse reactions in the present study was substantially higher than the average national incidence in china. this is likely due to the patients in our study being critically ill and thus treated in the department of tuberculosis intensive care unit. critically ill patients are likely to have more comorbidities and more severe disease, which would be expected to make them more susceptible to the development of adverse reactions induced by anti-tuberculosis drugs. to our knowledge, the incidence of adverse reactions to anti-tuberculosis drugs in critically ill patients has not been reported previously, either in china or elsewhere. clinical studies have found that the occurrence of adverse reactions to anti-tuberculosis drugs is related to a wide variety of factors, including patient age, patient gender, multidrug-resistance, smoking status, and comorbidities such as hiv co-infection, viral hepatitis and diabetes mellitus [ ] [ ] [ ] [ ] [ ] [ ] . in this study, logistic regression analysis identified copd as an independent factor associated with the withdrawal of anti-tuberculosis treatment in critically ill patients with tuberculosis. tuberculosis combined with copd is thought to be the leading cause of death due to respiratory infection [ ] . tuberculosis leads to decreased lung volume and airway stenosis, which are risk factors for the occurrence of copd, while long-term use of steroids for copd leads to decreased immune function, which is a risk factor for tuberculosis. thus, there is an association between tuberculosis and copd [ , ] . in this study, the number of patients with copd and the mortality rate were both significantly higher in the drug withdrawal group than in the therapy completion group, which would be consistent with comorbid copd increasing the risk of death in patients with tuberculosis, as has been reported previously [ , ] . the apache-ii scoring system is the most commonly used method for assessing the severity of critical illness, with a higher score indicating more severe disease. apache-ii score has been reported to be an independent factor associated with the prognosis of patients with ards caused by tuberculosis [ ] [ ] [ ] [ ] [ ] . in the present study, patients in the drug withdrawal group had a significantly higher apache-ii score than patients in the therapy completion group, and an apache-ii score > points was an independent factor associated with anti-tuberculosis drug withdrawal in critically ill patients with tuberculosis. therefore, we suggest that the apache-ii score may have certain clinical value for predicting whether a critically ill patient with tuberculosis will tolerate anti-tuberculosis treatment. patients in the drug withdrawal group had a significantly lower level of hemoglobin on admission than those in the therapy completion group, and a lower hemoglobin level was an independent factor associated with anti-tuberculosis drug withdrawal. tuberculosis-associated anemia is a recognized phenomenon but is usually mild and resolves following successful pharmacologic therapy [ ] . however, anti-tuberculosis drugs can cause hematologic abnormalities, including anemia, during intensive therapy [ ] . it is possible that patients with a lower baseline hemoglobin level before the initiation of treatment are more susceptible to reductions in hemoglobin level caused by therapy, which in turn would make drug withdrawal due to the manifestation of adverse events more likely. another possibility is that low hemoglobin levels impair the effectiveness of anti-tuberculosis therapy. consistent with this possibility, anemia was found to be associated with delayed sputum conversion after therapy, and it was suggested that the impaired response to treatment in those with low hemoglobin levels may be due to malnourishment and impaired t cell immunity secondary to zinc and iron deficiency [ ] . further research is needed to clarify the mechanism underlying the association between pre-therapy hemoglobin level and subsequent drug withdrawal. in this study, abnormalities of liver and kidney function, thrombocytopenia and disease deterioration accounted for around % of cases of drug withdrawal. hepatic injury was the most common adverse reaction caused by anti-tuberculosis drugs, as described in other publications [ ] . the incidence reported in previous studies vary greatly, from to % [ ] , possibly due to differences in the criteria used to evaluate liver dysfunction, population characteristics and treatment regimens. the rate of drug withdrawal due to liver damage in this study was . %, similar to that reported previously [ ] . rifampicin, isoniazid and pyrazinamide are the main anti-tuberculosis drugs that cause liver damage [ ] , which likely explains why the frequencies of withdrawal of these drugs were higher than those of the other drugs in this study. allergic reactions are also commonly encountered during the treatment of tuberculosis, and the incidence of allergic reactions in this study was similar to the incidence of . - . % reported during standard therapy [ ] . the allergic reactions reported in this study were mild and mainly manifested as urticaria and drug fever, with no cases of exfoliative dermatitis or bullous dermatitis. the incidence of thrombocytopenia as an adverse reaction of anti-tuberculosis drugs has been rarely reported, although it has been mentioned in case reports [ , ] . in patients with acquired immunodeficiency syndrome (aids), the rate of thrombocytopenia as an adverse reaction to anti-tuberculosis medication was . % [ ] , similar to the incidence observed in the present study. it is worth noting that patients with tuberculosis also have immune dysfunction [ ] , although the underlying mechanisms need further exploration. renal dysfunction was a relatively rare adverse reaction in our study, although its incidence was similar to that reported in the literature ( . %) [ ] . fungal infections cannot be ignored in critically ill patients (since this is known to increase the risk of death), and some patients in this study needed anti-aspergillus treatment with voriconazole. since rifampicin is an inducer of cyp , which reduces the blood concentration of voriconazole, it was discontinued during antifungal treatment. in addition, drug withdrawal in some patients was related to a continuous deterioration in their clinical condition, with worsening respiratory failure and hemodynamic instability. the other, less common reasons for drug withdrawal were likely not related to anti-tuberculosis medication and included gastrointestinal bleeding, malignant arrhythmias, psychiatric symptoms and the results of drug susceptibility testing. this study has some limitations. first, this was a retrospective analysis and so may have been prone to selection bias or information bias. some data are not very accurate, and some indicators cannot be unified. indeed, the reasons for treatment discontinuation were not reliably and consistently indicated in the medical charts and could not be used for analysis. in addition, because patients may have been visiting multiple departments or hospitals, the exact timing of drug start and discontinuation in relation to diagnosis could not be analyzed. the height of the patients was not reliably indicated in the charts and body mass index could not be calculated. second, other possible confounding factors not included in the analysis 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with respiratory failure requiring mechanical ventilation predictors of development and outcome in patients with acute respiratory distress syndrome due to tuberculosis a study of the prognostic factors associated with mortality in critically ill patients with tuberculous contributing factors to mortality rates of pulmonary tuberculosis in intensive care units profile of patients with active tuberculosis admitted to a respiratory intensive care unit in a tertiary care center of north india the prevalence and evolution of anemia associated with tuberculosis effect of anti-tuberculosis drugs on hematological profiles of tuberculosis patients attending at university of gondar hospital anemia at the initiation of tuberculosis therapy is associated with delayed sputum conversion among pulmonary tuberculosis patients in the causes of treatment cessation in the patients suffering from pulmonary tuberculosis an official ats statement: hepatotoxicity of antituberculosis therapy cutaneous adverse drug reactions caused by antituberculosis drugs a case of thrombocytopenia caused by rifampicin and pyrazinamide a case of pulmonary tuberculosis complicated with severe thrombocytopenia during treatment drug-induced complications of anti-tuberculosis drugs in hiv patients acute kidney injury due to anti-tuberculosis drugs: a five-year experience in an aging population the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. authors' contributions jq and cw conceived and supervised the study; jq and xp designed experiments; lp, xh and jx performed experiments; jq and mm analyzed data; jq, xp and lp wrote the manuscript; jx and xj made manuscript revisions. all authors reviewed the results and approved the final version of the manuscript. the clinical ethics committee of hangzhou red cross hospital approved the study. informed consent was waived due to the retrospective study design. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - eylgtbc authors: singh, david e.; marinescu, maria-cristina; carretero, jesus; delgado-sanz, concepcion; gomez-barroso, diana; larrauri, amparo title: evaluating the impact of the weather conditions on the influenza propagation date: - - journal: bmc infect dis doi: . /s - - -w sha: doc_id: cord_uid: eylgtbc background: predicting the details of how an epidemic evolves is highly valuable as health institutions need to better plan towards limiting the infection propagation effects and optimizing their prediction and response capabilities. simulation is a cost- and time-effective way of predicting the evolution of the infection as the joint influence of many different factors: interaction patterns, personal characteristics, travel patterns, meteorological conditions, previous vaccination, etc. the work presented in this paper extends epigraph, our influenza epidemic simulator, by introducing a meteorological model as a modular component that interacts with the rest of epigraph’s modules to refine our previous simulation results. our goal is to estimate the effects of changes in temperature and relative humidity on the patterns of epidemic influenza based on data provided by the spanish influenza sentinel surveillance system (sisss) and the spanish meteorological agency (aemet). methods: our meteorological model is based on the regression model developed by ab and js, and it is tuned with influenza surveillance data obtained from sisss. after pre-processing this data to clean it and reconstruct missing samples, we obtain new values for the reproduction number of each urban region in spain, every minutes during . we simulate the propagation of the influenza by setting the date of the epidemic onset and the initial influenza-illness rates for each urban region. results: we show that the simulation results have the same propagation shape as the weekly influenza rates as recorded by sisss. we perform experiments for a realistic scenario based on actual meteorological data from - , and for synthetic values assumed under simplified predicted climate change conditions. results show that a diminishing relative humidity of % produces an increment of about . % in the final infection rate. the effect of temperature changes on the infection spread is also noticeable, with a decrease of . % per extra degree.conclusions: using a tool like ours could help predict the shape of developing epidemics and its peaks, and would permit to quickly run scenarios to determine the evolution of the epidemic under different conditions. we make epigraph source code and epidemic data publicly available. seasonal influenza may not make headlines, but together with pneumonia, it is one of the top ten causes of death worldwide. influenza epidemics results in to million cases of severe illness a year, which puts a high burden on health providers and results in loss of productivity and absenteeism, such as mentioned by the world health organization in [ ] . it's been long known that in temperate climates these seasonal epidemics occur mostly in winter, and typical hypotheses assigned the blame to people being in closer proximity for longer periods of time, or lowered immune systems. in general, meteorological conditions affect virus transmission due to multiple effects: virus survival rates, host contact rates and immunity, and the transmission environment (except the case of direct or short-range contact). while these factors may have an influence, the solid evidence sustains the hypothesis that the virus's best surviving conditions are low temperatures and low absolute humidity. one of the goals of the current research in this field is to understand this relationship to be able to develop a more accurate seasonal influenza model for both temperate and tropical regions. as a motivation of this work, jt et al. [ ] conclude that environment factors may become more important for a future predictive model of the effects of climate change. in a previous paper [ ] , some of the authors of this paper studied the interaction of the spatio-temporal distribution of influenza in spain and the meteorological conditions during five consecutive influenza seasons. the work uses real influenza and meteorological data in combination with statistical models to show that there is a relationship between the transmission of influenza and meteorological variables like absolute humidity and amount of rainfall. in this work we use the same data sources (sisss and aemet agencies) following a different approach: we study some of these relationships from a simulation perspective, considering not only the existing influenza distributions but also the ones related to the climate change. in this work we extend epigraph [ ] , an influenza simulator, with a meteorological model (mm) starting from the model developed by ab and js [ ] . in their paper ab and js analyze monthly weather and influenza mortality data collected between and throughout all of the us urban counties. using a regression model, they conclude that there exist correlations between both absolute humidity and temperature with mortality. they report a quantitative assessment of the relation between mean daily humidity and temperature levels and mortality rates in different ranges. this is an extensive study and, as a result, we start from the assumption that their results are solid and appropriate to incorporate to epigraph in order to produce meteorological-dependent simulations based on real data. in this work we extend epigraph [ ] , an influenza simulator, with a meteorological model (mm) starting from the model developed by ab and js [ ] . in their paper ab and js analyze monthly weather and influenza mortality data collected between and throughout all of the us urban counties. using a regression model, they conclude that there exist correlations between both absolute humidity and temperature with mortality. they report a quantitative assessment of the relation between mean daily humidity and temperature levels and mortality rates in different ranges. this is an extensive study and, as a result, we start from the assumption that their results are solid and appropriate to incorporate to epigraph in order to produce meteorological-dependent simulations based on real data. regarding other influenza simulators that consider weather conditions, ps et al. presents an agent-based simulation model [ ] that evaluates the seasonal effects on the influenza propagation. although the reproductive rates are generated synthetically without considering actual meteorological data, this paper shows, in a similar way than our work, the impact of changing reproductive rates on the course of the influeza pandemic. in the article [ ] , js et al. simulate influenza transmission via a sirs model modulated by climate data to obtain the basic reproduction number r . both js et al. [ ] and acl et al. [ , ] study the effects of humidity on influenza transmission from the point of view of virus survival and conclude that aerosol transmission is most efficient in low humidity conditions. acl et al. [ , ] and bx et al. [ ] also conclude that aerosol transmission is more efficient at low temperatures. js et al. [ ] and jm et al. [ ] also deduce that virus survival increases with decreasing the humidity values. epigraph simulations use real data for modelling the population, the spatio-temporal distribution of influenza, and the meteorological conditions. this simulator consists of different components and data sources shown in fig. . the previous and novel components are represented in blue and orange colors, respectively. the simulator uses input data that is obtained from different sources including: ( ) the influenza data, that contains information about the initial individuals that are infected; ( ) the population data, that describes the individual interactions with others; ( ) the transport data, that contains information about the movement of individuals between different locations and ( ) the climate data, that contains the meteorological conditions existing during the simulated time span. this data feeds the different models implemented in the simulator. we briefly describe the three models that have been previously developed and presented in [ , ] . the epidemic model considers the propagation model of influenza extending the sir (as explained in [ ] by fb et al.) to include states for latent, asymptomatic, dead and hospitalized. the infective period has different phases which may affect the dissemination characteristics of the fig. overview of the data sources, processed data, and epigraph components influenza virus as ame et al. describe in [ ] . each individual has a slightly different length for each infection state. we adopt most of the concrete values for the model parameters from the existing literature on flu epidemics (see [ ] [ ] [ ] [ ] ). you can find them all in [ ] . the transport component models the daily commute of individual to neighboring cities (inter-city movement) and the long-distance travels for several days that represent commute of workers that need to reside at different locations or people that move at any distance for vacation purposes. the people mobility model is based on the gravity model proposed by cv et al. [ ] that uses geographical information extracted from google using the google distance matrix api service. the social model is an agent model that captures individual characteristics and specifies the interaction patterns based on existing interactions extracted from social networks. these patterns determine the close contacts of each individual during the simulation, which is a crucial element to model the spread of the infection. we extract interaction patterns from virtual interactions via email or social networks (enron and facebook) and scale them to approximate a physical connection of the whole network within an urban area. these connections are timedependent to realistically capture the temporal nature of interactions, in our case modeled depending on the day of the week and time of day. the distribution of the population is in terms of four group types: school-age children and students, workers, stay-home parents, and retirees. in this paper, as main contribution, we introduce a new component of the simulator (the meteorological model), that evaluates the impact of climate parameters on influenza propagation. this component is tuned with influenza surveillance data obtained from sisss to provide realistic simulations. as far as we know, this work is the first simulator that integrate real meteorological data to predict the spatio-temporal distribution of influenza. we think that this contribution will help to better understanding the influenza propagation in real environments. in the literature we can find different influenza simulators although none of the following consider meteorological factors in the simulation. examples of them is the work of kk et al. [ ] that presents an sirbased epidemic simulator that permits to parametrize both the population characteristics and the epidemic process. the goal of this work is to identify the turning point (peak of the infected population) of the infection. although the initial approaches for modelling the infection spreading across the contact network, our work consider a broader number of parameters and configuration of the network. he et al. [ ] analyze, by means of simulation, the relationship between social interaction patterns at workplaces and the virus transmission patterns during influenza pandemics. the main effort is geared towards the flexible specification of the different aspects involved in a simulation, such as intervention policies, social modelling, social organization of work, etc. sim-flu [ ] is different from most epidemic simulators in that it focuses on the discovery of most probable future influenza variants starting from virus sequences published by the national center for biotechnology information (ncbi). this work is complementary to the goal of most simulators, including ours, which is to understand and predict the spreading infection patterns of a known flu strand across a population. their methodology is based on observing directional changes in subtypes of influenza over time. js and ak present a framework [ ] to adjust an epidemic simulation based on real-time forecasts of infections from google flu trends. the paper focuses on prediction of the timing of peak infection, but other metrics could be predicted as well. the authors of [ ] simulate the spreading of influenza in an urban environment consisting of several close-by towns connected by trains. their goal is to be able to model and simulate intervention policies. epiwork [ ] was a european project in fp whose focus was to develop a tool framework for epidemic forecast. within this project's framework, wb et al. describe gleamviz [ ] , their tool for epidemic exploration which includes a simulator of transmission based on an accurate demographics of world's population over which they superpose a (stochastic) mobility model. db et al. [ ] use human mobility extracted from airline flights and local commute (based on the gravity model) to predict the activity of the influenza virus based on monte carlo analysis. sm and sm [ ] study the role of population heterogeneity and human mobility in the spread of pandemic influenza. in [ ] , the authors reconstruct contact and time-in-contact matrices from surveys and other socio-demographic data in italy and use this matrix for simulation. epigraph uses meteorological data provided by the spanish meteorological agency (aemet) to generate environment-dependent influenza simulations. the preprocessing stage is performed to obtain clean inputs for the meteorological model. first, the weather station nearest to each simulated urban region is identified. our simulations consider different urban regions with more than , inhabitants. in some cases, the station is within the city limits, while in others it is located in a nearby area (for instance at the region's airport). the data from each weather station is analyzed to reconstruct potentially missing samples. sometimes it is the case that some station data samples are missing because the station was not operational during a given time period. these represents just a small fraction of the overall samples, but they have to be properly addressed. figure shows an example of how the original missing data (shown in upper figure) is reconstructed producing a complete samplingclearpage (reconstructed values are shown in the lower figure in red color). in order to add the missing samples, we have used the reconstruct data algorithm (missdata) included in the matlab's system identification toolbox. this toolbox permits the construction of mathematical models for dynamic systems, starting from measured input -output data. the resulting data is then processed to filter nonrealistic values. some weather stations produce abnormal samples corresponding to non-realistic values that are too big or too small. figure shows an example of this kind of values around sample , . we have corrected these cases with a matlab algorithm we implemented to detect these peaks and correct them using an interpolation of the values from the previous days. these two steps are only performed once for each new meteorological input data and the results may be used for the rest of the process. this section describes how the r s are obtained from the meteorological conditions. in addition to the notations introduced in the introduction, for the rest of the paper we will use sh for the specific humidity and p * h o for the equilibrium water vapor pressure. a related value to sh is absolute humidity (ah), which is the mass concentration that describes the amount of water vapour per volume of air. previous studies [ , ] suggest that ah (and by extension sh) are one of the main factors affecting the influenza virus transmission. in epigraph we adopt the results of the regression model used by ab and js [ ] . in their paper, they analyze monthly weather and influenza mortality data collected between and throughout all the us urban counties. using regression, they conclude that there exists a strong correlation between absolute humidity and mortality, even when controlling for temperature, when the humidity drops below daily means of g/kg. temperature correlations also exist, mainly in the daily ranges between - . c and . c. in an earlier paper ( [ ] ) js et al. study the same dataset and simulate influenza transmission via a sirs model modulated by the data to obtain the basic reproduction number r . they also find bestfit parameter range combinations of r max between . and , and r min between . and . . we adopt the pair of (r max , r min ) that was found to be the best-fit parameter combinations they discover: r max = . , r min = . . from the definition of the specific humidity (sh) and relative humidity (rh)-see rhp and dwg [ ]-we know that: ( ) we also know from buck's equation that the equilibrium water vapor pressure can be calculated using the formula: where the temperature t is measured in degrees celsius. this formula works best for values of t in the range of - c to c. from known values rh, p, and t, and using eqs. ( ) and ( ), we can calculate the specific humidity. from laboratory experiments by js et al. [ ] we have: ( ) where a = − , b = log(r max − r min ) and q is the m above-ground specific humidity, which we approximate to sh at the given temperature. in this way, we obtain a value for p * h o in every sample (obtained every minutes) using eq. ( ) . from this value in combination with the values of rh and p we obtain the value of sh using eq. ( ). finally, eq. ( ) computes the new r values for each urban region. r s are, therefore, time-dependent values that determine, in a stochastic process, how many susceptible individuals of an infected person's connections could be potentially infected. this is the dynamic component of the infectivity of an individual with respect to the others. the other dynamic component is the stochastic transition between infective states [ ] , computed with variable probabilities. our model is not different for the different types / subtypes of influenza. the values of the model parameters (basic reproduction numbers for each stage of the disease) were chosen to fall in the ranges published by ab and js, which are based on actual data for all types of influenza, over years. we choose fixed r s within the ranges, although this is a parameter that can be configured to vary. on the other hand, the evaluation was performed over data from the - influenza season over the whole territory of spain, for all types of influenzas that were diagnosed. we consider that both the choice of r (based on exhaustive data) and the evaluation against real reported cases across spain are comprehensive enough to validate our results. the spanish influenza sentinel surveillance system (sisss) comprises networks of sentinel physicians (general practitioners and pediatricians) in of the spanish regions, as well as the network-affiliated laboratories, including the national influenza reference laboratory (national centre for microbiology, world health organization national influenza centre in madrid). more than sentinel physicians participated each season covering a population under surveillance of around one million-see [ , ] . sentinel physicians reported influenza-like illness (ili) cases-integrating virological data collected in the same population-detected in their reference populations on a weekly basis, following a definition based on the eu-ili, as described in [ ] . for influenza surveillance, they systematically swab (nasal or nasopharyngeal) the first two ili patients each week and sent the swabs to the network-affiliated laboratories for influenza virus detection. the information collected by the sisss includes data on demographics, clinical and virological characteristics, seasonal vaccination status, chronic conditions, and pregnancy. data is entered weekly by each regional sentinel network in a web-based application [ ] and analyzed by the national centre of epidemiology to provide timely information on the evolving influenza activity in spanish regions and at the national level. for example, during the - season, sentinel physicians and pediatricians participated to sisss and surveyed a total population of , , , which represents . % of the total population of spain. we obtained the sisss data from the national center of epidemiology, institute of health carlos iii of madrid (isciii). in order to produce realistic simulations, epi-graph has to be properly configured. this configuration process consists of setting up two parameters: the date of the epidemic onset and the initial influenza-illness rates for each urban region. the first parameter is the time of onset of the epidemics, which occurs during week of . at this time the national average incidence values for influenza are greater than cases per , inhabitants, which is the threshold determined by siss, based on data from the - seasonal epidemic, to be the start of the influenza season. in our simulation the exact date is the th of december of . the second parameter values were obtained from influenza surveillance data obtained from the sisss corresponding to the influenza season - . from this data we obtained the reported weekly ili rate at national and regional level in spain. the data for the murcia and galicia communities are not available and we approximated them based on the data from the nearest community. these rates allow us to approximate the initial number of (clinically) influenza-like-infected individuals using the following formula, based on the study published online (in march ) in the lancet respiratory medicine by ach et al. [ ] . where n report are the cases that demanded medical attention, as reported by the sisss, f pos is the fraction of positive cases, symp is the percentage of symptomatic individuals, and attend is the percentage of those with symptoms that see a doctor. for instance, for the reported n report = cases per , inhabitants in week , and with values f pos = % (empirical value for - in spain), and symp = %, attend = % (values taken from the cited study), we calculate that the total number of infected individuals is of approximately cases per , inhabitants -or . % of the total population. we use this value to set up the initial conditions of the simulation (described in this section), but also to validate its results. each community has a different n report , which leads to different numbers of initially infected individuals. epigraph allows modeling at the level of each individual, and thus can simulate the effect of vaccination policies. to produce realistic results, we use different influenza vaccination coverages by age group; for those older than we have used the vaccination ratios (per community) provided by the ministry of health, social services, and equality of spain. these values correspond to vaccination coverages collected by the national health system [ ] . for the rest of the population (individuals younger than ) we have used the data provided by the spanish statistical office, which is based on surveys done in each community. given that the data are available at community level, we assume that all the urban areas located in the same community have the same vaccination coverages. table shows these percentages per community and age. as input of the mobility model, we use % workers and % students for short distance travel, and % workers, % students, % retired individuals, and % unemployed for long distance travel. while epigraph accounts for many of the components that influence the spreading of the virus, the behavior of these parts and the values of the parameters (such as the initial infectious individuals or the vaccination rate) are unavoidably approximate. on their website, the world health organization reports that in annual influenza epidemics, - % of the population are affected with upper respiratory tract infections [ ] . we have therefore introduced a scaling factor which adjusts the infection propagation rate of each individual to produce, for each urban region, a final infection rate between % and % of the total population. these values are obtained in a pre-calibration phase of epi-graph for the real climate conditions-performed only once-and are then used for all subsequent simulation experiments. note that this is the only data -which is also based on real data-that we use for the calibration process. we do not calibrate the model to an existing epidemic curve. once calibration is done, we use data from sisss, which records influenza-like-illness cases that are not confirmed by laboratory tests, for setting the initial simulation conditions of each urban area. this fact doesn't affect the validity of our results because the purpose is to compare yearly/monthly numbers under different climate conditions rather than know the accurate number of infected individuals. we have performed different tests to validate our approach and simulator. we first validated the simulator against influenza surveillance data, then we evaluated two different environmental scenarios. we believe that our simulator can be useful to predict the short-and mediumterm spread of an infection, as well as to assess the effects that changes in climate can have over influenza epidemics worldwide. the first scenario involves real climate values from aemet and allows studying the short-and medium-term propagation for influenza strands. for the second set of scenarios we generate fictitious values of rh and t by scaling the real values. our idea is to study the effects of the changing climate conditions on influenza propagation. simulations occur across the largest cities in spain, which account for a population of , , inhabitants. the time span is months starting from the day identified as the onset date in our data -the th of december of . in our experiments we have used data from weather stations from the national network, distributed across the country. each weather station collects the values of temperature, atmospheric pressure, and relative humidity every minutes during the entire . these consists of about , data samples per station and . million data values in total. based on these values, we generate the basic reproduction numbers to obtain an r value per urban area at every minutes. with the previously determined initial influenza-like rates per region and (year-specific) date of onset, and after calibration, each urban region data -vaccination rates, individuals' characteristics, initial infective individuals, and r s values -are loaded from files. the validation of our simulator in terms of its capacity to predict qualitatively similar propagation results as those approximated from the influenza surveillance data recorded by sisss. the simulated values for each of the spanish regions are the aggregated values of all the urban regions belonging to it. figure shows the simulated and actual estimated data. the simulated values are scaled to make the largest simulated value to be the same as the maximum real value. this allows a comparison of the evolution of the influenza propagation for each community over time. we can observe that although not perfect, the prediction shows a similar evolution with those from real scenarios. note that the simulator considers an approximation of the real conditions during the simulated period, but producing a better (unlikely perfect) fit between the two domains would need to consider all the factors of the real world that affect the flu propagation at nation-level. some of these are possibly unknown, others are not currently measured, and yet others are not possible to measure. the reason for scaling the data is that the simulated and actual estimated data reflect the population rather differently. on one hand, the simulated values correspond to the overall number of individuals infected with influenza across the considered urban areas. these take into account all the individuals within the simulated areas but only include the largest urban regions (above , inhabitants); small cities, towns, and villages are not considered. on the other hand, the influenza surveillance data are only related to a small fraction of the existing clinical cases: sisss covers a representative but small percentage of the population, in addition to the fact that there are more cases than those reported due to people not seeking medical attention. in contrast, the number of cases are collected from the complete community (including both large and small populations). it is thus not possible to compare the absolute values of the two data sources, although they should be linearly related. figure shows an example of the value of these parameters for the urban region of terrasa (barcelona) over one year. we can observe strong variations of r that are related to the changing temperature, relative humidity, and pressure conditions. to evaluate the effect of both real and hypothetical meteorological climate changes on the spreading of influenza we evaluate temperature variations of t degrees and percentage variations of the relative humidity prh. t = and prh = . correspond to the initial scenario with the original climate conditions. studies show that climate change is producing increments in the average temperature (amplified by pollution) and, in southern europe, longer periods of drought. the idea is to evaluate the impact of these changes on the influenza propagation. in this section, we consider long-term meteorological climate changes, that is, changes in the climate conditions that extend to the entire simulated period of weeks. in this context, we evaluate two different scenarios, probably not as complex as future real climate changes. the first one corresponds to drought conditions, when the relative humidity values (rh) are smaller than current ones. we have considered a reduction of the relative humidity from % to % in increments of % (rh values half than the original ones). according to the infection model, influenza propagates easier for smaller rh values; we thus expect to observe a larger effect. figure shows the overall percentage of infected individuals per community predicted by epigraph. the diminishing rh has indeed a strong impact on the number of infected individuals. on average, . % of the population was infected in the base case (reduction factor equal to ), while the average infection rate for . factor is . %. we can observe that a percental reduction of rh of % produces an approximate increment of . % in the final infection rate. the second scenario evaluates the impact of an increase of temperature on the propagation. figure shows the final infection rate for an increment of the temperature between degrees (current case) and degrees celsius. we can observe that now there is a reduction in the infection rate when the temperature increases. now, an increment of degrees reduces the average infection rate from . % to . %-a decrement of . % per degree. both scenarios assume that the values of the parameters (rh, t) change one at a time. this is a simplification, and the idea behind this approach is to evaluate the impact of a single parameter variation on the overall influenza outcome. however, epigraph supports specifying any changing combination of climate conditions. in a more realistic scenario both parameters would change, and the climate specialists are those who should define what the concrete values are. figure shows the combined effect of temperature and relative humidity change on the average nation-wide infection rate. we have plotted two planes: the first one (colored) represents the average infection rates for different increments in the temperature values and percentile reductions in the relative humidity; the second one (green) displays the infection rate of the original scenario (without climate variation) for all the coordinates and represents the baseline case. the two planes intersect in the lowerleft point, where the temperature and rh have the original values. although both parameters influence the final infection rate, relative humidity has a larger effect than temperature. figure shows the effect of rh and temperature variations on the infection distribution for andalucia community. we can observe that the variation of both parameters changes the shape of the distribution, especially in terms of the peak values but also -more subtly -in terms of the propagation interval. the maximum and minimum % confidence intervals baseline scenario (no rh reduction nor temperature increment) ranges between . and . for urban areas in castilla la mancha and aragón, respectively. these results are produced by a simulator repeating the simulations times. note that there already exist uncertainty in the input data, both with respect to the number of initially infected individuals as well as from the point of view of the epidemic model. to evaluate the effect of short-term changes in climate conditions, we modify rh and the temperature exactly like described in the previous section, only for the first week of the simulation. the rest of the simulation uses the original climate parameters. figures and show the final infection rate for different variations of rh and temperature. we can observe that the impact on the overall percentage of infected individuals is still important, particularly for a decrease in rh of . or more and -less evidently -for an increase in temperature of degrees or more. for smaller changes in temperature the effect is less evident, but we believe that this is due to the fact that the short-term simulation of temperature increase is only one week. we achieve herd immunity in two ways: as result of vaccinating campaigns, and naturally when an individual that was infected goes to the recovery (or dead) state, in which case he becomes immune and starts acting as a propagation stopper. as a result, after certain threshold of infected vs susceptible individuals, the infection rate naturally goes fig. effect of long-term changes in the relative humidity (percentil reduction) and temperature (value increment in celsius) on the influenza propagation for the average nation-wide infection rates down. this occurs at the inflection point in the propagation graph, specifically at about weeks (in our data). the vaccination success rate during the - season was approximately % [ ] . given the parameters shown in fig. of [ ] for herd immunity for influenza, we consider that the r considered by our model takes into consideration this type of immunity. we do not model the level at which herd immunity starts acting as a parameter, although this phenomenon occurs naturally in the simulations. the simulator is flexible enough to support different daily contact patterns for each individual. the probability of an individual getting infected during an interaction also differs (it's a stochastic process), and thus the infection can be transmitted to individuals pertaining to different groups. recently, the work in [ ] suggests that rh should also be considered (together with the temperature) as a modulating factor in the influenza propagation. another study that analyses this relationship can be found in [ ] . this work provides a transmission risk contour map based on the temperature and rh humidity values. note that our work addresses the problem of evaluating the influenza propagation from a different perspective. instead of analyzing the propagation mechanisms of the virus and how they are related to the environment conditions, we focus on an empirical relationship between the virus's basic reproduction number and the outdoor specific humidity. the r values used in this work are the combination of both outdoor and indoor virus propagations, and provide an approximation of a real scenario. note also that the fig. effect of long-term parameter variation on the infection distribution shape for andalucía. in a different rh scales are evaluated ( % in red, % in green, % in blue and % in black); in b different temperature offsets are evaluated ( degrees in red, + degrees in green, + degrees in blue and + degrees in black) effect of short-term changes in the relative humidity on the influenza propagation for the different communities considered in the simulation: in color the average infection rates for different increments in the temperature values and percentile reductions in the relative humidity; in green the infection rate of the scenario without climate variation main goal of this work is to evaluate the impact of the weather conditions on the propagation. a possible limitation is that we only model the largest urban regions in spain; we could add more information related to smaller cities and towns, including rural regions. nevertheless we don't think this data would make a significant difference in the results, as the infection needs a large number of hosts to explode, and / or travel patterns between the infected areas. small town and village areas are arguably much less likely than cities to fulfill these roles. a second limitation is related to the meteorological factors affecting the infection propagation: the number and set of climate factors that the meteorological model takes into account, and the choice of the model itself. additional parameters that specialists mention as possible influencers in virus transmission are factors such as wind, precipitation, or pollution. fig. effect of short-term changes in the temperature on the influenza propagation for the different communities considered in the simulation one important thing to underline is that the data that the study [ ] (whose model we adopt) is based on is of real cases and spans years. interactions between meteorological trends and human behavior are therefore intrinsically reflected in the data, although the rules of behavior change are not explicitly specified for the agents (i.e. individuals) involved in the simulation. the case can be made that meteorological changes were not as extreme before , and that a regression model based on new data may change as well over time. while this is a definite possibility, we believe that its nature will not change in a fundamental way, such that we can still predict trends, if not absolute values. a third limitation is that we don't calibrate the model on an epidemic curve, which results in different timings of the flu peaks in some regions, such as in navarra and madrid. finally, to successfully simulate the flu epidemics requires leveraging many different types of data, most of them in large amounts, as input and calibration measurements for our tool (epigraph). for instance, we are using social network data from enron and facebook to set up the population interaction patterns, census data to extract the characteristics of the different types of individuals, google maps to initialize the transportation module, data from aemet to run simulations that are realistic from a meteorological viewpoint, and weekly ili rates obtained from the sisss to initialize and evaluate the simulator. this makes the implementation of epigraph more realistic, a strength that can lead to more accurate simulations. we have extended our simulator epigraph with a meteorological model that interacts with the rest of the system to better reflect the behavior of the influenza propagation through the entire population of spain. to produce realistic results we also take into account vaccination, with different ratios based on the individuals' ages. the simulator results are compared to real data on infection rates and across the whole country. the results for the prediction of the evolution of the influenza propagation for each community over time are similar in shape to the real data. after validating the simulator, we evaluate different scenarios that reflect changes in climate conditions, and show the predictions for variations in the relative humidity and temperature. lastly, we make epigraph's source code publicly available at [ ], to be used by the scientific community. as future work, an interesting, although independent, possibility is to investigate the potential of epigraph to simulate the evolution of the virus spread for different subtypes of influenza, once the propagation model parameters (e.g. incubating period, infectious period, basic reproduction numbers, etc.) are known, or to narrow down the possible subtypes in early phases of an infection. one could also investigate the impact of new meteorological factors on the evolution of the infection. world healh organization influenza (seasonal) global influenza seasonality: reconciling patterns across temperate and tropical regions climatic factors and influenza transmission, spain leveraging social networks for understanding the evolution of epidemics absolute humidity, temperature, and influenza mortality: years of county-level evidence from the united states modelling seasonality and viral mutation to predict the course of an influenza pandemic absolute humidity and the seasonal onset of influenza in the continental united states absolute humidity modules influenza survival, transmission, and seasonality influenza virus transmission is dependent on relative humidity and temperature high temperatures ( degrees c) blocks aerosol but not contact transmission of influenza virus climatological and geographical impacts on global pandemic of influenza a(h n ) role of absolute humidity in the inactivation of influenza viruseson stainless steel surfaces at elevanted temperatures towards efficient large scale epidemiological simulations in epigraph emergence of drug resistance: implications for antiviral control of pandemic influenza containing pandemic influenza with antiviral agents an influenza simulation model for immunization studies synchrony, waves, and spatial hierarchies in the spread of influenza coupling effects on turning points of infectious diseases epidemics in scale-free networks relevance of workplace social mixing during influenza pandemics: an experimental modelling study of workplace cultures simflu: a simulation tool for predicting the variation pattern of influenza a virus forecasting seasonal outbreaks of influenza parallel agent-based simulator for influenza pandemic the gleamviz computational tool, a publicly available software to explore realistic epidemic spreading scenarios at the global scale seasonal transmission potential and activity peaks of the new influenza a(h n ): a monte carlo likelihood analysis based on human mobility the role of population heterogeneity and human mobility in the spread of pandemic influenza little italy: an agent-based approach to the estimation of contact patterns-fitting predicted matrices to serological data absolute humidity modulates influenza survival, transmission, and seasonality global environmental drivers of influenza perry's chemical engineers' handbook sentinel surveillance system. characterisation of swabbing for virological analysis in the spanish influenza sentinel surveillance system during four influenza seasons in the period epidemiology of the influenza pandemic in spain. the spanish influenza surveillance system amending decision / /ec laying down case definitions for reporting communicable diseases to the community network under decision no / /ec of the european parliament and of the council comparative community burden and severity of seasonal and pandemic infl uenza: results of the flu watch cohort study coberturas de vacunación en mayores de años effectiveness of the - seasonal trivalent influenza vaccine in spain: cyceva study the vaccination coverage required to establish herd immunity against influenza viruses mechanistic insights into the effect of humidity on airborne influenza virus survival, transmission and incidence aerosol influenza transmission risk contours: a study of humid tropics versus winter temperate zone we would like to acknowledge all the sentinel general practitioners and pediatricians, epidemiologists, and virologists participating in the spanish influenza sentinel surveillance system. part of the input data used in this work have been obtained from the spanish influenza sentinel surveillance system and the meteorological information provided by spanish national meteorological agency, aemet, ministerio de agricultura, alimentación y medio ambiente. authors' contributions des., mcm. and jc. designed and implemented epigraph simulator. all authors conceived and designed the experiments. des. processed the input data and run the experiments. des. and mcm. wrote the paper. cd., dgb., and al. provided insights on the validity of our assumptions, recommended additional related work, and contrasted the results with their own findings. all authors review the different manuscript drafts and approved the final version for submission. the author(s) read and approved the final manuscript. this work has been partially supported by the spanish "ministerio de economía y competitividad" under the project grant tin - -p "towards unification of hpc and big data paradigms". the work of maria-cristina marinescu has been partially supported by the h european project growsmarter under project grant ref. . the role of both funders was limited to financial support and did not imply participation of any kind in the study and collection, analysis, and interpretation of data, nor in the writing of the manuscript. epigraph's user manual and source code are publicly available at [ ] and can be used by the scientific community. the dataset supporting the conclusions of this article is available in the [ ] repository. not applicable. not applicable. the authors declare that they have no competing interests. key: cord- -tgslabi authors: schnee, sarah valerie; pfeil, johannes; ihling, clara marlene; tabatabai, julia; schnitzler, paul title: performance of the alere i rsv assay for point-of-care detection of respiratory syncytial virus in children date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: tgslabi background: respiratory syncytial virus (rsv) is the most important cause of severe acute respiratory tract infection in young children. alere i rsv is a novel molecular rapid test which identifies respiratory syncytial virus in less than min. methods: we evaluated the clinical performance of the alere i rsv assay in a pediatric point-of-care setting during winter season / . test results from nasopharyngeal swab samples were compared to a real-time reverse transcription pcr reference standard. results: the overall sensitivity and specificity of the alere i rsv test assay was % (ci( ) % – %) and % (ci( ) % – %), respectively. alere i rsv performed well in children of all age groups. an optimal sensitivity of % (ci( ) % - %) and specificity of % (ci( ) % - %) was obtained in children < months. in children ≥ years, sensitivity and specificity remained at % (ci( ) % – %) and % (ci( ) % – %), respectively. false negative alere i rsv test results mostly occurred in samples with low viral load (mean ct value . ; ci( ) . – . ). the alere i rsv assay is easy to use and can be operated after minimal initial training. test results are available within min, with most rsv positive samples being identified after approximately min. conclusion: the alere i rsv assay has the potential to facilitate the detection of rsv in pediatric point-of-care settings. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. respiratory syncytial virus (rsv) is the most important cause of acute respiratory tract infection (arti) in neonates and young children worldwide [ ] . rsv is a frequent cause of hospitalization in young children and leads to significant morbidity in premature neonates and children with chronic lung or congenital heart disease [ ] [ ] [ ] . the clinical diagnosis of rsv is hampered by the mostly unspecific symptoms of rsv infection. early recognition of rsv infection is useful to optimize care management, minimize unnecessary antibiotic use [ ] and provide targeted infection control for children hospitalized with rsv infection [ ] . in addition, specific antiviral therapy for rsv infection is currently under early clinical evaluation [ ] , and early detection of rsv infection may become important for timely antiviral treatment in severely sick children. pediatricians therefore often apply rapid point-of-care rsv test assays. the major limitation of point-of-care rsv testing is the low sensitivity of commercially available rapid antigen detection tests (radt). radt sensitivity is strongly dependent on viral load, and therefore performs best in young infants with classical symptoms of rsv bronchiolitis. in older children and adults with low viral load, the sensitivity is poor and radt are not recommended in these age groups [ , ] . alere i rsv is a novel rapid molecular test assay which can identify rsv in less than min. in a previous analysis, we reported an alere i rsv sensitivity and specificity of % (ci - %) and % (ci % - %), respectively [ ] . this first analysis focused on young infants, and testing was done under laboratory conditions which may not reflect the performance in point-of-care settings. in the current study, we addressed these limitations and applied the alere i rsv test assay in a pediatric point-of-care setting on a larger study population across different pediatric age groups. the objective of this analysis was to report an estimate of the alere i rsv test performance in a pediatric point-of-care setting. between november and march , we prospectively collected nasopharyngeal swabs (nps) in the outpatient department of the center of childhood and adolescent medicine heidelberg, germany. study inclusion criteria were i) age < years, ii) clinical symptoms of an acute respiratory infection, and iii) indication for hospitalization according to the clinical judgment of the attending physician. patients with clinical symptoms of an acute respiratory infection included cases of upper respiratory tract infection (urti), otitis media, croup, bronchiolitis, bronchitis and pneumonia. nasopharyngeal swabs were collected by local staff in ml viral transport media (vtm; mswab; copan, brescia, italy). μl of the vtm were directly used for point-of-care testing with the alere i rsv assay. the remaining sample was transferred to the virology diagnostic laboratory, and stored in μl aliquots at − °c until further analysis. attending pediatricians prospectively reported medical information on a standardized data sheet, including the duration of clinical symptoms, demographic and clinical data. alere i rsv test assays were applied in the pediatric outpatient department. the test procedure followed the alere i rsv package insert [ ] . in brief, μl of the respiratory sample was added to the sample receiver containing . ml elution buffer. two μl volumes were added to the test base with the provided transfer cartridge for isothermal amplification. alere i rsv assays were conducted by attending physicians or nurses working in the pediatric outpatient department. all operators were allowed to carry out the test assay only after an initial hands-on-training. test results were printed and reported on the medical datasheet. invalid test results were re-tested immediately. the alere i rsv assay was evaluated against a cemarked real-time reverse transcriptase polymerase chain reaction assay (rt-pcr). for rt-pcr analysis, rna was extracted from μl respiratory specimen using the qiaamp® viral rna mini kit (qiagen, hilden, germany) according to the manufacturer's protocol. amplification and detection of viral rna was performed by ftd respiratory pathogens multiplex pcr (ftd , fast-track diagnostics ltd., sliema, malta) on a lightcycler® instrument ii (roche, mannheim, germany). the ftd assay can detect the following pathogens: influenza a, h n or b, rhinovirus, respiratory syncytial virus, bocavirus, adenovirus, parainfluenza , , or , coronavirus nl , e, oc or hku , parechovirus, enterovirus, human metapneumovirus a/b and mycoplasma pneumoniae. ftd results that did not correspond to the alere i test assay were verified by a second rt-pcr assay. for this purpose, rna was extracted from an independent sample aliquot and analyzed by altona realstar pcr (altona realstar rsv rt-pcr, altona diagnostics, hamburg, germany). to preclude possible discrepancies between the two rt-pcr methods, samples with different results in monoplex and multiplex pcr were again tested by the ftd assay (additional file : figure s ). both the ftd and the altona realstar assay were initially evaluated using defined rsv a and rsv b positive and negative samples from patients, from the german rsv reference laboratory and from the official german proficiency testing panel. results with a cycle threshold (c t ) value of < were considered positive. for sub-typing of rsv positive samples, sanger sequencing targeting the second hyper-variable region of the g gene was performed using primer pairs as previously described [ ] . resulting sequences were assembled and edited using the seqman ii software of the lasergene package (dnastar, madison, wi) and allocated to subtype rsv a or rsv b using the basic local alignment search tool (blast; http://blast.ncbi.nlm.nih.gov). statistical analyses were conducted using stata/ic . (statacorp. lp, college station, tx, usa). mann-whitney-u-test was applied to compare c t values in samples with true positive versus false negative alere i rsv result. the alere i rsv sensitivity in rsv a versus rsv b positive samples was compared using the χ -test. p values < . were considered statistically significant. data reporting was done according to stard recommendations [ ] . from november to march , nps were collected from children presenting with symptoms of acute respiratory tract infection. fifteen samples were excluded from the final analysis due to unavailability of sample aliquots for rt-pcr testing (n = ), duplicate sampling of patients during one hospital stay (n = ), or missed re-testing of an initially invalid alere i rsv test result (n = ). five hundred eighteen samples ( % of collected nps) from children were included in the final analysis (fig. ) . demographic and clinical characteristics of the study participants are summarized in table . alere i rsv was positive in % ( / ) and negative in % ( / ). in comparison to the rt-pcr reference standard, the alere i rsv test result was true positive in and true negative in samples, respectively. false positive test results were reported in patients, and patients were identified with false negative alere i rsv test outcome ( table ). the overall alere i rsv test sensitivity and specificity was % (ci % - %) and % (ci % - %), respectively. the mean ftd c t value of true positive samples was . (ci . - . ; range . - . ). nps with false negative alere i rsv result had a significantly higher mean c t value of . (ci . - . ; range . - . , p < . ; mann-whitney-u-test). we grouped the admission diagnoses in cases of urti (n = , including urti, otitis media and croup), lrti (n = , including bronchiolitis, bronchitis and pneumonia) and non-respiratory admission diagnoses (n = ). the latter includes children admitted for non-respiratory reasons (e.g. febrile convulsion, diarrhea) with concomitant acute rti from the clinical perspective, c t values were higher (and hence viral load lower) in respiratory specimens of older children and children admitted for non-respiratory reasons with concomitant respiratory tract infection (additional file : table s ). in consequence, the alere i rsv sensitivity was % (ci % - %) in children < months, % (ci % - %) in children - months, % (ci % - %) in children - months and % (ci % - %) in children above years of age (table ). in children hospitalized for urti or lrti, the alere i rsv test sensitivity was % (ci - %) and % (ci - %), respectively. in children who were admitted for non-respiratory reasons, we found a sensitivity of % (ci % - %) ( table ) . as illustrated in additional file : figure s , the relatively poor sensitivity in this group resulted from a high proportion of samples with low viral load (c t value > ), and coinfection with influenza a, parainfluenza, rhinovirus or coronavirus was detected in / cases. we did not undertake comprehensive analytical specificity testing but note that the nps with negative rt-pcr result included samples tested positive for influenza a, h n or b (n = ), rhinovirus (n = ), bocavirus (n = ), adenovirus (n = ), parainfluenza , , or (n = ), coronavirus nl , e, oc or hku (n = ), parechovirus (n = ), enterovirus (n = ), human metapneumovirus a/b (n = ) and mycoplasma pneumoniae (n = ). in rsv positive specimens, observed coinfections included rhinovirus (n = ), coronavirus (n = ), parainfluenza (n = ), bocavirus (n = ) and influenza (n = ). in cases, neither rsv nor any of the above-mentioned pathogens were detected. in our point-of-care setting, positive test results were identified after a mean duration of . min (ci . - . min; range . - . min). this included both sample pre-heating ( min) and the amplification reaction. after the first testing, invalid results were reported in % ( / ). these nps were directly retested, and valid (positive or negative) results were obtained in all cases. we found that the novel alere i rsv assay has a sensitivity of % (ci % - %) and a specificity of % (ci % - %) in a pediatric point-of-care setting. the test is user-friendly and test results are obtained in less than min, with most positive test results being identified after approximately min. no direct comparison of alere i rsv versus radt was done in our study. based on published data, the expected radt sensitivity in young children is approximately % [ ] . in our study setting, we previously found a radt sensitivity of % (ci - %) [ ] and % (ci % - %) [ ] over two different winter seasons. the alere i rsv sensitivity is clearly superior to the expected radt performance. in children aged - months with lower viral load, the use of radt is particularly limited with reported sensitivity of approximately % [ ] . in patients ≥ years of age, we found an alere i sensitivity and specificity of % and % in comparison to our rt-pcr reference standard, respectively. the alere i assay is suitable for point-of-care detection of rsv in children across all age groups. other sensitive rapid nucleic acid amplification assays are available for early detection of rsv infection. these assays require a testing time of at least min to more than h [ , ] . alere i rsv test results are available a limitation of our study is the use of vtm for alere i rsv testing instead of directly inserting the swab to the alere test base. using vtm was required to establish the rt-pcr reference standard. this procedure is in accordance with the alere i rsv package insert, but implies that samples are : diluted in comparison to directly inserting the swab. in our analysis, this could have provoked false negative results in samples with low viral load, and regular point-of-care users might prefer direct, non-diluted testing of swab specimens. second, we compared alere i rsv test results against a multiplex rt-pcr reference standard. only divergent results were further evaluated by monoplex rt-pcr. multiplex rt-pcr is usually slightly less sensitive than monoplex rt-pcr in samples with low viral load. [ ] in pediatric patients, viral loads are usually high and in fact, % of our rsv positive samples had a c t value < . we therefore believe that multiplex rt-pcr is a rigorous reference standard in our study cohort, but acknowledge that applying a monoplex rt-pcr reference standard might have resulted in a slightly lower sensitivity of the alere i rsv assay. in summary, we evaluated the novel alere i rsv assay in a pediatric emergency setting against a rt-pcr reference standard. the alere i rsv performed well in the point-of-care setting, and sensitive test results were obtained across all pediatric age groups within min. the assay requires a shorter test time than other currently available molecular test assays, and provides a significantly higher sensitivity than radt assays. the alere i rsv assay performs well in the pediatric point-of-care setting. the assay is easy to use, and the high sensitivity and specificity of test results help pediatricians to act appropriately both in patients with and without rsv infection. global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis respiratory syncytial virus and influenza are the key viral pathogens in children < years hospitalized with bronchiolitis and pneumonia in islamabad pakistan population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children hospitalization attributable to influenza and other viral respiratory illnesses in canadian children effect of rapid viral diagnosis on the management of children hospitalized with lower respiratory tract infection prospective controlled study of four infection-control procedures to prevent nosocomial infection with respiratory syncytial virus respiratory syncytial virus: prospects for new and emerging therapeutics. expert review of respiratory medicine lack of sensitivity of rapid antigen tests for the diagnosis of respiratory syncytial virus infection in adults diagnostic accuracy of rapid antigen detection tests for respiratory syncytial virus infection: systematic review and meta-analysis evaluation of alere i rsv for rapid detection of respiratory syncytial virus in children hospitalized with acute respiratory tract infection alere i rsv package insert circulation patterns of genetically distinct group a and b strains of human respiratory syncytial virus in a community stard : an updated list of essential items for reporting diagnostic accuracy studies screening for respiratory syncytial virus and isolation strategies in children hospitalized with acute respiratory tract infection reversetranscription loop-mediated isothermal amplification for rapid detection of respiratory syncytial virus directly from nasopharyngeal swabs host and viral factors affecting clinical performance of a rapid diagnostic test for respiratory syncytial virus in hospitalized children charnot-katsikas a. comparison of cepheid xpert flu/rsv xc and biofire filmarray for detection of influenza a, influenza b, and respiratory syncytial virus prospective and retrospective evaluation of the cepheid xpert(r) flu/rsv xc assay for rapid detection of influenza a, influenza b, and respiratory syncytial virus comparison of fast-track diagnostics respiratory pathogens multiplex real-time rt-pcr assay with in-house singleplex assays for comprehensive detection of human respiratory viruses we thank the physicians and nurses at the center for childhood and adolescent medicine for collecting respiratory samples and the technicians in the virology diagnostic laboratory for excellent technical assistance. alere i rsv and rt-pcr tests were partly provided by alere free of charge. alere had no influence on the study procedure and analysis of study results. s.v.s., c.i. and j.t. are financially supported by fellowships of the german center for infectious diseases (dzif). j.p. is the recipient of an hrcmm (heidelberg research center for molecular medicine) career development fellowship.availability of data and materials all data generated or analysed during this study are included in this published article and its supplementary information files. authors' contributions svs, jp and cmi conducted study experiments. svs and jp wrote the manuscript. all authors (svs, jp, cmi, jt and ps) contributed to the interpretation of study results and approved the final version of this manuscript.ethics approval and consent to participate this study was approved by the ethical research board of the university hospital heidelberg, germany (s- / ). all samples and medical information included in this study were obtained during routine medical care. written consent for the analysis and publication of the data included in this study was obtained from all parents or guardians. alere i rsv and rt-pcr test materials were partly provided by alere free of charge.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -cpl zf f authors: provoost, judith; valour, florent; gamondes, delphine; roux, sandrine; freymond, nathalie; perrot, emilie; souquet, pierre-jean; kiakouama-maleka, lize; chidiac, christian; lina, gérard; dumitrescu, oana; sénéchal, agathe; ader, florence title: a retrospective study of factors associated with treatment decision for nontuberculous mycobacterial lung disease in adults without altered systemic immunity date: - - journal: bmc infect dis doi: . /s - - -x sha: doc_id: cord_uid: cpl zf f background: nontuberculous mycobacteria (ntm) lung diseases are increasingly recognized as chronic opportunistic infections, occurring in individuals with a wide variety of underlying conditions. in the absence of systemic immunodeficiency, decision of ntm lung disease treatment must relies on a careful risk/benefit assessment, given the requirement of long-term administration of multidrug therapies supported by limited evidence. the primary objective was to identify the factors associated with anti-ntm treatment initiation. clinical and radiological outcome upon treatment were studied. methods: this retrospective, single center study ( – , months) addressed the criteria supporting treatment decision among adults with ntm lung disease without systemic immunodeficiency at our institution, with the assigned goal to harmonize the practice. all patients matched the current international definitions of ntm lung disease according to the american thoracic society criteria. factors associated with anti-ntm treatment were investigated by conditional logistic regression. clinical and radiological outcomes of treated and untreated ntm-disease cases were examined. mortality rate was assessed. an expert radiologist conducted a blinded computed tomography (ct)-scan review of the treated and untreated patients. results: among cases of ntm lung diseases, ( %) received anti-ntm treatment. in univariate analysis, a body mass index (bmi) < kg/m( ) (odds ratio (or), . [ % confidence interval (ci) . – . ]; p = . ), hemoptysis (or, . [ % ci . – . ]; p = . ), excavation(s) (or, . [ % ci . – . ], p = . ), prior anti-ntm treatment (or, . [ % ci . – . ]; p = . ), aspergillus spp. co-infection (or, . [ % ci . – . ]; p = . ) were associated with treatment initiation. in multivariate analysis, aspergillus spp. co-infection was the only independent determinant of treatment initiation (or, . [ % ci . – . ]; p = . ). twenty-one ( %) patients received ≥ anti-ntm drugs. median treatment duration and follow-up were . (interquartile range [iqr], . – . ) weeks and . (iqr, . – . ) months, respectively. regarding radiological outcome, ct-scans were reviewed, showing similar rates of regression or stabilization in treated and untreated patients. overall mortality rate was not different in treated and untreated patients. conclusion: the most relevant variable associated with anti-ntm treatment initiation was aspergillus spp. co-infection. radiological regression or stabilization of pulmonary lesions was not different between the treated and untreated patients. nontuberculous mycobacteria (ntm) are ubiquitous bacteria of environmental origin including a widely diverse number of species (> ), some of which cause disease in humans [ ] [ ] [ ] . prevalence of ntm lung diseases is unexpectedly increasing in industrialized countries, as consistently uncovered by recent studies [ , ] . the key issue remains to determine whether ntm are the true and single promoter of an evolving lung disease or chronic airway colonizers, among others. to standardize the diagnosis of ntm lung disease, the guidelines for ntm diagnosis of the american thoracic society (ats)/infectious disease society of america (idsa) and the british thoracic society (bts) require isolation and growth of the same ntm strain on at least two separate samples from the patient [ , ] . human host and pathogenic ntm relationship is still poorly understood, as ntm virulence is highly variable from one species to another. ntm lung disease is strongly associated with pre-existing pulmonary conditions such as chronic obstructive pulmonary disease (copd), cystic fibrosis, idiopathic bronchiectasis, prior active tuberculosis or pneumoconiosis [ ] . it is also frequently associated with genetic or acquired systemic immune deficiency such as defects in the pathways of inflammatory cytokines interleukin (il)- , tumor necrosis factor (tnf)-α or interferon (ifn)-γ, immunosuppressive treatments (including anti-tnf-α therapy or corticosteroids), solid-organ transplantation, or acquired immune deficiency syndrome (aids)/human immunodeficiency virus (hiv) infection [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, it may also occur in individuals without recognized severe immune local or systemic deficiency. in the absence of patent predisposition, ntm diseases are overrepresented among the specific morphotype of slender women with a low body fat [ ] . treatment decision for ntm lung disease is challenging. there is debate as to which patients should benefit the most from treatment according to medical background, comorbidities, clinical status, radiologic features and causal ntm strain. assessment of clinical, microbiologic, and radiologic response to treatment is not standardized as well. we focused on ntm lung diseases in adults without systemic immunodeficiency that met the ats criteria guidelines. based on the comparison of a group of treated and untreated patients, the primary objective was to identify the factors associated with physician decision of initiating anti-ntm treatment. secondary objectives were to study the outcome upon treatment and to propose a standardized evaluation for the diagnosis and decision making to treatment of ntm lung diseases in adults without systemic immunodeficiency. we conducted a retrospective, observational, single-center study between january and february ( months) among adults (≥ year-old) without systemic immunosuppression presenting ntm lung disease. exclusion criteria were hiv infection, cystic fibrosis, primary ciliary dyskinesia, active malignant disease, solid-organ transplantation or ongoing immunosuppressant treatments such as tnf inhibitor or high-dose corticosteroid (≥ mg/kg more than days). case identification was based on cross-referencing the databases of the mycobacteria laboratory and the departments of infectious and pulmonary diseases. patients eligible for inclusion in the ntm lung disease cohort were those who matched the criteria previously defined by the ats/idsa and the bts guidelines with the minimum requirement of clinical and microbiologic following criteria: (i) pulmonary symptoms associated with multifocal bronchiectasis with multiple small nodules on computed tomography (ct)-scan; (ii) proper exclusion of other diagnoses; (iii) ntm-positive culture results from at least two separate expectorated sputum samples or a ntm-positive culture result from at least one bronchial wash or lavage [ , ] . patients' characteristics at diagnosis were collected in order to perform analysis on selected variables: demographics; history of predisposing factors; underlying pulmonary diseases; comorbidities; pulmonary function testing; respiratory bacterial or mycological co-infection(s), which definition was similar to ntm criteria, namely positive culture isolation of the same species from at least two separate expectorated sputum samples or a positive culture result from at least one bronchial wash or lavage; immunologic status; nutritional status; clinical features; microbiologic assessment through identification of ntm species on positive ntm cultures and sample culture conversions; radiologic features on high-resolution ct-scans (fibrocavitary disease or nodular/bronchiectasis disease); prior treatment for ntm lung disease, treatment combination and duration; outcome. because of the retrospective observational nature of the study and the lack of any modification in patients' management, the need for informed consent was waived with the authorization of the ethics committee of lyon university hospital (comité d'ethique, hospices civils de lyon), which approved the study under the number - . an independent expert chest radiologist, blinded to the patient information, retrospectively reviewed the ct-scans performed without injection of intravenous contrast media, assigned in random order at diagnosis and six to months after treatment or during the follow up of the untreated patients. the number and size of cavity(ies) and their wall thickness were evaluated in the lung window setting. nodular opacity(ies) (≥ mm), cluster(s) of small nodules (≤ mm), the tree-in-bud pattern, the presence of bronchiectasis in any of the lobes or multifocal bronchiectasis were evaluated. based on the number and size of the lesions, the expert classified the lesions as improved, stable or worsening. the primary endpoint was to identify the factors significantly involved in the decision of initiating anti-ntm treatment by patient referent physician. secondary endpoints were the assessment of clinical and radiological outcomes upon anti-ntm treatment in comparison with no treatment. based on these findings, a standardized appraisal was proposed to assist diagnosis management and treatment decision for ntm lung diseases in adults without altered systemic immunity. descriptive data were used to estimate the frequencies of the study variables. there were expressed as count (percentage, %) for dichotomous variables and as medians (interquartile range [iqr]) for continuous values. the number of missing values was excluded from the denominator. non-parametric statistical methods fisher exact test, χ test and mann-whitney u test were used to compare groups, where appropriate. the probability of treatment initiation over time was evaluated by kaplan-meier survival curve analysis, with group comparison using the log-rank (mantel-cox) test. stepwise binary logistic regression analysis was used to assess the determinants for treatment initiation, expressed as odd ratios (or) with % confidence intervals ( % ci). after checking the variables for interactions, variables with medical meaning and with p-values obtained in the univariate analysis of < . were included in the final multivariate model. a value of p < . was considered significant. all analyses were performed using spss software version . (spss. chicago. il). out of patients eligible to ats/idsa ntm lung disease criteria, patients were included in the study, of who ( %) received and ( %) did not receive anti-ntm treatment (fig. ). the median age was (interquartile range [iqr], - ) year-old with a male/ female ratio of . . etiologic ntm agents were mycobacterium avium (n = , . %), m. chimaerae (n = , . %), m. xenopii (n = , . %), m. intracellulare (n = , . %), m. simiae (n = , . %), m. kansasii (n = , %), and m. abscessus (n = , %), with three patients having ≥ concomitant ntm lung diseases. importantly, the evidence of ntm lung disease has led to diagnose six underlying chronic lung diseases, of which a genetically documented cystic fibrosis in a year-old women. on descriptive analysis, patient's characteristics did not significantly differ, to the exception of a lower bmi (p = . ) and a higher number of previously known ntm lung disease (p = . ) in treated versus untreated patients (table ) . notable percentages of missing data at diagnosis have to be acknowledged for active tobacco smoking ( . %, n = ), respiratory functional testing ( . %, n = ) with a very few patients having a -min walk test, baseline arterial blood oxygenation levels ( . %, n = ), ct-scan follow up within months after diagnosis in untreated patients ( %, n = ). factors leading the patient referent physician to initiate anti-ntm treatment were assessed using bivariate analysis. table ). the probability of treatment initiation over time according to the presence or not of targeted variables was investigated. the probability was significantly higher in case of bmi < vs. > kg/m (p = . ), of hemoptysis vs. no hemoptysis (p = . ), of aspergillus spp. co-infection vs. no co-infection (p = . ), of pulmonary excavation(s) vs. no excavation (p = . ) (fig. a, b, c and d, respectively) . (table ) . finally, all-cause mortality was not different between treated and untreated groups, although lost to follow-up was high in the untreated group (n = , . %). regarding the four patients that deceased (n = in the treated group and n = in the untreated group), the cause of mortality was linked to the underlying diseases rather than ntm-related mortality. in the present study, treated patients were characterized by bmi < kg/m , presence of hemoptysis and excavation(s), aspergillus spp. co-infection, and prior anti-ntm treatment. aspergillus spp. co-infection was the only independent factor associated with treatment initiation. a single study from five english centers has recently addressed the factors that influence anti-ntm treatment initiation using similar retrospective design of treated and untreated cohort comparison inclusion criteria which allowed non-aids/hiv immunosuppressed patients ( and % in the treated and untreated subsets, respectively) to be evaluated. in the multivariate analysis, patients had increased odds of anti-ntm treatment in case of cavitation on ct imaging, night sweats and weight loss [ ] . here, three out of four criteria are part of guideline criteria leading to decision of anti-ntm treatment. they all reflect a degree of severity of ntm lung disease linked with progression of an impaired respiratory condition. the current problematic of ntm lung diseases shifts from distinguishing colonization from infection toward differentiating stable, poorly active vs. progressive active ntm disease, the latter being responsible for further structural lung damage(s). the risk/benefit analysis includes prescribing recommended long-term multidrug regimens with concerns over suboptimal cure rates and frequently reported drug-related side effects. to support these arguments, others have already shown that physician ats/idsa guideline [ ] . another study conducted in france has shown that among a cohort of ntm lung diseases, only ( . %) received appropriate treatment matching ats/idsa guidelines [ ] . inappropriate prescriptions were mostly related to shorter treatment duration ( months or less) and/or off-recommendation regimen, particularly those excluding macrolide from the combination or those using a single-drug macrolide regimen [ , ] . it has to be acknowledged that guidelines specify that treatment for mac-associated lung disease in hiv-negative individuals can be a three-times-weekly drug regimen upon culture conversion while on therapy for year, which may favor treatment compliance [ , , ] . patients with bronchiectasis and ntm lung disease have a higher prevalence of being sensitized to aspergillus than patients with ntm-free bronchiectasis [ ] . allergic airway manifestations in response to aspergillus are termed aspergillus-related lung diseases with a spectrum going from aspergillus-induced hypersensitivity to the severe allergic bronchopulmonary aspergillosis (abpa) [ ] . by itself, the coexistence of ntm and aspergillus in lung airway justifies the need for testing aspergillus serology, total serum immunoglobulin (ig)e and aspergillus-specific ige levels as well as mycological direct examination and culture of sputum or bronchial aspirates for presence of filamentous fungi in the diagnosis algorithm of patients with ntm lung diseases. active co-infections with ntm and aspergillus spp. have also been described, in which patients with ntm lung disease develop chronic forms of pulmonary aspergillosis which definitions and management have been revisited in recent updated guidelines [ ] [ ] [ ] [ ] . radiographic improvement may be hampered by concomitant lung disease and the limited potential for resolution of consolidated radiologic abnormalities. a previous study has investigated radiologic response to treatment showing consistent results with those found in the present study. although anti-ntm treatment led to an improvement or stabilization of lesions for a majority of patients, these modifications were not significantly different from the untreated group who went through ct-scan follow up indicating that anti-ntm treatment did not lead to radiological abnormalities reversion [ ] . the present study has strengths and limitations. the strength is the study of the largest cohort so far of ntm lung diseases in patients without systemic immunodeficiency with exhaustive data collection and blinded radiological assessment. we acknowledge the biases that contribute to mitigate conclusions from the study such as being conducted in a single center, the important differences in physician's management resulting in lack of consistency in treatment decision making, the number of missing data. in addition, the ct-scans were not performed at fixed intervals, particularly in the untreated subset of patients. finally, treatment duration and outcome criteria were not standardized, which prevent to properly assess treatment efficacy. useful consensus definitions for key outcome parameters to be used in the treatment of ntm lung diseases have been released very recently, which should harmonize data collection regarding ntm treatment [ ] . future researches are necessary to better define criteria associated with progressive active ntm disease in the immunocompetent setting. concretely, this preliminary study has led to implement in our institution a standardized appraisal for the diagnosis of ntm lung diseases in this particular setting ( table ). the aim is to provide a future basis for the development of a diagnosis scoring system supporting anti-ntm treatment decision. future studies should focus as well on the most relevant ct imaging variables associated with response to treatment over time that may be applied in future clinical trials to assess treatment outcome. in summary, the main factors supporting anti-ntm treatment decision in immunocompetent were low bmi, hemoptysis, lung excavation(s), prior anti-ntm treatment and aspergillus pp. co-infection, the latter being the only independent factor. anti-ntm treatment did not achieve radiological abnormalities reversion, as pulmonary lesions assessment showed no difference between the treated and the untreated patients. a diagnosis of ntm lung disease in an immunocompetent patient requires investigating the presence of a chronic pulmonary underlying disease. pulmonary disease caused by non-tuberculous mycobacteria nontuberculous mycobacterial lung disease: the top ten essentials leveraging advances in tuberculosis diagnosis and treatment to address nontuberculous mycobacterial disease pulmonary nontuberculous mycobacterial disease prevalence and clinical features: an emerging public health disease an official ats/idsa statement: diagnosis, treatment, and prevention of nontuberculous mycobacterial diseases british thoracic society guidelines for the management of non-tuberculous mycobacterial pulmonary disease (ntm-pd) pulmonary disease by nontuberculous mycobacteria-clinical management, unmet needs and future perspectives patients with nontuberculous mycobacterial lung disease exhibit unique body and immune phenotypes factors which influence treatment initiation for pulmonary non-tuberculous mycobacterium infection in hiv negative patients; a multicentre observational study lack of adherence to evidence-based treatment guidelines for nontuberculous mycobacterial lung disease are guidelines on the management of non-tuberculous mycobacteria lung infections respected and what are the consequences for patients? a french retrospective study from early results (at months) with intermittent clarithromycin including regimens for lung disease due to mycobacterium avium complex intermittent antibiotic therapy for nodular bronchiectatic mycobacterium avium complex lung disease nontuberculous mycobacterial disease and aspergillus-related lung disease in bronchiectasis nontuberculous mycobacterial lung infection complicated by chronic necrotising pulmonary aspergillosis chronic necrotizing pulmonary aspergillosis as a complication of pulmonary mycobacterium avium complex disease chronic pulmonary aspergillosisrationale and clinical guidelines for diagnosis and management treatment outcome definitions in nontuberculous mycobacterial pulmonary disease: an ntm-net consensus statement the authors gratefully acknowledge pr gilles devouassoux for helpful insights. no external funding was received for this study. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. authors' contributions jp contributed to conception and design of the study, acquisition of the data, interpretation of the data, drafted the manuscript and approved the final version; fv carried out the statistical analysis, participated in revision of the paper for important intellectual content, and approved the final version; dg carried out the blinded review of ct-scans with the examination of each imagery, she participated in revision of the paper for important intellectual content, and approved the final version; gl and od are in charge of the mycobacteria laboratory and have provided ntm strain identification and drug susceptibility tests. they participated in revision of the paper for important intellectual content, and approved the final version; sr, nf, ep, pjs, lkm, and cc contributed to acquisition of the data, revision of the paper for important intellectual content, and approved the final version; fa and as are the project initiators, contributed to conception and design of the study, drafted the manuscript and approved the final version. fa coordinated the project until the submission of the article. all authors read and approved the final manuscript. the ethics committee of lyon university hospital (comité d'ethique, hospices civils de lyon) approved the study under the number - . because of the retrospective observational nature of the study and the lack of any modification in patients' management, the need for informed consent was waived with the authorization of the ethics committee of lyon university hospital (comité d'ethique, hospices civils de lyon), which approved the study under the number - . not applicable. the authors have no relevant affiliations or financial involvement with any organization or entity with a financial interest in or financial conflict with the subject matter or materials discussed in the manuscript. no writing assistance was utilized in the production of this manuscript. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.author details département de pneumologie, hospices civils de lyon, lyon, france. key: cord- -ypli wtu authors: ma, zhan-ying; deng, hua; hua, li-dong; lei, wen; zhang, chang-bin; dai, qi-qiang; tao, wei-jing; zhang, liang title: suspension microarray-based comparison of oropharyngeal swab and bronchoalveolar lavage fluid for pathogen identification in young children hospitalized with respiratory tract infection date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: ypli wtu background: respiratory tract infection (rti) in young children is a leading cause of morbidity and hospitalization worldwide. there are few studies assessing the performance for bronchoalveolar lavage fluid (balf) versus oropharyngeal swab (ops) specimens in microbiological findings for children with rti. the primary purpose of this study was to compare the detection rates of ops and paired balf in detecting key respiratory pathogens using suspension microarray. methods: we collected paired ops and balf specimens from hospitalized children with respiratory illness. the samples were tested simultaneously for respiratory viruses and bacteria by suspension microarray. results: of paired specimens, patients ( . %) had at least one pathogen. balf and ops identified respiratory pathogen infections in ( %) and ( . %) patients, respectively (p > . ). the etiology analysis revealed that viruses were responsible for . % of the patients, whereas bacteria accounted for . % and mycoplasma pneumoniae for . %. the leading pathogens identified were respiratory syncytial virus, streptococcus pneumoniaee, haemophilus influenzae, mycoplasma pneumoniae and adenovirus, and they accounted for . % of etiological fraction. for detection of any pathogen, the overall detection rate of balf ( %) was marginally higher than that ( %) of ops (p = . ). the differences in the frequency distribution and sensitivity for most pathogens detected by two sampling methods were not statistically significant. conclusions: in this study, balf and ops had similar microbiological yields. our results indicated the clinical value of ops testing in pediatric patients with respiratory illness. respiratory tract infection (rti) in children is a leading cause of morbidity and hospitalization [ , ] . especially, severe pneumonia ranks the second most common cause of mortality in children younger than years worldwide according to a recent report [ ] . viral and bacterial infections are the primary etiology of rti. accurate and rapid identification of the etiologic agents has an essential role in ensuring the appropriate and effective treatment for patients with respiratory illness, which could avoid unnecessary usage of antibiotics, reduce the overall costs and shorten the period of hospitalization [ ] . laboratory diagnosis of respiratory infections is performed traditionally using culture and immunological assays. although microbial culture is regarded as the gold standard, it is time-consuming and labor-intensive. specially, some fastidious pathogens are difficult to cultivate. antigen/antibody detection is fast and simple, however, insufficient sensitivity limits its usage in clinical settings. nowadays, molecular techniques are becoming widely used for identification of respiratory etiologies in clinical practice. multiplex pcr can detect several targets at one time, however, design and optimization of dozens of primers and probes remain challenging. microarray has the benefits of high-throughput, speed and low-cost. suspension microarray enables simultaneously detect a number of pathogens in a single assay, it has faster hybridization kinetics and more flexibility in array construction compared to traditional solid-phase array. a variety of sampling methods have been used in detecting respiratory pathogens in clinical practice. recently, several studies have described the performance of nasal swab, nasal wash, nasopharyngeal aspirate, nasopharyngeal swab (nps), oropharyngeal swab (ops), sputum and bronchoalveolar lavage fluid (balf) samples in microbiological findings using qrt-pcr or multipathogen detection platforms [ ] [ ] [ ] [ ] [ ] . when testing for respiratory agents, the recommended and commonly collected sample is an upper respiratory swab because nasopharynx and oropharynx are two of the most common portals for the introduction of microbes into the respiratory tract. however, collection of nps is an uncomfortable sampling method for young children, particularly pediatric patients with nasal congestion. compared to nps, ops is less technically challenging and more acceptable to children because it is quick and simple [ ] . in addition, some reports have showed that ops can increase the number of viral infections identified by %, compared to the nps alone [ ] . specifically, it has been found to be significantly more sensitive than nps for the detection of certain viruses, such as adenovirus and pandemic influenza a (h n ) virus [ ] . so far, few studies have compared balf and paired ops samples from young children hospitalized with rti. in the present study, we used the suspension microarray, a multipathogen detection platform, to simultaneously detect viral and bacterial respiratory pathogens in matched ops and balf specimens from pediatric patients for comparison of the sensitivities between the two sample types. the study protocol was approved by the institutional ethics board of dongguan maternal and child health care hospital, china. written informed consent for the balf procedure and ops sampling was obtained from each parent or guardian. children aged month to years with signs and symptoms of rti admitted to the study hospital between october and september were enrolled into this study. ops and balf specimens were collected within - days of hospital admission. first, we collected the ops samples, a cottontipped swab was used to swab over the posterior pharynx and tonsils. balf specimens were obtained with bronchoscope according to the and guidelines of pediatric bronchoscopy in china. samples were stored at °c until analysis and all specimens were tested within h of collection. nucleic acid was isolated from balf and ops specimens using a magnetic bead-based blood total nucleic acid kit (magcares™, #m , genehar technologies inc., guangzhou, china) according to the manufacturer's protocol. dna and rna concentrations were measured using a quawell q uv spectrophotometer. to detect multiple respiratory pathogens in a single assay, suspension microarray was developed in-house based on the luminex xmap system. the procedure consisted of multiplex pcr, probe design, the attachment of probes to microspheres and hybridization as previously reported [ , ] . to assess the sensitivity for each sampling method, the presence of a pathogen in either of the specimens was deemed to be a true positive [ , ] . we compared the differences in sensitivity for each pathogen between two sample types using the chi-squared test or fisher's exact test, as appropriate. statistical analyses were conducted with the software graphpad prism . . a two-tailed p-value < . was considered statistically significant. a total of young children were subjected to respiratory pathogens detection in this project. clinical characteristics of the patients are summarized in table . the patients included males and females. the median age of children was months (range month to years), and . % of included children were younger than years old. according to the who definition of severe pneumonia and guideline for children with community acquired pneumonia in china ( version), patients were considered as severe respiratory tract infection (srti) and admitted to picu with clinical presentation of cough, difficulty in breathing/tachypnoea, and one or more of the general danger signs such as an inability to drink, persistent vomiting, convulsions, lethargy or unconsciousness, central cyanosis. to compare the ops and paired balf for pathogens detection in young children with rti, we tested the two sample types using suspension microarray. of the cases included in the analysis, patients ( . %) had at least one respiratory pathogen. balf and ops identified respiratory pathogen infections in ( %) and ( . %) cases, respectively ( table ). there was not a significant difference in detection rates between the two sample types (p > . ). of these, ( . %) had concordant ops and balf suspension array results: ( . %) had the same pathogens identified from the ops and balf suspension array, and ( . %) had concordant negative results. the overall distribution of the respiratory pathogens tested is shown in table . a total of pathogens were found in young patients. the etiology analysis revealed that viruses were responsible for . % of patients, whereas bacteria accounted for . % and mycoplasma pneumoniae for . %. the leading pathogens identified were rsv, sp, hi, mp and adv, and they accounted for . % of etiological fraction. for detection of any pathogen, the overall detection rate of balf ( %) was marginally higher than that ( %) of ops (p = . ). also, balf was more sensitive than ops for detecting moraxella catarrhalis (p < . ). the differences in frequency distribution and sensitivity for each single pathogen except moraxella catarrhalis of two sampling methods were not statistically significant. virus/virus and virus/bacterium co-infections were found in ops and balf (table ). in ops, a single pathogen was found in cases ( . %), two pathogens in cases ( . %), and three pathogens in cases ( . %). in balf, a single pathogen was identified in cases ( . %), two pathogens in cases ( . %), three pathogens in cases ( . %), and four agents in case ( . %). the most common co-infections observed was sp plus mp (n = ) in ops samples, rsv plus hi (n = ) and followed by rsv plus sp (n = ) in balf specimens. in the present study, we used suspension array to simultaneously detect multiple viral and bacterial pathogens in paired balf and ops specimens from symptomatic patients hospitalized with respiratory illness. to validate the reliability and accuracy of the multipathogen testing platform, we have compared the yield of suspension array with that of metagenomic next-generation sequencing for microbiological findings and highlighted the high concordance of targets between the two methods (manuscript in preparation). these results showed that our suspension array can reliably identify pathogens in patients with rti. the fast turnaround time (within - h) makes it possible to be a valuable tool in clinical settings. the reliability of the specimens taken via oropharyngeal/nasopharyngeal swab or wash compared to the deep samples such as balf is a matter of debate and it would be interesting to investigate if they are really useful. here, balf and ops had similar microbiological yields ( % vs. %, p > . ). the differences in the frequency distribution and sensitivity for most targeted pathogens except moraxella catarrhalis of two sampling methods were not statistically significant. selection of a sampling method for detecting respiratory pathogens must take into account its sensitivity, feasibility and costs. the collection of ops is relatively simple, quick and less invasive compared to other sampling methods. for these reasons, we consider the ops as the preferred method of respiratory tract sampling for pathogen detection. for moraxella catarrhalis, we detected cases in balf and none in paired ops specimens. in general, moraxella catarrhalis causes mainly upper respiratory tract infection (otitis media) in children and lower respiratory tract infection in adults with previously compromised airways such as chronic obstructive pulmonary ( ) hi + piv ( ) rsv + sp ( ) hi + adv ( ) sp + mp ( ) bp + rsv ( ) bp + rsv ( ) rsv + piv ( ) mp + rsv ( ) hbov+sp ( ) rsv + adv ( ) hbov+rsv ( disease [ , ] . however, some reports have demonstrated that moraxella catarrhalis may be involved in lower respiratory tract infections in children [ ] , which is consistent with our results. more studies are needed to investigate its role in respiratory illness in hospitalized children. among the patients, ( . %) had the same pathogens identified from the ops and balf suspension array, and ( . %) had concordant negative results. for the cases, they might be infected by some rare pathogens that are not covered by our suspension array. the top pathogens were rsv, sp, hi, mp and adv, accounting for . % of etiological fraction. our data are in agreement with other recent findings in multi-country case-control studies that found rsv was the most common cause of severe childhood pneumonia [ , ] . thus, rsv could be a primary target for children hospitalized with respiratory illness. in general, rsv is most commonly found in lower respiratory tract infections particularly in infants [ ] . however, the sensitivities of rsv between ops and balf was not significantly different in our testing. rsv infection and replication initiates in the nasopharynx. the virus could be found in both upper and lower airway via high-sensitivity molecular techniques when it spreads from the upper respiratory tract to the lower in individuals with compromised immunity. bordetella pertussis was rarely identified in infants perhaps due to high vaccination rates. in this study, we detected bordetella pertussis in young children. identification of bordetella pertussis in balf or ops specimens may provide predictive value for the outcome of respiratory illness at the individual case level [ ] . in terms of some pathogens such as influenza a, adenovirus, mycoplasma pneumoniae and streptococcus pneumoniae, relatively low concordance between balf and ops specimens for them may reflect different cell tropisms for different parts of the respiratory tract. notably, none of the cases was tested positive for hrv in this work, which is somewhat surprising given that this virus is often associated with upper respiratory infection. however, another project in our group showed that the detection rate of hrv was~ % in ops among pediatric outpatients with respiratory illness. here, the included children were inpatients. although hrv infections are frequent, they are mostly limited to the upper respiratory tract and generally cause relatively mild symptoms [ , ] . the contribution of hrv may vary by disease severity of included patients and other factors. cultivation is regarded as the gold standard in etiological identification. as shown in table , we have performed sputum culture in of cases. compared with pcr-based methods, the detection rate of it was significantly lower. one of possible explanations is the empirical antibiotic therapy in patients before sampling. here, we focused on comparison of ops and paired balf in detecting respiratory pathogens, rather than the sensitivity of nucleic acid-based array compared to the gold standard, i.e. cultivation. based on the same consideration, a healthy control group was not tested for ruling out false positives in this work because of our specific interest and aim. similarly, a prior study, patients enrolled and no healthy controls included, has also applied this strategy to compare the yields of bronchoalveolar lavage samples with that of nasopharyngeal swabs by using filmarray respiratory panel [ ] . this study has several limitations. first, our sample size was relatively small (n = ) because we focused on paired balf and ops specimens collected from hospitalized children. as a result, there was limited power to compare the sensitivities of balf and ops for specific respiratory pathogens in patients. further studies in a larger cohort may generate a relatively high degree of precision when performing comparative statistical analysis. second, although balf are regarded lower respiratory tract samples, oropharyngeal intubation for balf might result in potential contamination by upper respiratory tract "contaminants", particularly for bacteria/ viruses known to colonize the oropharynx. thus, the balf specimen might be actually both an upper and a lower airway combined sample. however, it does not affect our primary purpose that focuses on the microbiological findings of ops sampling. third, empirical antibiotic use in clinical practice may reduce the sensitivity of assays, particularly for bacteria. collectively, identification of a pathogen does not necessarily equate to the etiological agent, particularly in a multipathogen testing and laboratory results require further interpretation by experienced clinicians. in addition, we here focused on a subset of potential pathogens because the agents ( viruses and bacteria) are key respiratory pathogens in children based on previous epidemiological investigations in china. in fact, fungi are also important pathogens causing severe infections of the respiratory system. another project in our group by using metagenomic nextgeneration sequencing found that the infection rates of candida albicans, pneumocystis jiroveci and aspergillus fumigatus in young children admitted to picu with respiratory illness were . , . and . %, respectively. given the high prevalence and importance of the airborne fungal pathogens in respiratory infections, we plan to add fungal species to our upgraded in-house array, which would be a separate study due to many experiments and large undertakings. estimates of worldwide distribution of child deaths from acute respiratory infections acute lower respiratory infections in developing countries global, regional, and national causes of under- mortality in - : an updated systematic analysis with implications for the sustainable development goals impact of multiplex molecular assay turn-around-time on antibiotic utilization and clinical management of hospitalized children with acute respiratory tract infections comparison among nasopharyngeal swab, nasal wash, and oropharyngeal swab for respiratory virus detection in adults with acute pharyngitis concordance between rt-pcr-based detection of respiratory viruses from nasal swabs collected for viral testing and nasopharyngeal swabs collected for bacterial testing comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples improved detection of respiratory pathogens by use of high-quality sputum with taqman array card technology pneumonia methods working g: specimen collection for the diagnosis of pediatric pneumonia added value of an oropharyngeal swab in detection of viruses in children hospitalized with lower respiratory tract infection comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays applications of luminex xmap technology for rapid, highthroughput multiplexed nucleic acid detection multiplexed detection and identification of respiratory pathogens using the nxtag(r) respiratory pathogen panel comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection of pandemic influenza a/h n virus by real-time reverse transcriptase pcr comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction nosocomial transmission clusters and risk factors in moraxella catarrhalis prevalence and resistance pattern of moraxella catarrhalis in community-acquired lower respiratory tract infections moraxella catarrhalis: from emerging to established pathogen causes of severe pneumonia requiring hospital admission in children without hiv infection from africa and asia: the perch multi-country casecontrol study. the lancet microorganisms associated with pneumonia in children < years of age in developing and emerging countries: the gabriel pneumonia multicenter, prospective, case-control study viral and host factors in human respiratory syncytial virus pathogenesis pertussisassociated pneumonia in infants and children from low-and middleincome countries participating in the perch study rhinoviruses, allergic inflammation, and asthma role of rhinovirus load in the upper respiratory tract and severity of symptoms in lung transplant recipients publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. china. we used suspension-array to compare balf and paired ops specimens for detecting multiple pathogens in children hospitalized with respiratory illness. the similar sensitivities between the two sampling methods indicated the clinical value of ops testing in clinical settings. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the data set used and/or analyzed during the current study is available from the corresponding author on reasonable request. the study protocol was approved by the institutional ethics board of dongguan maternal and child health care hospital. written informed consent was obtained from each parent or guardian. not applicable. the authors declare that they have no competing interests.author details dongguan maternal and child health care hospital, dongguan , key: cord- -h cxlp authors: streng, andrea; prifert, christiane; weissbrich, benedikt; liese, johannes g. title: continued high incidence of children with severe influenza a(h n )pdm admitted to paediatric intensive care units in germany during the first three post-pandemic influenza seasons, / – / date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: h cxlp background: previous influenza surveillance at paediatric intensive care units (picus) in germany indicated increased incidence of picu admissions for the pandemic influenza subtype a(h n )pdm . we investigated incidence and clinical characteristics of influenza in children admitted to picus during the first three post-pandemic influenza seasons, using active screening. methods: we conducted a prospective surveillance study in picus in bavaria (germany) from october to september . influenza cases among children between month and years of age admitted to these picus with acute respiratory infection were confirmed by pcr analysis of respiratory secretions. results: a total of / / influenza-associated picu admissions were recorded in the post-pandemic seasons / / ; incidence estimates per , children were . / . / . , respectively. of all patients, % had influenza a, including % with a(h n )pdm . influenza a(h n )pdm was almost absent in season (incidence . ), but dominated picu admissions in seasons (incidence . ) and (incidence . ). clinical data was available for influenza patients; median age was . years (iqr . – . ). the most frequent diagnoses were influenza-associated pneumonia ( %), bronchitis/bronchiolitis ( %), secondary bacterial pneumonia ( %), and ards ( %). thirty-six patients ( %) had underlying medical conditions. median duration of picu stay was days (iqr – ). forty-seven per cent of patients received mechanical ventilation, and one patient ( %) extracorporeal membrane oxygenation; % were treated with oseltamivir. five children ( %) had pulmonary sequelae. five children ( %) died; all had underlying chronic conditions and were infected with a(h n )pdm . in season , patients with a(h n )pdm were younger than in season (p = . ), were diagnosed more often with bronchitis/bronchiolitis (p = . ), and were admitted to a picu later after the onset of influenza symptoms (p = . ). conclusions: active screening showed a continued high incidence of a(h n )pdm -associated picu admissions in the post-pandemic seasons and , and indicated possible underestimation of incidence in previous german studies. the age shift of severe a(h n )pdm towards younger children may be explained by increasing immunity in the older paediatric population. the high proportion of patients with underlying chronic conditions indicates the importance of consistent implementation of the current influenza vaccination recommendations for risk groups in germany. influenza is one of the most common vaccine-preventable viral diseases, with the highest morbidity reported for children and elderly patients [ , ] . influenza infections during childhood usually present as mild respiratory upper airway disease, but severe complications and fatalities also occur, especially in children less than years of age and in children with underlying chronic conditions [ ] [ ] [ ] [ ] [ ] [ ] . however, - % of influenza-associated fatalities occur in previously healthy children [ , ] . before the influenza a(h n )pdm pandemic in / , comparisons of clinical characteristics between patients infected with different influenza types (a vs. b) and between patients infected with different influenza a subtypes showed only small differences when controlling for age [ , ] . during the pandemic, however, some studies observed increased morbidity and mortality among children compared to previous seasonal influenza [ , [ ] [ ] [ ] , while other studies described the clinical features of a(h n )pdm as being similar or even milder [ , ] . acute respiratory distress syndrome (ards) and fatal viral pneumonia was observed more frequently during the pandemic [ ] . post-pandemic surveillance was recommended, as circulation of a(h n )pdm was expected to continue for several years, gradually assuming a seasonal influenza pattern [ ] . in germany, influenza sentinel surveillance on outpatients of all ages [ ] confirmed that the first postpandemic season / was dominated by influenza a(h n )pdm ( %), co-circulating with influenza b ( %) whereas a(h n ) was rare (< %). during the second season / , a(h n )pdm was rare ( %), whereas a(h n ) was diagnosed in % of cases and co-circulated with influenza b ( %). in the third season / , all three types/subtypes co-circulated in similar proportions ( % a(h n )pdm , % a(h n ), and % b). information on the incidence and clinical characteristics of severe paediatric influenza resulting in intensive care treatment and/or fatal outcome is still limited in germany, and post-pandemic data is thus far available only for the season / [ ] [ ] [ ] . based on cases recorded by a nation-wide paediatric intensive care unit (picu) reporting system, the pre-pandemic ( / - / ), pandemic ( / ) and post-pandemic ( / ) annual incidence of severe influenza cases per , children below or years of age was estimated as . , . - . , and . , respectively [ ] [ ] [ ] . the data so far available indicated a shift towards younger children in a(h n )pdm cases from the pandemic to the first post-pandemic season [ ] . in these studies, it remained unclear whether the higher pandemic and post-pandemic incidence in children was caused by higher influenza activity, heightened physician awareness, more frequent or more sensitive influenza testing, or a more severe course of disease of a(h n )pdm [ ] . furthermore, all these previous studies may have been affected by underreporting, as influenza cases were reported at the discretion of the picu physician without systematic screening for influenza in patients with severe acute respiratory infection. in the study presented here, we used active screening to estimate the incidence of laboratory-confirmed influenzaassociated picu admissions in one of germany's largest federal states during the first three post-pandemic seasons. furthermore, we described the clinical characteristics of influenza picu patients and compared patients with severe a(h n )pdm disease between the post-pandemic seasons. prospective, active surveillance was conducted in picus of paediatric hospitals in bavaria, germany. on december st , roughly , , children < years of age were registered in bavaria [ ] , representing % of the german population in this age group [ ] . the annual study population was defined as the sub-group of all children in bavaria at least month and < years of age. all paediatric hospitals in bavaria equipped for paediatric intensive care treatment of children older than month of age were invited to participate. these picus reported a total of intensive care beds (median , iqr [ ] [ ] [ ] [ ] [ ] [ ] , including beds (median , iqr - ) equipped with ventilation facilities. , all patients who fulfilled the following inclusion criteria were enrolled: i) admission to a participating picu with suspected acute respiratory infection (ari) of the upper or lower respiratory tract, with arirelated symptoms (for example, coryza, cough, or sore throat); ii) age at picu admission due to ari at least month and below years of age; iii) parental written informed consent. enrolled children with pcr-confirmed influenza were classed as influenza-associated ari. the picu physician documented demographic characteristics, underlying chronic medical conditions, influenza vaccination status, diagnostic findings, ari-associated diagnoses and complications, treatment, duration of hospital and picu stay, and outcome in a structured questionnaire. a respiratory sample, usually a flocked nasopharyngeal or pharyngeal swab, was collected on the day of picu admission for pcr-confirmation of influenza. microbiological testing for bacteria or fungi was at the discretion of the picu physician; pathogens detected at usually sterile sites or in tracheal aspirates were classified as bacterial or fungal co-infection. pcr confirmation of influenza was performed either at the local laboratories of the participating picus using influenza-specific pcr, or (in the majority of cases) at the central laboratory at the institute of virology and immunobiology of the university of würzburg using multiplex pcr for respiratory viruses. for the latter, respiratory samples were placed in a viral transport medium (mast diagnostica gmbh, reinfeld, germany). at the central laboratory, they were tested using the commercial multiplex pcr 'ftd® respiratory pathogens ' (fast track diagnostics, luxembourg) to screen for respiratory viruses (sensitivity and specificity of - % compared to singleplex pcr assays for all included viruses in clinical samples). pathogens detected by the test kit included influenza a and b viruses, respiratory syncytial virus (rsv), parainfluenza virus (piv) - , coronavirus (cov) nl , oc , hku , and e, human metapneumovirus (hmpv), human bocavirus (hbov), adenovirus (adv), rhinovirus (rhv), enterovirus (ev), parechovirus (pv), and additionally mykoplasma pneumoniae. samples positive for influenza a and b virus rna in the multiplex pcr were further analysed to determine the subtype and lineage, respectively. primers and probes specific for influenza a(h n )pdm were included in the 'ftd respiratory pathogens ' kit. all samples positive for influenza a virus rna but negative for influenza a virus (h n )pdm rna were tested by a pcr specific for influenza a virus h . all data was entered into a microsoft access database and transferred to ibm spss . for statistical analysis. data was analysed descriptively (percentages, or median with inter-quartile range, iqr). comparisons between groups were assessed for significance (p < . , twosided) using pearson's chi -test or fisher's exact test for categorical data, and the mann-whitney u-test for continuous data. the minimum incidence of influenza-associated picu admissions per , children < years of age was calculated for each season based on the observed number of influenza picu patients with a residential address in bavaria. to correct for non-participating picus, the estimates of the total number of picu influenza cases treated in all eligible picus in bavaria per season were derived taking into account the annual percentage of participating picus. the annual study population was used as denominator. a similar questionnaire and case definition had been used in previous studies on influenza-related picu admission [ , ] . key variables were extracted from these publications for comparison purposes. data from streng et al. [ ] and the present study were pooled for statistical comparison of pre-and post-pandemic seasons. the study was approved by the ethical committee of the medical faculty, university of würzburg, germany. based on the observed cases, the minimum incidence for pcr-confirmed influenza-associated picu admission per , children < years of age in bavaria was calculated as . / . / . for seasons / / , respectively. taking into account that the observed cases were based on data from %/ %/ % of all eligible picus, the total number of influenza-associated picu admissions in bavaria was estimated and corrected incidences were calculated as . / . / . per , children < years. subtype-specific corrected incidences were . (table ) . after onset of ari symptoms, children were admitted to hospital after a median interval of . days; % were transferred to the picu on the day of hospital admission or the following day (table ) . two long-term hospitalized children ( . %) required picu treatment due to ari and were diagnosed with suspected nosocomial influenza a(h n )pdm infection. median length of picu stay was . days and median length of total hospital stay was . days (table ) . underlying chronic medical conditions were reported for a total of influenza picu patients ( . %) ( table ) . chronic neurological diseases were most frequent ( . %), followed by chronic lung disease ( . %), preterm birth ( . %), cardiac malformations ( . %), obesity ( . %), genetic disorders ( . %), and immunocompromising conditions ( . %). of influenza picu patients with underlying chronic conditions, four ( . %) were too young (< months of age) to have been immunized against influenza, and for two patients ( . %), data on their influenza vaccination status was unavailable. twenty-nine ( . %) patients from this risk group had not been vaccinated against influenza although they would have been eligible. one immunocompromised child ( . %) had been vaccinated in october , but was diagnosed with a(h n ) in january . one or more specific influenza-associated diagnoses/complications were reported for ( . %) of the children ( table ). the most frequent diagnosis was influenzaassociated pneumonia ( . %), followed by bronchitis/ bronchiolitis ( . %), and secondary bacterial pneumonia ( . %). ards was reported for ( . %) and sepsis for six children ( . %); other complications were rare. thirty-nine of the patients ( . %) underwent a chest radiograph. in addition to influenza, laboratory-confirmed co-infections were reported for children ( . % out of ( )). the majority of the picu patients were treated intravenously with antibiotics ( . %), and with antipyretics ( . %) ( five children ( . %), infected with a(h n )pdm , died at an age of to years; four were male patients ( table ). four of these children suffered both from severe neurological conditions (two children with previous peripartal asphyxia and spastic tetraparesis; one child with cerebral paresis and tetraspasticity; one child with congenital cerebral disorder), and from chronic pulmonary conditions; two of these four children were also born pre-term. influenza-associated pneumonia was diagnosed in all four of these children; three additionally had secondary bacterial pneumonia, and one child also developed sepsis. for the fifth child, obesity was reported as the only risk factor; and sepsis and suspected encephalitis as complications. bacterial co-pathogens were detected in three of these five children and suspected in one child; two viral and two fungal co-infections were also reported. all five children received intratracheal ventilation, antibiotics and catecholamines; two were additionally treated with antiviral medication. death occurred , , , , and days after picu admission, with ards reported as cause of death in three children. sequelae were reported for five patients ( . %): state after surgery due to pleural effusion/empyema in two children; increased oxygen requirements in two children who had previously already received oxygen therapy at home; damage of the lung after high-pressure ventilation in one child. table ). figure shows the difference in age distribution between both seasons, and the high proportion of children below years of age as opposed to low proportions in all other age groups in season . after onset of symptoms, children were admitted to a picu after a significantly shorter period, with a median of days (iqr - ) in season compared to days (iqr . - . ) in season ( table ). in season , significantly fewer children were diagnosed with bronchitis/bronchiolitis (table ), and they tended to require cpap treatment less frequently than in season ( . % vs. . %, p = . , table ). in the pre-pandemic period, median duration of picu stay was longer ( days) , and children were more often diagnosed with encephalitis/encephalopathy ( %) and co-infections ( %) than in later periods ( table ) . the proportion of children with influenza-associated pneumonia was highest ( %) during the pandemic, whereas secondary bacterial pneumonia ( %), bronchitis/ bronchiolitis ( %) and sepsis ( %) were reported less frequently during the pandemic than in the pre-and post-pandemic seasons. oseltamivir treatment decreased significantly in the post-pandemic period (table ) . during the first three post-pandemic seasons / , / and / , active screening of children with acute respiratory infection admitted to paediatric intensive care units in bavaria identified a total of pcr-confirmed influenza cases, resulting in annual incidence estimates of . , . , and . influenza-associated picu admissions per , children, respectively. these figures would, by extrapolation, correspond to a total number of children with influenza-associated picu admission in germany within the -year post-pandemic period, with an annual average of children. this is almost times as high as the annual average of six to seven influenza-associated picu admissions detected by nation-wide picu surveillance in germany during three pre-pandemic years without active screening [ ] . furthermore, the incidence estimates for the subtype a(h n )pdm derived from our active screening study were higher in the first and third post-pandemic seasons ( . and . , respectively) than previous incidence estimates for picu patients in the pandemic ( . - . ) and the first post-pandemic (approximately . ) season in germany [ , ] . thus, our results indicate possible underreporting in previous studies, and show a continued high level of a(h n )pdm -associated picu admissions even years after the pandemic. in our study, the proportions of children with bacteriaassociated complications (secondary bacterial pneumonia, sepsis) were similar to the proportions observed during the pre-pandemic period, but appeared higher than those observed during the pandemic / [ ] . the lower proportions observed during the pandemic might be explained by the time shift of the peak of influenza cases, which was observed as early as november in germany [ ] . thus, the pandemic influenza peak did not coincide with the seasonal peak of streptococcus pneumoniae, the bacterial pathogen most frequently associated with community-acquired influenza [ ] . antiviral treatment patterns changed considerably during the post-pandemic period, with a decrease in the proportion of paediatric influenza cases receiving oseltamivir from previously % [ ] and % [ ] to %. oseltamivir is considered to be most advantageous when administered within the first h of influenza disease. the reduced use in the post-pandemic period may therefore be partly due to the fact that median time between onset of influenza symptoms and picu admission was longer than during the pandemic ( vs. days [ ] ). increasing uncertainty regarding the effectiveness of oseltamivir in the treatment of paediatric influenza may also have played a role [ , ] . post-pandemic oseltamivir treatment was associated with co-infections and longer picu stay, suggesting that it were mainly children with severe complications or with serious underlying conditions who received this medication. in our study, about two-thirds of influenza cases and all fatalities were a(h n )pdm -associated. during the postpandemic seasons / / , the proportion of a(h n )pdm cases among the picu patients was %/ %/ % and, thus, considerably higher than the proportion of this subtype reported among outpatients by national influenza surveillance ( %/ %/ %) [ ] . this observation suggests that a(h n )pdm may be associated more often with a severe course of influenza requiring picu treatment than other influenza types/ subtypes. similar observations on the proportion of a(h n )pdm -associated picu admissions have been reported in the united states [ ] . comparison of picu patients with a(h n )pdm between the post-pandemic seasons showed that median age was . years in the third season and, thus, significantly lower than in the first season. a significant age shift towards younger children, from a median age of to years, had already been observed in a comparison of the pandemic and the first post-pandemic season in germany [ ] . the continued shift towards younger patients in the third season is likely to be due to increasing immunity in the older paediatric population, after previous contact with a(h n )pdm . seroprevalence data from germany had already shown evidence for a(h n )pdm infection in as many as % of children aged - years and % of - year-old children for the pandemic season / [ ] . a similar shift towards younger hospitalized children [ , ] and towards younger children with severe paediatric a(h n )pdm -associated influenza from the pandemic season to the first post-pandemic season had also been detected in other european countries [ ] [ ] [ ] [ ] . in germany, paediatric influenza vaccination for pandemic influenza a(h n )pdm was recommended and funded for all children as monovalent vaccination from october to july [ ] . for seasonal influenza, however, paediatric influenza vaccination was and is currently recommended only for specific risk groups with underlying chronic conditions [ ] . vaccination uptake was low, even in this target group. pre-pandemic vaccination rates were % for all children and about % for children with chronic underlying conditions in / [ ] . for the pandemic and post-pandemic seasons, no data on vaccination rates is available for children, but vaccination rates as low as % ( / ) and % ( / ) were reported for adults, with a vaccination rate of only % even for risk group adults [ ] . in our study, more than % of influenzaassociated picu patients were children with underlying chronic conditions. analysis of their reported influenza [ ] ). data are given in %, by age group and season. season : oct -sep (n = ), season : oct -sep (n = ); season : oct -sep (n = ) is not shown vaccination status showed that among these were a high proportion of vaccine-eligible but unvaccinated children. patients with chronic conditions too young to be vaccinated and other paediatric risk groups, such as otherwise healthy children below years of age, are not covered by the current german recommendation. all these groups could profit considerably from universal influenza vaccination for children, either directly or by herd protection. in contrast to the situation in germany, in the united states universal influenza vaccination for all children older than months of age has been established, and vaccination coverage reached a level of approximately % in [ ] . compared to influenza-associated paediatric deaths observed in the united states during the pandemic / , only were observed in the strong a(h n )pdm season / [ ] . this might partly be explained by increasing immunity in children after previous a(h n )pdm infection, but may in part also be a result of the influenza vaccination program [ ] . in england, a universal childhood vaccination programme with a new live attenuated influenza vaccine (laiv) with intra-nasal application was started in the / influenza season [ ] . first results showed an overall uptake of % in primary school aged children, indicating a good acceptance of laiv, and suggesting direct and indirect impacts on disease incidence, including reduction of paediatric influenza-associated hospitalisations. to our knowledge, our study is the first in europe to investigate paediatric influenza in picu patients during the first three post-pandemic seasons after the / pandemic. the strengths of our study include the multicentre design covering the majority of picus in bavaria, the active screening for influenza in patients admitted to picus, and pcr-confirmation of all influenza cases. a limitation is that the corrected incidence estimates were based on the assumption that participating and nonparticipating picus treated a similar number of severe paediatric influenza patients. although picus of both groups were of similar size, some of the non-participating picus, where paediatricians indicated lack of time as reason for non-participation, may have treated a higher number of patients, or patients with higher acuity. further limitations include potential over-and underreporting in participating picus. on the one hand, due to different hospitalization rules some children may have been admitted to picus mainly for the purpose of monitoring their course of influenza disease more closely, thus resulting in an overestimate of severe cases. on the other hand, some parents of children with severe influenza may have refused study participation, or children with a fulminant course of influenza disease may have died before they were admitted to a picu [ ] . thus, the high incidence estimates derived in this study may still underestimate the true burden of severe influenza. [ ] b key data from this study were pooled with data from streng et al. [ ] and compared using fisher's exact test or mann-whitney u-test, respectively the incidence estimates of influenza a(h n )pdm associated picu admissions, derived from active screening of picu patients with acute respiratory infections, reached similarly high levels in the first and third postpandemic seasons. both incidence estimates were higher than those previously reported by nation-wide picu surveillance for the pandemic and the first post-pandemic season, suggesting possible underreporting in previous studies without active screening. comparison of the first and third post-pandemic seasons indicated an age shift of severe a(h n )pdm towards younger children, which might be explained by increasing immunity in the older paediatric population. the large proportion of children with underlying chronic conditions indicates the need for a more consistent implementation of the current recommendations for influenza vaccination of specific risk groups in germany. these children could also profit from herd protection, if universal influenza vaccination was successfully introduced in germany. authors' contributions as designed the study, coordinated data collection, performed the analysis, interpreted the data, and drafted the manuscript. bw and cp performed multiplex pcr and subtyping on laboratory specimens, interpreted virological data, and revised the manuscript. jgl supervised the study, supported data interpretation, and revised the manuscript. the clinical data were collected by the bavarian picu study group on influenza and other viral ari, from oct to september . all authors read and approved the final manuscript. group on influenza and other viral ari participants and their affiliations while participating during the study period städtisches klinikum münchen gmbh, klinikum harlaching städtisches klinikum münchen gmbh, klinikum schwabing christoph schmidtlein (kinderklinik dritter orden tobias trips (kliniken südostbayern ag missionsärztliche klinik ggmbh, kinderklinik; würzburg) references . heikkinen t. influenza in children the underrecognized burden of influenza in young children risk factors associated with severe influenza infections in childhood: implication for vaccine strategy influenzaassociated pediatric deaths in the united states identification of children at risk of influenza-related complications in primary and ambulatory care: a systematic review and meta-analysis the burden of seasonal and pandemic influenza in infants and children the burden of influenza illness in children with asthma and other chronic medical conditions implications for immunization recommendations patients hospitalized with laboratory-confirmed influenza during the - influenza season: exploring disease severity by virus type and subtype the burden of influenza b: a structured literature review hospitalized children with pandemic influenza a (h n ): comparison to seasonal influenza and risk factors for admission to the icu influenza a (ph n ) infection in children admitted to a pediatric intensive care unit: differences with other respiratory viruses clinical characteristics of pediatric hospitalizations associated with influenza a (h n ) in northern bavaria the role of infections and coinfections with newly identified and emerging respiratory viruses in children comparative analysis of clinical characteristics of pandemic influenza a/h n and seasonal influenza a infections in hospitalized children forward look risk assessment -likely scenarios and uncertainties in the / influenza season in europe and beyond bericht zur epidemiologie der influenza in deutschland saison / severe influenza cases in paediatric intensive care units in germany during the pre-pandemic seasons severe cases of pandemic (h n )pdm in children unchanged severity of influenza a(h n )pdm infection in children during first postpandemic season bavarian state office for statistics and data processing bacterial and viral infections associated with influenza. influenza other respir viruses neuraminidase inhibitors for preventing and treating influenza in healthy adults and children effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h n pdm virus infection: a meta-analysis of individual participant data post-pandemic seroprevalence of pandemic influenza a (h n )pdm infection (swine flu) among children < years in germany clinical features of influenza disease in admitted children during the first postpandemic season and risk factors for hospitalization: a multicentre spanish experience influenza in hospitalized children in ireland in the pandemic period and the / season: risk factors for paediatric intensive-care-unit admission critical care surveillance: insights into the impact of the / influenza season relative to the / pandemic season in england burden and characteristics of influenza a and b in danish intensive care units during the / and / influenza seasons first influenza season after the pandemic influenza: characteristics of intensive care unit admissions in adults and children in vall d'hebron hospital Änderung der empfehlung zur impfung gegen influenza analyse regionaler unterschiede der influenza-impfraten in der impfsaison influenza a(h n )pdm antibodies after pandemic and trivalent seasonal influenza vaccination as well as natural infection in update: influenza activity -united states influenza activity -united states, - season and composition of the - influenza vaccines uptake and impact of a new live attenuated influenza vaccine programme in england: early results of a pilot in primary school-age children, / influenza season the authors thank all participating hospitals, and picu and university staff involved in data collection and virological testing. karin seeger we thank for helpful comments on the manuscript. the study was supported by an unrestricted grant from glaxosmithkline gmbh & co. kg, munich, germany. apart from financial support, the company was not involved in any part of the study. the publication was funded by the german research foundation (dfg) and the university of wuerzburg in the funding programme open access publishing. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -erqmbi b authors: bugembe, daniel lule; ekii, andrew obuku; ndembi, nicaise; serwanga, jennifer; kaleebu, pontiano; pala, pietro title: computational mhc-i epitope predictor identifies % of experimentally mapped hiv- clade a and d epitopes in a ugandan cohort date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: erqmbi b background: identifying immunogens that induce hiv- -specific immune responses is a lengthy process that can benefit from computational methods, which predict t-cell epitopes for various hla types. methods: we tested the performance of the netmhcpan . computational neural network in re-identifying t-cell epitopes that had been previously independently mapped using the whole proteome ifn-γ elispot assays in hla class i typed ugandan individuals infected with hiv- subtypes a and d. to provide a benchmark we compared the predictions for netmhcpan . to mhcflurry . . and netctl . . results: netmhcpan . performed best correctly predicting of the experimentally mapped epitopes for a set length of -mer and matched hla class i alleles. receiver operator characteristic (roc) analysis gave an area under the curve (auc) of . . setting netmhcpan . to predict - mer length did not improve the prediction ( – of peptides) with an inverse correlation between the number of predictions and length set. late time point peptides were significantly stronger binders than early peptides (wilcoxon signed rank test: p = . ). mhcflurry . . similarly predicted all but of the peptides that netmhcpan . predicted and netctl . predicted only of the experimental peptides. conclusion: netmhcpan . class i epitope predictions covered % of the epitope responses identified in six hiv- infected individuals, and would have reduced the number of experimental confirmatory tests by > %. algorithmic epitope prediction in conjunction with hla allele frequency information can cost-effectively assist immunogen design through minimizing the experimental effort. computational algorithms are increasingly utilised in biological modelling and offer the potential to reduce the time and expense of immunological assays. computational algorithms were initially demonstrated as useful tools for predicting potential epitopes that might elicit quality t-cell responses [ , ] . computational algorithms that predict potential hla binding t-cell epitopes can facilitate the design of vaccines capable of inducing t-cell immunity against hiv- . the high variability of hiv- and the extensive genetic polymorphism of hla molecules can be managed in silico, allowing immunogen optimisation to increase breadth and magnitude of t cell responses in respect of hla allele frequencies and circulating virus strains in different populations. bioinformatics approaches were previously applied as proof of concept for an hiv- peptide-based vaccine for the env and gag genes [ ] in cynomolgus macaques for a broad spectrum of hiv- clades. computational optimisation of immunogens facilitates the development of the multivalent and mosaic vaccines [ ] necessary to control recombinant hiv- strains, an increasingly common occurrence in the epidemic in uganda [ ] . computational approaches aim to identify optimal epitopes relevant to vaccine development and are not isolated to hiv- only, but a wide range of pathogens, including ebola virus [ ] , therefore various statistical validation approaches have been applied for evaluation of these methods [ ] [ ] [ ] [ ] . for hiv- vaccine design purposes an important consideration for the suitability of a computational algorithm is the breadth of discrete number of t-cell epitopes it generates that could reach particular levels of coverage [ ] of circulating viruses. the higher the number of epitope variants the more the reduction in their requirements to attain optimum coverage levels for any epidemic. previous data has shown that breadth of tcell response is associated to viral set point in chronic hiv- infection [ ] [ ] [ ] [ ] [ ] [ ] . in order to translate the computational epitope prediction into vaccine design, the number of discrete epitopes computationally generated from particular hiv- proteins is an important metric for further investigation [ ] . a reliable pan-hla-specific algorithm netmhcpan . [ ] [ ] [ ] that has been improved by advances in hla binding data, covers mhc class i molecules from human (hla-a, b, c, e), mouse (h- ), cattle (bola), primates (patr, mamu, gogo) and swine (sla) [ , ] , and can also predict binding to alleles devoid of experimental data basing on similarity to known binders and non-binders [ , ] . this is an artificial neural network (ann) algorithm for predictions of - aa and capable of predicting epitopes for other hla alleles using data for similar alleles by positional similarity of residues in their binding motifs. netmhcpan . is considered to be the tool of choice for such predictions considering the benchmarking done against other related tools [ ] . nevertheless to have a conclusive outcome of the computational performance we compared netmhcpan . to both an older and recent tool, netctl . [ ] [ ] [ ] and mhcflurry . . [ ] respectively. the binding of ctl epitopes to mhc class i molecules is linear, anchoring at residues and ; hence the interface between ligand and ctl can be determined computationally [ ] . validation of such computational applications can be done by comparing their predictions with suitable experimental data. despite the paucity of data validating the performance of computational methods relative to wet laboratory experiments, a few have documented them to achieve an area under the curve auc of over % [ , , , ] by isolated experimental data. we have not come across a wet experiment that evaluated computational predictors to achieve a robust auc using a single set of wet laboratory experimental data. the previously reported % auc is largely based on positional specific scoring algorithms (pssm) for the collective isolated experiments alongside probability models used to establish affinity or binding scores. one study that explored the reliability of in-silico approaches in epitope prediction and its application for vaccine design reported a meagre , %, and relatively higher % match for three computational tools namely yfpeithi, ctlpred and iedb respectively [ ] . using experimental epitope mapping data generated from peptides tested on cells of early hiv- infected individuals at paired time points, we show that netmhcpan . can be useful for markedly reducing pooled peptide experiments as demonstrated by the % experimental and computational concordance. the data used was from an independent study that did not include this analysis in its objectives. experimental data of peptides previously mapped for hiv- epitope recognition of individuals for a separate study (table ) at time points each was used for comparison with the computationally predicted binders. these were from a ugandan early hiv- serodiscordant couple cohort approved by the uganda virus research institute (uvri), research and ethics review board and the uganda national council of science and technology (uncst). all participants provided informed consent. six ( ) participants whose experimental epitope recognition profile we evaluated were early hiv- infections (table ) , enrolled under the following criteria: (i) detection of hiv- p antigen with a simultaneous negative hiv- antibody [ ] .. the experimentally tested peptides totalled (fig. ) , were aa long, overlapping by aa and spanning the hiv- proteome consensus for subtypes a and d. cultured eli-spot assays using , cells/well as previously documented by obuku ae. et.al [ ] . and ex-vivo ifn-γ elispot assay using , cells/well were used for testing peptide pools and epitope mapping respectively. experimental positive pools were times the background wells and at least spot forming units per million cells. "deconvolute this" software [ ] was used to identify possible responding individual peptides from the pools or where it was not possible all the peptides in a pool were tested as single peptides. high resolution reference strand conformation analysis hla class i tissue typing for the early infected subjects was done using methods described elsewhere [ ] . hiv- subtyping determination was performed on the gag gene [ , ] using sanger method generated sequences. the sequences were input into the rega hiv- automated subtyping tool to determine the hiv- clade [ , ] . hiv- subtypes a and d consensus sequences were used as inputs for the computational epitope prediction. these peptide sequences were all for the year downloaded from the los alamos database (hiv.lanl.gov/content/sequence/newalign/align. html). the web version of netmhcpan . [ ] (http://www.cbs.dtu.dk/services/netmhcpan/) was configured to predict mer through mer epitopes for hla class i alleles ( table ) that were expressed by the hiv- infected donors. linux version mhcflurry . . [ ] was used to predict mer epitopes and an earlier tool netctl . was also used to predict mer epitopes for the hla class i alleles expressed by the study individuals. perl version . . was used to extract the binders from all the netmhcpan . predictions and also to compare the computational binders to the mapped experimental aa peptides for mer through mer hits using a sliding window. an experimental peptide was considered a hit if any of the computational mer through mer sequence was contained in the amino acid experimental peptide sequence as well as any of the hla-a, b or c expressed by the individual matched the netmhcpan . hla class i type(s). if multiple computational epitope predictions were contained in a single mer experimental peptide they were counted as a single hit. these were determined by a blast search of the computational binders against the derivative experimental peptides to determine computational predictions from the same test peptide. the accession numbers of the sequences used to determine the hiv- subtypes for of the study subjects are; kt , kt , kt , kt , kt , kt , kt , kt , kt , kt , kt , kt , kt , kt , kt , kt and kt . statistics computations and plots were generated using spss version . . . . the netmhcpan . computational performance was evaluated using a confusion fig. elispot peptide consort; the experimental peptide mapping data was generated by culture elispot of multiple peptide pools tested in duplicate wells per time point, followed by ex-vivo elispot of potential candidate epitopes. to experimentally map a single time point required at least assay wells matrix to classify true positives, true negatives, false positives and false negatives that were used for the receiver operator characteristic (roc) plot. the hit rate (sensitivity) and false hit rate (specificity) of binder predictions as determined by the netmhcpan . threshold of peptides within the top % (with a score of or less) were calculated and the strength of the model was determined by calculating the area under the curve, auc of the roc plot [ ] [ ] [ ] . pearson's correlation coefficient was used to evaluate the relationship between the number of epitopes with various hiv- genes. to evaluate if there were any differences in the early versus late time point peptides for the binding ranking of the experimentally mapped peptides as predicted by the computational score the wilcoxon signed rank test was used. to evaluate if hiv- subtypes a and d affected the number of computational predictions generated, fisher exact test was used. to determine whether multiple computationally predicted epitope sequences were derived from the same experimental peptide sequence, a local blast database was set up using geneious version . . . both hiv- clades a and d experimental consensus sequences were used separately each as a reference sequence for the blast. the computational peptide sequences were then aligned against the consensuses to evaluate those derived from a single amino acid experimental peptide sequence. where an experimental peptide was predicted by multiple or overlapping computational peptides, the average netmhcpan . score was assigned as the computational score for this peptide. this score was also used during the generation of the roc curve and the confusion matrix. to compare the association between elispot spot forming units and netmhcpan . scores or mhcflurry . . affinities and also the association between the values for experimentally mapped peptides for all participants and their cognate computational core -mer and a single -mer epitope sequence with scores. peptides shown in italic text were not algorithmically predicted as binders. multiple computational predictions contained in a single experimental peptide were counted as a single hit. participant's identifiers (id) beginning with e or l represent early or late time sampling points respectively the computational tools, pearson's correlation coefficient was used. number of experimental assays compared to computationally guided prediction assay projections to experimentally determine epitopes for peptides spanning the whole hiv- proteome for clades a and d as well as both time points of the individuals required a total of test assay wells. for each test subject these included antigen proliferation wells, culture elispot wells and an average of epitope mapping elispot wells (range; - test wells). using the hla alleles represented in the study subjects we were able to computationally predict % of the experimentally mapped epitopes. this approach could have reduced the test assays by eliminating all the t-cell antigen proliferation and culture elispot steps totalling to assay wells ( %) and leaving only ( %) epitope mapping assays required. applying a pooling strategy to the computational predictions similar to that used in the experimental pooling where each pool contained approximately peptides with a coverage of per peptide pool, the potential peptides ( % of experimental peptides for epitope mapping elispot derived from the ( %) eligible epitope mapping peptides) would make at most pools. consequently the computational prediction approach could have reduced the experimental assays by at least %. the core mer epitope sequence was similar across mer through mer set length except for one -mer peptide (hit in table ) magnitude of epitope predictions are variable across hla alleles, hiv- proteins and clades the input hiv- subtypes a and d consensus whole proteome sequences evaluated for potential , , , , and -mer binders to the hla alleles represented in the six patients, varied in the distribution of predicted binders across hiv- genes and hla alleles. all the peptide hits predicted for through -mer were also all predicted in the -mer set except for two -mer peptides. an expected positive correlation for hiv- protein length with number of epitopes predicted was observed as illustrated by spearman's rank order correlation; r s = . (fig. , a and b) . netmhcpan . predicted % ( / ) ( table ) of the experimentally mapped peptides as binders and missed % ( out of ) ( table ) for the time points of the participants. mhcflurry predicted % ( / ) of the experimental peptides and had a lot of similarity to netmhcpan . for the predicted hla. netctl was the least performing tool with only % ( / ) predicted experimental peptides ( table ) . comparison of the various epitope prediction length set showed that the mer setting was ideal for netmhc-pan . . the number of predictions were , , , , and hits out of for , , , , and -mer epitopes respectively. increasing the prediction length from mer through mer resulted in a smaller number of predicted binders as illustrated in fig. . since we held the assumption that our wet experimental data was the gold standard we evaluated the sensitivity and specificity of netmhcpan . .the computational predictor had more predicted binders than those determined by the experimental mapping as presented in the confusion matrix in table . the experimental positive's count also shown in table under column "hit no" shows the test peptide count ( through ) that contained the computational mer sequence. multiple computational epitopes may be contained in a single experimental peptide, as shown in the column "netmhcpan . -mer epitope prediction" in table . overall hiv- clade a -mer predictions were fewer in number than clade d (fig. , c) though the difference did not approach statistical significance. the experimental peptide mapping data was derived from a baseline time point corresponding to hiv- fiebig stages iv, v and vi (table ) and a later time point. ninety-three (n = ) epitopes were experimentally mapped of which were recognized at both baseline and later time points, only at baseline and only at the later time point. comparison of the ranked computational score for netmhcpan . binders of early (n = ) versus later peptides showed that the later time point predictions were stronger binders reaching statistical significance (wilcoxon signed rank p-value = . ) (fig. ) . netmhcpan . ranked binders as those predicted to false positive ( ) computational negative false negative ( ) true negative ( ) the total number of peptides experimentally tested were and these are broken down to show the fractions from both the experimental testing and netmhcpan . computational predictions fig. early versus late peptides. experimentally mapped peptides at baseline (n = ) and at least months later (n = ) were compared using the -mer computational netmhcpan . scores of the hits. the lower the computational score the stronger the predicted binding. late peptides were significantly stronger binders than early peptides (wilcoxon signed rank test, p = . ) be in the top % and assigned a score of . or below. any binder within the top . % and assigned a score of . or below was ranked as a strong binder. considering only the -mer computational predictions, peptides that were derived from the same mer experimental peptide were determined by a blast mapping to their derivative sequences. the -mer peptides were then classified into a confusion matrix (table ) as true positives, false positives, true negatives or false negatives. from the classification the true positive rate (sensitivity) was plotted against the false positive rate ( -specificity) using an roc curve and the auc attained reached . (fig. ) . only -mer length epitopes were considered in the roc analysis as increasing the length to -mer through mer netmhcpan . predictions neither raised the number of predicted binders nor improved the hit rate as all their predictions contained the sequence already predicted in the -mer set except , -mer peptide (hit in table ). comparison of the elispot magnitude of response (spot forming units) did not show any association to either netmhcpan . scores or mhcflurry . . affinity values. similarly a comparison of the latter computational predictors did not show any association between their assigned "affinity" values. netmhcpan . registered the highest concordance to the wet experiments followed by mhcflurry . . . in this analysis we showed that the computational method netmhcpan . predicted % of previously experimentally mapped hiv- epitopes in hiv- infected individuals expressing a total of different hla class i alleles. in our ifn-γ elispot assays we evaluated mer peptides overlapping by amino acids and covering the whole hiv- subtype a and d consensus proteomes. out of the experimentally determined epitopes missed by the algorithm (table ), were actually computationally predicted as binders but were not included for lack of concordance with the participant's hla alleles. about one third ( ) of total positive predictions were not experimentally supported in our tests. these do not necessarily represent false positives, as elispot detection depends on the frequency of specific t cells in the participant's repertoire, and we observed changes in dominant t cell specificities within a given participant between early and later time points after hiv- infection. a formal roc evaluation of the score generated by netmhcpan . as a classifier for peptides recognised/not recognised by pbmc in ifng elispot assays, produced an auc of . . thus experimental confirmatory tests cannot be dropped altogether, however the netmhcpan . algorithm could provide a considerable saving of time and resources in verifying just the predicted epitopes. as the participants had been enrolled in the acute/ early phase of hiv- infection and we had observed intra-participant changes in epitope recognition between early and late time points after infection, we compared the binding scores of confirmed epitopes at these time points and found a statistically significant change towards recognition of higher binding peptides as the infection entered the chronic phase. this might represent better support of the t-cell response directed at more stable hla/peptide complexes as the infection progresses into chronicity. the netmhcpan . algorithm, which is based on binding affinity and integrates data on eluted naturally processed ligands, reflected optimal hla class i binding for -mers, producing a decreasing number of predictions when the peptide size was increased from to amino acids. with a single exception, predicted binders between and amino acids included at least one mer predicted to bind on its own, suggesting a destabilizing effect of the extra amino acids beyond the canonical hla class i binding pockets at positions and could account for fewer predictions. important limitations are the lack of predictions of hla class ii restricted epitopes, which might have contributed to a fraction of ifn-γ elispot responses. approximately % of the computational predictions may be false positives that only increase the size of planned wet experiments and approximately % of true positives may also be missed. in this analysis, using netmhcpan . , mhcflurry and netctl to predict previously experimentally mapped epitopes, we demonstrate that the computational methods reliably predict an acceptable portion of binder epitopes. we recommend the use of such computational methods to reduce the size of experiments required cost associated. vaccine design for h n based on b-and t-cell epitope predictions methods for prediction of peptide binding to mhc molecules: a comparative study induction of broad cross-subtype-specific hiv- immune responses by a novel multivalent hiv- peptide vaccine in cynomolgus macaques first-in-human randomized controlled trial of mosaic hiv- immunogens delivered via a modified vaccinia ankara vector analysis of the history and spread of hiv- in uganda using phylodynamics mapping hla-a , −a and -b supertype-restricted t-cell epitopes in the ebolavirus proteome enhancing in silico protein-based vaccine discovery for eukaryotic pathogens using predicted peptide-mhc binding and peptide conservation scores discovering a vaccine against neosporosis using computers: is it feasible? vacceed: a high-throughput in silico vaccine candidate discovery pipeline for eukaryotic pathogens based on reverse vaccinology a combined prediction strategy increases identification of peptides bound with high affinity and stability to porcine mhc class i molecules sla- * : defining epitope coverage requirements for t cell-based hiv vaccines: theoretical considerations and practical applications control of human immunodeficiency virus replication by cytotoxic t lymphocytes targeting subdominant epitopes consistent cytotoxic-t-lymphocyte targeting of immunodominant regions in human immunodeficiency virus across multiple ethnicities cd t-cell recognition of multiple epitopes within specific gag regions is associated with maintenance of a low steady-state viremia in human immunodeficiency virus type -seropositive patients induction of multifunctional human immunodeficiency virus type (hiv- )-specific t cells capable of proliferation in healthy subjects by using a prime-boost regimen of dnaand modified vaccinia virus ankara-vectored vaccines expressing hiv- gag coupled to cd + t-cell epitopes dynamics of viral evolution and ctl responses in hiv- infection broad and gag-biased hiv- epitope repertoires are associated with lower viral loads netmhcpan, a method for mhc class i binding prediction beyond humans netmhcpan- . : improved peptide-mhc class i interaction predictions integrating eluted ligand and peptide binding affinity data pan-specific mhc class i predictors: a benchmark of hla class i pan-specific prediction methods a community resource benchmarking predictions of peptide binding to mhc-i molecules efficient peptide-mhc-i binding prediction for alleles with few known binders multipred: a computational system for prediction of promiscuous hla binding peptides automated benchmarking of peptide-mhc class i binding predictions an integrative approach to ctl epitope prediction: a combined algorithm integrating mhc class i binding, tap transport efficiency, and proteasomal cleavage predictions largescale validation of methods for cytotoxic t-lymphocyte epitope prediction reliable prediction of t-cell epitopes using neural networks with novel sequence representations mhcflurry: open-source class i mhc binding affinity prediction t-cell antigenic sites tend to be amphipathic structures emerging vaccine informatics prediction of peptide-mhc binding using profiles comparison of experimental fine-mapping to in silico prediction results of hiv- epitopes reveals ongoing need for mapping experiments dynamics of hiv viremia and antibody seroconversion in plasma donors: implications for diagnosis and staging of primary hiv infection macrophage inflammatory protein- beta and interferon gamma responses in ugandans with hiv- acute/early infections optimized determination of t cell epitope responses high resolution hla class i typing by reference strand mediated conformation analysis (rsca) profile of t cell recognition of hiv type consensus group m gag and nef peptides in a clade a -and d-infected ugandan population frequencies of gag-restricted t-cell escape "footprints" differ across hiv- clades a and d chronically infected ugandans irrespective of host hla b alleles a standardized framework for accurate, high-throughput genotyping of recombinant and nonrecombinant viral sequences an automated genotyping system for analysis of hiv- and other microbial sequences the meaning and use of the area under a receiver operating characteristic (roc) curve receiver operating characteristic (roc) curve: practical review for radiologists smallsample precision of roc-related estimates publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the study participants who provided specimen for the wet laboratory experiments, the aids information centre clinic kampala, uganda that steered the rubicon discordant couple cohort study, the late anthony kebba who initiated the rubicon cohort and christine watera who coordinated the recruitment clinic activities. we thank ruhena sargeant for hla typing, the late harr f. njai for hiv- elisa and western blot assays and deogratius ssemwanga for help with genbank submissions. this research is jointly funded by the uk medical research council (mrc) and the uk department for international development (dfid) under the mrc/dfid concordat agreement, the wellcome trust (grant wt ma), and edctp (project code: ta_ _ _ ). most of the relevant data to support the manuscript has been included in the write-up. if any addition data is required will be availed once requested. the rubicon study, from which we derived the experimental data, was reviewed and approved by the uganda virus research institute, research and ethics committee (uvri-rec) and the uganda national council of science and technology (unct). all participants provided informed signed consent accepting to freely participate in this study. not applicable. the authors declare that they have no competing interests. authors' contributions dbl performed the peptide mapping experiments. nn provided hiv- subtyping. dbl analysed the data together with pp. dbl wrote the manuscript with major contributions from pp, aeo, pk and js. all authors reviewed the manuscript and or provided useful contributions as well as approved the final manuscript. key: cord- -o dthlk authors: iwuji, collins c.; churchill, duncan; bremner, stephen; perry, nicky; to, ye; lambert, debbie; bruce, chloe; waters, laura; orkin, chloe; geretti, anna maria title: a phase iv randomised, open-label pilot study to evaluate switching from protease-inhibitor based regimen to bictegravir/emtricitabine/tenofovir alafenamide single tablet regimen in integrase inhibitor-naïve, virologically suppressed hiv- infected adults harbouring drug resistance mutations (pibik study): study protocol for a randomised trial date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: o dthlk background: currently recommended boosted protease-inhibitor (bpi) regimens may be associated with increased risk of cardiovascular or chronic kidney diseases; in addition, boosted regimens are particularly associated with drug-drug interactions. since both cardiovascular and renal disease, and polypharmacy, are common in ageing people with hiv, there is a need for alternative efficacious regimens. bpi-based regimens are often the treatment of choice for individuals with pre-treatment or treatment-acquired resistance but it is plausible that carefully selected hiv-positive individuals with drug resistance, who are virologically suppressed on their current bpi regimen, could maintain virological efficacy when switched to bictegravir, emtricitabine and tenofovir alafenamide (b/f/taf) fixed dose combination (fdc). methods/design: a phase iv, investigator-initiated, multicentre, open label pilot, randomised two-arm study to assess the safety and efficacy of switching from bpi regimen to b/f/taf single tablet regimen in integrase inhibitor-naïve, virologically suppressed adults with hiv- infection harbouring drug resistance mutations. eligible individuals will either continue on their bpi regimen or switch to b/f/taf fdc. after weeks, all participants in the bpi arm will be switched to b/f/taf and followed for a further weeks and all participants will be followed for weeks. the primary efficacy endpoint is the proportion of participants with hiv- rna < copies/ml at week using pure virologic response whilst the secondary efficacy endpoint is the proportion of participants with hiv- rna < copies/ml at week . other secondary outcome measures include between arm comparisons of drug resistance at virological failure, safety and tolerability and patient-reported outcome measures. discussion: we aim to provide preliminary evidence of the efficacy of switching to b/f/taf in patients with virological suppression on a bpi-based regimen who harbour select drug resistance mutations. trial registration: isrctn , registered june and eudract – - . boosted darunavir and atazanavir are recommended as preferred boosted protease inhibitors (bpi) in the british hiv association treatment guidelines. however, boosted darunavir was associated with increased cardiovascular risk in a large prospective observational study [ ] . although boosted atazanavir has not been associated with increased cardiovascular risk [ ] , cohort studies indicate an increased risk of chronic kidney disease [ ] . in clinical trials, atazanavir recipients experienced higher discontinuation rates from adverse events than darunavir [ , ] which were driven by hyperbilirubinaemia and renal events. ageing of people living with hiv is resulting in increasing prevalence of cardiovascular and renal diseases; in addition, polypharmacy is common due to comorbidities, pointing to an additional limitation for bpi due to their particularly high potential for drug-drug interactions [ , ] . there is evidence from clinical trials that switching from virologically suppressive bpi-based antiretroviral therapy (art) to regimens based on the newer strand transfer integrase inhibitors, bictegravir [ ] and dolutegravir [ ] is safe and efficacious. these studies however excluded individuals known to harbour hiv- drug resistance. it is well established that hiv-positive individuals that have either pre-treatment drug resistance (pdr) or limited drug resistance following failure of first-line art achieve virological suppression on regimens comprising a bpi plus two nucleos(t)ide reverse transcriptase inhibitor (nrtis) [ , ] . in contrast, in the switchmrk study, switching virologically suppressed individuals from a bpi to the integrase inhibitor raltegravir resulted in an increased risk of virological failure relative to individuals maintained on the bpi. a post-hoc analysis suggested that this effect might be mediated by prior virological failure compromising the activity of the nrti backbone [ ] . however, evidence indicates that second-generation instis, including bictegravir and dolutegravir, have an improved barrier to resistance relative to first-generation compounds and may overcome both pdr and limited treatment-associated drug resistance. in the dawning study, a second line switch to a regimen comprising the insti dolutegravir plus nrtis, where at least one nrti was predicted to be fully active based on resistance analysis at the time screening, was superior to a bpi plus nrtis at weeks in patients failing first line art [ ] . dawn ing study suggests second-generation insti like dolutegravir and by extension bictegravir are likely to be successful in switch strategies in the presence of either pdr or treatment-acquired drug resistance, including m v/i or thymidine analogue mutations (tams). a small, open label, single arm study switched patients ( % on a bpi-based regimen) harbouring the lamivudine ( tc) and emtricitabine (ftc) associated mutation m v/i to the fixed dose combination (fdc) of elvitegravir/cobiscitat/emtricitabine/ tenofovir alafenamide with no virological failures observed after weeks of follow up [ ] . another study investigated whether efficacy was maintained following a switch to bictegravir/emtricitabine/tenofovir alafenamide fdc (b/f/ taf) in individuals suppressed on either a pi-based regimen or the fdc of dolutegravir/abacavir/lamivudine (dtg/abc/ tc). amongst the participants on b/f/ taf, ( %) achieved virologic suppression (vl < copies/ml), with missing virologic data accounting for the majority of the remaining. of the participants, ( %) had baseline resistance data available determined by both historical genotypes and baseline proviral dna sequence; ( %) of whom had nrti associated resistance mutation present. / ( %) of those patients with archived f/taf resistance mutations maintained hiv- rna suppression through week [ ] . it should be noted that in virologically suppressed patients it is often possible to recover proviral sequences from cellular reservoirs in peripheral blood [ ] . the presence of archived drug resistance mutations identified by sequencing proviral dna is not necessarily reflective of the full range of resistant variants that may have emerged in an individual. furthermore, it does not necessarily correlate with an increased risk of failure as integrated provirus is often defective [ ] and reactivation of a particular virus is likely to be a stochastic event. in studies that examine drug activity in the presence of archived drug resistance, duration of follow up is crucial because the likelihood that a particular latent virus carrying a certain mutation would reactivate may increase with time, and the levels of adherence to art over time play a key modulating role. in the single arm switch study referred to earlier, drug resistance sequencing based on proviral dna missed half of m v/i mutation present in historical genotype [ ] . taken together these studies suggest that b/f/taf may be effective in maintaining virological suppression in patients with historical evidence of drug resistance mutations. in light of this, we hypothesize that switching hiv-positive patients who harbour selected drug resistance mutations and are virologically suppressed on bpi regimen to b/f/taf will maintain virological efficacy over weeks. the pibik trial is a phase iv, investigator-initiated, prospective, multicentre, open label pilot, randomised two arm study to assess the safety and efficacy of switching from a bpi-based regimen to b/f/taf single tablet regimen in insti-naïve, virologically suppressed hiv- infected adults harbouring drug resistance mutations. the allocation ratio is : (fig. ). subjects will be enrolled from seven sexual health clinics in england. these are: clinical staff in the hiv department will identify potential participants by any of the following methods: review of a clinic records/database, pre-identification of patients attending for routine care, review of notes during routine clinical follow up and posters/advertisements in the clinics to inform patients of the study. clinical staff identifying patients will be members of the direct care team or research nurses or doctors working within the same hiv multidisciplinary team. a medically qualified doctor on the study delegation log will confirm eligibility. anonymised information on participants who are not randomised / registered for consort ( ) reporting will include the reason, if they are not eligible for trial participation, or if they are eligible but declined. potentially eligible participants will be invited to attend for an appointment, having been provided with a participant information sheet (pis). adequate time will be allowed for questions and to consider the study before agreeing to participate. the investigator or designee will provide adequate explanation of the aims, methods, objectives and potential hazards of the study. it will also be explained to the individual that they are free to refuse or withdraw from the study for any reason without fig. trial design detriment to their future care or treatment. the investigator can decide to withdraw a subject from the study for urgent medical reasons or repeated non-compliance with the study protocol. once randomised, withdrawn subjects may be replaced if considered necessary by the chief investigator. years and above on a bpi-based art regimen with documented hiv- rna < copies/ml for at least months on current regimen and at screening (a switch from tenofovir disoproxil fumarate (tdf) to tenofovir alafenamide (taf), lamivudine ( tc) to emtricitabine (ftc), or splitting co-formulated tablets to their individual component or vice versa will not be considered true regimen changes) must have a historical genotype eligible drug resistance mutations in historical genotype include at least one of the following: a single repeat of a laboratory screening test will be allowed for test results that are unexpected based on documented prior laboratory results. provides written, informed consent to participate is willing to comply with the protocol requirements if a woman and of childbearing potential and are willing to continue practicing one of the following: o must be using effective birth control methods, that is has an expected failure rate of < % per year and willing to continue practicing these birth control measures during the trial and for at least days after the end of the trial. effective methods include iud, combined pill, contraceptive injection, implant, ius, contraceptive vaginal ring, contraceptive patches etc. o must be truly abstinent from penile-vaginal intercourse from weeks prior to administration of study drug, throughout the study, and for at least days after the end of the trial (when this is in line with the preferred and usual lifestyle of the subject.) [periodic abstinence (e.g., calendar, ovulation, symptothermal, post-ovulation methods), and withdrawal are not acceptable methods of contraception]. women who are postmenopausal for least years, women with a total hysterectomy, and women who have a tubal ligation are considered of nonchildbearing potential. if male, and sexually-active with female partners of child bearing potential, is using effective barrier contraception, and willing to continue using this during the trial and for at least days after the end of the trial. o presence of any of the following mutations: k r/n/e o presence of multidrug resistance mutations: t ins, q m with or without a v, v i, f l, f y o three or more tams (m l, d n, k r, l w, t f/y, or k q/e/n/r) individuals experiencing decompensated cirrhosis (e.g., ascites, encephalopathy, or variceal bleeding) an opportunistic illness within the days prior to screening active tuberculosis infection have been treated with immunosuppressant therapies or chemotherapeutic agents within months of study screening, or expected to receive these agents or systemic steroids during the study (e.g., corticosteroids, immunoglobulins, and other immune-or cytokine-based therapies) current alcohol or substance use judged by the investigator to potentially interfere with patients' adherence to study procedure. a history of malignancy of less than years or ongoing malignancy (including untreated carcinoma in-situ) other than cutaneous kaposi's sarcoma (ks), basal cell carcinoma, or resected, non-invasive cutaneous squamous carcinoma. individuals with biopsyconfirmed cutaneous ks are eligible but must not have received any systemic therapy for ks within days of day and are not anticipated to require systemic therapy during the study. active, serious infections (other than hiv infection) requiring parenteral antibiotic or antifungal therapy within days prior to day (except if the parenteral therapy is for syphilis infection) any other clinical condition or prior therapy that will, in the opinion of the investigator, make the patient ineligible any known allergies to the excipients of b/f/taf fdc females who are pregnant (as confirmed by positive urine pregnancy test) women who are breastfeeding women of childbearing age not using any reliable form of contraception (e.g. intrauterine device/ intrauterine system, long-acting contraceptive injection) acute hepatitis in the days prior to study entry, anyone with hepatitis c (hcv) who is likely to need direct acting antivirals in study any concomitant medications that cannot be administered with taf (i.e strong inducers of pglycoprotein) or bictegravir (dofetilide, rifampins) the investigational medicinal product in this trial is bik-tarvy® comprising fdc of b/f/taf. each film-coated tablet contains bictegravir sodium equivalent to mg of bictegravir, mg of emtricitabine, and tenofovir alafenamide fumarate equivalent to mg of tenofovir alafenamide. eligible individuals will either continue on their bpi regimen (arm ) or switch to b/f/taf fdc (arm ). after weeks, all participants in the bpi arm will be switched to b/f/taf arm and will be followed up for a further weeks whilst those immediately switched to b/f/taf at baseline will be followed up for weeks as illustrated in fig. . a participant is free to withdraw from the study at any time. in addition, the investigator may decide, for reasons of medical prudence, to withdraw a participant. if a participant discontinues study medication dosing, every attempt would be made to keep the participant in the study and continue to perform the required studyrelated procedures and follow-up procedures. if this is not possible or acceptable to the subject or investigator, the participant may be withdrawn from the study. study medication may also be discontinued in the following instances: if the participant withdraws his/her consent. if the investigator considers in the interest of the subject (i.e. intercurrent illness, unacceptable toxicity) that it is best for them to withdraw their consent. the participant fails to comply with the protocol requirements or fails to cooperate with the investigator. pregnancy during the course of the study. the date and reasons for the withdrawal will be clearly stated on the participant's ecrf and source document. every attempt should be made to arrange follow up visits for participants who are withdrawn from the trial (including where individuals fall pregnant). participant adherence b/f/taf and bpi will be assessed through: pill counting at each visit by a research team member and recording of the number of pills returned self-report using a visual analogue scale (vas) participants will bring in all pill bottles at each study visit. the total number of pills remaining at each visit will be counted and, then returned to the participant to take until the bottle is finished. the percentage of compliance for each participant will be calculated. participants will also be asked be asked to self-report their level of adherence at each visit using the vas in which they would be asked to indicate their level of adherence in the previous days on a scale ranging from to % in which represents no pill taken and represents every single dose had been taken. where adherence is < %, this will lead to likely withdrawal from the study although this will be at the discretion of the investigator. the primary endpoint is the proportion of participants with hiv- rna < copies/ml at week using pure virologic response (pvr). pure virologic response is defined as follows: we considered a number of sample size scenarios bearing in mind the pilot nature of the study (table ) . we will perform a futility analysis at weeks when assessing the primary outcome. at weeks, with participants in the trial, we will have % power for % significance to conclude non-inferiority of the b/f/taf arm assuming a non-inferiority margin of % and viral suppression in % of participants in both arms. for the study to continue beyond weeks, we need % ( / ) of the individuals randomised to the b/f/ taf arm to be suppressed with the lower limit of the confidence interval to just lie above %. recruiting participants per arm would achieve this at the % confidence level. the sample size required decreases as the level of confidence in our estimates decreases. we have allowed months to recruit participants over the seven sites involved in the trial. this requires recruitment of - participants per month per site which is deemed feasible. each site has been allocated a target of - subjects. in the case of slow enrolment, additional sites will be offered participation in the study. the web-based sealed envelope™ system will be used to allocate individuals randomly to arm and arm . the statistician will provide the randomisation list. each study site will be provided with a randomisation guide. the sealed envelope™ system will randomise subjects within strata as shown in table . the purpose of the stratification is to balance the treatment arms on important prognostic factors such as: the bpi used in the subject's baseline regimen (atazanavir or darunavir) use of lipid lowering therapy at study day (yes/no) number baseline mutations of the nrti class (< vs. ≥ ) investigators randomise patients by completing an onscreen form with patient details, stratification factors, inclusion and exclusion criteria. investigators are immediately shown the treatment allocation. trial managers have real-time access to recruitment statistics and are notified by email of every new randomisation. the randomisation application conforms to the requirements of fda cfr part , electronic records; electronic signatures and ich gcp. no-one can delete records from the randomisation database, so that all randomisations have to be accounted for. audit log files detailing all activity on the randomisation system are available to the trial manager. neither the investigators nor the participants will be blinded to the treatment allocation. the presence of resistance mutations will be determined using historical genotype results obtained in local laboratories in those eligible according to the inclusion and exclusion criteria. we would obtain more information on resistance mutations by sequencing cellassociated hiv- dna in peripheral blood mononuclear cells prior to commencing b/f/taf. the results of proviral dna sequence will not be available in real time and will not be used to inform treatment decisions but will further the understanding of the clinical importance of archived resistance mutations, if any, in individuals developing virological failure. the baseline visit will not exceed days after the screening visit. follow up of participants will continue until all participants have accrued weeks from their baseline visits. individuals who have completed week visit will be followed up days post cessation of trial treatment via a telephone call or a standard of care clinical appointment for performance of the following assessments; adverse events and symptoms review, hiv associated conditions, concomitant medications and for women of child bearing potential (wocbp), confirmation that contraception has been used in the previous days. study procedures, screening, randomisation and safety monitoring will be according to attached visit schedule in table . individuals with virological failure defined as a rebound in hiv- rna ≥ copies/ml, which is subsequently confirmed at the following scheduled or unscheduled visit. following the initial detection of virological rebound, subjects will be asked to return to the clinic for a scheduled or unscheduled blood draw ( to weeks after the date of the first measured rebound) for repeat viral load testing. if virological rebound is confirmed and the hiv- rna is ≥ copies/ml, the blood sample from the confirmation visit will be the primary sample used for hiv- genotypic testing. after a participant's first post-baseline resistance test, additional testing will be conducted on a case-by-case basis. any participant may be discontinued at the investigator's discretion or per local treatment guidelines. if no resistance is detected from the genotype, the participant may remain on study drugs and a repeat hiv- rna measurement should be performed ( to weeks after date of test with hiv- rna ≥ copies/ml). investigators should carefully evaluate the benefits and risks of remaining on study drug for each individual participant and document this assessment in the on-site medical record. data on patient reported outcome measures will be collected using the hiv-si and the psqi. the hiv-si is a validated, self-administered -item health-state questionnaire for use in clinical care and research amongst people living with hiv (plhiv)in order to identify and address common and bothersome symptoms associated with hiv treatment and disease [ ] . the instrument is considered to be the gold standard in contemporary hiv-symptom research [ ] . respondents will be asked about their experience with each symptoms during the past weeks using a -point likert scale. response options and scores are as follows: ) i don't have this symptom, ) i have this symptom and it doesn't bother me, ) i have this symptom and it bothers me a little, ) i have this symptom and it bothers me, ) i have this symptom and it bothers me a lot. the pittsburgh sleep quality index (psqi) is a selfrated questionnaire which assesses sleep quality and disturbances over a -day recall period [ ] . nineteen individual items generate seven component scores: subjective sleep quality, sleep latency, sleep duration, habitual sleep efficiency, sleep disturbances, use of sleeping medication, and daytime dysfunction. it uses a likert scale in with the following scores: ) not during the past month, ) less than once a week, ) once or twice a week, ) three or more times a week. the sum of scores for these seven components yields one global score ( to ). a total score of or greater is indicative of poor sleep quality. participants with early study termination from whatever cause will undergo the assessments outlined in table . a source data worksheet will be created to capture all the relevant information and will be filed as source documentation in the participants' notes. questionnaires and self-report vas scores will also be completed by the patients. a source data agreement will be completed prior to recruitment commencing to ensure all parties are aware which documents constitute source data. data from the source will be entered onto the electronic case report form (ecrf) on the web-based macro™ electronic data capture system. data entered will be checked by the data manager in accordance with the data management plan (supplementary appendix) and queries raised to the clinical sites via macro when appropriate. clinical sites will be responsible for the entry of data into the ecrf. patient data will be entered using study number only and no patient identifiable data will be seen by the data management team. direct access will be granted to authorised representatives from the sponsor, host institution and the regulatory authorities when appropriate to permit trial-related monitoring, audits and inspections. archiving will be the responsibility of the sponsor and all documentation will be archived for years, with the exception of the health records which will be kept in accordance with uk law and local policy. the sponsor named archivist will be responsible for ensuring the documentation is prepared in line with the relevant sponsor standard operating procedures. all randomised patients who received at least one dose of the study medication will be included in the analysis. summary statistics will be presented by trial arm using median with interquartile ranges for continuous variables with skewed distributions or mean with standard deviations for normally distributed variables. categorical variables will be summarised using frequencies and proportions. non-inferiority for the futility analyses will be concluded if the lower bound of a two-sided % ci for the difference in proportions (b/f/taf minus boosted pi) of patients with plasma hiv- rna < copies/ml at week is greater than − %. primary efficacy endpoint proportions of individuals with hiv rna < copies/ml at weeks will be estimated using pvr. the percentage of participants with pvr for hiv- rna cut-off at copies/ml at week will be summarized. differences between trial arms will be estimated together with % confidence intervals. secondary efficacy endpoint proportions of individuals with hiv rna < copies/ml at weeks will be estimated using pvr. the percentage of participants with pvr for hiv- rna cut-off at copies/ml at week will be summarized. differences between trial arms will be estimated, together with % confidence intervals. the proportion of patients with hiv- rna < copies/ml at weeks and using pvr in those with any archived resistance detected in proviral dna will be estimated. the proportion of patients with any emergent drug resistance following virological rebound will be estimated. the analysis of the following secondary safety outcomes will be presented as the estimated difference and % confidence interval between arms from baseline to and weeks mean total cholesterol, ldl, hdl and triglycerides will be estimated. mean hba c mean weight and bmi laboratory and clinical adverse events will be described and summarised using percentages. treatment differences for patient reported outcomes using the hiv-si module and the psqi will be compared by using the prevalence of symptoms reported by each method and presented with % confidence intervals. consistent with prior analyses on hiv-si [ , ] we would dichotomise symptoms into not bothersome (scores of or ) or bothersome (scores of , and ). the overall bothersome symptom count at baseline will be generated by counting the number of individual symptoms scored as bothersome. reported poor sleep quality scores on the psqi will be summarised by the seven components as well as the global scores by arm. the global scores will be dichotomised into poor sleep quality (score of < ) and good sleep quality (≥ ) and an exact % binomial confidence interval presented for the difference in prevalence between arms. . in the event of missing data, only available data will be included in the analyses and missing data will be quantified but not imputed. for missing data relating to primary and secondary efficacy endpoints, using the principle of pvr, the last known measured viral loads will be carried forward if data is missing at the and week time points. a three-member independent data safety monitoring board (dsmb) has been established comprising specialists in clinical infectious diseases, hiv medicine and a clinical trial statistician. the role of the dsmb will be to safeguard the interest of the trial participants, assess safety and efficacy of the intervention and to monitor the overall conduct of the trial. the dsmb should receive and review the progress and accruing data of the trial and provide advice on the conduct of the trial to the trial steering committee (tsc). the dsmb would perform an interim review of the trial's progress including updated figures on recruitment, data quality, main outcomes and safety data and will have responsibility on the decision whether to stop or continue the trial. the dsmb will meet six-monthly but the frequency of meetings may depend on recruitment rates or other trial events. further details on the dsmb charter can be found in the supplementary appendix. information on all adverse events (aes), adverse reactions (ars), serious adverse reactions (sars), suspected unexpected serious adverse reactions (susars) and serious adverse events (saes) will be documented in the case report forms. aes, saes, ars, sars and susars may be directly observed, reported spontaneously by the participant or by questioning the participant at each study visit. these will be followed up until they are resolved or the participant's participation in the study ends (i.e. until the final crf is completed for that participant). any untoward event that may occur subsequent to the reporting period that the investigator assesses as related to the study drug medication will also be reported as an adverse event. the adverse event reporting period will be from consent until the participant's final study visit. after informed consent, but prior to initiation of study treatment, all saes and adverse events related to protocol-mandated procedures would be reported on the crfs. following initiation of study treatment, all aes, regardless of cause or relationship until days post cessation of trial treatment would be reported on the crfs. in addition, all serious adverse events assessed by the investigator as related to the investigational medication would continue to be followed even after participation in the study is over. such events would be followed until resolution, or until no further change can reasonably be expected. this study is registered with the international standard randomised controlled trials number registry ( ) and with the european union drug regulating authorities clinical trials database ( - - ). the main study findings will be reported in accordance the consolidated standards of reporting trials (consort) statement [ ] . the study received ethical approval from the health research authority ( /lo/ ) and will be conducted in accordance with the declaration of helsinki. written, informed consent will be sought from participants by an appropriate member of the research team identified on the delegation log and this is mandatory prior to any study procedures. participants would be made aware that they may not continue to be prescribed b/f/taf after the end of the trial unless they are eligible according to nhs england prescribing criteria for tenofovir alafenamide. in this situation, participants would be switched to alternative efficacious art combination decided by the local principal investigator. all investigators and trial site staff will comply with the requirements of the general data protection regulation (gdpr) [ ] with regards to the collection, storage, processing and disclosure of personal information and will uphold the act's core principles. personal information will be collected, kept secure, and maintained. this will involve the creation of coded, depersonalised data where the participant's identifying information is replaced by an unrelated sequence of characters, secure maintenance of the data and the linking code in separate locations using encrypted digital files within password protected folders and storage media and limiting access to the minimum number of individuals necessary for quality control, audit, and analysis. the confidentiality of data will also be preserved when the data are transmitted to sponsors and co-investigators by using only pseudonymised codes rather than personal identifiable information. trial data will be stored for years and the principal investigator at site is the data custodian. it is plausible that carefully selected hiv-positive individuals with pre-treatment or treatment-acquired resistance who are virologically suppressed on their current pi-based regimen could maintain virological efficacy when switched to b/f/taf fdc. the hypothesis is based on a consideration of each component of b/f/taf. taf, like the earlier version tenofovir disoproxil fumarate (tdf), is a prodrug of tenofovir. however, taf yields -fold higher intracellular concentrations of the active moiety tenofovir diphosphate (tfv-dp) in hiv target cells than tdf, despite much lower plasma drug concentration [ ] .. since taf and tdf produce the same active metabolite, they have similar resistance profiles but it could be proposed that the higher intracellular concentrations of tfv-dp yielded by taf could be beneficial in resistant viral isolates [ ] . furthermore, the selective conversion of taf to tfv-dp within hiv target cells and lower levels in plasma is associated with less renal and bone toxicity [ ] . hiv- strains harbouring the m v/i mutation, which causes high-level resistance to tc and ftc, display increased or restored susceptibility to tenofovir [ ] . m v/i mutants also display a loss of fitness that reduces their replication capacity and may account for the partial residual activity of tc in the presence of the mutation. in art-experienced individuals who developed virological failure whilst treated with either zidovudine or stavudine, the stepwise accumulation of tams resulted in increasing resistance to tenofovir, with three or more tams being associated with markedly reduced tenofovir susceptibility [ ] . this cross resistance to tenofovir is more marked for the tam- pathway of mutations (m l, l w, and t y) than the tam- pathway of mutations (d n, k r, k /e/n/q/r, and t f). as a result, we have allowed a maximum of tams when assessing eligibility for inclusion in the trial. the revertant mutations t s/c/d/e/i/v/n/a/l which arise from viruses that once harboured t y/f do not directly impact nrti susceptibility [ ] . however, both in vitro and in vivo, the effect of tams on tenofovir is partially reversed by the presence of the m v/i mutation [ , ] with tc maintaining residual activity in viruses harbouring this mutation [ ] . bictegravir has potent in vitro activity against laboratory strains and clinical isolates of hiv- , a higher genetic barrier to resistance development than raltegravir (ral) and elvitegravir (evg), and a statistically improved resistance profile compared to ral, evg, and dtg against a set of patient derived insti-resistant viral isolates [ ] . in the earnest [ ] , and mobidip [ ] studies, bpi given with nrti in individuals with previous virological failure and predicted limited nrti activity due to resistance (mainly m v/i and tams) achieved high rates of virological suppression. in the dawning study [ ] , dtg a high genetic barrier insti, demonstrated superior virological efficacy over a bpi. hence there is a strong scientific plausibility for bictegravir demonstrating high rates of virological efficacy in the presence of a limited pattern nrti mutations when switching patients from bpi regimen to b/f/taf. we do not foresee a challenge to recruiting the participants required for this trial, however recruitment will be monitored closely and if sluggish, we would activate additional sites for participation in the study. since all sites will be utilising standardised study documents and procedures, the number of study sites should not affect data quality. furthermore, all sites will be closely monitored for compliance with the study protocol. the trial started enrolling participants on september and it is anticipated that enrolment will continue until september . cardiovascular disease and use of contemporary protease inhibitors: the d:a:d international prospective multicohort study metabolic effects of darunavir/ritonavir versus atazanavir/ritonavir in treatment-naive, hiv type -infected subjects over weeks development and validation of a risk score for chronic kidney disease in hiv infection using prospective cohort data from the d:a:d study efficacy and tolerability of nonnucleoside reverse transcriptase inhibitor-sparing antiretroviral regimens for treatment-naive volunteers infected with hiv- : a randomized, controlled equivalence trial combination antiretroviral therapy and the risk of myocardial infarction contemporary protease inhibitors and cardiovascular risk efficacy and safety of switching to fixed-dose bictegravir, emtricitabine, and tenofovir alafenamide from boosted protease inhibitor-based regimens in virologically suppressed adults with hiv- : week results of a randomised, open-label, multicentre, phase , non-inferiority trial switching from a ritonavir-boosted protease inhibitor to a dolutegravirbased regimen for maintenance of hiv viral suppression in patients with high cardiovascular risk second-line antiretroviral treatment successfully resuppresses drug-resistant hiv- after first-line failure: prospective cohort in sub-saharan africa effectiveness of protease inhibitor/nucleos(t)ide reverse transcriptase inhibitor-based second-line antiretroviral therapy for the treatment of human immunodeficiency virus type infection in sub-saharan africa: a systematic review and meta-analysis switch to a raltegravir-based regimen versus continuation of a lopinavir-ritonavir-based regimen in stable hiv-infected patients with suppressed viraemia (switchmrk and ): two multicentre, double-blind, randomised controlled trials superior efficacy of dolutegravir (dtg) plus nucleoside reverse transcriptase inhibitors (nrtis) compared with lopinavir/ritonavir (lpv/r) plus nrtis in second-line treatment: -week data from the dawning study a phase b open-label pilot study to evaluate switching to elvitegravir/ cobicistat/emtricitabine/tenofovir alafenamide (e/c/f/taf) single-tablet regimen in virologically-suppressed hiv- infected adults harboring the nrti resistance mutation m v and/or m i (gs-us- - ): week results resistance analyses of bictegravir/emtricitabine/tenofovir alafenamide switch studies defective proviruses rapidly accumulate during acute hiv- infection development and validation of a self-completed hiv symptom index patient reported outcome instruments used in clinical trials of hiv-infected adults on nnrti-based therapy: a -year review the pittsburgh sleep quality index: a new instrument for psychiatric practice and research patientreported symptoms over weeks among participants in randomized, doubleblind, phase iii non-inferiority trials of adults with hiv on co-formulated bictegravir, emtricitabine, and tenofovir alafenamide versus co-formulated abacavir, dolutegravir, and lamivudine vacs project t. patient-reported symptoms on the antiretroviral regimen efavirenz/ emtricitabine/tenofovir statement: extension to randomised pilot and feasibility trials guide to the general data protection regulation (gdpr) antiviral activity, safety, and pharmacokinetics/pharmacodynamics of tenofovir alafenamide as -day monotherapy in hiv- -positive adults characterization of hiv- resistance to tenofovir alafenamide in vitro tenofovir alafenamide: a novel prodrug of tenofovir for the treatment of human immunodeficiency virus relationships among various nucleoside resistance-conferring mutations in the reverse transcriptase of hiv- genotypic and phenotypic predictors of the magnitude of response to tenofovir disoproxil fumarate treatment in antiretroviral-experienced patients stanford university hiv drug resistance database. marvel on rt mutations at position tenofovir resistance and resensitization phenotypic susceptibilities to tenofovir in a large panel of clinically derived human immunodeficiency virus type isolates boosted protease inhibitor monotherapy versus boosted protease inhibitor plus lamivudine dual therapy as second-line maintenance treatment for hiv- -infected patients in sub-saharan africa (anrs / mobidip): a multicentre, randomised, parallel, open-label, superiority trial antiviral activity of bictegravir (gs- ), a novel potent hiv- integrase strand transfer inhibitor with an improved resistance profile assessment of second-line antiretroviral regimens for hiv therapy in africa publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank all the members of the pibik study group in all the participating sites, the data safety and monitoring board, the trial steering committee and the research and enterprise department of the university of sussex. received: april accepted: july supplementary information accompanies this paper at https://doi.org/ . /s - - -y.additional file . authors' contributions ci conceived the study and drafted the manuscript. dc, co, lw, sb, np, amg contributed to the design of the study and assisted in drafting the manuscript. dl, cb developed the data management plan and data collection tools for the study and assisted in drafting the manuscript. yt is responsible for the coordination of the study and contributed to the manuscript draft. ci obtained funding for the study. all authors have read the final manuscript and give approval for it to be published. availability of data and materials not applicable. this trial protocol has been approved by the health research authority, uk ( /lo/ ). all patients in the trial will provide written informed consent to participate. competing interests ci has received honoraria, support to attend conferences and research funding (paid to university of sussex) from gilead sciences. dc has received honoraria and support to attend conferences from gilead sciences and viiv. lw has received speaker/advisory fees or conference support from gilead, viiv, janssen, msd, cipla and mylan. co has been a recipient of grants, speaker's bureau and travel sponsorship from gilead, msd, viiv, gsk and janssen. np has received honoraria for services rendered to gilead sciences. amg receives personal consultancy fees from roche pharma, provides consulting services to gilead sciences, janssen, and viiv (paid to the university of liverpool) and is the recipient of research funding from gilead, roche pharma, and viiv (paid to the university of liverpool). all other authors declare that they have no competing interests. key: cord- -p hhd ed authors: Şahar, esra atalay; can, hüseyin; İz, sultan gülçe; döşkaya, aysu değirmenci; kalantari-dehaghi, mina; deveci, remziye; gürüz, adnan yüksel; döşkaya, mert title: development of a hexavalent recombinant protein vaccine adjuvanted with montanide isa v and determination of its protective efficacy against acute toxoplasmosis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: p hhd ed background: toxoplasma gondii is an obligate intracellular parasite that can infect almost all warm-blooded animals, avian species and humans. toxoplasmosis is asymptomatic in healthy individuals, whereas it may lead to death in immune suppressed or deficient patients. a vaccine against t. gondii is required to prevent consequences of the infection. the aim of this study is to generate a multivalent recombinant protein vaccine against t. gondii. methods: previously discovered antigenic proteins of t gondii were evaluated by their expression level in e. coli and by comprehensive bioinformatics analyses to determine antigenic epitopes. based on these analyses, six vaccine candidate proteins were selected to generate a hexavalent recombinant protein vaccine adjuvanted with montanide isa v. humoral and cellular immune responses were determined by flow cytometry and elisa. vaccinated mice were challenged with t. gondii ankara strain tachyzoites. results: in mice vaccinated with hexavalent vaccine, strong total igg (p < . ) and igg a (p < . ) responses were induced compared to controls, the ratio of cd (+) and cd (+) t lymphocytes secreting ifn-γ increased, and significantly higher extracellular ifn-γ secretion was achieved compared to the controls (p < . ). the survival time of the vaccinated mice increased to . ± . days which was significantly higher than controls (p < . ). conclusions: altogether, these results show that the hexavalent vaccine which is developed for the first time against t. gondii induced strong and balanced th and th immune responses as well as conferred significant protection against challenge with lethal toxoplasmosis in murine model. toxoplasma gondii is an obligate intracellular parasite that causes toxoplasmosis in all warm blooded animals and humans. it is reported that one third of the world's population is estimated to be infected with t. gondii. t. gondii usually causes an asymptomatic infection in healthy people, but can be life-threating in immunecompromised patients (organ transplant recipients, acquired immunodeficiency syndrome patients, and cancer patients). congenital toxoplasmosis may cause abortion, neonatal death or foetal abnormalities in the foetus [ ] . farm animals such as sheep, goats, and pigs have also been shown to be susceptible to t. gondii infection. toxoplasmosis infection in farm animals causes significant economic losses as a result of prenatal death, abortion, and neonatal death. moreover, t. gondii is linked to mental disorders and may affect human behaviour, personality, and other phenotypic traits [ ] . cats and other felines are the definitive hosts of t. gondii [ ] . in the life cycle of t. gondii the main infective forms are tissue cysts (containing bradyzoites) and oocysts (containing sporozoites). the infection sources for humans are consumption of vegetables, fruits or water contaminated with faeces of infected cats containing oocysts and raw or undercooked meat contaminated with tissue cysts [ ] . for these reasons, development of a safe and protective vaccine against t. gondii infection that can be used in animals and humans has utmost importance. recombinant protein vaccines are safe and efficient, and have a great potential for prevention or eradication of diseases. one of the most important issues during a recombinant protein vaccine development is the selection of the vaccine candidate antigen(s) [ ] . antigens to be used in vaccine formulations against toxoplasmosis should actively induced strong immune response, produce long-lasting immunity, and be antigenic in each stage of the parasite [ ] . in our study group's previous studies, we used protein microarray containing candidate exon products of t. gondii to screen well-characterized sera from acute and chronic toxoplasmosis human cases and murine model infected orally with oocysts or tissue cysts. during these studies, screening human sera prioritized antigenic proteins and screening these antigens with murine sera prioritized proteins based on their immunogenicity [ ] [ ] [ ] . in this study, we selected six proteins based on their antigenic epitopes using bioinformatics and protein expression level in e. coli. thereafter, we developed a hexavalent recombinant protein vaccine adjuvanted with montanide isa v and administered to a murine model to determine its immunogenicity and protection efficiency against lethal toxoplasmosis. animals - week old female healthy swiss outbred mice were obtained from the bornova veterinary control institute animal production facility and used during the experiments. animals were housed under standard and suitable conditions. specifically rooms had ambient temperature and humidity, adequate light cycle, and diet was specific for each animal type. all animals were checked for humane endpoints every day such as rapid weight loss more than~ % of gross body weight, inability to assess water or food, or loss of skin elasticity indicative of dehydration. in any of these circumstances, we pre-euthanized the animals with ketamine hydrochloride ( mg/kg) and % xylazine ( mg/kg) and then euthanized with cervical dislocation. based on the data obtained from previous studies [ ] [ ] [ ] , proteins were selected based on their immunogenicity in humans and murine sera (table ). in order to determine the vaccine candidate antigens, expression levels of these proteins were analysed using western blot and moreover, a comprehensive bioinformatics analyses was performed. the gene sequences of the different genes were accessible from the toxoplasma genomic resource (http:// www.toxodb.org/toxo/). the plasmids expressing these recombinant proteins were constructed as previously described [ ] [ ] [ ] . thereafter, the plasmids were cloned into chemically competent escherichia coli (e. coli) bl star (de ) plyss cells according to the manufacturer's protocol (invitrogen, usa). e. coli bl star (de ) plyss cells containing different plasmids were incubated at °c with shaking at rpm for - h until the optical density (od ) reached an absorbance of , ng/μl. then, recombinant protein expression was induced with . mm iptg (isopropyl-d-thiogalactopyranoside) and the bacterial cultures were incubated for h, °c with shaking at rpm. next, cell cultures were harvested by centrifugation at ×g for min. the pellets were homogenized with lysis buffer [ . % triton x- , mm tris-cl and . m nacl (ph: . )] and were freeze-thawed times. the homogenates were incubated on a rotator at room temperature for min and then were centrifuged at . ×g for min. the supernatants were incubated with ml ni-nta superflow beads (qiagen, usa) for min with shaking at rpm. at the end of incubation, the suspension was centrifuged at rpm for min and the supernatant was discarded. then, the ni-nta beads were washed with mm tris-cl and . m nacl and mm imidazole (ph: . ) for min with shaking at rpm. thereafter, the suspension was centrifuged at rpm for min and the supernatant was discarded. next, ni-nta beads were incubated with mm tris-cl and . m nacl and . m imidazole (ph: . ) for min with shaking at rpm. finally, the suspension was centrifuged at rpm for min and supernatants were analysed with western blot as described below to determine the expression level of recombinant proteins. the most abundantly expressed recombinant proteins were selected for vaccine development. the protective immune response against toxoplasmosis is conferred by mainly by cellular immune response and through humoral immune response [ , ] . in addition, the expression of recombinant proteins of t. gondii will be performed in e. coli. t. gondii is a eukaryotic obligate intracellular parasite. protein post-translational modifications are common events in most eukaryotes, such as glycosylation. glycosylation of peptide effects protein immunogenicity and major histocompatibility complex (mhc) binding [ ] . thus, apart from the determination of the expression levels, proteins will analysed by bioinformatics tools to determine the presence of antigenic epitopes as well as glycosylation sites. at the end of the bioinformatics analyses, we aimed to select the proteins that have cellular and humoral immune response inducing epitopes with the least glycosylation sites. since t. gondii is an intracellular parasite, immunemediated protection against toxoplasmosis is mainly conferred by interaction between cd + t cells and mhc class i and cd + t cells and mhc class ii [ ] [ ] [ ] [ ] . antigenic sites recognized by cd + t cells and cd + are peptides containing to and amino acids associated with the mhc class i (mhc-i) and mhc-ii molecules, respectively [ ] [ ] [ ] . predicted epitopes of cd + t cells table the plasmids encoding selected t. gondii antigenic proteins toxodb a name plasmid name toxodb a name plasmid name tgme _ _ pa tgme _ _ pb tgme _ _ pb tgme _ _ pc tgme _ _ pc tgme _ _ pd tgme _ _ pd tgme _ _ pe tgme _ _ pe tgme _ _ pf tgme _ _ pf tgme _ _ pg tgme _ _ pg tgme _ _ ph tgme _ _ ph tgme _ _ pa tgme _ _ pa tgme _ _ pb tgme _ _ pb tgme _ _ pc tgme _ _ pc tgme _ _ pd tgme _ _ pd tgme _ _ pe tgme _ _ pe tgme _ _ pf tgme _ _ pf tgme _ _ pg tgme _ _ pg tgme _ _ ph [ ] [ ] [ ] [ ] [ ] [ ] . the potential linear b-cell epitopes of antigenic proteins were also analysed by support vector machine (svm) which has been utilized by combining the tri-peptide similarity and propensity scores (svmtrip; http://sysbio.unl.edu/ svmtrip/prediction.php) [ ] . in many eukaryotic pathogens, proteins may have nand o-linked glycosylation sites through post translational modification which may affect their interaction with their host organisms. protein glycosylation is common in t. gondii as it is in other eukaryotes [ ] . in this study, e. coli was used as the expression system and we aimed to select the vaccine candidate proteins among the recombinant proteins with least glycosylation sites in predicted antigenic epitopes. the potential n-glycosylation and oglycosylation sites of antigenic proteins were analysed using netnglyc . server (http://www.cbs.dtu.dk/services/netnglyc/) netoglyc . server (http://www.cbs. dtu.dk/services/netoglyc/) [ ] . according to protein expression levels and bioinformatics results, proteins were [ph (tgme _ _ ), pa (tgme _ _ ), pe (tgme _ _ ), pd (tgme _ _ ), pe (tgme _ _ ), ph (tgme _ _ )] were selected as vaccine candidate. thereafter, e. coli bl star (de ) plyss cells containing the ph , pa , pe , pd , pe , and ph stocks were inoculated into individual ml lb broth medium supplemented with ampicillin ( μg/ml) and incubated overnight at °c with rpm shaking. next day, the overnight cultures were inoculated into the bioreactor (bioflo , new brunswick, usa) containing . l enrichment medium supplemented with ampicillin ( μg/ml). the dissolved oxygen and ph levels were maintained at - and . ± . with vigorous mixing ( rpm) at °c until od reached . . then, the cell cultures were induced at a final concentration of . mm iptg and incubated for h at °c. the cells were centrifuged at ×g for min and the pellet was resuspended with ml pre-chilled loading buffer ( mm tris-cl, . m nacl, ph . ) and homogenized with a blender for s (waring, usa). then, the homogenized cells were disrupted twice using a microfluidizer processor (microfluidics m- l pneumatic, usa) at a low temperature under internal pressure of , psi and centrifuged at , g for ½ h at °c. the homogenates were centrifuged at , ×g for min at °c. the clarified supernatants were filtered using . μm pore filter (corning, usa). the filtered samples were purified with akta fast protein liquid chromatography (fplc) system, controlled by uni-corn™ software (ge health, usa), using ml hitrap ni + chelating hp column (ge health, usa). approximately ml filtered supernatant was loaded to the hitrap ni + chelating hp column. after binding, the column was washed with buffers containing increasing concentrations of imidazole ( mm, mm, mm). the recombinant proteins (rh , ra , re , rd , re , and rh ) were eluted by raising the imidazole concentration to . m. purity and identity of the purified proteins were analysed by % (sodium dodecyl sulfatepolyacrylamide gel electrophoresis) sds-page and western blot analysis and concentrated with vivaspin (sartorius, germany). the proteins were further purified by fplc on a superdex / - gl [ - , molecular weight cut-off (mwco)] column (ge health, usa) to remove excess endotoxin. the subsequent protein fractions were pooled, concentrated and quantitated by bradford method (pierce, usa). to observe the expression levels, purity and immunoreactivity, proteins were separated by % sodium dodecyl sulfate-polyacrylamide gel (sds-page). the separated proteins were transferred to polyvinylidene difluoride (pvdf) transfer membrane (immobilon-p, millipore, ma), blocked by . % non-fat dry milk for h at room temperature. the membranes were probed with a / dilution of monoclonal anti-polyhistidine antibody (sigma-aldrich, usa) for . h at room temperature. then the membranes were probed with a / dilution of alkaline phosphatase-conjugated goat anti-mouse igg (h + l) antibody (sigma-aldrich, usa) for h at room temperature. the blot was developed with diethanolamine buffer ( % diethanolamine, m hcl ph: . , the amount of endotoxin in the purified vaccine candidate recombinant proteins were determined with limulus ameobocyte lysate (lal) gel-clot test using pyrotell single test vials according to the manufacturer's protocol (cape cod inc., usa). the test sensitivity was . endotoxin units (eu)/ml. e. coli o :h control standard endotoxin (cape cod inc., usa) was used for positive control and lal reagent water was used for negative control. the concentration of endotoxin-depleted, purified recombinant protein samples were calculated with bradford method (pierce, usa) and stored at − °c until use. vaccination and t. gondii challenge infection - weeks old female swiss webster outbred mice were randomly divided into four groups, each group consisting of eleven mice (n: ). mice were vaccinated intraperitoneally (i.p.) twice at weeks intervals. vaccines and control groups as shown in table . the first group was immunized with hexavalent ( antigens) recombinant proteins adjuvanted with montanide isa v (seppic, france) prepared according to the manufacturer's protocols. montanide isa v was used as adjuvant due to its efficiency in inducing both humoral and cellular immune response. three groups were considered as control; one control group was administered only with hexavalent recombinant proteins without montanide isa v adjuvant. the adjuvant control group was inoculated with only montanide isa v [ μl montanide isa v adjuvant + μl phosphate buffered saline (pbs)] and the last control group was inoculated only pbs ( μl pbs). tail bleeds were performed weeks after each vaccination. nine weeks after first vaccination, eight mice from all four groups were challenged intraperitoneally with × tachyzoites of t. gondii local strain called ankara [ ] . thereafter, the infected mice were observed for the symptoms of toxoplasmosis such as loss of fur brightness and appetite and survival times were recorded on a daily basis. detection of total igg and igg subclass antibody response using rec-elisa determination of t. gondii specific total igg, igg and igg a antibodies in vaccinated mice were performed by recombinant enzyme-linked immunosorbent assay (rec-elisa) as described [ , ] . in brief, each well of microplates (nunc, usa) were coated with μl of recombinant proteins solution (containing μg/ml of each rh , ra , re , rd , re and rh in × pbs) and incubated overnight at °c. next day, plates were washed times with μl pbs-t ( . % tween in pbs) and then blocked with % nonfat dry milk containing . % pbs-t for h at room temperature. mice sera were diluted to / with blocking buffer supplemented with e. coli lysate at a final concentration of mg/ml protein to block anti-e. coli antibodies and incubated for min at °c. then, the mouse sera were added to the wells in duplicate and incubated for h at °c with gentle shaking. after three washes with μl pbs-t, the plates were incubated with μl of anti-mouse igg to evaluated cytokine production, three mice from per group were euthanized weeks after the prime vaccination and their spleens were removed. single-cell suspensions of splenocytes were prepared as previously described [ , ] . aliquots of × viable splenocytes in growth medium [ × rpmi supplemented with % fcs (nbcs, hyclone, thermo fisher scientific, usa), mm l-glutamine (gibco, invitrogen,usa) penicillin ( u/ml) and streptomycin ( μg/ml) (sigma-aldrich, usa), . mm non-essential amino acid and thereafter, the cells were permeabilized and labelled with pe conjugated rat anti-mouse ifn-γ (bd biosciences, usa) or pe conjugated rat anti-mouse il- antibodies (bd biosciences, usa) according to the manufacturer's recommendations. antibodies were diluted in perm/wash solution (bd biosciences, usa) for intracellular staining. all antibodies were used at a final concentration of . μg/ cells. t cell populations of cells, gated with cd + positive expression, were analysed to quantify: the percentage of rh , ra , re , rd , re and rh proteins specific cd + t lymphocytes secreting ifn-γ and cd + t lymphocytes that secreted il- and ifn-γ using facs diva software (bd biosciences, usa). all data were obtained on a bd facsaria flow cytometer (facsaria; bd biosciences, usa). data obtained during the experiments were processed using microsoft excel software and prism . program (graphpad, san diego, ca). a two-tailed unpaired t-test with % confidence interval was used to determine the significance between the vaccination groups. kaplan-meier survival curves were constructed to illustrate protection from lethal toxoplasmosis. humoral and cellular immune responses and survival time were expressed as mean ± standard deviation (s.d.). the expression of recombinant proteins were induced with . mm iptg when growing cells reached an absorbance of . ng/μl at od nm. the cell cultures were harvested h after induction, homogenized with lysis buffer and purified by ni-nta beads. then, the recombinant protein expression levels were assessed by western blotting and rh (tgme _ _ ), ra (tgme _ _ ), re (tgme _ _ ), rd (tgme _ _ ), re (tgme _ _ ), and rh (tgme _ _ ) had detectable bands at . kda, . kda, . kda, . kda, . kda, and . kda, respectively. bioinformatic analyses to predict mhc-i, mhc-ii, and b cell epitopes of the recombinant proteins using immune epitope database and analysis resource (iedb) and svmtrip showed that there were , , , , , and predicted mhc-i epitopes (fig. a) ( table ) , , , , , , and predicted mhc-ii epitopes (fig. b) (table ) and , , , , , and predicted b-cell epitopes (fig. c) (table ) in re , rd , rh , re , ra and rh , respectively. the n-glycosylation and o-glycosylation sites of antigenic proteins were predicted using netnglyc . server and netoglyc . server. moreover, n and oglycosylation sites on the predicted mhc-i, mhc-ii, and b-cell epitopes were also analysed ( table ) . the results showed that rh , re , rd , re , ra , and rh have ( . %), ( . %), ( . %), ( . %), ( . %), and ( . %) o-glycosylation sites, respectively (fig. a) ( table ) . regarding n-glycosylation, rd , ra , re and rh proteins have ( . %), ( . %), ( . %), ( . %) sites respectively, while rh and re proteins didn't have n-glycosylation site (fig. b) (table ) . next, we next focused on the potential glycosylation sites inside predicted mhc-i, mhc-ii, and b cell epitopes of re , rd , rh , re , ra , and rh . the results showed that in the mhc-i epitopes, re and rh have only one n-glycosylation site, re , re , rh , and rd have , , and o-glycosylation sites, respectively (table ). in mhc-ii epitopes, re and rh have and one n-glycosylation site, re , rd , rh , re , and rh have , , , , and o-glycosylation sites, respectively (table ) . on the other hand, b-cell epitopes of re , rd , rh , re , and ra have , , , and oglycosylation sites but did not contain any n-linked glycosylation site (table ) . based on the protein expression levels and mhc-i, mhc-ii, and b cell epitope prediction results, rh , ra , re , rd , re , and rh were grown in big batches of lb using a bioreactor, purified in on a hitrap ni + chelating column and then polished on a gel filtration column to remove excess endotoxin. the purity of rh , ra , re , rd , re and rh were assessed by sds-page and western blotting as shown in fig. a and b. the purified rh , ra , re , rd , re and rh had apparent molecular weight of approximately . kda, . kda, . kda, . kda, . kda, and . kda, respectively. some of the proteins gave several bands possibly due to multimerization of recombinant protein, degradation or stalling of protein synthesis in e. coli. to determine total igg, igg and igg a antibodies against rh , ra , re , rd , re , and rh proteins in adjuvanted and control group mice serum samples, rec-elisa was performed. according to the results, total igg response detected at day was significantly higher in sera of mice administered with hexavalent recombinant protein mixture (+) montanide isa v vaccine and the control group administered with only hexavalent recombinant protein mixture compared to the pre-vaccination sera (p < . , ***). in control groups administered with pbs or montanide isa v didn't induce a significant total igg response. significantly high levels of total igg immune response was detected in the hexavalent recombinant protein mixture (+) montanide isa v when compared with the mice administered with hexavalent recombinant protein mixture (p = . , **) (fig. ) . to assess the polarization of igg /igg a which is a preliminary marker of whether vaccine induced the th or th immune response, the igg and igg a response was analysed by rec-elisa. the polarization of igg and igg a response is shown in fig. . hexavalent recombinant protein mixture (+) montanide isa v vaccine induced significantly high levels of igg and igg a at day compared to the controls groups (p < . , **). overall, in mice administered with hexavalent recombinant protein mixture (+) montanide isa v, igg a and igg levels showed a strong and balanced immune response. in control groups, pbs or montanide isa v didn't induce a significant igg or igg a response. (fig. ) . single-cell suspensions of splenocytes obtained from mice administered with hexavalent recombinant protein mixture (+) montanide isa v and controls were stimulated with purified rh , ra , re , rd , re , and rh proteins. concentration of extracellular cytokine il- and ifn-γ were determined using elisa. according to the results, ifn-γ level was significantly higher in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to mice vaccinated with only montanide isa v (p = . ,***) or pbs (p = . ,***) (fig. a) . on the other side, il- level was also significantly high in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to control groups administered with pbs (p = . , *) or hexavalent recombinant protein mixture (p = . , *) (fig. b) . flow cytometry analysis was used to determine the ratio of cd + t lymphocytes secreting ifn-γ and cd + secreting ifn-γ and il- in vaccinated and control groups. protection against intracellular parasite like t. gondii is primarily achieved by cd + t lymphocytes secreting ifn-γ. for this reason, cd + t cell response is important for an ideal vaccine against toxoplasmosis [ , , ] . t cell population has been gated with cd + staining. in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v vaccine, the ratio of cd + t lymphocytes secreting ifn-γ increased , . and . times compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. a) . besides, the ratio of cd + t lymphocytes secreting ifn-γ increased . and . times in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. b) . on the other hand, the ratio of cd + t lymphocytes secreting il- in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v increased . , . , and . times compared to control groups vaccinated with pbs, montanide isa v, and only hexavalent recombinant protein mixture (fig. c) . cd + t cells from all experimental groups proliferated to comparable ratios in response to cona (data not shown). protection against lethal toxoplasmosis in mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v was determined using t. gondii ankara strain tachyzoites. eight from each the results are summarized in fig. . recently, our study group has discovered some t. gondii proteins that can be used as vaccine candidate against toxoplasmosis using in silico and immunoscreening approaches based on protein microarrays [ , ] . we selected the antigens from this screening approach because antigens selected without a screening approach ended with disappointing clinical results such as the malaria vaccine rts,s which is a hybrid protein particle designed in s. the major surface glycoprotein (msg) containing hybrid protein was formulated in a multi-component adjuvant (as ) and showed % protection in east african children in [ ] . for these reasons, development of a multivalant recombinant protein vaccine using some of these discovered proteins was the aim of this study. for this purpose, we used the data from protein microarray screening and prioritized proteins based on their immunogenicity. to further analyse these proteins for their availability for vaccine development against toxoplasmosis, we conducted small scale protein expression experiments in conjunction with bioinformatics analyses. according to protein expression levels proteins were suitable to be used in the vaccine development. among them, rh (tgme _ _ ) is a dnak family protein, ra (tgme _ _ ) is rop , re (tgme _ _ ) is a sag-related sequence srs a, rd (tgme _ _ ) is an fig. extracellular a ifn-γ and b il- levels elicited by hexavalent recombinant protein mixture (+) montanide isa v and control groups. the red bars represent ifn-γ response and the green bars, il- response. each bar represents the mean ± sd value of ifn-γ and il- responses of mice from each group. each bar represents the mean ± sd value of ifn-γ and il- responses of mice from each group. single cell suspensions were stimulated with rh , ra , re , rd , re , and rh recombinant proteins with a final concentration of μg/ml. in figure, *** represent p ≤ . and * represent p ≤ . ubiquitin carboxyl-terminal hydrolase, re (tgme _ _ ) is a hypothetical protein, and rh (tgme _ _ ) is a plectin. the protection against t. gondii infection is dependent on cd + t-cytotoxic lymphocytes which play a significant role in cell mediated protection as well as b cells which is important in humoral immune response [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . for this reason, mhc-i, mhc-ii, and b-cell epitopes of the vaccine candidate proteins were predicted by bioinformatics. svmtrip online service was used to analyse the b-cell epitopes of re , rd , rh , re , ra , and rh proteins. as shown in fig. c , the presence of b-cell epitopes on the vaccine candidate proteins suggests that they have a strong potential to act as a bcell antigen. we also used the online service iedb to analyse mhc-i and mhc-ii epitopes of re , rd , rh , re , ra , and rh proteins and found mhc-i and mhc-ii epitopes on the vaccine candidate proteins ( fig. a and b) . protein glycosylation has great importance in terms of protein stability, three-dimensional structure, surface expression, activity, and antigenicity [ ] . previously, it was shown that t. gondii contains n-linked glycosylated proteins and o-linked glycosylated proteins by lectin-probed western blot analysis. moreover, it is reported that n and o-linked glycosylated proteins are found throughout the secretory pathway of the t. gondii and n-linked glycosylation of proteins is essential for the survival of parasite [ ] . for this reason, nand o-linked glycosylation sites of antigenic proteins, which may be candidates for vaccination, was predicted by bioinformatics. the results suggest that the vaccine candidate proteins contain n-linked glycosylation sites except for the rh and re proteins (fig. b) . o-linked glycosylation sites are found on re , rd , rh , re , ra , and rh proteins (fig. a) . at this stage, we further examined the n-and olinked glycosylation sites in b cell, mhc-i, and mhc-ii epitopes of the vaccine candidate antigens by bioinformatics. the results demonstrate that b-cell epitopes of vaccine candidate proteins were not containing n-linked glycosylation sites. b-cell epitopes of re , rd , rh , re , ra were o-glycosylated ( table ). the n-linked glycosylation site in the mhc-i epitopes were only found in the re and rh proteins and the o-linked glycosylation is detected in the re , re , rh , and rd proteins ( table ). the n-linked glycosylation site in the mhc-ii epitopes were similarly found in the re and rh proteins and the o-linked glycosylation is detected in the re , rd , rh , re , and rh proteins (table ). these results show that the vaccine candidate proteins and their epitopes are glycosylated at various ratios and their antigenicity is high. thereafter, we developed a hexavalent recombinant protein protein vaccine adjuvanted with montanide isa v which has shown to induce strong cellular and humoral immune response. after vaccination of swiss webster outbred mice, strong total igg, igg and igg a responses were detected in mice administered with hexavalent recombinant protein mixture (+) montanide isa v compared to control groups vaccinated with only montanide isa v or pbs (p < . ) indicating strong and balanced th and th responses (figs. and ) . the production of extracellular ifn-γ was significantly higher in mice vaccinated with the hexavalent recombinant protein mixture (+) montanide isa v compared to mice vaccinated with only montanide isa v or pbs (p < . ) (fig. a) . flow cytometry results were also in compatible with extracellular elisa in which hexavalent recombinant protein mixture (+) montanide isa v showed increment in ratio of cd + and cd + t lymphocytes secreting ifn-γ ( fig. a and b) . cd + t lymphocytes secreting il- were also increased according to flow cytometry results as well as there was an increase in extracellular il- secretion according to elisa (figs. b and c) . flow cytometry results, specifically cd + t lymphocytes secreting il- cell ratio is bigger than cd + t lymphocytes secreting ifn-γ which contradict with elisa results in which ifn-γ was higher than il- secretion. this discrepancy can interpreted as ifn-γ levels detected by elisa can be related to cd + t lymphocytes secreting and macrophages other than cd + t lymphocytes. on the other side, the main protective cells against toxoplasmosis are conferred by cd + t lymphocytes secreting ifn-γ which has increased with recombinant protein mixture (+) montanide isa v compared to controls. the protective efficacy of hexavalent recombinant protein mixture (+) montanide isa v against lethal toxoplasmosis was evaluated by infecting mice intraperitoneally with t. gondii ankara strain tachyzoites. t. gondii ankara strain is africa genotype and causes death in mice in approximately - days [ ] . challenging study showed that survival was prolonged from to days which was observed in control group mice administered with only montanide isa vand pbs to more than days in two mice vaccinated with hexavalent recombinant protein mixture (+) montanide isa v (fig. ) . in this study, montanide isa v was selected as an adjuvant due to its efficiency in inducing both humoral and cellular immune responses. montanide isa v was used as adjuvant in previous studies against bovine herpesvirus , boophilus microplus, foot and mouth disease virus (fmdv), and leishmania major [ ] [ ] [ ] [ ] . in the vaccine trial against fmdv, boophilus microplus, and leishmania major, montanide isa v induced significant levels of protective cytokine production and/ or antibody response. during the vaccine trial with recombinant glycoprotein d of bovine herpesvirus , a mixed th /th response was elicited [ ] . in this study, hexavalent recombinant protein mixture (+) montanide isa v showed strong and balanced th and th responses. overall, the th part of the immune response elicited by hexavalent recombinant protein mixture (+) montanide isa v induced significant levels of cd + and cd + t lymphocytes secreting ifn-γ and conferred significant protection in swiss outbred mice challenged with lethal dose of t. gondii ankara strain tachyzoites. in literature, multivalent recombinant protein vaccines have been developed against toxoplasmosis. in these studies, surface related antigens rsag , rsag , rsag , rsrs , rp , rsrs , and rsrs ; dense granule proteins rgra , rgra , rgra , rgra , rgra , and rgra ; rhoptry proteins rrop , rrop , and rrop as well as rtgpi- , rmag and rbag have been used [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in all of the recombinant protein or dna vaccine studies against toxoplasmosis, murine models are being used to determine the immunity and protection conferred by vaccinations. michima et al., vaccinated mice with a mixture of rsag , rsag , rsag , rsrs , and rp proteins adjuvanted with freund and achieved % survival up to days after i.p. challenging with t. gondii beverley strain bradyzoites [ ] . golkar et al., vaccinated mice with a mixture of rgra and rgra proteins adjuvanted with monophosphoryl lipid a and achieved . % decrease in brain cyst formation after i.p. challenging with t. gondii pru strain cysts [ ] . in one study, rrop , rgra , rgra proteins and cholera toxin were administered through intranasal route to mice and oral challenge with veg cysts resulted in . % decrease in brain cysts compared to controls [ ] . in a study that used antigenic epitopes of sag , gra , and mag proteins adjuvanted with freund decreased the brain cysts formation by % [ ] . dziadek et al., , evaluated rrop , rrop , rgra , and rsag in three vaccine formulations adjuvanted with incomplete freund administered subcutaneously to mice and challenge with t. gondii dx cysts results with to % decrease in parasite burden [ ] . in another study, a synthetic peptide, generated using b-cell and two t-cell epitopes derived from sag , gra , and gra antigens was adjuvanted with freund and challenge with gjs tachyzoites increased survival time [ ] . in other study, mice were vaccinated with the mixture of rrop and rsag adjuvanted with freund and challenged with the t. gondii rh strain. the results showed that t. gondiispecific igg antibodies levels and lymphocyte proliferative responses are increased in vaccine group and conferred more efficient protection compared to the control groups [ ] . sun et al., vaccinated mice with a mixture of rbag , rsrs , and rsrs proteins adjuvanted with freund or recombinant mindin. the results showed that vaccine using mindin as an adjuvant efficiently stimulated humoral and cellular responses, including antigen-specific igg and igg a, as well as lymphocyte proliferation. also the improved protection against t. gondii infection was observed in the mindin adjuvanted vaccine group compared with the other controls [ ] . in another study, t-and b-cells epitopes of ama , ron , and ron proteins was used to develop multivalent peptide vaccine formulations. the iga levels were increased in the mice immunized with single rron , while the igg levels were higher in the mice immunized with rama (+) rron. significant level of ifn-γ was detected in mice immunized with rama (+) rron . infection with t. gondii is naturally occurs through the oral route by water or food contaminated with tissue cysts (containing bradyzoites) or oocysts (containing sporozoites). interestingly, in this study, the mice were challenged orally with × tachyzoites of t. gondii rh strain. it was reported that the control mice died within days. the mice immunized with a + r achieved % survival rates. the mice immunized with ama , a + r + r , and ron achieved , , and % survival rates, respectively [ ] . another vaccine trial using t-and b-cells epitopes of sag , gra , gra , and rop proteins resulted with higher levels of igg and igg a subclass titters, significant production of ifn-γ, percentage of t lymphocyte subsets and longer survival times against intraperitoneal challenge with t. gondii rh strain tachyzoites [ ] . rgra and rbag were used in a multivalent recombinant protein vaccine adjuvanted with alum. significant increase in igg, igg a subclass titters as well as significant increment in ratio of ifn-γ secreting cd + and cd + t lymphocyte subsets were achieved. mice were challenged orally with - t. gondii pru strain tissue cysts and the amount of tissue cysts in vaccinated group decreased . % compared to control groups. in sum, multistage and multivalent rbag and rgra vaccine increased immune response but induced partial protection against toxoplasmosis [ ] . picchio et al., vaccinated mice with rtgpi- , rrop , and rgra proteins ajuvanted with alum intradermally or with cpg-odn intranasally and mice were orally challenged with the t. gondii me tissue cyst. p + r + g vaccine formulations induced significant decreases in the number of cysts per brain compared to the control group. according to the levels of igg and igg a subclasses p + r + g vaccine groups showed a mixed th /th immunity [ ] . overall, in the present study a hexavalent recombinant protein vaccine adjuvanted with montanide isa was first time developed and administered to mice to protect against lethal toxoplamosis. moreover, the immunogenic and protective efficiency of rrop , dnak family protein, srs a, ubiquitin carboxyl-terminal hydrolase, and plectin was first time tested in an animal model. in addition, montanide isa v was first time used as an adjuvant in mice model against toxoplasmosis. apart from these, multiplexing recombinant proteins induced strong and balanced th and th immune 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toxoplasma gondii in balb/c mice protective immunity induced by peptides of ama , ron and ron containing t-and b-cell epitopes via an intranasal route against toxoplasmosis in mice toxoplasma gondii: vaccination with a dna vaccine encoding t-and b-cell epitopes of sag , gra , gra and rop elicits protection against acute toxoplasmosis in mice vaccine potential of antigen cocktails composed of recombinant toxoplasma gondii tgpi- , rop and gra proteins against chronic toxoplasmosis in c h mice publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions eaŞ, hc, ayg, md conceived the study and participated in its design. eaŞ, hc, sgİ, add, mkd, md participated in in vitro and in vivo studies. eaŞ, hc, rd, ayg, md helped in discussion of results. eaŞ and md performed the statistical analyses. eaŞ, hc, ayg, md interpreted the results and drafted the manuscript. all authors read and approved the final manuscript. this study was supported by the scientific and technological research council of turkey (tubitak) grant s to ayg. the funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. the dataset analyzed during the current study is available from the corresponding author on reasonable request. the animal study was performed under the instructions and approval of the institutional animal care and use committee (iacuc) of ege university for animal ethical norms (permit number: - ). not applicable. the authors declare that they have no competing interests. key: cord- -cu atqvr authors: morikane, keita; suzuki, shoko; yoshioka, jun; yakuwa, jun; nakane, masaki; nemoto, kenji title: clinical and microbiological effect of pulsed xenon ultraviolet disinfection to reduce multidrug-resistant organisms in the intensive care unit in a japanese hospital: a before-after study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: cu atqvr background: no-touch environmental disinfection using ultraviolet devices has been highlighted in the past several years to control the transmission of multidrug-resistant organisms (mdros). however, its effectiveness in non-us healthcare settings is yet to be examined. this study aimed to evaluate the effectiveness of disinfection by portable pulsed xenon ultraviolet (px-uv) devices in controlling transmission of mdros in a non-us healthcare setting. methods: all patients admitted in the intensive care unit in a -bed tertiary referral hospital in japan from august to february were enrolled. during the study period, px-uv disinfection was added to manual terminal cleaning after every patient transfer/discharge. for microbiological evaluation, surfaces were selected for sampling by contact plates before/after manual cleaning and after px-uv. after overnight incubation, colonies on the plates were counted. results: the incidence of newly acquired methicillin-resistant staphylococcus aureus (mrsa) declined significantly ( . to . per , patient days, incidence rate ratio . , p = . ), as well as that of newly acquired drug-resistant acinetobacter ( . to . , . , p < . ). the percent reduction of the microbiological burden by manual cleaning was %, but a further % reduction was achieved by px-uv. conclusions: px-uv is effective in further reducing the microbial burden and controlling mdros in a non-us healthcare setting. infection remains a significant cause of morbidity and mortality in the healthcare setting, despite international initiatives in infection control and prevention. pathogens such as clostridioides difficile, vancomycin-resistant enterococci (vre) and multidrug-resistant acinetobacter are especially difficult to deal with. these pathogens can easily reside in the healthcare environment [ ] and are difficult to remove or eradicate by conventional environmental cleaning, typically by manual wiping with disinfectants and cloths [ ] . ultraviolet light disinfection has recently been used as an adjunct to terminal cleaning, and many studies have shown its effectiveness in reducing environmental contamination [ ] [ ] [ ] and healthcare-associated transmission of these pathogens [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, most of these studies [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] have been performed in the united states, where the majority of rooms are private. to our knowledge, there are no published studies investigating the clinical effectiveness of this novel technology in healthcare settings outside the united states. in yamagata university hospital, there has been sporadic identification of two-drug resistant acinetobacter baumannii ( dra), which is resistant to two classes of antimicrobial: carbapenem and quinolone. in order to halt the transmission of this pathogen, interventions such as enhanced terminal cleaning by hypochlorous acid and strict contact precaution were implemented. this was partially successful; however, it did not lead to the eradication of transmission. therefore, we decided to implement further intervention using pulsed xenon ultraviolet light (px-uv). the objective of this study was to evaluate the effect of px-uv on the transmission of healthcare-associated pathogens and on the environmental contamination within a japanese healthcare setting. this study was conducted at yamagata university hospital, a -bed academic tertiary referral hospital in yamagata, japan. the yamagata university school of medicine institutional review board approved this study with a waiver of informed consent. a px-uv device (xenex disinfection services, san antonio, tx, usa) was introduced in the study hospital in november . it was decided that this device would be used in the intensive care unit (icu), since most of the newly detected dra infections occurred in the icu patient population. after education to clinical engineering staff, the device was deployed in the icu environment. the icu has six rooms and beds. one room has independent walls and positive/negative air pressure. the other rooms are in an open space separated by curtains. portable drapes, which protect uv and visible light from leaking outside the designated room, were used when operating px-uv in the open space rooms. the device came into full operation by january . terminal cleaning after every patient discharge or transfer to the ward was performed using cloths soaked with diluted sodium hypochlorite solution, which were then applied to every possible touchable surface in the room as well as portable and non-portable equipment. this type of cleaning was already implemented at the beginning of the baseline period and continued throughout the study period. next, the room was covered by portable drapes, which were hung inside of the curtains of the room. after hanging the portable drapes, two -min disinfection cycles using px-uv were run following the manufacturer's recommendation. the machine was operated by clinical engineering staff, and sequentially placed on two opposite corners of the room. for each operation, the operator's name, date and time, and duration (in seconds) of uv irradiation was recorded on the device's cloud-based reporting system. every patient admitted in the icu was screened by nasal sampling for methicillin-resistant staphylococcus aureus (mrsa) and dra colonization on the day of the admission and every tuesday and thursday, until transfer or discharge from the unit. new acquisition of mrsa or dra was defined as the identification of these pathogens from the screening specimen or clinical culture specimen on or after the third day of admission to the icu, with at least one preceding negative screening result. microbiological contamination of the environment was evaluated by using replicate organism detection and counting (rodac) plates (p tryptic soy agar with lecithin and tween , hardy diagnostics, santa maria, ca, usa). ten frequently touched surfaces were selected for sampling: the bed rail (inside and outside near patient's head, near patient's feet), touch panel of cardiopulmonary monitor, ventilator control panel, intravenous fluid pump control panel, glove hanger, workstation keyboard, workstation cart handle and water basin. samples were collected by gently stamping rodac plates onto the site for s. the roll plate method was used for nonflat surfaces such as bedrails. sampling occasions included ( ) immediately after patient discharge, before terminal cleaning ( ) immediately after terminal cleaning ( ) after px-uv disinfection. plates were then cultured at degrees celsius for h. colony-forming units (cfu) were counted and reported as cfu per cm [ ] . poisson regression model analysis was used to estimate the incidence of mrsa and dra infection, which were expressed as the number of acquisitions per , patient days. for the statistical analysis, we assumed that the pre-intervention period was the baseline, which means the event (acquisition of mrsa or dra) occurs in a probability calculated by using the data in the preintervention period. to analyze the cfu data a shapiro-wilk test was conducted to determine the skewness of the cfu data. the results suggested the cfu data were skewed (p < . ), indicating the need for a nonparametric test. the dunn's test of multiple comparisons using a rank sum was used to assess the difference between environmental sampling timepoints. this test is a nonparametric multiple comparison test, that uses a wilcoxon rank sum that allows for more than a twogroup comparison. the dunn's test used a bonferroni correction to adjust for multiple comparisons. the statistical analyses were conducted using the poisson and dunn's test package in stata . (college station, tx, usa). the baseline incidence of mrsa and dra in the icu were . and . per , patient days, respectively. the incidence dropped to . and . , resulting in a decrease of and %, respectively ( table ). the reductions in both mrsa and dra were statistically significant (p = . and p < . , respectively) ( table ) . notably, in the intervention period, new acquisition of dra in the icu was observed only in the first six months, with no new acquisition for a seven month period. this also led to the eradication of new acquisition of dra throughout the hospital in august . environmental sampling was performed after patient discharges. some of the samplings were not performed due to time constraints by waiting patients or unique features of certain rooms. in total, sites were sampled. the total colony count was: (before manual cleaning), (after manual cleaning, before px-uv) and (after px-uv). compared to pre-cleaning, a statistically significant decrease in colony count was observed after manual cleaning (p < . , wilcoxson rank sum test) (table ) . also, compared to post-manual cleaning, a statistically significant decrease in colony count was observed after px-uv disinfection (p = . ) ( table ) . there are a number of studies which have demonstrated the effect of ultraviolet light disinfection in reducing environmental microbiological contamination [ ] [ ] [ ] and healthcare-acquired infections by mrsa [ , ] , vre [ , ] and clostridioides difficile [ ] [ ] [ ] [ ] [ ] [ ] . however, all but one study [ ] was performed in the united states, where most of the patient rooms are private. furthermore, that study [ ] did not evaluate clinical outcomes. our study is the first hospital-acquired infection outcome study evaluating the clinical effectiveness of the px-uv device in a non-us healthcare setting with an open-style icu. in this setting, patient beds were separated by privacy curtains, not by walls. px-uv emits intense visible light and creates a sound while disinfecting. the light and sound can be seen and heard outside the privacy curtains. this was initially not well tolerated by healthcare staff, some of whom raised concerns about sensitivity to the light and sound. we also experienced faults from the pulse oximeter when px-uv was used adjacent to patient beds. to overcome these challenges, we provided goggles and earplugs to healthcare staff and ordered blackout curtains from the px-uv vendor that hung inside the privacy curtains during px-uv operation. the blackout curtains worked well and eliminated the problems stated above. adding px-uv disinfection to routine terminal cleaning after patient discharge increased the turn-around time of icu beds by approximately min. manual cleaning by sodium hypochlorite solution took about min, so the increase in time by adding px-uv was not significant and was well accepted by icu staff and physicians as a routine workflow. the effectiveness of px-uv is expected to be maximized when environmental contamination is a major factor in transmission of the specific pathogen. in this context, pathogens such as clostridioides difficile and multidrug-resistant acinetobacter are more likely to be controlled. the effectiveness of px-uv in controlling transmission of clostridioides difficile is well studied and demonstrated [ ] [ ] [ ] [ ] [ ] [ ] , but that of acinetobacter has not been well investigated. sporadic transmission of dra has been observed in our icu for the last five years. in japan, antimicrobial resistance of acinetobacter is not as serious. according to the japanese national microbiological surveillance report, , and % of acinetobacter isolates were reported to be susceptible to meropenem, amikacin and levofloxacin, respectively [ ] . therefore, most of the newly identified dra in our hospital seemed to be acquired by horizontal transmission. until mid- , this situation has not been well controlled, despite our efforts for elevated compliance to hand hygiene, strict contact precaution of patients with this pathogen, terminal cleaning using bleach, and in some occasions, restriction of new admission to the icu. however, by introducing the px-uv, transmission of dra in the icu halted. as of june , no new isolation of dra from patients in the icu has been observed for months (august to june ). generally, terminal cleaning by bleach is effective in preventing transmission of pathogens via environmental route, however, the result from the environmental sampling in this study, approximately % reduction in cfu by manual cleaning, indicated that cleaning by bleach is partially effective in reducing the bioburden in the patient care area. the halt of transmission of dra in our icu after introducing px-uv suggests its adjunctive effect. the effectiveness of ultraviolet light disinfection is maximized when performed after every patient discharge from the targeted ward. in the only multicenter, randomized controlled study conducted by anderson et al. [ ] , ultraviolet disinfection was performed in only isolation rooms occupied by known mdro or c. difficile colonization or infection. they did not observe any statistically significant effect on the incidence of mrsa and multidrug-resistant acinetobacter. the effect of the addition of px-uv to terminal cleaning by bleach was not observed in the study by anderson et al. [ ] . the differences between our study result and the results from andersen et al. could be due to differences in the healthcare setting (proportion of private rooms), in the way the device is implemented (disinfecting isolation rooms in multiple units vs every discharge/transfer on single unit), or in the intervention fidelity (proportion of eligible rooms that are disinfected). we operated px-uv after every patient discharge or transfer from the icu, regardless of their colonization status, and obtained a statistically significant reduction in the incidence of healthcare-associated transmission of mrsa and dra. this difference may be because undetected carriers of mrsa or acinetobacter could serve as a source of environmental contamination even under the operation of ultraviolet light disinfection in the targeted rooms. no new isolation of dra from patients in the non-icu ward has been observed for months (september to june ). we believe that px-uv successfully decreased or eradicated the environmental acinetobacter bioburden of not only the targeted ward (icu), but also of the other wards, perhaps indirectly through the decrease in bioburden throughout the hospital and also of colonized/infected patients with dra. anderson et al. experienced a similar decrease in the transmission of mrsa and vre throughout the hospital by using ultraviolet light disinfection in only isolation rooms after patients with targeted pathogen were discharged [ ] . our study has several limitations. first, the effect of px-uv was evaluated using historical controls when we were not using this technology. we have only one icu in our hospital, so we were not able to have a nonintervention arm in this study. second, not all mrsa or dra identified were necessarily acquired in the icu by horizontal transmission. however, we screened all patients on the day of their admission into the icu and if they were positive for mrsa and/or dra we regarded it as prior acquisition and excluded them from the acquisition in the icu. therefore, we believe that most of the identified mrsa and dra in this study were acquired by horizontal transmission. third, the microbiological effect of px-uv was evaluated by comparing the number of colonies from sampling the same frequently touched surface, but with different sites adjacent to each other. if there were significant differences in contamination between sites on the same surface, the result may not accurately reflect the effect of cleaning and px-uv. we, however, believe that by sampling over surfaces, we can minimize the effect by the heterogeneity of environmental contamination. fourth, we did not measure the environmental contamination of mrsa or dra by using selective media to sample the environment. for this reason, we are unable to demonstrate the direct relationship between environmental disinfection by px-uv and the decrease/eradication of new isolation of mrsa or dra. however, it is well known that contamination of the patient care area by the same pathogen that is isolated from the patient is common [ ] . we have previously observed contamination by dra on the keyboard in a patient care area where dra was isolated from the patient (data not shown). furthermore, mrsa, dra and other common environmental flora are susceptible to bleach and px-uv disinfection. therefore, it is plausible that environmental contamination by mrsa and dra was controlled by adding px-uv as an adjunct to manual cleaning, which led to the decrease/eradication of the transmission of these pathogens. the addition of px-uv to terminal cleaning successfully decreased the bioburden in the healthcare environment and led to the decrease of mrsa and drug-resistant acinetobacter transmission in the icu as well as in other wards of our hospital. the role of environmental cleaning in the control of hospitalacquired infection reduction of clostridium difficile and vancomycin-resistant enterococcus contamination of environmental surfaces after an intervention to improve cleaning methods evaluation of a pulsed-xenon ultraviolet room disinfection device for impact on hospital operations and microbial reduction is the pulsed xenon ultraviolet light no-touch disinfection system effective on methicillin-resistant staphylococcus aureus in the absence of manual cleaning? evaluation of a pulsed xenon ultraviolet light device for isolation room disinfection in a united kingdom hospital impact of a multi-hospital intervention utilizing screening, hand hygiene education and pulsed xenon ultraviolet (px-uv) on the rate of hospital associated methicillin resistant staphylococcus aureus infection implementation and impact of ultraviolet environmental disinfection in an acute care setting enhanced terminal room disinfection and acquisition and infection caused by multidrug-resistant organisms and clostridium difficile (the benefits of enhanced terminal room disinfection study): a cluster-randomised, multicentre, crossover study the effect of portable pulsed xenon ultraviolet light after terminal cleaning on hospital-associated clostridium difficile infection in a community hospital clostridium difficile infections before and during use of ultraviolet disinfection utilization and impact of a pulsed-xenon ultraviolet room disinfection system and multidisciplinary care team on clostridium difficile in a long-term acute care facility a trial of pulsed xenon ultraviolet disinfection to reduce clostridioides difficile infection ministry of health labour and welfare of japan website environmental contamination due to methicillin-resistant staphylococcus aureus; possible infection control implications publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank dr. deborah passey at xenex disinfection services for her generous assistance in the statistical analysis, and mr. tatsuro takezawa authors' contributions km collected and analysed all of the data in this manuscript. km, ss, jy (jun yoshioka), jy (jun yakuwa), mn and kn established the operational logistics. jy and jy operated and maintained the px-uv device. all authors read and approve the final manuscript. no funding was obtained for this research, except that the rodac plates were supplied from xenex disinfection services. the datasets generated and/or analysed during the current study are available in a github repository, https://github.com/keitamorikane/bmcid. the yamagata university school of medicine institutional review board approved this study (reference number: h - ) with a waiver of informed consent, based on this study's design which contains no direct intervention on patients and collects no data regarding patient identification. not applicable.competing interests km declares that he has received honorarium from terumo corporation, which sells the px-uv devices in japan. the other authors declare that they have no competing interest.author details key: cord- -nroflfmc authors: deng, wang; guang, tian-wen; yang, mei; li, jian-rong; jiang, de-peng; li, chang-yi; wang, dao-xin title: positive results for patients with covid- discharged form hospital in chongqing, china date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: nroflfmc background: since december , over , patients with coronavirus disease (covid- ) have been confirmed in china. with the increasing number of recovered patients, more attention should be paid to the follow-up of these patients. methods: in the study, patients with covid- discharged from hospital in chongqing, china from january , , to march , were evaluated by viral nucleic acid tests for severe acute respiratory syndrome coronavirus (sars-cov- ) to determine if they could be released from quarantine. among the patients, patients ( . %) had positive rt-pcr test results of sars-cov- . we aimed to analyze the demographics, clinical characteristics and treatment of patients. results: these positive patients were characterized by older age, chronic medical illness and mild conditions. ( . %) patients who were asymptomatic without abnormalities on chest radiographs were found in the positive with covid- . also, they showed positive results of stool or sputum specimens with negative results of nasal and pharyngeal swab specimens. the median duration of positive result of sars-cov- was varied from days to days in the patients discharged from hospital with no family member infection. conclusions: multi-site screening of sars-cov- including nasal and pharyngeal swabs, stool and sputum specimens could be considered to improve the diagnosis, treatment and infection control in patients with covid- . our findings provide the important information and clinical evidence for the improved management of patients recovered from covid- . increasing number of recovered patients discharged from hospital by regular follow-up and medical observation, some medical professionals were found to be positive for covid- after hospital discharge [ ] , and more attention should be paid to the follow-up of non-medical patients. severe acute respiratory syndrome coronavirus (sars-cov- ) can be detected in the specimens of upper respiratory tract, stool and lower respiratory tract [ ] . currently, a small number of positive results of sars-cov- in some recovered patients have been reported [ , , ] , but the management of these positive patients still remains an unsolved problem. in addition, there is a lack of clinical characteristics, the potential impact and significance of positive patients recovered from covid- , which makes it difficult to provide clinical evidence and experience for the management of patients with covid- in the recovery period. the objectives of this study were to investigate the clinical characteristics of patients discharged form hospital with positive results of sars-cov- . in the study, patients with covid- discharged from hospital were retrospectively analyzed in chongqing, china. among them, patients had positive results of sars-cov- by real-time reverse transcriptase polymerase chain reaction (rt-pcr) test, which provided the important information and clinical evidence for the improved management of patients recovered from covid- . five hundred seventy-six patients with covid- discharged from chongqing public health medical center, yongchuan affiliated hospital of chongqing medical university, and wanzhou general hospital, chongqing, china, from january , , to march , were evaluated by rt-pcr assay for sars-cov- according to the manufacturer's protocol (jiangsu perfectus biotechnology co., ltd., patch no. jc - n) to determine if they could be released from quarantine at home. recovered patients discharged from hospital or discontinuation of quarantine should meet the following criteria [ ] : ( ) normal temperature for more than days, ( ) significant improvement of respiratory symptoms, ( ) significant absorbtion of acute exudative lesions on chest radiograph and ( ) two consecutively negative results by rt-pcr assay of nasal and pharyngeal swabs with at least day interval. we extracted the clinical symptoms information, laboratory findings and radiologic abnormality form electronic medical records. patients were recommended quarantine at home after hospital discharge and returned to hospital for viral nucleic acid detection by rt-pcr. nasal and pharyngeal swab specimens were collected as previously described [ ] . stool and sputum specimens were also collected for viral nucleic acid detection by rt-pcr. two consecutively tests by rt-pcr of nasal and pharyngeal swab specimens, stool and sputum specimens were performed with at least day interval combined with chest radiograph during the quarantine period. continuous variables were presented as mean (sd) or median (iqr) and categorical variables as count (%).spss (version . ) was used for all analysis. the study was approved by the ethics committee of the second affiliated hospital of chongqing medical university and the need for informed consent was waived. the patients have not been reported in any other submission by anyone else. among the patients discharged from hospital, ( . %) patients had positive results of sars-cov- by rt-pcr tests. none of the patients were medical professionals. demographics, clinical characteristics and treatment of patients were shown in table . the median age of patients was . years, including ( %) female patients and ( %) male patients. ( . %) patients had chronic diseases, including chronic obstructive pulmonary disease, hypertension, diabetes, digestive system disease, cerebrovascular disease, chronic kidney disease and chronic hepatitis. ( . %) patients were asymptomatic. the most common symptoms were fever ( . %), cough ( %), sputum production ( . %), and sore throat ( . %), while the less common symptoms were headache ( . %), shortness of breath ( . %), fatigue ( . %) and diarrhea ( . %). on admission, leucocytes were above the normal range in ( . %) patients and below the normal range in ( %) patients. ( . %) patients had neutrophils above the normal range. lymphocytes and platelets were below the normal range in ( . %) patients and ( . %) patients respectively. nine patients had different degrees of liver function abnormality, with the increase in alanine aminotransferase or aspartate aminotransferase (table ) . most patients ( . %) had hypoproteinemia. fourteen patients had different degrees of renal function damage, with the elevation of blood urea nitrogen or serum creatinine. chest computed tomography (ct) scan of symptomatic patients showed local patchy shadowing ( . %), groundglass opacity ( . %), bilateral patchy shadowing ( . %) and interstitial abnormalities ( . %). normal ct imaging was showed in asymptomatic patients. the severity of disease was mild status in ( . %) patients. sixty-one patients were received treatment for days (iqr . - . ) and had two consecutively negative results of nasal and pharyngeal swab specimens by rt-pcr before discharge. they were required to continue the currently, the diagnosis of sars-cov- is dependent on viral nucleic acid detection. sixty-one patients with covid- had positive results by rt-pcr that fulfilled the criteria for hospital discharge during or more than the -day quarantine period. the underlying mechanisms of positive results of sars-cov- in recovered patients with covid- remain unclear. the major factors including the different sampling tissues [ ] , false negative of rt-pcr test [ ] , immunological status [ ] , viral load and intermittent shedding [ ] , and viral distribution [ ] are currently considered for the possible reasons of re-detectable positive. in the study, though both orf b gene and n gene of sars-cov- were detected using commercial kit, false-negative of test kit may partially account for the reason as previously reported [ ] . according to our results, the rate of falsenegative in virus detection was lower than a recent study reported by xiao et al [ ] . we found most recovered patients had hypoproteinemia, suggesting nutritional status probably involved for the positive results. in the study, most patients ( . %) with sputum production had positive results of sars-cov- after discharge. in addition, the median duration of positive result of sars-cov- was varied from days to days after hospital discharge, suggesting the intermittent shedding of virus might occur in recovered patients. fourteen patients had positive results of stool specimens for more than days with negative results of nasal and pharyngeal swabs, suggesting viral shedding from the digestive system lasting longer than that from the respiratory tract. therefore, stool or sputum specimen-testing might be benefit for the detection of sars-cov- in determining the diagnosis, treatment and termination of quarantine [ , ] . positive results of sars-cov- determined by stool and sputum also indicated that viral distribution in different sampling tissues and multiple shedding routes [ , ] . positive results occurred in most recovered patients with covid- might not be caused by virus recurrence or second virus infection. sampling tissues of multiple sites could be considered for recovered patients with covid- . in this study, patients with two consecutively negative results of nasal and pharyngeal swabs were shown positive results of stool or sputum specimens using rt-pcr test after discharge, indicating the necessity of stool and sputum specimens by rt-pcr adding to the criteria for discharge or discontinuation of quarantine. the positive rate of specimen detection is limited by the level of viral nucleic acid [ ] . the detection of virus rna was dependent on viral load, suggesting the potential sars-cov- replication in different sampling tissues [ ] . viral nucleic acid was detected by orf b gene and n gene of sars-cov- in the study. it was difficult for rt-pcr to distinguish the viral activity. despite the compliance with discharge criteria, viral rna remained positive in nasal and pharyngeal swabs, stool and sputum specimens in the recovered patients with covid- . viral residual and delay in clearance of viral rna might be considered as the potential factors [ ] . further studies should address isolation of sars-cov- in tissue specimens of recovered patients to identify the viral activity. also, the igm-igg combined assay in blood samples could be considered for the potentially rapid screening of sars-cov- infection in patients [ ] . for a small proportion of patients who had positive results of sars-cov- for more than days, virus carrier status probably existed. appropriate prolongation of isolation period should be also further proposed. the transmissibility of covid- is mainly dependent on the high level of sars-cov- shedding in the upper respiratory tract, even among presymptomatic patients [ ] . more than half of residents with positive results of sars-cov- were asymptomatic in a skilled nursing facility reported by arons et al. [ ] the viral load of asymptomatic patients with positive results of sars-cov- was similar to that in symptomatic patients, indicating the potential transmission [ ] . therefore, asymptomatic infection may play an important role in the spread of sars-cov- .in our study, the screening protocol of asymptomatic patients during quarantine period for viral nucleic acid detection was necessary for management and control transmission. surprisingly, non-infected family members were reported during home quarantine in the study. the management of asymptomatic patients with covid- requires further investigation. patients with symptoms, abnormalities on chest radiograph and abnormal laboratory results were received antiviral treatment in time. convalescent plasma therapy was performed in severe patients. the study has a limitation of small sample of patients with covid- discharged form hospital. also, the lack of serum-specific antibody levels testing in the recovered patients with covid- was due to the shortage of testing load and medical resource and clinical workload in the frontline during the outbreak. a larger cohort study is necessary to investigate the prognosis and transmission risk of recovered patients with covid- . the study revealed the clinical features of recovered patients with the recurrence of positive results of sars-cov- .multi-site screening including nasal and pharyngeal swabs, stool and sputum specimens could be considered to improve the diagnosis, treatment and infection control in patients with covid- . these findings provide the important information and clinical evidence for the management of recovered patients with covid- . criteria for hospital discharge or discontinuation of quarantine would be updated with the progress of clinical evidence and experience accumulation. a novel coronavirus from patients with pneumonia in china who characterizes covid- as a pandemic predictors of mortality for patients with covid- pneumonia caused by sars-cov- : a prospective cohort study clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study positive rt-pcr test results in patients recovered from covid- guidelines for laboratory diagnosis of coronavirus disease (covid- ) in korea persistence and clearance of viral rna in novel coronavirus disease rehabilitation patients positive result of sars-co- in sputum from a cured patient with covid- china national health commission. guidelines for the diagnosis and treatment of covid- pneumonia (trial version ). clinical features of patients infected with novel coronavirus in wuhan positive rectal swabs in young patients recovered from coronavirus disease (covid- ) false-negative results of real-time reverse-transcriptase polymerase chain reaction for severe acute respiratory syndrome coronavirus : role of deep-learning-based ct diagnosis and insights from two cases clinical and immunological features of severe and moderate coronavirus disease quantitative detection and viral load analysis of sars-cov- in infected patients evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov- infection stability issues of rt-pcr testing of sars-cov- for hospitalized patients clinically diagnosed with covid- false-negative of rt-pcr and prolonged nucleic acid conversion in covid- : rather than recurrence comparison of throat swabs and sputum specimens for viral nucleic acid detection in cases of novel coronavirus (sars-cov- )-infected pneumonia (covid- ) molecular and serological investigation of -ncov infected patients: implication of multiple shedding routes washington state -ncov case investigation team. first case of novel coronavirus in the united states enteric involvement of coronaviruses: is faecal-oral transmission of sars-cov- possible? virological assessment of hospitalized patients with covid- development and clinical application of a rapid igm-igg combined antibody test for sars-cov- infection diagnosis asymptomatic transmission, the achilles' heel of current strategies to control covid- presymptomatic sars-cov- infections and transmission in a skilled nursing facility sars-cov- viral load in upper respiratory specimens of infected patients publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank dr. judy zhao from crystal run healthcare, new york, usa for revising and reading the manuscript. all data analyzed during this study are included in this published article. the raw datasets used for the analysis are available from the corresponding author on reasonable request. the study was approved by the ethics committee of the second affiliated hospital of chongqing medical university and the need for informed consent was waived. the data used in this study was anonymised before its use. not applicable. the authors declare that they have no conflicts of interests. key: cord- - bakci authors: zhang, peng-jun; li, xiao-li; cao, bin; yang, shi-gui; liang, li-rong; gu, li; xu, zhen; hu, ke; zhang, hong-yuan; yan, xi-xin; huang, wen-bao; chen, wei; zhang, jing-xiao; li, lan-juan; wang, chen title: clinical features and risk factors for severe and critical pregnant women with pandemic h n influenza infection in china date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: bakci background: pandemic h n (ph n ) influenza posed an increased risk of severe illness among pregnant women. data on risk factors associated with death of pregnant women and neonates with ph n infections are limited outside of developed countries. methods: retrospective observational study in severe or critical pregnant women admitted to a hospital with ph n influenza from sep. , to dec. , . rrt-pcr testing was used to confirm infection. in-hospital mortality was the primary endpoint of this study. univariable logistic analysis and multivariate logistic regression analysis were used to investigate the potential factors on admission that might be associated with the maternal and neonatal mortality. results: pregnant women were included, were infected with ph n in the third trimester. had pneumonia, and died. a pao( )/fio( )≤ (odds ratio (or), . ; % confidence interval (ci), . - . ) and higher bmi (i.e. ≥ ) on admission (or, . ; % ci, . to . ) were independent risk factors for maternal death. of deliveries, neonates survived. premature delivery (or, . ; % ci, . - . ) was associated neonatal mortality. among patients who received mechanical ventilation, patients were treated with non-invasive ventilation (niv) and were successful with niv. the death rate was lower among patients who initially received niv than those who were initially intubated ( / , . % vs / , . %; p = . ). septic shock was an independent risk factor for failure of niv. conclusions: severe hypoxemia and higher bmi on admission were associated with adverse outcomes for pregnant women. preterm delivery was a risk factor for neonatal death among pregnant women with ph n influenza infection. niv may be useful in selected pregnant women without septic shock. pregnant women are at an increased risk for contracting influenza and its complications associated with influenza [ ] . like previous epidemic and pandemic diseases, pandemic h n (ph n ) influenza posed an increased risk of severe illness among pregnant women [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a report from the first month of the ph n outbreak noted that the rate of hospitalization among pregnant women was approximately four times the rate in the general population in the usa [ ] . as reported by the california department of public health (cdph), a total of % of the patients who were hospitalized or died from the ph n influenza were pregnant [ ] . according to the ministry of health (moh) of the people's republic of china, pregnant women accounted for . % of deaths associated with ph n influenza [ ] . pregnant women with influenza appear to have an increased risk of miscarriage, premature birth and stillbirth [ , , ] . reports from victoria in australia [ , ] , new york [ ] , and california [ ] , demonstrate that ph n infection was associated with substantial maternal and fetal morbidity and mortality. however, information is limited concerning the risk factors for maternal and neonatal death when pregnancy is complicated by severe or critical illness related to ph n influenza. in this report, we described the characteristics of ph n influenza in pregnant women and the risk factors for maternal and neonatal death. all patients who were admitted to hospitals with confirmed ph n influenza from sep. to dec. , from chinese provinces were screened if they fulfilled the diagnostic criteria for severe or critical cases. a confirmed case was a person whose ph n virus infection was verified by real-time reverse-transcriptase polymerase chain reaction (rrt-pcr) with or without the presentation of other clinical symptoms. patients were excluded if they had been treated as outpatients or in emergency rooms or duration of hospitalization < h, or if they had incomplete records of clinical outcomes. severe and critical cases were defined according to the h n clinical guidelines (third edition, ) released by the moh (additional file : table s ). our research retrospectively collected the patient's clinical information and did not involve the patient's personal information and samples, so there was no informed consent. the case report form included demographic information, underlying conditions, gestational age, vaccination status, treatment, intensive care unit (icu) admission, complications, and maternal and neonatal outcomes. body mass index (bmi) was calculated using height and weight recorded in the case report form, patients with bmi ≥ were categorized as obesity. indications for applying noninvasive ventilation (niv): pregnant women who complained shortness of breath or blood gas analysis confirmed hypoxemia pao to fio < . one nonpulmonary major organ dysfunction or unconsciousness was contraindications for niv. indications to change from niv to invasive ventilation: a cautious trial of niv was attempted and response to niv was monitored after the first hour or two. if there was a deterioration of oxygenation, invasive ventilation was considered. definition of successful niv: pao to fio improved and respiratory rate decreased during one or two hour niv therapy. the patients successfully weaned off niv and survived. definition of failed niv: during the one or two niv trial, a deterioration of oxygenation was observed and invasive ventilation was needed. data collection and analysis were coordinated by the moh. a standard data collection form was used for each study site. site investigators were primarily infectious disease physicians closely involved in taking care of such patients at their centers. the data were entered in duplicate into a computerized database. patient confidentiality was maintained by recording only patient date of birth and gender on the data collection form. the research ethics board at beijing chao-yang hospital and the first affiliated hospital, school of medicine, zhejiang university approved the study. we analyzed the reported demographic characteristics, underlying conditions, symptoms, treatments, complications, clinical course and maternal and neonatal outcomes. means (standard deviations, sd) or medians (interquartiles, iqr) were calculated as summaries of continuous variables. for categorical variables, percentages of patients in each category were calculated. we compared clinical characteristics and clinical outcomes by using an anova test, chi-square test, or fisher's exact test or wilcoxon rank-sum test as necessary. the primary outcome was in-hospital mortality. we performed univariable logistic analysis to investigate the potential factors on admission that might be associated with the maternal mortality. factors with statistical significance (p < . ) in the univariate analyses were included in the multivariate logistic regression analysis. a p value of less than . was considered to indicate statistical significance. all analysis was carried out using spss for windows (release . ). clinical description of cohort severe or critical cases were screened and cases involved pregnant women ( figure ). demographic characteristics, underlying conditions, symptoms, and lab findings of the pregnant women are illustrated in diseases were rare in this analysis. none of the patients had been immunized against seasonal influenza or ph n . the median apache ii score was . (iqr, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . at the time of admission, patients ( . %) had pneumonia with an abnormal chest radiography or chest computed tomography. the most common symptoms were cough ( ; . %) and dyspnoea ( ; . %). the median pao /fio on admission was . (iqr, . - . ) ( table ). of the hospitalized patients, ( . %) were admitted to an icu at a median of days from onset of illness (iqr to ; table ). medication ( . %) patients received oseltamivir. the median time from onset of illness to oseltamivir therapy was days (iqr to ), among them only patients ( . %) received oseltamivir within h of onset of illness. out of patients received antibiotics. received traditional chinese medicine. corticosteroid therapy was administered to patients ( table ) . the most commonly reported complication in this study was acute respiratory disease syndrome (ards) ( ; . %) ( table ) . ( . %) women delivered at a median of days (iqr to ) after ph n symptom onset. out of women delivered prematurely (additional file : table s ). the most common delivery method was cesarean delivery ( patients, . %) ( table ). among live-birth deliveries for which the gestational age was known, were premature (additional file : table s ). among the pregnant women in the study, died (table ) , out of the patients who died were in their third trimester. the main cause of death was refractory hypoxemia ( patients, . %). of patients with secondary infection, three patients had acinetobacter baumannii, one patient had aspergillus spp, and one patient had both acinetobacter baumannii and aspergillus spp. . % of women included in the study required intensive care and . % required mechanical ventilation. (table ). the first case of ph n virus infection in china was documented on may , the virus has rapidly spread throughout the mainland. a total of , confirmed cases were reported by mar , , including patients severe and patients died. among all these severe cases, about . % of patients were pregnant women [ ] . in this large study of pregnant women who were hospitalized with severe ph n influenza, the clinical characteristics were similar to those reported by others [ , , , ] . . % of patients were infected in the second or third trimester. in our study, the most common comorbidities were cardiovascular diseases ( . %), diabetes mellitus ( . %), respiratory diseases ( . %), and obesity ( . %). in our study, the prevalence of underlying diseases was much lower than reports from the united states ( . %) [ ] , % in australia [ ] , % in california [ ] , . % in brazil [ ] , and % in france [ ] . in those studies, the main cause of underlying disease was asthma. a study compared asthma prevalence of chinese adolescents living in canada and in china. the authors found that for girls, the range of asthma was . % in guangzhou to . % in canadian-born chinese adolescents. these results suggest that the lower prevalence of pre-existing asthma in our samples reflects prevalence of the disease in the chinese population [ ] . the mortality rate for severe or critically infected pregnant women in our study was %, similar to what was reported in canada, mexico, and new zealand [ ] [ ] [ ] [ ] , but higher than in france ( % death in icuhospitalized pregnancy women) [ ] . risk analysis showed that a pao /fio ≤ and higher bmi (i.e. ≥ ) on admission were risk factors for maternal death. pregnancy and ards are associated with increased oxygen consumption, which can result in hypoxemia in the mothers and the neonates. we reported that a higher bmi was associated with maternal mortality after adjusting for baseline clinical factors. observations of a high prevalence of obesity in severe and fatal cases of ph n infection have been reported in chile, canada, the united kingdom and mexico [ , , ] . as observed in australia, % of patients had a bmi of more than and % of patients more than , while the corresponding proportions in the general australian pregnant population was % and % respectively [ ] . however, our research retrospectively collected the patient's clinical information recorded in crfs. proportion of obesity has been overestimated based on bmi in the rd trimester of pregnancy. data from previous pandemics and seasonal influenza epidemics suggested that the risk of complications associated with influenza might be higher in the second and third trimester of pregnancy than in the first trimester [ , , ] . we also observed a higher proportion of maternal death occurring in the second and third trimester. during the h n influenza pandemic, in the united states, the rate of premature birth ( . %) was higher than the rate of premature births ( %) reported [ ] , consistent with data demonstrating a higher rate of premature delivery during previous pandemics [ ] . among women in our study for whom data on pregnancy outcomes was available, the rate of premature birth was . %. in a multivariable analysis, preterm delivery contributed to fetal mortality. delivery in severe and critically infected women after weeks' of gestation had improved neonatal outcomes compared to similar patients who delivered before weeks of gestation. evidence on the useful role of niv in pregnant patients with ards secondary h n viral infection was lacking. dr. amit banga [ ] reported a -year-old pregnant female with ards (pao /fio ) due to community-acquired severe pneumonia who successfully treated with niv. in , dr. michel djibre and collegues [ ] reported a -year-old pregnant woman at weeks' gestation with pao /fio who was successfully treated with niv. in our study, the success rate among pregnant women with h n infection for niv was . %. a recent prospective multicenter survey also found that when niv was used as first-line therapy for selected ali/ards patients (those with organ failures, hemodynamic instability, or encephalopathy were excluded), % avoided intubation and had excellent outcomes [ ] . apart from previous findings that major organ dysfunction and obtunded sensorium would obviously be unsuitable candidates for niv, we found that pregnant women complicated by septic shock were less likely to be successfully treated by niv. our data also support that cautious selection of appropriate patients is important for successful application of niv. patients should be monitored closely for signs of niv failure until stabilized. if there are signs of niv failure, patients should be intubated promptly before a crisis develops. our investigation has several limitations. firstly, we only evaluated pregnant women admitted to a hospital who fulfilled the diagnostic criteria of severe or critical cases. secondly, it was an observational study, and could therefore only demonstrate associations and could not infer cause. thirdly, we lacked follow up visits for maternal and neonatal outcomes. lastly, despite the use of a standardized data-collection form, not all information was collected for all patients. the clinical data reported herein is consistent with previous studies that demonstrate that pregnant women with influenza are at an increased risk of serious illness and death. our novel findings included: ) niv was useful for some selected pregnant women with ph n virus infection complicated by respiratory failure, but septic shock should be considered a contraindication; ) a pao /fio ≤ and higher bmi (i.e. ≥ ) on admission were independent risk factors for maternal death; ) premature delivery was an independent risk factor for neonatal death. additional file : the diagnosis criteria for severe and critical cases. additional file : maternal and neonatal outcomes by different delivery methods in different trimesters. data are presented as no. (%)/total no.(%), if otherwise stated. percentages are based on patients with complete information in the respective categories. * two patients missed the detailed information in maternal outcomes. neonatal outcomes were unknown in four cases. ** one patient missed the detailed information in maternal outcomes. neonatal outcomes were unknown in two cases. pandemic influenza and pregnant women influenza occurring in pregnant women h n influenza virus infection during pregnancy in the usa french experience of a/h n v influenza in pregnant women deaths from asian influenza associated with pregnancy impact of influenza on acute cardiopulmonary hospitalizations in pregnant women novel influenza a (h n ) virus infections in three pregnant women-united states prevention and control of seasonal influenza with vaccines: recommendations of the advisory committee on immunization practices (acip) h n influenza in pregnancy: cause for concern factors associated with death or hospitalization due to pandemic influenza a(h n ) infection in california progress in prevention and treatment of the h n pandemic of pregnancy women best practices in perinatal care: prevention and treatment of novel influenza a (h n ) virus during pregnancy and the immediate postbirth period bonar be: pandemic influenza and pneumonia in a large civilian hospital the anzic influenza investigators and australasian maternity outcomes surveillance system: critical illness due to a/h n influenza in pregnant and postpartum women: population based cohort study h n influenza a and pregnancy outcomes in severity of pandemic influenza a (h n ) virus infection in pregnant women severe h n influenza in pregnant and postpartum women in california official report of ministry of health, people's republic of china pandemic influenza a(h n ) virus illness among pregnant women in the united states outcomes for pregnant women infected with the influenza a (h n ) virus during the pandemic in porto alegre, brazil prevalence of asthma among chinese adolescents living in canada and in china critically ill patients with influenza a(h n ) infection in canada critically ill patients with influenza a(h n ) in mexico the anzic influenza investigators: critical care services and h n influenza in australia and new zealand factors associated with death or intensive care unit admission due to pandemic influenza a (h n ) infection epidemiology of fatal cases associated with pandemic h n influenza uk obstetric surveillance system pregnancy outcome in south australia births: preliminary data for use of non-invasive ventilation in a pregnant woman with acute respiratory distress syndrome due to pneumonia non-invasive management of acute respiratory distress syndrome related to influenza a (h n ) virus pneumonia in a pregnant woman a multiple-center survey on the use in clinical practice of noninvasive ventilation as a first-line intervention for acute respiratory distress syndrome all authors made substantial contributions to conception and design, acquisition of data, or analysis and interpretation of data, reviewed and approval of the final manuscript. drs. pjz, xll, bc and sgy contributed equally to this article. cw and lj l, the principal investigator, takes full responsibility for the integrity of the submission and publication, and was involved in the study design as part of the steering committee, had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. drs z pj, l xl, cb, y sg had full access to all of the data in the study, and they take responsibility for the integrity of the data and the accuracy of the data analysis and draft of the manuscript. drs l lr and glwere involved in the study design as part of the steering committee. drs xz, hk, z hy, y xx, h wb, cw, z jx were responsible for the patient enrollment and the data collection. the authors declare that they have no competing interests. key: cord- -hsqvkm i authors: matsuda, kentaro; narita, mitsuo; sera, nobuyuki; maeda, eriko; yoshitomi, hideaki; ohya, hitomi; araki, yuko; kakuma, tatsuyuki; fukuoh, atsushi; matsumoto, kenji title: gene and cytokine profile analysis of macrolide-resistant mycoplasma pneumoniae infection in fukuoka, japan date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: hsqvkm i background: recent epidemiologic data suggest that the prevalence of macrolide resistant mycoplasma pneumoniae (mr-m. pneumoniae) is increasing rapidly worldwide. this study assessed the present status of m. pneumoniae infection in japan and clinical end-points to distinguish children with mr-m. pneumoniae. methods: during an outbreak of m. pneumoniae infections in fukuoka, japan in – , a total of children with clinically suspected m. pneumoniae infection were enrolled. m. pneumoniae was analyzed for macrolide resistance in domain v of the s rrna gene. sixty -five patients with pcr positive for m. pneumoniae were analyzed with regard to clinical symptoms, efficacy of several antimicrobial agents and several laboratory data. results: causative pathogens were detected in . % ( of ) and m. pneumoniae was identified . % ( of ). the resistance rate of m. pneumoniae was . % ( of ) in this general pediatric outpatient setting. patients infected with mr-m. pneumoniae showed longer times to resolution of fever and required frequent changes of the initially prescribed macrolide to another antimicrobial agent. we observed three different genotypes of m. pneumoniae including the rarely reported a t mutation (a g: strains, a t: strains, no mutation: strains). drug susceptibility testing showed different antimicrobial susceptibility profiles for each genotype. serum ifn-gamma, il- and ip- levels were higher in patients with mr-genotypes than in those infected with no-mutation strains (p < . ). conclusions: macrolide resistance is more common than previously thought and a small epidemic of rarely reported a t mutation was observed in fukuoka, japan. furthermore our results reveal the possibility that levels of certain inflammatory cytokines may be a candidate to predict mr-m.pneumoniae infection. mycoplasma pneumoniae (m. pneumoniae) is a common bacterial cause of upper and lower respiratory tract infections in children and adolescents. recent advances in antimicrobial agents, including macrolide antibiotics [ ] , tetracyclines and fluoroquinolones, have enabled its treatment on an outpatient basis for most children with community-acquired pneumonia. since the time when macrolide resistant (mr) m. pneumoniae was first isolated from a pediatric patient in [ ] , the drug resistance trend has been documented all over the world [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , especially in children. extremely high rates of macrolide resistance have been reported mainly in asian countries [ , ] . according to the continuous surveillance in japan, the frequency of mr-m. pneumoniae cases has increased year by year, and nowadays > % of m. pneumoniae isolates were shown to be macrolide resistant [ ] . however, most of these reports included the data in secondary or tertiary care facilities; thus very little information is currently available on the prevalence of mr-m. pneumoniae infection, especially in outpatient settings. macrolide-resistant genotypes can be defined by the detection of specific point mutations in the domain v of the single-operon derived s rrna gene of m. pneumoniae. while most frequent mutations that induce high levels of macrolideresistance included an a-to-g transition at position or [ , ] , recent epidemiologic studies have suggested that distribution of mr-m. pneumoniae genotypes has been changing [ , ] . the primary objective of our study was to clarify the present status of mr-m. pneumoniae. for the present, it is difficult to make an early identification of which children being infected by mr-m. pneumoniae. thus, secondly, we evaluated children with m. pneumoniae infections with regard to clinical symptoms, efficacy of several antimicrobial agents and several laboratory data to investigate the predictive factors of mr-m. pneumoniae infections. between september and december , a total of patients with clinically suspected m. pneumoniae infection were recruited from matsuda children's clinic (onojo-city, fukuoka prefecture) after informed consent was obtained from both the children and their parents. nasopharyngeal swab specimens and blood samples were obtained from all patients at the time of enrollment, and the throat swabs were sent to the laboratory of virology, fukuoka institute of health and environmental sciences. laboratory-confirmed m. pneumoniae infection was defined as detection of m. pneumoniae dna from the throat swab by pcr methods. multiplex pcr was carried out as described previously [ , ] . m. pneumoniae, rhinoviruses (rv), respiratory syncytial virus (rsv), coronaviruses, human metapneumovirus (hmpv), human bocavirus, influenza a, and b viruses, adenovirus and parainfluenza virus (piv - ). ultimately, data on children ( girls, boys; mean age . years) with positive for m. pneumoniae were analyzed. this study was approved by the ethical committee of kurume university school of medicine and the national research institute for child health and development. to evaluate the disease activity of m. pneumoniae infection, we performed general blood tests, including white blood cell (wbc) counts, c-reactive protein (crp), erythrocyte sedimentation rate (esr) at the time of throat swab collection. serum concentrations of chemokines and cytokines were measured using luminex xmap technology (milliplex map kits; millipore, billerica, ma, usa) according to the manufacturer's instructions. the detection limits are . , . , . , . , and . pg/ml for il- , il- , ifn-gamma, ip- and il- , respectively. clinical records of the patients with m. pneumoniae infection were examined retrospectively. precise quantitative data such as total duration of fever and the usage of antibiotics which were obtained from the clinical charts and the clinical courses of patients infected by each of the three different genotypes (a g, a t, and no mutation) were compared. a febrile day was defined as a day during which the body temperature exceeded . °c. as an indicator of the severity of mycoplasma infection, the number of patients requiring hospitalization was also assessed. the chemotherapeutical agent was often changed from a macrolide to minocycline (min) or tosufloxacin (tfx) according to clinical conditions such as persistent fever and cough as well as chest x-ray findings. the attending physician (k. matsuda) had no information about the susceptibility to m. pneumoniae at the time of clinical decision-making. the mutations associated with resistance to macrolides were detected by sequencing the targeted domain v region of the s rrna gene as described by matsuoka et al. ( ) using the following primers for domain v of s rrna (forward: ′-gcagtgaagaagaacgagg gg- ′, reverse: ′-gtcctcgcttcggtcctctcg- ′) which were synthesized as reported by lucier et al. [ ] . the sequences were aligned using the clustalw option within the software molecular evolutionary genetics analysis (mega) . . (http://www.megasoftware. net/) and compared to the registered sequence of m. pneumoniae m (accession no. × ). minimal inhibitory concentrations (mics) of antibiotics were determined by a broth microdilution method based on the national committee for clinical laboratory standards (clinical and laboratory standards institute) as described elsewhere [ , ] . the following antibiotics were tested: erythromycin (ery), clarithromycin (clr), azithromycin (azm), josamycin (jos), rokitamycin (rki), clindamycin (cli), tetracycline (tet), min, levofloxacin (lvx), ciprofloxacin (cpx), tfx, sparfloxacin (spx) and gatifloxacin (gfx). briefly, serial twofold dilutions of antibiotics were prepared in the mycoplasma broth base inoculated by to cfu/ml of m. pneumoniae in -well microplates and incubated at °c. the mic was determined as the lowest concentration of antimicrobial agent at which the color of the control medium changed. the degree of resistance was evaluated according to the japanese standard (</= μg/ml; susceptible, μg/ml; intermediate, > /= μg/ml; resistant, for ery, clr, azm, jos, rki, cli) [ ] . statistical analysis was performed using the computing environment r (r development core team, ). the data in table are presented as medians ( st and rd quartiles). the kruskal-wallis test or fisher's exact test was used for multiclass comparison (a g, a t, no mutation). if the results were significant, then the wilcoxon rank-sum test was used for pairwise comparisons (a g vs no mutation, a t vs no mutation, and a g vs a t, respectively). p-values less than . were regarded as statistically significant. during this study period, a total of children were enrolled in this study. the causative pathogens identified were; m.pneumoniae (n = , . %), rv (n = , . %), hmpv (n = , . %), influenza b virus (n = , . %), piv (n = , . %), rsv (n = , %), adeno virus (n = , %). mixed viral infections were found in ( . %) cases. sixty -five patients ( . %) with pcr positive for m. pneumoniae were analyzed. the patients' clinical characteristics are shown in table . all patients were outpatients at the time of onset and had no severe underlying illnesses, nor a history of receiving systemic corticosteroids prior to enrollment. macrolide-resistant mutations in domain v of the s rrna gene were detected in . % ( / ) of the children with m. pneumoniae infection, while the remaining ( . %) were found to harbor no mutation known to be associated with resistance in that region. thirty-one of the macrolide-resistance mutations ( . %) showed the a g transition; the remaining ( . %) showed the a t transversion in domain v of the s rrna gene. the three groups (a g, a t, no mutation) did not differ significantly in age, gender distribution or number of days between the onset of initial symptoms and the examination performed (table ) . detailed information concerning the antimicrobial agents prescribed at the clinic is shown in table . briefly, fiftythree of ( . %) patients were initially treated with the -membered-ring macrolide clr or the -memberedring macrolide azm. in a total of patients ( . %) these antibiotics were eventually changed to min or tfx because of persistent fever, cough, and worsening of chest x-ray findings. a significant difference was observed in the number of patients needing to change to another antimicrobial agent among the three groups (p = . ). specifically, patients with the a g transition ( . %) were more likely to have had the initially prescribed macrolide changed to another antimicrobial agent other than a macrolide in comparison not only to macrolide-sensitive patients ( . %, p = . ) but also to those with a t transversion ( . %, p = . ). fever disappeared dramatically within hr after changing to other drugs such as min or tfx in all patients whose clinical findings had failed to improve or whose chest radiographic findings had worsened during the initial treatment. in two patients, symptoms resolved spontaneously without any antibiotics (one with no mutation and the other with a t). with regard to the timing of sample collection, of the patients with m. pneumoniae had already been treated with antimicrobial agents before nasopharyngeal specimens were collected. there were no statistically significant differences in the incidence of macrolide resistance as to whether sample was collected before or after antimicrobial agents administration (in of ( . %) collected before administration, and in of ( . %) collected after administration, p = . ). the total number of febrile days was significantly greater in patients infected with either of the macrolide-resistance genotypes than in those infected with the no-mutation strains (a g: . days, a t: . days, no mutation: . days; comparison of a g vs no mutation: p = . , a t vs no mutation: p = . , and a g vs a t: not significant). two patients (one with a g and the other with a t) required hospitalization because of the progression to dyspnea and the development of a secondary bacterial infection ( table ) . no significant differences were observed among each genotype group with regard to the results of any acute phase general infection markers (wbc, crp, esr). whereas serum il- and il- levels were not different among the three groups, significant differences were observed in the levels of some serum inflammatory cytokines among the three groups. serum ifn-gamma, il- and ip- levels were significantly higher in patients infected by the macrolide-resistance genotypes than in those infected by the no-mutation strains (comparison of a g vs. no mutation: p < . , a t vs no mutation: p < . and a g vs a t: not significant, table ). mics could be measured exclusively for clinical strains obtained between november and december (a g; strains, a t; strains, table ) for the reason of performed facilities' difficulties. both the a g mutation and the a t mutation yielded a high degree of resistance to ery and clr (mic range, to > μg/ml), though both mutated genotypes exhibited no elevation of mics for min and tfx. although the strains with the a g mutation exhibited variable resistance (mics - μg/ml) to the -membered macrolide azm, all strains with the a t mutation were susceptible to azm (mic . μg/ml). in addition, we performed the multiplelocus variable-number tandem-repeat analysis (mlva) for these strains according to the previously described method ( ) and found different mlva types ( types for the strains of a g and other types for the strains of a t, data not shown). there was no obvious link between the mlva type and the patient's age and residential area, or resistance to macrolides. in the present study, we have clarified . % of pediatric m. pneumoniae infections were caused by either of two macrolide-resistant genotypes of m. pneumoniae. the incidence of mr-m. pneumoniae in this general pediatric outpatient setting was nearly as high as those described in continuous surveillance in japan [ ] . thirty-one of the mr-m. pneumoniae strains ( . %) harbored an a-to-g transition mutation at position in the s rrna genes, which was consistent with previously reported findings [ ] that the a g transition was the most frequent mutation. a striking finding in our present work is that the remaining samples ( . %) showed an a-to-t transversion at position in the s rrna genes. the a t mutation was first reported in china during - [ , ] but has been rarely reported thereafter. however, recent reports from japan have revealed that the a t transversion have been increasing [ , ] . a community outbreak of a t transversion was reported recently in yamagata, japan, in [ ] . in this point, the resistant strains in our study were polyclonal in origin, which is consistent with the previous report [ ] . although the precise mechanisms of outbreak of the a t transversion mutation are not yet known, in general, transversion mutations that change the chemical structure dramatically are thought to affect bacterial growth more severely and in consequence to be less common than transition mutations. for example, an in-vitro study revealed that the mutation rate of a-to-t transversion is less than one-tenth that of a-to-g transition in e. coli cells, coinciding with the spontaneous misinsertion rate of its dna polymerase iii [ ] . another important finding in our study is the profile of serum cytokines and chemokines. it is true that gene amplification methods such as real-time pcr [ ] and pyrosequencing [ ] must be a best way to detect resistance, however, these methods are confined to very few specialized laboratories in japan. for reason of that, we expect some additional information on pathogenesis of mycoplasmal infection through measurement of cytokines and chemokines. higher levels in serum were found for some inflammatory cytokines such as ifngamma, ip- and il- in patients infected by the macrolide-resistant strains than in those infected by the strains with no-mutation, while no difference was observed in the levels of il- and il- between the two groups. it has been thought il and il- play a pivotal role in the pathogenesis of m. pneumonia infection according to the disease severity [ , ] . there are several explanations for this discrepancy. first, disease severity was not so high in our patients infected with macrolideresistant strains. indeed we did not experience apparent treatment failure or serious illness. these findings were consistent with previous reports which showed that drug resistance in m. pneumoniae does not always lead to severe clinical manifestations [ , ] . second, the timing of blood sampling might alter the levels of these cytokines as reported by narita et al. [ ] , since we only performed blood sampling at the time of enrollment. on the other hand, the higher levels of ifn-gamma, ip- and il- in the patients with mr-m. pneumoniae infection might reflect, as a non-specific inflammatory marker, the prolonged low degree of inflammation which is represented by slightly longer duration of fever due to the drug resistance. our results imply the possibility that in addition to the lower clinical efficacy of macrolide treatment, measurement of levels of certain inflammatory cytokines may be a strong candidate that allows the clinicians to suspect mr-m. pneumoniae infection. strains with the a g transition were highly resistant to -and -membered-ring macrolides as previously described [ , ] and the patients infected by the strains with this mutation were more likely to have had the initially prescribed macrolide changed to a non-macrolide table mics of selected antimicrobial agents for strains of m. pneumoniae isolated from clinical samples antimicrobial agent when compared with the patients infected not only by macrolide-sensitive strains (p = . ) but also by a t transversion strains (p = . ). as shown by the susceptibility testing, all four strains with the a t mutation showed lower mics for azm ( . μg/ml), and actually, the eight patients with a t mutation who were treated with azm showed complete resolution of clinical symptoms. thus our data showed that the in-vitro susceptibility directly predicts the clinical efficacy as previously reported [ , , ] . the reason why azm is effective in patients with an a t transversion is unclear. one plausible explanation is a difference in chemical structures. that is, while the binding of -membered-ring macrolides to a is affected both by the g transition and by the t transversion, the binding of -membered-ring macrolides to a is not affected by t transversion. nevertheless, since the a t transversion mutation must be related to more profound damage to growth efficiency, the reason remains unknown why so many strains with the rarely reported a t mutation, a total of , were isolated in this study (in the fukuoka area). interestingly, the a t mutation slightly but significantly raises the mics for rki ( μg/ml), in contrast to the fact that the strains with the a g mutation can be considered susceptible to this macrolide ( μg/ml). this suggests that a g transposition does not affect the binding of rki to the domain v while a t transversion affects the binding to some degree. the limitations of the present study is that the study was a single-center study conducted over one year; accordingly, the sample number was limited, and the statistical power may therefore have been insufficient to elucidate the linkage between drug susceptibility and genotype of m. pneumonia. further studies including larger sample sizes to investigate the predictive factors of macrolide-resistance are necessary. practical implications of our present study are that the prevalence of mr-m. pneumoniae is increasing more rapidly than we thought, even in general outpatient settings and drug susceptibility may be different for each genotype. moreover, our results reveal the possibility that levels of certain inflammatory cytokines such as il- , ip- and ifn-gamma may be a candidate to predict mr-m. pneumoniae infection at the early stage of infection. in view of these findings, treatment should be tailored to each patient or at least taking into account which patient is infected by the particular genotype, and the treatment strategies must be optimized for each genotype. vitro and in vivo activities of macrolides against mycoplasma pneumoniae. anti agents chem characteristics of macrolide-resistant mycoplasma pneumoniae strains isolated from patients and induced with erythromycin in vitro high prevalence of macrolide resistance in mycoplasma pneumoniae isolates from adult and adolescent patients with respiratory tract infection in china nationwide surveillance of macrolide-resistant mycoplasma pneumoniae infection in pediatric patients macrolide resistance of mycoplasma pneumoniae first report of macrolide-resistant strains and description of a novel nucleotide sequence variation in the p adhesin gene in mycoplasma pneumoniae clinical strains isolated in france over years detection of macrolide resistance in mycoplasma pneumoniae by real-time pcr and high-resolution melt analysis occurrence of macrolideresistant mycoplasma pneumoniae strain in germany rising rates of macrolide-resistant mycoplasma pneumoniae in the central united states macrolide-resistant mycoplasma pneumoniae in humans characterization and molecular analysis of macrolide-resistant mycoplasma pneumoniae clinical isolates obtained in japan emergence of macrolide-resistant mycoplasma pneumoniae with a s rrna gene mutation community outbreak of macrolide-resistant mycoplasma pneumoniae in yamagata development of three multiplex rt-pcr assays for the detection of respiratory rna viruses rapid identification of nine microorganisms causing acute respiratory tract infections by single-tube multiplex reverse transcription-pcr: feasibility study transition mutations in the s rrna of erythromycin-resistant isolates of mycoplasma pneumoniae proposed antibiotic breakpoint on mycoplasma pneumonia clinical isolates concerning macrolide and lincosamide antibiotics acute respiratory diseases study group increased macrolide resistance of mycoplasma pneumoniae in pediatric patients with community-acquired pneumonia nested pcrlinked capillary electrophoresis and single-strand conformation polymorphisms for detection of macrolide-resistant mycoplasma pneumoniae in beijing development of multiple-locus variable-number tandem-repeat analysis for molecular typing of mycoplasma pneumoniae dna replication errors produced by the replicative apparatus of escherichia coli macrolide resistance determination and molecular typing of mycoplasma pneumoniae by pyrosequencing significant role of interleukin- in pathogenesis of pulmonary disease due to mycoplasma pneumoniae infection role of interleukin- and t-helper type cytokines in the development of mycoplasma pneumoniae pneumonia in adults macrolide-resistant mycoplasma pneumoniae: characteristics of isolates and clinical aspects of community-acquired pneumonia clinical evaluation of macrolideresistant mycoplasma pneumoniae late increase of interleukin- levels in blood during mycoplasma pneumoniae pneumonia a comparative clinical study of macrolide-sensitive and macrolide-resistant mycoplasma pneumoniae infections in pediatric patients gene and cytokine profile analysis of macrolide-resistant mycoplasma pneumoniae infection in fukuoka this work was supported in part by grants of national center of child health and development ( a- to k.m.). no of strains ery clr azm jos rki cli tet min lvx cpx tfx spx gfx a g > key: cord- - xl isee authors: andrei, stefan; ghiaur, alexandra; brezeanu, lavinia; martac, cristina; nicolau, andreea; coriu, daniel; droc, gabriela title: successful treatment of pulmonary haemorrhage and acute respiratory distress syndrome caused by fulminant stenotrophomonas maltophilia respiratory infection in a patient with acute lymphoblastic leukaemia – case report date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: xl isee background: stenotrophomonas maltophilia-induced pulmonary haemorrhage is considered a fatal infection among haematological patients. the outcome can be explained by the patients’ immunity status and late diagnosis and treatment. case presentation: we present the rare case of successful outcome in a -year-old female who developed alveolar haemorrhage and acute respiratory distress syndrome days after a chemotherapy session for her acute lymphoblastic leukaemia, in the context of secondary bone marrow aplasia. stenotrophomonas maltophilia was isolated in sputum culture. the patient benefitted from early empirical treatment with colistin followed by trimethoprim/sulfamethoxazole, according to the antibiogram. despite a severe initial clinical presentation in need of mechanical ventilation, neuromuscular blocking agents infusion, and ventilation in prone position, the patient had a favourable outcome and was discharged from intensive care after days. conclusions: stenotrophomonas maltophilia severe pneumonia complicated with pulmonary haemorrhage is not always fatal in haematological patients. empirical treatment of multidrug-resistant stenotrophomonas maltophilia in an immunocompromised haematological patient presenting with hemoptysis should be taken into consideration. stenotrophomonas maltophilia is an anaerobic nonfermentative bacteria that does not cause infections in immunocompetent hosts, but might be fatal in patients with weakened immune system [ ] . several case reports and case series describing pulmonary haemorrhage caused by respiratory infection with stenotrophomonas maltophilia have been reported in adult patients with haematological diseases [ ] . severe pulmonary haemorrhage due to stenotrophomonas maltophiliahas also been reported in neonates [ ] . the literature presents a variety of cases in adults with allogeneic stem cell transplantation [ , ] or secondary to chemotherapy-induced pancytopenia, particularly myeloid leukaemia [ , ] . in all these cases, the mortality was very high; mori et al. reported no survival in one review of haematological adult cases [ ] . we report the case of successfully treated severe stenotrophomonas maltophilia respiratory infection complicated with pulmonary haemorrhage in a chemotherapyinduced pancytopenia patient diagnosed with acute lymphoblastic leukaemia. a -year-old female was diagnosed in december with philadelphia chromosome-positive acute lymphoblastic leukaemia. her past medical history was significant for hypertension and type diabetes mellitus for more than years. at the moment of diagnosis, the total white blood cell count was × /l (wbc) with peripheral blood smear showing % blasts, haemoglobin . g/dl, and × /l platelets count (plt). the bone marrow smears showed hypercellularity with more than % blast cells. the flow cytometry revealed % blast cells compatible with common b cell acute lymphoblastic leukaemia. the cytogenetic and fluorescence in situ hybridization analysis have identified the translocation t ( ; )(q ;q )(philadelphia chromosome). the molecular analysis was positive for bcr-abl fusion gene (p isoform), and the bcr-abl/abl ratio was . %. the patient received treatment according to graaph protocol with imatinib mg/ day. the patient achieved complete morphological remission after the induction, with a molecular response bcr-abl/abl ratio of . % is. the months follow-up assessment showed a deep molecular response (molecular analysis ratio bcr-abl/ abl: . % -mr . log is). the cytogenetic analysis ,xx [ ] was consistent with a complete cytogenetic response, and the bone marrow aspiration showed trilineage dysplasia. her treatment was continued with the same regimen. on day eight after consolidation therapy with cycle (methotrexate g/m at day , cytarabine mg/m bid at day and day ), a severe aplasia developed ( . × /l wbc, × /l absolute neutrophil count (anc), × /l plt), and she became febrile with a mild cough and a small area of right perihilar infiltration on chest x-rays ( fig. ) , despite prophylactic treatment with levofloxacin. sputum culture and peripheral blood cultures were performed, and the patient was started on empirical broad-spectrum antibiotics (piperacillin-tazobactam, amikacin) and fluconazole. over the next days, the respiratory status worsened, and the patient developed severe cough and hemoptysis, dyspnea and an increased in the oxygen demand. considering the worsening clinical status, the risk of multidrugresistant (mdr) germs infection, as well as severe bone marrow aplasia with multiple hospitalizations, the antibiotic treatment was escalated to meropenem, linezolid, amikacin, and fluconazole was switched to voriconazole. the patient received platelets and subcutaneous granulocyte colony-stimulating factor treatment. four days after the onset of fever ( days after chemotherapy), the patient was admitted to icu for type i respiratory failure and pulmonary haemorrhage. on icu admission, the patient was conscious and oriented, very dyspneic and tachypneic with a respiratory rate of /min, and a spo % on l per minute (lpm) o on facial mask, and bilateral auscultatory pulmonary crackles. the blood pressure was / mmhg, and the heart rate was beats per minute, sinus rhythm. there were no signs of peripheral hypoperfusion. the temperature was . o celsius. the arterial blood gases revealed a ph . , pao mmhg, paco mmhg, lactate . mmol/l. the antibiotic cover was broadened with the addition of colistin. after h on high flow nasal oxygen (fio , flow lpm) and in the absence of clinical improvement (persistent respiratory effort), mechanical ventilation ( ml/kg, positive end-expiratory pressure titrated at cm h o, plateau pressure cm h o), sedation, and continuous muscle paralysis were initiated. one-hour post intubation, the pao /fio ratio was to , on % o . the decision to initiate ventilation in prone position for h was made at this point, with poor respiratory responsiveness (pao /fio ratio ). the chest x-rays at this moment is shown in fig. . the acute respiratory distress syndrome (ards) diagnosis was established. within the next h in the icu (day after chemotherapy), the patient recovered from aplasia ( . × /l wbc, . × /l anc, × /l plt). the neuromuscular blockade was discontinued after h. the clinical state and blood results prior to icu admission are summarized in fig. . the sputum culture was positive for stenotrophomonas maltophilia (cultured on day after chemotherapy, days before the icu admission). therefore, the antibiotic treatment has been de-escalated to colistin ( . miu, bid, after initial bolus dose of miu) and trimethoprim/sulfamethoxazole ( mg/ mg, qid). after other days, the antibiogram showed a germ susceptible to trimethoprim/sulfamethoxazole (tmp/smx), intermediary susceptible to levofloxacin, and resistant to all beta-lactams and aminoglycosides (no determined mic available). the antibiotic treatment has been further de-escalated the next day to tmp/smx only for a total duration of days. unfortunately, the laboratory was logistically unable to test the susceptibility to colistin at this point. all blood cultures were reported negative during the hospital stay. the respiratory improvement allowed for a bronchoscopy with bronchoalveolar lavage, days after icu admission, which showed a non-haemorrhagic mucosa, and numerous blood clots. the cytologic examination was very rich in red blood cells with some macrophages, rare lymphocytes and neutrophils. respiratory cultures from the bronchoalveolar lavage were negative. the clinical course was marked by a failed extubation at day of icu, explained by icu-induced neuromyopathy and ventilator-associated pneumonia (vap) with mdr pseudomonas aeruginosa susceptible only to colistin (mic ), and resistant to carbapenems and other antibiotics. treatment with iv and nebulized colistin was introduced for a total duration of weeks. the respiratory status progressively improved, allowing the successful extubation on day in icu. the chest x-rays post extubation is shown in fig. . the patient was transferred to the haematology ward after days of icu stay and days after chemotherapy. the patient was discharged home soon after, on family request, and her clinical condition and respiratory function were excellent at months follow up. we reported a case of severe acute respiratory distress syndrome (ards) with alveolar haemorrhage and a good response to treatment. to our knowledge, this is the first reported case of pulmonary haemorrhage and ards caused by a fulminant stenotrophomonas maltophilia respiratory infection in eastern europe, and it seems to be a rare case of positive outcome in a patient with haematological malignancy. the patients diagnosed with acute leukaemia have a high risk of infection with opportunistic pathogens. the pulmonary haemorrhage caused by stenotrophomonas maltophilia is a rare condition with a poor outcome. several reports have shown the presence of predisposing factors like severe thrombocytopenia, severe and prolonged neutropenia, the previous use of quinolones, corticosteroids, and immunosuppressive therapy [ , , ] . our patient had grade iv thrombocytopenia and neutropenia secondary to chemotherapy. she also had a history of prolonged hospitalization for chemotherapy and complications related to chemotherapy, and she was treated with multiple classes of antibiotics, including quinolones, and she had prolonged corticosteroids use. in the published literature, we found haematological patients with stenotrophomonas maltophilia and pulmonary haemorrhage [ , ] . most patients ( out of ) were diagnosed with acute myeloid leukaemia, and the mortality rate was %, and the survival length was - days. it is important to notice the high early death rate: almost % of patients died in the first days after the onset of the respiratory symptoms. another the particularity of our case is that the patient recovered very soon from severe aplasia after icu admission (day ) permitting the respiratory stabilization in the absence of recurrent hemoptysis. also, we speculate that the empirically introduced colistin at the moment of icu admission for ards, associated with neutrophil count recovery, might have been efficient in stopping the bacterial proliferation. the only available options to treat the stenotrophomonas maltophilia infection, in this case, were tmp/smx and colistin -which has not been tested because of laboratory unavailability of the antibiogram kit. the literature-reported resistance of stenotrophomonas maltophilia to colistin was < % [ ] . there is no standard therapy for the treatment of severe stenotrophomonas maltophilia pneumonia with pulmonary haemorrhage, but combination antibiotic therapy represents an alternative to consider in critical situations [ ] . in our case, the patient received colistin and tmp/smx combination for days, the time between the germ identification and the antibiogram results, followed by further de-escalation to tmp/smx only. the decision to continue iv colistin has been taken considering the fact that the patient has been already exposed to levofloxacin, the possibility of a mdr germ and the unknown bioavailability of enterally administered tmp/smx in a severely ill patient with reactive digestive ileus and gastric stasis (the iv form is not available in our country). some retrospective studies have shown that, during icu stay, the absence of neutropenia recovery and the presence of organ failure are associated with poor outcome in the critically ill patient with malignancy [ , ] . furthermore, according to other reports, survival was higher for patients who underwent a first-line chemotherapy, had lobar ards and who received antibiotic treatment active on difficult-to-treat bacteria like pseudomonas aeruginosa and stenotrophomonas maltophilia [ ] . the duration of neutropenia seems to be correlated with short-term mortality, while -day mortality is affected by organ dysfunction [ , ] . one explanation for the high mortality in this clinical situation could be that the patients do not survive the first days of icu because of the difficulty of proper pathogen isolation and its corresponding antibiogram, allowing targeted antibiotic treatment [ ] . indeed, days were needed to identify the pathogen in the respiratory samples, and days to have complete cultures and susceptibilities. the consequence of initial wide spectrum antibiotic treatment was the development of a late vap with mdr pseudomonas aeruginosa. we are not able to provide a full explanation for this pneumonia with a mdr germ susceptible to colistin in a patient who already received colistin, as a serum colistin level was not measured (logistically unavailable). nevertheless, we may speculate that this might have happened due to changes in the volume of distribution in the patient with prolonged icu fig. chest x-rays after extubation (day of icu). the regression of the bilateral infiltrates and mild bilateral pleural effusion can be noticed hospitalization. the efficacy of associated nebulized colistin might be an argument for an increased active concentration in the targeted organ. overall, the prolonged treatment with colistin was well tolerated with no neurotoxicity, nor nephrotoxicity. the clinical management of this case had limitations and debatable aspects, such as the unavailable quantitative sputum culture, a long delay in obtaining germ identification and antibiogram, the unavailable colistinresistance kit for stenotrophomonas maltophilia, the unavailable iv form for tmp/smx, or the impossibility to determine the colistin serum levels. the rapid escalation in the antibiotic cover might be partially explained by our national known struggle and local experience with bacterial resistance. infections with multi-resistant opportunistic pathogens in haematological patients treated with chemotherapy is a complication that associates therapeutic challenges and high mortality rate. this clinical case highlights the severity and rapid progression of stenotrophomona smaltophilia pneumonia in a patient with acute lymphoblastic leukaemia. the patient had a good outcome following treatment with colistin and tmp/smx, associated with rapid recovery cell count after the pneumonia onset. this case might be an argument that the clinician might consider the empirical covering of stenotrophomonas maltophilia, particularly in the immunocompromised haematological patient presenting with hemoptysis, as it proves that stenotrophomonas maltophilia is not always lethal in fragile haematological patients. stenotrophomonas maltophilia: an emerging global opportunistic pathogen life-threatening hemorrhagic pneumonia caused by stenotrophomonas maltophilia in the treatment of hematologic diseases haemorrhagic pneumonia caused by stenotrophomonas maltophilia in two newborns stenotrophomonas maltophilia infection during allogeneic hematopoietic stem cell transplantation: a single-center experience stenotrophomonas maltophilia infection in hematopoietic sct recipients: high mortality due to pulmonary hemorrhage lethal pulmonary hemorrhage caused by a fulminant stenotrophomonas maltophilia respiratory infection in an acute myeloid leukemia patient fatal hemorrhagic pneumonia: don't forget stenotrophomonas maltophilia clinical characteristics of rapidly progressive fatal hemorrhagic pneumonia caused by stenotrophomonas maltophilia infections caused by stenotrophomonas maltophilia in recipients of hematopoietic stem cell transplantation prevalence and antibiotic resistance of stenotrophomonas maltophilia in respiratory tract samples: a -year epidemiological snapshot prognosis of acute respiratory distress syndrome in neutropenic cancer patients sepsis and septicshock in patients withmalignancies: a groupe de recherche respiratoire en réanimation onco-hématologique study impact of neutropenia duration on short-term mortality in neutropenic critically ill cancer patients publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations none. authors' contributions sa and ag reviewed the published literature, drafted the manuscript, and managed the case. lb, cm and an were involved in data collection and clinical management. dc and gd provided critical reviewing of the manuscript and clinical expertise. all authors approved the final version of this manuscript. the authors received non funding for this publication.availability of data and materials supplementary medical data are available at request. this case report has the approval of the ethics committee of our university hospital according to local and national rules. written informed consent was obtained from the patient for publication of this case report. the authors declare no conflict of interest. key: cord- - x rkor authors: honkpehedji, yabo josiane; adegnika, ayôla akim; dejon-agobe, jean claude; zinsou, jeannot fréjus; mba, romuald beh; gerstenberg, jacob; rakotozandrindrainy, raphaël; rakotoarivelo, rivo andry; rasamoelina, tahinamandranto; sicuri, elisa; schwarz, norbert g.; corstjens, paul l. a. m.; hoekstra, pytsje t.; van dam, govert j.; kreidenweiss, andrea title: prospective, observational study to assess the performance of caa measurement as a diagnostic tool for the detection of schistosoma haematobium infections in pregnant women and their child in lambaréné, gabon: study protocol of the freebily clinical trial in gabon date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: x rkor background: schistosoma antigen detection in urine is a valuable diagnostic approach for schistosomiasis control programmes because of the higher sensitivity compared to parasitological methods and preferred sampling of urine over stool. highly accurate diagnostics are important in low schistosoma transmission areas. pregnant women and young children could particularly benefit from antigen testing as praziquantel (pzq) can be given to only confirmed schistosoma cases. this prevents the unborn baby from unnecessary exposure to pzq. we present here the protocol of a diagnostic study that forms part of the freebily project. the aim is to evaluate the accuracy of circulating anodic antigen (caa) detection for diagnosis of schistosoma haematobium infections in pregnant women and to validate caa as an endpoint measure for anti-schistosoma drug efficacy. the study will also investigate schistosoma infections in infants. methods: a set of three interlinked prospective, observational studies is conducted in gabon. the upconverting phosphor lateral flow (ucp-lf) caa test is the index diagnostic test that will be evaluated. the core trial, sub-study a, comprehensively evaluates the accuracy of the ucp-lf caa urine test against a set of other schistosoma diagnostics in a cross-sectional trial design. women positive for s. haematobium will proceed with sub-study b and will be randomised to receive pzq treatment immediately or after delivery followed by weekly sample collection. this approach includes comparative monitoring of caa levels following pzq intake and will also contribute further data for safety of pzq administration during pregnancy. sub-study c is a longitudinal study to determine the incidence of s. haematobium infection as well as the age for first infection in life-time. discussion: the freebily trial in gabon will generate a comprehensive set of data on the accuracy of the ucp-lf caa test for the detection of s. haematobium infection in pregnant women and newborn babies and for the use of caa as a marker to determine pzq efficacy. furthermore, incidence of schistosoma infection in infants will be reported. using the ultrasensitive diagnostics, this information will be highly relevant for schistosoma prevalence monitoring by national control programs as well as for the development of medicaments and vaccines. trial registration: the registration number of this study is nct (clinicaltrials.gov, date of registration: december ). schistosomiasis is one of most widespread human parasitic diseases worldwide [ , ] . infections with blood flukes of the genus schistosoma are endemic in countries with more than million individuals being infected and % of infections are occurring in sub-saharan africa [ ] . schistosoma haematobium is highly prevalent in africa [ ] resulting in chronic morbidities, particularly affecting urogenital organs. women in child-bearing age suffer two-fold from schistosoma infections: directly from immune-pathological effects induced by worm infection, and indirectly from potential adverse birth outcomes during pregnancy [ ] . currently, there is no vaccine available against schistosomiasis and control is based on a single drug -praziquantel (pzq) for chemotherapy and prevention. the who roadmap on neglected tropical diseases calling for schistosomiasis elimination initiated a tremendous increase in pzq deployment [ ] . this fuelled the ever-growing concern of emergence of pzq resistant schistosoma, particularly since schistosomes with reduced susceptibility have been identified sporadically in sub-saharan africa [ ] [ ] [ ] [ ] . however, drug efficacy outcomes largely depend on the accuracy of the surrogate endpoint measures that reflect treatment effects in clinical trials [ ] . clinical evaluation of pzq efficacy, of new pzq formulations (e.g. paediatric) [ ] or of new drug candidates relies on reliable diagnostic instruments to correctly judge a drug's potency to kill schistosoma infections in humans. egg microscopy is still the reference standard widely used in schistosomiasis diagnosis but the methodology lacks sensitivity and cannot precisely measure drug efficacy due to huge variation in egg counts intrinsically associated with egg excretion that varies from day-to-day and occurs in clusters in stool [ ] [ ] [ ] [ ] [ ] . egg reduction rates vary considerably in a study cohort necessitating a huge sample size in clinical trials to be able to identify significant differences between the investigational product and the control. furthermore, available pzq efficacy data may be limited [ , ] . pregnant women and their infants are two vulnerable population groups, particularly in sub-saharan africa, who -amongst other infectious agents -are heavily exposed to schistosoma infections. infants were thought not to be exposed enough to get infected in their first months of life, however meanwhile high infection rates in infants aged as young as months have been demonstrated [ ] . schistosoma infection causes nutritional and hematologic deficits and overt pathology caused by chronic inflammation to eggs trapped in the bladder and urogenital tissues and can lead to granulomatosis and progress to fibrosis and cancer at the late stage. approximately million women are suffering from female genital schistosomiasis (fgs) which is caused by embolized eggs in tissues of the uterus, cervix and the lower genital tract associated with bleeding and pain. fgs is a significant risk factor for hiv infection [ , ] . detrimental effects of maternal schistosomiasis during pregnancy on the foetus may lead to poor birth outcomes including low birth weight, preterm delivery and maternal anaemia [ , , ] . schistosomiasis is a large contributor to anaemia and undernutrition and is per se unfavourable for the developing foetus. in , the who recommended that all pregnant women positive for schistosoma should be treated with pzq either individually or as part of mass drug administration (mda) programs during the second and third trimester [ , ] . adoption of the recommendation and implementation by national disease control programs was however delayed in most african countries, due to the side-effects of pzq and the lack of safety data in pregnant women and unborn babies. first results from randomised controlled trials with pzq in pregnancy meanwhile have provided evidence for the safety of pzq also in newborns [ , ] . nonetheless, pregnant women, unborn babies and newborns are vulnerable populations and there is a reluctancy to include them in pzq (mass) treatment campaigns as this would imply unnecessary exposure to pzq (even of pregnant women and infants that are not infected). in addition, logistical and financial obstacles have delayed implementation of pzq preventive chemotherapy in pregnant women at country levels. accordingly, innovative patient-centred approaches, combining accurate schistosomiasis diagnosis and targeted treatment during pregnancy, are needed to control schistosomiasis in this population. in gabon, s. haematobium is the major prevalent schistosoma species and s. intercalatum [ ] is present only at a minimal level. according to the recommended who strategy, in a total of . individuals would have required preventive schistosomiasis treatment in gabon alone [ ] . despite being an upper middle-income country [ ] , there is no effective national control program for schistosomiasis established in gabon, but rather the national program for neglected tropical diseases (ntd) which includes schistosomiasis and other neglected diseases. a recently published study indicated that approximately % of the pregnant women living in lambaréné and fougamou vicinities tested positive for s. haematobium infection with increased odds (or . , % ci . - . ) for low birth weights of the newborns [ , ] . as it is true for most of the observational and interventional studies on schistosomiasis, the power of the study was weakened due to the low sensitivity of the reference schistosomiasis diagnosis applied (egg microscopy), and one might assume that a considerable proportion in the control group were misclassified as negative. diagnostic tests that are highly sensitive and specific are essential for the detection of schistosoma infections and are urgently needed in a variety of applications such as a test-and-treat strategy to control schistosomiasis in pregnancy and as tools to determine efficacy of new interventions in clinical trials. detection of schistosoma circulating antigens is becoming an important schistosoma diagnostic strategy. circulating anodic antigen (caa) and circulating cathodic antigen (cca) are constantly released by schistosomes into the host circulation and day to day fluctuation of caa in serum is relatively little [ ] . furthermore, caa levels are correlating with the number of worms [ , ] . as caa and cca have been shown to clear within a few days or weeks after pzq treatment [ , , ] , assays measuring antigen levels are promising techniques to more accurately assess drug efficacy. particularly, the detection of circulating anodic antigen (caa) by the up-converting phosphor-based lateral flow (ucp-lf) technology has proven to be a highly sensitive and specific diagnostic tool for schistosomiasis [ ] [ ] [ ] . the high sensitivity and specificity of the ucp-lf caa test is warranted by the uniqueness of the antigen to the schistosoma genus, in combination with specific, high affinity monoclonal antibodies, a carbohydrate specific sample pre-treatment step and a unique background free reporter technology [ , , ] . the overall aim of this study is to evaluate the accuracy of the ucp-lf caa urine test for the detection of s. haematobium infections in pregnant women and to validate caa as an endpoint measure for pzq efficacy. this study is part of the fast and reliable easy-to-use diagnostics for eliminating bilharzia in young children and mothers (freebily) project which aims to thoroughly evaluate the use of cca and caa antigen tests for the diagnosis of schistosoma infections in pregnant women and their newborns [ ] . the freebily project is complemented by an additional trial performed in madagascar that investigates a cca-based test-and-treat strategy integrated into routine maternal and child primary health care programmes. the freebily gabon-study consists of a set of interlinked prospective, observational sub-studies (a-c), each targeted to assess a specific objective (see fig. for a schematic trial design, procedures and stages). sub-study a is a prospective, cross-sectional, observational study conducted in pregnant women in lambaréné and surroundings to determine the sensitivity and specificity of the ucp-lf caa test (index test) for the detection of s. haematobium infections in urine samples. sampling will include standardized urine collection on consecutive days for s. haematobium detection by egg microscopy and ucp-lf caa testing; qpcr and poc-cca will be done on one urine sample. to control for concomitant parasite infections one stool sample (within the days of urine sampling) will be collected to screen for s. intercalatum and soil-transmitted helminths infections, as well as one blood sample will be taken for filaria and plasmodia spp. detection. the blood sample will also be used to measure anti-schistosoma antibodies and to provide blood cell counts and haemoglobin levels. the primary objective of sub-study a is to compare the ucp-lf caa urine test against a composite diagnostic reference including egg microscopy, qpcr, and serology in order to determine ucp-lf caa test performance. sub-study a will allow to identify participants for sub-study b. sub-study b is an observational, follow-up study of at least pregnant women identified in sub-study a as being infected with s. haematobium (by egg microscopy and/or urine ucp-lf caa test). schistosoma-infected women will be randomised : ( : ) for immediate pzq treatment during the pregnancy (single dose of mg/kg) or delayed pzq treatment after delivery (control group). the control group will not be treated during pregnancy (to monitor "natural" caa level fluctuation in a participant if not treated), but months after delivery. the primary objective is to determine the kinetics of caa after treatment during pregnancy compared to the control group. the control group serves also as a safety control of pzq administration to pregnant women and exposure of their offspring (s). therefore, both groups (treatment and control) will be actively followed-up and urine will be collected on day , day and day after pzq treatment and then once a week until both egg microscopy as well ucp-lf caa assay become negative but no longer than weeks after pzq treatment. the secondary objective is to collect data on safety of pzq during pregnancy. for details on the safety investigation, see below (treatment, adverse events and safety assessment). sub-study c will follow up infants born to mothers included in sub-study a and their mothers for a maximum of months (n = ). this is an observational, longitudinal study with the primary objective to determine the incidence of s. haematobium infection and age of first s. haematobium infection in infants. therefore, after delivery, mother and her infant will be asked to provide urine every months until the ucp-lf caa test becomes positive for s. haematobium in the infant. children who test s. haematobium positive will be in addition, a customized questionnaire addressing the knowledge, attitude and practice (kap) of schistosomiasis of mothers and infants will be asked. health-related quality of life (hrqol) will also be measured by using a standardized tool: this will allow filling a wide literature gap, where only very few studies have attempted to measure hrqol in schistosomiasis endemic areas [ ] . to our knowledge, this study will be the first one collecting information on hrqol among pregnant women in areas endemic of schistosomiasis and of other parasitic infections such as malaria. the study is planned to take place in two areas located in the center of gabon: lambaréné including vicinities (bindo, makouké and nzilé-pk villages) and fougamou. both regions are endemic for s. haematobium with the prevalence ranging from to %, depending on the area or the population age group [ , [ ] [ ] [ ] . the study population are pregnant women and their newborns. the study protocol and informed consent forms (icf) were approved by the national ethics committee of gabon (prot n° / /sg/cne) and by the institutional ethics committee at cermel/gabon (n° / ). written informed consent will be sought from any volunteer or the legal guardian of minor volunteers before any study procedure will be performed. prior to enrolment, the study will be explained to the pregnant womenand legal guardian if minor -presenting to the antenatal care centres (anc) in lambaréné (albert schweitzer and regional georges rawiri hospitals). those willing to participate will be provided with the informed consent form (icf) for signature for themselves and for their unborn child. particular emphasis is given to information on schistosoma transmission, diagnosis, associated pathologies and risk factors for schistosoma infection in particular, and to intestinal helminths in general. inclusion criteria are pregnancy at gestational age between and weeks (based on last date of menses), providing signed icf, willing to deliver in one of the two maternities in the study areas and no plans to move out of the study area during the following months. exclusion criteria are reporting of complicated previous pregnancy, known chronic infections or diseases as hiv, hbv, hbc infections. following the icf process, a kap questionnaire will be administered to collect standard demographic, health history, obstetrical data and information on schistosomiasis related knowledge, attitude and practice.. economic and healthrelated quality of life (hrqol) questionnaire will be included as well. the questionnaire will be used to measure hrqol among all pregnant women at enrollment and to monitor hrqol among the positive women over time. for this, the hrqol questionnaire will be administered again to positive women week after enrollment when test results are available and treatment provided (at a ) and a few months later, well after treatment completion (at c ). physical examination will be done by the study physician; participants will be asked to provide urine (midstream urine will be collected in a clean container), stool and blood samples for parasite diagnostics and health care service. at delivery, newborns will be weighted, and gestation age will be recorded. the newborn's anthropometry parameters, apgar score at and min, maternal bleeding volume, placenta quality and weight will be collected. for details on study visits over time with respect to the study phases and details of sample collection see additional file . the index test is the ucp-lf strip test detecting and quantifying caa levels. lf strips can be analysed with poc care readers [ ] or, as used in this study, with a multistrip benchtop reader (upcon; labrox oy, turku, finland) capable of analysing strips at a time. the ucp-lf caa test was developed at leiden university medical center (leiden, the netherlands) [ , ] . so far, the tests are produced in-house at lumc in a batch-wise manner (ca. tests per batch), fully quality-controlled using standardized reference samples, ensuring specificity and sensitivity. a dry reagent format is available that can be stored and transported at ambient temperature [ ] . here, μl of fresh or stored (− °c) urine will be tested by the ucp-lf caa urine test following published procedures [ ] including a concentration step (centrifugal amicon ultra . ml filters with a kd cut-off; merck millipore) after pretreatment of the urine with / volume of % trichloroacetic acid and (ucaaht format) [ ] . all measurements will include a set of caa-spiked standards into a negative urine to determine the threshold of positivity that is set to ≥ pg/ml. schistosoma egg detection by microscopy is commonly used for the diagnosis of schistosomiasis as recommended by who and diagnostic procedures will follow standardized protocols [ ] . urine ( ml) will be passed through a μm filter (whatman nucleopore) using a syringe. the filter will then be placed on a glass slide and read under a light microscopy ( x magnification). every slide will be read by two independent microscopists. the test will be considered positive if at least one s. haematobium egg is found and egg counts will be given (n eggs/ ml urine). qpcr schistosoma egg dna will be detected by real-time pcr (qpcr) on urine samples following a published protocol [ ] . we will use the established schistosoma genusspecific primers (ssp f and ssp r) and the dual labelled probe ssp t to amplify a -bp fragment of the internal transcribed spacer- (its ) sub-unit. an internal control will be added to monitor efficient dna extraction. dna extraction from urine and qpcr assay will be performed. antibodies against s. haematobium will be measured using a lateral flow assay (ucp-lf antibody assay) that is based on the same technology as the ucp-lf caa test and can be evaluated using the same ucp labrox reader. the serology test is designed to detect antibodies against soluble egg antigen (sea) and soluble crude cercarial antigen preparation (scap) in sera of participants [ , ] . the point-of-care (poc) cca urine test is a commercially available poc rapid test for the detection of cca in urine (rapid medical diagnostics, south africa). this lf immuno-chromatography test delivers qualitative detection of active schistosoma infection, and is recommended and generally applied to detect s. mansoni infections performing less in s. haematobium settings [ , ] . it is ideally suited for field conditions as it does not require laboratory facilities or highly trained personnel. to control for the impact of concomitant other parasite infections on the accuracy of ucp-lf caa, stool samples will be investigated for s. intercalatum (as well as any other schistosoma species) and for other highly prevalent helminths such as ascaris lumbricoides, trichuris trichuria, hookworms, and strongyloides stercoralis. stools will be examined by kato-katz and coproculture techniques [ , ] . therefore, approximately mg of fresh stool will be processed. microscopy reading ( x magnification) is done by two independent readers and numbers of eggs per gram of stool will be reported according to who guidelines [ , ] . remaining stool will be stored for later confirmation by respective qpcr [ ] . blood samples will be examined for infections with malaria parasites (plasmodium falciparum, non-falciparum) and filarial parasites (loa loa, mansonella perstans) by standard diagnostic procedures (giemsa-stained thick blood smears, saponin leucoconcentration assay followed by microscopy, respectively) [ , ] . women infected with schistosoma (positive by egg microscopy and/or ucp-lf caa) will be treated with pzq either during pregnancy (as recommended by who) or after delivery. single dose pzq ( mg/kg) will be administered by an unblinded member of the study team under observation. safety of pzq administration in women will be assessed verbally within h (after h and h later) to record all adverse events occurring after drug administration. all adverse events will be assessed by the study physician, following local guidelines and will be graded as mild, moderate or severe. all investigators (study physicians and lab technicians) will be blinded to the treatment allocation. other parasitic infections will be treated following national treatment guidelines. this work will be done in collaboration with the gabonese national program for the fight against ntds who will contribute to the pzq supply. maternal health outcomes, maternal anaemia (defined as hb < g/dl) at inclusion and at delivery will be investigated. in offspring, birthweight (with low birthweight defined as weight at birth < . kg) and small for gestational age (used as an indicator for possible intrauterine growth restriction) will be investigated. in addition, the exposure to pzq during pregnancy will be evaluated in infants at the age of and months. time to sit, crawl, stand and walk, incidence of illnesses and vaccine coverage during the follow-up will be recorded. an independent data safety and monitoring board (dsmb) has been established which will review safety data in the sub-study b during the study. analysis of the ucp-lf caa (ucaaht ) test performance will be compared against a composite diagnostic reference (i.e. by latent class analysis). assuming a % egg positivity rate in the study population [ ] , about pregnant women should be tested to obtain schistosoma positive women. assuming further a sensitivity of the index test (ucp-lf caa) between and % with infected women, statistical precision (two-sided % confidence limit) of ± . % will be achieved if a sensitivity of % is observed and a lower limit of . % if a sensitivity of % is observed. due to the limited sensitivity of the egg detection test, to estimate the specificity, only study participants negative for reference tests should be included. furthermore, assuming a true schistosoma prevalence of % (based on a conservative estimate of % sensitivity of the egg test) and assuming a false positive rate of % of the other two reference tests (pcr and ucp-lf antibody test), at least women will enter the naïve estimate of specificity. this will lead to a statistical precision of between ± . and ± . % depending on the observed specificity. for sub-study b, the analysis is based on the assumption of a non-inferiority delta of . , identical probabilities of positivity and negativity at the end of the sub-study b for both tests (ucp-lf caa and egg microscopy) and a discordance of %, with pregnant women, the power will be % to show non-inferiority (type error: . % one-sided). all data will be collected on paper forms which will be entered into a redcap database [ ] . initial data will be double entered in redcap to ensure their accuracy. each participant included into the study will receive a unique identifier. the case report from (crf) is the source documents for personal data, clinical data and laboratory result sheets will be considered as source documents for laboratory results reported in the crf. analysis of the ucp-lf caa (ucaaht ) test performance will be compared against a composite diagnostic reference by using latent class analysis or other bayesian approaches which will allow to compare the estimated sensitivity, specificity, positive predictive value (ppv), negative predictive value (npv) and the accuracy values for detecting schistosoma in the sample. the model will be fitted to determine the bayesian information criteria (bic), akaike information criteria (aic), the chi square test and the standardized residuals. to assess the effect of pzq treatment on birth weight, analysis of covariance (ancova) will be used. this is a fundamental statistical method of analysis for both randomised and non-randomised studies. it is a regression analysis which includes parameter estimates for observed covariates in addition to the treatment effects we are ultimately interested in estimating. there is no vaccine against schistosomiasis so far, and mda of pzq remains important for the control of schistosomiasis but is not sufficient when moving towards the goal of schistosomiasis elimination. the ideal test for test and treat strategies would be an easy to use point of care test, which is highly sensitive and specific for all schistosoma species (without the necessity to distinguish between the species as the treatment is the same for all species) and used material that does not require invasive procedures (e.g. urine or saliva instead of blood). microscopic methods lack sensitivity and require a lot of resources time and trained personnel. serology cannot distinguish between an acute, ongoing and past infection and pcr methods require advanced microbiological facilities and capabilities. antigen tests, however can provide easy to use, field suitable tests [ , , ] . there is a need of sensitive and accurate schistosoma diagnostics to support communities in need and also control programs. the freebily gabon study will comprehensively evaluate the performance of the ucp-lf caa laboratory test with the focus on pregnant women and its extended usefulness as a read-out measure for pzq efficacy. the ucp-lf-caa test is highly sensitive and specific and also performs well for urogenital schistosomiasis. unfortunately, this test is not currently available as a point of care test. the assessment of this test in pregnant women in a s. haematobium endemic regions with weekly follow up of initially positive pregnant women as well as in infants to validate the caa antigen test as an endpoint for measuring pzq efficacy. determining the true efficacy of pzq against different schistosoma infections is important for schistosomiasis control programs. it informs on the susceptibility of local schistosoma infections towards pzq as well as providing information for future efficacy trials for paediatric formulations of pzq. furthermore, using the ucp-lf-caa test minimizes sample size in future efficacy trials as caa levels are relatively constant in serum or urine, whereas the number of eggs is subjected to highly random daily fluctuations. the ucp-lf-caa test clearly demonstrates that prevalence of active schistosoma infections is underestimated by a factor of up to ten when compared to egg microscopy. especially in low-endemic settings, the ucp-lf-caa test is superior over parasitological methods. this test can easily identify individuals with very low levels of infection as well as early infections and is well-suited for determining efficacy (or even failure) of treatment. the test is ready to be taken up by industry for upscaling and deployment at large scale. so far, there is no alternative for evaluating long-term mda programmes, and moving towards post-mda surveillance or elimination. beyond the study, this work will prepare for the development of caa rapid diagnostic tests, that can play a role in the future as a test-based schistosomiasis treatment strategy in pregnancy in endemic regions. furthermore, the study tackles a neglected disease (schistosomiasis) and provides a concept to overcome the neglect of pregnant women and small children by schistosomiasis control programmes. schistosomiasis and water resources development: systematic review, meta-analysis, and estimates of people at risk reassessment of the cost of chronic helmintic infection: a meta-analysis of disability-related outcomes in endemic schistosomiasis human schistosomiasis the global status of schistosomiasis and its control schistosomiasis 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a threonine-linked polysaccharide consisting of --> )-(beta-d-glcpa-( --> ))-beta-d-galpnac-( --> repeating units fast and reliable easy-to-use-diagnostics for eliminating bilharzia in young children and mothers: the freebily project health-related quality of life among school children with parasitic infections: findings from a national cross-sectional survey in côte d'ivoire schistosoma haematobium effects on plasmodium falciparum infection modified by soil-transmitted helminths in school-age children living in rural areas of gabon assessment of the effect of schistosoma haematobium co infection on malaria parasites and immune responses in rural populations in gabon: study protocol demography, maternal health and the epidemiology of malaria and other major infectious diseases in the rural department tsamba-magotsi feasibility of a lateral flow test for neurocysticercosis using novel up-converting nanomaterials and a lightweight strip analyzer improved sensitivity of the urine caa lateral-flow assay for diagnosing active schistosoma infections by using larger sample volumes circulating anodic antigen (caa): a highly sensitive diagnostic biomarker to detect active schistosoma infections-improvement and use during score basic laboratory methods in medical parasitology . world health organization application of a circulating-cathodic-antigen (cca) strip test and real-time pcr, in comparison with microscopy, for the detection of schistosoma haematobium in urine samples from ghana refining diagnosis of schistosoma haematobium infections: antigen and antibody detection in urine who | bench aids for the diagnosis of intestinal parasites. who a five-country evaluation of a point-of-care circulating cathodic antigen urine assay for the prevalence of schistosoma mansoni a modified agar plate method for detection of strongyloides stercoralis who | prevention and control of schistosomiasis and soil-transmitted helminthiasis: who technical report series n° . who studies on the laboratory diagnosis of human filariasis: preliminary communication limit of blank and limit of detection of plasmodium falciparum thick blood smear microscopy in a routine setting in central africa research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support evaluation of circulating cathodic antigen (cca) urine-cassette assay as a survey tool for schistosoma mansoni in different transmission settings within bugiri district notes on the use of urine-cca dipsticks for detection of intestinal schistosomiasis in preschool children publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we acknowledge support by open access publishing fund of university of tübingen. the freebily-consortium (www.freebily.eu) consists of partner institutions, including from africa, and from europe, represented by the fol- received: july accepted: september recruitment of study participants has started in april and is expected to be finished by april : follow-up of sub-study b is expected to be finished by july and of sub-study c by the end of . supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file . overview of study visits over time with respect to the study phase and details of sample collection, describes the study phases and procedures. this work is supported by the european and developing countries clinical trials partnership (edctp-ria mc- ). the funders had no role in study design, decision to publish the study protocol, or preparation of the manuscript. we acknowledge support by open access publishing fund of university of tübingen. open access funding enabled and organized by projekt deal.availability of data and materials not applicable. the study (study protocol version , june ) was reviewed and approved by the national ethics committee of gabon (prot n° / /sg/ cne) and by the institutional ethics committee at cermel/gabon (n° / ). only participants given written informed consent (or the legal representative if minor) are enrolled in the study. the centre de recherche médicale de lambaréné, gabon, has the trial's sponsorship. not applicable. the authors declare that they have no competing interest.author details centre de recherches médicales de lambaréné, lambaréné, gabon. institut für tropenmedizin, universität tübingen, tübingen, germany. department of parasitology, leiden university medical center, leiden, the netherlands. german center for infection research (dzif), tübingen, germany. department of infectious diseases, division of internal medicine, academic medical center, university of amsterdam, amsterdam, the netherlands. key: cord- -i e g te authors: liu, wen-kuan; liu, qian; chen, de-hui; tan, wei-ping; cai, yong; qiu, shu-yan; xu, duo; li, chi; li, xiao; lin, zheng-shi; zhou, rong title: epidemiology of hbov infection and relationship with meteorological conditions in hospitalized pediatric patients with acute respiratory illness: a -year study in a subtropical region date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: i e g te background: human bocavirus (hbov ) is an important cause of acute respiratory illness (ari), yet the epidemiology and effect of meteorological conditions on infection is not fully understood. to investigate the distribution of hbov and determine the effect of meteorological conditions, hospitalized pediatric patients were studied in a subtropical region of china. methods: samples from , hospitalized pediatric patients (≤ years old), with ari were tested for hbov and other common respiratory pathogens using real-time pcr, between july and june . in addition, local meteorological data were collected. results: of the , patients tested, ( . %) were positive for at least one respiratory pathogen. two hundred forty-eight of , ( . %) were positive for hbov infection. co-infection was common in hbov -positive patients ( . %, / ). a significant difference in the prevalence of hbov was found in patients in different age groups (p < . ), and the peak prevalence was found in patients aged – months ( . %, / ). two hbov prevalence peaks were found in summer (between june and september) and winter (between november and december). the prevalence of hbov was significantly positively correlated with mean temperature and negatively correlated with mean relative humidity, and the mean temperature in the preceding month had better explanatory power than the current monthly temperature. conclusions: this study provides a better understanding of the characteristics of hbov infection in children in subtropical regions. data from this study provide useful information for the future control and prevention of hbov infections. human bocavirus (hbov ), which belongs to family parvoviridae, was firstly identified in respiratory secretions of children with respiratory tract disease in [ , ] . hbov has been confirmed as an important respiratory pathogen and is found in respiratory infections in children and adults worldwide. the prevalence of hbov nucleic acid detection varies from . to % in patients with acute respiratory illness (ari), according to different studies [ ] [ ] [ ] [ ] [ ] . serological and nucleic acid test results are generally consistent [ ] [ ] [ ] [ ] , showing hbov infection is very common. hbov can cause both upper respiratory illness (uri) and lower respiratory illness (lri) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . infection with hbov can lead to development of a cough, rhinitis, fever and other common clinical symptoms [ , ] . in some cases, it can cause respiratory distress, hypoxia, wheezing and other severe respiratory symptoms [ , ] . clinical diagnosis is mainly pneumonia, bronchitis, pneumothorax, mediastinal emphysema and otitis media and other complications [ ] [ ] [ ] [ ] [ ] . in some cases, patients develop severe respiratory injury symptoms, which can be fatal [ , ] . hbov can be detected in fecal samples [ ] , blood samples [ , ] , urine [ , ] , cerebrospinal fluid [ ] [ ] [ ] , river water [ ] and sewage [ , ] , indicating that hbov may be associate with a variety of diseases. current in vitro studies modeling tissue-like airway epithelial cells cultures show hbov infection can lead to disruption of the tight-junction barrier, loss of cilia and epithelial cell hypertrophy [ ] [ ] [ ] , similar to lung injury tissue changes in vivo. there is currently no vaccine or specific treatment for this virus; prevention and treatment of hbov -related diseases still require further research. the prevalence of respiratory viruses is associated with many factors, including local climate, which may impact the survival and spread of the viruses [ ] . studying the epidemiology of hbov and its relationship with meteorological conditions will improve diagnosis, treatment, control and prevention of this virus. in this study, we investigated the epidemiology of hbov infection in children (≤ years old) hospitalized with ari in a subtropical region in china over a -year period. in addition, we collected climate data to determine if there was a relationship between hbov prevalence and meteorological conditions. this study will add to existing epidemiological data on hbov and its relationship with climate conditions in subtropical regions and will play a positive role in hbov control and prevention. the study sites were three tertiary hospitals in guangzhou, southern china (longitude: e ° ′ to e ′; latitude n ° ′ to n ° ′). inclusion criteria were pediatric patients (≤ years old) who presented with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, rhinitis, dyspnea or who were diagnosed with pneumonia by chest radiography during the previous week. chest radiography was conducted according to the clinical situation of the patient. throat swab samples were collected from the enrolled patients between july and june for routine screening for respiratory viruses, mycoplasma pneumoniae (mp), and chlamydophila pneumoniae (cp). the samples were refrigerated at - °c in viral transport medium, transported on ice and analyzed immediately or stored at − °c before analysis, as described previously [ , ] . meteorological data for guangzhou, were collected from july to june , from the china meteorological administration, including the monthly mean temperature (°c), mean relative humidity (%), rainfall (mm), mean wind speed (m/s), mean air pressure (hpa), mean vapor pressure (hpa), sunshine duration (h). real-time pcr for hbov and common respiratory pathogen detection dna and rna were extracted from the respiratory samples using the qiaamp dna mini kit and qiaamp viral rna mini kit (qiagen, shanghai, china), respectively, in accordance with the manufacturer's protocols. taqman real-time pcr for hbov was designed based on the conserved region of the np gene, as described previously [ ] . common respiratory pathogens, including respiratory syncytial virus (rsv), influenza a virus (infa), influenza b virus (infb), four types of parainfluenza (piv - ), adenovirus (adv), enterovirus (ev), human metapneumovirus (hmpv), four strains of human coronavirus (hcov- e, oc , nl and hku ), human rhinovirus (hrv), mp and cp were detected simultaneously as previously reported [ ] . data were analyzed using chi-squared test and fisher's exact test in spss . (spss inc., chicago, il, usa). correlation with climate data was analyzed using multiple linear regression analysis. all tests were two-tailed and a p value < . was considered as statistically significant. eleven thousand three hundred ninety-nine pediatric patients (≤ years old) hospitalized with ari were enrolled in the study between july and june . the male-to-female ratio was . : ( : ) and the median age was . years (interquartile range . - . ). overall, . % ( / ) of patients were under the age of years. all the , patients were tested for all pathogens mentioned, and ( . %) were positive for one or more of those pathogens (table ) , and had a median age of . years (interquartile range . - . ). the male-to-female ratioes were . : ( : ) in pathogen-positive patients and . : ( : ) in pathogen-negative patients (p = . ). two hundred forty-eight of , patients ( . %) tested positive for hbov infection. of the hbov -positive patients, ( . %) were co-infected with other pathogens, most frequently with rsv ( . %, / ) ( table ). the median age was year (interquartile range . - . ). the male-to-female ratio was . : ( : ) in hbov -positive patients and . : ( : ) in hbov -negative patients (p = . ). to clarify the age distribution of hbov , patients were divided into seven age groups; - months, - months, - months, - years, - years, - years and - years old. there was a significant difference in the prevalence of hbov in patients in different age groups (p < . ) and the peak prevalence was found in patients aged - months ( . %, / ) (fig. ) . in this study, we monitored the prevalence of hbov in patients (≤ years old) hospitalized with ari from july we collected meteorological data for guangzhou, including monthly mean temperature, mean relative humidity, rainfall, mean wind speed, mean air pressure, mean vapor pressure and sunshine duration for a -year period, to explore the correlation between meteorological conditions and prevalence of hbov . guangzhou, which is located in southern china (longitude ° ′ to ° ′, latitude ° ′ to ° ′), has a maritime subtropical monsoon climate. between july and june , the mean temperature was . ± . °c (mean ± standard deviation), humidity was . ± . %, sunshine duration was . ± . h, wind speed was . ± . m/s, rainfall was . ± . mm, air pressure was . ± . hpa and vapor pressure was . h ± . hpa. between and , the mean temperature from may to september was greater than °c (fig. ) . for multiple linear regression analysis of hbov prevalence and meteorological conditions correlation, independent variables of mean air pressure (adjusted r = . , p < . ) and mean vapor pressure (adjusted r = . , p < . ), which linearly associated with mean temperature, and rainfall (adjusted r = . , p < . ), which strongly correlated with mean relative humidity, were excluded. the independent variables for the final multiple linear regression analysis included mean temperature, mean relative humidity, mean wind speed and sunshine hours. the effect of temperature had a delay therefore mean temperature in the preceding month (mean temperature month before) was also included as an independent variable in the analysis ( table ) . both regression models were established (p < . ) and the adjusted r values were . and . in the mean temperature in the preceding month model and the current monthly temperature model, respectively. hbov prevalence was positively correlated with temperature (coefficient = . in the current temperature model (p = . ), coefficient = . in mean temperature in the preceding month model (p < . )). conversely, hbov prevalence was negatively correlated with relative humidity (coefficient = − . in the current temperature model (p = . ), coefficient = − . in the temperature delay model (p = . )) ( table ). ari is one of the most common human diseases, predominantly caused by different respiratory viruses [ , ] . one of these viruses, hbov infection, causes global epidemics, has a high public health burden and circulates with different patterns in different areas [ ] [ ] [ ] [ ] [ ] ] . in general, the prevalence of viruses varies because of factors such as multiple linear regression analysis was performed using hbov monthly prevalence as the dependent variable, monthly mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed and sunshine duration as the independent variables data captured in bold are highly significant geographical location, climatic conditions, population and social activity [ ] . epidemiology of hbov in temperate regions has been described in more detail and a high incidence of infection has been observed in children under the age of years in winter and spring [ , , , ] . to describe the epidemiology of hbov in guangzhou, we collected throat swabs from , children (≤ years old), hospitalized with ari and monitored hbov and other common respiratory pathogens over a -year period (table ). in the current study, . % ( / ) of patients were under the age of years, with a median age of . years, indicating that infants and young children were most at risk of ari, consistent with previous reports [ , ] . overall, . % ( / ) of patients tested positive for one or more respiratory pathogens, . % ( / ) of patients were tested with hbov infection (table ) . a higher prevalence of hbov was detected in male patients compared with female patients (p = . ), consistent with previous reports [ , , , ] . co-infection with hbov and other pathogens is common [ , ] . in our study, . % ( / ) of hbov -positive patients also tested positive for other pathogens (table ). this may be partly caused by coinciding epidemics of hbov and other pathogens. in our study, the hbov seasonal distribution and total positive pathogen distribution were consistent, confirming this inference (fig. ) . current research shows that hbov infection can lead to the collapse of the first line of defense of airway epithelium [ ] [ ] [ ] , which may lead to a higher susceptibility to other pathogens, explaining the high rate of co-infection. whether co-infection leads to more severe disease is currently unknown and more research is needed to determine this. the characteristics of the hbov infection are likely to be a good model for studying the effects of co-infections. in this study, there was a significant difference in prevalence of hbov in patients of different ages (p < . ). the majority of hbov infections occurred in patients under years old and the peak frequency of hbov infection occurred in patients aged - months (fig. ) , consistent with previous serological and epidemiological reports on the virus [ - , , , , ] . this might be because children's immune systems are still under development and maternal antibodies gradually disappear in this age group. the distribution of hbov in patients of different ages will provide important reference for future vaccines and new drug research and development, as well as providing important data for disease prevention and control. many factors affect the epidemiology of pathogens, such as geographical location and local climate. guangzhou, a central city and main transport hub in southern china, is located in a subtropical region. guangzhou is hot and has high annual rainfall, long summers, short winters and the annual precipitation and high temperature are almost in the same period (fig. ) . in this study, two hbov peaks were observed (fig. ) . the large prevalence peaks of hbov infection occurred between june and september of each year, which are the summer months in guangzhou, with mean temperatures of higher than °c (fig. ) . small peaks of hbov infection occurred in winter, between november and december of each year. this seasonal distribution is similar to the prevalence in subtropical regions reported previously [ ] , but different from the hbov epidemics in temperate regions, which mostly occur in winter and spring [ , , , ] , as well as from tropical regions, such as india, where no obvious epidemic season has been found [ ] . to analyze the correlation between hbov prevalence and meteorological conditions, multiple linear regression analysis was performed, with hbov monthly prevalence as the dependent variable and mean temperature (or mean temperature in the preceding month), mean relative humidity, mean wind speed and sunshine duration as the independent variables (table ) . both regression models were established (p < . ) and the adjusted r value ( . ) of the temperature dorp month model was greater than the adjusted r value ( . ) of the current monthly temperature model, indicating that the temperature dorp month model had better explanatory power than the current monthly temperature model. both of the models showed that the prevalence of hbov was significantly correlated with temperature and relative humidity ( table ). in detail, hbov prevalence was positively correlated with temperature, that is consistent with previous reports [ , ] . conversely, hbov prevalence was negatively correlated with relative humidity, this was different from a previous report in suzhou [ ] , which may be related to guangzhou high humidity (mean monthly relative humidity was . ± . %) (fig. ) . it is common for pathogen prevalence to fluctuate over time because of a variety factors. in this study, hbov prevalence was relatively low in to . it might be partly related to the relatively higher mean relative humidity during this period (fig. ) . climate conditions may impact the survival and spread of respiratory viruses, however no significant linear relationship between hbov infection and wind speed or sunshine duration were found in this study (p > . ) ( table ) . some limitations of this study should be noted. first, because our study mainly focused on hbov circulation in hospitalized patients with ari, hbov in outpatients and the asymptomatic population were not included. second, many factors can affect virus epidemics, meteorological data analysis alone may not serve as a final conclusive interpretation. third, the study was only conducted in three hospitals and may not be representative of the overall population. our study has provided a better understanding of the epidemiology of hbov in subtropical regions, specifically correlations with climate data; these data will be helpful for future control and prevention of hbov infections. cloning of a human parvovirus by molecular screening of respiratory tract samples human parvoviruses virological and clinical characterizations of respiratory infections in hospitalized children detection of human bocavirus type infection in panamanian children with respiratory illness detection of human bocavirus in nasopharyngeal aspirates versus in broncho-alveolar lavage fluids in children with lower respiratory tract infections human bocavirus detection in nasopharyngeal aspirates of children without clinical symptoms of respiratory infection detection of bocavirus in saliva of 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bocaviruses in the cerebrospinal fluid of children hospitalized with encephalitis in china detection of human bocavirus in the cerebrospinal fluid of children with encephalitis detection and quantification of human bocavirus in river water mixed viral infections causing acute gastroenteritis in children in a waterborne outbreak frequent detection of highly diverse variants of cardiovirus, cosavirus, bocavirus, and circovirus in sewage samples collected in the united states in vitro modeling of human bocavirus infection of polarized primary human airway epithelia establishment of a reverse genetics system for studying human bocavirus in human airway epithelia human bocavirus infects commercially available primary human airway epithelium cultures productively are meteorological parameters associated with acute respiratory tract infections? epidemiology of acute respiratory infections in children in guangzhou: a three-year study epidemiology and clinical presentation of the four human parainfluenza virus types global burden of childhood pneumonia and diarrhoea viral etiology of hospitalized acute lower respiratory infections in children under years of age -a systematic review and meta-analysis human bocavirus capsid messenger rna detection in children with pneumonia human bocavirus- primary infection and shedding in infants respiratory virus surveillance and outbreak investigation respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology clinical and epidemiological profiles of lower respiratory tract infection in hospitalized children due to human bocavirus in a subtropical area of china human bocavirus infection in children with acute respiratory tract infection in india clinical and epidemiologic profile of lower respiratory tract infections associated with human bocavirus we thank the study volunteers for their generous participation. we thank yinghua zhou, haiping huang, jing zhang and jing ma for their technical assistance. . the sponsors of the study had no role in the study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. key: cord- - t xyayf authors: he, hangyong; wang, hao; li, xuyan; tang, xiao; sun, bing; tong, zhaohui title: successful management of refractory respiratory failure caused by avian influenza h n and secondary organizing pneumonia: a case report and literature review date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: t xyayf backgroud: organizing pneumonia (op) is a rare complication of influenza infection that has substantial morbidity. we report the first case of op associated with avian influenza h n infection that had significant improvement with corticosteroid treatment. case presentation: a -year-old male admitted to intensive care unit because of respiratory failure. he was diagnosed as severe pneumonia caused by avian influenza h n viral infection. after initial clinical improvement supported by extracorporeal membrane oxygenation (ecmo), the patient’s condition worsened with persistent fever, refractory hypoxemia. chest x-rays and computed tomographies showed areas of consolidation and ground glass opacification. although op was suspected and mg/kg methylprednisolone was used, the patient’s condition didn’t improved considerably. an open lung biopsy was performed, and histopathological examination of the specimen was compatible with op. the patient was treated with methylprednisolone . mg/kg for days. ecmo was weaned on day , and he was discharged on day with good lung recovery. conclusions: to the best of our knowledge, this was the first case of successful management of refractory severe respiratory failure caused by avian influenza h n infection complicated with op. refractory hypoxia with clinical manifestation and radiological findings compatible with op, a differential diagnosis should be considered among patients at the second or third week of influenza h n infection, especially in patients with clinical condition deteriorated after the primary influenza pneumonia was controlled. and a steroid dose of methylprednisolone . mg/kg may be suggested for treatment of op associated with avian influenza h n infection. human infected with avian influenza a h n virus were first confirmed on march th, in china [ , ] , with high incidence of severe respiratory failure, high intensive care unit (icu) admission and mortality. the development of organizing pneumonia (op) has been reported in patients with influenza a, h n and influenza b, and a high incidence of more than % of op in influenza a h n was reported in one case series [ ] [ ] [ ] . in this report, we describe the first case of op associated with avian influenza h n infection. a year-old male, non-smoker, with a history of poultry contact days before, was admitted to emergency room with fever and cough for days (considered as day for the case timeline). physical examination showed bilateral moist crackles. laboratory tests showed white blood cell count (wbc) was . × /l (and it became to . × /l two days later), c reactive protein (crp) was . mg/l, and procalcitonin (pct) was< . ng/ml. chest x-ray and chest computed tomography (ct) showed bilateral ground-glass opacities (ggo) and consolidation (fig. ) . moxifloxacin mg daily was administered for two days. and his condition deteriorated with dyspnea and severe respiratory failure, and the blood gas analysis showed pao was mmhg under oxygen mask with a fio of . . he was transferred to our intensive care unit (icu) supported with noninvasive ventilation (niv) and intubated h later. mechanical ventilation with peak inspiratory pressure (pip) of cm h o, positive end expiratory pressure (peep) of cm h o and fio of . could not maintain the oxygenation. as his pao /fio ratio less than mmhg lasted for h, the venovenous-extracorporeal membrane oxygenation (vv-ecmo) was established. the microscopic examination, culture and galactomannan detection from serum and bronchial-alveolar lavage fluid (balf) for virus pcr, fungal, and the culture for bacteria and the microscopic examination for bacteria and tuberculosis were done at icu admission. the nucleic acid polymerase chain reaction (pcr) for influenza h n virus of sputum specimen turned out to be positive and oseltamivir phosphate was initiated ( mg twice daily for weeks after the pcr of the virus were negative for two consecutive tests.). as the pct rose to . ng/ml, and the galactomannan detection was positive ( . from serum sample and . from balf sample), vancomycin, imipenem cilastatin and caspofungin were applied. hydrocortisone mg daily for days was also used for septic shock. ecmo was weaning off on day (the time of ecmo supporting was days) when his blood flow of ecmo was decreased to less than l/min with a significant improvement on his chest x-ray. the patient turned to high fever in the following days. repeated pcr for h n virus were tested and show continuous negative in the lower respiratory samples after a week of icu admission. advanced antibiotics and antifungal agents were administered, no positive pathogenic result was emerged, and pct level remained downtrend. the chest ct on day shows bilateral ggo with aggravating consolidation on new areas (fig. ) , compatible with organizing pneumonia (op). considering no underlying cause of op existed other than virus infection, therefore, op associated with h n influenza virus infection was suspected. methylprednisolone mg ( mg/kg) daily was applied on day for days with tapering. with clinical improvement, the patient was extubated on day , and supported with niv with a fio of . . the chest ct on day showed obvious remission of consolidation with patchy ggo and fibrotic changes. however, the clinical condition of the patient deteriorated again on day with high fever to °c, refractory hypoxemia (pao :fio = ) and a mild leukopenia (wbc was . × /l). the patient was reintubated and supported with invasive mechanical ventilation. methylprednisolone mg daily was applied at the beginning as a suspicion of the relapse of op. and chest ct on day revealed progression of consolidation especially in the lower lobe. as the patient's respiratory failure and condition did not improve after days of daily use of methylprednisolone mg, histological examination was done via open lung biopsy (olb) on day , and op was confirmed with the presence of intraluminal plugs of granulation tissue within alveolar ducts and surrounding alveoli associated with chronic inflammation of the surrounding lung parenchyma. the therapy of steroid was changed to methylprednisolone mg ( . mg/kg) for days, mg for days, mg for days. the oxygenation improved, and the patient was extubated on day and discharged on day . a time line of the steroids use, white cell count and ratio of pao /fio is illustrated in fig. . the following-up for months from onset of primary virus infection showed gradually improvement, with mild interlobular septal thickening, traction bronchiectasis and consolidation in chest ct on the ninth month ( fig. ) . as shown in tables , previously published cases and the current case of op associated with influenza virus infection were reviewed [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . with available information, the age range was -year-old to year-old with % ( / ) female. influenza a constituted the majority ( %, / ), with cases were identified to h n , and the current case, h n . the other one case was op complicated with co-infection of influenza b and streptococcus. respiratory failure associated with op was reported in % ( / ) patient. fever ( %, / ), dyspnea ( %, / ), cough ( %, / ) were the most common symptoms reported. especially, clinical condition deteriorated after controlling of primary disease can be found in % ( / ) patients. ggo and consolidation were the main findings on high resolution computed tomography (hrct), shown in %( / ) and %( / ) of cases, with release of primary opacity associated with influenza infection in some cases. autopsy were applied at about two weeks and most transbronchial lung biopsy (tblb) or open lung biopsy (olb) were applied over three weeks. steroid was the main treatment, varying from prednisolone mg/day to methylprednisolone mg/day pulse therapy, with or without tapering. most patients react well to the treatment with clinical and radiological improvement, excepting the relapsing of op in our case. since the cases infected with avian influenza h n virus were first confirmed in in china [ , ] , five seasonal epidemics were observed, with an earlier start and a steep increase in infected number in the latest epidemic [ ] . in a clinical study including cases confirmed h n virus infection, . % were admitted to an icu and % died. the median time from the exposure to disease onset was five days, then, . days to acute respiratory distress syndrome (ards). in dead cases, the median time from disease onset to death was days [ ] . half of the patients required mechanical ventilation and % need ecmo for severe respiratory failure [ , ] . however, refractory severe respiratory failure caused by op secondary to avian influenza h n virus infection was first reported in this case. op was originally described by davison et al. in [ ] ( ), and in , epler et al. described the same entity as the term "bronchiolitis obliterans organizing pneumonia" (boop) [ ] . now, for avoiding confusion with bronchiolitis obliterans (bo), the term organizing pneumonia (op) is preferred [ ] . in fact, op is an nonspecific inflammatory response from human body towards acute lung injuries. it can be with idiopathic considering symptomatology, physical signs, laboratory and pulmonary function tests, radiologic or histomorphological findings, there is no obvious difference between cop and secondary op, excepting the latter may have higher mortality [ ] . op has been linked to multiple influenza viral infections including influenza a and b, but this is the first report of avian influenza h n -associated op with relapse occurrence and severe respiratory failure. the main clinical features of our avian influenza h n -associated op is similar to the cases of influenza a cases. in these cases reported, op onset mostly at the second to third week in the course of influenza, and occurred after the releasing of primary virus infection; and the op is complicated with respiratory failure, and no evidence of other pathogen was found; and the main findings on hrct for this kind of op were ggo and consolidation. biopsy were done via tblb or olb at the third week. in cases with influenza associated op, other than common findings of op, diffuse alveolar damage (dad), alveolar hemorrhage and edema and bronchiolitis can also be found, showing the lung injury of primary virus infection. however, in our case, the patient present with high fever as a main manifestation of op, and had a relapse of respiratory failure associated with op. a differential diagnosis of op should be considered at the second to third week after the primary infection among patients with influenza h n infection. furthermore, there was no clear evidence of bacterial and fungus infection during the beginning of hospitalization. however, the levels of pct and galactomannan showed significant increase after the patient was established with ecmo. the op may also be caused by the nosocomial infection which was frequently complicated with severe influenza pneumonia. the majority of patients with op show rapid responding to steroids. the introduced initial dose is prednisone . - . mg/kg, with tapering over - month. however, up to one-third patients may relapse in tapering period. for the current patient with avian influenza h n associated op, methylprednisolone mg ( mg/kg) daily was applied for days with tapering in the beginning of suspicion of op. the patient showed rapid clinical and radiological improvement and was extubated on the fifth day of steroids applying. however, the patient deteriorated with high fever and refractory hypoxemia days later. with the confirmation of histological examination, steroid dose was increased to methylprednisolone mg ( . mg/kg) daily for days. and op was finally controlled without relapsing in follow-ups. insufficient initial dose of steroid may contributed to the relapsing of op before final diagnosis. therefore, post avian influenza h n infection op may responsive to a brief course (weeks) of moderate-to-high dose prednisone therapy. to the best of our knowledge, this was the first case of op associated with avian influenza h n infection. with clinical manifestation and radiological findings compatible with op, a differential diagnosis should be considered among patients with influenza h n infection at the second or third week after the initial viral infection, especially in patients with clinical condition deteriorated after controlling of primary influenza pneumonia. and a steroid dose of methylprednisolone . mg/kg maybe suggested for treatment of op associated with avian influenza h n infection. our case provide clinical insight into refractory respiratory failure with lung involvement due to avian influenza h n . op should be considered on the differential diagnosis of patients with fever and respiratory failure after severe influenza a h n infection, where steroids might be useful. human infection with a novel avian-origin influenza a (h n ) virus human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome organizing pneumonia in patients with severe respiratory failure due to novel a (h n ) influenza influenza a-associated bronchiolitis obliterans organizing pneumonia mimicking wegener's granulomatosis influenza b/streptococcal coinfection complicated by organizing pneumonia chest ct findings of influenza virus-associated pneumonia in adult patients. influenza other respir viruses clinicopathological findings of four cases of pure influenza virus a pneumonia influenza a (h n ) virus-associated pneumonia: high-resolution computed tomography-pathologic correlation influenza a (h n ) organising pneumonia organizing pneumonia associated with swine-origin influenza a h n viral infection sudden increase in human infection with avian influenza a(h n ) virus in china clinical findings in cases of influenza a (h n ) virus infection cryptogenic organizing pneumonitis bronchiolitis obliterans organizing pneumonia european respiratory society international multidisciplinary consensus classification of the idiopathic interstitial pneumonias organizing pneumonia: a kaleidoscope of concepts and morphologies publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations no acknowledgement. all authors made substantial contributions to conception and design, or acquisition of data, or analysis and interpretation of data; reviewed and approved the final manuscript; and contributed significantly to this study. hh takes full responsibility for the integrity of the submission and publication, and was involved in study design. hw and hh had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis, and was responsible for data verification, analysis and drafting of the manuscript. bs, zt had full access to all the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. xt and xl were responsible for the data collection. all authors read and approve the final manuscript. not applicable.availability of data and materials not applicable. we did not commence any experimental use of a novel procedure or tool in this case, and all therapy was approved by an ethics committee of our hospital. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor of this journal. the authors declare that they have no competing interests. key: cord- -cxqzac a authors: huang, weiwei; yang, yinhui; zhang, xinlei; zhao, changan; yin, aihua; zhang, xiaozhuang; he, zhengxin; jiang, yongqiang; zhang, liang title: an easy operating pathogen microarray (eopm) platform for rapid screening of vertebrate pathogens date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: cxqzac a background: infectious diseases emerge frequently in china, partly because of its large and highly mobile population. therefore, a rapid and cost-effective pathogen screening method with broad coverage is required for prevention and control of infectious diseases. the availability of a large number of microbial genome sequences generated by conventional sanger sequencing and next generation sequencing has enabled the development of a high-throughput high-density microarray platform for rapid large-scale screening of vertebrate pathogens. methods: an easy operating pathogen microarray (eopm) was designed to detect almost all known pathogens and related species based on their genomic sequences. for effective identification of pathogens from eopm data, a statistical enrichment algorithm has been proposed, and further implemented in a user-friendly web-based interface. results: using multiple probes designed to specifically detect a microbial genus or species, eopm can correctly identify known pathogens at the species or genus level in blinded testing. despite a lower sensitivity than pcr, eopm is sufficiently sensitive to detect the predominant pathogens causing clinical symptoms. during application in two recent clinical infectious disease outbreaks in china, eopm successfully identified the responsible pathogens. conclusions: eopm is an effective surveillance platform for infectious diseases, and can play an important role in infectious disease control. the frequent invasion of microorganisms, including viruses, bacteria, fungi, parasites, and other eukaryotic and prokaryotic organisms, has threatened and will continue to threaten the life and health of humans and other vertebrates. in recent years, mutant or new forms of some existing pathogens have been identified as the causative agents of a number of outbreaks that have endangered public health in china [ ] . severe acute respiratory syndrome (sars), caused by a coronavirus, spread throughout guangdong province in , followed by a worldwide epidemic. during the epidemic, % of the sars cases were reported in china, resulting in human deaths [ ] . in , an outbreak of hand, foot, and mouth disease (hfmd) infected persons and caused the death of three children in linyi city, shandong province, china [ ] . the influenza a (h n ) pandemic affected more than , human patients, leading to deaths in china alone [ ] . because of its large and highly mobile population, the emergence of infectious diseases in china is relatively more frequent. therefore, a system implemented by the medical community and government for the monitoring of pathogens that could have a significantly negative impact on public health is urgently required in china. china has an established hospital-based surveillance system for infectious diseases. all clinical and hospital reports of both suspected and confirmed cases of notifiable infectious disease must be sent to local centers for disease control (cdc). the information is then sent to the china cdc headquarters in beijing through the national infectious diseases monitoring information system database, which was established in . the hierarchical administrative organization of the surveillance system ensures a rapid and efficient upward flow of epidemic information [ ] . based on this system, development of effective diagnostic platforms can greatly enhance the prevention and control of infectious diseases in china. the predominant techniques for identification of microbial pathogens depend on conventional clinical microbiology monitoring approaches. although well established, these approaches usually require culture of the pathogens, followed by susceptibility tests, which are time-consuming and laborious. in addition, many microbes are difficult to culture, and may be undetectable by culture-based approaches. molecular approaches for microbial surveillance and discovery have emerged as a very promising alternative for early diagnosis of infectious diseases. currently, molecular approaches include traditional sanger dna sequencing, polymerase chain reaction (pcr), oligonucleotide microarrays, and next generation sequencing (ngs). among these four technologies, the former two can identify a few known pathogens that must then be confirmed individually, and thus cannot cover a wide range of pathogens. the latter two methods cover a broad range of pathogens, and are therefore suitable for identifying unknown or even novel pathogens in infectious outbreaks. although ngs produces the most in-depth, unbiased information, and can reveal completely novel organisms, it is time-consuming and expensive, especially for the analysis of complex samples [ ] . derisi and colleagues developed the first generation of microarray platform, called virochip, to detect a wide range of viruses [ ] . in , the virochip helped to characterize sars as a novel coronavirus [ ] . since then, virochip has also been used to detected a human metapneumovirus [ ] , a novel influenza virus [ ] , and a novel adenovirus [ ] . more recently, greenechip and mda microarrays have been developed, which are broader spectrum approaches that can detect several thousand pathogenic viruses, bacteria, fungi, and protozoa [ , ] . the aforementioned three platforms all used long oligonucleotide probes and random amplification of nucleic acids. in this study, we report the construction of a high throughput pathogen microarray platform, named easy operating pathogen microarray (eopm), for largescale pathogen surveillance and discovery in china. the platform uses similar technical features to previous methods, but will be more useful for clinical applications because of its user-friendly analysis software. the eopm was designed based on the latest versions of nucleic acid sequence resources for microbes. clinical application of the microarray system confirmed that it can correctly identify the pathogens responsible for infectious disease. release of the european molecular biology laboratory (embl, http://www.embl.org/) database (march ) was used to establish our vertebrate viral sequence database. the terms at the family level that describe the host as a vertebrate animal were extracted from the "virus taxonomy list " (http://ictvonline.org/virustaxonomy.asp? version= ), compiled by the international committee on taxonomy of viruses (ictvdb). we only considered viruses under these taxonomy nodes. we also downloaded the sequences of fungi and parasites from embl. s rrna sequences were extracted using the cds tag. finally, we obtained bacterial s rrna sequences from the ribosomal database project (rdp . , http://rdp.cme.msu. edu). the final integrated dataset included , , viral sequences representing complete and partial viral genomes, , , bacterial s rrna sequences, , fungal s rrna sequences, and , , s rrna sequences from parasites. the eopm chip distinguishes all , known vertebrate virus species (involving genera, families), bacterial genera (involving families), fungal genera (involving families), and genera of parasites (involving families). considering that bacterial s rrna genes show a relatively high level of homology, and that bacteria require the presence of active virulence genes for pathogenesis, virulence genes were selected, including rfbe, slt- , ipaa, and katg, and probes were designed against these gene sequences. the basic design of the viral probes included as many different genomic target regions as possible for each species of vertebrate virus in the embldb. first, probes were targeted to conserved regions in areas encoding the structural proteins. the protein families database (pfam, http://pfam.sanger.ac.uk/) of multiple sequence alignments was used to cluster the functionally related sequences [ ] . the regions tagged as ′ utr, ′ utr, and ltr were also extracted and used as candidate sequences for the following probe design. second, candidate probes were screened according to the following criteria: probes with a length of nt, no repeats exceeding a length of nt, no hairpins with stem lengths exceeding nt, gc content between - %, and tm from - °c. third, we used blast analysis to select the conserved viral probes at the genus level from all of the candidate probes. the extent of conservation was evaluated for each probe, and all were found to detect the majority of species in each genus. a target species was considered to be represented if a probe matched it with at least % sequence identity. probes conserved at the genus level were selected based on a flexible threshold because the sequence conservation between species belonging to different genera is quite variable. finally, we aligned the sequences of all the candidate probes against the nt database, which was downloaded from ncbi ftp in august . probes with high sequence similarity to non-target genomes were eliminated. both species-specific and genus-conserved probes were included in the final probe set. the identification of bacterial, fungal, and parasite probes was similar, but only focused on the s and s rrna sequences. in addition, probes were also designed to target host immune response genes as a potential index to pathogenesis. the -mer oligonucleotide probes were synthesized on a mm × mm glass slide by applying an inkjet deposition system (agilent technologies, palo alto, ca). a total of eight sub-arrays with , distinct -mer probes in one slide were customized. all hybridizations involved a fluorescently-labeled synthetic oligonucleotide that was complementary to a positive control probe, which was replicated for more than , spots scattered in different zones of each sub-array. this ensured that signals appeared in every zone of each sub-array to facilitate data extraction from hybridization figures. microbial nucleic acids were extracted from serum, plasma, throat swabs, nasal lavage, feces, cerebrospinal fluid, and other body fluid using a tianamp virus dna/ rna kit (tiangen biotech., beijing, china). the carrier rna from the kit was applied to extract virus nucleic acid with low molecular weight. the kit can be used to extract the nucleic acid from both rna and dna viruses (like adenovirus), as well as bacteria, fungi, and parasites. a previously described random pcr amplification strategy [ ] with minor modification was applied to amplify extracted nucleic acids and label amplified products with fluorescent dye. in brief, the first cdna strand was reverse transcribed with a random decamer heeled with a pcr primer ( ′-gtttcccagtcacgatcnnnnnnnnn- ′). the first strand cdna was then synthesized to double-stranded dna using the same primer and klenow dna polymerase (takara, dalian, china). double stranded cdna from both patients and normal controls was pcr amplified using the heel primer. resultant pcr amplicons were then purified and labeled with cy -dctp or cy -dctp for the normal controls and patient samples, respectively, using klenow polymerase (takara). labeled dna was mixed with μl of hybridization buffer and added to the × , eopm arrays for hybridization overnight at °c in a hybridization oven (agilent). the eopm arrays were then washed with × ssc, . % triton x- at room temperature for min, followed by a second wash with . × ssc at °c for min. the arrays were then scanned using a dual-laser scanner (agilent) and the images were extracted and analyzed using feature extraction software (agilent). the normal distribution of microbes in the human body should be considered when using eopm to identify pathogens that are responsible for obvious clinical symptoms. we used two strategies to eliminate the background of normal microflora. firstly, at the experimental level, we always compared the suspected clinical sample with a normal sample of the same type, i.e. serum vs. serum or feces vs. feces. secondly, on a database level, we compared clinical samples with the same type of samples from a database that included more than different samples from a normal population, such as serum, feces, cerebrospinal fluid, and throat swabs. the second aspect may avoid unexpected issues in the experimental normal control. under the above strategy, each clinical sample was first compared with a normal control, and then with the normal sample database, so that potential pathogens should be identified based on their increased distribution compared to the normal human samples. to facilitate the application of eopm in multiple surveillance sites for infectious diseases, we designed software with a user-friendly interface, which is supported by a statistical analysis method based on a comprehensive microbial sequence identification database. in microbial diagnostic microarrays, only a few probes are designed for each targeted microbe, and each probe should be confirmed with specific positive and negative samples. in the pan-microbial microarrays, many probes are designed for one pathogen, and there is no way to confirm each probe. however, the majority of the probes targeting an expected pathogen are likely to be positive, and not hybridize with other non-target microbes. we applied a hypergeometric distribution to calculate a p-value for each species as an assessment of statistical significance. whether a pathogen was significantly present was determined using a complex interpretation method. the formula of hypergeometric distribution function is as follows: where c stands for the combination formula; n is the whole number of microbial probes on an array; m is the number of probes for a target microbe; n is the number of probes for which the intensity is positive on an array; and m is the number of probes whose intensity is positive for a target microbe. the probes were ranked by the signal of the cy fluorescent dye that was used to label the patient sample. in the user-interface of the eopm software, the proportion of probes can be chosen by the user according to the sample types. a small p-value indicates that there is a very low likelihood that a mistake has occurred in the multi-probe analysis, and correspondingly, that there is a high probability of the existence of the target microbe. finally, the p-value is adjusted using benjamini and hochberg's fdr correction [ ] . because the probes were designed to both the species and genus levels, results will be given accordingly. in eopm analysis, when there were at least three positive probes for a specific species of pathogen and an enrichment p-value < . , the given species could be considered positive for further investigation, including the clinical symptom coincidence analysis. molecular detection methods, including pan-microbial microarrays and unbiased high throughput sequencing, traditionally rely on random amplification, and so have lower sensitivity than specific pcr [ ] . clinical samples usually contain host nucleic acid which may interfere with the sensitivity of microarray analysis. to determine the sensitivity of epom, we spiked viral rna into human rna, mimicking the actual clinical samples. enterovirus (ev ), a single-stranded rna virus, was cultured with vero cells. the rna from the culture supernatant medium was extracted and quantitatively determined using a qrt-pcr standard curve. then, - ev molecules were spiked into rna extracted from human hela cells. the rna was then randomly amplified and hybridized with the eopm microarray as described above. in parallel, rt-pcr using a pair of specific primers to amplify ev was performed to compare the sensitivity of the two methods. known pathogens, including cell-cultured viral reference strains, cultured bacteria, and fungi, were used to verify eopm performance. clinical samples were all from patients with obvious infectious disease symptoms and which obtained negative results with routine diagnostic methods. following detection by eopm, the screened pathogens that caused similar clinical symptoms to those of the patients from which the clinical samples were collected were pcr amplified with species-or genusspecific primers. pcr-positive samples were then sequenced. this study obtained ethical approval from ethical committee of guangdong women and children's hospital. informed consent was not required because clinical samples were screened for potential pathogens in vitro. original microarray data have been submitted to the gene expression omnibus with the platform access number gpl . high throughput microarrays with long oligonucleotide probes, such as the virochip and greenechip systems, have proved effective for pathogen screening [ , , , ] . the eopm technique described here also uses long oligonucleotide probes and random pcr amplification. several known viruses, bacteria, and fungi were used to evaluate the accuracy of eopm. dengue virus was used as a test subject to determine whether the eopm method could detect the virus from an infected c / cell culture (tables , , and ). as shown in table , among the top ranked probes, eight targeted dengue virus specifically, while a further four probes targeted related flavivirueses such as phnom penh bat virus, tembusu virus, and deer tick virus. we also carried out enrichment analysis of the positive probes at both the species and genus level. notably, only dengue virus or closely related species showed significant enrichment (table ) , and only flavivirus showed significant enrichment at the genus level (adjusted p-value< . ) ( table ). both results were consistent with the known cultured dengue virus. by following a similar procedure, we successfully tested eopm on a panel of other known pathogens, including an rna virus, a dna virus, bacteria, fungi, and parasites (listed in table ). in terms of detection sensitivity, eopm could reliably detect ev when > copies of ev rna were mixed into copies of hela cell rna, while copies of spike virus rna could be detected in copies of host rna by specific rt-pcr following agarose gel electrophoresis. therefore, we inferred that when there was a high level of background nucleic acid, the detection sensitivity of random primer amplification was three orders of magnitude lower than specific primer amplification. most adenovirus infections cause similar symptoms to those induced by some respiratory viruses and mycoplasmas, making it difficult to identify the pathogens by traditional clinical diagnostic procedures. in february of , an outbreak of disease caused by an unknown pathogen occurred in baoding city, hebei province. patients presented with obvious infectious symptoms, such as high fever, coughing, throat congestion, lung tissue necrosis, and bronchopneumonia. initially, influenza virus, sars virus, and mycoplasma, known causes of these clinical symptoms, were suspected, but pcr tests were negative for all three pathogens. to rapidly identify the unknown pathogen, eopm chips were selected to screen the possible pathogens responsible for these infections. nucleic acid was extracted from patient serum samples to be used for eopm analysis. nucleic acid from normal serum was used as a control. one scanned microarray image is shown in figure , and the enrichment results for the top-ranked pathogens at species and genus level are listed in tables and respectively. adenoviruses were found to be significantly enriched, as were the top five species results (tables and ). we further verified adenovirus as the causative agent by pcr targeted to a conserved region of mastadenovirus genomic sequence (see additional file ). hand-foot-and-mouth disease (hfmd) is a common viral illness that predominantly affects infants and children younger than years old. hfmd epidemics usually occur in china in late spring and early summer. the pathogens responsible for hfmd are mainly coxsackie a virus (cva ) and enterovirus (ev ), both of which belong to the enterovirus genus. the routine hfmd clinical diagnosis includes three qrt-pcr kits targeting the enterovirus genus, cva , and ev species respectively. in may of , many children were found to have clinical symptoms of "hand-foot-and-mouth diseases" at guangdong women and children's hospital, located in southern china. although most patients were diagnosed as having cva or ev infections by the qrt-pcr analysis, some were negative for enterovirus. to identify the pathogens responsible for enterovirus-negative hfmd children, samples from each of the patients were subjected to eopm analysis. about mg of a feces sample was used to extract rna, using a tianamp virus dna/rna kit, and labeled with cy following random amplification. in the probes were ranked by ratio of cy /cy intensity. non-infected cell samples were labeled with cy , and virus infected cell samples were labeled with cy . parallel, rna extracted from normal feces was labeled with cy and used as a control. the enrichment analysis at the species level identified theiler's-like cardiovirus as the most probable pathogen responsible for the hfmd infection in these patients ( table ) . analysis of the enrichment results at the genus level revealed cardiovirus as the number one match, showing significant enrichment ( the microarray raw data of other symptom-causing pathogens, such as streptococcus and mycoplasma, identified by eopm in peripheral blood in infectious patients, were also submitted to the geo database. the primary purpose of developing the eopm was to facilitate the rapid identification of unknown pathogens in regional surveillance centers in china when emergent pathogen-causing incidents occur. when considering the application of microarray technology, data analysis is a significant obstacle to users without specialized knowledge in bioinformatics analysis of microarray data and nucleic toxoplasma gondii toxoplasma parasite figure hybridization picture of eopm in the adenovirus outbreak case. rna/dna from patients was labeled with red cy fluorescent dye, and nucleic acid from normal control serum was labeled with green cy dye. acid sequences. therefore, we implemented the statistical enrichment analysis in a user-friendly interface (figure ). the software can support a large-scale search of probe hits against a comprehensive microbial sequence database. we believe this software will greatly facilitate the installation of the eopm platform in different infectious surveillance system laboratories in china. the software can be accessed at http://www.genestone.com.cn: /microbial/index.jsp. since the first application of a high-throughput, rapid, and unbiased microarray for detecting viral pathogens in [ ] , several pan-microbial microarray platforms with different degrees of coverage of various pathogens have been established. these microarray platforms use long oligonucleotide probes ( - -mer) and random pcr amplification, and have successfully identified unexpected pathogens in infectious disease outbreaks, even discovering novel viruses with homology to known species [ , ] . in this study, we constructed a high-density eopm array for screening all known viruses, bacteria, fungi, and parasites that could become vertebrate pathogens. based on the sequence data available for vertebrate pathogens, we have designed , -mer oligonucleotide probes targeting , vertebrate virus species (involving genera, families), bacterial genera (involving families), fungal genera (involving families), and parasite genera (involving families). the -mer oligonucleotide probes can cross-hybridize with similar but non-identical sequences, allowing the detection of novel pathogens that are related to known species. the eopm probes designed to detect bacteria, fungi, and parasites were targeted to s rrna or s rrna sequences. whereas rrna sequences are relatively conserved in the same genus or family, eopm can distinguish bacteria, fungi, and parasites at either the genus or family level, which has already been successfully applied in a clinical setting for confirmation and treatment. in the sensitivity study of eopm, we designed experiments to compare the sensitivity of random amplification and specific amplification, while not considering the effect of other issues, such as clinical sample collection and nucleic acid extraction, on the sensitivity of eopm. eopm showed -fold lower sensitivity than specific target pcr amplification, which was consistent with a previous report [ ] . the lower sensitivity was due to the random pcr amplification adopted in the eopm sample preparation, which was not as efficient as specific pcr for amplification of a particular species. despite having lower sensitivity than target-specific pcr, the eopm platform is sufficiently sensitive to identify the pathogens causing clinical symptoms in infectious outbreaks, in which symptom-causing pathogens should be highly enriched. the sensitivity can be further improved in practice if acellular samples with minimal host nucleic acid contamination, such as serum and throat swabs, are used for pathogen screening. for example, greninger and colleagues had used virochip microarray to identify influenza a/h n in nasal swab samples showing a comparable sensitivity with rt-pcr [ ] . in the sample preparation for the eopm method, all rna and dna extracted from samples are firstly reverse transcribed. rna viruses are converted into cdna, and dna viruses keep its dna status in the reverse transcription reaction, then the dna, including the reverse-transcribed cdna and original dna viruses, were transformed to double strand dna for the subsequent random amplification procedure. therefore, eopm can detect both rna viruses and dna viruses in the same standard protocol. for bacteria, fungi, and parasites, eopm detects s rrna or s rrna copies encoded by rrna genes located in the genomic dna. because rrna genes are highly transcribed, detecting rrna molecules instead of rrna genes should achieve higher sensitivity. with the dual color strategy used by the eopm method, one normal sample without infectious symptoms was always analyzed in parallel. despite this, the "normal" sample may possess its own clinical characteristics. for example, we have found torque teno virus and human endogenous retroviruses in some normal blood samples. these viruses do not cause obvious clinical symptoms, and should not interfere with the aim of eopm analysis, which is to determine the possible pathogens causing the symptoms in the test patients. eopm data analysis consists of two steps. first, we screened for significantly enriched microbes in the target sample compared with the normal sample using the dual color chip. second, the predicted microbes identified in the first step were compared with a database compiled from the normal population mentioned above, to eliminate the background microbes that also exist in normal samples without infectious symptoms. pan-microbial screening microarrays differ from nucleic acid-based microbial diagnostic technologies, such as qpcr and low density microarrays. these diagnostic technologies are merely aimed at identifying one or a few types of microbes using target-specific probes that should be confirmed with specific positive and non-specific samples. moreover, diagnostic low-density microarrays usually use short oligonucleotides of about -nucleotides as specific probes, similar to taqman probes in qpcr technology [ , ] . the very limited number of short probes/primers targeting a pathogen could fail to detect sequences with mutations located in the regions targeted by the probes/primers. however, over a dozen long oligonucleotide probes were designed for each pathogen in the eopm method, allowing reliable identification of a pathogen based on a statistical enrichment analysis of the probe group, instead of one individual probe. moreover, eopm can effectively narrow down the potential pathogens and even identify novel pathogens in complex clinical infection situations. in addition to the pathogen sequences, host immune response genes were also included in the eopm database. during eopm analysis of clinical samples, the immune response genes show dramatic up-or downregulation in the target samples compared with the normal reference (data not shown). so far we have not found any reliable relationships between the immune response genes figure user interface of the eopm software. users will generally only need to input raw microarray data for pathogen identification. and the pathogen categories. the overall clinical information for patients and normal controls should also be comprehensively analyzed. human immune related genes in peripheral blood show dramatic differences in expression even in a normal population, with differences correlated with sex, age, and sampling time, amongst other factors [ , ] . until now, the available genome-wide technologies to detect unknown pathogens in infectious outbreaks primarily consisted of microarrays and ngs. although ngs can provide the most in-depth, unbiased information, and can reveal completely novel pathogens, it is timeconsuming when the sample contains hundreds of microbial species that require comprehensive data processing. therefore, ngs cannot meet the short time requirement for infectious disease control. however, the most complicated step in eopm technology is probe design, which can be undertaken by a core bioinformatics team in the development phase. once probe design is complete, and the whole microarray procedure is optimized as a standard procedure, pathogen screening results can be interpreted in less than hours. therefore, eopm is more suitable for applications requiring detection of unknown pathogens during infectious outbreaks. in addition, with the rapid increase in microbial metagenomic sequence data produced by ngs, the probes used for eopm can easily be upgraded, and the eopm version can be updated due to the in situ synthesis technology replacing the spotting technology in microarray fabrication. in conclusion, eopm is a very powerful pan-microbial detection microarray platform, which can detect almost all known pathogens and related species. in several clinical test applications, we found that eopm technology is sensitive enough to detect the pathogens causing evident clinical symptoms. eopm is designed for easy operation, with detection software containing a user-friendly interface, facilitating its application in molecular laboratories. infectious disease epidemics emerge frequently in china, and we believe that the use of eopm in main pathogen surveillance sites across the country could play an important role in infectious disease control in china. changing health in china: re-evaluating the epidemiological transition model isolation and characterization of viruses related to the sars coronavirus from animals in southern china an outbreak of hand, foot, and mouth disease associated with subgenotype c of human enterovirus in shandong characterization of the h n highly pathogenic avian influenza virus derived from wild pikas in china web-based infectious disease reporting using xml forms direct metagenomic detection of viral pathogens in nasal and fecal specimens using an unbiased high-throughput sequencing approach microarray-based detection and genotyping of viral pathogens viral discovery and sequence recovery using dna microarrays diagnosis of a critical respiratory illness caused by human metapneumovirus by use of a pan-virus microarray a metagenomic analysis of pandemic influenza a ( h n ) infection in patients from north america cross-species transmission of a novel adenovirus associated with a fulminant pneumonia outbreak in a new world monkey colony a microbial detection array (mda) for viral and bacterial detection panmicrobial oligonucleotide array for diagnosis of infectious diseases pfam: clans, web tools and services controlling the false discovery rate in behavior genetics research diagnostics and discovery in viral hemorrhagic fevers detection of respiratory viruses and subtype identification of influenza a viruses by greenechipresp oligonucleotide microarray microarray detection of human parainfluenzavirus infection associated with respiratory failure in an immunocompetent adult circulation of lineages of a novel saffold cardiovirus in humans nucleic acid amplification strategies for dna microarray-based pathogen detection detection of m. tuberculosis using dna chips combined with an image analysis system biochip system for rapid and accurate identification of mycobacterial species from isolates and sputum individual-specific variation of gene expression in peripheral blood leukocytes individuality and variation in gene expression patterns in human blood an easy operating pathogen microarray (eopm) platform for rapid screening of vertebrate pathogens we gratefully acknowledge professor taijiao additional file : two pairs of specific primers for amplifying adenovirus, and the sequence of pcr products from clinical case .additional file : sequence of nested rt-pcr primers for cardiovirus, and the pcr product sequence from clinical case . there are patents pending by the authors related to the probe design methods and array data statistical enrichment methods. in addition, software copyright is pending related to pathogen interpretation.authors' contributions lz and yj conceived the study and analyzed the data. lz drafted the manuscript. wh and yy conducted the microarray experiments, pcr, and sequencing confirmation. xz and hl designed probes and software. xz, ay, cz, and zh participated in the sample collection and array data analysis. all authors read and approved the final manuscript.submit your next manuscript to biomed central and take full advantage of: key: cord- -e a suk authors: rhim, jung-woo; lee, kyung-yil; youn, you-sook; kang, jin-han; kim, ji-chang title: epidemiological and clinical characteristics of childhood pandemic h n virus infection: an observational cohort study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: e a suk background: there was a pandemic influenza around the world in including south korea since last pandemic occurred four decades ago. we aimed to evaluate the epidemiological and clinical characteristics of this infection in childhood. methods: we evaluated the epidemiologic characteristics of all the subjects infected with the h n influenza a virus ( , patients, ≤ years of age), and the clinical and laboratory findings of the inpatients ( patients, had pneumonia) between september and january in a single hospital throughout the epidemic. results: the age distribution of all the subjects was relatively even. over % of cases occurred during a two-month period. two hundred and five patients ( . %) received oseltamivir within h of fever onset, and % of inpatients defervesced within h of medication. the group with pneumonia included more males than females, and had higher leukocytes counts with lower lymphocyte differentials than the group without pneumonia. the white blood cell count and lymphocyte differential were associated with the severity of pneumonia. corticosteroid treatment for severe pneumonia patients was highly effective in preventing disease progression. conclusion: children of all ages affected with even rates of infection, but males were predominant in pneumonia patients. pneumonia patients showed lymphopenia and its severity was associated with the severity of illness. our results suggest that the mechanism of lung injury in h n virus infection may be associated with the host immune response. although influenza virus infection has been a major global concern since the pandemic 'spanish flu', there have been no pandemic influenzas for near four decades after the 'hong kong flu'. the pandemic h n influenza a ( h n ) virus infection was reported first in mexico in february , and then the virus spread rapidly worldwide, including in south korea [ ] . it has been reported that the majority of h n patients were children and young adults and the mortality rate was not higher than that of seasonal influenza [ ] [ ] [ ] . the majority of patients affected by the h n virus infection recovered uneventfully, but some previously healthy patients developed a rapidly progressive pneumonia, leading to acute respiratory distress syndrome (ards), multi-organ failure, and death [ ] [ ] [ ] . with this enigma, the pathogenesis of acute lung injury (pneumonia) in influenza infections remains unknown [ , , ] . in south korea, the first patient with h n virus infection was reported in may, . the number of patients gradually increased until mid-october, when the number of patients was overwhelming for a month, and then gradually decreased, with few people becoming infected after february, . owing to sensational reports of childhood fatality in the mass media and a new diagnostic tool, the real-time reverse transcriptase-polymerase chain reaction (rt-pcr), we had the opportunity to evaluate patients with h n virus infection from the onset of their illness. in addition, during the study period we observed a dramatic effect of early treatment with corticosteroids and oseltamivir for patients with severe pneumonia including rapidly progressive pneumonia [ , ] . in this study, we evaluated the epidemiological, clinical and laboratory features of children with h n virus infections in a single hospital throughout the epidemic. we retrospectively evaluated all patients with h n virus infection during the pandemic ( , patients) for epidemiologic characteristics, and for clinical characteristics, we reviewed the medical records and chest radiographic findings of children admitted to the catholic university of korea, daejeon st mary's hospital between september and january . the diagnosis of patients depended on positive results for the h n virus rt-pcr (accupower ™ in korea, bioneer, alameda, ca, usa) through throat swabs. although indications for admission were not clearly defined in this study, the majority of the inpatients were those who were suspected to have severe disease such as pneumonia and to have risk factors for severe disease such as infants and bronchial asthma. however, it might be possible that excessive concern of parents on fatality of this infection in part affected on admission of the uncomplicated cases. among the inpatients, we selected patients with pneumonia and patients without pneumonia, based on the chest radiographs. the chest radiographic patterns of admitted patients were reviewed and classified by two pediatricians (ky lee and jw rhim) and one pediatric radiologist (jc kim). the patients with chest radiographic patterns that showed increased nodular densities along the bronchial trees unilaterally or bilaterally, were designated the bronchopneumonia group ( patients). patients with a distinctive large patch of infiltration, segmental or lobar consolidations were designated the segmental/lobar pneumonia group and regarded as having a severe pneumonia ( patients). the first day of fever was regarded as the first day of illness. among pneumonia patients, we tried corticosteroid treatment for patients with severe pneumonia. as for indication of corticosteroids (methyprednisolone, mp or prednisolone), the subjects had severe respiratory distress with hypoxemia at presentation ( cases) or during hospitalization ( cases) requiring o therapy ( , ). we compared the clinical and laboratory characteristics of the different groups. the study was approved by the institutional review board of the catholic university of korea, daejeon st mary's hospital. statistical analyses were performed using the statistical package for the social sciences for windows version . (spss, chicago, il, usa). continuous variables are reported as means ± standard deviations. statistical significance was assessed using the student's t-test and the paired t-test for continuous variables, and using the χ test for categorical variables. a p value of < . was considered significant for the statistical tests. during the study period, , children (aged months- years) with influenza-like illness were seen at our hospital. among them, the , patients were positive by rt-pcr, and patients were admitted to the isolation wards. the hospitalization rate was . %. the mean age of the patients ( , cases) was . ± . years of age and the male-to-female ratio was . : ( , / , ). the age distribution of the patients is shown in figure . children of all age groups except infants had a relatively even rate of infection ( figure , gray bars). the numbers of new patients each week is shown in figure . there was an explosive pattern, with over three quarters of the cases occurring during a single month (the rd- th weeks of ; october to november). the age distribution and the weekly case rate of the patients admitted to hospital ( cases) are also shown on figure and (black bars). these demonstrated similar patterns to those of the total patient cohort. for hospitalization rates, younger children ( - years, . %, / ) showed higher admission rates than older children ( - years, . %, / and - years, . %, / ). in the hospitalized children, the mean age was . ± . years, and the male-to-female ratio was . : ( / ). almost all the patients had a high fever ( . %) and cough ( . %) suggestive of severe infection such as pneumonia at the time of admission. most of the patients were previously healthy although some had underlying diseases and only patients had underlying disorders ( . %), including chronic pulmonary diseases ( patients with bronchial asthma and one with past history of brochopulmonary dysplasia), neuromuscular disorders (two with epilepsy and two with cerebral palsy), one with chronic liver disease, and one with nephrotic syndrome. the outcomes for these patients were uneventful, except for two asthmatic patients who developed a mild pneumonia. all inpatients received oseltamivir of the recommended doses for body weight and a broad-spectrum antibiotic (ampicillin/sulbactam). two hundred and five patients ( . %) received oseltamivir within h of fever onset. the mean duration of fever before admission (including the day of admission) was . ± . d and patients ( %) defervesced on the next day. only seven patients had fever that persisted > h after oseltamivir treatment. during hospitalization, five patients showed progressive pneumonia despite early antiviral therapy. according to the initial chest radiographs, patients had pneumonia and were divided into two groups: the bronchopneumonia group ( patients) and the segmental/lobar pneumonia group ( patients). in pneumonia patients, pneumonic infiltrations appeared within h of fever onset in patients ( %). when we analyzed the inpatients according to age ( - years, patients; - years, patients; and - years, patients), the rates of pneumonia in each age group were . % ( / ), . % ( / ) and . % ( / ), respectively. the severe pneumonia (segmental/lobar type) was predominant in the older age groups ( . % ( / ), . % ( / ) and % ( / ), respectively). among the pneumonia patients, patients showed severe respiratory distress with hypoxemia at presentation ( cases) and during their hospital stay ( cases). arterial blood gas analysis was done in patients, and patients showed hypoxemia (po < mmhg in the room air). these patients received additional corticosteroids as soon as possible when indicated; patients received intravenous mp ( mg/kg/day, divided two doses, at presentation, mg/kg/day at next day and then tapered off within a week) and patients received oral prednisolone ( mg/kg/day, divided doses, for days tapered off within a week). six patients received early mp with osetalmivir which is recommended early use, before the positive rt-pcr results. interestingly, all patients except one (age years) were among the - years age group, and male patients were predominant ( / ). we performed serial chest radiographs of some cases in these patients. a -year-old male patient complained of one day fever and cough, and his initial chest radiograph showed few pulmonary infiltrations. on the following morning, he complained of severe dyspnea and showed chest radiographic infiltrations on right lower lung and left hilar regions. he had received two doses of oseltamivir before the mp treatment ( figure a -c). a year-old male patient was presented with a day fever and severe cough and had a rapidly progressive pneumonia in which initial patchy infiltrates on left upper lobe progressed to total left lung consolidation within h after admission. he was given three doses of oseltamivir before the mp treatment ( figure a-g) . these two patients showed dramatic improvements in their clinical symptoms and radiographic findings within h after mp treatment. a -year-old male patient with lobar pneumonia was treated with oseltamivir, mp and intravenous immunoglobulin (ivig) treatment. he was admitted with fever, cough and progressive dyspnea of days, and after mp ( mg/ kg/day, divided doses) infusion, he showed persistent dyspnea and slightly aggravated pneumonic consolidation ( figure c ). on the following morning, high-dose ivig ( g/kg) was infused for hours. at the time of termination of ivig, his clinical symptoms disappeared and a dramatic improvement of radiographic findings was observed within hours after ivig termination ( figure a-f) . there was no adverse or rebound reaction in any patient treated with corticosteroids. the clinical symptoms of all patients improved within a day and their pneumonic infiltrations, regardless of severity, ceased immediately after corticosteroid treatment and disappeared within several days without adverse reactions. extrapulmonary manifestations (complications) of h n infection were observed as four cases of febrile seizure, two cases of urticaria, one case of hepatitis and one case of myositis, and no cases of encephalopathy. when we compared the patients with and without pneumonia, and the patients with segmental/lobar pneumonia versus those with bronchopneumonia, there were significant differences in certain parameters between the groups. compared with the group without pneumonia, the males were over-represented in the pneumonia group (p = . ) and longer hospitalizations (p < . ), higher values for hemoglobin (p = . ), wbc (p < . ) and crp (p < . ) and lower values for lymphocyte differential (p < . ) ( table ). the patients with the more severe type of pneumonia (segmental/lobar pneumonia) showed a higher mean age (p = . ) and leukocyte count (p = . ) and lower lymphocyte differential values (p < . ) compared with the group with bronchopneumonia (table ). in addition, the severe pneumonia patients who received corticosteroids ( cases) had the highest leukocyte counts and crp levels, and the lowest lymphocyte differentials ( ± /mm , . ± . mg/dl, and . ± . %, respectively) [ ] . in this study, we found that children of all ages except infants had a relatively even rate of infection with the h n influenza virus. this is in agreement with other studies showing that few children and young adults have immunity against a new viral infection [ , , ] . our study of all h n virus-infected children, in a single hospital, throughout the epidemic may have epidemiological implications, along with other studies based on data from admitted patients or gathered during a portion of the pandemic period [ ] [ ] [ ] [ ] . it has been reported that younger children (< - years old) with h n virus infection tended to be admitted to hospital more frequently than older children or adults, and our results are compatible with these studies [ , , ] . it has also been reported that certain risk factors are related to the likelihood of developing severe illness, including a younger age and particular underlying diseases, including obesity [ , , , ] . in this study, we did not analyze obesity and young age as risk factors, and only patients ( . %) had underlying diseases. although the hospitalization rate was higher among the younger children ( - years of age), in this study, pneumonia and severe pneumonia were more prevalent in the - -year-old group than in younger children. the explosive increase in the infection rate during two months (mid-october to mid-december) of the study period may be a typical pattern of spread of an acute respiratory viral infectious disease that has a short incubation period ( - days). this pattern is similar to those reported in other regions of korea and other countries [ , , ] . vaccination against the h n influenza virus in korea started on november, for school-aged children, on december, for children aged months to years and on december, for adults with risk factors, which was after the peak of the epidemic. the rapid disappearance of the spread of infection might be the effect of vaccination and the strengthened individual hygiene, but the epidemiologic pattern suggests that an unknown herd immunity against a new viral infection might also be responsible [ , ] . it is reported that patients with h n virus infections had a relative leukopenia with lymphopenia [ , ] . however addition to this finding, we found in the present study that the patients with pneumonia had a higher wbc count with lower lymphocyte differential, and the more severely affected patients had the highest wbc with the lowest lymphocyte differential in the early stages of the infection (within two days of fever onset). previous human studies of h n virus infections have revealed that a lower lymphocyte count was associated with a poor outcome [ ] , and mice infected with influenza viruses showed a lymphopenia and the h n subtype was associated with marked lymphopenia with total lymphoid depletion [ ] . therefore peripheral lymphocyte may be associated with the pathogenesis of acute lung injury in influenza virus infections [ ] . altered crp and esr values were not prominent in h n virus infections, but higher crp values were associated with a more severe illness. the male-to-female ratio was . : among all patients, and . : among the admitted patients. furthermore, male patients were predominant among those with pneumonia ( : , / ) and those with respiratory distress who received corticosteroids ( . : , / ). other epidemiologic studies on children have reported a male predominance [ ] , but some studies on inpatients have reported a female predominance [ , ] . these findings suggest that genetic factors and possibly environmental factors are regarding the pathogenesis of lung injury in influenza virus infections, it has been believed that the viruses from upper respiratory tract spread to lower lung tissues and elicit the cytopathic reaction. however, some clinical and experimental studies have suggested that the innate and/ or cell-mediated immune reaction (t cell) with excessive production of cytokines of the host may also be responsible for the lung injury in influenza virus infection [ , [ ] [ ] [ ] [ ] . we experienced no intensive care patient in this series despite large subjects of the study, and it may be, at least in part, explained by a rapid use of corticosteroids on the patients with severe pneumonia [ , ] . because this pandemic occurred over years after last pandemic (hong kong flu), there have been no controlled-clinical trials for the efficacy of corticosteroids on influenza virus infections, although yearly seasonal influenza and small cases of sporadic h n avian influenza virus infection have occurred during inter-pandemic period. the beneficial effect of corticosteroids in pneumonia caused by influenza virus infections may have resulted from reduction of systemic inflammation caused by immune cells and cytokines [ , ] . our treatment policy which needs to be proven by controlled clinical studies in coming pandemics or in other influenza virus infections, may help to reduce the morbidity and possibly prevent the progression to fatal pneumonia [ , ] . we have reviewed the rationale of our corticosteroid treatment and the host immune responses to viral insults in influenza virus infections, and proposed a new concept for the pathogenesis of acute lung injury in influenza virus infections, using a 'protein homeostasis system' of the host [ ] . it has been reported that antiviral therapy (oseltamivir) is effective in the acute stages of influenza infection, including h n virus infection in humans and experimental animals [ , ] . in agreement with this, we found that the majority of patients ( %) defervesced within h after medication and most pneumonic infiltrations in pneumonia patients had improved at discharge. there are some limitations to this study. compared with other studies, we had many uncomplicated inpatients owing to flexibility of our admission policy. because we did not perform extensive microbiological testing, such as viral cultures, paired-sample serologic studies and pcr for other pathogens, we cannot rule out the possibility of coinfection with other respiratory pathogens. in pandemic h n virus infections, children of all ages were evenly affected, and males were predominant in pneumonia patients. early antiviral treatment was very effective in inducing rapid defervescence for the patients. the patients with h n infections showed lymphopenia, and its severity was associated with the severity of the illness in the early stages. together this finding, the rapid improvement in clinical signs and the prompt resolution of severe pneumonic consolidations after immunemodulator (corticosteroid and ivig) treatment suggest that the mechanisms of lung injury in this infection may be associated with the cell-mediated immune response of the host, rather than virus-induced cytopathies. emergence and pandemic potential of swine-origin h n influenza virus and composition of the - influenza vaccine australia's winter with the pandemic influenza a (h n ) virus impact of the novel influenza a (h n ) during the autumn-winter season in a large hospital setting in wriging commitee of the who consultaton on clinical aspect of pandemic (h n ) influenza: clinical aspects of pandemic influenza a (h n ) virus infection the anzic influenza investigators: critical care services and h n influenza in australia and newzaeland critically ill patients with influenza a (h n ) infection in canada the cotton rat as a model to study influenza pathogenesis and immunity hyperactive immune cells (t cells) may be responsible for acute lung injury in influenza virus infections: a need for early immune-modulators for severe cases early corticoid treatment for severe pneumonia caused by h n influenza virus novel swine-origin influenza a (h n ) virus investigation team: emergence of a novel swine-origin influenza a (h n ) virus in humans incidence of pandemic influenza a h n infection in england: a cross-sectional serologic study pandemic influenza a (h n ) virus hospitalizations investigation team: hospitalized patients with h n influenza in the united states pandemic influenza in canadian children: a summary of hospitalized pediatric cases h n ) working group: factors associated with death or hospitalization due to pandemic influenza a(h n ) infection in california risk factors and outcoms among children admitted to hospital with pandemic h n influenza pandemic influenza a (h n ) breakthrough infections and estimates of vaccine effectiveness in germany relative lymphopenia and monocytosis may be considered as a surrogate marker of pandemic influenza a (h n ) swine-origin influenza a (h n ) in indian children human infection with highly pathogenic avian influenza virus (h n ) in northern vietnam depletion of lymphocytes and diminished cytokine production in mice infected with a highly virulent influenza a (h n ) virus isolated from humans fatal outcome of human influenza a (h n ) is associated with high viral load and hypercytokinemia effect of antilymphocyte serum on influenza virus infection in mice ( ) effect of low-and highpassage influenza virus infection in normal and nude mice delayed clearance of viral load and marked cytoline activation in severe cases of pandemic h n influenza virus infection hydrocortisone infusion for severe community-acquired pneumonia. a preliminary randomized study pro: the illegitimate crusade against corticosteroids for severe h n pneumonia treatment options for h n influenza: evaluation of the published evidence there was no funding for this study. we thank drs. sung-ku kim, kyung-won bang, hyun-oh kim, ji-jung lee and ja-young hwang for patient care during the pandemic, and drs. jae-wook lee and joon-sung lee for careful review of this paper. the authors declare that they have no competing interests. all authors read and approved the final manuscript. kyl had primary responsibility for concept, design of the study and writing the manuscript; jwr participated in the preliminary data collection, data analysis and writing the manuscript; ysy participated in patient care and data analysis; jhk contributed to the interpretation of the data and editing of the manuscript and supervised the design and execution of the study; jck participated in reading of chest radiograph of the patients key: cord- -s qp a e authors: wei, yiping; zeng, weibiao; huang, xiangyun; li, junyu; qiu, xingting; li, huadong; liu, dinghua; he, zhaofeng; yao, wenzhong; huang, ping; li, chao; zhu, min; zhong, chunlan; zhu, xingen; liu, jiansheng title: clinical characteristics of hospitalized patients with coronavirus disease in zengdu district, hubei province: a single-center descriptive study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: s qp a e background: we aimed to report the epidemiological and clinical characteristics of hospitalized patients with coronavirus disease- (covid- ) in zengdu district, hubei province, china. methods: clinical data on covid- inpatients in zengdu hospital from january to march , were collected; this is a community hospital in an area surrounding wuhan and supported by volunteer doctors. all hospitalized patients with covid- were included in this study. the epidemiological findings, clinical features, laboratory findings, radiologic manifestations, and clinical outcomes of these patients were analyzed. the patients were followed up for clinical outcomes until march , . severe covid- cases include severe and critical cases diagnosed according to the seventh edition of china’s covid- diagnostic guidelines. severe and critical covid- cases were diagnosed according to the seventh edition of china’s covid- diagnostic guidelines. results: all hospitalized covid- patients, (median age: . years), were enrolled, including non-severe and severe patients. the proportion of patients aged over years was higher in the severe group ( . %) than in the non-severe group ( . %, p < . ). approximately a quarter of the patients ( . %) had at least one comorbidity, such as hypertension, diabetes, or cancer, and the proportion of patients with comorbidities was higher in the severe group ( . %) than in the non-severe group ( . %, p < . ). common symptoms included fever ( . % [ / ]) and cough ( . % [ / ]). . % ( / ) of the patients had a fever at the time of admission. most patients ( . % [ / ]) were cured and discharged; . % ( / ) deteriorated to a critical condition and were transferred to another hospital. the median covid- treatment duration and hospital stay were . and . days, respectively. conclusions: most of the covid- patients in zengdu had mild disease. older patients with underlying diseases were at a higher risk of progression to severe disease. the length of hospital-stay and antiviral treatment duration for covid- were slightly longer than those in wuhan. this work will contribute toward an understanding of covid- characteristics in the areas around the core covid- outbreak region and serve as a reference for decision-making for epidemic prevention and control in similar areas. in december , a series of pneumonia cases with similar symptoms were reported in wuhan, hubei province, china [ ] . that pneumonia was later named coronavirus disease (covid- ) by the world health organization (who) [ ] . the causative pathogen was identified as a novel enveloped rna beta coronavirus named severe acute respiratory syndrome coronavirus (sars-cov- ) [ ] . covid- is highly contagious and spreads rapidly through human-to-human transmission [ ] [ ] [ ] . as of march , , there were , confirmed covid- cases and deaths in china, including , confirmed cases and deaths in hubei province, and , confirmed cases and , deaths worldwide. however, many infected people have not been counted owing to a lack of timely diagnosis. covid- is a global pandemic. therefore, a comprehensive and in-depth understanding of the epidemiological and clinical characteristics of covid- is imperative for controlling the pandemic as soon as possible. the number of covid- cases in wuhan was large, the spread was fast, and the fatality rate was high. most of the clinical characteristics of covid- have been summarized from the samples of patients in wuhan [ ] . controlling the epidemic in the areas around the core covid- outbreak region is an important link in blocking the spread of the disease. the chinese government has enlisted many volunteer doctors to support hospitals in these key areas. however, there are few reports on the clinical characteristics of covid- inpatients in these areas [ , ] . thus, this study collected clinical data for covid- inpatients in zengdu hospital, a community hospital supported by volunteer doctors and nurses from jiangxi province (about miles from zengdu district). we describe the epidemiology, clinical features, laboratory findings, imaging features, and outcomes of covid- inpatients in zengdu district, which is a -h drive from wuhan city. we hope that our work will contribute toward an understanding of covid- characteristics in the areas around the core covid- outbreak region and provide a decision-making reference for epidemic prevention and control in similar areas. the study was approved by the institutional ethics board of suizhou zengdu hospital, which was established by the chinese government to treat covid- patients in zengdu district. all the patients diagnosed with covid- , according to the interim guidance from the who [ ] , in zengdu hospital from january to march were admitted and included in this study. the patients were followed up for clinical outcomes until march , . only laboratory-confirmed cases that were defined as positive based on the results of highthroughput sequencing or real-time reversetranscriptase-polymerase chain reaction (rt-pcr) assay of nasal and pharyngeal swab samples were included. these confirmatory assays for sars-cov- were performed at the suizhou cdc in accordance with the guidelines developed by the who [ ] . medication and treatment measures were selected according to the scheme recommended in the guidelines and each patient's condition [ ] . a team of doctors who had treated these patients extracted the recent exposure history, clinical symptoms, laboratory findings, radiologic manifestations, and clinical outcomes from patients' medical records. all patients underwent at least one chest computed tomography (ct) scan, and data were extracted after the scans were reviewed by a dedicated imaging physician. all laboratory tests were performed according to treatment needs. the researchers obtained the outcome data of transferred patients by contacting the hospitals that received these patients, and also contacted the patients by phone if anything was unclear or information necessary for the study was missing from the medical record. according to the national treatment guideline, covid- severity was defined as mild, moderate, severe, or critical [ ] . the mild type was defined as mild clinical symptoms and no radiological manifestations of pneumonia. the moderate type was defined as respiratory symptoms and pneumonia on imaging. the disease was defined as severe if one of the following criteria was met: respiratory rate of ≥ beats per minute; finger oxygen saturation of ≤ % at resting state; and arterial blood oxygen partial pressure (pao )/oxygen concentration (fio ) of ≤ mmhg. the critical type was defined as respiratory failure or shock and requirement of mechanical ventilation or intensive care unit (icu) monitoring and treatment. accordingly, the patients were divided into a non-severe group (mild or moderate disease type) and severe group (severe or critical disease type). due to limited medical facilities at the zengdu hhospital, critical patients were transferred to hospitals with superior treatment facilities. the incubation period was defined as the interval between the patient's earliest date of exposure to the transmission source and the date of the initial symptom. for patients who had recently visited wuhan, the earliest date of exposure was estimated as the median date of their stay in wuhan; for patients who had been in contact with people returning from wuhan, the earliest date of exposure was considered to be the earliest contact date, the earliest date of exposure was considered to be the earliest contact date. fever was defined as an axillary temperature of ≥ . °c. lymphopenia, eosinopenia, and thrombocytopenia were defined as lymphocyte, eosinophil, and platelet counts of less than , , and , of the corresponding cells per cubic millimeter. the smoking index was equal to the product of the number of cigarettes per day and smoking years. the length of covid- treatment was defined as the time interval from patient admission to the meeting of the cure and discharge criteria of the chinese management guidelines for covid- (version . ) [ ] . the cure and discharge criteria were as follows: normal body temperature for more than days; significantly improved respiratory symptoms; significantly improved acute exudative lesions on pulmonary imaging; and two consecutive negative results of the nucleic acid tests of sputum, nasopharyngeal swabs, and other respiratory tract samples. the primary composite end points were discharge from the hospital owing to being cured and transfer to another hospital because of condition deterioration. the secondary end points were cure or discharge rate and the length of hospital stay. statistical analyses were performed with spss (v. . ; spss inc., chicago, il, usa). continuous variables are described as median values and interquartile ranges (iqrs), and categorical variables are reported as numbers and percentages. we used the mann-whitney u test, χ test, or fisher's exact test to compare differences between the two groups. a two-sided α of less than . was considered statistically significant. we obtained data on the demographic characteristics, symptoms, and outcomes for patients hospitalized in suizhou zengdu hhospital as of march , . the severe group included ( . %) patients while the nonsevere group included ( . %) patients. the demographic and clinical characteristics of the patients are shown in table . forty-three ( . %) of the patients had visited wuhan within days before the study enrollment; . % ( / ) of the patients had come into contact with people who had travelled to wuhan or were diagnosed with covid- . the remaining patients reported they had not been to wuhan, and it was unclear how these patients had been exposed to the transmission source; none of the patients had a history of exposure to the huanan seafood wholesale market or a wild animal. the incubation period calculated based on the data from patients with a known exposure time was days (iqr, - days). the longest incubation period was days. a nurse in the fever clinic of suizhou zengdu hhospital was the only medical staff included in the study. the median age of the patients was years (iqr, - years). the patients in the severe group were significantly older than those in the non-severe patients ( had at least one comorbidity; this percentage was significantly higher than that among the non-severe patients ( . %, / ). table shows the results of radiology and laboratory tests at admission. all patients underwent ct at admission, and abnormal results were obtained for . % ( / ) of the patients. the most common chest ct findings were bilateral patchy shadows figure shows typical ground-glass shadows and bilateral patchy shadows in two patients. according to the results of the first examination after admission, . % ( / ), . % ( / ), and . % ( / ) of the patients had leukopenia, lymphocytopenia, and thrombocytopenia, respectively. lymphocytopenia and thrombocytopenia were more obvious in the severe group compared to that in the non-severe group. the c-reactive protein levels were elevated in . % ( / ) of the patients; the erythrocyte sedimentation rate of . % ( / ) of patients and d-dimer levels of . % ( / ) of patients were also elevated. elevated procalcitonin, creatine kinase, alanine aminotransferase, aspartate aminotransferase, and myoglobin levels were observed in . , . , . , . , and . % of patients, respectively. as shown in table , most of the patients ( . %, / ) were cured and discharged from the hospital. ten out of ( . %) patients, all of whom belonged to the severe group, showed condition deterioration to a critical status and were transferred to suizhou central hospital, a superior hospital. eventually, five of them died and five survived. all five people who died received endotracheal intubation; one of the five survivors received endotracheal intubation, which was removed days later. the remaining four only received non- understanding the clinical characteristics of covid- inpatients in the areas around the core covid- outbreak region is very important for controlling the spread of covid- and decision-making for epidemic control. our study on inpatients in zengdu hhospital confirms that covid- patients in the areas surrounding the core covid- outbreak region showed mainly mild and moderate illness with fever and lymphocytopenia as the main clinical features. older patients (age > years) or those with underlying comorbidities are at higher risk of deteriorating to critical status. the length of hospital-stay and antiviral treatment duration for covid- were slightly longer than those in wuhan. all patients who tested positive for covid- by rt-pcr in the study region were admitted to the hospital, regardless of the severity of the patients' condition. there were several reasons why our hospital established such an admission standard. first, at that time, the outbreak was still in the early stage. the understanding of the epidemic situation in zengdu district, a residential a lymphocytopenia was defined as a lymphocyte count less than per cubic millimeter. eosinopenia was defined as an eosinocyte count of less than per cubic millimeter. thrombocytopenia was defined as a platelet count of less than , per cubic millimeter. these are results of the first examination after admission resources to treat all the diagnosed patients. our admission criteria were formulated under such special circumstances, although this admission standard was different from the current international standard. moreover, compared to studies in which only seriously ill covid- patients were admitted, our admission criteria better reflect the disease characteristics in the area around the outbreak point, so as to provide a decision-making reference for hospitals in the residential area to decide which patients should stay at home for observation and which high-risk patients should be hospitalized in a timely manner. the patients in zengdu area show mainly showed mild and moderate illness, with a few patients showing severe and critical illness. in wuhan, as the site with the most serious covid- infection in china, many patients did not get timely diagnosis and treatment initially, and medical resources were insufficient to accommodate the sudden burst of patients. as a result, the proportion of severe cases reached . - . % [ , ] , while the rate of severe disease in other regions was - % [ , ] , similar to . % in this study. this may be because, with the deepening of the understanding of covid- and the formulation of relevant guidelines [ , ] , many patients were diagnosed and treated in a timely manner without deteriorating into severe disease. besides, the difference in admission criteria was also a reason why the rate of severe disease in this study was significantly lower than that in wuhan or abroad. the early common symptoms of covid- patients include fever, cough, sputum, and other symptoms of lower respiratory tract infection. as the most common symptom, in general, more than % of patients have a fever, but only . % of the patients had a fever at the time of admission, which shows that the fever in many patients was intermittent. it also means a large number of patients with intermittent fever will be set free if instant body temperature readings are the only measure used for screening [ , ] . the proportion of fever in critically ill patients increases significantly after hospitalization, and most of these new fever cases may be caused by secondary infection, so it is necessary for severe patients to receive antibiotics to prevent secondary infection [ ] . covid- patients over years old were more likely to show deterioration into critical illness. previous studies on severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) have confirmed that age was an important predictor of poor prognosis [ , ] , and similar conclusions were obtained for covid- [ ] . data obtained by nanshan zhong et al. [ ] and zhongliang wang et al. [ ] showed that the age of severe patients was significantly older than that of non-severe patients. consistent with these findings, among the patients we collected, the median age of severe patients was years, while that of non-severe patients was years. in addition, about . % of the severe patients were more than years old. these studies have shown that older covid- patients have a poor prognosis. covid- patients with comorbidities were also likely to show deterioration [ ] . the studies by nanshan zhong et al. and daweiwang et al. [ ] both showed high proportions of comorbidities in severe patients. a who survey reported that people with comorbidities had a higher risk of severe disease [ ] . in a recent retrospective study of death cases with covid- [ ] , all of the deceased patients have comorbidities, which were considered to be one of the most important risk factors for death. in this study, . % of the severe patients had comorbidities. this may be due to abnormal immune function and increased susceptibility to sars-cov- in patients with comorbidities [ , ] . in addition, covid- damage to the lungs can aggravate some comorbidities, such as chronic obstructive pulmonary disease. antiviral drugs and glucocorticoids also have limited benefits for patients with comorbidities. in terms of laboratory tests, % of patients had lymphopenia, and more obvious findings were noted in severe patients. the novel coronavirus can induce a cytokine storm and inhibit the generation of lymphocytes [ , ] , so lymphopenia is very common in patients with covid- . the low absolute value of lymphocytes can be used as a reference indicator for clinical diagnosis of novel coronavirus infections [ ] . lymphocytes showed a pronounced decline in severe patients than in non-severe patients, indicating that the degree of lymphocyte decline can be used to assess the severity of the disease [ ] , and that continuous decline of lymphocytes is also one of the indicators of disease deterioration [ ] . in the absence of nucleic acid detection and ct, this can be an important tool for determining the severity of the disease. the length of hospital stay in this study was slightly longer than that in wuhan, which was - days [ , ] . this contradicted the finding that the length of hospitalization is positively related to disease severity because covid- severity in this study was significantly lower than that in wuhan. however, the allocation of medical resources is also an important factor affecting the length of hospitalization. the number of infected patients in wuhan was large and medical resources were scarce, so the hospital had to discharge inpatients as soon as possible to treat newly admitted patients. the inpatient data collected in this study were from a community hospital that was supported by many jiangxi doctor volunteers and medical supplies, which ensured sufficient medical resources. the characteristics of inpatients under this special medical setup were different from those at other hospitals. in particular, after the local epidemic is mostly controlled, some wastage of medical resources may be inevitable. for example, patients were allowed to stay in the hospital for some time to recover even after meeting the discharge criteria for covid- , which was not possible in the hospital in wuhan. this was also the reason why the length of treatment for covid- ( days) is significantly shorter than the length of hospitalization ( days). in addition, hospitals in wuhan only accept patients who have been diagnosed as showing covid- , while hospitals outside wuhan admitted many patients who were not diagnosed at admission and were also hospitalized for the - days it took for nucleic acid test results to arrive. three studies from regions with sufficient medical resources [ ] [ ] [ ] , namely taizhou, guangdong, and shenzhen, can support our hypotheses since their median hospital stays were days, days, and days, respectively, which were close to the results of this study. this study has several limitations. first, since it is a retrospective study with a limited number of patients, some conclusions need to be verified by studies with more rigorous design and larger samples. second, zengdu hhospital was a community hospital, and most of the critically ill patients had to be transferred to superior hospitals for treatment. we are temporarily unable to get information on the followup treatment and complications of these patients. third, when calculating the incubation period, we excluded the unclear contact date, resulting in fewer patients included, and the potential memory bias will also affect our results. fourth, our admission criteria were different from the current internationally recognized criteria, which limits comparability with other studies. however, our admission criteria were set in high-risk areas at the early stage of the epidemic to avoid the spread of the epidemic, which was essential and important. in addition, only pcr-confirmed covid- patients were included in this study and asymptomatic infections without pcr confirmation were omitted, so the characteristics we described are only suitable for pcr-confirmed covid- patients. most of the covid- patients in zengdu area had mild disease. older patients with underlying comorbidities had a high risk of progressing to severe disease. a large number of patients with intermittent fever will be omitted by the temperature checks that are currently widely being deployed. the length of hospitalization and antiviral treatment for covid- were slightly longer than those in the wuhan area. this work will contribute to our understanding of the disease characteristics in the areas around the covid- core outbreak point and provide reference data for decision-making for epidemic prevention and control in such special areas. department of respiratory medicine, jiangxi province hospital of integrated chinese and western medicine, nanchang, china. department of general practice, the first people's hospital of fuzhou, fuzhou, china. department of pediatric neurology, ganzhou women's and children's hospital of jiangxi province coronavirus infections-more than just the common cold clinical management of severe acute respiratory infection when novel coronavirus ( -ncov) infection is suspected: interim guidance. in: world health organization who. coronavirus disease (covid- ) outbreak. journey of a thai taxi driver and novel coronavirus transmission of covid- in the terminal stage of incubation period: 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peripheral blood of hospitalized patients with novel coronavirus pneumonia (ncp). medrxiv clinical and immunologic features in severe and moderate coronavirus disease t-cell immunity of sars-cov: implications for vaccine development against mers-cov clinical course and outcome of patients infected with the novel coronavirus, sars-cov- , discharged from two hospitals in wuhan clinical characteristics of discharged cases of novel coronavirus-infected pneumonia in taizhou, china clinical outcomes of covid- cases and influencing factors in guangdong province covid- in a designated infectious diseases hospital outside hubei province publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank all the medical staff who participated in treating patients and all the patients enrolled in this study. the data of these patients have been published for the first time. all the authors who are not from zengdu hospital are volunteers from different hospitals in jiangxi province, and these volunteers work with doctors and nurses from zengdu hospital to fight against covid- . special thanks to the other volunteers from jiangxi province for their contributions. not applicable. authors' contributions ljs, zxg, wyp and hxy designed the study. hp, lc, zm, lhd, and ldh were responsible for collecting the epidemiological and clinical data; qxt examined the ct images and extracted the data; hzf, ljy, and zcl were responsible for processing the statistical data; and zwb, ywz, and lhd wrote the paper. zxg participated in the design and revision of the manuscript. all authors read and approved the final manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. this study was approved by the ethics committee of suizhou zengdu hospital. written informed consent has been obtained from all participants. the data used in this study was anonymized before its use. not applicable. the authors declare that they have no competing interests to disclose. key: cord- -xz twqx authors: vorobieva s. jensen, v.; furberg, a-s; slotved, h-c; bazhukova, t.; haldorsen, b.; caugant, d. a.; sundsfjord, a.; valentiner-branth, p.; simonsen, g. s. title: epidemiological and molecular characterization of streptococcus pneumoniae carriage strains in pre-school children in arkhangelsk, northern european russia, prior to the introduction of conjugate pneumococcal vaccines date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: xz twqx background: the -valent pneumococcal conjugate vaccine (pcv- ) was introduced in the national immunization programme (nip) schedule in russia in march . previously, the -valent pneumococcal conjugate vaccine (pcv- ) was marketed in russia in but has never been offered for mass vaccination. a carriage study was performed among children in arkhangelsk in . the objective was to determine the prevalence of carriage, serotype distribution, antimicrobial susceptibility and the molecular structure of streptococcus pneumoniae strains before marketing and introduction of pcv- . methods: a cross-sectional study was conducted on a cluster-randomized sample of children and a self-administrated questionnaire for parents/guardians. nasopharyngeal samples were collected from children younger than years attending nurseries and kindergartens in the arkhangelsk region, russia. detailed demographic data, as well as information about the child’s health, traveling, exposure to antimicrobials within the last months and anthropometric measurements were collected for all study subjects. variables extracted from the questionnaire were analysed using statistic regression models to estimate the risk of carriage. all pneumococcal isolates were examined with susceptibility testing, serotyping and multilocus sequence typing. results: the overall prevalence of asymptomatic carriage was high and peaking at months with a rate of %. pcv- covered . % of the detected strains. high rates of non-susceptibility to penicillin, macrolides and multidrug resistance were associated with specific vaccine serotypes, pandemic clones, and local sequence types. nine percent of isolates represented three globally disseminated disease-associated pandemic clones; penicillin- and macrolide-resistant clones norway(nt)- and poland( b)- , as well as penicillin- and macrolide-susceptible clone netherlands( )- . a high level of antimicrobial consumption was noted by the study. according to the parent’s reports, . % of the children used at least one antimicrobial regime since birth. none of the hypothesised predictors of s. pneumoniae carriage were statistically significant in univariable and multivariable logistic models. conclusions: the study identified a high coverage of the pcv- -vaccine, but serotype replacement and expansion of globally disseminated disease-associated clones with non-vaccine serotypes may be expected. further surveillance of antimicrobial resistance and serotype distribution is therefore required. streptococcus pneumoniae is a bacterial pathogen causing disease among all age groups. despite the introduction of effective vaccines, invasive pneumococcal disease (ipd) is associated with high mortality and morbidity [ ] [ ] [ ] . as was anticipated, the introduction of the pneumococcal conjugate vaccines (pcvs) in the national immunization programmes has substantially reduced pneumococcal-related deaths worldwide [ ] . immunization by conjugate pneumococcal vaccines has now been implemented in countries [ ] . still, according to the most recent report based on data from the world health organisation (who) and the maternal and children epidemiology estimation collaboration, in , pneumococci were estimated to have caused , (uncertainty range , - , ) deaths for both hiv-infected and hiv uninfected infants and young children in the age - months globally [ ] . the epidemiology of pneumococcal disease prior to the introduction of pneumococcal vaccines was dominated by the spread of global disease-causing epidemic clones, both multidrug-resistant (mdr) and antimicrobial susceptible clones [ ] . the success of epidemic clones, though not well understood, has been linked to certain capsular types [ , ] , carriage of a pilus islet [ ] and various virulence factors [ ] . mass vaccination has reduced the occurrence of mdr pneumococcal molecular epidemiology network (pmen) clones with serotypes covered by the vaccine. reports from countries dating to the post-pcv era show a rapid reduction of pcv-serotype-related pmen-isolates. however, some sequence types (st) st , st , st and other highly successful clones with non-vaccine related serotypes rapidly replace the disease-associated endemic clones shortly after the introduction of pcv-vaccines [ ] . capsule serotype replacement in clones targeted by pcvs has also been demonstrated [ ] , such as a switch from f to a in the disease-associated high-level penicillin-resistant endemic clone taiwan f - [ ] . russia is a large country with an estimated infant and child population aged up to years of . million in [ ] . the immunization programme for infants and children in the russian federation presently includes ten less expensive vaccines, while, for example, the haemophilus influenzae type b conjugate vaccine has not been available for mass vaccination [ ] . the pcv- was marketed in russia in but has never been offered for mass vaccination. the extended pneumococcal conjugate vaccine with serotypes was licenced in russia in (pcv- , prevenar , wyeth pharmaceuticals inc., marketed by pfizer inc.), and included in the russian national immunization programme (nip) schedule in march [ , ] . immunizations are administered in a + -dose schedule, with two primary immunizations given at and . months and a booster at months of age [ ] . no additional catch-up immunization has been offered for the rest of the child population [ ] . national immunization coverage data are only partially available, but a sharp increase of pcvcoverage was reported by the who/ united nations international children's emergency fund reporting system in the years after the introduction [ ] . in , the rates of pcv- coverage were and % for the nd and the rd doses, respectively, while the rates for the st dose remain unknown since [ ] . neither national nor regional surveillance of incidence for ipd cases exists in russia [ , ] . the overall incidence of pneumococcal meningitis in russia was estimated at . per , cases for all age groups, and % of all cases were presented by children under years. the low rates of pneumococcal meningitis have been associated with suboptimal diagnostics and antimicrobial treatment preceding laboratory examinations [ , ] . the present study was conducted in the arkhangelsk region in the northwest part of russia where no data about the pre-pcv carriage are available. in order to determine pneumococcal carriage at baseline [ ] and evaluate possible effects of the introduction of pcv- in the russian immunization schedule, the authors performed a cross-sectional study of asymptomatic nasopharyngeal s. pneumoniae carriage in healthy pre-school children attending daycare centres (dccs) years before the introduction of pcv- . all pneumococcal isolates were analysed with regard to serotypes, phenotypic antimicrobial resistance patterns and population structure based on multilocus sequence typing (mlst). children and parents/guardians from ten dccs were invited to participate in the study. all the dccs were public childcare institutions that belonged to small towns and suburbs of the arkhangelsk region and located within a range of to km from the city of arkhangelsk. each dcc consisted of a nursery and a kindergarten and was attended by to children. besides, randomly chosen healthy children living in the centrum of arkhangelsk were sampled. children and parents/ guardians were invited to participate by the announcement in the local newspaper. all children were sampled during the last week of november by one otolaryngologist. none of the children experienced symptoms of a common cold like cough, runny or stuffy nose at the date of examination. the body temperature was normal for all children on the day of sampling. the questionnaires were filled out by parents or guardians for all participants in the study. each questionnaire included questions concerning the child's health, length of breastfeeding, travelling abroad or outside of the arkhangelsk region within the last months, smoking habits of family members, as well as the household size and the number of siblings and family members. information on the use of antimicrobials agents during the last months prior to sampling was also collected for all children. anthropometric measurements were taken for all participants on the day of sampling. all parents/guardians were informed about the study by informational letters and a majority of the parents/ guardians participated in informational meetings. the written informed consents were filled out by parents or guardians for all participants of the study. ethical approval was obtained from the ethics research committee of north norway, the reference number of the approval . . from the rd of june and the ethics research committee of the northern state medical university of arkhangelsk, the reference number for the approval / from the th of june . permission to conduct the study was also obtained from the health and educational services of the arkhangelsk region. the european intervention study (euris) manual was used for isolation of bacterial strains [ , ] . nasopharyngeal samples were transferred to the laboratory using transport media swabs (copan c, copan diagnostics, inc., corona, usa), and inoculated within to h after the arrival. samples were cultured on % defibrinated sheep blood agar (oxoid ltd., uk) supplemented with gentamicin ( mg/l) and incubated at - °c under anaerobic conditions for to h. samples were also cultured on sheep blood agar (oxoid ltd., uk) with optochin disks (ab biodisk, sola, sweden) ( μg) and incubated in % co at - °c for - h. strains were identified as s. pneumoniae by colony morphology, negative catalase reaction, optochin susceptibility, agglutination in the pneumo-kit slidex test (biomèrieux, missouri, usa), and by the bile solubility test [ ] . isolates were serotyped by the quellung reaction using serotype-specific antisera (ssi diagnostica, denmark). strains were tested for antimicrobial susceptibility by disk diffusion on iso-sensitest agar (isa) (oxoid ltd., basingstoke, uk) supplemented with nicotinamide adenine dinucleotide (mast diagnostics merseyside, uk) and % defibrinated sheep blood. antimicrobial paper disks (oxoid ltd., uk) containing μg oxacillin (oxa), μg erythromycin (ery), μg tetracycline (tet), μg trimethoprim-sulfamethoxazole (sxt) or μg norfloxacin (nor) were used. oxa-resistant isolates (inhibition zone < mm) were further examined by penicillin g (pen), cefuroxime (cxm), cefotaxime (ctx) and meropenem (mem) etests according to the manufacturer's instructions (ab biodisk). unless otherwise stated, the breakpoints defined by the norwegian working group for antibiotics (nwga) were used. nor-resistant isolates were examined by etest for their susceptibility to ciprofloxacin (cip), nor, moxifloxacin (mxf) and levofloxacin (lvx), using breakpoints from the swedish reference group for antibiotics (srga) [ ] [ ] [ ] . multidrug-resistance (mdr) was defined as resistance to three or more antimicrobial classes [ ] . the double-disk diffusion (ddd) test with ery and clindamycin (cli) (oxoid ltd., uk) was used for characterization of inducible macrolides, lincosamides, streptogramines (imls) resistance as described [ ] . blunting of the cli inhibition zone indicated imls b -resistance, resistance to both ery and cli indicated constitutive resistance (cmls b ), whereas susceptibility to cli and resistance to ery indicated m-type resistance. detection of the macrolide resistance determinants ermb and mefa was performed by pcrs as previously described [ ] . all strains were examined by mlst as described by enright et al. [ ] and assigned to sts based on a combination of alleles at seven housekeeping loci. the seven housekeeping genes used for mlst were aroe, gdh, gki, recp, spi, xpt, and ddl. alleles were identified and isolates were assigned into sts using the pubmlst database (https://pubmlst.org/spneumoniae/). the phyloviz® programme was used for assigning the isolates into clonal complexes (ccs), defined as clusters sharing six out of seven common alleles. rstudio© version . . {https://www.rstudio.com/} and r version . . for windows were used for calculation of odds ratios (or), confidence intervals ( % ci), and p-values using two-tailed fisher's exact test. carriage rates were calculated as incidence rate ratios (irrs) with % ci. p-values < . were considered significant. we used fisher's exact test and univariable and multivariable logistic regression models to examine potential risk factors for pneumococcal carriage including sex, age, early life variables (weight and length at birth, breastfeeding length, living in arkhangelsk since birth), family and socioeconomic status (parents' education, having siblings < years at the moment of examination, number of rooms at home), medication and disease (having had rhinitis, otitis or pneumonia since birth, average number of respiratory tract infections per year since birth, regular medication, any disease within a month prior to the examination, receiving antimicrobials within months prior to the examination), and lifestyle factors (body height, body weight, body mass index (bmi), passive smoking). out of children attending the ten selected dccs, ( . %) agreed to participate in the study and nasopharyngeal swabs were collected from all these children. the demographic data for all children are given in table . the percentage of parents or guardians who agreed to the participation of their children varied from . to . % between institutions and the number of isolates ranged from to between institutions. samples were gathered from non-vaccinated healthy children aged to months (mean age . months), and . % of the children were boys. bmi was in the range between and kg/m for % of the children and greater than for % of the children on the day of examination. only % of the children had a bmi of less than on the day of sampling. the overall prevalence of antimicrobial consumption was high. according to the parent's reports, . % of the children used at least one antimicrobial regime since birth. additionally, . % ( / ) of the children with recognized carriage have been treated with antimicrobials within the last months before sampling. pneumococci were isolated from children (mean age . months), giving an overall carriage rate of . % (ci . - . %). the highest rate of carriage was found among the children aged from to months and it was lowest at the age less than months ( table ). the carriage rate peaked at the age of months ( . %). the carriage rate of s. pneumoniae for males and females were compared with univariable and multivariable regression models to estimate the risk of carriage (odds ratios) and did not display any statistically significant differences. sex did not influence the carriage rate of s. pneumoniae significantly ( table ) . none of the hypothesised predictors of s. pneumoniae carriage, including sex, breastfeeding, number of rooms at home, respiratory tract infections and illness, were statistically significant in univariable and multivariable logistic regression models (table s ). receiving antimicrobial therapy months before to sampling was not significantly associated with carriage of penicillin non-susceptible s. pneumoniae (or . with % ci . - . ). twenty-four different serotypes were detected in the pneumococcal collection, and isolates were nontypeable (nt) ( table ) . serotypes f (n = ; . %), f (n = ; . %), a (n = ; . %) and b (n = ; . %) were most prevalent (tables and ). the most diverse serotype composition was observed in children in the age groups to months and to months with a total number of different serotypes in each group. the diversity of serotypes in other age groups varied from eight (age group to months) to (age group to months). most isolates ( / , . %) with serotype were found in samples taken from children aged less than months of age. pcv- , including serotypes , , , , a/b, f, v, , c, a/f, and f, would cover . % ( / ) of the isolates in the study (table ). in contrast, pcv- provided coverage for only . % ( / ) of the isolates in our collection. the coverage rate for the valent pneumococcal polysaccharide vaccine (ppv) was . % ( / ). the rates of non-susceptibility were as follows: sxt (n = ; %), tet (n = ; %), oxa (n = ; %), and ery (n = ; %). characterization of penicillin nonsusceptible pneumococci (pnsp) and macrolide nonsusceptible pneumococci (mnsp) is given below. only a single strain was resistant to fluoroquinolones (< %). mdr was detected in strains ( %). only % ( / ) of nasopharyngeal carriage strains were susceptible to all examined antimicrobials (oxa, ery, sxt and tet). the isolates displayed different sts (table ) . fortythree sts comprising isolates and representing . % of the entire population were assigned into clonal complexes. clonal complex (cc) represented by different sts (st , st , st , and st ) was found to be the most prevalent complex ( . %, n = ). ten percent of all sts were represented by single isolates. thirty-five of the sts were identified for the first time in arkhangelsk. the majority of the pneumococcal isolates in each st was related to a single serotype. two sts, st and (table ) . three additional strains expressed serotype b and were associated with poland b - . the mlst-based phylogeny for all penicillin non-susceptible isolates is given in fig. . the expected rate of pcv- coverage for mnsp isolates was % ( / ). ten pneumococcal isolates displayed non-susceptibility to pen, but were susceptible to macrolides. seven of these strains expressed serotype f and belonged to st and were also resistant to stx. the three remaining isolates were non-serotypeable (st ; st and st ) ( table ). the analysis of all macrolide non-susceptible isolates by ddd method revealed the following phenotypes: imls b (n = ), cmls b (n = ), and m-type resistance (n = ). the results were confirmed by ermb and mefa-pcrs. macrolide non-susceptibility was associated with two globally disseminated clones. six of the macrolideresistant isolates belonged to cc (st and st ), expressed the ermb-gene and were associated with the international poland b - clone. six of the isolates belonged to st -norway nt - and possessed both mefa (n = ) and ermb (n = ) determinants. the other macrolide-resistant isolates were represented by unrelated st , st , st , and st and were connected to ermb (n = ) and mefa (n = ) genes. this is one of the largest carriage surveys prior to the introduction of pneumococcal vaccines in russia, where serotyping, antimicrobial susceptibility testing and mlst were performed on whole strain collection [ ] . moreover, the present study provides information regarding the population structure of s. pneumoniae carriage isolates in pre-school children of the arkhangelsk region between separately located dccs. the serotype distribution was diverse in the area, but globally reported epidemiological features, such as age-dependence, carriage rates a . % overall frequency of pneumococcal carriage rate was found in non-vaccinated pre-school children. the carriage rate among children aged months was as high as . %. previous carriage studies in dccs in russia have described even higher overall frequencies of asymptomatic s. pneumoniae colonization [ ] [ ] [ ] [ ] [ ] . overall, we found no significant difference in the carriage rates among children with birth weights < g, birth heights < cm, none or < months of breastfeeding, as well as living with siblings < years. our study found an average rate of pneumococcal carriage similar to what has previously been described for populations in upper-middle-income countries at a baseline period [ ] . the prevalence of carriage is independent of geographical region but strongly associated with accumulated risk factors, such as young age, high-density living conditions, and poor health conditions [ ] [ ] [ ] . the rates of asymptomatic carriage varied markedly between different age groups in our study, and also the diversity of serotypes displayed age variation. these findings have previously been observed by others [ ] [ ] [ ] . young children aged - months expressed the highest rates of asymptomatic colonization and the widest range of serotypes. isolates with serotype (paediatric serotype) were linked to children younger than months in our study. the age-dependence analysis showed a low frequency of pneumococcal colonization up to months and a peak incidence at the age of months with a stable decline from the age of months. this tendency has previously been discovered for children living in developed and upper-middle-income countries [ ] , but not for children living in low-income countries [ ] . the seven most common serotypes ( , b, f, f, a, v, c) from our study were previously described in the group of the ten most common serotypes of ipd cases globally [ ] , and they are a part of pcv- . three other pcv- associated serotypes , and f are not frequently detected among pneumococcal carriage isolates in russia [ ] [ ] [ ] nor in other geographical areas [ , ] but were generally related to cases of ipd in infants and young children [ ] [ ] [ ] . none of the isolates from our study collection expressed serotype a reported as the eighth most prevalent globally [ ] and the most common serotype in childhood ipd following pcv- introduction [ , ] . still, . % of the carriage isolates belonged to the pen and macrolide-susceptible taiwan f - cluster previously associated with serotype f to a replacement [ ] . serotype a is strongly associated with pen-resistant cases of ipd and was commonly described shortly after the introduction of pcv- in vaccination schedules [ , ] , leading to the inclusion of serotype a in the -valent vaccine. a high incidence of ipd due to serotype a has been associated with a limited number of clonal complexes (cc , cc , and cc ). contrary to that, a study from russia carried out by mayanskiy et al. [ ] demonstrated that local serotype distribution in our study, two isolates ( . %) belonged to serotype a, which is not included in pcv- . this serotype was previously associated with mdr and was isolated from most ipd cases in the post-pcv era in several post-industrial countries [ ] [ ] [ ] . our two serotype a isolates were pen and macrolide susceptible and were st , which was not previously associated with globally disseminated clones. a single carriage isolate from our collection displayed serotype b, which is a non-vaccine serotype associated with high capacity for biofilm production [ ] . the isolate was susceptible to pen and macrolides, but showed resistance to tet and was associated with st /none-cc in contrast to previous reports [ , ] . the expansion of serotype b associated with both ipd and non-ipd cases in paediatric populations has been reported in several countries after the introduction of pcv- [ , ] . since the pneumococcal disease is preceded by asymptomatic colonization, the distribution of antimicrobial resistance patterns in nasopharyngeal s. pneumoniae carriage strains may predict rates of resistance in invasive isolates [ , ] . rates of nonsusceptibility among invasive and carriage isolates changed dramatically after the introduction of pcvs in industrialized countries [ ] [ ] [ ] ] . furthermore, non-susceptibility to pen in invasive pneumococcal isolates after the introduction of pcvs was strongly associated with an increased mortality rate in infants and children, as well as in the elderly [ ] . the study found significantly higher rates of pen and macrolide non-susceptibility than previously reported from russia before the vaccine implementation [ , ] . high rates of mdr carriage was discovered during the survey. similar to the intermediate rate of carriage, an intermediate rate of pnsp was found. treatment with antimicrobials months before sampling was not a significant risk factor for carriage of pnsp in this cohort. a high concordance between nonsusceptibility to pen and macrolides and genotypes was also noted. remarkably, a recently published study from russia demonstrated a significant rise in resistance to oxacillin, erythromycin and clindamycin in disease-associated nasopharyngeal isolates in response to pcv- implementation. the growing resistance was explained by the expansion of mdr endemic clone st with serotype [ ] . contrary to the reported low rates of sxt consumption in the area [ ] the study found a much higher rate of resistance to sxt than previously reported [ , , ] and a high rate of non-susceptibility to tet. the all-russia survey cited non-susceptibility rates of and % to tet for the european and asian parts of the country, respectively [ ] . rates of resistance are strongly associated with rates of antimicrobial consumption in local settings. according to the report from the european surveillance of antimicrobial consumption (esac) network [ ] , the rates of outpatient antimicrobial consumption in russia in were the lowest among all participating countries. however, the low rates of antibiotic consumption at outpatient level do not agree with the present study. on the contrary it was found that children were intensively treated with antimicrobials prior to sampling. the low level of consumption published by esac should be regarded with caution due to possible bias in the reported sales and self-medication data [ ] . we observed a high prevalence of various locally disseminated sts in the area. although the arkhangelsk region has rather low rates of migration and tourism and does not border any other countries, we found a close clonal relationship with the major globally disseminated pandemic clones, thus indicating possible import. our study found st with serotype f to be associated with pen non-susceptibility. a study carried out in siberia found a high rate of st carriage isolates which were susceptible to pen thus contrasting to the arkhangelsk isolates [ ] . differences in the susceptibility profiles among strains within the country may suggest the acquisition of resistance in response to local antimicrobial prescribing practices. besides, . % of macrolideresistant isolates were associated with st and st . to the authors' knowledge, these sts have never been associated with macrolide resistance before, which is also may be linked to a local treatment choice. we found that pcv- could be effective against % of the pneumococcal population and thus reduce the majority of penicillin, macrolide, and multidrug-resistant strains as previously shown in other countries [ ] [ ] [ ] [ ] . however, . % of macrolide-resistant isolates were associated with non-vaccine serotypes f and a ( f-st and a-st -cc ), that may replace the pcv- -vaccine associated serotypes. st is a singleton not previously related to any clonal complexes, whereas st is part of the st -cluster, which has previously been linked to pen resistance and several ipd-associated serotypes [ ] . a high proportion of both penicillin and macrolide non-susceptible isolates in our study was found to be related to nt isolates. the recently published meta-analysis of estimated invasive disease potential for individual pneumococcal serotypes showed a low disease potential for non-serotypeable pneumococci [ ] . however, all non-serotypeable isolates in our study were closely related and belonged to the st -cluster, the globally disseminated pmen clone norway nt - , vaccination against which is so far unavailable. the high effectiveness of pcvs against ipd in infants and young children has been proven in countries with well-established national surveillance. according to several reports [ ] [ ] [ ] [ ] a sharp decrease of both carriage and ipd cases with vaccine-associated serotypes has occurred shortly after the introduction of pcvs with a subsequent decline in morbidity and mortality rates associated with these serotypes. it is too early to estimate the vaccine impact in russia based on serotype distribution only. reliable national and regional surveillance of invasive and non-invasive cases is needed to determine the effectiveness of the pcvs introduction and suggest strategies concerning vaccination schedules, the choice of pcv and vaccination coverage targets. the present study has documented several important aspects of the local pneumococcal epidemiology specific for the north of european russia. the s. pneumoniae population was found to be highly diverse and had common features such as age-dependency, dominant serotypes and the presence of major epidemic clones. high rates of resistance were linked to high rates of antimicrobial consumption in the area. clonal expansion of several globally distributed pandemic clones was identified in a remote part of north european russia with low rates of migration and tourism. the effectiveness of pcv- introduction cannot easily be predicted. according to international experience, clonal expansion due to replacement 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changing serotype distribution and resistance patterns among pediatric nasopharyngeal pneumococci collected in moscow a point prevalence survey of hospital-acquired infections and antimicrobial use in a paediatric hospital in north-western russia european surveillance of antimicrobial consumption (esac): outpatient antibiotic use in europe the inventory of antibiotics in russian home medicine cabinets effect of introduction of the pneumococcal conjugate vaccine on drug-resistant streptococcus pneumoniae nationwide surveillance of streptococcus pneumoniae in greece: patterns of resistance and serotype epidemiology impact of -valent pneumococcal conjugate vaccination in invasive pneumococcal disease incidence and mortality impact of a pneumococcal conjugate vaccination program on carriage among children in norway emergence of penicillinnonsusceptible streptococcus pneumoniae clones expressing serotypes not present in the antipneumococcal conjugate vaccine publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank all study participants for their involvement. we would like to thank the administration of the northern state medical university in arkhangelsk, russia for support during sample collection. we would also like to thank the administration of the regional clinical hospital in arkhangelsk for providing facilities of the microbiological laboratory for primary sample isolation. the authors acknowledge nadezda semenova, oksana lebedeva and bettina aasnaes for contribution to collection of samples. the authors also gratefully acknowledge bettina aanaes for excellent laboratory support. the authors also thank highly skilled laboratory staff of the norwegian institute of public health, oslo for assisting with serotyping and sequencing of pneumococcal isolates. authors' contributions gss, as and vvsj conceived the study. vvsj, asf, tb, as and gss designed the study. vvsj, asf, bch, as and gss contributed to the protocol writing. vvsj and tb recruited the study subjects. vvsj, gss, bch and tb contributed to collection of clinical samples. vvsj and bch carried out laboratory analysis. vvsj, asf, hcs, pvb, hcs and dac carried out data analysis. all authors wrote the manuscript and approved its publication. vvsj was granted by the quota scheme of the norwegian state education loan fund. hcs is involved with projects supported by pfizer. all other authors were funded by their individual departments. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. the data and materials are available on request from the corresponding author (veronika vorobieva solholm jensen, dept. of virus and microbiological special diagnostics, statens serum institute; artillerivej , dk- copenhagen s, denmark. email:veronika.v.vorobieva@gmail.com), but restrictions apply under licence for the current study. the data may be publicity available upon reasonable request and with permission of the northern state medical university, arkhangelsk, russia. the permission to carry out the study was sought from the authorities of the arkhangelsk region. all parents/ guardians were informed about the study by informational letters and a majority of the parents/guardians participated in informational meetings. the written informed consents were signed by parents or guardians for all participants of the study. there was a questionnaire that parents or guardians filled out for all participants of the study. consent for publication does not apply. hans-christian slotved is involved with projects supported by pfizer. all other authors had declare that they have no competing interests. key: cord- -id u br authors: beran, jiří; Šalapová, eva; Špajdel, marian title: inosine pranobex is safe and effective for the treatment of subjects with confirmed acute respiratory viral infections: analysis and subgroup analysis from a phase , randomised, placebo-controlled, double-blind study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: id u br background: inosine pranobex (isoprinosine®) is an immunomodulatory drug approved in several countries for the treatment of viral infections. this study compared the efficacy and safety of inosine pranobex versus placebo in subjects with clinically diagnosed influenza-like illness, including subjects with laboratory-confirmed acute respiratory viral infections. subgroup analyses evaluated the efficacy of inosine pranobex compared to placebo in otherwise healthy (without related ongoing disease) subjects that were less than years of age and healthy subjects that were at least years of age. the effect of body mass index (bmi) was evaluated in subjects less than years of age. methods: a total of subjects were randomly assigned to receive inosine pranobex (n = ) or placebo (n = ) in this phase , randomised, double-blind, multicentre study. the primary efficacy endpoint was time to resolution of all influenza-like symptoms present at baseline to none. safety was evaluated through analysis of adverse events, vital signs, and physical examinations. results: the difference in time to resolution of all influenza-like symptoms between treatment groups was not statistically significant but showed a faster improvement in subjects in the inosine pranobex group versus those in the placebo group - hazard ratio = . ; ( % ci: . – . ). p-value = . . in the subgroup analysis for subjects less than years of age, statistically significant differences in time to resolution of influenza-like symptoms that favoured the inosine pranobex group over the placebo group were observed in those without related ongoing disease and those who were non-obese (bmi < kg/m( )). the differences between the inosine pranobex and placebo groups in subjects at least years of age without related ongoing disease and in subjects less than years of age who were obese (bmi ≥ kg/m( )) were not statistically significant. inosine pranobex was generally well tolerated, and no deaths were reported. conclusions: the study results indicate the safety of inosine pranobex for the treatment of subjects with confirmed acute respiratory viral infections and confirm the efficacy of inosine pranobex versus placebo in healthy non-obese subjects less than years of age with clinically diagnosed influenza-like illnesses. trial registration: ewo-iso- / , eudract - - ; date of registration: apr ; detail information web link: https://www.clinicaltrialsregister.eu/ctr-search/trial/ - - /results electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory infection is a serious infection that is responsible for approximately . million deaths per year and is one of the leading causes of morbidity and mortality worldwide [ ] . acute respiratory infections are categorized as either upper or lower respiratory infections and are caused by well-recognised viral pathogens, including but not limited to influenza virus (types a and b), parainfluenza virus, respiratory syncytial virus (rsv), metapneumovirus (types a and b), coronavirus, rhinovirus, enterovirus, reovirus, bocavirus, and adenovirus, and bacterial pathogens, primarily streptococcus pneumoniae and haemophilus influenza [ , ] . influenza is an acute viral respiratory infection that affects % to % of the global adult population per year and results in approximately . to . million deaths and to million cases of severe illness worldwide annually [ , ] . influenza-like illnesses (ili) are considered a subset of acute respiratory infections and result in the sudden onset of symptoms such as fever (body temperature greater than °c), cough, and sore throat in patients [ ] . physicians have difficulty with the treatment of ili because determining the aetiology is generally not possible solely on a clinical basis. current pharmacological interventions for the prevention and treatment of respiratory viral infections are primarily limited to vaccines and antivirals for influenza. available antiviral treatments for influenza infection are m ion channel inhibitors (eg, amantadine and rimantadine) and neuraminidase inhibitors (eg, oseltamivir and zanamivir) [ ] . if the infection is caused by bacterial pathogens, treatment can involve antibiotics and also medications that provide symptomatic relief. however, in case of viral aetiology, only medication for symptomatic treatment can be provided. [ ] . vaccination for seasonal influenza remains one of the standard approaches for prevention of the disease. however, immunisation rates for seasonal influenza remain low in many european countries even though the influenza vaccine is part of the national immunisation schedule in these countries [ ] . moreover, the vaccination is effective only when most of the circulating influenza viruses in a given season are similar to the viruses that were included in the influenza vaccine. the vaccine does not offer any clinical benefit against other pathogens that circulate during a season [ ] . inappropriate use of antibiotics for the treatment of acute respiratory infections has been observed during winter months, even though the majority of infections are caused by viral pathogens and are self-limiting. this practice may result in side effects and the development of antibiotic resistance in pathogens as well as an increased cost burden for the healthcare system [ , , ] . inosine pranobex (isoprinosine®), a combination of the p-acetamidobenzoate salt of n,n-dimethylamino- -propanol and inosine in a : molar ratio, is an immunomodulatory antiviral drug that has been licensed since in several countries worldwide for the treatment of viral infections [ , ] . inosine pranobex stimulates a nonspecific immune response that is independent of the specific viral antigen responsible for the ili. in clinical studies, inosine pranobex has been shown to induce a type t helper cell-type response in mitogen-or antigenactivated cells, and this response initiates t-lymphocyte maturation and differentiation and potentiates induced lymphoproliferative responses ( ) ( ) ( ) . similarly, the drug modulates t-lymphocyte and natural killer cell cytotoxicity and cd + suppressor and cd + -helper cell functions and increases the number of immunoglobulin g and complement surface markers ( , ) . inosine pranobex also increases cytokine interleukin (il)- production and il- production and upregulates the expression of the il- receptor in vitro [ , ] . the safety profile of inosine pranobex has been established through clinical trials for several indications and populations [ , [ ] [ ] [ ] . a rapid increase in the number of mononuclear cells after the first dose of inosine pranobex was observed in % of the subjects, and this increase was consistent with clinical observations of rapid resolution of common cold symptoms [ , ] . this phase study aimed to compare the efficacy and safety of inosine pranobex with placebo in subjects with laboratory-confirmed acute respiratory viral infections in order to evaluate the clinical use of inosine pranobex for the treatment of acute respiratory viral infections. the primary efficacy endpoint was comparison between inosine pranobex and placebo groups in terms of the time to resolution of all influenza-like symptoms present at baseline to none. in a subgroup analysis of subjects with clinically diagnosed ili, the study further evaluated the efficacy of inosine pranobex compared to placebo in healthy (without related ongoing disease) subjects less than years of age and in those at least years of age. the study also evaluated the effect of body mass index (bmi) in subjects who were less than years of age and were non-obese (bmi < kg/m ) or obese (bmi ≥ kg/m ). in addition, the study evaluated the efficacy of inosine pranobex in subjects less than years of age for the time to resolution of all influenza-like symptoms present at baseline to mild or none (i.e. score of or on the influenza-like symptoms assessment scale). this was a phase , randomised, placebo-controlled, double-blind, multicentre study in subjects with clinically diagnosed ili, including subjects with laboratoryconfirmed acute respiratory viral infections due to influenza a or b virus, rsv, adenovirus, or parainfluenza virus or . the study was conducted at study sites in the czech republic ( study sites) and slovakia ( study sites), with enrolment occurring between december and april , and the last subject visit was on june . detailed primary efficacy endpoints are provided in the additional file . male and nonpregnant female subjects aged to years with a clinical diagnosis of ili were included in this study. influenza-like illnesses were defined as an oral temperature of at least °c observed at the study site with at least respiratory symptom of cough, sore throat, or nasal obstruction and at least constitutional symptom of fatigue, headache, myalgia, or feverishness. the respiratory and constitutional symptoms were required to be considered by the subject as moderate or severe in intensity (a score of more than on the point influenza-like symptoms assessment scale). the subjects were required to have experienced the onset of ili no more than h prior to screening, where onset is defined as the time when the subject experienced fever and at least respiratory symptom and at least constitutional symptom. the full inclusion criteria and influenza-like symptoms assessment scale are detailed in the additional file . subjects were excluded from participation in this study if they met any of the following criteria: had an immunosuppressive disorder or were receiving immunosuppressive therapy; were undergoing treatment with xanthine oxidase inhibitors or uricosuric agents or treatment with thiazide or loop diuretics; had chronic renal dysfunction or severe liver function impairment; were lactose intolerant; had cancer in a nonremission stage; were undergoing treatment with zidovudine; were pregnant or lactating/ breastfeeding; had received a dose of inosine pranobex, oseltamivir, zanamivir, amantadine, or rimantadine during this occurrence of ili; or had been administered an investigational drug or investigational vaccine within days prior to screening. detailed exclusion criteria are provided in the additional file . a sample size of subjects ( subjects in each treatment group) in the modified intent-to-treat (mitt) analysis set with a total of randomly assigned subjects was calculated using the log-rank test (inputting the median survival times). sample size calculations were performed using pass software version (ncss, llc, kaysville, utah, usa) and considered a statistical power of % to detect a clinically relevant difference between . days in the treatment group and days in the placebo group. a % dropout rate was also considered, which meant that a final sample size of subjects ( subjects in each treatment group) would be required for the study. however, because of the challenges faced during enrolment, which included a late influenza alert and an unexpectedly mild influenza season, the decision was made to continue enrolment until april . a total of subjects were randomly assigned, and of these, only subjects met the criteria for inclusion in the mitt analysis set. all analyses were conducted using sas® software version . (sas institute inc, cary, north carolina, usa). all statistical tests were -sided hypothesis tests performed with a % level of significance, which resulted in % ( -sided) confidence intervals (cis). no adjustments for multiplicity were made. the hazard ratios (hrs) and % cis were estimated using a proportional hazards model (hr > indicated a benefit to inosine pranobex compared with placebo). the safety analysis set consisted of all subjects who received at least dose of any study drug. all analyses using the safety analysis set grouped subjects according to the treatment that the subjects had actually received. the mitt analysis set consisted of all randomly assigned subjects who had a positive laboratory confirmation of acute respiratory viral infection due to influenza a or b virus, rsv, adenovirus, or parainfluenza virus or . this set was used for the primary and secondary efficacy analyses. detailed primary efficacy endpoints are provided in the additional file . all analyses using the mitt analysis set grouped subjects according to the randomised treatment. the intent-to-treat (itt) analysis set included all subjects who were randomly assigned to receive double-blinded study drug and was used for the subgroup analyses. on day , eligible subjects were randomly assigned to receive either inosine pranobex or placebo in a : allocation ratio with no stratification. the active drug and matching placebo tablets were provided in identical cartons that were identified with a kit number, such that all study site staff and subjects remained blinded throughout the study. only personnel with access to the interactive web response system and clinical supplies were unblinded and had access to the treatment assignments; all other parties involved in the study were fully blinded. inosine pranobex or placebo -mg tablets were selfadministered by the subjects for days ( tablets orally times daily). the first dose was taken immediately after randomisation at the study site, and the remaining doses were to be self-administered at home. doses were taken approximately h apart but consistent with the subject's lifestyle, ie, scheduling of dosing did not disturb the subject's usual sleep patterns. the subjects were provided with kits containing (randomised) medication sufficient for subject for days of treatment. subjects were instructed to consume no more than tablets for the specified duration and were required to return the excess study drug tablets at the end-of-treatment (eot) visit. adherence to study drug administration was good and was monitored as part of the study. the total study duration was days (± days) for each subject and consisted of a -day dosing period (day to day ), day for the eot visit (day + day), and a -day follow-up period (day ± days) after the eot visit. prior to randomisation on day , a nasopharyngeal swab sample was collected to test for the presence of influenza a or b virus, rsv, adenovirus, and parainfluenza virus or using the appropriate polymerase chain reaction analyses. the results were used to identify the subjects to be included in the mitt set and consequentially identify the subjects to be included in the primary endpoint analysis. a detailed procedure for the study visits is included in the additional file . the primary efficacy endpoint was the time to resolution of all influenza-like symptoms present at baseline to none (ie, a score of , defined as the complete absence of symptoms, on the influenza-like symptoms assessment scale [details provided in additional file ]). the secondary endpoints included time to resolution of respiratory symptoms (cough, sore throat, and nasal obstruction); time to absence of fever (oral temperature of ≤ . °c for at least consecutive readings that were at least h apart); time to resumption of normal activity (ie, score of on daily activities assessment scale); and frequency of viral respiratory infection complications, defined as hospitalisation, death due to ili or complications of ili, or requirement of antibiotic treatment for secondary bacterial infection. a subgroup analysis was conducted for time to resolution of all influenza-like symptoms present at baseline to none in subjects with clinically diagnosed ili. this was conducted in subjects less than years of age and subjects at least years of age without related ongoing disease (related ongoing disease was any medical condition with the preferred terms of asthma, bronchitis, chronic bronchitis, or chemical bronchitis that was ongoing at the start of the study). in addition, an analysis was conducted in subjects less than years of age who were non-obese (bmi < kg/m ) or obese (bmi ≥ kg/m ). an additional analysis was conducted for time to resolution of all influenza-like symptoms to mild or none for subjects less than years of age. safety was evaluated during the study through analysis of adverse events (aes), vital signs, and physical examinations. the study was performed in accordance with ethical principles that have their origin in the declaration of helsinki, international council for harmonization e (r ), and all applicable regulations. study was approved before study start by two multicentre ethics committees (mec). one mec in the university hospital brno approved study for all study centres in the czech republic and the second one mec of košice regional office approved study for all study centres in slovakia. all potential subjects signed an informed consent form prior to randomisation on day before any study-related procedures were performed. the study included a total of subjects who were randomly assigned to receive either inosine pranobex (n = ) or placebo (n = ), and . % of subjects completed the study (fig. -flow chart) . there were subjects who discontinued the study; the reasons included protocol noncompliance (n = ), aes (n = ; rhinopharyngitis and pleuropneumonia), and withdrawal of consent (n = ). overall, subjects ( . %) had positive nasopharyngeal swab test results and were included in the mitt analysis set (inosine pranobex, n = ; placebo, n = ). the details of the demographic and baseline characteristics are presented in the additional file . the demographic characteristics were similar between the treatment groups. the overall mean age was . years, and the overall mean bmi was . kg/m (range: . to . kg/m ). the majority of subjects in this study were less than years of age. at baseline, most of the subjects presented with at least influenza-like symptom (cough, sore throat, nasal obstruction, fatigue, headache, myalgia, or feverishness) with a score of , , or in severity. medical histories were reported for subjects ( . %) in the inosine pranobex treatment group and subjects ( . %) in the placebo group, and the most commonly reported medical histories were vascular disorders and surgical and medical procedures. medical histories of gastrointestinal disorders ( . % in the inosine pranobex group and . % in the placebo fig. flow-chart of enrolment, placement in treatment and placebo arms, division into the different subgroups and mitt and itt analysis sets group) and hepatobiliary disorders ( . % in the inosine pranobex group and . % in placebo group) were also reported. overall, the mean reported dose compliance was high, and it was similar between treatment groups ( % in each treatment group). in the majority of subjects in both treatment groups, dose compliance ranged from to % (inosine pranobex, n = [ . %]; placebo, n = [ %]). the difference in time to resolution of all influenza-like symptoms between treatment groups was not statistically significant but showed a trend towards improvement in subjects in the inosine pranobex group compared with subjects in the placebo group (hr: . ; % ci: . , . ; p = . ) (fig. ) . a substantial decrease in the proportion of subjects with all influenzalike symptoms was observed in the inosine pranobex group after days while a decrease to similar proportions occurred only after days for subjects in the placebo group. the differences in time to resolution for the secondary endpoints also showed a similar trend towards improvement for subjects in the inosine pranobex group compared with subjects in the placebo group, but these secondary endpoint differences were not significant. the detailed results are provided in the additional file . in the subgroup analysis, for subjects less than years of age without related ongoing disease, the difference in time to resolution of all influenza-like symptoms between treatment groups was statistically significant (p = . ) and showed faster improvement in subjects in the inosine pranobex group compared with subjects in the placebo group (hr: . ; % ci: . , . ) (fig. and table ). however, for subjects at least years of age without related ongoing disease, the difference in time to resolution of all influenza-like symptoms between treatment groups was not statistically significant (hr: . ; % ci: . . , . ; p = . ). for non-obese (bmi < kg/m ) subjects less than years of age, the difference in time to resolution of all influenza-like symptoms between treatment groups was statistically significant (p = . ) and showed a faster improvement in subjects in the inosine pranobex group compared with subjects in the placebo group (hr: . ; % ci: . , . ). however, for obese (bmi ≥ kg/ m ) subjects less than years of age, the difference in time to resolution of all influenza-like symptoms in subjects less than years of age, the difference in time to resolution of all influenza-like symptoms to mild or none between treatment groups was statistically significant (p = . ) and showed a faster improvement in subjects in the inosine pranobex group compared with subjects in the placebo group (hr: . ; % ci: . , . ). the proportion of subjects experiencing treatmentemergent aes (teaes) and the number of the majority of teaes were mild or moderate in severity and were unrelated to study drug, in the opinion of the investigator. overall, severe teaes were reported in subjects ( . %). severe teaes of rhinopharyngitis, maxillary sinusitis, and vertebrogenic pain syndrome were reported in subject ( . %) each in the inosine pranobex group, and the severe teae of pleuropneumonia was reported in subject ( . %) in the placebo group. no deaths were reported during the study. overall, subjects ( . %) in the inosine pranobex group and subjects ( . %) in the placebo group experienced teaes that led to study drug discontinuation. three treatment-emergent serious aes (saes) were reported in subjects ( . %). severe rhinopharyngitis and severe vertebrogenic syndrome were reported in subject in the inosine pranobex group, and subject in the placebo group reported severe pleuropneumonia. these saes resulted in permanent discontinuation of the study drug and discontinuation of both subjects from the study. all saes resolved, and none of the saes in the opinion of the investigator were related to the study drug. the mean changes in vital sign and physical examination values from baseline were small, and no clinically the "bold data" are statisticly significant significant trends were observed between treatment groups. no pregnancies were reported during the study. this was a phase , randomised, placebo-controlled, double-blind, multicentre study that evaluated the efficacy of inosine pranobex in subjects with clinically diagnosed ili, including subjects with laboratory-confirmed acute respiratory viral infections due to influenza a or b virus, rsv, adenovirus, or parainfluenza virus or . the study also evaluated the efficacy of inosine pranobex in subgroups of subjects less than years of age who were without related diseases (such as asthma, bronchitis, chronic bronchitis, and chemical bronchitis) and who were non-obese (bmi < kg/m ) or obese (bmi ≥ kg/m ) as well as a subgroup of subjects at least years of age without related ongoing disease. in addition, a subgroup analysis was conducted in subjects less than years of age for time to resolution of all influenza-like symptoms to mild or none. in the current study, the analysis of the primary endpoint of time to resolution of all influenza-like symptoms showed a faster improvement in subjects treated with inosine pranobex compared with subjects administered placebo, although the difference between treatment groups did not reach the threshold of statistical significance. the results were similar for the secondary efficacy endpoints of time to resolution of respiratory symptoms (cough, sore throat, and nasal obstruction), time to absence of fever, and time to resumption of normal activity. the difference in the occurrence of viral respiratory infection complications between treatment groups was not statistically significant. immunosenescence, ie, the age-related decline of the immune system, and obesity play an important role in the efficacy of the immune response to pathogens [ , ] . older subjects show a diminished immune response to pathogens, which increases their risk for severe infection and compromises their ability to adequately combat viral infections. this phenomenon was observed with split-virus influenza vaccines; a low response to the vaccine was observed in older adults, whereas the vaccine was effective in younger subjects. this low response resulted in increased susceptibility to influenza and associated complications in older adults compared to younger adults who typically benefit from a higher response [ , ] . obesity has also been identified as an independent risk factor for increased susceptibility to influenza virus infection; this susceptibility results from diminished cd + and cd + t-cell responses and lower influenza vaccine antibody levels [ , , ] . obesity may also increase the risk of pneumonia or other infections by restricting lung volume [ ] . immunosenescence and obesity can bias efficacy studies because of the impaired response of the immune system to pathogens, as the risk of complications is increased in such individuals. in the subgroup analysis of the current study, in subjects less than years of age who were without related ongoing disease and in those less than years of age who were non-obese (bmi < kg/m ), statistically significant differences in time to resolution of influenzalike symptoms favoured the inosine pranobex group over the placebo group. statistically significant differences were not observed between the inosine pranobex and placebo groups in subjects at least years of age without related ongoing disease or in subjects less than years of age who were obese (bmi ≥ kg/m ). thus, the efficacy of inosine pranobex was improved in nonobese subjects compared with obese subjects, probably because the immune system in the former is more capable of defending against pathogens and is not negatively affected by obesity-related complications. older patients have a decreased immune response to pathogens as a result of immunosenescence; therefore, they may take longer to recover from illnesses such as influenza and antiinfluenza drugs may not be as effective. in an additional analysis, in subjects less than years of age, statistically significant differences in time to resolution of influenzalike symptoms to mild or none favoured the inosine pranobex group over the placebo group, thus indicating that the improvement of symptoms was better with inosine pranobex than placebo in this subset of subjects (hr: . ; % ci: . , . ). a substantial decrease in the proportion of subjects with all influenza-like symptoms was observed in the inosine pranobex group after days while a decrease to similar proportions occurred only after days for subjects in the placebo group. this difference could be a result of the time necessary for activation of the immune system, as inosine pranobex acts indirectly by stimulating the immune system and does not directly attenuate the symptoms. this result is also consistent with the results observed in a study in healthy volunteers in which inosine pranobex showed immunomodulating properties through an increase in serum levels of interferon-γ, il- , il- , and tumour necrosis factor-α from to days [ ] . the approved dose and treatment duration of inosine pranobex (two -mg tablets orally times daily) were used in this study, and the administered treatment did not vary according to weight or symptom duration. from the subgroup analysis, the posology of inosine pranobex ( g/day orally) in this study is most suitable for subjects less than years of age without related ongoing disease and subjects less than years of age who were non-obese (bmi < kg/m ). for certain subjects, such as those at least years of age and those who are obese, different dosing strategies could be more appropriate; varying the dosing regimen requires further evaluation. the use of a placebo-only group in this study was justified because ili is generally mild and self-limiting and no other treatments are approved for acute respiratory viral infections other than influenza. in addition, the use of influenza-specific antivirals (neuraminidase inhibitors or amantadine) is not a component of routine medical management of ili in many countries, including the countries in which the study was conducted. performing a high-quality efficacy trial for ili is challenging because of epidemiologic considerations from the influenza outbreak period; the influenza season cannot be predicted in advance and can vary from year to year [ ] . furthermore, the enrolment of subjects with symptoms that have been present for less than h is necessary, as the first h is the period of maximal viral replication and antiviral medication is expected to have the most benefit during this time. the current study was anticipated to be performed in influenza season in the northern hemisphere, between october and april , and enrol the required number of subjects in each group during this timeframe. however, enrolment was challenging, as it could not be commenced because of a low attack rate and was only started after a late alert was issued by national public health authorities in the first week of december regarding the statistically higher incidence of acute respiratory viral infections. a low density of circulating viruses was present until the end of january , which resulted in the enrolment of subjects until april in order to maximize the number of completed subjects. approximately % of the randomised subjects were expected to have a positive laboratory confirmation of acute respiratory tract infection. however, only subjects met the criteria for inclusion in the mitt analysis set, which was subjects fewer than the subjects expected. the effects of the absence of a significant influenza outbreak adversely affected the statistical power, thus reducing the power of the study, which could potentially explain the lack of statistical significance for the primary and secondary efficacy endpoints. a slightly longer duration of treatment or a different dosing strategy may have influenced the observed efficacy of inosine pranobex, as proof of therapeutic effect requires a high attack rate, ie, a higher number of sick patients during one influenza season. the attack rate is difficult to predict, and studies, which will adjust the sample size calculations to account for the possibility of a below-average flu season or studies with longer duration, e.g., those that include or more influenza seasons to account for lower than predicted attack rates, are necessary to achieve the desired results [ ] . in addition, . % of enrolled subjects in the inosine pranobex group reported a medical history of gastrointestinal disorders and . % reported hepatobiliary disorders. pharmacokinetic parameters were not measured in this study, but it is possible that the presence of these disorders at baseline may have affected the absorption, distribution, and metabolism of the study drug and may have influenced the study results, including the efficacy results. furthermore, age, comorbidities, and the obesity of enrolled subjects, particularly in subjects more than years of age, may also have affected the outcome of the study. the safety analysis demonstrated that inosine pranobex treatment was well tolerated, and no major differences in safety profiles were observed between treatment groups. treatment-emergent saes were reported in subjects, and none were considered to be related to study drug by the investigators. no subjects died during the study. no significant changes were observed in vital signs and physical examinations in either study group. the study results indicate the safety of inosine pranobex for the treatment of subjects with confirmed acute respiratory viral infections and confirms the efficacy of inosine pranobex versus placebo in healthy non-obese subjects less than years of age with clinically diagnosed influenza-like illnesses. the results of this study were affected by epidemiologic considerations, which included a late influenza alert and a low density of circulating viruses. further studies may be important to define predictors of treatment success, including the potential of different dosing strategies in certain patient populations, such as those with underlying conditions that may impact drug plasma levels and related drug effects. additional file : the primary efficacy assessment; secondary efficacy endpoints; detailed primary endpoint; inclusion and exclusion criteria; procedures and table s . influenza-like symptoms assessment scale; table s . demographic and other baseline characteristics (itt analysis set); secondary endpoints; table s . time to resolution of respiratory symptoms and resumption of normal activity (mitt analysis set); table s . treatment-emergent adverse events (safety analysis set); table s . treatment-emergent ae occurring in at least % of subjects overall by system organ class per treatment (safety analysis set); figure s 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cell subsets obesity is associated with impaired immune response to influenza vaccination in humans immunosenescence: implications for response to infection and vaccination in older people effects of aging on human leukocytes (part i): immunophenotyping of innate immune cells overweight and obese adult humans have a defective cellular immune response to pandemic h n influenza a virus obesity and influenza infection severity the impact of obesity on the immune response to infection challenge of conducting a placebo-controlled randomized efficacy study for influenza vaccine in a season with low attack rate and a mismatched vaccine b strain: a concrete example all authors acknowledge support from dr richard south, dr sandra palleja, and priyanka tiwari of ppd for their involvement in the main stages of the trial and preparation of the clinical study report and manuscript. all authors also thank saurabh aggarwal, phd, senior medical writer at ppd, for his significant contributions to this manuscript. the design of the study, collection, analysis, and interpretation of data and writing the manuscript was funded by ewopharma international. the datasets generated during and/or analysed during the current study are available on eudract website: https://www.clinicaltrialsregister.eu/ctr-search/ trial/ - - /results. patient data are provided as summary data in anonymized form. authors' contributions jb participated in the entire study as an investigator at one of the centres. he evaluated the data and clinical study report and contributed to the manuscript. es participated as the medical officer of the study and contributed to the manuscript. she is a medical director in ewopharma international. ms participated as a biostatistics specialist. he assessed the data and clinical study report, suggested additional analyses, and contributed to the manuscript. all authors read and approved the final version of manuscript. es is an employee of ewopharma international. jb received grants for performing the study at his centre. ms has no competing interests. not applicable. the study was performed in accordance with ethical principles that have their origin in the declaration of helsinki, international council for harmonization e (r ), and all applicable regulations. study was approved before study start by two multicentre ethics committees (mec). one mec in the university hospital brno approved study for all study centres in the czech republic and the second one mec of košice regional office approved study for all study centres in slovakia. all potential subjects signed an informed consent form prior to randomisation on day before any study related procedures were performed. key: cord- -wgigtgl authors: dube, felix s.; kaba, mamadou; robberts, f. j. lourens; tow, lemese ah; lubbe, sugnet; zar, heather j.; nicol, mark p. title: respiratory microbes present in the nasopharynx of children hospitalised with suspected pulmonary tuberculosis in cape town, south africa date: - - journal: bmc infect dis doi: . /s - - -z sha: doc_id: cord_uid: wgigtgl background: lower respiratory tract infection in children is increasingly thought to be polymicrobial in origin. children with symptoms suggestive of pulmonary tuberculosis (ptb) may have tuberculosis, other respiratory tract infections or co-infection with mycobacterium tuberculosis and other pathogens. we aimed to identify the presence of potential respiratory pathogens in nasopharyngeal (np) samples from children with suspected ptb. method: np samples collected from consecutive children presenting with suspected ptb at red cross children’s hospital (cape town, south africa) were tested by multiplex real-time rt-pcr. mycobacterial liquid culture and xpert mtb/rif was performed on induced sputa obtained from each participant. children were categorised as definite-tb (culture or qpcr [xpert mtb/rif] confirmed), unlikely-tb (improvement of symptoms without tb treatment on follow-up) and unconfirmed-tb (all other children). results: amongst children with a median age of months (interquartile range, [iqr] – months), ( %) had definite-tb, ( %) had unconfirmed-tb and ( %) were classified as unlikely-tb. moraxella catarrhalis ( %), streptococcus pneumoniae ( %), haemophilus influenzae spp ( %) and staphylococcus aureus ( %) were the most common bacteria detected in np samples. other bacteria detected included mycoplasma pneumoniae ( %), bordetella pertussis ( %) and chlamydophila pneumoniae ( %). the most common viruses detected included metapneumovirus ( %), rhinovirus ( %), influenza virus c ( %), adenovirus ( %), cytomegalovirus ( %) and coronavirus o ( . %). both bacteria and viruses were detected in , and % of the definite, unconfirmed and unlikely-tb groups, respectively. there were no significant differences in the distribution of respiratory microbes between children with and without tb. using quadratic discriminant analysis, human metapneumovirus, c. pneumoniae, coronavirus , influenza virus c virus, rhinovirus and cytomegalovirus best discriminated children with definite-tb from the other groups of children. conclusions: a broad range of potential respiratory pathogens was detected in children with suspected tb. there was no clear association between tb categorisation and detection of a specific pathogen. further work is needed to explore potential pathogen interactions and their role in the pathogenesis of ptb. electronic supplementary material: the online version of this article (doi: . /s - - -z) contains supplementary material, which is available to authorized users. lower respiratory tract infection (lrti) is a leading cause of mortality and morbidity in children under five years, accounting for approximately million deaths in globally [ ] . pulmonary tuberculosis (ptb) is an important cause of lrti and may present as acute or chronic disease [ ] . tb is increasingly recognised as a primary cause or as a comorbid condition in children hospitalized with pneumonia, particularly in settings endemic for human immunodeficiency virus (hiv) and tb [ ] . diagnosis of ptb in children is largely made based on clinical and radiological features, which may be nonspecific. therefore ptb cannot be easily differentiated from other causes of acute or chronic lrti [ ] . recent studies have reported detection of polymicrobial infections in children with lrti [ ] . further, ptb has increasingly been reported in children presenting with acute pneumonia; culture confirmed tb was reported in % of south african children hospitalized with acute pneumonia with no difference by hiv status [ ] . a recent meta-analysis confirmed mycobacterium tuberculosis in . % of childhood pneumonia cases in tb endemic areas [ ] . there are limited published data on the role of other respiratory pathogens amongst patients suspected to have tb [ ] . in africa, only one study, conducted in botswana, has addressed this question amongst adult ptb suspects. this study reported microbiologically confirmed tb in / ( %); mycoplasma pneumoniae infection in / ( %) and pneumocystis jirovecii infection in / ( %) of ptb suspects [ ] . co-infection with two or more pathogens was documented in % of patients [ ] . in our studies of south african children hospitalised with suspected ptb, % of children hospitalised with suspected ptb had microbiologically-confirmed ptb, % were classified as unconfirmed-ptb and % were classified as unlikely-ptb (children in whom tb was excluded and who improved in the absence of tb therapy) [ ] . approximately half of the children were treated for ptb, including all children with definite ptb and most with unconfirmed ptb [ ] . these data raise several questions around potential co-infections including the extent to which these may contribute to disease or severity in ptb, the aetiology of lrti amongst children with symptoms suggestive ptb and the role of other potential respiratory pathogens in those without tb. we have recently shown [ ] that specific pathogens (including bordetella pertussis, influenza virus, respiratory syncyntial virus [rsv], adenovirus, parainfluenzavirus, bocavirus) are detected significantly more frequently from the nasopharynx (np) of children with pneumonia than age-matched controls, and so are likely to play an important role in pneumonia aetiology. we therefore conducted a preliminary study to investigate the diversity of potential respiratory pathogens in the np of children hospitalised with suspected ptb. the population studied in this study has been previously described [ ] . briefly, we enrolled children under years of age suspected of ptb presenting (between july and may ) at red cross war memorial children's hospital (rch), a specialist referral paediatric hospital in cape town, south africa. verbal assent was obtained from children older than seven ( ) years of age and informed consent was obtained from a parent or legal guardian for all children. suspected tb was defined as having history of a cough and one of the following: i) a household contact with tb within the previous three months, ii) loss of weight or failure to gain weight in the previous three months, iii) a positive tuberculin skin test (tst) to purified protein derivative (ppd; tu, ppd rt , staten serum institute, denmark, copenhagen), or iv) a chest radiograph suggestive of ptb. a positive skin test was defined as mm or more of transverse induration in children with hiv infection or mm or more in children without hiv infection [ ] . children were excluded if the child was on tb treatment or tb prophylaxis for more than h, and if they could not be followed-up (not resident in cape town). all patients with laboratory confirmed tb and those diagnosed with tb based on clinical and radiological criteria were referred for tb therapy at a local clinic in accordance with south african national guidelines [ ] . children were followed up at and months to assess response to therapy or improvement without tb treatment. all children received care in the public health system that includes free expanded programme on immunisation (epi) for diphtheria, pertussis (whooping cough), and tetanus (dpt), haemophilus influenza type b, and streptococcus pneumoniae ( -valent pneumococcal conjugate vaccine [pcv] from , replaced with -valent pcv in ). two paired induced sputa and a nasopharyngeal (np) swab specimens were collected from each child and transported to the laboratory within h of collection. np swabs were obtained before sputum induction using nylon flocked swabs (copan italia, brescia, italy) by trained study staff [ ] . each np swab was immediately placed into . ml primestore® transport and stabilization medium (primestore® mtm, longhorn vaccines and diagnostics, san antonio, tx) and stored at − °c within h of collection until further batch processing. samples were randomly selected from a convenience subset of children, over a -year period for testing of np specimens for other microbes. as the volume obtained on induced sputum specimens was small, the entire specimen was required for detection of m tuberculosis, for optimal management and as this study was nested within a broader study investigating better diagnostics for tb in children [ ] . we did not want to compromise this primary aim and only np samples were available for study of other respiratory pathogens. induced sputum specimens were submitted to the national health laboratory services (nhls) medical microbiology laboratory at groote schuur hospital (cape town, south africa) for mycobacterial liquid culture (bactec mgit, becton dickinson microbiology systems, cockeysville, md) and nucleic acid amplification testing (xpert mtb/rif, cepheid, sunnyvale, ca). children were categorised as 'definite-tb' (i.e. culture or xpert mtb/rif positive for m. tuberculosis), 'unlikely tb' (i.e. no clinical diagnosis of tb with improvement on follow-up without tb treatment) and 'unconfirmed tb' (all others) [ ] . multiplex pcr testing of nasopharyngeal samples np swabs were thawed at room temperature ( °c) and vortexed for s. thereafter, μl of each sample was transferred to a zr bashingbeadstm lysis tube (zymo research corp., irvine, ca) and subjected to mechanical lysis on a tissuelyzer lt (qiagen, hilden, germany) [ ] . the lysed samples were then centrifuged at , × g for min to pellet all cellular debris. aliquots of μl of the supernatant were transferred to a ml sterile tube (sarstedt, nümbrecht, germany) and μl of an exogenous internal control (equine arteritis virus) was added to each sample prior to automated total nucleic acid extraction on the qiasymphony sp instrument using the qiasymphony® virus/bacteria mini kit (qiagen, hilden, germany). total nucleic acid was eluted in μl elution buffer and stored at - °c until further processing. nucleic acid amplification was performed using the ftd resp kit according to the manufacturer's instructions (fast-track diagnostics, luxembourg). the assay comprises eight multiplex real-time pcr reactions for the detection of nucleic acid targets (additional file : table s ). results were interpreted according to manufacturer's instructions using the ftd resp analyser, an in-house java based program (available at http://www.gematics. com/analyser.html). exploratory statistics were performed using stata software (stata corporation, college station, tx), whilst the openly available statistical environment r, version . . [ ] was used for more detailed analyses. pearson's chisquared test was used to compare the occurrence of each microbe between children with definite tb and unlikely tb with yates' continuity correction. permutation tests were used to determine which microbe pairs were statistically concurrent. briefly, for each pair of microbes, x and y, the observed number of concurrences (m) was counted. the null hypothesis was that there was no relationship between microbes x and y and that the co-occurrences were purely random. the null hypothesis was tested by generating random permutations of the occurrences of x and y. the number of concurrences under these random conditions was then counted, m . by repeating the permutation process times, random concurrences were obtained as follows: m , m ,…, m . the shape of the null-distribution, the distribution of co-occurrence counts under purely random conditions, was estimated from the observed values of the permutation test. the achieved significance level (asl) was computed as the number of permuted co-occurrences that were equal to or greater than the observed number of concurrence (tail probability under the null-distribution). this can be interpreted as a nonparametric p-value [ ] . linear discriminant analysis (lda) and quadratic discriminant analysis (qda) [ ] were used to optimally discriminate respiratory microbes occurring in relation to tb status. the lda is visually represented in a canonical variate analysis (cva) biplot and the qda in a qda biplot [ ] . the visualizations provide information on how the different tb groups overlap and to what extent the detection of microbes differs between the groups. for all the tests, a p-value less than . was used as the limit of statistical significance. the human research ethics committee (hrec / ) of the faculty of health sciences, university of cape town, south africa approved this study. hiv-infected, with similar hiv prevalence by tb category. immunization records were available for out of ( %) children included in this study. among children for whom data on immunization profile was available, the vaccination status was up to date in % of children, while among the remaining % ( / ), at least one scheduled immunization was missing. none of the risk factors considered in table was associated with the occurrence of any microbes pertussis even after adjusting for potential confounders (additional file : table s ). nucleic acid of at least one of the targeted respiratory microbes was detected in % of np specimens. the most common bacteria detected were moraxella catarrhalis ( %), s. pneumoniae ( %), h. influenzae spp ( %) and staphylococcus aureus ( %) ( table ). m. pneumoniae ( %), b. pertussis ( %) or c. pneumoniae ( %) were detected less frequently. the most frequently detected viral targets were human metapneumovirus (hmpv) ( %), rhinovirus ( %), influenza c virus ( %), adenovirus ( %), cytomegalovirus ( %) and coronavirus o ( . %) ( table ) . seasonal patterns were observed for hmpv, rhinovirus, enterovirus and influenza viruses with peak prevalence in late winter (august) and spring (november). in addition, a seasonal pattern was detected for tuberculosis (additional file : figure s ). no distinct seasonal patterns were observed for other microbes. a single bacterial target was detected in / ( %) of samples tested. two bacterial targets were detected in / ( %), and three bacterial targets detected in / ( %). a single viral target was detected in / ( %) of samples, two viral targets in / ( %) and three or more viral targets in / ( %) specimens. bacteria alone were detected in / ( %) of samples, viruses alone were detected in / ( %) of samples and both viruses and bacteria in / ( %) of samples. a detailed overview of all possible pairs of co-occuring respiratory microbes, irrespective of tb category (additional file : table s ). these co-occurences were further tested for significance (table ). significant bacterial-bacterial associations included interactions between m. catarrhalis and each of m. pneumoniae, s. pneumoniae and h. influenza spp. viral-viral associations included: bocavirus and influenza a virus, parainfluenza virus and coronavirus nl as well as hmpv and enterovirus. significant viral -bacterial associations were common particularly between h. influenza (type b or non-type b) and a range of viruses, including enteroviruses, hmpv, influenza c, influenza a and cytomegalovirus. when the unconfirmed tb group is excluded from the lda analysis, the lda biplot is reduced to a onedimensional plot (additional file : figure s ). in this case, the presence of rhinovirus, coronavirus , adenovirus, parainfluenza , hmpv, bocavirus, c. pneumoniae, s. pneumoniae, h. influenzae type b, m. catarrhalis, influenza virus c virus, and b. pertussis best discriminated cases with definite-tb from those with unlikely tb. quadratic discriminent analysis did not identify any significant association between definite-tb and unlikely-tb groups. however, visual inspection of the qda biplot (fig. ) showed that hmpv, coronavirus , influenza c virus, rhinovirus, cytomegalovirus and c. pneumoniae formed the dominant microbial profile associated with definite-tb cases. in contrast, m. pneumoniae, h. influenzae, p. jirovecii, enterovirus, influenza b virus and rsv a/b were associated with the unlikely-tb category. this is the first comprehensive detailed study of the diversity of respiratory microbes detected in children presenting with suspected ptb in a tb endemic setting and showed that multiple potential pathogens are present in th nasopharynx of such children. the ftd multiplex real-time pcr detected at least one of the microbial targets in % of np swabs from children suspected of ptb. detection of multiple bacterial and viral targets was common. bacterial species frequently found as commensals in the nasopharynx were most commonly detected [ , ] . they include m. catarrhalis ( %), s. pneumoniae ( %), h. influenzae spp ( %), and s. aureus ( %). in addition, potential pathogenic organisms were detected in the nasopharynx including rsv, c. pneumoniae and b. pertussis. the prevalence of b. pertussis was % in our study. similar detection rates ( - %) were reported in other south african settings in children with lrti including at our study site, - years post transition from whole-cell vaccines to acellular vaccines (south african infants are vaccinated with dtap-ipv/hib; pentaxime®, sanofi pasteur) [ ] . the prevalence of some clinically relevant viral targets (rsv, bocavirus and adenovirus) in this cohort is lower than that previously reported in children with lrti [ ] [ ] [ ] . the observed differences may be explained by our enrolment criteria which targeted symptoms suggestive of ptb. however, viral pcr positivity of hmpv, enterovirus and influenza virus is similar to a recent case-control study that also showed their association with community acquired pneumonia [ ] . as with bacteria, care needs to be taken with the interpretation of molecular detection of some viruses in np specimens, since target nucleic acid may be detected for some time after resolution of symptoms, and from otherwise healthy children [ , ] . in this study, some microbes showed no association with any of the tb categories. these included m. catarrhalis, and s. pneumoniae. a randomised controlled trial of the efficacy of pcv in south african children showed decreased rates of culture-confirmed and clinically diagnosed tb in pcv recipients hospitalised with lrti compared with placebo recipients (relative risk reduction %) [ ] . this suggests that coinfection with m. tuberculosis and s. pneumoniae causes severe infection requiring hospitalization. although common in our cohort, s. pneumoniae did not cluster together with the tb or unlikely-tb groups, however we measured np colonization which is likely to be an inaccurate measure for the contribution of s. pneumoniae to lrti. we have recently shown [ ] that specific pathogens (specifically b. pertussis, influenza virus, rsv, adenovirus, parainfluenzavirus, bocavirus) are detected significantly more frequently from the np of children with pneumonia than age-matched controls. in this study we detected all of these organisms, irrespective of tbclassification, suggesting that these pathogens may play a role in the exacerbation of symptoms in children with tb as well as accounting for the respiratory illness of a subset of the children without tb. whilst we detected multiple significant co-occurrences between different microbes in this study, these require more detailed assessment in a larger group of children. for example, the co-occurrence between m. catarrhalis, s. pneumoniae and h. influenzae, may reflect age-specific colonization patterns as previously reported [ , ] . other microbial co-occurrences, such as the association between s. aureus and p. jorovecii, were unexpected. we are currently conducting a larger, longitudinal study to better understand these co-occurences [ ] . we were unable to identify significant associations between individual nasopharyngeal microbes and tb classification. discriminant analysis identified that the presence of c. pneumoniae, hmpv, coronavirus o , influenza c virus, rhinovirus and cytomegalovirus best discriminated children with definite tb. the significance of co-detection of these microbes in children with tb is unclear, and needs to be further assessed. one possibility is that the relative immune suppression or lung pathology associated with ptb may render the host susceptible to other respiratory infections, or alternatively, that intercurrent infection may be immunosuppressive, predisposing to an accelerated clinical course or likelihood of symptoms in children with ptb. active tb is associated with suppression of cellular immune responses, which are critical for the control of intracellular infections [ ] such as many of those associated with definite tb in this study. however, in this study, individual microbes were each only detected in small numbers of children which limits our ability to draw firm conclusions in this regard. a recent south african study has shown an increased risk of death in adults with tb-influenza a virus coinfection (adjusted relative risk ratio [arrr] . ) compared to tb infection alone [ ] . in contrast, de paus et al. did not find a correlation between the seroprevalence of influenza antibodies and the development of clinically active tb in an indonesian cohort [ ] . they did however show an association between elevated antibody titres against influenza a and the clinical stage of tb lung disease suggesting recent re-infection with influenza precedes clinical presentation with ptb [ ] . a limitation of this study is the lack of a control group of children without lower respiratory symptoms. we are therefore unable to infer whether the pathogens detected played a role in the development or exacerbation of symptoms in this cohort. further limitations include sampling of the nasopharynx rather than the lower respiratory tract, limiting the ability to infer causality for lung co-pathogens. klebsiella pneumoniae, legionella spp and salmonella targets were excluded from analysis due to problems with assay specificity for these targets. in conclusion, this study describes the detection of multiple respiratory microbes in the nasopharynx of children hospitalised with suspected ptb. whilst there was no clear separation between the pathogens present in the airways of children with and without ptb, c. pneumoniae, hmpv, coronavirus o , influenza c virus, rhinovirus and cytomegalovirus formed the dominant microbial profile in children with ptb but this failed to reach statistical significance on testing of each individual microbe. in contrast, p. jirovecii, h. influenzae spp, rsv, m. pneumoniae, influenza b virus and enteroviruses were more consistently detected in children without tb although not statistically significant. this pilot work may signal broader differences in the microbial ecology of the upper respiratory tract of these children, which warrants further study. additional file : figure s . seasonal distribution of viruses and bacteria. figure s . canonical variate analysis (cva) biplot depicting the spread of respiratory pathogens in the definite tb (red line) and not tb (blue line) groups only. observations under each group are denoted by "+" signs and the median of each group by the red and blue ovals. table s . target pathogens in the ftd respiratory pathogens multiplex realtime pcr assay. table s . summary of all paired pathogen co-occurrence counts *. table s . risk factors associated with the occurrence of each microbes. (pdf kb) global, regional, and national causes of child mortality in - , with projections to inform post- priorities: an updated systematic analysis understanding latent tuberculosis: a moving target tuberculosis as a cause or comorbidity of childhood pneumonia in tuberculosis-endemic areas: a systematic review advances in the diagnosis of pulmonary tuberculosis in hiv-infected and hiv-uninfected children aetiology of childhood pneumonia in a well vaccinated south african birth cohort: a nested case-control study of the drakenstein child health study role of streptococcus pneumoniae in hospitalization for acute community-acquired pneumonia associated with culture-confirmed mycobacterium tuberculosis in children: a pneumococcal conjugate vaccine probe study etiology of pulmonary infections in predominantly hiv-infected adults with suspected tuberculosis accuracy of the xpert mtb/rif test for the diagnosis of pulmonary tuberculosis in children admitted to hospital in cape town, south africa: a descriptive study urine lipoarabinomannan testing for diagnosis of pulmonary tuberculosis in children: a prospective study national tuberculosis management guidelines detection of streptococcus pneumoniae from different types of nasopharyngeal swabs in children evaluation of tuberculosis diagnostics in children: . proposed clinical case definitions for classification of intrathoracic tuberculosis disease. consensus from an expert panel a comparison of the efficiency of five different commercial dna extraction kits for extraction of dna from faecal samples r: a language and environment for statistical computing an introduction to the bootstrap visualisation of quadratic discriminant analysis and its application in exploration of microbial interactions associations between pathogens in the upper respiratory tract of young children: interplay between viruses and bacteria clinical characteristics of children with lower respiratory tract infections are dependent on the carriage of specific pathogens in the nasopharynx incidence and diagnosis of pertussis in south african children hospitalized with lower respiratory tract infection respiratory viruses associated with community-acquired pneumonia in children: matched case-control study etiology of community-acquired pneumonia in hospitalized school-age children: evidence for high prevalence of viral infections viral and atypical bacterial detection in acute respiratory infection in children under five years clinical epidemiology of bocavirus, rhinovirus, two polyomaviruses and four coronaviruses in hiv-infected and hiv-uninfected south african children identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections dynamics of nasopharyngeal colonization by potential respiratory pathogens early acquisition and high nasopharyngeal co-colonisation by streptococcus pneumoniae and three respiratory pathogens amongst gambian new-borns and infants investigating the early-life determinants of illness in africa: the drakenstein child health study immunosuppression during active tuberculosis is characterized by decreased interferon-gamma production and cd expression with elevated forkhead box p , transforming growth factor-beta, and interleukin- mrna levels influenza virus infection is associated with increased risk of death amongst patients hospitalized with confirmed pulmonary tuberculosis in south africa the influence of influenza virus infections on the development of tuberculosis we thank the national health laboratory service diagnostic microbiology at groote schuur hospital (cape town, south africa) for the microbiological tb testing. we further wish to thank lesley workman, charmaine barthus, widaad zemanay, layla hendricks, nchimunya hapeela, whitney barnett and the rest of the study team for their help and technical assistance. we thank the western cape health department and the staff at red cross children's hospital for their support of the study. informations in our database is confidential, however, data used for the analysies is available upon request. authors' contributions mpn, mk and hjz conceptualised and supervised this study. mpn and hjz obtained funding. fsd performed the experiments and analysed data with supervision from sl, who performed the linear and quadratic discriminant analysis (lda and qda). mk, mpn, hjz, fjlr, sl and lat contributed to experimental design, data analysis and manuscript preparation. all authors reviewed, contributed to, and approved the final manuscript. the authors declare that they have no competing interests. submit your next manuscript to biomed central and we will help you at every step: key: cord- -joz gie authors: weber, katharina l.; lesassier, danielle s.; kappell, anthony d.; schulte, kathleen q.; westfall, nicole; albright, nicolette c.; godbold, gene d.; palsikar, veena; acevedo, carlos a.; ternus, krista l.; hewitt, f. curtis title: simulating transmission of eskape pathogens plus c. difficile in relevant clinical scenarios date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: joz gie background: the prevalence of healthcare-acquired infections (hai) and rising levels of antimicrobial resistance places significant economic and public health burdens on modern healthcare systems. a group of highly drug resistant pathogens known as the eskape pathogens, along with c. difficile, are the leading causes of hais. interactions between patients, healthcare workers, and environmental conditions impact disease transmission. studying pathogen transfer under varying contact scenarios in a controlled manner is critical for understanding transmission and disinfectant strategies. in lieu of human subject research, this method has the potential to contribute to modeling the routes of pathogen transmission in healthcare settings. methods: to overcome these challenges, we have developed a method that utilizes a synthetic skin surrogate to model both direct (skin-to-skin) and indirect (skin-to fomite-to skin) pathogen transfer between infected patients and healthy healthcare workers. this surrogate material includes a background microbiome community simulating typical human skin flora to more accurately mimic the effects of natural flora during transmission events. results: we demonstrate the ability to modulate individual bacterial concentrations within this microbial community to mimic bacterial concentrations previously reported on the hands of human subjects. we also explore the effect of various decontamination approaches on pathogen transfer between human subjects, such as the use of handwashing or surface disinfectants. using this method, we identify a potential outlier, s. aureus, that may persist and retain viability in specific transfer conditions better than the overall microbial community during decontamination events. conclusions: our work describes the development of an in vitro method that uses a synthetic skin surrogate with a defined background microbiota to simulate skin-to-skin and skin-to fomite-to skin contact scenarios. these results illustrate the value of simulating a holistic microbial community for transfer studies by elucidating differences in different pathogen transmission rates and resistance to common decontamination practices. we believe this method will contribute to improvements in pathogen transmission modeling in healthcare settings and increase our ability to assess the risk associated with hais, although additional research is required to establish the degree of correlation of pathogen transmission by skin or synthetic alternatives. healthcare-acquired infections (hais) and increasing levels of antimicrobial resistance, particularly among hai-associated pathogens, represent a significant source of morbidity and mortality cost in the us healthcare system [ ] . understanding the dynamics of pathogen transmission and transfer between patients, healthcare workers, and surrounding environmental surfaces is critical to evaluate potential intervention methods, identify possible environmental hai reservoirs or fomites, and to delineate pathogen-specific transmission behaviors [ ] [ ] [ ] [ ] . ethical restrictions on human subject research complicates experimental analysis of human-to-human pathogen transfer. retrospective studies on hai outbreaks do not provide experimentally changeable parameters. thus, there is a need for in vitro methods to study these types of transmission scenarios. the eskape pathogens (enterococcus faecium, staphylococcus aureus, klebsiella pneumoniae, acinetobacter baumannii, pseudomonas aeruginosa, and enterobacter species) [ ] are a group of grampositive and gram-negative bacteria characterized by drug-resistance [ , ] . taken together with clostridioides difficile, which is often caused by antibioticinduced disruption of the native gut microbiota [ ] , these pathogens (here termed eskape+c) collectively are the leading cause of nosocomial infections [ , ] . these pathogens employ diverse mechanisms of drug resistance, such as drug inactivation, active site modification, efflux pumps, and biofilm formation, leading to multidrug-resistant (mdr) strains and a need for novel antibiotics [ , , , ] . as a result of their clinical impact, the centers for disease control and prevention (cdc), with the food and drug administration (fda), maintains the antibiotic resistance (ar) isolate bank which maintains and curates multiple, well-characterized strains of eskape pathogens and isolate panels, a resource that was utilized for this effort [ ] . previous studies have investigated surface transfer of individual eskape+c pathogens, focusing on outcomes of possible transmission events. arinder et al. utilized vitro-skin to show transfer rates of s. aureus to and from various fomites, but the study did not include relevant background microbiota [ ] . dyer et al. investigated c. difficile transmission from hospital fomites by directly applying spores to the fomite surface, which may not be representative for most transmission scenarios [ ] . these studies take diverse approaches to investigating microbial transfer, making comparison of findings and transmission rates challenging. a standardized approach that is broadly applicable to relevant hai pathogens would be beneficial in modeling pathogen transmission in healthcare-like scenarios. in the current study, an in vitro method to enable investigation of both direct (skin-to-skin) and indirect (skin-to fomite-to skin) transmission scenarios of eskape+c pathogens is presented. this method utilizes a commercially available synthetic skin surrogate, vitro skin® n- , to mimic human skin and allow more accurate assessment of bacteria transmission behavior under various direct and indirect scenarios. we describe the inclusion of a set of background organisms commonly found on human skin, recapitulating the native microbiota present during such transmission events. further, washing approaches for both vitro-skin and relevant porous and nonporous surfaces (i.e., stainless steel, nitrile, and cotton) are presented. using this method, eskape+c pathogens were found to transfer at different rates and the inclusion of washing methods for indirect, but not direct, scenarios generally reduced the transfer burden. the described approach is highly customizable, allowing researchers to investigate a specific surface, disinfectant methods, or pathogen(s) as desired. bacterial transmission can occur through either "direct" skin-to-skin contact, such as between an infected patient and a healthcare worker, or an "indirect" skin-to fomite-to skin transfer. we constructed a workflow to mimic these contact scenarios in order to investigate direct and indirect eskape+c pathogen transfer utilizing vitro-skin, a synthetic skin material (fig. ). as pathogen transfer does not occur in isolation, a set of "background" bacteria were included to represent the native skin microbiota that could be present on human hands. table lists the eskape+c and background organisms used for this work. to simulate direct contact, a primary vitro-skin coupon (i.e., infected patient) was briefly touched to a secondary vitro-skin coupon containing only background organisms (i.e., healthy healthcare worker with a healthy skin flora) with or without a simulated handwashing step (fig. a) . for indirect contact, the primary vitro-skin coupon was touched to an intermediate fomite (i.e., cotton, nitrile glove, or stainless steel). the surface was either washed or not washed before contact with the secondary vitro-skin coupon (fig. b) . in both scenarios, viable pathogens were then recovered from the secondary coupon and enumerated. total bacterial loadings on the initial simulated skin coupon were quantified to ensure concordance with previously reported bacterial concentrations on the human hand. published reports describing typical total microbial content on human hands in healthcare settings generally range between . × and . × colony forming units (cfu)/cm [ ] . to better simulate a range of pathogen transmission scenarios, different amounts of pathogen were added to a consistent background microbiome community. the pathogens were grouped into three mixes to better enable analysis by plating on selective media ( table ) . the "high" concentration samples contained approximately equivalent amounts of each microbial component ( cfu/cm or cfu per cm coupon). pathogen amounts in the "low" concentration samples were - fold less abundant in comparison. the amount of each pathogen, the background community, and the total amount of bacteria loaded onto the simulated skin coupon is shown in fig. . fig. approach for the assessment of eskape+c pathogen transmission for direct and indirect transmission events. a for the direct contact scenario, the primary vitro-skin coupon is inoculated with a mix of pathogen and background bacteria, representing a contaminated patient hand (step ). the inoculated vitro-skin is either washed (step a) or not washed (step b) and then touched to a new, secondary vitro-skin with only background microorganisms (step ), simulating the touch transfer of bacteria from a sick patient to a clean healthcare worker. the secondary vitro-skin with any transferred bacteria (step ), representing the contaminated healthcare worker, is then harvested for downstream analysis (step ). b for the indirect contact scenario, the primary vitro-skin coupon is inoculated with a mix of pathogen and background bacteria, representing a contaminated patient hand. the inoculated vitro-skin is touched to a surface (nitrile, stainless steel, or cotton), simulating bacterial transfer to the fomite (step ). the fomite then either undergoes a surface-appropriate wash (step a) or does not ( b). a new, secondary vitro-skin with only background microorganisms, representing a clean health worker hand, touches the fomite (step ) where bacteria on the surface may transfer to the secondary vitro-skin (step ). the secondary vitro-skin with any transferred bacteria is then harvested for downstream analysis (step ) the overall level of eskape+c pathogen transfer from the primary vitro-skin coupon to a secondary vitro-skin coupon following direct contact was established by measuring the total cfu recovery from the secondary coupon. figure shows the total cfu (log ) of each pathogen following transfer for each bacterial mix ( a) or individual pathogen ( b). each individual replicate is plotted (n = ). the total inoculum on the initial coupon is indicated for comparison. a. baumannii, e. aerogenes, e. faecium, k. pneumoniae, p. aeruginosa, and s. aureus had significantly greater direct transfer with smaller log differences at lower inoculum ranging from . to . compared to higher inoculum with log differences ranging from . to . (p < . ). c. difficile (p = . ) and e. cloacae (p = . ) had no significant differences in direct transfer rates between the high and low inoculum used in this study. while the high inoculum samples generally transferred a greater number of total cfu in comparison with the lower inoculum samples (with the novel exception for e. faecium), higher pathogen concentrations led to lower overall pathogen transfer rates from the primary to the secondary coupon. similar to the direct transfer testing described above, total cfu counts were measured for each mix or individual eskape+c pathogen recovered from the fig. representative bacterial loadings on the initial vitro-skin coupon. the eskape+c pathogens were grouped into three separate mixes to facilitate culture-based screening at either high or low levels relative to a consistent background microbial community. total loadings (black squares) for both the high and low inoculum levels fell within equivalent reported ranges (denoted by black dotted lines) of bacterial loads on the hands of healthcare workers (between . × and . × cfu / cm ) [ ] secondary vitro-skin coupon following indirect transfer. indirect transfer involved transmission to a fomite (cotton, nitrile, or stainless steel) from the initial vitro-skin coupon followed by transmission from the fomite to the secondary vitro-skin surface. as with the direct transfer data, total cfus were plotted for both high and low pathogen innocula (fig. ) . initial pathogen inoculum levels are indicated by lines for estimation of overall transfer rates across each fomite type. overall, indirect transmission led to significantly lower transmission rates than direct transfer (fourway anova including the factor for type of transfer: f ( , ) = . , p < . ) with cfu counts generally one to three orders of magnitude for high concentration innocula. a three-way analysis of variance (anova) was conducted on the influence of inoculum, organism, and fomite type on the change in log concentration during indirect transfer of different mixtures of pathogen from vitro-skin to a fomite to a vitro-skin. the main effects inoculum (f ( , ) = . , p < . ), organism (f ( , ) = . , p < . ), fomite (f ( , ) = . , p < . ), and the interaction term of inoculum and organism (f ( , ) = . , p < . ) were statistically significant. post-hoc tukey honest significant difference test indicated significant differences between indirect transfer with cotton fomite compared to nitrile gloves (p = . ) and stainless steel (p < . ), while there was no significant difference in transfer between stainless steel and nitrile gloves (p < . ). similar to direct transfer of pathogens, there was no significant difference in log differences between high and low inoculum of c. difficile (p = . ) and e. cloacae (p = . ). the other pathogens, a. baumannii, e. aerogenes, e. faecium, k. pneumoniae, p. aeruginosa, and s. aureus had significantly lower differences in log cfu at lower inoculum, ranging from . to . , than higher inoculum, ranging from . to . , indicating greater relative abundance transfer (p < . ). the three-way interaction term of all main effects (f ( , ) = . , p = . ) and interaction terms including fomite with inoculum (f ( , ) = . , p = . ) or organism (f ( , ) = . , p = . ) were not statistically significant. s. aureus represented the exception in the indirect transfer data, transferring at a higher rate across all three surfaces than the other eskape+c pathogens. fig. direct transfer of eskape+c organisms performed in mixture with high and low initial inoculum. a total amount of pathogen recovered from secondary vitro-skin coupon following direct transfer. the cfu transferred (log ) within mixtures are represented by the sum of the cfu for each pathogen averaged across three replicates. b total amount of individual eskape+c organisms within the microbial community mixture recovered from the secondary vitro-skin coupon following direct transfer. each pathogen is represented by the mean total cfu for each pathogen across three replicates. black or gray lines above each point indicate initial pathogen spike levels on the primary vitro-skin coupon. dotted-line represents the limit of detection. points below the dotted line result from averaging between replicates when one replicate had zero cfu the artificial skin method described herein provides opportunities to evaluate many facets of skin-to-skin pathogen transmission, including the effectiveness of hand washing or fomite decontamination methods. the effect of handwashing on direct transfer rates was simulated by manual scrubbing and immersion into a series of tubes containing phosphate buffered saline (pbs) to approximate rinsing. overall, the effect of simulated hand washing was minimal, with washed samples generally demonstrating less than -fold reduction in transferred pathogen compared to non-washed samples (fig. ) . a three-way anova was performed on the influence of inoculum, organism, and washing on the change in log change during direct transfer of mixtures of pathogen between vitro-skin. the main effects inoculum (f ( , ) = . , p < . ), organism (f ( , ) = . , p < . ), and washing (f ( , ) = . , p < . ), and the interaction terms with inoculum and organisms (f ( , ) = . , p < . ) or washing (f ( , ) = . , p = . ) were statistically significant. post-hoc tukey honest significant difference test indicated a significantly greater log difference, less transfer, after washing compared to unwashed scenarios (p < . ). post-hoc testing also indicated that washing the high inoculum samples significantly increased log difference and led to less transfer, compared to not washing (p < . ). there were no significant differences at low inoculum between washing and not washing (p = . ). the three-way interaction term of all main effects (f ( , ) = . , p = . ) and the interaction term of washing and organisms (f ( , ) = . , p = . ) were not statistically significant. indirect transfer of eskape+c organisms performed in mixture with high and low initial inoculum. a total amount of pathogen recovered from secondary vitro-skin coupon following indirect transfer across cotton, nitrile, and stainless steel surfaces. the cfu transferred (log ) within mixtures is represented by the sum of the cfu for each pathogen averaged across three replicates. b total amount of individual eskape+c organisms within the microbial community mixture recovered from the secondary vitro-skin coupon following indirect transfer across the three surface types. each pathogen is represented by the mean total cfu for each pathogen across three replicates. black or gray lines above each point indicate initial pathogen spike levels on the primary vitro-skin coupon. dotted-line represents the limit of detection. points below the dotted line result from averaging between replicates when one replicate had zero cfu the effect of fomite decontamination was also investigated (fig. ). cotton samples were agitated in a soap solution to simulate laundering, while nitrile and stainless steel surfaces were swabbed with a disinfectant wipe. cotton surfaces that were washed experienced a ≥ log cfu reduction in bacterial abundance compared to the unwashed samples for the high spike-in. a four-way anova was performed on the influence of inoculum, organism, fomite type, and washing on the differences in log change during indirect transfer of mixtures of pathogens from vitro-skin to fomite to vitro-skin. the main effects inoculum (f ( , ) = . , p < . ), organism (f ( , ) = . , p < . ), fomite type (f ( , ) = . , p < . ), and washing (f ( , ) = . , p < . ) were statistically significant. additionally the interaction terms of inoculum and washing (f ( , ) = . , p < . ), organism and washing (f ( , ) = . , p < . ), the three-way interaction term inoculum, organism, and washing (f ( , ) = . , p < . ), and three-way interaction term organism, fomite type, and washing(f ( , ) = . , p = . ) were also statistically significant. at low inoculum, the pathogens a. baumannii, c. difficile, k. pneumoniae, p. aeruginosa, and s. aureus had less indirect transfer and greater log change after washing (p < . ). c. difficile, e. cloacae, e. aerogenes, and p. aeruginosa at high inoculum had less indirect transfer after washing (p < . ). the indirect transfer of e. faecium at high and low inoculum was not statistically significantly different (p > . ) after washing. washing was most effective against the indirect transfer of c. difficile from all three surfaces examined (p < . ). washing also significantly reduced the indirect transfer of k. pneumoniae on cotton surfaces (p < . ) and p. aeruginosa on stainless steel (p < . ). similar to direct and indirect transfer without washing, the pathogens had significantly smaller log changes and higher overall relative transfer rates with low inoculum compared to high inoculum (p < . ) when washed, with the exception of s. aureus which demonstrated no statistically significant difference (p = . ). to assess the overall effectiveness of the decontamination methods utilized in this study, the percent of total pathogen transferred from the initial to the secondary coupon was calculated. all raw data used for these calculations, including total bacterial loadings and total pathogen transferred for each replicate are included in supplemental table . table s includes the calculated transfer rate for each pathogen and each replicate. these transfer rate values were averaged for each pathogen at each condition (n = ) and plotted. regression analysis was performed to compare percent transfer between washed and unwashed samples. in this case, a slope of y = would suggest no effect of washing on pathogen transfer, while a slope of y = would demonstrate complete pathogen removal or decontamination. figure a suggests that, in high inoculum samples, the success of decontamination was limited, especially on nitrile and stainless steel surfaces. essentially equivalent amounts of pathogen were transferred across nitrile or stainless steel fomites regardless of the use of disinfectant wipes. laundering the cotton surface or hand washing the direct contact samples decreased overall pathogen transmission rates approximately %. however, the indirect transfer results were heavily skewed by a single pathogen, s. aureus. removing the s. aureus outlier (fig. b) reveals that the surface decontamination methods were reasonably effective for each of the other eskape+c pathogens, reducing overall pathogen transmission by at least % in each case and nearly eliminating pathogen transmission across the laundered cotton fomite. these results align with previous findings regarding s. aureus and fig. effect of simulated fomite decontamination on indirect pathogen transfer. a total amount of pathogen recovered from secondary vitro-skin coupon following indirect transfer across cotton, nitrile, and stainless steel surfaces in the presence or absence of a simulated decontamination or washing event. the cfu transferred (log ) within mixtures is represented by the sum of the cfu for each pathogen averaged across three replicates. b total amount of individual eskape+c organisms within the microbial community mixture recovered from the secondary vitro-skin coupon following indirect transfer across the three surface types with or without simulated decontamination or washing. each pathogen is represented by the mean total cfu for each pathogen across three replicates. black or gray lines above each point indicate initial pathogen spike levels on the primary vitro-skin coupon. dotted-line represents the limit of detection. points below the dotted line result from averaging between replicates when one replicate had zero cfu decontamination approaches on skin and related surface types [ , , ] . the removal of s. aureus did not alter the overall direct transfer rates, suggesting the higher rate of s. aureus transfer in indirect scenarios arises from resistance to the fomite decontamination methods used. figure c demonstrates similar results for the low concentration pathogen samples. at low pathogen inoculum levels, fomite decontamination was largely effective, reducing pathogen transfer rates between and %. in contrast to the high concentration results, s. aureus was not an outlier at low inoculum levels. however, the transfer rate of p. aeruginosa was an outlier at the low inoculum level specifically for direct transfer, demonstrating nearly equivalent transfer rates in washed versus unwashed scenarios. excluding p. aeruginosa from the fig. percent pathogen transfer from initial to secondary coupon at high and low pathogen concentration. individual transfer scenarios (direct vs. indirect) are shown. the percent transferred when no wash or decontamination step was included is represented on the x-axis, with the percent transferred following a wash or decontamination step represented on the y-axis. values for individual pathogens were averaged (n = ) prior to plotting. linear regression lines were plotted with the slope of each line indicated. a percent pathogen transfer at high initial pathogen concentration. s. aureus outlier results are indicated with gray arrows. b) percent pathogen transfer at high initial pathogen concentration excluding s. aureus. b percent pathogen transfer at high initial pathogen concentration excluding s. aureus. c percent pathogen transfer at low initial pathogen concentration. p. aeruginosa outlier results are indicated with a gray arrow. d) percent pathogen transfer at low initial pathogen concentration excluding p. aeruginosa analysis (fig. d) decreased overall pathogen transfer rates for low inoculum direct transfer to levels approximating the high inoculum levels (compare yellow line between fig. a and d) . p. aeruginosa transfer is commonly linked to hand hygiene and these results may be indicative of bacterial persistence on the hand following washing [ ] [ ] [ ] . exclusion of p. aeruginosa did not substantially affect the overall transfer rates observed in the indirect contact scenarios. the prevalence of healthcare-acquired infections (hai) and rising levels of antimicrobial resistance places a significant economic burden on modern healthcare systems [ , ] . epidemiological studies are commonly used to model or track pathogen transmission within the healthcare environment [ ] , but these studies are often limited by the amount of available data. relevant synthetic systems, such as the one we describe, are critical to model and understand the transmission of pathogens in clinical settings because they do not require the involvement of human subjects. such systems permit the generation of precise, quantitative data regarding pathogen transmission rates without risking the health of human subjects. the degree to which synthetic alternatives mimic the properties of human skin remains an open question worthy of further research. the use of surrogate skin to understand pathogen transfer is not new, but such approaches may not be broadly relevant to the clinical setting due to the contrived nature of the sample [ , ] . by producing and evaluating a method that incorporates a background microbial flora in addition to specific pathogens of interest, we have provided an additional tool in the arsenal of epidemiologists to study pathogen transmission within the healthcare setting in a way that could potentially be used to mimic the biology of the human hand. additional work is required to establish the degree to which the vitro-skin substrate mimics the human hand. while our method incorporates a background commensal skin microbial community to better model that of a human hand, adhesion of this community to the artificial surface may be different than adhesion to human skin. the properties and pressures of microbial growth on human skin over time likely cannot be fully recapitulated by the artificial surface. future studies are needed to establish whether the artificial surface can support sustained microbial growth. if so, it may be possible to more accurately model the sudden introduction of pathogen (e.g., c. difficile) onto a hand with an established skin microbiome community. by incorporating each of the eskape+c pathogens into this testing, it was possible to compare relative transfer rates in both direct and indirect scenarios. s. aureus demonstrated a consistently higher transfer rate in the indirect contact scenarios, including persistence during decontamination or washing. it is unclear if this finding arises from surface properties of s. aureus that promote transfer or more robust fitness in comparison to the other pathogens used in the study resulting in greater viability during culture [ , ] . e. faecium transferred at a higher rate in the low inoculum samples, with the total transferred cfu values nearly identical in direct contact scenarios despite the different input amounts (fig. ) . additional study is necessary to determine whether these findings are related to the biology of e. faecium or represent an artifact of the transfer and recovery method used in this study. several patterns emerged when evaluating pathogen transfer and the relative effectiveness of decontamination across transfer scenarios. as expected based on previous studies, s. aureus proved resistant to simulated decontamination on fomite surfaces, especially when present at high initial concentrations on nitrile and cotton (fig. b and fig. ) [ , , ] . p. aeruginosa persisted in a robust manner following washing or decontamination across direct and indirect transfer (fig. b) . this pattern emerged in a particularly clear fashion when comparing transfer rates of all pathogens for direct transfer at low concentration where p. aeruginosa appeared as an outlier in the overall trend of decontamination rates (fig. c and d) . it is not clear whether these results reflect underlying biological characteristics of these specific pathogens or could be due to artifacts arising from the specific decontamination procedures or artificial skin system used herein. previous studies suggest that the properties of vitro-skin may not perfectly align with those of human skin as it relates to the transfer of viruses [ ] . it remains to be determined if the patterns observed herein are based on the intrinsic cellular properties of the pathogens and are not due to artifacts of vitro-skin, the decontamination approaches, or insufficient statistical power for the study. while these variables must be taken into account in future studies, the artificial skin system we describe appears to show promise for the discovery of molecular mechanisms by which these pathogens persist and transfer in healthcare settings. pathogen transfer rates were significantly higher in low inoculum samples than in high. while it is unclear what factor or factors caused this difference, it is possible that higher loadings led to the formation of clumps of bacteria instead of individual cells or thin layers. this may have prevented much of the bacteria present on the coupon from contacting the secondary coupon, limiting the amount transferred. future efforts will be required to characterize the deposition of different amounts of bacteria on the surface of vitro-skin and to devise methods to achieve even distribution of bacteria across the surface in a way that recapitulates a human skin sample. while this study investigated various decontamination methods relevant to pathogen transfer in healthcare settings, this study was not intended to serve as an evaluation of these methods or to provide recommendations on their effectiveness. instead, a limited number of relevant decontamination methods were included to show the value of this model in future studies and to grapple with the influence of the inclusion of a background flora on transfer and decontamination rates. the limitations imposed by our simulated handwashing method were apparent. by not duplicating the force of water pressure on the artificial skin surface, a significant amount of pathogen transmission still occurred in the "washed" samples; generally, only a - % reduction in transmission when compared to the unwashed controls. hand washing procedures specified by the cdc instruct individuals to wash their hands with running water, where the force of the water has been shown to reduce bacterial loads. without the force of running water in our scenario, more bacteria can remain on the hands even with the friction applied while rubbing and soaking in water afterwards [ , , ] . additionally, as described in the materials and methods, the vitro-skin was not dried completely after the simulated-hand washing. this may have aided in bacterial transfer, similar to how residual moisture on hands has the potential to transfer bacterial loads more than dried hands [ , ] . the low contact time associated with the application of antimicrobial soap may have also played a role in the relative low success of hand washing observed in this study. ultimately, uncovering challenges with the decontamination methods utilized in this study highlights the utility of the vitro-skin system for the evaluation of factors relating to the effectiveness of handwashing and decontamination in future studies. here, we describe the development of an in vitro method that utilizes a synthetic skin surrogate in addition to a defined, customizable microbial community mimicking the human skin flora. this approach allows various pathogen transmission pathways to be quantified, including skin-to-skin and skin-to fomite-to skin contact scenarios, in a manner that does not involve human subject testing. our results illustrate the value of simulating a holistic microbial community for transfer studies by elucidating differences in different pathogen transmission rates and resistance to common decontamination practices. indeed, multiple differences in transmission rates between individual eskape+c pathogens were identified suggesting areas for future research focus. ultimately, we believe this method will contribute to improvements in pathogen transmission modeling in healthcare settings. future work should be performed to establish the concordance between pathogen transfer on hands and synthetic matrices, and to verify that the relative transmissibility of individual pathogens described herein is extensible to the clinical environment. microorganisms used for this effort were sourced from atcc or the cdc & fda antibiotic resistance isolate bank (table ) [ ] . all microorganisms were propagated according to cdc and atcc guidelines ( table ). an environmental protection agency (epa) standard operating procedure (sop) was used as a guideline for the preparation of c. difficile endospores [ ] . greater than % of the bacterial preparation were spores. all media and media supplements were acquired from teknova and anaerobic equipment from bd. to facilitate rapid viability screening using culture methods, three separate mixtures were created for testing ( table ). the viability of each eskape+c pathogen was assessed on three selective medias: mannitol salt agar (msa), eosin methylene blue (emb) agar, and c. difficile agar, with the indicated antibiotics included (acros organics for gentamicin and alfa aesar for ampicillin) (see table ). plates were supplemented with antibiotics and allowed to dry overnight prior to testing. bacteria were enumerated by serial dilution in pbs (teknova) and subsequent plate count following incubation for - h at °c. c. difficile growth occurred under anaerobic conditions. vitro-skin n- (ims inc.) was used to simulate both skin-to-skin (i.e., direct) and skin-to fomite-to skin (i.e., indirect) transmission. all vitro-skin sheets were cut into × cm coupons prior to hydration. vitro-skin was used within h of hydration. stainless steel coupons ( × cm) (home depot) were cleaned with % isopropyl alcohol, rinsed with deionized (di) water, and allowed to dry. after drying, surfaces were steam sterilized by autoclaving prior to testing. nitrile gloves (vwr) and cotton surfaces were cut to × cm, placed in a sterile container exposed to uv light for approximately h. sterilized surfaces were used within h. cotton surfaces were cut from a new laboratory cotton/polyester blend coat. negative controls (vitro-skin coupons not inoculated with bacteria) were included to confirm that no eskape+c pathogens were present on sterilized fomite surfaces. at the time of testing, pathogenic and background cultures were pelleted by centrifuging at x g for min (excluding anaerobic microorganisms or cultures prepared in sheep's blood) and resuspended in ml of pbs. resuspended bacteria were combined per the appropriate mix (table ) at the appropriate spike-in level and brought up to ml using pbs. spike in target levels were~ cfu per vitro-skin for "high"~ cfu for "low." actual spike in values were measured and reported (fig. ) . on the day of testing, one mix containing pathogens plus background and one mix containing only background microorganisms was prepared. once the appropriate pathogen and background mixes were prepared, . ml of a bacterial mix was added per hydrated vitro-skin coupons and spread evenly using a sterile spreader, avoiding edges. the inoculated coupons were dried at °c for min or until visibly dried. for the no-wash direct contact scenario, a primary vitro-skin coupon containing a pathogen mix was touched to a secondary vitro-skin coupon containing only background microbiota by evenly applying pressure for s using sterile forceps. the secondary vitro-skin coupon was then added to ml pbs and vortexed for min to release the bacteria. recovered bacteria was then appropriately diluted and plated on selective media. for simulated hand washing, a procedure was adapted from arinder et al. [ ] to a pathogeninoculated vitro-skin coupon, . ml of hospitalgrade hand soap (thermo scientific softcide hand soap) was applied to the bacterial-laden surface. a second pathogen-inoculated vitro-skin coupon was pressed evenly onto the first, allowing the soap to spread evenly between both coupons. the coupons were rubbed against each other times to achieve the same amount of foam as normal hand washing. after washing, the secondary vitro-skin was rinsed sequentially using two separate ml conical tubes containing sterile pbs. following the second rinse, the coupon was rinsed in ml of dey-engley (d/e) broth. after the d/e broth rinse, the washed vitro-skin was touched to a new vitro-skin coupon that contained only background microbes by evenly applying pressure for s using sterile forceps. the new vitro-skin with the transferred bacteria was added to ml of pbs, vortexed for three minutes, and appropriately diluted and plated on selective media. indirect contact events were performed in a similar manner as direct contact events (fig. b) . dried and inoculated vitro-skin samples were prepared and touched to one of the three different surface types (i.e., nitrile gloves, cotton cloth to represent a patient bedsheet, and stainless steel to represent medical equipment). this testing occurred with and without a decontamination step on the surface following the initial transfer across two pathogen spike-in levels. the cleaning procedure for stainless steel and gloves was performed with a fresh, broad spectrum hospital disinfectant wipe per manufacturer instructions (metrex caviwipes bleach®). the cloth surfaces were cleaned with a simulated hot-water cleaning to replicate how textiles are cleaned in a hospital washing machine. afterward, a secondary coupon containing only the background microbiome was touched to the cleaned or noncleaned surface. the simulated hot water cloth cleaning procedure was adapted from "a sanitary study of commercial laundry practices," recommended by the cdc [ ] . all replicates of soiled cotton coupons were placed in a sterile beaker with a sterile stir bar containing ml of sterile water to ensure all cloth replicates were submerged and were rinsed for min at °c. after first rinse, the water was dispensed into a waste container to be autoclaved. sterile water ( ml; . μl/ml stock detergent (all® free & clear) was added to a new, sterile beaker to ensure all cotton replicates were submerged and allowed to stir to provide agitation for min at °c. afterward, % bleach was added to the mixture and was allowed to stir to provide agitation for min at °c. the water was then removed and placed in a waste container for autoclaving. sterile water ( ml) was added to a new, sterile beaker to ensure all cotton replicates were submerged and was allowed to stir for min at °c. the water was removed appropriately and placed in a waste container for autoclaving. the wash step for min at °c was repeated twice. after the final rinse, each cotton replicate was placed in an appropriate sterile container and placed in an oven at °c for min or until visibly dried. once dried, each replicate was added to ml pbs, vortexed for min, and enumerated per standard spread plating technique. for each pathogen mixture and simulated contact scenario, the following controls were harvested and plated: one primary inoculated vitro-skin as the pre-transfer control; one clean vitro-skin (i.e., no background skin microbiome or eskape+c pathogens) as the negative control; one clean or sterile surface (i.e. fomite) per surface type as a negative control. one sterile agar plate per agar type was incubated alongside test plates to ensure plate sterility. each pathogenic microorganism per mixture type per testing simulation was struck for isolation to ensure there was no contamination. a volume of . ml of each dilution buffer, harvesting buffer, etc. was plated onto sterile tsa to ensure no contamination of each respective buffer at the time of testing. following the transmission event simulations, each replicate was aseptically placed into separate conicals containing ml of pbs and vortexed at max speed for at least min. afterward, individual ml aliquots (n = ) were plated directly onto the appropriate selective media and incubated at the appropriate temperatures. colonies were counted manually to quantify the number of cfus. equations and below were used for culturedependent method analysis. equation was used to determine the number of viable bacteria of a given strain on the surface of a vitro-skin carrier following elution in ml of buffer. eq. was used to generate the average cfu per carrier across multiple experimental replicates. re-estimating annual deaths due to multidrug-resistant organism infections evidence that contaminated surfaces contribute to the transmission of hospital pathogens and an overview of strategies to address contaminated surfaces in hospital settings acquisition of spores on gloved hands after contact with 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a comparison of healthcare networks transfer of enteric viruses adenovirus and coxsackievirus and bacteriophage ms from liquid to human skin an update on clinical burden, diagnostic tools, and therapeutic options of staphylococcus aureus surface adhesins of staphylococcus aureus outbreaks where food workers have been implicated in the spread of foodborne disease. part . factors contributing to outbreaks and description of outbreak categories residual moisture determines the level of touch-contact-associated bacterial transfer following hand washing epa mlb sop mb- : procedure for the production and storage of spores of clostridium difficile for use in the efficacy evaluation of antimicrobial agents a sanitary study of commercial laundry practices r: a language and environment for statistical computing austria: r foundation for statistical computing publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank jim gibson for his help creating the contact scenario figure. all data generated or analyzed during this study are included in this published article and its supplementary information files.ethics approval and consent to participate not applicable. not applicable. statistical analysis of log difference between the control vitro-skin and the resulting vitro-skin from transfer of the eight esckape+c pathogens were performed by anova using multiple factors as described. the analysis was performed using the 'aov' function in 'r' statistical package through 'rstudio' [ ] . the anova was followed by a tukey honest significant difference test utilizing the 'tukeyhsd' function. the detection limit for cfu counts were used for values below that limit. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file . the authors declare that they have no competing interests.received: january accepted: may key: cord- -w scpc authors: amariei, raluca; willms, allan r; bauch, chris t title: the united states and canada as a coupled epidemiological system: an example from hepatitis a date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: w scpc background: hepatitis a (ha) is a low-incidence, non-endemic disease in canada and the united states (us). however, a large difference in ha incidence between canada and ha-endemic countries has made travel an important contributor to hepatitis a prevalence in canada. there is also a (smaller) incidence differential between canada and the us. although the us has only moderately higher ha incidence, the volume of travel by canadians to the us is many times higher than travel volume to endemic countries. hence, travel to the us may constitute a source of low to moderate risk for canadian travelers. to our knowledge, travel to the us has never been included as a potential risk factor for ha infection in canadian epidemiologic analyses. the objective of this study was to use dynamic models to investigate the possible effects on hepatitis a incidence in canada due to ( ) implementing vaccination in the us, and ( ) varying the volume of travel by canadians to the us. methods: we developed and analyzed age-structured compartmental models for the transmission and vaccination of hepatitis a, for both canada and the us. models were parameterized using data on seroprevalence, case reporting, and travel patterns. the potential effect of hepatitis a prevalence in the us on hepatitis a prevalence in canada was captured through a term representing infection of canadians due to travel in the us. results: the model suggests that approximately % of ha cases in canada in the mid s may have been attributable to travel to the us. a universal vaccination programme that attained % coverage in young children in the us in the mid s could have reduced canadian incidence by % within years. conclusion: since not all necessary data were available to parameterize the model, the results should be considered exploratory. however, the analysis shows that, under plausible assumptions, the us may be more important for determining ha prevalence in canada than is currently supposed. as international travel continues to grow, making vaccination policies ever more relevant to populations beyond a country's borders, such multi-country models will most likely come into wider use as predictive aids for policy development. wealth varies dramatically across countries, and with it, the disease burden for many infectious diseases [ ] . one example is hiv, where prevalence is % in sub-saharan africa but only . % in western europe [ ] . a less striking but still significant example is hepatitis a (ha). ha is a non-endemic, low-incidence disease in the us and canada, but is highly endemic in many other countries [ ] [ ] [ ] [ ] . the average reported ha incidence in canada was . per , per year from to [ ] , and the average reported ha incidence in the us was . per , per year from to [ ] . by comparison, in , reported incidence ranged from to per , per year in africa and the middle east (depending on the country), to in asia, and to in central and south america [ ] . moreover, reported incidence significantly underestimates actual incidence due to underreporting and subclinical infection [ , , ] . because subclinical infection is more common in children, who are infected more frequently in developing countries than developed countries, the global differential in true infection levels is much higher than for reported incidence. this incidence differential between canada and haendemic countries, combined with increasing air travel, makes travel by canadian residents to ha-endemic countries a significant source of ha infection in canada [ , ] . travel to endemic countries is also a source of infection in the us, with % of reported infections attributable to travel in ha-endemic countries in [ ] . hepatitis a vaccine has been available in canada and the us since [ ] . in canada, the average reported incidence from to , while a targeted vaccination programme was in place, declined to . per , per year [ ] . the vaccination policy in canada is still targeted and includes high-risk groups, such as men who have sex with men, intravenous drug users, members of first nations communities, and travelers to endemic countries, among others. in the us, after vaccination was implemented (with universal vaccination in the states with highest incidence), the reported incidence had declined to . per , per year by [ , ] . the true incidence of infection (including both clinical and subclinical infection) has been underestimated by approximately fold in canada and -fold in the us [ , ] . there also exists an incidence differential between canada and the us, with the us having somewhat higher incidence ( figure ). hepatitis a incidence tends to rise and fall at the same time in the us and canada. in fact, the reported incidence in the two countries is positively correlated with a correlation coefficient + . ( figure ) [ , ] . interestingly, outbreaks of hepatitis a in men who have sex with men (msm) in montreal often follow outbreaks in msm in new york by or months (vladimir gilca, institut national de santé publique du québec, pers. comm.). the two countries are also bound together by very high travel volume. for instance, in , the number of person-trips by canadian residents returning to canada from the us was times the number to all other countries combined, and times the number made to all ha-endemic countries combined [ ] . similarly, on average from - , outbound travel from canada to the us was times that to all other countries combined [ ] . given these observations, it is worth posing the question: how does ha epidemiology in the us influence ha epidemiology in canada? this issue has implications for policy, since it implies that health interventions in one country may potentially influence health outcomes in other countries. this question also has implications not only for hepatitis a but for many diseases such as sars, as burgeoning air travel turns local problems into global problems. in this paper, we develop mathematical (agestructured compartmental) models of hepatitis a transmission and vaccination in canada and the us. we use travel data to couple the two countries epidemiologically through travel. we focus on these two countries (rather than attempting a global model) because of the relatively good availability of data for the us and canada and the close relationship of canada to the us. the coupled model allows us to analyze how transmission and vaccination in the us may be affecting ha incidence in canada. we begin with a description of hepatitis a epidemiology, which will motivate our choice of mathematical model. in canada and the us, unlike most developing countries, hepatitis a is transmitted mostly by person-to-person contact, by the fecal-oral route [ ] . unlike in many countries, foodborne outbreaks are very infrequent in canada [ ] . children play an important role in transmission due to their higher rates of subclinical infection and poor hygiene [ ] . clinical illness typically lasts four weeks, there is no chronic state of infection [ ] , and natural immunity is lifelong. although % of infected post-adolescents develop jaundice, many do not seek medical attention [ , ] . individuals with symptomatic ha infection experience nausea, loss of appetite, fatigue, fever, abdominal pain and jaundice [ ] . hepatitis a infection is more severe in older individuals or those with co-morbidities such as chronic liver disease [ , ] . the most serious possible complication of hepatitis a infection is fulminant hepatic failure. the rate of mortality attributable to ha varies from . % in symptomatic young adults to . % in symptomatic individuals years and older [ ] . given the predominance of person-to-person contact, lifelong immunity, and the importance of children in transmission, a suitable mathematical model is an agestructured compartmental model. this widely-used class of models has been shown to be particularly useful in assessing the effects of universal vaccination programmes against diseases with acquired immunity transmitted horizontally through person-to-person contact, and has been shown to provide good agreement with pre-and post-vaccination age stratified case reports and seroprevalence surveys for infectious diseases such as measles [ , ] . our age-structured seirv compartmental model stratifies individuals according to epidemiologic status (susceptible-exposed-infectious-recovered-vaccinated) and age class (ages - , - , - , - , - , - maternal immunity is short-lived and affects relatively few individuals in a non-endemic country such as canada, so we do not include it [ ] . since ha in canada and the us is spread primarily person-to-person, we do not model foodborne or waterborne outbreaks [ ] . the model equations appear in appendix a and the parameterization is described in appendix b. a diagram of the model appears in figure . the united states and canada are both large countries and one may consider that a model by states would be more appropriate. however, despite their close proximity, there is more travel within canada than between canada and the us: from to , there were . times as many person-trips made within canada (across provinces) as between canada and the us [ ]. table gives the parameter values used for the us model and their data sources [ , , , [ ] [ ] [ ] [ ] . demographic and epidemiologic parameter values are from the pre-vaccine era, - . demographic parameters such as birth rates and age-specific death rates were taken from demographic data. clinical and epidemiologic literature on hepatitis a were used to determine the durations of latent and infectious periods, vaccine efficacy, and duration of vaccine-derived immunity. the rate at which a susceptible person is infected due to travel in endemic countries ( ), and the rate at which a susceptible person is infected by infectious persons due to domestic us transmission ( ) were computed simultaneously using: ( ) published data on the true incidence of hepatitis a in the us, adjusted for under-reporting and the probability of jaundice [ ] , ( ) data on the age-specific proportion of cases attributable to travel in endemic countries [ ] , and ( ) an assumed form for a "who acquires infection from whom" matrix consisting of the parameters [ ] . this method of computation, which uses the model equations and does not require explicit knowledge of the force of infection or seroprevalence data (although those were necessary to estimate the true incidence in this particular case), is described in appendix b. hepatitis a is not endemic in the us, and the us incidence of ha is only modestly higher than that of canada. however, there is such a greater volume of travel to the us than to ha-endemic countries that it makes sense to make allowance for infection due to travel to the us in the where y us (resp. y end , y c ) is the incidence in the us (resp. endemic countries, canada) where (resp. ) is the annual volume of travel by individuals in age class i to the us (resp. endemic countries), and where (resp. ) is the average duration of stay by travelers in the us (resp. endemic countries). these parameters can be obtained from published data [ ] or from travel data available on government agency websites [ ] . the difference between canadian incidence and us/endemic incidence is used since that is proportional to the marginal increase in infection risk to canadian residents traveling in other countries. for instance, if canadian incidence were per , per year, and us incidence changed from per , per year to per , per year (due to more foodborne outbreaks in that country, for example), then the additional risk of infection per year that canadian residents assume upon themselves due to travel to the us would double. we note that equation does not take all possible factors into account. for instance, individual behaviour may vary, since canadian residents are perhaps more risk-averse when traveling in an endemic country than when traveling in the us. the canadian parameters and are estimated using the same method as for the corresponding us parameters and , except that the force of infection due to travel to the us is first subtracted from the total force of infection, and canadian seroprevalence, case reporting, table . the age-structured model for canada has identical structure to the us model except for the additional term representing infection attributable to travel in the us (see appendix a). we wish to make the force of infection attributable to travel to the us a function of the number of infectious individuals at any given time in the us, in order to study the effects of differing vaccine coverage in the us and differing travel volume to the us. hence, the function therefore couples the two countries and reflects our assumption that when the number of infected individuals in the us increases (resp. decreases), the number of canadian individuals becoming infected due to travel in the us also increases (resp. decreases). the demographic and epidemiological parameter values for canada are listed in table . parameter values relating to disease progression in infected individuals are the same as those for the us and so are not listed. some parameters (such as the birth rate) are very similar in the two countries. the number of residents of the us who become infected while traveling in canada is likely very small due to the relative population sizes of the two countries, and ( ) rate at which an individual in age class i dies , , , , , . , . per year [ ] rate at which a susceptible individual in age class i becomes exposed due to travel in endemic countries us so a similar term was not introduced in the us equations. however, we note that this assumption could become invalid under certain situations. for instance, if us vaccination coverage is high and canadian vaccination coverage is low, then travel to canada could, in principle, be a risk factor for us residents (particularly those living close to the border). however, figure suggests that this is unlikely in practice, as canadian incidence has remained below us incidence before and after the vaccine was licensed in both countries in the mid s. here we describe the predicted incidence of hepatitis a in canada under various vaccination scenarios in the us, and for various volumes of travel to the us. the adjusted incidence values reported here are the predicted incidence of reported cases adjusted for subclinical infection and under-reporting. hence, the adjusted incidence represents the true incidence of all ha infections. if reported incidence were plotted instead, the qualitative results would be the same and the quantitative results would be similar except for a scaling due to the adjustment for underreporting and subclinical infection. figure shows the adjusted incidence in canada at the equilibrium state of the dynamic model as a function of vaccination coverage in the - age class in the us. as the vaccine coverage in the us increases, the adjusted ha incidence in canada decreases significantly. for instance, universal vaccination in the us at % coverage in the - age class causes a % decline in the adjusted canadian incidence, across all age classes. hence, this allows us to infer that approximately % of canadian incidence was attributable to travel in the united states, in the years for which the model was parameterized ( to ). implementing a universal vaccination programme in the us soon shows its effects in canadian incidence. figure shows the adjusted incidence in the us and canada when, initially, there is no vaccination in either country, but in , a strategy of vaccinating % of children in the us in the - age class begins. within a few years of the start of the us vaccination programme, adjusted incidence has also declined in canada significantly. the choice of as the year that vaccination begins is motivated by the fact that was the last year before ha vaccine became widely available in the us and canada. we also note that the model was parameterized using data from (the earliest year of availability for certain data) to inclu- figure shows the adjusted us and canadian incidence, stratified by age class, before and after implementing universal vaccination in at % coverage in the - age class in the us. in the us, incidence declines rapidly not only in the - age class, but also in the other unvaccinated age classes due to the indirect protective effects of herd immunity. likewise, incidence declines in all age classes in canada upon initiation of universal vaccination in the us. similarly, the effect of an instantaneous % increase in the us adjusted incidence in is soon reflected in a % increase in canadian incidence ( figure ). although this scenario of such a rapid increase in incidence is only hypothetical, the example serves to illustrate how closely coupled the countries are. the time difference between the us peak and the canadian peak in figure is about days. as noted already, the observed time delay between outbreaks of hepatitis a in gay men in new york with outbreaks in gay men in montreal is - months (vladimir gilca, inspq, pers. comm.). other scenarios where the effects of fluctuating us incidence on canadian incidence are studied, such as sinusoidal variation in the us, give rise to lags between us and canadian incidence peaks of approximately months. as the annual volume of travel by canadian residents to the us increases, the adjusted incidence in canada also adjusted incidence by age classes in the us (top) and canada (bottom) upon initiation of a universal vaccination pro-gramme in the us at % coverage in all age classes in adjusted incidence in canada versus us vaccination coverage (in the - age class), at the equilibrium state of the model dynamics figure adjusted incidence in canada versus us vaccination coverage (in the - age class), at the equilibrium state of the model dynamics. the adjusted incidence is the reported incidence adjusted for asymptomatic infection and under-reporting. the effect of implementing universal vaccination in the united states on the incidence in canada figure the effect of implementing universal vaccination in the united states on the incidence in canada. universal vaccination is implemented in in the united states by vaccinating % of individuals in the - age class. the adjusted incidence is the reported incidence adjusted for asymptomatic infection and under-reporting. increases in almost direct proportion (figure ) . the adjusted incidence in canada when there is no travel to the us is % less than the adjusted incidence in canada at the actual volume of travel in (indicated in figure ). hence, approximately % of infected individuals in canada in may have acquired the disease through travel to the us, insofar as equation is correct. i.e., the combination of parameters and such that the incidence can be reduced most quickly. although ha incidence is much lower in the us than in ha-endemic countries, it is still somewhat higher than in canada. this, coupled with the enormous annual volume of canadian travel to the us compared to endemic countries, means that the us could be a more significant source of travel-related infection (particularly for hepatitis a) than previously recognized. indeed, the results in the previous section illustrate the potential impact of hepatitis a transmission and vaccination in the us on ha prevalence in canada. simulation results based on the assumptions in equation show that a significant proportion ( %) of ha incidence in the mid 's, before vaccination was introduced, may have been attributable to travel in the us. hence, some of the declines in ha incidence observed after in canada ( figure ) may partly be due to the start of universal vaccination in the higher-incidence regions of the us in the mid- s. we speculate that detecting travel to the us as a risk factor is difficult because ( ) the incidence is only moderately higher in the us than in canada (hence the risk to an individual is only modestly increased when traveling to the us), and ( ) due to high travel volume, travel to the us becomes a commonplace and under-reported event in the lives of many canadians. however, this modelling study suggests that future epidemiological studies of risk factors for ha infection should include travel to the us as a variable in risk factor analysis. adjusted incidence in canada at the equilibrium state of the model dynamics, as a function of the total annual volume of canadian travel (number of trips per year) to the united states figure adjusted incidence in canada at the equilibrium state of the model dynamics, as a function of the total annual volume of canadian travel (number of trips per year) to the united states. the "x" denotes the actual volume of travel to the us in . adjusted incidence in the us and canada after a sudden increase in us incidence figure adjusted incidence in the us and canada after a sudden increase in us incidence. in , the number of infected individuals in the united states is instantaneously increased by %. there are several limitations to the methodologies used in this paper. firstly, these results assume the contribution due to travel in the us is similar to the contribution due to travel to endemic countries, adjusted for the total passenger-days in those respective destinations as well as the difference in incidence between canada and the respective destination (equation ). moreover, here we have neglected cohort effects for the sake of simplicity [ ] . in reality, the age-structured seroprevalence profile for hepatitis a exhibits a cohort effect, whereby the seroprevalence in older age classes is higher than can be explained by the current force of infection (thus implying that the force of infection was higher in the past). the existence of a cohort effect influences how the disease can be modeled. in particular, if the cohort effect is neglected and it is assumed that transmission rates have always been constant, then the dynamic model will overpredict the average population incidence both before and after vaccination, and will also overpredict the percentage reduction in incidence due to vaccination [ ] . finally, there is social heterogeneity within countries in risk factors and transmission patterns for hepatitis a that may be important for modelling certain aspects of disease transmission. geographical heterogeneity in travel destinations of canadian traveling to the us may also be important. the actual ha incidence in the us and canada appeared to oscillate on a non-seasonal seven-year cycle before the vaccine era ( figure ) . however, our model solutions do not oscillate. non-seasonal oscillations in models are often associated with endemic diseases, and the period of oscillation (time between peaks) can even be predicted successfully from models [ ] . in the case of these models, setting the travel transmission rates to zero for both the us and canada caused the infection to die out, suggesting that hepatitis a was not endemic in these two countries for the period - , the time for which the model was parameterized. the fact that ha incidence surface plot of adjusted incidence in canada, versus vaccination rate and travel-related transmission rate figure surface this would allow the model to capture, for instance, us residents who return from endemic countries and travel to canada shortly thereafter. however, the data requirements for such a model would be significantly greater. there have been at least eight hepatitis a transmission models in recent years that have assessed hepatitis a transmission and/or vaccination in various populations and have included herd immunity effects [ , , , [ ] [ ] [ ] [ ] [ ] . like the present model, these models have mostly been deterministic, age-structured compartmental models. some have structured the population along social [ ] or, like the present study, geographic lines [ , ] . those models predicting or estimating the effects of universal vaccination report declines in incidence due to universal vaccination that are similar in magnitude to those found by the present model [ , , , ] . several of the models also included a cost-effectiveness or cost-utility analysis [ , ] . van effelterre and colleagues included transmission across regions in the united states in a preliminary way, as part of the sensitivity analysis of their model of us hepatitis a transmission and vaccination [ ] . they found that the benefits of universal vaccination across the entire us, compared to the benefits of region-specific strategies according to regional hav incidence, were less important with transmission among regions than without. however, there were still benefits in terms of the number of cases averted by universal vaccination across the entire us with transmission among regions. they did not incorporate travel data into their model. to our knowledge, the present model is the first to incorporate transmission of hepatitis a between countries due to international travel by residents. the worldwide sars coronavirus outbreaks exemplified how a public health problem in one population can quickly become a problem in others, due to strong travel connections between countries. the example of hepatitis a transmission in canada and the us represents the (significantly less spectacular) flipside to that of sars: the decline of hepatitis a in canada may partly be attributable to universal vaccination in the us. other modelling work illustrates how nonvaccinators in a population can "freeride" by taking advantage of the herd immunity provided by vaccinators [ ] [ ] [ ] [ ] . this has been compared to a prisoner's dilemma (wherein vaccinators are "cooperators" and nonvaccinators are "defectors") and analyzed using game theory [ ] [ ] [ ] . in the same way, entire countries can also "free-ride" by benefiting from vaccination programmes carried out in and funded by other countries. as international air travel continues to increase, vaccination policies and public health policies in one country will become increasingly important to other countries. in the future, multi-country or multi-regional models may come into more common usage. this study illustrates that changes in hepatitis a vaccination or incidence in the us, or changes in the volume of travel by canadians to the us, may all have significant and rapidly-realized impacts on the prevalence of hepatitis a in canada. the possibility of such a connection is also supported by other evidence, such as the positive correlation in hepatitis a incidence in the us and canada from to ( figure ). hence, declines in reported incidence since the mid- s observed in canada may be partially attributable to vaccination in the us. future epidemiological studies of risk factors for ha infection should include travel to the us as a variable in risk factor analysis. should travel to the us be found as a significant risk factor, then it should be included as such in vaccine recommendations. ha: hepatitis a us: united states seirv: susceptible-exposed-infectious-recovered-vaccinated the author(s) declare that they have no competing interests. all authors contributed to the modelling, parameterization, analysis and/or the writing of the manuscript. ra programmed and simulated the dynamic model and wrote early drafts of the manuscript. aw conceived of the method of parameterization for transmission rates described in appendix b and wrote early drafts of the manuscript. ctb conceived the study, contributed background material, and finalized the manuscript. all authors read and approved the final manuscript. this study is based on ra's msc thesis in mathematics, university of guelph, . the model equations for the us are: the definitions of the variables and parameters are given in table . note that a s = bn to represent recruitment into the youngest age class through birth, and a = . the simulations took as initial conditions s = e = r = v = and i > , however, equilibrium solutions are analyzed throughout the results section and hence the initial values are not relevant to the analysis. the canadian equations are identical except for the susceptible and exposed compartmental equations: the model was simulated in matlab, and the fourth-order runge-kutta method was used to numerically integrate the equations. the model is parameterized using incidence and demographic data from to , since seroprevalence data is readily available for years after , and since vaccination introduced in altered outbreak patterns and hence transmission probabilities. the size of each age class from the us census data [ ] is approximately n = n = , , , n = n = n = n = , , , n = , , . the number of births in the us in was approximately , , [ ] . the ageing parameter, a i , is simply the inverse of the time spent in each age class, hence a = a = / = . year - , a = a = a = / = . year - , a = / = . year - , a = year - . the death rates are obtained by requiring the size of each age class to remain constant over time (by balancing the inflow and outflow for each age class). this is expressed by the equations solving these equations using the above values for b us , , and a i yields . we note that, in principle, it would be possible to include demographic parameters that change over time according to real-world patterns. for instance, the changing sizes of age classes might be incorporated. however, because the primary purpose of the model is to illustrate the effects of travel coupling between the us and canada rather than to exactly predict future incidence, the introduction of extraneous processes corresponding to the additional parameters may make the model output more difficult to interpret. clinical and epidemiological literature on hepatitis a was used to estimate the durations of the latent and infectious periods. the mean duration of the latent period, /δ, is approximately weeks [ , ] . the mean duration of the infectious period for the different age groups is /γ = . weeks, /γ = . weeks, /γ = /γ = /γ = /γ = /γ = . weeks [ , , ] . the longer durations in younger age classes reflect the fact that virus is shed for longer in children than adults. let be the total force of infection (the probability per year that a susceptible person in age class i becomes infected, or approximately, the number of infected individuals in a given year in age class i divided by the susceptibles in that age class at the start of that year). in , . % of infected ha cases in the us acquired the disease through international travel [ ] . here it is assumed that, when the disease was acquired through international travel, it was acquired by traveling to endemic countries. to obtain age-specific values of the proportion of cases attributable to travel in endemic countries for (the values to be used in the model), the available age-specific number of cases for [ ] were adjusted for the population sizes of age classes [ ] and the resulting incidence values by age for were multiplied by the ratio of the overall proportion of cases attributable to travel in compared to , yielding . to estimate the number infectious at any given time dur- ing the year, , we adjust the reported incidence per year per , in age class i, , for subclinical infection and under-reporting, yielding the adjusted incidence . this is then modified using the typical duration of infectiousness to obtain . the reported incidence per year per , for the years - is, by age class, [ ] . the incidence in each age class is then divided by the probability of jaundice by age class p i : p = . , p = . , p = . , p = p = p = p = . [ ] and multiplied by the under-reporting factor of . [ ] to obtain the adjusted incidence values ( = . × observed incidence/p i ). finally, we must modify once again since it represents the number of cases per , per year, not the total number of cases at a given time during the year. for this, following formula is used, . in other words, the number of infectious individuals at any given time during the year is the annual incidence adjusted for the size of the age group divided by the average number of intervals of infectiousness that can occur within a year. therefore thus the system of linear equations (system b ) can be solved to obtain the values for us transmission rate attributable to travel to endemic countries (see table for values). next, is found from equation b . since there are more unknowns ( × = ) than knowns ( ), we make additional assumptions about the values: , etc. similar assumptions have been made for other diseases modeled with age-structured compartmental models [ ] . expressed as a matrix, this becomes a "who acquires infection from whom" matrix. the values obtained by solving the linear system of equations (b ) are found in table . the rate of waning vaccine-derived immunity was obtained by fitting an exponential curve to the estimated proportion of vaccinated individuals retaining immunity after , , , and years ( %, %, %, %, and % respectively), obtained using the delphi method [ ] , yielding f = . year - . in our simulations, only individuals in the first age class were vaccinated, hence . to determine such that % of individuals are vaccinated at a given age each year in the - age class, we set f = , , applied the condition to system a and solved it analytically to obtain = / year - . the average size of the age classes from - in canada were approximately [ ] . the average number of births per year during this time was approximately , [ ] . the death rates, ageing parameters, and duration of the latent infectious periods are the same as in the us model. the canadian rate of infection due to travel to endemic countries ( ) was computed using the same method as for the us, except that there is an additional contribu-tion to the travel transmission rate, , from travel to the us. hence, the total force of infection in canada is again using the fact that , we have that is found following the same method as for the us case, but using canadian incidence and travel data. the values appear in table . the parameter is obtained from equation . the volume of travel (annual number of trips) by canadian residents using any mode of transport in to the us and endemic countries is available from published data [ ] . estimates of age-specific travel volumes to the us ( ) and endemic countries ( ) are obtained from these data by assuming travel volume to be distributed across the age classes according to the sizes of the age classes [ ] , yielding the results in table . the average duration of travel to the us and to endemic regions are = . nights and = . nights respectively [ ] . the incidence per , per year in the us, canada and endemic countries, adjusted for under-reporting and subclinical infection, are estimated as y us = . , y c = . , y end = . [ , , ] . all these values are substituted into equation to obtain the values appearing in table . the parameter is found using the same methods as for the us model, yielding values for (see table ). the rate of loss of vaccine derived immunity is the same as for the us model, f = . year - [ ] . the vaccination policy in canada is currently targeted vaccination toward high-risk groups. a previous study estimates that, to date, about % of the canadian population has been vaccinated under this programme (which also vaccinates travelers to endemic countries) (bauch et al, unpublished data). hence, we assume in this paper a % coverage rate for each age class. although the actual coverage rates across age classes under the current targeted policy may be dissimilar, the available data do not allow us to stratify the vaccine coverage rates by age. hence, we have assumed the same vaccination rate applies to each age class. we note that there are also other heterogeneities in vaccine coverage (e.g., social) that the present model was not designed to address. the values of g i are obtained from imposing f = , as for the us case, as well as the constraints on system a , and solving system a to yield g c = . year - . .. the global burden of disease, - unaids: aids epidemic update the effects of socioeconomic development on worldwide hepatitis a seroprevalence patterns hepatitis a: old and new epidemiological patterns of hepatitis a in different parts of the world hepatitis a in latin america: a changing epidemiologic pattern public health agency of canada (phac): notifiable diseases online hepatitis a virus infections in the united states: model-based estimates and implications for childhood immunization global impact of hepatitis a virus infection changing patterns hepatitis a vaccines: the growing case for universal immunisation of children ineffectiveness of the current strategy to prevent hepatitis a in travelers hepatitis a virus in urban children -are preventative opportunities being missed? hepatitis surveillance report quantifying the impact of hepatitis a immunization in the united states a dynamic model for assesssing universal hepatitis a vaccination in canada touriscope: international travel; travel between canada and other countries . historical supplement statistics canada: cansim table - public health agency of canada (phac): canadian immunization guide centers for disease control and prevention: prevention of hepatitis a through active or passive immunization kelley pw: frequency of illness associated with epidemic hepatitis a virus infection in adults relapsing hepatitis a. review of cases and literature survey hav infection in chronic liver disease: a rationale for vaccination clinical spectrum and natural history of viral hepatitis a in a shanghai epidemic the cost-effectiveness of adolescent hepatitis a vaccination in states with the highest disease rates predicting the impact of measles vaccination in england and wales: model validation and analysis of policy options effects of long-term retinoic acid treatment on epidermal differentiation in vivo: specific modifications in the programme of terminal differentiation hepatitis a in the united states prevention of hepatitis a with the hepatitis a vaccine another vaccine preventable disease continues to emerge annual population estimates by sex, race and hispanic origin, selected years from infectious diseases of humans: dynamics and control oxford statistics canada: international travel. travel between canada and other countries cohort effects in epidemic models: an example from hepatitis a the spread and persistence of disease in structured populations cost-effectiveness of hepatitis a immunization of children and adolescents in germany a mathematical model of hepatitis a transmission in the united states indicates value of universal childhood immunization cost-utility of universal hepatitis a vaccination in canada individual versus public priorities in the determination of optimal vaccination policies imitation dynamics predict vaccinating behaviour vaccination and the theory of games group-interest versus selfinterest in smallpox vaccination policy trends in characteristics of births by state: united states controle de l'hépatite a par l'immunisation au quebec. (final report results from the national sentinel health unit surveillance system, - aw and ctb were supported by discovery grants from the natural sciences and engineering research council of canada (nserc). the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -iyh d mj authors: ding, lin; zhao, yu; li, xuyan; wang, rui; li, ying; tang, xiao; sun, bing; he, hangyong title: early diagnosis and appropriate respiratory support for mycoplasma pneumoniae pneumonia associated acute respiratory distress syndrome in young and adult patients: a case series from two centers date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: iyh d mj background: mycoplasma pneumoniae (m. pneumoniae) is one of the most common causes of community acquired pneumonia (cap). establishing an early diagnosis of m. pneumoniae pneumonia in patients with acute respiratory distress syndrome (ards) may have important therapeutic implications. methods: we describe diagnosis and management of m. pneumoniae pneumonia induced ards in a case series of adults and youth hospitalized with radiographically confirmed cap prospectively enrolled in an observational cohort study in two university teaching hospitals, from november to october . results: in all patients, early and rapid diagnosis for severe m. pneumoniae pneumonia with ards was achieved with polymerase chain reaction (pcr) or metagenomic next-generation sequencing (mngs) testing of samples from the lower respiratory tract or pleural effusion. the average pao( )/fio( ) of all patients was mmhg. of the cases, cases had moderate ards ( mmhg ≤ pao( )/fio( ) < mmhg) and cases had severe ards (pao( )/fio( ) < mmhg). high flow nasal cannula (hfnc) was applied in all patients, though only two patients were sufficiently supported with hfnc. invasive mechanical ventilation (imv) was required in patients. high resistance (median l/cmh( )o/s) and low compliance (median ml/cmh( )o) was observed in cases. in these cases, recruitment maneuvers (rm) were applied, with patient demonstrating no response to rm. prone positioning were applied in cases. two cases needed ecmo support with median support duration of . days. no patient in our case series received corticosteroid therapy. all patients were survived and were discharged from hospital. conclusions: early and rapid diagnosis of severe m. pneumoniae pneumonia with ards can be achieved with pcr/mngs tests in samples from the lower respiratory tract or pleural effusion. in our case series, half of m. pneumoniae pneumonia induced ards cases were adequately supported with hfnc or niv, while half of cases required intubation. rm and prone position were effective in % of intubated cases, and % needed ecmo support. when early anti-mycoplasmal antibiotics were given together with sufficient respiratory support, the survival rate was high with no need for corticosteroid use. mycoplasma pneumoniae (m. pneumoniae) is one of the most common causes of community acquired pneumonia (cap) often seen in children and young adults, and accounts for - % of all cases of adult cap cases [ , ] . m. pneumoniae pneumonia is typically mild and characterized by a persistent dry cough or self-limiting pneumonia that resolves with no medication [ ] . however, respiratory failure and severe acute respiratory distress syndrome (ards) occur in . - % of all m. pneumoniae pneumonia cases and primarily affect young adults [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the rates of intensive care unit (icu) admission of hospitalized m. pneumoniae pneumonia patients are reported as % in the us and . % in europe [ , ] . the rate of icu admission is even higher at . % in patients older than years, compared to % in patients older than years [ ] . in one retrospective study from our hospital, . % of m. pneumoniae pneumonia patients needed icu admission for acute respiratory failure in the setting of an epidemic [ ] . severe ards and fatal outcome as a result of m. pneumoniae pneumonia may be the result of unclear clinical features [ ] , delayed diagnosis, inappropriate respiratory support, and/or insufficient initial treatment. when acute nonbacterial pneumonia progresses, m. pneumoniae must be considered as a possible cause, and appropriate diagnosis, respiratory support and therapeutic measures should be promptly instituted. previous studies suggest that m. pneumoniae infection should be included in the differential diagnosis of ards, and that establishing an early diagnosis may have important therapeutic implications [ ] . in recent years, rapid diagnostic methods have been developed, allowing for early diagnosis of m. pneumoniae pneumonia. detection of m. pneumoniae using fluorescence-quantatitive pcr in respiratory samples [ , [ ] [ ] [ ] [ ] and metagenomic next-generation sequencing (mngs) has increased [ ] ; these methods are especially useful for early detection of rare, atypical, and slow-growing microbes. case reports have also described using new forms of respiratory support for m. pneumoniae pneumonia induced ards, such as highflow nasal cannula (hfnc) [ ] , non-invasive ventilation (niv) [ ] and veno-venous extracorporeal membrane oxygenation (ecmo) [ , , ] . however, there has not yet been a full evaluation of the new available diagnostic and therapeutic measures in m. pneumoniae pneumonia induced ards. the aim of our study was to describe a case series of patients with m. pneumoniae pneumonia induced ards and provide an overview of available modalities for diagnosis and treatment. we describe the epidemiological, clinical, imaging, and laboratory features of our patients, review the available procedures for early diagnosis, and evaluate available respiratory support techniques in clinical practice in order to highlight the importance of rapid recognition and appropriate treatment. we retrospectively identified all cases of young and adult patients (age over years) with ards caused by m. pneumoniae pneumonia who were admitted to the respiratory icu in two teaching hospitals (beijing chao-yang hospital and beijing luhe hospital, beijing, china) with radiographically confirmed cap from november to october . the study was approved by the institutional review boards (irb) at each institution. written informed consent was obtained from all participants, where participants are children (under years old) from their parent or guardian. case definitions for m. pneumoniae pneumonia induced ards criteria for diagnosis of m. pneumoniae pneumonia were based on ) clinical signs and symptoms (cough, fever, productive sputum, dyspnoea, chest pain or abnormal breath sounds); ) radiographic pulmonary abnormalities that were at least segmental and were not explained by pre-existing or other known causes; and ) positive detection of m. pneumoniae nucleic acid by pcr or mngs from lower respiratory tract secretion (sputum and balf), which were considered as microbiological evidence of infection. all cases of pneumonia occurring more than days after hospitalization were considered nosocomial and were excluded. patients with hiv infection, neutropenia, or who were receiving immunosuppressive chemotherapy were excluded. the diagnosis of ards was assigned to patients who met the berlin definition criteria: ) presence of acute hypoxemic respiratory failure; ) onset within days of insult, or new (within days) or worsening respiratory symptoms; ) bilateral opacities on chest x-ray or ct not fully explained by effusions, lobar or lung collapse, or nodules; and ) cardiac failure not the primary cause of acute respiratory failure. we categorized patients into mutually exclusive classes of ards severity using previous definitions based on degree of hypoxemia: ) mild ( mmhg ≤ pao /fio < mmhg); ) moderate ( mmhg ≤ pao /fio < mmhg); and ) severe (pao /fio < mmhg). patient specimens, including sputum, whole blood, and serum samples, were collected upon admission and during hospitalization for bacterial and viral testing. microbiological tests were performed at the department of infectious disease and clinical microbiology laboratories in our centers. throat swabs, sputum, endotracheal aspiration or balf were collected for m. pneumoniae pcr assay. all severe ards patients had m. pneumoniae infection confirmed by pcr assay or mngs from lower respiratory tract secretion (sputum and balf). pcr of pleural effusion fluid were also tested in some patients for the detection of m. pneumoniae. clinical information collected included the following: characteristics (age and sex), comorbidities, clinical symptoms (fever, cough, sputum, dyspnea, chest pain, rash, nausea, vomiting, abdominal pain, diarrhea and headache), clinical signs (body temperature, heart rate, respiratory frequency, blood pressure and crackles in the lungs), laboratory tests (whole-blood cell count and blood chemistry), and microbiological findings and images of the lung, including chest x-ray (cxr) and high resolution computed tomography (hrct). concomitant medications, respiratory support (hfnc, niv, invasive mechanical ventilation, prone position and ecmo), complications, and outcomes were also recorded. pooled epidemiological, clinical, imaging, and laboratory data are shown as median with range for quantitative variables and as absolute and relative frequencies for qualitative variables. the enrolled patients were divided into two groups based on use of invasive mechanical ventilation. continuous variables were compared using the mann-whitney u-test, whereas categorical data were compared using the chi-squared test or the fisher's exact test, where appropriate. all comparisons were performed using the spss statistics package version . . differences were considered statistically significant when p was < . . between november and october , patients met criteria of severe m. pneumoniae pneumonia. of the patients, one patient was excluded due to diagnosis of lymphoma combined with adenovirus pneumonia. therefore, immunocompetent patients were included in the final analysis. the age range of our patients was to (median ) years. there were male and female patients. all cases were admitted in different months of the year except for january, february and september. only one patient (case ) had diabetes mellitus. the other patients had no underlying diseases (table ) . seven ( %) and ( %) patients had positive serum m. pneumoniae igg and igm, respectively. m. pneumoniae pcr of the sputum was performed in ( %) cases, and was positive in all cases. three of the cases had m. pneumoniae pcr from balf at the same time, and all cases ( %) were positive. another patient was diagnosed with m. pneumoniae pneumonia through pcr of pleural effusion fluid. five cases had mngs from balf, and all these cases were positive for m. pneumoniae (table ) . acinetobacter baumannii was detected in patients ( %) who were transferred from another hospital after icu admission, but these were isolated from the lower respiratory tract (lrt) samples collected after more than days of their icu stay, and therefore were not considered as causative agents of ards together with m. pneumoniae (table ) . all patients had cough and fever at the onset of illness. they presented with a high fever, with a median body temperature of . °c (range, . °c to . °c). eight patients ( %) had dry cough and two patients had productive cough. four patients ( %) had diarrhea and one patient ( %) had abdominal pain (table ) . acute respiratory deterioration occurred to (median ) table ). nine patients were tested for cell-mediated immunity, immunoglobulins (serum igg, iga and igm), and components (attached file , e- table ). table ). all patients had cxrs. cxrs revealed bilateral multilobular or segmental consolidation in nine ( %) patients. one patient's cxr showed diffuse peribronchial infiltration. all patients underwent chest hrct. unilateral or bilateral consolidation and infiltration were found on hrct scans of patients ( %). large areas of consolidation within a single lobe or several lobes ( %), followed by pleural effusion ( %), were the most common findings on hrct (fig. ) . only the hrct of case showed peribronchial infiltration without consolidation and pleural effusion. the average pao /fio of all patients was mmhg. four ( %) cases had moderate ards ( mmhg ≤ pao /fio < mmhg), and three cases ( %) had severe ards (pao /fio < mmhg). hfnc was applied in all patients ( %), with a median gas flow of l/min ( - l/min) and fio . ( . - . ), but only two patients were sufficiently supported with hfnc. niv was used in four patients with a median duration of ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) h, and one patient had niv failure and needed intubation ( table ) . invasive mechanical ventilation (imv) was carried out in patients ( %). high airway resistance (median l/cmh o/s) and low respiratory system compliance (median ml/cmh o) was observed in all cases. recruitment maneuver (rm) was applied in cases, with patient not responsive to rm, and cases were put into prone position. the maximum peep was cmh o (range, - cmh o). two cases ( %) needed ecmo support with median support duration of . days ( table ). as shown in table , case had the worst compliance and was unresponsive to rm, and ecmo was established. case , case , and case were responsive to rm and prone position. all patients did not receive fluoroquinolones at the onset of their illness, and switched to fluoroquinolones . ± . days after onset. all patients received β-lactams treatment as first therapy, and patients received treatment combined with macrolides before their admission to icu. after icu admission, moxifloxacin were given to all patients. no patients in our case series received corticosteroid therapy. all patients survived in the icu and were discharged from the hospital. the mortality of our cases was %. the average length of icu stay was days, and the average length of hospital stay was days. as shown in table , compared to non-intubated patients, patients in intubated group were younger, were less likely to be male, had lower pao /fio and higher apache ii scores, and had higher procalcitonin and neutrophil proportion at icu admission. to our knowledge, our study is the first and largest case series to evaluate diagnostic and therapeutic modalities in severe m. pneumoniae pneumonia induced ards. our main findings are as follows: ) early and rapid diagnosis for severe m. pneumoniae pneumonia with ards was achieved with pcr/mngs test of samples from the lower respiratory tract and pleural effusions; ) ct findings mainly showed alveolar patterns with bilateral consolidations rather than interstitial patterns; ) respiratory mechanics showed low respiratory system compliance and high airway resistance; ) % of m. pneumoniae induced ards were adequately supported with hfnc or niv, % required intubation, rm and prone position were effective in % intubated cases, and % needed ecmo support; ) when early anti-mycoplasmal drugs together with sufficient respiratory support are given, the survival rate was high with no need for corticosteroids; and ) younger patients with lower pao / fio and apache ii scores, and higher pct and higher neutrophil cell proportion at icu admission were more likely to require intubation. in our study, the clinical manifestations of severe m. pneumoniae pneumonia induced ards were primarily dry cough, high fever, and acute respiratory failure with bilateral consolidations on radiologic examination. respiratory failure occurred a median of days (range, to days) after onset of symptoms, similar to the previous descriptions [ , ] . however, these clinical features are not specific for early recognition and diagnosis of severe m. pneumoniae pneumonia. therefore, early and precise laboratory detection of m. pneumoniae infection is essential to prevent deterioration. previous methods, such as mycoplasma culture and serological tests, which may require several weeks, are not practical. as presented in our study, early definitive diagnosis is now dependent on pcr or mngs [ ] , which had high specificity and sensitivity. thus, further development of these relatively new diagnostic tools is warranted, and should be applied in cases of severe cap induced ards with suspected m. pneumoniae infection. furthermore, our study found that most of our cases had pleural effusion, and pcr was positive for m. pneumoniae in pleural effusion fluid. similar findings were also reported in a previous case report [ ] . therefore, in patients with dry cough and difficulty obtaining a lower respiratory sample, early pcr/mngs for m. pneumoniae using pleural effusion fluid may be an option. as ards is a clinical syndrome with many different causes and may induced by some less common pathogens, mngs is used in our icu for early detection of possible unknown etiology, and we found that mngs had a good value in diagnosis for m. pneumoniae in our cases. however, in most cases mngs is more appropriate to be used for patients with unknown etiology, and may not be suitable for routine examination of some common pathogens such as adv, rsv, and m. pneumoniae. thus, once commercial pcr kits are available for diagnosis of m. pneumoniae infection, it is not necessary to detect m. pneumoniae using mngs as a primary option. in both our case series and previously reported cases of m. pneumoniae associated ards, deterioration of the clinical state presumably due to a significant period of inadequate antibiotic treatment [ , ] . in a review of severe or fatal m. pneumoniae pneumonia, the average duration from onset of infection to the development of respiratory failure was . days (range, - days) [ ] . chan and miyashita et al. reported durations of - and . days, respectively, from onset to first administration of appropriate anti-mycoplasma agents [ , ] . the duration of an average of days to change the treatment from our study was similar to the previous studies. therefore, our management would still be considered as late intervention, and the delay as a risk factor for development of respiratory failure and ards. earlier recognition of m. pneumoniae in the differential diagnosis and earlier initiation of appropriate antibiotics would potentially prevent m. pneumoniae pneumonia from progressing to ards. furthermore, more awareness is needed on the emergence of macrolide-resistant m. pneumoniae infection in adults [ , ] . a previous report from our center found the rate of resistance to macrolides was . % of the isolated m. pneumoniae, and all resistant strains harbored a g mutations. the isolated macrolide-resistant m. pneumoniae were resistant to erythromycin, and also showed cross-resistance to clarithromycin and azithromycin. all isolates were sensitive to tetracyclines and fluoroquinolones. moxifloxacin was more active than ciprofloxacin and levofloxacin [ ] . however, sequencing of macrolide resistance genes is not a routine test in clinical practice in our centers, and we did not perform sequencing for macrolide resistance genes in our cases. we speculated that we have the similar high rate of resistance and similar type of resistant genes in our case series. thus, early fluoroquinolones were considered as first line treatment for m. pneumonia induced severe ards cases in adults. a previous epidemiological study from one of our centers (beijing chao-yang hospital) that routinely screened for m. pneumoniae in outpatients during - determined that only patients out of patients ( . %) with m. pneumoniae infection needed icu admission [ ] . however, after that study concluded, m. pneumoniae infection was only routinely screened in patients with a diagnosis of community acquired pneumonia who were hospitalized in our general ward or admitted to our icu. during our study period, of severe cap were admitted to our icu and were diagnosed with m. pneumoniae pneumonia ( . %). additionally, as shown in table in our study, the first lrt sample for m. pneumoniae was collected on an average of ± days after the onset of symptoms. the higher rate of m. pneumoniae pneumonia in our case series suggests that early detection for the pathogen may be needed to start an early intervention and proper treatment. the patients with mild ards in our study were successfully supported by hfnc and niv without intubation. one patient with moderate ards was successfully supported with a combination of hfnc and awake prone positioning, which proved safe and effective in moderate ards patients by our team in a prospective study [ ] . hfnc or niv, combined with early prone positioning, may be a new support strategy for acute respiratory failure in m. pneumoniae indunced mild to moderate ards patients. although the radiologic findings showed a diffuse alveolar pattern with consolidations and the respiratory mechanics showed decreased respiratory system compliance, most intubated patients were responsive to rm and prone positioning during invasive ventilation, with a maximum peep of - (median ) cmh o was applied. however, two cases deteriorated to severe hypoxia despite anti-mycoplasmal therapy and invasive ventilation, eventually requiring ecmo support. in a recent case report and literature review for use of ecmo in m. pneumoniae associated ards, the mean ecmo run was h/ . days [ ] , similar to that of our cases. the overall survival rate for cases of m. pneumoniae requiring ecmo with reported outcome was . % ( / ) , demonstrating that ecmo may be safely and effectively used to treat severe ards caused by m. pneumoniae infection [ ] . previous reports support the hypothesis that the severity of the disease and pulmonary infiltrates may be directly correlated with the level of the individual immune response. however, in our study, we did not observe significant increases of cell or humoral immunity as demonstrated by t cell subset cell count or immunoglobulin levels in more severe disease. most interestingly, we found that with appropriate respiratory support and anti-mycoplasmal therapy, all patients had a rapid clinical improvement. therefore, no corticosteroids were given, and all patients finally recovered from ards without corticosteroid use. prolonged or inappropriate use of corticosteroids may cause excess downregulation of cell-medicated immunity and result in immunosuppression, making individuals more susceptible to more severe m. pneumoniae infection or opportunistic infections. a recent case report revealed that m. pneumoniae associated ards had no elevated pulmonary vascular permeability, and was successfully treated using low-dose short-term hydrocortisone, suggesting that pulmonary infiltration in ards caused by m. pneumoniae does not match the criteria of permeability edema observed in typical ards [ ] . therefore, careful consideration is required when deciding whether to use high dose corticosteroid in the future cases similar to ours. there are several limitations for our study. first, performing statistical analysis on a small sample size was prone to bias, potentially yielding spurious findings. increasing the sample size and collecting more cases in a further study may avoid this kind of limitation. second, this study is a retrospective study with the associated limitations on complete data collection. in conclusion, early and rapid diagnosis for severe m. pneumoniae pneumonia with ards can be achieved by pcr/mngs test in 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institutional affiliations we thanked ann andee wang m.d. (from feinberg school of medicine, northwestern university, chicago, il, usa) for her advices and help for the manuscript revision. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file . e- table . the laboratory findings for the patients with severe m. pneumoniae pneumonia on the first day of admission.additional file . e- table . the result of pleural effusion biochemistry and pleural effusion routine of the severe m. pneumoniae pneumonia.additional file . e- table . cell-mediated immunity and humoral immunity on the immunocompetent patients with severe m. pneumoniae pneumonia on the first day of admission. authors' contributions ld, yz, xl, rw, yl, xt, bs and hh carried out the treatment, collected analyzed the data and wrote the manuscript. hh and ld conceived of the study, and participated in its design and coordination and draft the manuscript. all authors read and approved the final manuscript. no. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. this study is approved by the irb of beijing chao-yang hospital and beijing luhe hospital. written informed consent was obtained from all participants (where participants are children under years old, from their parent or guardian). written informed consent was obtained from all participants for their data to be used for research and publication. written informed consent for participation in the study was obtained where participants are children (under years old) from their parent or guardian. we confirm that the patients, and in the case of minors their parents/guardians, provided written informed consent for the publication of potentially identifying images and clinical details. the authors declare that no conflicts of interests. key: cord- -v u mr i authors: bonnin, paul; miszczak, fabien; kin, nathalie; resa, cecile; dina, julia; gouarin, stephanie; viron, florent; morello, remy; vabret, astrid title: study and interest of cellular load in respiratory samples for the optimization of molecular virological diagnosis in clinical practice date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: v u mr i background: respiratory viral diagnosis of upper respiratory tract infections has largely developed through multiplex molecular techniques. although the sensitivity of different types of upper respiratory tract samples seems to be correlated to the number of sampled cells, this link remains largely unexplored. methods: our study included upper respiratory tract specimens of which negative and positive for viral detection in multiplex pcr. all samples were selected and matched for age in these groups. for the positive group, samples were selected for the detected viral species. results: among the factors influencing the cellularity were the type of sample (p < . ); patient age (p < . ); viral positive or negative nature of the sample (p = . ); and, for the positive samples, the number of viral targets detected ( . < p < . ) and viral species. conclusion: the cellular load of upper respiratory samples is multifactorial and occurs for many in the sensitivity of molecular detection. however it was not possible to determine a minimum cellularity threshold allowing molecular viral detection. the differences according to the type of virus remain to be studied on a larger scale. associated with particular diseases (respiratory syncytial virus and bronchiolitis, parainfluenza virus and laryngitis, rhinovirus and common cold, influenza virus and flu syndrome), there is no evidence for a clinical specificity, and only the virological diagnosis provides an accurate identification of the ari [ , ] . detection of respiratory viruses is of little interest in general practice, in that the infection does not present a risk of severity for the patient. however, virological confirmation of ari is needed in severe clinical presentations, requiring hospitalization in intensive care units and occurring in vulnerable subjects [ , ] . the goal of early virological diagnosis would be an optimization of patient care, which could lead to reduction in length of hospital stay, a saving of antibiotics, and complementary examinations [ ] . virological tests allow for the establishment of accurate diagnosis of infection, assessment of evolving risks (bacterial infection, acute respiratory distress syndrome), and the establishment of measures to limit its spread (isolation, wearing gloves and masks). pandemics of severe acute respiratory syndrome (sars, (sars, - and influenza a-h n ( ) lead to the development of molecular biological techniques applied to virological diagnosis, mainly based on pcr (polymerase chain reaction). performances of molecular methods in respiratory virology are so significant that they have replaced conventional techniques (culture, detection of viral antigens) as a reference method [ ] [ ] [ ] [ ] . multiplex pcr techniques are particularly suited to medical diagnosis because they can detect multiple viral targets in the same time, avoiding the virologist a selection of viral targets to search. there are now many commercial kits for the detection of a range of to respiratory viruses and some intracellular bacteria [ , , , ] . molecular techniques (real-time pcr) also make it possible to achieve a semi quantification of the viral molecular material present in the sample, giving additional information about the respiratory viral load (interest in therapeutic monitoring and infection transmission risk) [ ] . a normalized viral load can be obtained by adding a cell quantification step. the primary site for replication of respiratory viruses is the ciliated airway epithelium. the sample must be taken as soon as possible after the onset of symptoms. this is usually a nasal swab or nasopharyngeal aspiration (especially realized in children under ) [ ] . these samples are easily accessible and especially adapted to upper ari. if a rich cell collection appears to be an important prerequisite for the quality of respiratory viral diagnosis, there is currently no information on a possible cellularity threshold that would validate the result of the viral molecular detection. the main objective of this work is the study of cellularity in respiratory specimens previously characterized virologically. the results should help to define the concept of "cellular richness" and determine the factors that influence it. eight hundred respiratory samples were included in this study. all were collected between september , and february , in different departments of the university hospital of caen (france), and immediately sent to the virology department for a respiratory viral diagnosis. respiratory samples were divided into nasal swabs corresponding to nasopharynx sampling (posterior nares), collected on universal viral transport medium (utm) and nasal aspirates. after receipt in the laboratory, each sample underwent a pre-analytical step including division into aliquots: one was immediately used for the viral detection and the other two were stored frozen at - °c. one of its two frozen fractions was used for this study. this complementary diagnostic study was then conducted on residual clinical specimens, in french law, the right to use the end of the samples is written in the code of public health : code de la santé publique -article l - . these aliquots were selected in the laboratory samples bank according to their results in virological diagnosis: positive and negative for molecular detection of respiratory panel using the respifinder ® smart_ _fast technique (pathofinder, maastricht, netherlands). this kit allows for the detection of rna and dna viral targets and intracellular bacterial targets in respiratory specimens (tables and ). a total of age groups reflecting the distribution observed in practice in the laboratory were indicated as follows: infants (age < ; %; n = ), children (aged from to ; %; n = ), adults (aged from to ; . %; n = ) and elderly (age ≥ ; . %; n = ). each group is composed of half positive and half negative in molecular viral detection, and so as to be matched for age. within the group of positive samples, the distribution of detected viral species was modeled on that observed in the routine activity of the laboratory (tables and ) . after thawing the samples, the extraction of nucleic acids was performed using the plc qiasymphony® (qiagen, hilden, germany). the extraction was performed with μl of sample using qiasymphony_dsp_virus/ cell quantification was achieved by amplification and detection of a human household gene in real-time pcr (hypoxanthine phosphoribosyl transferase- ) [ , ] . cell quantification was performed on a lightcycler ii® platform (roche, meylan, france). the reaction mixture included μl of amplification premix cell_control r-gene® (argene/biomerieux, lyon, france) and μl of nucleic acid extract. each manipulation included two negative controls: one undergoing all the analytical steps, called ec (extraction control) and one introduced prior to the pcr reaction, called "negative control". cell quantification standard (qs : × cells/pcr and qs : × cells/pcr) was ready to use in the kit. an external standard curve was performed using these two standards and additional dilutions, respectively containing × and cells/pcr. the reading of the results was carried out directly from the plc software, which displays the ct values (cycle threshold) obtained and the corresponding number of cells/pcr-reaction (i.e. μl of extract). this number was converted to "cells/ml" and the final results were expressed in logarithmic scale (log /ml). the quantification kit performances were verified in our laboratory by quantifying mrc cells (human embryonic fibroblasts) of known concentration (rd-biothech, besancon, france). a range of -fold serial dilutions was made from initial mrc cell suspension in viral transport medium (utm). the nucleic acids were extracted from these cell suspensions, and cell quantification reaction was carried out under the same conditions as for the respiratory samples. descriptive statistics were used to show the characteristics of the different variables. quantitative variables were described using means and standard deviation. qualitative variables were described using frequencies and percentages. the relationships between qualitative variables were studied using the chi-square test or fisher's exact tests. the anova was used to compare the means of quantitative variables in two or more independent groups with the bonferroni post hoc test. the relationship between two quantitative variables was assessed using the spearman correlation coefficient (ρ). to look for a diagnostic threshold to divide positive and negative samples, a receiver operating characteristic curve (roc curve) was used. all the tests were two-tailed and their level of significance (p) was defined as p < . . ibm®-spss® . for windows® was the statistical software used. the -fold serial dilution range from initial mrc suspension had expected cellularity values between log/ ml (concentration given by the manufacturer) and . log/ml (last dilution). the measured cellularity values were consistent with those expected for the concentrations between and . log. the last dilutions deviated more than . log of the expected value, corresponding to concentrations between . and . log ( table ). the mean deviation from expected values was . log/ml. regarding our samples population, cell quantification was negative (no hprt- dna detection) for of the positive and for of the the negative in viral detection. comparative study of sample "swabs" and "nasal aspirates" among the cell-quantified respiratory samples, were nasal aspirates and were nasal swabs. cellularity these results are presented in table . regarding nasal swabs only, average cellularity were not significantly different between the age groups except for children compared with adults (p < . ). comparison of cellularity among the positive (n = ) and negative (n = ) samples in viral detection as the subjects were matched for age, the age distribution is identical in the two groups positive and negative (p = . ). these two groups are comparable, as expected. the average cellularity was . (+/- . ) log/ml for the positive group and . (+/- . ) log/ ml for the negative group. this difference was significant (p = . ). the results of comparison between the age groups according to the result of the viral detection (positive or negative) are presented in fig. b . within a single age group (infants, children, adults, elderly), the differences between positive and negative samples were not significant (p = . , p = . , p = . and p = . respectively). based on the results of the comparison between positive and negative samples, a roc (receiver operating characteristic) curve was performed. no minimum cellularity threshold could be defined for molecular viral detection (fig. ) . aspirates swabs fig. average cellularity of respiratory specimens depending on the sample type and age group. a nasal aspirate was the sample type that provides, on average, more epithelial cells for patients under the age of ( . log/ml for aspirates versus . log/ml for swabs). b although having the same shape, the average cellularity curve of positive samples was always located above the negatives among the selected samples, viral detection was negative in , were positive for viral target, were positive for targets and were positive for targets. the average cellularity was . (+/- . ) log/ml, . (+/- . ) log/ml, . (+/- . ) log/ml, and . (+/- . ) log/ml for these groups respectively. the average cellularity in negative samples was significantly lower than in cases of mono (p = . ), bi (p = . ) or tri-detection (p = . ). a significant tendency was observed between positive samples for one viral target and those positive for or virus (p = . ), this trend was confirmed by a spearman correlation (ρ = ) indicating a strong correlation between sample cellularity and the number of viruses detected. molecular detection, including multiplex techniques, is currently the gold standard for viral respiratory diagnosis. we have very powerful molecular tools, ensuring a quality respiratory viral diagnosis, available for all clinicians supporting hospitalized patients. one factor limiting this diagnosis is represented by the collected respiratory specimens. the main objectives of this work have been to study the cellularity of these clinical respiratory specimens, to propose a possible definition of what is commonly called "cellular richness," and to measure the impact of this marker on the molecular viral diagnosis. very few published studies have been completed in this area. however, a number of facts are commonly accepted within the medical community: respiratory specimen should be "rich" to allow for "good" viral diagnosis, the "good" samples are obtained almost exclusively in infants and children. in total we identified studies published in international journals between and , whose objective was to compare the various upper respiratory samples, in terms of sampling equipment (flocked swabs versus rayon swab), in terms of sampling site (nasal, oropharyngeal, nasopharyngeal, combining sites), and sampling modality (swab, wash, aspiration) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the number of target cells and/or the number of extracellular viral particles collected could define the respiratory sample quality during sampling. it is not unreasonable to consider that the majority of the viral sequences detected by molecular techniques are mainly located in the intracellular compartment. however, several factors could modulate this distribution: the viral species, the cytopathic effect induced in vivo, the inflammatory responses, and the sampling delay from the onset of the symptoms. this study is retrospective; it was therefore not possible to collect data from the time between sampling and clinical symptoms in a standardized way. this matter is nonetheless very interesting and remains to be explored. for this study, we considered cell quantification, or cellular richness, as the main marker of diagnostic efficacy for a sample. the studies referenced above used indirect measurement of sample richness through the virus detected in it: used molecular detection methods, and one was an antigen detection using rapid diagnostic test evaluate the number of infected and uninfected cells deposited on the slide [ ] . overall, the results are consistent and show a superiority of nasopharyngeal swab versus oropharyngeal and a superiority of flocked swab versus classical ones [ , , , ] . the superiority of the "wash" versus the "swab" is not found by all authors, and comes well counteract the widespread idea that washing is always greater than swabbing [ ] . alsaleh et al. ( ) performed a molecular cell quantification using real time pcr in order to validate viral detection on nasal swabs. the cellularity of these samples was assessed using the quantification of a human endogenous retrovirus (erv ) known to be present in two copies per diploid cell [ ] . the authors report their results by comparing the ct obtained upon erv detection and not in terms of cellular load [ ] . in our study, we made the choice to use a direct detection method for assessing the number of cells in respiratory samples. to the extent that we needed a large number of samples characterized by many markers for statistical analysis (age of the sampled patient, detected viral target, etc.), a prospective study on freshly sampled respiratory specimens could not be performed. we therefore used previously characterized and stored frozen (- °c) respiratory samples. the definition of cellular richness is not affected by freezing or thawing samples since it theoretically does not change the amount of hprt nucleic acid, i.e. copy per haploid cell, whether or not lysed. similarly, no distinction between living and dead cells was made before the freezing process because such a distinction does not affect the quality of molecular detection by pcr (detection of viral genome into infected cells, living or not). the analysis of samples, divided into positive and negative in viral detection, failed to establish a minimum cellularity threshold to invalidate the negative results in viral molecular detection. the roc curve shows that the cellularity is not a quantifiable predictor of the outcome of the virus detection. however, our work has yielded interesting results concerning the factors influencing the cellularity of samples and the impact of cellularity on the result of molecular detection of respiratory viruses. we have clearly demonstrated that nasal aspirates allowed us to collect more cells than with nasal swabs and this only in the age group under years old. this result must be tempered by the fact that % ( / ) of the nasal aspirations of the study were from children under years of age, since the distribution was random at inclusion of samples in the study. this reflects a common practice of sampling methods in clinical departments: nasal aspirates are rarely performed in adults and the elderly over years old. this gesture is considered invasive and unpleasant. our results support the idea that, de facto, it would not be appropriate to perform this type of sampling in this group. for all types of samples (swabs and/or nasal aspirates), the age of the sampled patient remains an important marker influencing cellularity, with a maximum average cellularity under the age of , a minimum average cellularity in adults group, and an intermediate average cellularity in the elderly group. the reasons for this lack of cellularity in respiratory samples from adults remain obscure. regarding the influence of cellularity on the result of viral detection, it should be noted that negative samples present, all age groups combined, an average cellularity lower than that obtained for the positive samples, even though it has not been possible to establish a predictive relationship. this difference was tenuous for the children group. this observation leads to two possible explanations: either in the positive group the infection increases the sample cellularity by promoting epithelial desquamation and, to a lesser extent, the mucus capture, containing free viral particles; or, in the negative group, there are false-negatives in virus detection, induced by a lack of cells in the sample. this result was obtained from all samples (aspirates and swabs). insofar as aspirates are evenly distributed in both positive and negative groups, we think that the result of comparison is not biased given that aspirates are richer samples. considering viral detection in its entirety, without analysis of the viral species detected, it is interesting to note that the two factors, "cellularity" and "viral codetection" (detection of or viruses) are associated positively. this suggests that the detection of several viruses is facilitated in the context of a rich sample. in cases of viral co-detection, the question of whether these are the same cells that are infected or not is not resolved, even if it is conventionally accepted that an already infected cell is less permissive to a second viral infection. it should also be noted that viral co-detection can be either a co-infection, or two or more sequential infections. such phenomena have already been discussed in the works of alsaleh et al. which showed that the positive samples in viral detection had, on average, a greater amount of genetic material of human origin than negative ones. similarly, samples where the gene erv was not detected had lower viral detection. finally, they also found that the positive samples for several viruses were also those in which cellular loads were highest [ ] . the analysis of the potential impact of cellularity on the specific detection of various viruses included in the "respiratory panel" showed results that should be confirmed with larger numbers in each group. indeed, on the one hand, the highest average cellularity was obtained in the hmpv positive samples, equally detected among adults and children groups; on the other hand, the lowest average cellularity is obtained in the rsv positive samples, mostly detected in infants and children groups. these results are surprising in that the two viruses, rsv and hmpv, belong to the same virus family (paramyxoviridae) as well as to the same virus subfamily (pneumovirinae), and are genetically close. they have many similarities in the circulatory mode and in the pathophysiology of the infection they cause. yet the cellularity of hmpv positive samples is significantly greater than that of the rsv positive specimens. the quality of samples dramatically affects the quality of results provided to clinicians. it is important that a better understanding of the sample characteristics goes along with technological developments. this work is uncommon. he tries to give answers to a trivial scientific question: respiratory specimens should they be « rich in cells » to ensure optimal virological molecular diagnosis? the cellular load is multifactorial and occurs for many in the sensitivity of molecular detection. however, it was not possible to determine a minimum threshold allowing molecular viral detection. adv, adenovirus; ari, acute respiratory infection; ct, cycle threshold; dna, deoxyribonucleic acid; ec, extraction control; erv , human endogenous retrovirus ; flu, influenzae virus; hbov, human bocavirus; hcov, human coronavirus; hmpv, human metapneumovirus; pcr, polymerase chain reaction; piv, parainfluenza virus; rhv/ev, rhino/entero virus; rna, ribonucleic acid; roc, receiver operating characteristic; rsv, human respiratory syncytial virus; sars, severe acute respiratory syndrome; utm, universal transport medium department of virology epidemiology of viral respiratory infections bronchiolitis viruses the underrecognized burden of influenza in young children viral epidemiology and severity of respiratory infections in infants in : a prospective study techniques actuelles de diagnostic des infections virales respiratoires en réanimation cost analysis of multiplex pcr testing for diagnosing respiratory virus infections superiority of reverse-transcription polymerase chain reaction to conventional viral culture in the diagnosis of acute respiratory tract infections in children increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms quantitation of respiratory syncytial virus rna in nasal aspirates of children by real-time rt-pcr assay molecular diagnosis of respiratory viruses comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness comparative evaluation of six commercialized multiplex pcr kits for the diagnosis of respiratory infections identification of respiratory viruses in adults: nasopharyngeal versus sampling development of an efficient qrt-pcr assay for quality control and cellular quantification of respiratory samples guideline to reference gene selection for quantitative realtime pcr comparison of flocked and rayon swabs for collection of respiratory epithelial cells from uninfected volunteers and symptomatic patients nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control swabbing for respiratory viral infections in older patients:a comparison of rayon and nylon flocked swabs comparison among nasopharyngeal swab, nasal wash, and oropharyngeal swab for respiratory virus detection in adults with acute pharyngitis improved detection of respiratory viruses in pediatric outpatients with acute respiratory illness by real-time pcr using nasopharyngeal flocked swabs comparison of nasopharyngeal and oropharyngeal swabs for the diagnosis of eight respiratory viruses by real-time reverse transcription-pcr assays evaluation of the quidel quickvue test for detection of influenza a and b viruses in the pediatric emergency medicine setting by use of three specimen collection methods a quantification of human cells using an erv- real time pcr assay not applicable. this study was supported by national reference laboratory for measles and respiratory paramyxoviridae. we have no additional data to communicate and have incorporated article all data, tables and figures necessary for the understanding of the study.authors' contributions av and rm designed the study. pb and av wrote the main manuscript text and prepared figures. rm was responsible for statistics analysis and wrote the statistics part of the methods. cr provided protocoles for the use of cell quantification kit. av, fm, nk, jd, sg and fv participated in the successful conduct of experiments, construction of the methodology and the reading of the manuscript. pb conducted all experiments. all authors have read and approve of the final version of the manuscript. the authors have declared that no competing interests exist. not applicable. this is a complementary diagnostic study conducted on residual clinical specimens, in french law, the right to use the end of the samples is written in the code of public health : code de la santé publique -article l - . this provision permits to work on the remaining clinical specimen sampled for diagnostic test (in our study, samples were upper respiratory specimen). it was possible to perform an additional analysis (in our study, it is the cell quantification of sampling) unless the patient, previously informed, does not express its refusal. the approval of an ethics committee was therefore not necessary for this kind of study. • we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -z bbz b authors: lee, seung hun; shin, na-ri; kim, choon-mee; park, sungdo; yun, na ra; kim, dong-min; jung, dong sik title: first identification of anaplasma phagocytophilum in both a biting tick ixodes nipponensis and a patient in korea: a case report date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: z bbz b background: human granulocytic anaplasmosis (hga) is a tick-borne infectious disease caused by anaplasma phagocytophilum. to date, there have been no reported cases of a. phagocytophilum infection found in both the biting tick and the patient following a tick bite. case presentation: an -year-old woman presented with fever following a tick bite, with the tick still intact on her body. the patient was diagnosed with hga. the tick was identified as ixodes nipponensis by morphological and molecular biological detection methods targeting the s rrna gene. the patient’s blood was cultured after inoculation into the human promyelocytic leukemia cell line hl- . a. phagocytophilum growth was confirmed via culture and isolation. a. phagocytophilum was identified in both the tick and the patient’s blood by anaplasma-specific groel- and anka-based nested polymerase chain reaction followed by sequencing. moreover, a four-fold elevation in antibodies was observed in the patient’s blood. conclusion: we report a case of a patient diagnosed with hga following admission for fever due to a tick bite. a. phagocytophilum was identified in both the tick and the patient, and a. phagocytophilum was successfully cultured. the present study suggests the need to investigate the possible incrimination of i. nipponensis as a vector for hga in korea. supplementary information: supplementary information accompanies this paper at . /s - - - . human granulocytic anaplasmosis (hga) is a tick-borne infectious disease caused by anaplasma phagocytophilum, an obligate intracellular bacterium, which grows in membrane-bound vacuoles of humans and animals [ ] . the annual incidence of this disease was reported to be . cases per million person-years between and in the united states [ ] . ixodes scapularis, a vector for lyme disease or babesiosis, is known to be the primary vector of hga in the united states, whereas ixodes pacificus (western black-legged tick) and ixodes ricinus (castor bean tick) are the presumed vectors across the western united states and europe, respectively [ , ] . in korea, following the first report of anaplasmosis in , hga has been described as an emerging infectious disease [ ] . however, to date, no studies have investigated the vectors of hga in korea. we therefore performed molecular detection and isolation of a. phagocytophilum from the blood of a patient who presented with fever after a tick bite, with the tick still attached to the body. case an -year-old woman was hospitalized with a chief complaint of fever. she developed fever, headache, and vomiting days prior to admission and was treated conservatively at a local hospital. three days prior to admission, her fever symptoms recurred and were accompanied by several vomiting episodes, abdominal pain, and shortness of breath. consequently, the patient visited the emergency room of a local hospital and was misdiagnosed with cholecystitis, based on the results of abdominal computed tomography, and tenderness in her right upper quadrant on examination. she underwent laparoscopic cholecystectomy the next day and received antibiotic therapy; however, her fever persisted. her guardian identified a mass-like lesion on the right side of her neck where an adhesive patch had been applied and notified the medical staff. upon confirming the presence of a tick, the tick was removed (may ). a single dose of mg doxycycline was administered, and the patient (along with the tick) ( fig. a ) was transferred to the chosun university hospital in gwangju city, korea (fig. a) . on admission (may ), physical examination indicated that the patient was febrile, with a blood pressure of / mmhg and body temperature of . °c. she looked acutely ill on inspection, and her pulse and respiratory rates were beats/min and breaths/min, respectively. skin examination confirmed a tick bite lesion on the right side of the neck (fig. ) . laboratory investigations further revealed the following findings: white blood cell count, , / fig. gross findings of the tick removed from the right neck area of the patient. dorsal view (a) and ventral view (b) of the tick. identification of ixodes nipponensis from the phylogenetic tree analysis based on the s rrna gene of the tick of -bp amplicons produced from the conventional polymerase chain reaction (c) mm ( . % polymorphonuclear leukocytes); hemoglobin level, . g/dl; platelet count, , /mm ; aspartate aminotransferase level, . u/l; alanine aminotransferase level, . u/l; and creatine phosphokinase level, u/l. the patient indicated that she was a resident of a rural area and lived close to a cattle farm with approximately cows; the farm was separated from her house by only a single wall. the patient had a vegetable garden at her home, where she spent a considerable amount of time working almost every day both in the morning and evening. during the medical history interview on admission, the patient reported that she first noted discomfort in her right side of neck on april , , which was prior to the initial appearance of symptoms. when her husband was asked to inspect the area, he mentioned that it appeared to be a wart. at that time, she developed a papular lesion, accompanied by itching and persistent discomfort, for which she began applying adhesive patches on may , . we suspected that the patient was bitten by the tick at least or days prior to showing the site to her husband on april . a. phagocytophilum was identified from the patient specimen through polymerase chain reaction (pcr). subsequently, the patient was treated with mg doxycycline twice daily for days. the patient showed improvement in the c-reactive protein level and pancytopenia, normalization of liver function test results, and symptom improvement with treatment. she was discharged accordingly. the developmental stage and species of the tick were morphologically identified by microscopy, using standard taxonomic keys (https://shire.science.uq.edu.au/parasites/arachnids/ticks/ticks-identification.php). after morphological identification, the tick was washed in % ethanol for - times, washed again in sterilized distilled water for times, and dried by placing on a sterile filter paper. the tick was then placed in the mk hard tissue grinding tube (bertin technologies, rockville, md, usa), μl of phosphate-buffered saline ( % fetal bovine serum, % penicillin/streptomycin) was added, and ground by using fastprep®- classic instrument (mp biomedicals, solon, oh, usa). the genomic dna of the tick was then extracted, using a g-spin total dna extraction kit (intron biotechnology, seongnam, korea), following the manufacturer's protocol. total rna was isolated using the viral gene-spin™ viral dna/rna extraction kit (intron biotechnology, seongnam, korea). for molecular biological identification of the tick, genomic dna was subjected to the mitochondrial s rrna gene-targeted pcr assay [ ] . pcr detection for vector-borne infectious disease pathogens pcr was performed on the tick lysate and the patient's blood to detect for vector-borne infectious pathogens. dna was extracted from the patient's initial blood specimen and from the tick, using the qiaamp blood and tissue mini kit (qiagen, hilden, germany), following the manufacturer's instructions [ ] . anaplasma-specific ankaand groel-based nested pcrs (n-pcrs) were performed; the primers used for groel were hs /hs (for the first pcr) and hs /hs (for the npcr), while those for anka were ank-f /ank-r (for the first pcr) and ank-f /ank-r (for the npcr) [ , ] . to detect rickettsia spp., rickettsia-specific ompa fragment was amplified using primers r . f/rr . r (for the first pcr) and r . f /rr . r (for the n-pcr) [ , ] . for the detection of borrelia spp., pyrg n-pcr was performed by amplifying the ctp synthase genes, using primers pyrg- f/pyrg- r (for the first pcr) and pyrg- f/pyrg- r (for the n-pcr) [ ] . the gene encoding the orientia tsutsugamushi -kda antigen was amplified using the designed primers bo- f/ bo- r (for the first pcr) and bo- f/ bo- r (for the n-pcr). to assess for severe fever with thrombocytopenia syndrome virus rna, we performed reverse transcription pcr, as previously described [ ] . the genomic dnas of a. phagocytophilum kz_a , rickettsia conorii, borrelia burgdorferi, and the o. tsutsugamushi karp strain served as positive controls for anaplasma-specific, rickettsia-specific, borrelia-specific and o. tsutsugamushi-specific targets, respectively. in each pcr run, the reaction mixture without template dna served as the negative control. all primer sequences and pcr cycling conditions are shown in table . positive pcr products from the patient's blood and the tick were sequenced and aligned to the genbank database to identify revealed bacterial agents at the species level and to compare our sequences with published sequences using blast analysis and clustalw alignment (www.clustal.org). dna sequences were identified and analyzed using dnastar lasergene v (dnastar, madison, wi, usa). a phylogenetic tree was constructed by the neighbor-joining method using clustalx version . (www.clustal.org/), based on sequences of the amplified anaplasma-specific groel and anka gene fragments from the specimen and from genbank. a bootstrap analysis was performed using replicates to improve the confidence level of the phylogenetic tree. a thick peripheral blood smear was examined for the presence of a. phagocytophilum in the neutrophils. a drop of the patient's blood was smeared onto a glass slide, air-dried, and stained with diff-quik solution (sysmex corporation, kobe, japan), following the standard procedure. the smear was observed under a microscope at a magnification of x. for a. phagocytophilum culture and isolation, the human promyelocytic leukemia cell line hl- (kclb- ) was maintained in rpmi medium (gibco, thermo fisher scientific, ma usa), supplemented with % fetal bovine serum (gibco, thermo fisher scientific, ma usa) and mm l-glutamine at °c and under % co conditions. the buffy coat or tick lysate solution was inoculated into hl- cells. infected cells were cultured under the same conditions as above, with regular medium changes (cell density of - × cells/ml). they were then stained with diff-quik at - -day intervals and were subsequently cytocentrifuged for microscopic examination. an indirect immunofluorescence assay (ifa) was performed, following the standard procedure and/or manufacturer's instructions (fuller laboratories, fullerton, ca, usa) [ ] . in-house ifa commenced with antigen fixation of the heavily-infected hl- cells onto tefloncoated slides with acetone. an anti-a. phagocytophilum serum (fuller laboratories, fullerton, ca, usa) was then added to the antigen slides, and the slides were incubated in a humidity chamber at °c for min. dylight -labeled goat anti-human igg or igm (fuller laboratories, fullerton, ca, usa) was used as a secondary antibody under the same conditions. the antigen slides were counterstained with . % evans blue and mounted for fluorescence microscopy. manufacturer's slides containing infected hl- cells (fuller laboratories, fullerton, ca, usa) and the human serum were used as positive and negative controls, respectively. for the serological diagnosis of scrub typhus, murine typhus, and lyme disease, igm and igg antibodies against the standard o. tsutsugamushi antigen (gilliam, karp, kato, and boryong strains), rickettsia typhi, and b. burgdorferi were assessed using in-house ifa [ ] . to investigate a. phagocytophilum infection, the cytoplasmic area of occupied vacuoles was explored by electron microscopy at the ultrastructural level. the cultured cells were stained with diff-quik at - -day intervals, cytocentrifuged to confirm the infection level, and then examined, using scanning and transmission electron microscopy (sem and tem, respectively). morphological analysis confirmed that the tick was a female adult i. nipponensis (fig. a, b) . this was further confirmed by molecular identification and phylogenetic tree analysis of the -bp s rrna amplicons produced using conventional pcr (c-pcr) followed by sequencing (fig. c) . on initial peripheral blood smear analysis, the wright-giemsa-stain showed findings suggestive of morulae (fig. b) , whereas diff-quik confirmed this by revealing intracytoplasmic inclusion bodies in the neutrophils (fig. c, d) . the n-pcr was performed on the patient's buffy coat and the tick by using the -bp amplicons of a. phagocytophilum-specific primers targeting groel. sequence alignment indicated % homology between the patient and the tick (ncbi accession no. mh , mh ), which in turn shared % homology with a. phagocytophilum isolates d-se- (accession no. ku ) and s-dd- (accession no. ku ), previously identified in dogs and cats, respectively, in korea. sequences were cut to a size of -bp for the phylogenetic tree analysis (fig. a) and were shown to form clusters with a. phagocytophilum strain d-se- (accession no. ku ), identified in dogs in korea, anaplasma spp. (accession no. jq ), identified in a japanese tick, and a. phagocytophilum strain kh-ip (accession no. hm ), identified in a russian tick ( bootstraps). in terms of the anka gene sequence, sequence alignment indicated % homology between the patient and the tick (ncbi accession no. mh , mh ), which also shared % homology with a. phagocytophilum isolates kza (accession no. kt ) and gw (accession no. kj ). sequences were cut to -bp for the phylogenetic tree analysis (fig. b) results and confirmed the presence of the same a. phagocytophilum bacterial strain identified in the patient's blood (named a. phagocytophilum kz_a ). the infection stages adhesion, replication, invasion, and release of isolated a. phagocytophilum kz_a were visualized within hl- cells using tem (fig. a) . reticulate cells were characterized by dispersed nucleoids (fig. b) , a smoother outer membrane than that observed with densecored cells (fig. c) , and pleomorphism ( fig. a-d) . infected cells were further examined by sem at the ultrastructure level (fig. e, f) , which showed the replication of a. phagocytophilum kz_a within large vesicles of the cell, with a grape-like cluster appearance. ruptured vesicles were also observed, revealing a cluster of a. phagocytophilum (fig. f) . infectious dense-cored cells were predominantly coccoid bacteria at the late stages of infection (fig. e, f) . a. phagocytophilum culture with tick lysates hl- cell culture at days post-inoculation with anaplasma spp. isolated from the tick lysate showed unexpected microbial growth, which was later observed in other cell culture assays (supplementary figure ) . the intracellular morphology was distinct from that of anaplasma spp., and was similar to that of rickettsia spp. with diff-quik staining, the unexpected bacteria replicated faster than anaplasma spp.; however, anaplasma spp. was detected in infected cells using inhouse ifa, and fluorescent bacteria were observed following incubation with an a. phagocytophilum antiserum. this prompted a re-evaluation of the infection by this agent (supplementary figure ) . pcr with rickettsia genus-specific primers of -bp amplicons targeting the outer membrane protein a (ompa) was performed on the tick. sequence alignment indicated a % identity to the r. monacensis strain mt (accession no. jx ) isolated from a korean tick. we confirmed that the unexpected intracellular bacteria, detected during tick lysate culturing, were r. monacensis. although anaplasma spp. was detected during tick lysate culture, its isolation was unsuccessful, possibly due to coinfection. o. tsutsugamushi, r. typhi, and borrelia spp. were not found in the tick specimen. a -fold or greater rise in titer between acute and convalescent sera represented evidence for infection. ifa results of the patient serum showed that both igm and igg antibodies to a. phagocytophilum were negative at the time of admission ( days after symptom onset). the levels of igg ( : ) and igm ( : ) antibodies increased days after symptoms onset; similarly, the levels of antibodies to o. tsutsugamushi and r. typhi were also elevated. however, when the patient's admission blood specimen was used for ompa-targeted pcr to detect r. typhi and r. monacensis and for pcr to detect o. tsutsugamushi and borrelia spp., all results were negative ( table ). a. phagocytophilum is transmitted by i. scapularis in new england and the north-central united states, i. pacificus in the western united states, i. ricinus in europe, and i. persulcatus in asia [ ] . in japan, which is in close proximity to korea, a. phagocytophilum has been identified in i. persulcatus and i. ovatus and was first reported in [ ] . furthermore, . % of i. persulcatus isolated from northern china was reported to have tested positive for a. phagocytophilum [ ] . kim et al. [ ] reported that among ticks ( haemaphysalis longicornis, i. persulcatus, and i. turdus) collected from korean provinces, h. longicornis and i. persulcatus were confirmed to be positive for a. phagocytophilum, whereas i. persulcatus tested positive for ehrlichia chaffeensis. in a molecular detection study of anaplasma spp. infection rates in ticks collected from migratory birds on hongdo island, korea, a. phagocytophilum was detected in only i. nipponensis nymph, among a total of ticks ( haemaphysalis flava, h. longicornis, i. turdus, i. nipponensis, and i. ornithophilia) [ ] . thus, it is speculated that h. longicornis, i. persulcatus, and i. nipponensis may act as anaplasmosis vectors in korea. a. phagocytophilum has previously been identified in three tick species (h. longicornis, i. persulcatus, and i. nipponensis) in korea, but no direct transmission from ticks to humans has been confirmed. since the first report on the serological and molecular detection of hga in korea in , numerous studies on a. phagocytophilum vectors have been performed [ ] however, there are no reported cases that have characterized the species of the biting tick or that have detected the presence of an identical a. phagocytophilum strain in both the biting tick and the patient, particularly with a tick that was intact on the patient's body. in the present study, the identical a. phagocytophilum strain was identified in not only the patient who was diagnosed with hga following admission for fever symptoms after a tick bite but also in the tick itself. moreover, a. phagocytophilum was successfully cultured from both the patient's blood and the biting tick, follow-up antibody test results of the tick dna sample showed elevated antibodies not only for a. phagocytophilum but also for o. tsutsugamushi, r. typhi, b. burgdorferi and r. monacensis monacensis. therefore, the possibility of coinfection or sequential infection could not be dismissed. in our previous study on patients with scrub typhus, . % showed elevated igm antibodies ( : ) within month of symptom onset, whereas . % continued to test positive for igm antibodies at the -month follow-up test [ ] . a recent infection would have correlated with elevated levels of igm antibodies; however, this was not observed in the present case. moreover, all pcr results on the patient's blood and the tick were negative for o. tsutsugamushi, r. typhi, and b. burgdorferi. thus, we believed that the probability of a past infection, sequential infection, or antibody cross-reactivity may be higher than that of a coinfection. similarly, the cross-reactivity between e. canis and a. phagocytophilum has been reported previously in experimental animal [ ] . however, additional studies are needed specifically to clarify the cross-reactivity between anaplasmosis spp. and b. burgdorferi. the studies showed that the full engorgement of ticks require to days of attachment on the host before shedding off to continue with their life cycle. however, it has also been suggested that some ticks may require days or longer [ ] . nonetheless, the literature has shown that the incubation period for human anaplasmosis ranges from to days [ ] . a limitation of this study was that the analyzed tick specimens were completely engorged by the blood of the infected woman, and the positivity to a. phagocytophilum observed in the dna of the tick specimen could have come from the patient's blood and not from the salivary glands of the tick. therefore, we could not confirm that i. nipponensis is a vector for a. phagocytophilum. furthermore, since the patient came from an area with a high risk of tick bites, we could not eliminate the likelihood of undetectable tick bites at other sites of her body. in conclusion, we presented a case involving a patient diagnosed with hga following admission for fever due to a tick bite. an identical a. phagocytophilum strain was identified in both the patient and the tick, and a. phagocytophilum was successfully cultured from the patient's blood. nevertheless, further studies are warranted to determine whether i. nipponensis can act as a possible vector for anaplasmosis in korea. case report: polymerase chain reaction testing of tick bite site samples for the diagnosis of human granulocytic anaplasmosis human granulocytic anaplasmosis in the united states from to : a summary of national surveillance data human granulocytic ehrlichiosis anaplasma phagocytophilum in questing ixodes ricinus ticks in southwestern finland human granulocytic anaplasmosis, south korea discrimination between haemaphysalis longicornis and h. qinghaiensis based on the partial s rdna and the second internal transcribed spacer (its- ) clinical usefulness of eschar polymerase chain reaction for the diagnosis of scrub typhus: a prospective study isolation and propagation of the ap-variant strain of anaplasma phagocytophilum in a tick cell line pcr amplification and comparison of nucleotide sequences from the groesl heat shock operon of ehrlichia species genotypic identification of rickettsiae and estimation of intraspecies sequence divergence for portions of two rickettsial genes severe fever with thrombocytopenia syndrome virus in ticks collected from humans, south korea differentiation of spotted fever group rickettsiae by sequencing and analysis of restriction fragment length polymorphism of pcr-amplified dna of the gene encoding the protein rompa improved culture conditions for the growth and detection of borrelia from human serum serological response of patients suffering from primary and recrudescent typhus: comparison of complement fixation reaction, weil-felix test, microimmunofluorescence, and immunoblotting adaptation of a microimmunofluorescence test to the study of human rickettsia tsutsugamuskh antibody human ehrlichiosis and anaplasmosis anaplasma phagocytophilum-infected ticks prevalence of anaplasma phagocytophila and borrelia burgdorferi in ixodes persulcatus ticks from northeastern china identification of ehrlichia chaffeensis, anaplasma phagocytophilum, and a. bovis in haemaphysalis longicornis and ixodes persulcatus ticks from korea molecular detection of anaplasma, bartonella, and borrelia species in ticks collected from migratory birds from hong-do island serologic and molecular detection of ehrlichia chaffeensis and anaplasma phagocytophila (human granulocytic ehrlichiosis agent) in korean patients a serosurvey of orientia tsutsugamushi from patients with scrub typhus serological detection of anaplasma phagocytophilum, borrelia burgdorferi sensu lato and ehrlichia canis antibodies and dirofilaria immitis antigen in a countrywide survey in dogs in poland guidelines for the diagnosis of tick-borne bacterial diseases in europe publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank our collaborator kim jee-woong, from the division of evaluation and control, for performing the electron microscope analysis. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . the same description has been provided as a word file, as per the journal requirements. the datasets analyzed during the current study are available at national center for biotechnology information (ncbi) repository. (accession numbers; mh , mh , mh , mh , ku , ku , jq , hm , kt , kj , kt , kj , jx ). the study was approved by the ethics in human research committee of chosun university hospital (irb no. - - - ). the patient provided written informed consent to participate in the study. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor of this journal upon request. the authors do not have any commercial interests or other associations, which might pose a conflict of interest. key: cord- -jtvf r authors: liao, qiuyan; cowling, benjamin j; lam, wendy wt; ng, diane mw; fielding, richard title: anxiety, worry and cognitive risk estimate in relation to protective behaviors during the influenza a/h n pandemic in hong kong: ten cross-sectional surveys date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: jtvf r background: few studies have investigated associations between psychological and behavioral indices throughout a major epidemic. this study was aimed to compare the strength of associations between different cognitive and affective measures of risk and self-reported protective behaviors in a series of ten cross-sectional surveys conducted throughout the first wave of influenza a/h n pandemic. methods: all surveys were conducted using questionnaire-based telephone interviews, with random digit dialing to recruit adults from the general population. measures of anxiety and worry (affective) and perceived risk (cognitive) regarding a/h n were made in serial surveys. multivariate logistic regression models were used to estimate the cognitive/affective-behavioral associations in each survey while multilevel logistic models were conducted to estimate the average effects of each cognitive/affective measure on adoption of protective behaviors throughout the ten surveys. results: excepting state anxiety, other affective measures including “anticipated worry”, “experienced worry” and “current worry” specific to a/h n risk were consistently and strongly associated with adoption of protective behaviors across different survey periods. however, the cognitive-behavioral associations were weaker and inconsistent across the ten surveys. perceived a/h n severity relative to sars had stronger associations with adoption of protective behaviors in the late epidemic periods than in the early epidemic periods. conclusion: risk-specific worries appear to be significantly associated with the adoption of protective behaviors at different epidemic stages, whereas cognitive measures may become more important in understanding people’s behavioral responses later in epidemics. future epidemic-related psycho-behavioral research should include more affective-loaded measures of risk. understanding relationships between psychological state and protective behaviors during respiratory infectious disease epidemics (rides) can inform risk communication and interventions addressing behavior change [ , ] . studies of behavioral change during rides usually assess risk perception as an affect-neutral cognitive ("cognitive") process, commonly using measures such as perceived personal probability of infection or perceived severity of the illness [ ] [ ] [ ] , or as a more affect-active process, by assessing worry and anxiety [ ] [ ] [ ] [ ] [ ] . the latter are frequently referred to as "affective" dimensions of risk, though worry is often considered a cognitive dimension of anxiety [ ] . the dual-process theory proposes that responses to external stimuli involve two different processing systems, one being deliberate, slow and rule-based, the other being experiential, quick and intuitive [ ] . these two systems may reflect distinct response pathways to risk: risk-asanalysis (cognitive estimates) and risk-as-feeling (affective estimates) [ ] . the affect heuristics and risk-as-feeling hypotheses imply that affect quickly and more efficiently guides cognitive risk analysis and behavior [ ] [ ] [ ] . previous studies found that in the ride situation when personal threat is highly uncertain, affective measures of risk more powerfully predict protective behavior uptake than do cognitive measures [ , ] . therefore, both cognitive and affective components of risk appear to be relevant to understanding rides-related population behavior [ ] . in the early epidemic stage when uncertainty about the epidemic characteristics, treatment and prevention is higher, affective responses may be optimal for guiding behavioral change [ , , ] but cognitive risk responses should increasingly drive behavior as the epidemic evolves. we term these "psycho-behavioural" associations. given this, the question arises: should studies or assessments done early in the epidemic emphasize affect-based assessments of risk, whereas those performed later in the epidemic emphasize cognitive-based measures, in order to optimally predict behaviors? otherwise, it is possible that research conducted in different stages of an epidemic may observe different strengths for the same psycho-behavioral association and misattribute these. observed variation in the strength of specific psycho-behavioral associations across an epidemic introduces avoidable measurement error in the target cognitive/affective measure which will subsequently influence its association with behavioral change, reducing the apparent reliability of risk assessments as predictors of behavior change during rides. this raises questions about whether the same or similar associations would be repeatedly identified in surveys conducted in different epidemic periods within the same population. however, very few studies appear to have examined the consistency of these psycho-behavioral associations across different ride stages [ ] . we therefore performed secondary data analyses for data collected in a series of ten consecutive cross-sectional surveys spanning the epidemic wave of pandemic influenza a/h n in hong kong [ ] . the objectives of this study were to compare the strength and stability of associations between affective and cognitive measures of risk and the adoption of ride-related health protective behaviors. this was assessed by comparing the associations between health protective behaviors against a/h n and different cognitive/affective measures of risk used for each of the ten cross-sectional surveys. most psycho-behavioral studies of new communicable respiratory disease outbreaks were rapidly implemented [ ] . consequently, many used unrefined questionnaires, with several suffering from minimal theoretical support for the inclusion of specific psychological variables, items of limited utility in understanding behavioral change or items that may have posed task difficulty for respondents [ , ] , and multiple items, which increase interview load, thereby reducing interview efficiency and the accuracy of results. to inform future item selection, we therefore also sought to assess the difficulty respondents faced in answering different question measures of risk perception. this was done by examining proportions of missing data for different psychological measures as an indirect reflection of task difficulty. our null hypotheses were: . cognitive and affective measures of risk will not differ in terms of stability of association with adoption of protective behaviors across the ten cross-sectional surveys; . for the same associations measured at different epidemic periods, strength of associations between affective/cognitive measures and adoption of health protective behaviors will not decline/increase across epidemic stage; . there will be no difference in proportions of missing data for cognitive estimation items such as estimates of the likelihood of contracting influenza infection than other risk assessment formats reflecting no differences in the difficulties posed to respondents by such items [ , ] . between april and november , we monitored population psycho-behavioural responses to the influenza a/h n pandemic using cross-sectional surveys (s -s ) covering the entire first wave of the a/h n pandemic in hong kong [ ] . during the survey period, approximately % of the hong kong population were infected with this new virus [ ] . here we report data from of these (s -s and s -s ). the first two surveys (s and s ) conducted between april and may were excluded from this study because of incompatibility with later surveys and because the local a/h n epidemic did not start until s was conducted. survey s was excluded because of sample insufficiency. all surveys utilized identical methods involving random household telephone interviews based on randomly computergenerated landline telephone numbers of all hong kong households. one adult aged or above within each household was randomly selected based on a kish grid and invited for the telephone interview. sampling details have been published elsewhere [ ] . the study received ethical approval from the institutional review board (irb) of the university of hong kong. the irb waived written informed consent in lieu of verbal consent given the format of these ten telephone surveys. all participating respondents gave verbal consent for telephone interviews. the sample sizes for each of the ten surveys (s -s , s -s ) ranged between , - , , with response rates of . %- . % [ ] . surveys were conducted every two weeks with data collection completed within - days for each survey. the ten surveys covered different a/h n epidemic stages in hong kong. specifically, s (jun - , ) was conducted when local a/h n human cases were first identified in hong kong; s (sep [ ] [ ] [ ] [ ] ) was conducted when the local epidemic reached peak activity and s (nov - , ) when epidemic activity had declined substantially (additional file : figure s ). core items for the questionnaires used in the ten surveys were retained throughout. minor changes were made on one measure of risk perception (the comparator "perceived relativesusceptibility relative to others" was made more precise by specifying age and gender at s ) and two new items (current worry and infectivity relative to seasonal flu) were added in later surveys to refine measurement and during the surveys. table details psychological measures associated with risk covered by different survey periods. four measures (state anxiety, anticipated worry, experienced worry and current worry about a/h n infection) were classified as affective measures. four other measures (perceived absolute susceptibility and perceived relative susceptibility to a/h n infection, perceived a/ h n severity relative to sars and perceived a/h n infectivity relative to seasonal influenza) were classified as cognitive estimates of risk. the definitions, questions and response scales for these measures are detailed below and in the additional file : table s . respondents' anxiety level was assessed with a previously validated state-anxiety scale of the state-trait anxiety inventory (stai) wherein respondents' rate their feelings in response to ten general statements [ , ] . positive feeling statements were reversely coded and then the mean scores of the ten items (possible range - ) were calculated for subsequent analyses to overcome the problems of randomly missing items. (additional file : table s ). respondents were asked to rate their worry about possibly developing a/h n symptoms within the next hours (additional file : table s ). hence this measure was prospective. respondents were asked to recall whether they had experienced any worry over the past week about contracting a/h n (additional file : table s ). this measure was retrospective. starting from survey ( table ) respondents were asked about their current level of worry related to a/h n (additional file : table s ). this measure was current. respondents estimated their personal likelihood of contracting a/h n in the coming months throughout the ten surveys (additional file : table s ). this measure was prospective. in the earlier surveys, respondents estimated their personal likelihood of contracting a/h n relative to another table psychological measures and their proportions of missing data throughout the surveys s s s s s s s s s s missing rang% c totally missing% note "√" indicates that the measure was covered in the survey. a the measure of anxiety state included ten items asking about ten general feeling statements and thereby the proportions of missing data for anxiety in the table were the highest proportion of missing data of the item among the ten statement items. b these two items were combined as "perceived relative susceptibility" in the analysis. c the range of missing proportions across the covered surveys. (unspecified) person in the general population. in later surveys (s -s ), this item was slightly changed to personal likelihood of contracting a/h n relative to another person of similar age and sex in the general population (additional file : table s ). perceived a/h n severity relative to sars throughout the selected surveys, respondents estimated the perceived severity of a/h n infection relative to sars (additional file : table s ). starting from survey (table ) , this item was added in the surveys to assess the infectivity rate of a/h n relative to seasonal influenza, serving as an additional measure to assess the perceived severity of a/h n . the frequencies of three protective behaviors against a/h n were polled throughout the ten surveys. these were avoiding crowded places, maintaining good indoor ventilation and disinfecting the household frequently. all three protective measures were recommended by the hong kong government to minimize the transmission of influenza during the epidemic [ ] . respondents were asked whether they had adopted any of these three protective behaviors over the past seven days, and if so, whether the behaviors were adopted for a/h n prevention. these behavioral outcomes were dichotomized as " " (adopted for preventing a/h n ) and " " (not adopted or adopted for reasons other than preventing a/h n ) for subsequent analyses. previous analyses showed trends for psycho-behavioral associations were similar across the responses range on all the above risk measures [ ] . therefore, these responses were dichotomized as either above or below a threshold for subsequent analyses in order to facilitate comparison, and the process detailed in the additional file : table s . first, the proportions of missing data for all psychological measures associated with risk were calculated. then, multiple imputation was used to generate ten values for each missing value, the mean of which was substituted for the missing value. for each survey, one multiple logistic regression model calculated the associations between each of three protective behaviors (avoiding crowded places, maintaining good indoor ventilation and disinfecting household frequently) and each psychological variable (psycho-behavioral association) plus the corresponding % confidence interval. the psychobehavioral association was adjusted for respondents' age, gender, education, marital status and birth place in each logistic regression model because all these demographics are potential confounders of these psycho-behavioral associations [ ] . i (an index of variability) based on q-statistic was calculated to quantify heterogeneity of these psycho-behavioral associations across the ten surveys and to determine the appropriateness of combining the data from ten surveys to calculate averaged effects. i produces values ranging between and %, indicating the percentage of the total variation across surveys due to heterogeneity rather than chance [ ] . values of %, % and % arbitrarily indicate low, medium and high heterogeneity, respectively [ ] . all studied psycho-behavioral associations had either low or low-medium heterogeneity except that the associations between experienced worry and disinfecting the household frequently and between perceived severity relative to sars and disinfecting the household frequently had medium-tohigh heterogeneity across the ten surveys. therefore, random-effect multilevel logistic regression models were used to estimate the pooled effect of each psychobehavioral association across the ten surveys. for these multilevel models, individual responses were specified as the first level while survey periods were specified as the second level. all multilevel logistic regression models were adjusted for age, gender, education, marital status and place of birth. to minimize potential interactions (moderation or mediation) between different psychological measures [ , ] , only one psycho-behavioral association was assessed in each model. all analyses were conducted based on data excluding the small proportions ( . %- . %) of subjects who reported having had influenza-like illness (ili: fever plus cough or sore throat) within the two weeks prior to each survey. p-values < . were considered to be statistically significant. all analyses were conducted using stata software (version . ; stata corp., college station, tx). the ten surveys included a total of , subjects after excluding ( . %) subjects with ili. the effect sizes for differences between the sample characteristics (age, gender, education and place of birth) of each survey and the hong kong population were small, indicating good sample representativeness [ ] . missing data for the risk-related psychological measures table reports the proportions of missing data for each psychological measure and survey. among all the measures, perceived absolute susceptibility and perceived relative susceptibility to a/h n infection had the highest proportions (totally missing . % and . %, respectively) of missing data throughout the surveys, followed by perceived a/h n infectivity relative to seasonal influenza ( . %) and perceived a/h n severity relative to sars ( . %). affective measures generally had few missing data (below %, table ). figures , and show forest plots describing the associations of different risk-related psychological measures with avoiding crowded places (figure ) , maintaining good indoor ventilation ( figure ) and household disinfection (figure ) , respectively, throughout the ten surveys. the patterns of psycho-behavior associations were similar for the three types of health protective behaviors. for each of the three figures, the upper four forest plots illustrate the associations between affective measures and adoption of protective behaviors while the lower four illustrate the associations between cognitive measures and adoption of protective behaviors. averaged effects of different perceptions on adoption of each of the three protective behaviors are indicated by the lower diamond of each forest plot and in table . overall, all risk-related psychological variables were positively and significantly associated with all three heath protective behaviors except for the association between perceived absolute susceptibility and household disinfection ( table ) . figures , and suggest that all affective measures excepting state anxiety are more strongly associated with adoption of protective behaviors than are cognitive measures, these associations being consistently positive and statistically significant across the ten surveys. in particular, current worry and experienced worry had the strongest associations with adoption of protective behaviors among the eight risk-related psychological measures. state anxiety was only significantly associated with avoiding crowds in s and s (figure ) , with maintaining good indoor ventilation in s (figure ) , and with household disinfection in s , s , s and s (figure ). perceived absolute susceptibility was only weakly and significantly associated with avoiding crowds in s and s ( figure ) and maintaining good indoor ventilation in s , s and s (figure ) but not with household disinfection across the ten surveys ( figure ). perceived relative susceptibility seemed to have stronger associations with avoiding crowds and household disinfection than did perceived absolute susceptibility (figures and ) . no change was seen in associations between perceived relative susceptibility compared to another person, and adoption of protective behaviors in s -s when the refined measure of perceived relative susceptibility specified "a general person of similar age and gender". perceived higher a/h n severity relative to sars was more likely to be significantly associated with adoption of protective behaviors in later (s -s ) than earlier surveys, a pattern not found for other cognitive measures (figures , and ) . perceived a/h n infectivity relative to seasonal influenza was generally significantly associated with adoption of health protective behaviors but the associations were relative weaker than the associations between perceived a/h n severity relative to sars and adoption of health protective behaviors (figures , and ). our findings were mostly consistent with those hypothesized and the null hypotheses were largely rejected. the main finding is that affective measures of risk perception generally had stronger associations with reported adoption of health protective behaviors during the a/h n pandemic than did cognitive measures. this finding is consistent with those from other studies conducted during both sars [ ] and pandemic a/h n [ , ] , suggesting that affective components contribute significantly to adoption of protective behaviors in response to the threat during epidemics over and above simpler cognitive risk estimates. while previous studies were mainly conducted in early epidemic periods [ , ] , this study examined affectivebehavioral associations across the entire epidemic wave of a/h n in hong kong and found that the association between affect-loaded risk measures and adoption of protective behaviors were consistently strong and positive across different epidemic periods. studies of the anxiety-behavior association throughout the sars epidemic found consistently significant and positive associations during the early epidemic phase surveys but mostly non-significant associations in late epidemic phase surveys [ ] . the present study did not duplicate this pattern for any of the four affective measures. reported anxiety level was inconsistently associated with adoption of health protective behaviors in these surveys. one possible reason could be that the measure we used assessed general anxiety only rather than anxiety specific for a/h n . furthermore, overall reported state anxiety levels remained quite stable and consistently low throughout the a/h n epidemic [ ] , indicating a floor effect, suggesting that a low level of anxiety has little effect on these behaviors. other affective measures including anticipated worry, experienced worry and current worry generally involve less intense affective components compared with anxiety and thereby are more likely to covary with behavioral change. in particular, our study found that experienced worry and current worry seemed to have stronger associations with adoption of protective behaviors than did anticipated worry. one possible reason could be that the actual affective experience or associated processing may be more strongly associated with behavioral change than its anticipation, which may be subject to forecasting errors [ ] . cognitive risk assessments, in particular perceived susceptibility to a/h n (either absolute or relative susceptibility) had weak associations with adoption of protective behaviors. this suggests that cognitive-behavioral models such as the health belief model [ , ] that rely primarily on purely cognitive estimates of risk to predict behavioral change should perform relatively more poorly at predicting the adoption of protective behaviors during rides. cognitive-behavioral models generally assume rational processing of external information to inform action. however, during rides particularly in the early stages, uncertainty is usually widespread and poses high [ ] or ambiguous personal threat. consequently, people may face difficulties when attempting to quantify the probabilities of their risk of acquiring the infection and the severity of associated disease. whether it is threat ambiguity, task difficulty in determining risk magnitude or a primary affective response that modifies cognition, that leads to affect-related measures dominating remains unclear. this study found that the proportions of missing data for purer cognitive risk perception measures, particularly perceived absolute/relative susceptibility to a/h n were greater than for affect-loaded measures, suggesting that respondents may face greater figure associations between psychological responses and disinfecting household frequently during a/h n pandemic. task difficulties in comprehension and/or responses to such questions under epidemic circumstances. further study is needed to confirm the extent of this effect. perceived relative susceptibility seemed to have stronger associations with adoption of protective behaviors than perceived absolute susceptibility. perceived susceptibility measured in this relative way involves social comparison and accommodates the influences of optimistic bias [ ] and therefore probably involves more cognitive processing. more cognitive processing is associated with greater risk estimates and psychological distress [ ] . this might account for the more substantial associations with behavioral change than seen for simple personal risk estimates. associations between cognitive risk perception measures and protective action were quite inconsistent across the ten selected surveys in this study. previous reviews concluded associations between cognitive risk perception and adoption of protective behaviors during rides were inconsistent [ ] . our evidence suggests a major reason for this inconsistency lies in these studies being conducted in different epidemic stages [ , , ] . our hypothesis was that cognitive factors were more important in changing human behaviors in the later epidemic stage when people had more knowledge and less uncertainty about the threat. this study found that the associations between perceived a/h n severity relative to sars and adoption of each of the three protective behaviors became significantly and consistently positive starting from survey after the a/h n case confirmations had peaked, consistent with our hypothesis. however, this pattern of associations was not found for perceived susceptibility. perceived a/h n infectivity relative to seasonal flu, though not measured before survey s had weaker associations with adoption of health protective behaviors, than did perceived a/h n severity relative to sars in each survey and overall. however, these two measures assessed different aspects of a/h n severity with the former focused on the infectivity rate of a/ h n while the latter may primarily focus on the fatality rate of a/h n . further study is needed to confirm which aspects of disease severity could be more important in motivating behavior change. study limitations include the serial cross-sectional design and thereby reverse-causality remains a possible explanation. nonetheless, it is difficult to think of plausible mechanisms whereby, for example, disinfecting one's home will lead to greater worry regarding infection. alternatively, the associations could be spurious but this is unlikely given the consistent pattern of the associations in separate samples. it therefore seems most likely that the protective behaviors are consequential on the risk perceptions, and not vice versa. examining psychobehavioral associations using longitudinal data during rides is difficult due to their often-rapid evolution and the short lead-time compared to the need to obtain and retain large cohorts for follow-up surveys. conducting a series of consecutive cross-sectional surveys to investigate the psycho-behavioral associations is a better option than using a single cross-sectional survey. there may be concerns about the generalizability of our findings to more severe rides. for example, during the initial phase of the sars epidemic, population state anxiety regarding the epidemic was much higher and thereby had strong association with protective behavioral change [ ] . however, sars was the first of the new wave of rides, and a degree of risk fatigue may have subsequently set in. considering the common situation during rides, we believe that most of the findings in this study could be all data represent odds ratios and their corresponding % confidence intervals (in parentheses). all odds ratios were adjusted by age, gender, education, marital status and birth place. **p< . ; *** p < . . applicable in other rides. finally, because all data were self-reported the results may reflect social desirability bias. this study raises important implications for future respiratory communicable disease-related psycho-behavioral research and public health interventions. first, affective responses improve understanding of behavioral responses throughout different ride periods and must form part of measures in relevant studies. however, intense but nonspecific affect such as generalized state anxiety is probably less useful for understanding public behavioral responses during most epidemics where perceived milder threat fails to arouse such affect. less intense, specific affective responses to a identifiable, if uncertain threat that currently activates or has in the past activated worry may be more likely to show strong and consistent effects on behavioral change across different epidemic periods. second, cognitive risk estimates during the early epidemic stage may be poor at predicting human behavioral change and present task difficulties to respondents. however, cognitive risk estimates may inform individual behavioral change later in the ride epidemic trajectory and should be included in studies conducted during these phases. relative measures of perceived susceptibility appear superior to perceived absolute susceptibility in predicting behavioral change and thereby are preferable where questionnaire brevity is an issue. from a public health perspective, recognizing that the public may not show expected "rational" behaviors during rides is important. therefore, risk probabilities alone are unlikely to be sufficient to motivate protective behaviors. what affective strategies to use to best motivate behavioral change awaits clarification. risk perceptions related to sars and avian influenza: theoretical foundations of current empirical research demographic and 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prospective study worry: a cognitive phenomenon intimately linked to affective, physiological, and interpersonal behavioural processes the self-regulation of health and illness behaviour risk as analysis and risk as feelings: some thoughts about affect, reason, risk, and rationality the affect heuristic risk as feelings community psychological and behavioral responses through the first wave of the influenza a(h n ) pandemic in hong kong risk perceptions: assessment and relationship to influenza vaccination risk perception measures' associations with behavior intentions, affect, and cognition following colon cancer screening messages epidemiological characteristics of (h n ) pandemic influenza based on paired sera from a longitudinal community cohort study state trait anxiety inventory: a test manual/test form center for health protection hong kong government: general guideline: a guide to personal, home and environmental hygiene: keep clean be healthy measuring inconsistency in meta-analyses emotions and preventive health behavior: worry, regret, and influenza vaccination avoidance behaviors and negative psychological responses in the general population in the initial stage of the h n pandemic in hong kong the health belief model and personal health behaviour the health belief model in the prediction of dietary compliance: a field experiment do moderators of the optimistic bias affect personal or target risk estimates? a review of the literature the function of credibility in information processing for risk perception factors associated with uptake of vaccination against pandemic influenza: a systematic review 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 impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong anxiety, worry and cognitive risk estimate in relation to protective behaviors during the influenza a/h n pandemic in hong kong: ten cross-sectional surveys additional file : figure s . the a/h n pandemic curve in hong kong and timeline of the surveys.additional file : table s . questions for measuring anxiety, worry and risk perception in the study and their associated response scales. the authors declare that they have no competing interests.authors' contributions ql participated in the study design, analyzed the data, interpreted the data and drafted the manuscript. bjc supervised the research, contributed to study design, data interpretation and amended the manuscript. wwtl contributed to study design, data interpretation and amended the manuscript. dwmn contributed to questionnaire design, coordinated data collection and amended the manuscript. rf conceived of the study, designed the questionnaire, interpreted data and amended the manuscript. all authors read and approved the final manuscript. key: cord- - x f t authors: lin, feng; muthuraman, kumar; lawley, mark title: an optimal control theory approach to non-pharmaceutical interventions date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: x f t background: non-pharmaceutical interventions (npi) are the first line of defense against pandemic influenza. these interventions dampen virus spread by reducing contact between infected and susceptible persons. because they curtail essential societal activities, they must be applied judiciously. optimal control theory is an approach for modeling and balancing competing objectives such as epidemic spread and npi cost. methods: we apply optimal control on an epidemiologic compartmental model to develop triggers for npi implementation. the objective is to minimize expected person-days lost from influenza related deaths and npi implementations for the model. we perform a multivariate sensitivity analysis based on latin hypercube sampling to study the effects of input parameters on the optimal control policy. additional studies investigated the effects of departures from the modeling assumptions, including exponential terminal time and linear npi implementation cost. results: an optimal policy is derived for the control model using a linear npi implementation cost. linear cost leads to a "bang-bang" policy in which npis are applied at maximum strength when certain state criteria are met. multivariate sensitivity analyses are presented which indicate that npi cost, death rate, and recovery rate are influential in determining the policy structure. further death rate, basic reproductive number and recovery rate are the most influential in determining the expected cumulative death. when applying the npi policy, the cumulative deaths under exponential and gamma terminal times are close, which implies that the outcome of applying the "bang-bang" policy is insensitive to the exponential assumption. quadratic cost leads to a multi-level policy in which npis are applied at varying strength levels, again based on certain state criteria. results indicate that linear cost leads to more costly implementation resulting in fewer deaths. conclusions: the application of optimal control theory can provide valuable insight to developing effective control strategies for pandemic. our findings highlight the importance of establishing a sensitive and timely surveillance system for pandemic preparedness. emerging influenza is threatening the world with the next pandemic [ ] . the current swine flu caused by a novel h n virus has infected a documented , humans, killing , from april to august [ ] . the world health organization (who) declared the outbreak to be a pandemic because of growing worldwide cases [ ] . currently, the severity of the outbreak is moderate as most people recover from infection without the need for medical care [ ] . however, if the virus mutates and achieves the ability to cause severe illness, it will kill more people and overwhelm the health system. vaccination is the most effective means of pandemic mitigation. vaccine production is a complex multi-step process which involves development, manufacturing, and delivery processes and current levels of vaccine production capacity are inadequate. thus many uncertainties exist in every step and effective vaccines are typically available well after the viral strain has emerged [ ] [ ] [ ] [ ] . for instance, vaccines against the h n strain are still under development and will remain in short supply by november [ ] . current stockpiling of antiviral drugs will also be in short supply and their efficiency will be limited once a pandemic occurs [ , ] . public health systems need to be prepared for cases when effective pharmaceutical interventions are unavailable. non-pharmaceutical interventions (npis) are necessary to delay and dampen the pandemic before pharmaceuticals become available [ ] . recommended npis include: ( ) social distancing: school closure, workplace distancing, restricted public gathering and travel; ( ) case containment measures: voluntary case isolation, voluntary quarantine of members of households with ill persons; and ( ) infection control measures: hand hygiene, cough etiquette, and mask/respirator usage [ , ] . npis were implemented during the pandemic and more recently during the severe acute respiratory syndrome (sars) outbreak of . although research on these events confirms the importance of npis, suboptimal triggering during the pandemic rendered npis only moderately effective at reducing mortality [ ] [ ] [ ] . during sars, sheltering and quarantine were found to be effective [ , ] , while border screening was not [ ] . during the current h n outbreak, infection control is recommended to prevent spread of the virus among humans. public health authorities are developing action plans which may request social distancing actions depending on the severity of the outbreak [ ] . mathematical models are often used to study disease spread, with the susceptible-infectious-recovered (sir) model being preferred for diseases spread via droplet and aerosol. the sir model has been used to study pandemic flu [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , seasonal flu [ ] [ ] [ ] , sars [ , [ ] [ ] [ ] , and smallpox [ ] [ ] [ ] [ ] . these papers use sir to simulate the disease outbreak and evaluate the effectiveness of selected control measures under various predefined scenarios. they do not provide optimal controls for initiating implementation, and thus we will not review them here. sir literature most directly relevant to this work includes [ ] [ ] [ ] [ ] [ ] [ ] . these authors use the sir model to study optimal controls, i.e., controls that minimize a prescribed objective function. most show "bangbang" controllers to be optimal (in the sense of minimizing a specified objective function). these policies apply no control until the occurrence of a triggering event and then apply controls at maximum strength. sethi derived optimal closed-form results for isolation and immunization policies [ , ] using an si model. with this model, the population is partitioned into two parts, susceptible and infectious. the control is to either isolate and vaccinate at a maximum rate or do nothing. infectious individuals who recover become susceptible once again, and thus immunity due to infection and subsequent recovery are not considered. clancy [ ] studied the properties of optimal policies for isolation and immunization assuming that all infectious individuals can be immediately isolated and all susceptible individuals can be immediately immunized. the policy takes no action when the number of infectious is below an optimal threshold and immediately isolates and/or immunizes when the number exceeds the threshold. however, they can only obtain optimal policies when the state space is small. morton and wickwire [ ] developed optimal control policies for immunization assuming an infinite pandemic terminal time. however their switching curve derivation has an error in the derivatives (eqs. a and b of [ ] ) and thus their results are unclear. behncke [ ] derived mathematical properties of optimal vaccination programs under the following assumptions: ) the time when vaccine becomes available is known; ) infectious individuals can be immediately and completely isolated; and ) the time horizon of the pandemic is infinite. overall, we think the underlying assumptions of currently published results are questionable. it is not the case that all infectious can be immediately identified and isolated. further, planners do not know when vaccines will become available, and even when available, it is not true that mass prophylaxis is instantaneous. finally, during a pandemic, people will die. the current models do not account for mortality, which could be significant for viral strains such as h n . in this work, we use an expanded sir model to develop triggers for npi implementation to minimize expected person-days lost resulting from influenza related deaths and npi implementation. npi policies are derived for a deterministic control model. results are compared with the most relevant optimal control papers discussed above. multivariate sensitivity analyses based on latin hypercube sampling are performed to investigate the effects of input parameters on the control policy structure and the mean cumulative deaths. additional studies investigate the effects of departures from the modeling assumptions, which include exponential terminal time and linear npi implementation cost. in this section, we formulate an optimal control problem with an expanded epidemic model to compute npi implementation strategy. to understand the following discussion, the reader is referred to figure , which illustrates the compartmental model, and to table , which provides a summary of notation. to construct the model, we make six assumptions: . at any time, t ≥ , the community is composed of s(t) susceptible, i(t) infectious, r(t) recovered, and d(t) deceased individuals. the population is closed, ignoring the demographic turnover or immigration, i.e. s(t) + i(t) + r(t) + d(t) = n. to make the analysis independent of population size, we normalize the model by letting s t . thus, any community can be described by state variable x(t) = (s(t), i(t), r(t), d(t)). . the population is homogeneously mixed and people make contact at random. . people susceptible are able to get infected when they contact infectious people. once infected, they move into the infectious compartment. people infected can either recover at a constant rate, g, or die at a constant rate, τ. people recovered are assumed to be immune within our study horizon. . npi implementation is modeled by the decision variable u(t), where ≤ u(t) ≤ b <b. npi implementation reduces the rate of contact between susceptible and infectious individuals, and thus dampens the infection rate from b to a lower level, bu(t). in this paper, we only consider npis that will disrupt the normal societal functions and result in significant economic impact, such as school closure, work space distancing and voluntary isolation and quarantine. we assume these npis have the same effect in reducing disease spread. . we let person-days lost due to death be unit cost, while c ≤ will be person-days lost due to npi implementation. the cost c is a weight of npi cost relative to death. it measures the loss of productivity (persondays) due to implementing npis. to determine the value of c, the public health officials need to consider many factors, such as culture of the community, perceptions to death, consequences of pandemic and of figure expands the classic susceptible-infectious-recovered (sir) model to capture the mortality. table model notation. provides a summary of notation. x(t) = (s (t), i(t), r(t), d(t)) state that describes the disease status of a community x( ) = (s ( ), i( ), r( ), d( )) initial disease state of a community proportion of the control space hamilton-jacobi-bellman equation the npis, economic constraints, and etc. we assume that cost of npi implementation increases with implementation strength. in particular, we consider two cost structures, a linear structure in which cost is proportional to npi strength, and a quadratic structure in which the change in cost with npi strength is more variable. this paper is focused most extensively on the linear structure. . the optimization horizon t is assumed to be the time when effective vaccine becomes available to the community. although the production of the first doses of vaccine is estimated to take - months once the virus strain can be identified, nobody knows exactly when circulating virus can be identified. along with vaccine development, there are issues regarding manufacturing, delivery, and deployment. to capture the uncertainty in vaccine arrival time, we let t follow an exponential distribution. we can derive a model with a more general vaccine arrival time, but the problem is not currently numerically tractable, that is, algorithms for the general terminal time case present significant challenges and yet to be developed and tested. the exponential assumption ensures we get the discounted value function, which allows for a solution and some insights about the policy. in later sections we tested the model sensitivity to the exponential assumption. the probability density function (pdf) of t is written as f(t, Φ) = Φe -Φt , where t ≥ . because npi implementation disrupts daily activities vital to a productive society, they should only be used when they will be effective. thus, in developing triggers, npi cost must be balanced with npi effectiveness. in this work, we use person-days lost to measure npi cost and effectiveness. npis cause person-day losses by shutting down vital activities, but mitigate person-day losses by reducing mortality. by capturing the dynamic relationship between npi implementation, mortality, and associated person-day losses, our model provides a mechanism for finding an npi triggering scheme that helps minimize total person-days lost during the time period before vaccines become available. the dynamics of the controlled epidemic are given by the non-linear differential equations (dots denote time the total person-days lost for a pandemic under a policy which prescribes an admissible control, u, will be denoted by the value function v (x, u), which is defined as: the objective is to minimize expected person-days lost over the time horizon, t, which is assumed to be the time when effective vaccine becomes available to the community. an admissible control, u*, which minimizes v (x, u) will be called optimal, and the corresponding value function is denoted by: from eqs. ( ) and ( ) we can see that the knowledge of the state variables s(t) and i(t) determines the other two, thus we focus our analysis on the susceptible and infectious compartments. this reduces the system to two dimensions, s and i. thus, we study the control system in the (s, i)-plane, defined as Ω = {(s, i), s, i ≥ , s + i ≤ }. we let (s, i) represent the initial state x( ) = (s( ), i( )), and v (x, u) = v ((s, i); u) be the associated value function, which captures total person-days lost starting in state (s, i) Ω and operating under control u. the expected value of v*(s, i) is calculated by integrating v ((s, i), u)·f(t; Φ) over the range of t: by changing the order of integration, the problem is converted to an infinite horizon discounted problem: based on pontryagin's maximum principle [ ] , which gives necessary conditions for optimal control, we can derive a first-order partial differential equation satisfied by the optimal value function, v*. this is called the hamilton-jacobi-bellman (hjb) equation: eq. ( ) is a linear function of u, thus the optimal control will be "bang-bang" control [ ] , i.e., the candidate control, u*, should satisfy: where the switching curve ψ (s, i) is the coefficient of u in eq. ( ), defined as: the optimal control u* is pushed to its lower/upper bound depending on the sign of its coefficient, the switching curve ψ (s, i), because the hjb equation, eq. ( ), is linear in the control, u. however, if the hjb is nonlinear in u, the optimal control will not be bangbang. an example will be the linear quadratic optimal control problem, where the optimal policy is a linear state feedback [ ] . by the bang-bang nature of candidate u*, the npis should be implemented at the maximum level when ψ (x) < . to understand this, assume the community is in state x a = (s, i) and consider exerting an instantaneous and small control, δ u , which will move the system to state x b = (s + δ u si, i-δ u si) instantaneously. the exertion will cost csδ u person-days lost. if v*(x a )-v* (x b ) exceeds csδ u , then it is cost-effective to implement the npis. hence, indicates that exerting control reduces person-days lost. we keep implementing npis until ψ (x) > . due to the complexity of the eq. ( ), the switching curve, ψ (x), has no closed form. to find ψ (x), we use an algorithm based on dynamic programming [ ] , which requires discretization over time and space. the uniqueness and convergence of the solution is guaranteed by the viscosity solution concept developed in [ ] . the optimal control, u*, is obtained by solving the hjb equation (eq. ( )). figure shows u* for two infection rates, . and . , given a recovery rate of . and a death rate of . (taken from [ ] ). person-days lost from npi implementation, c, is set to . ( % of the cost of a single death). the maximum impact of npis on the infection rate is assumed to be a % reduction. note that the basic reproductive number r without any control is given by figures (a) and (b) indicate when to trigger npi implementation. when the system state falls in region Ω , npis should be implemented at maximum strength; in contrast, npis should not be implemented when the state falls in Ω . for example, in the influenza scenario of figure (a), npis should be implemented when % remains susceptible and % of the population is infected. however, if % remains susceptible and % is infected, it is better not to trigger the npis. we compare our policy against the most relevant optimal isolation policies derived in [ ] . figures (c) and (d) show these isolation policies under the different infection rates. the control either isolates at a maximum rate when the number of infectious exceeds a threshold or does nothing. for a pandemic with b = . and r = . , figure (d) tells us not to act until the percent infectious exceeds %; while figure (b) tells us to implement npis at an earlier stage of the outbreak, for example % susceptible and % infectious. we illustrate cases for which r > , i.e., the uncontrolled infection spreads rather than dying out. figures (a) and (b) compare the epidemic curves with and without npis, starting from a state % susceptible and % infected. according to figures (a) and (b) , the npis should be triggered at this state. in figure (a) , npi implementation not only reduces the total death by %, but also eliminates the peak of the outbreak. overall, npi implementation saves % of the average person-days lost. in figure (b) , where a more severe pandemic is considered, the reduction in total deaths is % and npi implementation reduces and delays the peak of outbreak, which allows additional time for vaccine development. figures (c) and (d) compare the epidemic curves with and without npis starting from states that fall on the control thresholds of [ ] shown in figures (a) and (d) . the proportions of recovered and dead population were set to because they could not be differentiated from infectious people in an si model. it is still best to implement npis at these states but the impact is limited. we also computed control policies for systems assuming quadratic control cost instead of linear cost, i.e., the value function is written as , while the system dynamics still follow eq. ( ). based on pontryagin's maximum principle [ ] , we can derive the hamilton-jacobi-bellman (hjb) equation for the new problem: eq. ( ) is now a quadratic function of u, thus the optimal control will not be "bang-bang" control. the candidate control u* should satisfy: by solving eq. ( ), we obtained the npi policies for systems assuming quadratic control cost shown in figure shows the optimal control policies for two infection rates, . and . , given a recovery rate g = . and a death rate τ = . . (a) presents the optimal npi control for b = . and r = . . (b) presents the optimal npi control for b = . and r = . . (c) presents the optimal isolation policy derived in [ ] for b = . and r = . . (d) presents the optimal isolation policy derived in [ ] for b = . and r = . . influenza characterized by b = . and r = . . figure (a) shows a multi-level policy in which npis are applied at varying strength levels based on certain state criteria. note that "red" indicates > % npi strength, "yellow" indicates % npi strength, and so forth. the level of npi policy decreases from % of maximum npi level to % as system state traverses from the upper to the lower part and from the right corner to the interior region. the contour line at u* ≈ % of maximum impelmentation level is very similar in shape and location to the boundary between Ω and Ω in figure (a). as another demonstration, figures (b) and (b) show the control policies under both control costs with influenza characterized by b = . . the two boundaries between Ω and Ω in figure (b) resemble the contour lines u* ≈ % of maximum impelmentation level in figure (b). the two regions of high level of u* in the upper corner and lower right corner of figure (b) correspond to two control regions Ω in figure (b). in addition, table compares the means of the expected person-days lost per person due to death, d , and control intensity, u *, between the linear and quadratic models. the overall npi strength of the linear model is higher than that of the quadratic model, while the expected person-days lost due to death is lower. the linear model tends to implement npis more intensely and save more lives while having a higher overall cost. a multivariate uncertainty and sensitivity analysis was performed to study the effects of input parameters on the control policy for the linear cost model. this analysis investigated the effects of five inputs (r , g, τ, c, b) on a performance measure, ω, defined as the proportion of the control space to the total state space, i.e., . there is no well-defined performance measure to evaluate the npi policy, especially when the policy is defined in a -dimensional state space. we figure epidemic curves of infectious and dead population with and without npi implementation. figure shows the impact of optimal control on pandemic severity, peak, and total deaths, when npis are triggered at different initial states. (a) compares the epidemic curves with and without npis, starting from a state % susceptible and % infected when b = . . (b) compares the epidemic curves with and without npis, starting from a state % susceptible and % infected when b = . . (c) compares the epidemic curves with and without npis, starting from a state % susceptible and % infected when b = . . (d) compares the epidemic curves with and without npis, starting from a state % susceptible and % infected when b = . . chose ω because it captures the overall intensiveness of npi implementations. in addition, we investigated the effect of these parameters on the outcome of applying the control policy, defined as the mean cumulative death, d t . we simulated the sird system under the optimal policy starting from all state (s , i , r , d ), where s > %, i < %, and d = . the simulation was terminated at a randomly selected exponential terminal time, and we recorded and analyzed the cumulative number of deaths. the mean cumulative death was calculated by taking the average of cumulative deaths over all tested initial states. table summarizes the estimated probability distribution functions (pdfs) of five input parameters, assuming the input parameters are statistically independent. the pdfs of influenza transmission characteristics (r , /g, and /τ) are estimated based on the pandemic [ ] . note that the infection rate b can be written as r (g + τ). the effect of npis, b, was found to reduce the infection rate, b, by up to - % in and in many cases the effect of npis was very limited [ ] ; thus we assume the impact of npi implementation, b, follows uniform( , %). we are not able to find any empirical data on the cost of npi, c. this value is a relative cost, which depends on decision makers' perceptions of saving lives versus maintenance of daily societal functions. in our analysis we let c take the value from to . uniformly, which would imply that the cost of four sessions of maximal npi implementation is equivalent to the cost of one death. we sampled ranges of the parameters times using latin hypercube sampling (lhs) to generate scenarios [ ] [ ] [ ] . then we conducted multivariate uncertainty and sensitivity analysis to determine the uncertainty in the performance measure that was due to the uncertainty in estimating the input parameters. the descriptive statistics for ω and d t are given in table , which lists the mean, variance, median, minimum, and maximum of ω and d t . figure shows the empirical cumulative distribution functions (cdfs) of ω and d t obtained from lhs scenarios and table provides the partial rank correlation coefficients (prccs) for the performance measures and each parameter. the cdf of ω revealed a wide range of estimates due to the uncertainty in estimating the values of the five input parameters. sixty percent of ω estimates are less than . %, with a minimum of % and a maximum of . %. for ω, the prccs are all statistically significant, i.e. p < . . the cost of npi implementation, c, the time when a death occurs after infection, /τ, and the time for an infectious person to recover, /g, are the most statistically influential (|prcc| > . ). an increase in c or /τ corresponds to a decrease in ω; while an increase in the infection period /g corresponds to an increase in ω. the cdf of d t shows that the mean of cumulative death is . %, with a minimum of . % and a maximum of . %. for d t , the prccs are are all statistically significant, i.e. p < . . the most statistically influential inputs are /τ, r , and /g (|prcc| > . ) while the other two parameters are lessinfluential. a decrease in /τ corresponds to an increase in d t ; while an increase in r or /g corresponds to an increase in d t . to test the sensitivity of our control policy to the terminal time assumption, we simulated the disease propagation and studied the outcome of applying our npi policy to settings with an exponential and a gamma terminal time. for each flu scenario specified in the sensitivity analysis, a corresponding npi policy can be obtained. we randomly selected a value from exponential( . ) and a value gamma( , . ) as the vaccine arrival time (or simulation terminal time). the sampling was repeated times for each scenario. then, we simulated the sird system starting from an initial state applying the corresponding npi policy. the simulation was terminated at the sampled vaccine arrival times and the cumulative deaths were recorded. a total of initial states were selected, where s ≥ % and i ≤ %, for a total of , simulations. we studied the difference in cumulative deaths under these two vaccine arrival assumptions. table lists the descriptive statistics of percentage difference in cumulative deaths for the same initial states at two terminal times. overall, the difference in cumulative deaths under exponential and gamma terminal times is small (mean = . %). the distribution of difference in cumulative deaths is left-skewed, with . % of these differences being less than %. there are a few cases where the cumulative deaths differ significantly (≥ %). these cases all started from initial states where only a small table lists the partial rank correlation coefficients (prccs) for the performance measure ω and d t . table lists the summary statistics of difference in cumulative deaths at exponential and gamma terminal time. proportion people are infectious, i.e., i ≤ %, and the difference between the selected gamma and exponential terminal time exceeds % of the maximum of these two. for example, in a case where i = %, s = %, gamma terminal time = days and exponential terminal time = days, the difference in cumulative death is . %. effect of npi policies on the epidemic npis reduce and delay the spread of pandemic by moderating social contact between susceptible and infectious people. because npis disrupt daily societal functions, it is important that they be implemented judiciously. this requires identifying effective initiating triggers, which is a challenging research task. implementing npis will impede influenza spread; on the other hand, normal societal functions will be interrupted. the optimal control method takes both aspects into account and tries to find the best balance between them, given the decision makers relative weighting of the two. based on figures and , early implementation for moderate and severe pandemic is very important for npis to have impact on the outbreak and the impact is effective only if npis are implemented early. late npi implementation might still be optimal, but the impact is much less. for a severe pandemic, it is optimal to trigger npis at the beginning stage when susceptible population is large and infectious population is small. if we miss the beginning stage, it is not optimal to implement npis until the outbreak is significantly progressed. this is because once the pathogen achieves a certain level of infection, npis are not effective against it, and thus are not worth the cost. that is, the benefit of npis at a stage when the disease has progressed significantly is less than the cost of npi implementation. this finding supports the cdc pandemic mitigation guidelines, which state that when the pandemic is category or , all npis are recommended for early implementation [ ] . furthermore, earlier npi implementation reduces and delays the peak of the outbreak as illustrated figures (a) and (b), which allows additional time for vaccine development. if a severe pandemic occurs, hospitals will experience an overwhelming influx of patients and need to operate at their surge capacities. earlier npi implementation can reduce the magnitude of infectious at the peak, which relieves some of the burden on hospitals and other health care infrastructures. in contrast, npis are not nearly as effective if disease has already spread into the community as the cases shown in figures (c) and (d). in both cases, npis are triggered after the peak of the outbreak, where hospitals might have already been operating at their surge capacities for a few weeks. both cases start at states falling on the control thresholds recommended in figures (c) and (d) [ ] . this finding indicates that the additional complexity of our model is warranted when compared with the si model used in [ ] . timely and sensitive surveillance systems are key to successful application of the optimal control method as knowledge of both the pathogen characteristics and the community state are assumed. the surveillance systems should be able to identify the virus quickly and provide accurate estimates for parameters which characterize the severity of an influenza. the effectiveness of the control policy depends on the accuracy of these estimates, which include infection rate b, death rate τ and recovery rate g. once the control policy is computed, we also need to track the community state to determine if npis should be triggered. as early npi implementation is found to be much more effective, we do not want to miss the beginning stage of the outbreak. thus, the surveillance system should also estimate the community state, including the size of the infectious and susceptible populations. our sensitivity analysis identified three important input parameters for determining the overall npi intensity. the important parameters are the npi cost, death rate, and recovery rate. for high npi costs, it is not worthwhile implementing npis because the benefit is less than the cost. for higher death rates, the policy sacrifices daily societal functions to save lives. in contrast, when recovery rate is small, infected people recover more slowly and continue infecting susceptible people. thus, more npi implementation is required. these results suggest that an influenza virus with a high death rate and a small recovery rate requires early intensive npi implementation, particularly when the community places a high value on avoiding death. cumulative death was most affected by the death rate, the basic recovery number and the recovery rate. for higher death rates, a higher proportion of infected people will die. for higher basic reproductive number, more people will be infected, resulting in more deaths even when the death rate is smaller. for lower recovery rate, infected people recover at a slower rate, and thus more people will be infected. this suggests that an influenza virus with a high death rate, a high basic reproductive number and a small recovery rate is less affected by npi implementation. npi cost does not seem to affect the cumulative death. however, npi cost was identified as the most influential (prcc = - . ) in determining the intensity of npi implementation. different communities have different perspectives of death versus disruption of daily societal functions. the range of c should be determined by decision makers after carefully evaluating the demographic, cultural, and economic characteristics of the community. as a performance measure, ω does not capture the complete structure of the control policy. it only captures the overall control intensity, but subtle differences between control policies, such as distribution and shape of Ω in the state space, is missing. policies that have different structure in the state space might have same ω value. therefore, continued effort should be made in selecting more refined measures for the control policy performance. linear v.s. quadratic cost function if the cost function is linear, the control policy is bangbang, which suggests implementing npis at the maximum strength or not implementing at all as shown in figure . if the cost function is nonlinear, for example the quadratic cases presented in figure , the control policy has multiple levels, which requires varying the npi strengths as the system evolves from one state to another. it is easily shown from eqs. and that if the linear model indicates npi implementation for a state x, i.e., u* = b for state x, then the quadratic model also indicates some implementation in state x, i.e., u* > for state x. but the inverse is not true. this property can be proved easily. if u* = b in the linear model, we have ψ(s, i) < , for all (s, i) Ω according to eq. . so together with eq. , we have u* > for these states in the quadratic model. thus, a quadratic cost structure will require npis to be implemented in more states but with much lower intensity in those states. overall, a linear cost structure leads to higher average npi intensity than the quadratic cost. in the examples observed, the boundaries between control and non-control regions in linear model resemble the quadratic contour lines u* ≈ % of maximum implementation. moreover, the linear cost model appears to place more weight on death, thus implementing more control and saving more lives as shown in table . in cases with quadratic cost structure, using a linear model might result in over-control which would not be marginally effective. however, quadratic cost introduces additional complexity in parameter estimation, computation, and policy interpretation. for example, we must determine how to implement npis at x% of maximum. this requires determining which npis will be implemented for each control level. the bang-bang policy, on the other hand, has only two levels, which is easier to understand and implement. finally, it is not clear that a direct comparison between policies obtained under linear and qudratic cost structures is appropriate, because the value functions are defined differently. more research needs to be done to better define the npi levels and interpret the policy if a non-linear cost function is chosen. we are currently not able to derive an optimal control policy for a general terminal time assumption. although the exponential optimal policy will not be optimal for cases with general terminal time, results shown in table indicate that the expected cumulative deaths predicted by the exponential optimal policy will be close to those occurring in a more general terminal time case. this suggests that the impact of the optimal exponential policy on virus spread is not always sensitive to terminal time distribution. although the exponential assumption might not be completely realistic, making this assumption allows us to obtain the npi policy, which then seems to provide desirable impact for cases with nonexponential terminal times. our model was limited in several ways. first, although hjb can be derived for models assuming general terminal time (e.g., gamma), so far the control policy can only be computed assuming exponential terminal time. second, the present modeling framework does not capture uncertainty in parameter estimation, i.e. the model accuracy relies on accurate estimation of input parameters. in practice, collection of accurate data and estimation of input parameters from data can be challenging and time consuming. third, the present modeling framework assumes equal effect of various npis in a homogeneously-mixed population, while different npis will have distinct impacts for disparate population groups. finally, bang-bang control must be further refined since it is not clear that on/off implementation is realistic for larger communities. to better apply optimal control methods in disease control problems, continued efforts should be made to refine the present model and to better estimate the input parameters. to conclude, we have considered a problem of nonpharmaceutical intervention (npi) implementation for pandemic control using optimal control theory to develop triggers that minimize expected person-days lost associated with infection related death and npi implementation over an exponential time horizon. the best control strategy for the model depends on the transmission characteristics of the influenza virus, the state of the pandemic, and the cost and implementation levels of npis. we present the computed policies under different transmission characteristics, where it is optimal to activate all npis when the system state falls in the control region, Ω . the optimal policy can be calculated for any combination of flu and cost parameters. we compare the impacts of npis triggered at different states, which supports the idea of early containment. for comparison, we present the npi policies assuming quadratic control cost. the quadratic cost assumption introduces additional complexity into parameter estimation, computation and policy interpretation, thus more research needs to be done to better define the npi levels and interpret the policy. we perform multivariate sensitivity analysis, which identifies important parameters that affect the intensity of control and the outcome of applying the policy. the findings highlight the importance of establishing a sensitive and timely surveillance system. finally, we study the outcome of applying our npi policy under exponential and gamma terminal times, and find small difference in the cumulative death. many uncertainties exist in estimating flu parameters, future research directions include developing a model that allows using stochastic rather than deterministic inputs and updates the control polices in real time. since npi implementation is not mandatory, compliance to npi requirements is crucial for successful implementation. community engagement, job security, and disruption of daily life affect compliance to npi implementation [ ] . moreover, prolonged outbreak might result in compliance fatigue. thus, in future work, we will integrate time-based compliance models into the system dynamics. other important research directions include consideration of population heterogeneity, stochasticity and partial observability in disease outbreak, and developing methods for general terminal time distributions. world health organization (who): who strategic action plan for pandemic influenza situation updates -pandemic (h n ) current who phase of pandemic alert world now at the start of influenza pandemic will vaccines be available for the next influenza pandemic nonpharmaceutical interventions for pandemic influenza international measures centers for disease control and prevention: intreim pre-pandemic planning guidance: community strategy for pandemic influenza world health organization (who) writing group: non-pharmaceutical interventions: their role in 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epidemic models optimal quarantine programmes for controlling an epidemic spread optimal immunization rules for an epidemic with recovery on the optimal control of a deterministic epidemic optimal control of deterministic epidemics dynamic programming and optimal control athena scientific introduction to optimal control theory springer optimal control and viscosity solutions of hamilton-jacobi-bellman springer what is the best control strategy for multiple infectious disease outbreaks sensitivity and uncertainty analysis of complex models of disease transmission: an hiv model, as an example uncertainty and sensitivity analysis of the basic reproductive rate: tuberculosis as an example modelling an outbreak of an emerging pathogen pre-publication history the pre-publication history for this paper can be accessed here an optimal control theory approach to non-pharmaceutical interventions the authors thank the indiana state department of health for supporting several pandemic planning projects that led to this research. the authors are also grateful to the editors and referees for very insightful and helpful comments. authors' contributions lf conducted the research, including model design, acquisition of data, analysis and interpretation of data, and manuscript drafting. km provided important guidance in model design and methods. he also revised the manuscript critically for important intellectual content. ml supervised the study. he had actively involved in model design and interpretation of data. he also revised the manuscript critically for important intellectual content. all the authors have given approval of the version to be published. the authors declare that they have no competing interests. key: cord- -s gnwb s authors: li, tiegang; feng, jing; qing, pengzhe; fan, xiaomei; liu, weisi; li, meixia; wang, ming title: attitudes, practices and information needs regarding novel influenza a (h n ) among employees of food production and operation in guangzhou, southern china: a cross-sectional study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: s gnwb s background: as of may , human infections with avian influenza a (h n ) had been reported in chinese cities. on may , because a chicken infection with h subtype avian influenza virus was detected in guanzhou, guangzhou became the th city to conduct emergency response operations. the goal of this study was to identify attitudes, practices and information needs among employees of food production and operation in guangzhou. methods: a cross-sectional survey of face-to-face interviews was used during – june . all adults seeking health examination in guangzhou center for disease control and prevention who had lived in guangzhou for at least months, were engaged in food production and operation, and agreed to participate were interviewed. results: of , participants, . % worried about being infected with the a/h n and . % stated that they had searched for information about a/h n . the internet ( . %), television ( . %), and newspapers ( . %) were the main methods of obtaining information; the use of these methods differed significantly by various demographic variables (p < . ). more than one-fifth of participants complained that the information was not timely enough ( . %) and was intentionally concealed by the government ( . %). nearly one-third ( . %) did not believe that the government could control the a/h n epidemic. most participants ( . %) reported washing hands more frequently than before, while over one-third ( . %) stated no longer buying poultry. a total of . % indicated a willingness to receive an a/h n vaccine, and the primary reason for not being willing was concern about safety ( . %). a history of influenza vaccination and worry about being infected with the a/h n were significantly associated with intention to receive an a/h n vaccine (p < . ). conclusions: our findings provide insight into the attitudes and practices of employees of food production and operation months after the first human a/h n case reported in china, and month after infected chickens were identified in guangzhou. distrust in the health department should be addressed, and more effort should be made to improve compliance of proper preventive measures to reduce panic among the public. the information needs should be taken into account in the next step of health education. in early , a novel strain of avian influenza a (h n ) virus was detected in humans in shanghai, eastern china. the virus had never been reported in humans, and the world health organization is taking this novel a/h n seriously. most h n patients have presented with respiratory tract infection with progression to severe pneumonia and breathing difficulties [ ] . as of may, a total of human infections had been reported in chinese cities [ ] , of whom died [ ] . this yields a case fatality rate of . %, which is substantially higher than seasonal influenza viruses, pandemic a/h n virus [ ] , and other subgroups of h influenza a viruses (subtypes h n , h n , and h n ) [ ] in china. although no person-toperson transmission or epidemiologic link between any of the cases has been identified, infection seemed to have involved contact with infected poultry [ ] . the viral isolates from some patients were very similar to those from epidemiologically linked market chickens [ ] . furthermore, detection of more than cases in months compared with roughly human cases of avian influenza a/h n infections in a decade suggests that h n is already more transmissible from poultry to humans than h n [ ] . as the largest trading city of southern china, guangzhou had a large burden of both severe acute respiratory syndromes(sars) in and pandemic influenza a (h n ) (ph n ) in [ ] . although no human infections with the avian influenza a (h n ) were reported in guangzhou, human cases have been identified in jiangxi, hunan, and fujian. these three provinces all border guangzhou, and have frequent population movement and agricultural trade with guangzhou ( figure ). on may , the guangzhou agriculture department announced that chicken samples from a poultry market tested positive for h subtype avian influenza virus. this led to guangzhou being the th city in china to conduct emergency response operations related to novel influenza a (h n ). public cooperation in complying with infection control measures is required to minimize the spread of infectious diseases. previous studies have demonstrated the positive correlation between a willingness to adhere to the recommendations around infection control practices and perceived infectiousness and severity of the disease [ ] [ ] [ ] , perceptions about the effectiveness of control measures [ ] , and trust in the information being provided by national and international public health authorities [ ] . therefore, learning more about the concerns, attitudes, and behaviors of the public during an infectious disease epidemic is crucial to improve communication efforts by public health officials [ ] . because a considerable number of h n patients engaged in food-related work before symptom onset (including chef, food transporter, poultry seller and slaughter), we conducted face-to-face interviews on attitudes, practices and information needs among employees of food production and operation in guangzhou, with an effort to assess the preparedness and response of the public, and to facilitate the development of effective prevention strategies against h n infection. guangzhou is , square kilometers in size, situated at ° ′n - ° ′n and ° ′e - ° ′e. as of the census, the city had over . million registered inhabitants and a floating population (such as migrant workers) of . million. it is the largest trading city in southern china, and is about km north-northwest of hong kong and north-northeast of macau ( figure ). in china, according to the public places health management regulations and implementing rules promulgated by the chinese government, employees of food production and operation must accept a health examination every year, and it is illegal to engage in food-related work without a valid health certificate. there are health examination centers available to the public in guangzhou, and the centrally-located guangzhou center for disease control and prevention (cdc) health examination center is the largest. it is typically the first choice for employees, because people with a health certificate from guangzhou cdc can legally work in all districts of the city. in this study, subjects were recruited by convenience sampling from the guangzhou cdc health examination hall. all adults seeking health examination between - june , who had lived in guangzhou for at least months, engaged in food production and operation, and agreed to participate in the investigation were interviewed face-to-face to complete a three-page questionnaire. interviewers consisted of epidemiologists and dialect interpreters, and spent an average of hours at a randomly chosen time of day to recruit participants. each interview lasted about minutes. all interviewers attended a hour pre-training before conducting interviews. because some questions in this study were about the government's work, the responses might be untruthful if participants knew that this investigation was conducted by guangzhou cdc; therefore, we masked our occupation when explaining the nature of this study to interviewees. pilot surveys were conducted prior to the study, to confirm that participants could understand the survey questions and to ensure the validity of the questionnaire content. using the results of this pilot study, the survey questionnaire was amended to create a final version (additional file ). all questions were either closed-ended or multiple-choice. nine questions were used to assess attitudes toward a/ h n . one item was "do you worry about getting h n ?", and response options for this ranged from = "absolutely not" to = "very worried". six items related to "what do you think about the h n information published by the government?". each of these six items was phrased as a statement, with response options "agree" and "disagree". one item was related to "satisfaction evaluation on measures taken by the government", and response options for this ranged from ="very dissatisfied" to ="very satisfied". one item was "do you believe that government can control the h n epidemic?", and response options for this were "yes", "hard to say", and "no". participants were asked questions about their recent practices. one question was "did you recently search for information about h n ?" with response options of "yes" and "no". if the response was "yes", a supplementary question of "what methods or ways did you use?" was asked. each method/way was phrased as a statement, and response options were "yes" and "no". seven items related to "preventive measures that have been taken after the emergence of h n "; all were phrased as "over the past months, i have … because of h n ", and the response options were "yes" and "no". two items related to vaccines. participants were asked "have you received an influenza vaccine in the past three years?", with response options of "yes" and "no". participants were also asked "if the h n vaccine is available, would you like to receive it". if the response was "no", we asked "why?" as the supplementary question; there were three response options to this supplementary question. participants were asked to report any information needs or concerns if large numbers of human infections with h n cases emerge or a h n pandemic occurs in guangzhou. there were items for participants to respond with "yes" or "no": four related to the epidemic situation, one related to vaccination, three related to preventive measures, three related to drug treatment, and one related to poultry safety. demographic variables consisted of sex, age, household income, educational level, marital status, birthplace, living area, and length of time live in guangzhou. epi info™ . . . , a free statistical software package produced by the u.s. centers for disease control and prevention, was used for data entry, cleaning, and initial analysis. descriptive statistics, such as percentages, means, and % confidence intervals, were calculated. a χ test and/or fisher's exact test were used to compare proportions of different groups. multivariate logistic regression analyses were used to clarify the relationship between different methods of obtaining h n information and demographic variables, and to identify the significant independent predictors of acceptance of a h n vaccine, by calculating odds ratios (or) after controlling for gender, age, and other demographics. these regression analyses were conducted using spss version . (spss inc. ). p < . was considered statistically significant for all analyses. this study was approved by the guangzhou center for disease control and prevention. of , subjects approached, , participants accepted and successfully completed the interview, yielding a response rate of . %. the age ranged from to years old (mean: . years) ( table ). the proportions of participants under age , - , and over age were . %, . %, and . %, respectively. there were more females (n = , . %) than males (n = , . %). the majority of participants ( . %) lived downtown, and more than half ( . %) have lived in guangzhou for more than years. nearly all ( . %) participants had a middle school diploma or higher. half of the participants had an annual household income per capita less than $ , (table ) . at the time of this study, which took place months after the first a/h n human infection was reported and month after the government announced that a chicken in guangzhou market was infected, the majority of participants ( . %) worried about being infected with the a/h n (table ) . when participants were asked about their opinions on h n information published by the government, . % reported "accurate and transparent". however, more than a fifth ( . %) reported "not timely enough", and . % thought "some information was intentionally concealed by the government". with regard to satisfaction on the measures taken by the government, . % of participants chose "satisfied", "more satisfied", or "very satisfied". a total of . % of participants believed that the government could control the h n epidemic. however, nearly one-third ( . %) stated "hard to say" or "no" ( table ) . after the emergence of h n , . % of participants stated that they had searched for information about h n (tables , ). this differed by sex, with . % of males and . % of females (p < . ) reporting searching for information. the most common method of obtaining information was "use internet" ( . %), followed by "watch tv" ( . %), and "read newspaper" ( . %). males, younger participants, and those with a higher education level were more likely to choose "use internet" (p < . ). however, females and older are more likely to choose "watch tv" and "read newspaper" (p < . ). compared with the middle-or high-income groups, the low-income group had a significantly higher (p < . ) proportion of "watch tv" ( . % vs. . %). approximately one-fifth ( . %) of participants chose "ask friends". this differed between sex, with . % of males, and . % of females reporting "inquire friends (p < . ). in addition, the proportion of "ask friends" between those under years old, - years old, and over years old was . %, . %, and . %, respectively. this trend reached statistical significance (trend χ = . , p = . ). a total of . % of participants reported "listen to the radio" to get h n information. this differed among age groups, with . % of those under years, . % of those aged - years, and . % of those over years old reporting "listen to the radio" (trend χ = . , p = . ). moreover, . % of participants chose "consult doctor", which was significantly higher in males than in females ( . % vs. . %). regarding the preventive measures, . % of participants reported washing hands more often than usual, which was significantly higher (p < . ) in females ( . %) than in males ( . %). in addition, a total of . % reported ventilating rooms (by opening windows and doors) more frequently than before. roughly half of the participants ( . %) cancelled or postponed their social events, and this was significantly higher in females than in males ( . % vs. . %). of particular note, over one-third ( . %) of participants reported no longer buying chickens, ducks, geese and other poultry, and this was higher in females than in males ( . % vs. . %) ( tables , ) . regarding vaccination, . % of participants reported that they would accept an a/h n influenza vaccine if it is available. the primary reason for not accepting the new vaccine was "worry about safety" ( . %), followed by "not necessary, i would not be infected with h n " ( . %), and "do not want spend money to immunize, if it is free, i will" ( . %) (figure ). when participants were asked about their information needs if there were an outbreak of h n in guangzhou, the highest proportion of responses was "how to protect my family from infection?" ( . %), followed by "what is the current epidemic situation?"( . %), "is the vaccination available? is it safe?" ( . %), "is there any effective drug treatment?" ( . %), and "how to conduct home disinfection?" ( . %) ( table ) . because different methods of obtaining h n information varied significantly with some of demographic variables (indicated by the chi-square test on univariate analysis; tables , ), we conducted multivariate analyses to determine which independent variables are significant in logistic regression models, with non-significant variables also entered into the models. the results are shown in tables , . in model a, sex did not remain significant on multivariate analysis (p > . ). younger (≤ years) and more education were significantly associated with higher likelihood of using the internet (p < . ). model b indicated that females and those with a higher income had a significantly higher likelihood of watching tv (p < . ), and model c indicated that females had a significantly higher likelihood of reading the newspaper (p < . ). model d indicated that being male and younger (under years) was significantly with higher likelihood of inquiring friends (p < . ), model e indicated that older participants (over years) were significantly more likely to listen to the radio (p < . ), and model f indicated that being male was significantly associated with a higher likelihood of consulting a doctor (p < . ). in addition, because sex and age were both significant in model d, we further conducted an interaction analysis. the interaction term (sex*age) was found to not be significant (p > . ), so we excluded it from the final model. we also conducted multivariate analysis of willingness to accept h n vaccine, with possible predictor variables and demographics (including sex, age group, marital status, education level, living area, annual income, and place of birth) included in the logistic regression model. influenza vaccination within the past years (or = . , p < . ) and worry about being infected with the a/h n (compared to "not worried", or for "worried" = . , or for "more worried" = . , and or for "very worried" = . ; p < . for all three) were significantly associated with willingness to receive an h n vaccine (table ). to our knowledge, this is the first study that focuses on understanding the public's awareness of and attitudes toward influenza a (h n ). in a previous study designed to assess the implications of public understanding of avian influenza, researchers found that the majority of participants did not believe a pandemic would occur, and believed that dealing with the disease was the responsibility of the government [ ] . opinions about the credibility of health information varied from distrust to belief in the credibility of information released by the local health department [ ] . our current study demonstrated that . % of participants worried about being infected with the a/h n . despite the fact that the majority of participants ( . %) thought the h n information published by the government was accurate and transparent, over one-fifth ( . %) complained it was not timely enough, and . % thought some information was intentionally concealed by the government. furthermore, when asked "do you believe that the government can control the h n epidemic?", nearly one-third of participants ( . %) responded with "hard to say". these findings reflect some distrust in the announcements of the health department. this may be because the public did not accept the long interval between the time when cases were identified and the time that information was released to public, as well as the increasing number of cases and infected areas. we found that after the emergence of h n , a majority of participants actively searched for information about h n , and the primary method of obtaining information was using the internet. this result is consistent with other studies that indicated that the internet is increasingly used by the public as the most important source of healthrelated information [ ] . furthermore, our data also indicate that different methods of obtaining information were significantly associated with different demographic variables. for example, younger and more educated participants were significantly more likely to use the internet, while female and higher-income participants were significantly more likely to watch tv. females were also more likely to read a newspaper for information. although few participants ( . %) chose "listen to the radio", older participants were more likely to get information from the radio than younger participants. this suggests that the transmission of heath information should consider the demographic characteristics of the target audience in determining which methods to emphasize. in addition, it is worth noting that among the young participants, over one-fifth chose "ask friends" rather than seeking a more formal information source. this should be addressed in targeting intervention efforts specifically at young people. our study demonstrated that most subjects ( . %) reported washing their hands more often than usual. similar findings were also reported at the beginning of the h n influenza pandemic in hong kong [ ] , and in the united kingdom, % of subjects reported changing their hand washing behavior as a result of h n influenza [ ] . we also found that of participants . % reported ventilating rooms more frequently than before, and nearly half ( . %) cancelled or postponed their social events because of a/h n . when viewed together, the data from these studies implies that preventative health behaviors become more prevalent during infectious disease epidemics. furthermore, our data revealed [ ] [ ] [ ] . therefore, men need special targeting for health education, especially to improve their knowledge of influenza, because knowledge of influenza and perceived effectiveness of personal hygiene practices (phps) has been shown to be associated with phps [ ] . we found that more than one-third of participants stated that after the emergence of h n , they no longer bought chickens or other poultry to eat. a previous study has revealed that perceived theoretical threat from poultry was associated with less buying of live poultry [ ] . we should be aware that the prolonged warning that a future pandemic could be sparked by avian influenza viruses is likely to cause pandemic fatigue in the public, and would probably not change their perception of avian influenza risk and associated protective behavior [ ] . some causal beliefs and lay perceptions of avian influenza contradicted public health efforts at control [ ] . therefore, more effort should be made to improve compliance of proper preventive measures and reduce panic among the public. in addition, because the guangzhou population faces risks from the high prevalence of exposure during purchase and poultry rearing [ ] , better management for raising and selling poultry in guangzhou is needed. the high proportion ( . %) of participants indicating a willingness to receive h n vaccine if it is available bodes well for influenza prevention through vaccination in guangzhou. of those whose response was "no", the primary reason for unwillingness to accept a vaccine was concern about the safety of the new vaccine. a similar finding was also observed during the ph n pandemic in hong kong, which indicated that perceived risk from the ph n vaccine could inhibit ph n vaccine uptake [ ] . these results suggest that some participants lacked an understanding of the process of developing influenza vaccine based on the probability of strains. while we only studied a small subset of the population in guangzhou, if these results were found to be representative, educational materials distributed about the novel influenza vaccine should focus on its safety record, manufacturing, and the similarities between seasonal influenza vaccination and the h n vaccine. these efforts could help to dispel these fears, considering that we found that participants who had received influenza vaccine within recent three years were nearly two times more likely to accept h n vaccine compared with those who had not. although the interviewees responded a relative high willingness to receive h n vaccine, in the past three years, only . % actually received the seasonal influenza vaccine. this implied that the current high willingness of accepting vaccine may be attributed to the high case fatality rate of h n reported, and a high proportion of participants fearing they will be infected. as indicated in this study, worry was found to be the strongest predictor of vaccination uptake. consistent with our finding, liao et al. also reported that perceived low risk from ph n could inhibit ph n vaccine uptake [ ] . that means if the public believes that the severity of a/h n is lower, the acceptance rate may decline. during the early stage of the ph n pandemic (may-june, ), international studies assessing willingness to receive the ph n vaccine indicated rates that ranged from . % [ ] to . % [ ] . however, national data from australia collected in november and december , when the public believed that the pandemic was coming to an end [ ] , showed that there had only been a % uptake of the vaccine [ ] . therefore, combining our finding with previous published literatures suggests that when levels of worry are generally low, acting to increase the volume of mass media and advertising coverage is likely to increase the perceived efficacy of recommended behaviors, which, in turn, is likely to increase their vaccination uptake. we showed that in response to "once h n outbreaks in guangzhou", participants' main concerns included "how to protect my family from infection?", "what is the current epidemic situation?", "is the vaccination available? is it safe?", "is there any effective drug treatment?", and "how to conduct home disinfection?". this is similar to aihua et al., who reported that during the h n influenza pandemic, the public's primary concern was effective and easy-to-operate preventive measures [ ] . therefore, these information needs should be taken into account in future health education campaigns. some limitations of this study must be acknowledged. first, our subjects were employees of food production and operation, and we recognize the limitations of applying the results of this study to the general population. second, this survey measured the participants' views at a specific point in time; therefore, the attitudes and practices reflected the information available at that time. third, some of the questions in our questionnaire had likert-type response options, which restricted the preferences of participants to a few options. a fourth limitation is inherent to the study design: the use of convenience sampling -as opposed to random sampling -imposes some inherent selection bias and diminishes the internal validity. taken together, despite these limitations, our study provides valuable insight into attitudes and practices related to h n influenza among employees of food production and operation just three months after the first human infection case reported in china, and one month after chickens were found to be infected in guangzhou. we found that: ) the majority of participants worried about being infected with the a/h n and took initiative to find information about h n . the internet, television, and newspapers were the main methods of obtaining information, and the methods differed by sex, age, and some other demographic variables. ) quite a number ( . %)of participants complained that the information was not timely enough, and . % believed information was intentionally concealed by the government. nearly one-third of participants did not firmly believe that the government could control the h n epidemic. these results reflect some distrust in the health department. ) most participants took positive measures to prevent infection; however, more than one-third reported no longer buying chickens and other poultry to eat. a majority of participants indicated a willingness to receive an a/h n vaccine, and the primary reason for unwillingness to receive a vaccine was concern about safety. a history of influenza vaccination and worry about being infected with the a/h n were significantly associated with the intention to receive an h n vaccine. this suggests that more effort should be made to improve compliance of proper preventive measures and reduce panic among the public. in addition, we also reported the public's main information needs if a human h n outbreak occurs in guangzhou. these findings should be used to improve health education and develop a correct strategy for h n control and prevention. written informed consent was obtained from the patient for the publication of this report. additional file : questionnaire on investigation of attitudes, practices and information needs regarding novel influenza a (h n ). human infection with a novel avian-origin influenza a (h n ) virus deaths associated with avian influenza a(h n ) virus in china national health and family planning commission of the people' s republic of china: overview of people infected with avian influenza a (h n ) in china avian influenza a(h n ) virus infections human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome influenza a (h n ): from anxiety to preparedness a two-year surveillance of pandemic influenza a (h n ) in guangzhou, china: from pandemic to seasonal influenza? why do i need it? i am not at risk! public perceptions towards the pandemic (h n ) vaccine the community's attitude towards swine flu and pandemic influenza swine flu") early assessment of anxiety and behavioral response to novel swine-origin influenza a(h n ) public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey awareness, attitudes, and practices related to the swine influenza pandemic among the saudi public implications of public understanding of avian influenza for fostering effective risk communication is internet search better than structured instruction for web-based health education? stud health technol inform widespread public misconception in the early phase of the h n influenza pandemic monitoring community responses to the sars pandemic in hong kong: from day to day factors influencing the wearing of facemasks to prevent the severe acute respiratory syndrome among chinese in hong kong longitudinal assessment of community psychobehavioral responses during and after the outbreak of severe acute respiratory syndrome in hong kong the influence of social-cognitive factors on personal hygiene practices to protect againstinfluenzas: using modelling to compare avian a/h n and pandemic a/h n influenzas inhong kong avian influenza risk perception and live poultry purchase in changing perception of avian influenza risk what causes h n avian influenza? lay perceptions of h n a etiology in south east and east asia live poultry exposure factors affecting intention to receive and self-reported receipt of pandemic (h n ) vaccine in hong kong: a longitudinal study public perceptions in relation to intention to receive pandemic influenza vaccination in a random population sample: evidence from a cross-sectional telephone survey does receipt of seasonal influenza vaccine predict intention to receive novel h n vaccine: evidence from a nationally representative survey of adult vaccination survey provisional topline results for h n vaccination uptake. canberra: australian government department of health and ageing investigation on information needs towards influenza pandemic among public in guangzhou submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported by the research fund from the health bureau of guangzhou (grant number a ), science and technology bureau of guangzhou (grant number y - ) and guangdong science and technology program (grant number b ). all enrollees who participated in the study are appreciated. we wish to give special thanks to dr. doug thoroughman, dr. john poe, dr. sara robeson, and dr. kraig humbaugh from the kentucky department for public health (usa) for their epidemiology guide. the authors declare that they have no competing interests. all authors contributed to the design and execution of the study and analyses. tgl contributed to the conception of study and interpretation and writing of the manuscript. jf participated in the conception of the study and drafting of the manuscript. pzq participated in the design and data collection. wsl participated in the design and statistical analysis. mxl participated in the design and data collection. xmf contributed to the manuscript writing. mw contributed to the study design, interpretation, and manuscript writing. all authors read and approved the final manuscript. key: cord- -uu uipfy authors: hasan, mohammad rubayet; sundaram, manu somasundaram; sundararaju, sathyavathi; tsui, kin-ming; karim, mohammed yousuf; roscoe, diane; imam, omar; janahi, mohammad a.; thomas, eva; dobson, simon; tan, rusung; tang, patrick; lopez, andres perez title: unusual accumulation of a wide array of antimicrobial resistance mechanisms in a patient with cytomegalovirus-associated hemophagocytic lymphohistiocytosis: a case report date: - - journal: bmc infect dis doi: . /s - - -z sha: doc_id: cord_uid: uu uipfy background: infections with multidrug-resistant organisms (mdro) pose a serious threat to patients with dysregulated immunity such as in hemophagocytic lymphohistiocytosis (hlh), but such infections have rarely been comprehensively characterized. here, we present a fatal case of hlh secondary to cytomegalovirus (cmv) infection complicated by both anti-viral drug resistance and sepsis from multiple mdros including pandrug-resistant superbug bacteria. case presentation: a previously healthy, six-year-old boy presented with a -day history of fever prior to a diagnosis of hemophagocytic lymphohistiocytosis and hemorrhagic colitis, both associated with cmv. on hospital admission, the patient was found to be colonized with multiple, multidrug-resistant (mdr) bacteria including vancomycin-resistant enterococci (vre) and carbapenamase-producing organisms (cpo). he eventually developed respiratory, urine and bloodstream infections with highly drug-resistant, including pandrug-resistant bacteria, which could not be controlled by antibiotic treatment. antiviral therapy also failed to contain his cmv infection and the patient succumbed to overwhelming bacterial and viral infection. whole genome sequencing (wgs) of the mdr bacteria and metagenomic analysis of his blood sample revealed an unusual accumulation of a wide range of antimicrobial resistance mechanisms in a single patient, including antiviral resistance to ganciclovir, and resistance mechanisms to all currently available antibiotics. conclusions: the case highlights both the risk of acquiring mdr superbugs and the severity of these infections in hlh patients. antimicrobial resistance (amr) is one of the most important public health challenges of current times as the burden of infectious diseases with multidrug-resistant organisms (mdro) is increasing at an alarming rate. globally, approximately , people die each year due to drug-resistant infections and, if not controlled, these deaths are predicted to exceed million by [ ] . particularly vulnerable are patients with immune deficiency or dysregulation whose inability to fight infections leads to increased risk of disseminated infection and greater dependency on antimicrobial therapy [ ] . hemophagocytic lymphohistiocytosis (hlh) is a potentially life-threatening condition characterized by overactivation of lymphocytes and macrophages that results in dysregulation of inflammatory responses [ ] . while these factors likely put hlh patients at high risk of infection with mdros, no such reports has been published to date and the clinical course of such infections in these patients remains unknown. here we report severe infections with multiple mdros in a patient with cytomegalovirus (cmv)-associated hlh. we also show an unusual accumulation of a wide-array of antiviral and antibiotic resistance mechanisms in a single patient based on data generated by shotgun metagenomic sequencing of blood from the patient and whole genome sequencing (wgs) of mdros isolated from the patient. a previously healthy -year-old boy presented at our hospital in august with a -day history of intermittent, high-grade fever without a clear source, accompanied by loss of appetite, weight loss, painful tongue ulcers, diffuse abdominal pain and intermittent left calf muscle pain. he was a resident of qatar who had just returned from a family holiday in india, where he was hospitalized twice due to fever of unknown origin (fuo) and received intravenous (iv) ceftriaxone and amikacin without any improvement. on examination, he looked moderately ill and pale, and was found to have mouth ulcers and splenomegaly. initial laboratory investigations revealed neutropenia, thrombocytopenia, normocytic normochromic anemia with high ferritin, elevated liver enzymes and c-reactive protein (crp). on admission, surveillance cultures for mdros were positive for vancomycin-resistant enterococcus faecium (vre) and carbapenemase-producing escherichia coli (ndm ) and klebsiella pneumoniae (oxa- ) (fig. ) . he was also found to be positive for cytomegalovirus (cmv) igm with a viral load of , iu/ml. bone marrow examination performed twice showed hypocellularity with myeloid preponderance and no morphologic evidence of malignancy. immunological workup revealed severe reduction in cd + b-cells and cd + cd + nk cells. genetic testing (invitae, san franscico, usa) with a primary immunodeficiency (pid) panel comprised of genes revealed three variants of uncertain significance. his liver enzymes were elevated. liver needle biopsy and electron microscopy revealed mild steatosis with steatohepatitis. patchy sinusoidal dilation with hepatocellular plate atrophy was noted. wilson's disease was excluded by genetic testing. all bacterial, mycobacterial and fungal cultures of bone marrow were negative. extensive infectious disease workup during the course of his hospitalization were negative except that the respiratory pathogen pcr panel was positive for adenovirus and rhinovirus and that vre and candida spp. were isolated from his urine culture. antiviral therapy for cmv viremia was started with iv ganciclovir for weeks. cmv viral load dropped to iu/ml. patient showed symptomatic improvement and was discharged with oral valganciclovir for another weeks. four months post discharge, he developed fever and a productive cough. he had several visits to the emergency department, and infectious disease and hematology outpatient clinics, and was briefly hospitalized again for fever, recurrent respiratory and candida infections, recurrent thrombocytopenia and suspected immunodeficiency. after discharge, he continued to have intermittent fever, and his parents took him to india for further evaluation. he was diagnosed with hemophagocytic lymphohistiocytosis (hlh) secondary to cmv infection, and started treatment with dexamethasone and cyclosporine in february . in order to manage the cmv viremia, he was also treated with multiple antivirals including cidofovir and foscarnet. the child was re-admitted to sidra medicine after month of therapy for hlh with a one-week history of fever, hematochezia and fatigue. on the same day, he developed massive rectal bleeding with blood clots and required resuscitation and was transferred to the pediatric intensive care unit (picu). the rectal bleeding and hemodynamic instability required blood and platelet transfusions and urgent colonscopy, which showed erosion and signs of colitis. cmv-induced hemorrhagic colitis was suspected, with the possibility of transmural infiltration of the bowel with hemophagocytic cells. he was intubated on day two. on picu day three, the patient became hypotensive during interventional radiology and required resuscitation. blood culture was positive with multidrug-resistant e. coli. prolonged antiviral therapy and the lack of response led to a suspicion of cmv ganciclovir resistance, so foscarnet was started. he was extubated days after intubation but on picu day , he was reintubated after developing pulmonary hemorrhage. the chest x-ray showed worsening pulmonary hemorrhage and he developed catecholamineresistant shock that required initation of steroids. also, he continued to have bleeding per rectum requiring continuous support with blood products. a ct angiogram showed bleeding from the terminal ileum and colitis in the large intestine. he was put on total parenteral nutrition. surgical intervention for the gastrointestinal bleeding was considered too risky. thus, he was conservatively managed with octreotide and esomeprazole infusion and blood product transfusions. he also developed acute kidney injury likely from the combination of nephrotoxic medications, hematuria and infection, and continued to be anuric, requiring continuous renal replacement therapy. he also developed microangiopathic hemolytic anemia requiring cycles of plasmapheresis. for hlh, his treatment was escalated to increasing doses of dexamethasone, intravenous immunoglobulin (ivig) and cyclosporine. microbiological findings during this time were remarkable for multiple cultures positive for mdros. the first was a positive blood culture on picu day with an extensively drug resistant, carbapenemase-producing e. coli, which prompted the addition of colistin to his antimicrobial treatment (fig. ). this was followed by multiple positive blood cultures with elizabethkingia meningoseptica, e. faecium and pandrug-resistant k. pneumoniae, and a positive urine culture with vre. whole genome sequencing (wgs) was performed on several multi-dug resistant bacteria isolated from the patient at various time points showing accumulation of resistance mechanisms to almost all classes of antibiotics used to treat these bacteria ( table , supplemental methods). shotgun metagenomic sequencing performed on serum on day , revealed an a v mutation in the ul gene of cmv (fig. , supplemental methods), which is known to confer van vancomycin *bacterial whole genome sequencing (wgs) was performed as described in the supplemental methods and data were analyzed as described previously [ ] resistance to ganciclovir. as a result, ganciclovir was discontinued leaving foscarnet as his anti-viral treatment. iv levofloxacin and vancomycin was added to cover e. meningoseptica and vre, along with prophylactic trimethoprim/sulfamethoxazole and antifungal treatment. his cmv titres continued to be high in spite of antiviral therapy. over the course of the last few days of picu, the patient continued to deteriorate clinically: pulmonary bleeding persisted with intermittent respiratory acidosis despite maximal ventilator support. he continued to be hypotensive despite inotropic support, remained anuric and continued to have gastrointestinal (gi) bleeds. the patient passed away on picu day from multiorgan failure associated with sepsis with highly drug-resistant bacteria. hlh is characterized by dysregulation of the immune system whereby hematopoietic cells are phagocytosed by activated macrophages and lymphocytes [ ] . primary or familial hlh may be inherited in an autosomal recessive manner and are grouped into types based on the affected gene. type hlh is caused by a defect in chromosome , while types , , and are known to be caused by mutations in familial hlh (fhlh) genes prf , unc d, stx and stxbp genes, respectively [ ] . at the initial presentation to our hospital in june , with cmv viremia, the patient did not meet criteria for hlh. this diagnosis was made when abroad in india, and treatment started there. subsequent review identified that the patient developed / hlh- criteria [ ] . in our patient, the history of recurrent infection, the cmv viremia, the clinical diagnosis of hlh and marked reduction in b-cell and nk-cell numbers could suggest an underlying primary immunodeficiency (pid) disorder. however, a sample sent to a referral laboratory to test for pid genes including above hlhassociated genes did not identify significant mutations in known pid genes, suggesting that hlh in our patient was cmv-associated. hlh in an immunocompetent patient secondary to cmv infection is extremely rare but has been reported in the literature [ ] . genotype and antiviral resistance profiles of the cytomegalovirus strain. nucleic acid extract from patient serum was subjected to ngs library preparation using nextera xt kit (illumina, usa) and sequencing was performed on a miseq (illumina). paired sequence reads were mapped to ul and ul sequences (gene id and genbank accession abv . , respectively) to obtain corresponding gene sequences from the patient's cmv strain. the sequences were then analyzed by using an online mutational resistance analyzer (mra) available from the university of ulm, https://www.informatik.uni-ulm.de/ni/mitarbeiter/hkestler/mra/app/index.php?plugin=form [ ] . ganciclovir resistance was confirmed by the presence of the a v mutation in ul [ ] what was unique in our patient compared to other reported cmv-associated hlh cases was the overwhelming infection with mdros. on hospital admission, the patient was found to be colonized with multiple mdros including vre, and carbapenamase-producing enterobacteriaceae, which may have been acquired during his previous hospital course in india. wgs revealed that the vre isolate harbored the vanhax gene cluster that encodes vana, providing high level resistance to vancomycin and teicoplanin. apart from vancomycin resistance, the isolate also possessed genes encoding resistance to macrolides, lincosamides, tetracyclines and aminoglycosides (table ) . phenotypically, the isolate was also resistant to linezolid but we were unable to identify the genetic determinant. the e. coli and k. pneumoniae isolates harbored genes encoding a wide array of antimicrobial resistance mechanisms affecting the vast majority of antibiotic classes, including ctx-m- and ndm β-lactamases, the most common extendedspectrum β-lactamases and carbapenemases, respectively, found in enterobacterales in india [ , ] . notably, the e. coli isolate had different modifying enzymes conferring resistance to aminoglycosides, which has rarely been reported [ ] . there were also several narrow host range plasmids belonging to the incf family (replicons fia, fib and fii) in the e. coli and k. pneumoniae strains (data not shown) [ ] . since these plasmids can simultaneously harbor most of the genes detected and can be transferred both within the same species and between both species, it is presumed that they played a role in the acquisition and subsequent exchange of resistance determinants among these isolates. consequently, our patient developed respiratory, urine and bloodstream infections with highly drug-resistant, including pandrug-resistant, bacteria, which could not be controlled by antibiotic treatment. although the patient initially responded to the antiviral drug ganciclovir as reflected by a drop in cmv viral load (fig. ) , he later became non-responsive to antiviral treatment. due to the severity of the cmv infection despite ganciclovir treatment, real-time metagenomic analysis was performed, revealing ganciclovir resistance and allowing for tailoring of the antiviral therapy. although the ul mutation detected in this case is well described in the literarture for its association with ganciclovir resistance, the reduced absorption of the drug because of cmvassiciated enterocolitis may have contributed to the development of resistance as well [ ] . by wgs, the vre isolate from his urine and the e. coli and k. pneumoniae isolates from his blood culture were found to be of different sequence types compared to the isolates the patient was previously found to be colonized with, but the later clinical isolates harbored both the resistance mechanisms found in the colonizing strains as well as newly acquired resistance mechanisms. in particular, the pandrug-resistant k. pneumoniae isolated from the patient's blood had ndm β-lactamases in addition to oxa- β-lactamases. phenotypically, the isolate was resistant to all currently used antibiotics including the last resort antibiotic, colistin. we were unable to detect the mcr gene that is commonly known to encode for colistin resistance and the genetic determinants of colistin resistance remained unknown in this study. while the exchange of resistance determinants via horizontal gene transfer is a possibility, the invasive superbug infections in our patient may have been facilitated by his travel history as well as his cmv-associated enteropathy, which eventually culminated with untreatable sepsis, multiorgan failure and the untimely death. our study has a few limitations. although the patient was tested for a panel of genes for pid, wgs was not performed to look for other genetic causes of hlh or pid, outside of the commonly known mutations. also, the clinical course of our patient in india was not well documented and details regarding antibiotic and antiviral treatment of the patient are unknown. however, it appears most likely that the patient acquired mdros while seeking medical treatment in india, where mdro infection is frequently associated with hospitalization [ ] [ ] [ ] . therefore, the risks of mdro infections associated with medical tourism to regions with high rates of antibiotic resistance should be discussed with patients, who are at higher risk for complications from these organisms. it is important that the travel and other relevant history be elicited from patients so that appropriate screening measures for mdros can be implemented. strict infection control measures are necessary to reduce nosocomial transmission of mdros, especially in centers caring for immunocompromised or critically ill patients. in our institution, we perform risk-based screening for mdros, implement appropriate isolation measures to prevent spread to other patients and health care workers, and perform wgs to monitor for nosocomial transmission. this case depicts the dire circumstances associated with severe infection with mdr superbugs in a particularly vulnerable patient, and underscores the need for urgent measures to prevent the development of antibiotic resistnace through appropriate use of antimicrobials and to prevent the spread of mdros through surveillance and implementation of appropriate infection control measures. supplementary information accompanies this paper at https://doi.org/ . /s - - -z. additional file . supplemental methods. review on antimicrobial resistance: tackling drug-resistant infections globally pulmonary infections in immunocompromised patients an overview of hemophagocytic lymphohistiocytosis draft genome sequences of two streptococcus pneumoniae strains causing invasive infections in children in qatar extended pairwise local alignment of wild card dna/rna sequences using dynamic programming characterization of multiple cytomegalovirus drug resistance mutations detected in a hematopoietic stem cell transplant recipient by recombinant phenotyping treatment of hemophagocytic lymphohistiocytosis with hlh- immunochemotherapy and bone marrow transplantation acute cytomegalovirus (cmv) infection associated with hemophagocytic lymphohistiocytosis (hlh) in an immunocompetent host meeting all eight hlh diagnostic criteria the role of epidemic resistance plasmids and international high-risk clones in the spread of multidrug-resistant enterobacteriaceae prevalence and clonality of extended-spectrum betalactamases in asia emergence of five kinds of aminoglycoside-modifying enzyme genes simultaneously in a strain of multidrug-resistant escherichia coli in china cytomegalovirus viremia and resistance patterns in immunocompromised children: an -year experience antimicrobial resistance: the next big pandemic carbapenemase-producing klebsiella pneumoniae, a key pathogen set for global nosocomial dominance high reported rates of antimicrobial resistance in indian neonatal and pediatric blood stream infections publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions mrh designed the study, analyzed data and drafted the manuscript; mss collected clinical data and prepared the case report; ss collected sequence data and revised the manuscript; kmt analyzed sequence data and revised the manuscript; myk, dr, oi, maj, et, sd, rt, pt and apl interpreted data and substantially revised the manuscript. all authors read and approved the final version of the manuscript. bacterial whole genome sequencing (wgs) and metagenomic sequencing were performed as part of other research projects supported by sidra medicine, qatar (grant no. sirf_ to m.r.h. and sirf_ to a.p.l.). bacterial whole genome sequence (wgs) data and metagenomics data are available under bioprojects prjna and prjna in ddbj/ena/ genbank with the accession numbers srx , srx , srx , srx , srx , srx , srx and srr , respectively.ethics approval and consent to participate not applicable the parents of the patient provided written informed consents to publish their child's personal or clinical details along with any identifying images. the authors declare that they have no competing interests.received: november accepted: march key: cord- - sdt zz authors: andrews, denise; chetty, yumela; cooper, ben s.; virk, manjinder; glass, stephen k; letters, andrew; kelly, philip a.; sudhanva, malur; jeyaratnam, dakshika title: multiplex pcr point of care testing versus routine, laboratory-based testing in the treatment of adults with respiratory tract infections: a quasi-randomised study assessing impact on length of stay and antimicrobial use date: - - journal: bmc infect dis doi: . /s - - -z sha: doc_id: cord_uid: sdt zz background: laboratory-based respiratory pathogen (rp) results are often available too late to influence clinical decisions such as hospitalisation or antibiotic treatment due to time delay in transport of specimens and testing schedules. ward-based i.e. point of care (poc) testing providing rapid results may alter the clinical management pathway. methods: filmarray® rp polymerase chain reaction (pcr) systems were placed in three in-patient and out-patient medical areas. patients presenting with influenza-like illness /upper respiratory tract infection +/− lower rti were recruited between january–july . filmarray® poc testing occurred on even days of the month (intervention) or routine, laboratory-based rp pcr testing +/− atypical serology on odd days (control). the primary outcome was length of hospital stay. the secondary outcomes were impact on the use of antimicrobials, readmissions, all-cause mortality, length of ward stay and turn-around time (tat) (time to result from admission). results: of eligible patients, ( . %) were included; in the control arm and in the intervention arm. % of control arm patients and % of intervention arm patients had an rp detected. poc testing was not associated with the primary outcome measure, length of stay, but reduced the tat from . h to . h, p < . . only the prescribing decision differed between study arms, p < . . when antivirals were given, the intervention was associated with a reduction in the median time to the first dose of h and allowed appropriate treatment of mycoplasma infection. conclusions: we found no association between respiratory pcr poc testing and length of stay or most of the secondary outcomes except the antimicrobial prescribing decision. this was probably due to a delay in initiating filmarray® testing. despite this, poc testing allowed time-critical antivirals to be given significantly faster, appropriate mycoplasma treatment and results were available considerably faster than routine, laboratory-based testing. ward-staff of all grades performed poc testing without difficulty suggesting potential use across many divergent healthcare settings. further studies evaluating the implementation of rapid respiratory pcr poc testing and the effect on length of stay and antimicrobial use are required. trial registration: isrctn , retrospectively registered, / / . respiratory tract infections (rti) place a significant burden on health systems globally, particularly during the annual respiratory season epidemics [ ] . diagnostic tests for respiratory pathogens (rp) are usually laboratorybased with an inherent delay in time to result relating to specimen in transit to the laboratory and laboratory testing schedules, for example once a day, and/or not performed on weekends and holidays. for this reason or due to the nature of the test (culture, serology or batch molecular testing) results are rarely available to the clinician when the patient is first assessed. consequently, though respiratory viruses are frequently isolated in community acquired pneumonia (cap) [ ] and are reported to be responsible for . % of cap cases admitted to uk hospitals [ ] , the decision to manage as a viral rti or treat for bacterial infection including mycoplasma pneumoniae or chlamydia pneumoniae ('atypical bacteria') is based upon the clinical scenario and severity criteria such as the curb- score. thus a proportion of infections will be inappropriately managed with antibiotics or the result may arrive too late for influenza treatment to be effective [ ] . the world health organization (who) states that antimicrobial resistance threatens the effective prevention and treatment of an ever-increasing range of infections [ ] . the centre for evidence based medicine highlights the considerable number of new diagnostic technologies in development to underpin the rational prescribing of antibiotics [ ] , which extends to antivirals. point of care (poc) tests eliminate the need for specimen transportation to the testing laboratory and can be performed on demand by ward staff. by providing faster results, poc tests may influence early treatment decisions such as hospital admission and allow earlier discharge, targeted antimicrobial prescriptions and better antimicrobial stewardship. poc testing should also reduce cross-transmission and subsequent nosocomial outbreaks related to viral rti cases that are undiagnosed and patients not placed in appropriate isolation. however, current poc tests for respiratory viruses are generally antigen detection tests, usually only detect influenza and respiratory syncytial virus (rsv) and their sensitivity can be suboptimal [ , ] . the biofire filmar-ray® respiratory panel (biofire diagnostics, salt lake city, ut, a biomerieux company) detects respiratory viruses: influenza virus types a and b (with influenza a subtyping), adenovirus, coronaviruses hku , nl , e and oc , human metapneumovirus, human rhinovirus/enterovirus, parainfluenza virus types - and rsv and bacteria: mycoplasma pneumoniae, chlamydia pneumoniae and bordetella pertussis. filmarray® lends itself to poc testing as it is a small, desktop, fullyautomated nested multiplex pcr in an enclosed disposable pouch requiring only min of hands-on time with results available in about hour [ ] . though more expensive than single or low multiplex laboratory or poc tests, clinical outcomes such as a reduced length of stay and reduction in inappropriate antimicrobial usage resulting from the rapid and extended panel may offset test costs. thus understanding filmarray®'s clinical utility as a poc test early in a patient's admission is important. we undertook a study to assess the filmarray® rp panel as a poc test compared to routine, laboratory-based detection methods in order to assess the impact on length of stay and antibiotic utilization. it is the first study, to our knowledge, in which ward-staff were performing the fil-marray® rp panel as a poc test. the aim of the study was to determine whether in adults presenting with upper respiratory tract infection (urti)/ influenza-like illness (ili) +/− lower respiratory tract infection (lrti), filmarray® rp panel poc testing, when compared to the routine, laboratory-based rp testing was associated with length of hospital stay or antimicrobial use. the study took place in a bedded teaching and tertiary referral site of a two-site bedded acute nhs hospital trust in london.four filmarray® systems were placed in side-rooms across three adult (> years) wards: two acute medical units (amus) and the medical assessment centre (mac). the amus are and bedded short-stay, acute medical wards to which patients are admitted from the emergency department (ed). the mac is an area to which out-patients can be referred for review by clinicians. it is open daily from am to pm. mac patients are assessed and then admitted to an amu or discharged. a quasi-randomised trial design was used such that patients were enrolled in to the control arm on odd days of the month and in to the intervention arm on even days of the month. this was the most pragmatic design that could be implemented on the study wards. the study was not blinded. study-ward staff were educated about the study and consent taking, given a staff information sheet and trained to use the filmarray® respiratory panel as per manufacturer's instructions. competency assessments were conducted. all staff were required to don personal protective equipment when performing each assay. filmarray® tests were to be ordered and performed by study-ward staff, however if they were unable to do the testing e.g. due to clinical duties, the study investigators, who worked on weekdays until pm and a half-day on weekends and bank holidays, performed the filmarray® test. in order to interpret the filmarray® results out of hours, study-ward staff consulted a standard operating procedure describing each pathogen, the type of diseases associated with it, the groups at risk of severe infection, any medical management and the infection control precautions required, if at all. they also had direct contact numbers for the study investigators who were available at all hours. eligible patients were identified by ward staff or study staff. written informed consent was obtained by ward staff before patient participation. in the control arm, combined nose and throat flocculated swabs (copan diagnostics, italy), were placed in viral transport medium (vtm, copan) which was transported to the laboratory by hospital porters as is routine. the standard, routine diagnostic assays for viral pathogens used in the control arm are in-house developed real-time pcrs with separate multiplex assays (influenza a (h n ) pdm matrix gene rna and h rna, influenza a virus rna, influenza b virus rna, rhinovirus rna, rsv subgroup a and subgroup b rna, parainfluenza viruses , and rna and human metapneumovirus rna) and an adenovirus monoplex [ ] [ ] [ ] . outside this study, there is no rapid/poc rp testing at this hospital. if requested by the clinical team, mycoplasma pneumoniae and chlamydia pneumoniae were tested for in the control arm using the laboratory's routine, complement fixation tests (cft) (launch diagnostics, kent, uk and tcs biosciences, buckingham, uk). a comparison of the pathogens detected by the filmar-ray® and the routine diagnostic tests is shown in table . the routine tests were performed on site by qualified health care scientists. pcr results were available at in the intervention arm, combined nose and throat flocculated swabs (copan diagnostics, italy), were placed in viral transport medium (vtm, copan) and thereafter a rehydration buffer and μl of the vtm was introduced into the filmarray® pouch by ward-based staff (poc testing) on even days of the month (intervention arm). filmarray® was validated against the routine method to ensure satisfactory performance characteristics but a head to head comparison was not included as part of this study. external (quality control for molecular diagnostics [qcmd], glasgow, uk) and internal quality control specimens were tested by filmarray® during the study. the use of the filmarray® was approved by the trust point of care testing committee. all results were uploaded to the hospital results reporting system. positive results for both study arms were telephoned by a microbiologist or virologist to the health care provider. antimicrobial stewardship activities did not change during the study. all other diagnostic specimens e.g. for bacterial culture were sent to the laboratory as usual for both study arms. these results were not included in the analysis. the inclusion criteria were that patients were ≥ years of age, with mental capacity to give written informed consent and presenting with urti/ili defined as symptoms including fever or feeling feverish (chills), cough, sore throat, runny or stuffy nose, muscle-aches or body-aches, headaches, fatigue (tiredness) and possibly vomiting or diarrhoea [ ] +/− lrti. patients who did not meet inclusion criteria or with evidence or suspicion of bacterial infection affecting sites other than the respiratory tract were excluded from the opportunity to participate in the study. patients who requested withdrawal from the study were excluded from the analysis. the primary outcome studied was the length of hospital stay defined as the time between hospital admission and hospital discharge. the filmarray® rp panel is relatively expensive (£ -£ for the consumables and £ , -£ , for the capital purchase of the system depending upon the country) compared to currently employed routine tests, (£ per test for our standard laboratory method). however the average cost for managing pneumonia in the community is estimated at £ per episode compared with £ - for hospitalised patients in the uk [ ] and $ in the us [ ] . thus if a filmarray® poc result allows earlier discharge, the consequent financial savings from the shorter hospital stay may offset the extra test costs and prove cost-beneficial, hence our choice of primary outcome. the secondary outcomes were antimicrobial prescription/s (antibiotics: any versus none, duration, time to prescription within the first h of the patient's stay and prescribing decisions within h after the the diagnostic results under investigation: start, stop, de-escalation, escalation and continued use, as assessed by the chief investigator), readmission rates and all-cause mortality (both within days of the test) and length of studyward stay (i.e. removing the length of stay on subsequent wards to which the patient was transferred, where relevant). escalation was defined as the addition of an antibiotic/s to the existing antibiotic therapy or the substitution of the current agent with a broader agent. de-escalation was the cessation of ≥ antibiotic when > antibiotic was prescribed or the substitution with a narrower agent. we collected these data prospectively and retrospectively from the electronic patient record (epr) and the electronic patient medicines administration system (epma). in some cases, enrolment of the patient into the hospital administrative system occurred after examination and initiation of antibiotic therapy by the physician on the ward or in the ed. therefore, a negative time to antibiotic administration on the ward was corrected to zero hours to antibiotics. we defined the turn-around time (tat) of the tests as the time between hospital admission and the time of the result on the fil-marray® system (intervention) or the time of the result on the epr (control). for each patient, we collected demographic data, charlson co-morbidity score, and at the time of admission, curb- score for patients with a diagnosis of community-acquired pneumonia, an early warning score (ews) [ ] , peripheral white cell count (wcc), creactive protein (crp) and the day of the week of admission. the type of lrti e.g. infective exacerbation of chronic obstructive pulmonary disease (copd), bronchitis, pneumonia was not recorded. the power calculation was based upon length of stay, which had previously been observed to be . days on the study wards. rapid pathogen identification has been associated with a reduction of mean hospital length of stay, in one study from . days to . days (approx. %) (p = . ) [ ] . for a % reduction in length of stay (los) in the intervention arm, sample size calculations indicated that sample sizes of cases in each arm were required to achieve % power to detect a difference of . between the null hypothesis that both group means are . days ( . h, based upon data collected from the study wards) and the alternative hypothesis that the mean of group is . days ( . h) with known group standard deviations of . and . and with a significance level (alpha) of . . the primary outcome was analysed with a linear regression model (after log-transformation of length of stay data) according to a pre-specified analysis plan with an individual patient taken as the unit of analysis. secondary outcomes were analysed using linear regression models for continuous outcome data, logistic regression for binary outcomes, and negative binomial regression for count outcome data. for all these patient-related outcomes we adjusted for multiple pre-specified potential confounders (age, sex, charlson score, ews, wcc, crp). pearson's chi-squared test was used to test for differences between the arms in categorical antibiotic prescribing decisions with p-values calculated by , monte carlo replicates (to avoid problems associated with small cell counts associated with the usual asymptotic p-values). a t-test was used to compare the time to test between the two arms. a planned subgroup analysis was performed as above for primary and secondary outcomes excluding patients who had infection proven elsewhere after enrolment as it is plausible that a respiratory pathogen poc result would not alter los or antibiotic use when the patient had another infective diagnosis made. analysis was conducted in r [ ] . multiple imputation was used to account for missing data using the package mice [ ] . the study ran from th january until st july as planned. no changes were made to the study protocol. during this time patients met eligibility criteria (fig. ). sixty-one ( . %) of these patients were not included ( in the intervention arm, in the control arm) because were discharged before enrolment, an interpreter was unavailable for three, declined participation, one patient died before being approached, one patient consented but the test was not performed and information is missing for two patients. no patients withdrew from the study. thus ( . %) patients were enrolled and included in the analysis, in the control arm and in the intervention arm. all statistical analyses were pre-specified; there were no post-hoc analyses. baseline characteristics were similar between the two study arms (table ) . curb- score was missing for . % of patients for whom it was relevant and was omitted from the analysis. one hundred and sixty-five ( %) patients had a negative time to antibiotics changed to h (median − . h [iqr − . to − . ]). the median time to result from admission was substantially shorter in the intervention arm compared to the control arm (control arm . h (iqr . - . ), intervention arm . h (iqr . - . ) ), two-sample ttest assuming unequal variances, p < . . ward staff of all grades performed % of poc tests, % of the samples were tested by study investigators and there is no record for %. no adverse events were reported. overall, ( . %) of the patients had a positive result (table ) , ( . %) in the control arm and ( . %) in the intervention arm. the viruses and bacteria detected are shown in table . every virus on the panels was identified except parainfluenza virus type , type and type . only single pathogens were detected by routine testing but filmarray® detected dual infections in five samples. filmarray® also detected coronaviruses, not detected using standard tests. there were three and four invalid tests in the control and intervention arms respectively, the remaining tests were negative ( . % control, . % intervention). m. pneumoniae was the only bacterium on the panels which was identified. four patients in the control arm had an elevated mycoplasma cft ( : , : , : , > : ). convalescent serology was not sent, rendering results uninterpretable. all of these results were available after ward discharge, three after hospital discharge, and did not influence management. the tat was - days. five patients in the intervention arm had m. pneumoniae detected by filmarray®. antibiotics were started for of these cases and extended in after discussion with the microbiologist. there was no evidence that the length of hospital stay, the primary endpoint, was reduced by poc testing. the median length of hospital stay was . h (iqr . - . ) in the control arm and . h (iqr . - . ) in the intervention arm. in the linear model (for log-transformed length of stay data, adjusting for potential confounders) the rapid test was associated with an absolute difference in the natural logarithm of the length of stay of . ( % ci [− . , . ]; p = . ). this corresponds to an % ( % ci [− %, %]) increase in length of stay associated with the rapid test arm. six of patients tested on the mac in the control arm ( %) and of patients tested in the mac in the intervention arm ( . %) were discharged without admission to a hospital ward. for all but one of the secondary outcomes, there was no evidence that the intervention had an effect (table ) . only the prescribing decision within h following the diagnostic results under investigation showed evidence of a difference between study arms (table , p < . , pearson's chi-squared test, p-value calculated by , monte carlo replicates). charlson score a ( - ) ( - ) white cell count (× /l) a . ( . - . ) . ( . - . fifty-one patients had influenza a virus and/or influenza b virus detected by either the routine assay ( ) or by the filmarray® ( ) . of these patients, of ( %) in the control arm and of ( %) in the intervention arm were given antivirals. the time to the first dose from the time of admission was known for all but one patient in each arm and was considerably reduced in the intervention arm: median of . h in the control arm (iqr . - . ) and h in the intervention arm (iqr . - . ). only one patient in each arm was given empiric antivirals but had a no viruses detected. the planned subgroup analysis excluding patients who had infection proven elsewhere after enrolment e.g. urinary tract infection (n = ) did not substantially alter any of the above results. we found no evidence for an association between respiratory multiplex pcr (biofire filmarray®) poc testing and length of hospital stay when compared to our routine, laboratory-based respiratory pcr and serology testing. there was an association between poc testing and the antibiotic prescribing decision within h after the result. poc testing also produced results considerably faster than laboratory-based assays. we did not investigate whether the poc results actually influenced decision making and the association with the prescribing decision may only reflect that the filmarray® result was available before the antibiotics were prescribed and not that the prescriber considered the filmarray® result in their decision making. similar percentages of patients analysed with a negative binomial regression all models adjusted for age, sex, charlson and potts scores, day of week, and admission values of wcc and crp and used multiple imputation to account for missing data. the curb- score was not available for % of patients, and was therefore not adjusted for in statistical models. there was less than % missing data for all covariates. missing outcome variables ) antibiotics within h and ) antibiotics at any time: missing data for patients in the control arm and patients in the intervention arm, ) time to antibiotics in the first h: missing data for patient in the intervention arm, ) duration of antibiotics: missing data for patients in the control arm and in the intervention arm, ) readmission: missing data for patient in intervention arm, ) mortality: missing data for patient in intervention arm control arm de-escalate: stop ≥ antimicrobial, substitution of beta-lactam with narrower spectrum beta-lactam. intervention arm de-escalate: stop ≥ antimicrobial, substitution of beta-lactam with narrower spectrum beta-lactam, substitution of beta-lactam with narrower spectrum beta-lactam and atypical agent was stopped. control arm escalate: add antibiotic to existing antibiotics (all agents against atypical pneumonia). intervention arm escalate: add antibiotics to existing antibiotics ( agents against atypical pneumonia, addition of agents against 'typical pneumonia' to atypical agent e.g. beta-lactam or teicoplanin with ciprofloxacin if penicillin allergic), substitution of beta-lactam with broader spectrum beta-lactam received antibiotics in both study arms. there were no significant differences for the remaining secondary outcomes between the two study arms. the hypothesis was that poc testing would reduce the length of stay. however, though the poc test was day faster than laboratory-based testing, the results were available later than anticipated. this was not due to testing performance of filmarray®, which took only min but related to a delay in the processing of the specimen by the clinical staff on the ward. sixty-eight percent of poc tests were performed by study investigators reflecting the fact that the study protocol was not initiated by clinical staff as soon as the patient was admitted to the study ward in many cases. instead testing was delayed until the study investigators visiting the study wards initiated the study protocol. this resulted in a significant delay in time to results. this is in contrast to a trial of mrsa poc screening by our amu ward staff that had a tat from admission of . h [ ] . mrsa testing is mandatory and most patients were eligible for inclusion. the delay in the present study may be related to screening for more complex eligibility criteria than the mrsa study and an additional reliance on study investigators present on the ward to perform the test. if this is the case, the tat would be faster if filmar-ray® was embedded as a routine, diagnostic poc test. when ward staff did perform the test, they were from all grades and they performed it without incident. others report a tat of . h for filmarray® poc testing, though the tat was from the time of decision to test to the time of result and testing was conducted by trial staff [ ] . thus filmarray® poc testing can be successfully implemented but this study failed to achieve the optimum tat. poc testing was associated with a reduction in time to antivirals for those identified with influenza virus. antivirals were given a day and a half quicker in the intervention arm and within a day of admission. given that these drugs are of clinical benefit only if administered within h of symptom onset [ ] , this is a key, clinical outcome. poc testing allowed changes to therapy for the appropriate treatment of mycoplasma infection; in the control arm, positive results were uninterpretable and were predominantly available after discharge. the ability of filmarray® to detect coronaviruses allowed for a diagnosis to be made in samples that would have been missed using routine methods. routine testing only identified single pathogens as opposed to filmarray® which identified dual infection in patients, of importance for infection control and virus surveillance. parainfluenza virus types , and , chlamydia pneumoniae and bordetella pertussis were not detected during the course of this study which did not span the entire winter. the positive impact on antivirals of the faster time to detection of influenza with filmarray® was reported by an observational study of paediatric patients, % of who were given oseltamivir in a timely manner, which was not possible with the comparator test [ ] . an observational study reported a faster time to a negative influenza result with filmarray® compared to another rt-pcr ( . h versus . h) which shortened unnecessary oseltamivir use by days with an estimated cost saving per patient of $ . us [ ] . seventy-five percent of the poc results were negative, providing no information about the aetiology of the infection or the predicted clinical course. negative results would not be expected to expedite discharge or antibiotic cessation, the main outcomes under consideration here. a paediatric observational study found that patients with a positive respiratory virus pcr result had a % shorter duration of intravenous antibiotics [ ] . a retrospective study noted a reduced length of stay in children with a positive filmarray® result reported within h, not seen with negative tests [ ] . the reduced time to detection of influenza with laboratorybased filmarray® was associated with significantly lower odds ratios for admission, length of stay, duration of antibiotics and chest x-rays when compared to positive routine tests in a retrospective study of adults [ ] . a randomised trial noted that patients with positive fil-marray® poc results received shorter courses of antibiotics and had shorter hospital stays than those with negative poc results [ ] . our - % positive yield may be because our study was seasonally limited and extended in to the summer months. further, we did not record the types of rti, which may have resulted in this lower than expected percentage. another uk, adult study between september and february identified viruses in % of patients with lower rti [ ] . the poc result was too slow to influence initial antibiotic decision making as the median time to antibiotics from admission was h. this rapid initiation of antibiotics was also found in a randomised trial of filmarray® poc testing [ ] and is consistent with guidelines that recommend antibiotic treatment for cap within h of presentation to hospital [ ] . therefore, we would expect almost all patients to be initially started on an antibiotic. however with the delay in poc testing we would not expect this parameter to be impacted, i.e. if testing had been done in an appropriate time frame (< h after patient evaluation) the subsequent initiation or discontinuation may have been significantly influenced. some of this reflects a continuation on the study wards of antibiotics started in the ed. even with a positive poc result, amu doctors may be dissuaded on safety grounds from stopping or de-escalating antibiotics that were started on the basis of a clinical assessment that they did not witness in the ed. our findings are consistent with a randomised trial of filmarray® poc testing, which found that the mean duration of antibiotics did not differ between the filmarray® and control arms. however that group identified that a greater proportion of patients in the intervention arm (with a poc result) than in the control arm received only a single dose of antibiotics or < h of antibiotics [ ] , something that we did not assess in the present study. other trials of rp diagnostics in adults, including filmarray®, have found that pcr detection of only a viral pathogen coupled with a low procalcitonin level led to antibiotic cessation in only % of cases [ ] or a trend towards fewer days of antibiotic treatment off-set by only / patients having their antibiotics stopped [ ] . the authors of the latter study advocate real-time stewardship with rp results, which was omitted from the intervention in the present study. rapid pathogen identification with antimicrobial stewardship has been associated with a significant reduction of hospital costs for adult in-patients [ ] . this study has other limitations, nearly all due to limited resources. we employed a pragmatic quasirandomised design, allocating patients to the intervention arm on even days making the study vulnerable to bias due to differences in patients allocated to the study arms. though we found no evidence for such differences, and the outcome analysis adjusted for several prespecified potential confounders, a fully randomised design would have provided a stronger level of evidence. there were more patients in the intervention arm suggesting that the patient recruitment processes in the two arms were not equivalent and this may reflect enthusiasm for the filmarray®. this may also reflect increased disease severity in the intervention arm and a need to identify the cause of the disease, thereby resulting in a biased use of the filmarray® in this cohort. curb- scores were not assessed and therefore an accurate comparison of the severity of the pneumonia in the two arms of the study could not be determined. further, we did not collect data on the type of rti; a randomised trial of filmarray® poc testing recorded a shorter duration of antibiotics for patients with asthma and copd who were in the intervention arm versus the control and a shorter length of stay for copd patients in the filmar-ray® arm [ ] . the ed does not routinely use epma and so we do not know exactly how many patients received antibiotics there. a negative time to antibiotics, due to administrative errors, was changed to h in % of patients however the median was small (− . h) and this phenomenon occurred in both study arms. as in other similar studies on this subject [ ] we did not include routine bacteriology results in the analysis however the hypothesis had greater dependence upon the predominantly viral panel results under investigation and bacteriology results would not be expected to differ between the study arms and thus influence results. finally, the number of eligible patients admitted to the study wards was less than predicted by the data used to plan the study and hence the number of cases recruited fell short of the statistical calculation that required patients in each arm to detect a fall of . days. it is not clear why this was the case but may be related to patients bypassing the study wards during the busy winter months of january to march and due to the inclusion of some summer months in the study. we have selected for patients who required admission and possibly antibiotics by placing the filmarray® systems on hospital wards. a panel which includes common bacterial causes of lower rti would probably have identified more pathogens in this setting. a recent study in the uk identified bacteria in lower respiratory tract specimens from % of patients with pneumonia [ ] . though we did not record the type of rti in this study, as in-patients, most patients probably had a lower rti. a study in the ed might have tested a greater number of patients with a viral illness. there, poc results could provide reassurance that discharge is reasonable. with a rapid result and the broader rp panel afforded by fil-marray®, it is plausible that safety-netting antibiotic prescriptions would have reduced. due to the mandated maximum -h wait for patients in english eds [ ] and a perceived lack of understanding of these results vocalised by our ed staff (because with laboratory-based testing the patient has left ed when results are available), we moved the study one step in to the hospital. this highlights an important knowledge gap. an ed-based study of poc testing incorporating decision making support is therefore advisable. we found no association between respiratory multiplex pcr (biofire filmarray®) poc testing and length of hospital stay when compared to our routine, laboratorybased respiratory pcr and serology testing. this result was most likely influenced by the delay in the rapid poc testing. poc testing produced results considerably faster than the routine tests but the results were not rapid as designed to be. this was not the fault of the poc test, but highlights the fact that new technology itself is not enough: the correct systems must be in place in order to reap their benefits. patients who had the poc test received time-critical antivirals for influenza significantly faster and appropriate therapy for mycoplasma infection, not seen in the control arm. ward staff of all grades performed the poc test without incident meaning that this test has potential across a range of healthcare. further studies are required that focus on implementing respiratory multiplex pcr poc testing with rapid results, in order to fully assess the impact on length of stay and antibiotic use. surveillance of influenza and other respiratory viruses, including novel respiratory viruses community-acquired pneumonia guidelines for the management of community acquired pneumonia in adults: update pride consortium investigators. effectiveness of neuraminidase inhibitors in reducing mortality in patients admitted to hospital with influenza a h n pdm virus infection: a meta-analysis of individual participant data who antimicrobial resistance fact sheet position paper on anti-microbial resistance diagnostics -centre for evidence-based medicine rapid diagnostic testing for influenza: information for health care professionals influenza: epidemiology, clinical features, therapy, and prevention. semin respir crit care med an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection detection of nine respiratory rna viruses using three multiplex rt-pcr assays incorporating a novel rna internal control transcript pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr cdc protocol of real-time rtpcr for influenza a(h n ) community-acquired pneumonia: the annual cost to the national health service in the uk factors associated with hospitalization costs for patients with community-acquired pneumonia standardising the assessment of acute illness severity in the nhs integrating rapid pathogen identification and antimicrobial stewardship significantly decreases hospital costs r: a language and environment for statistical computing. r foundation for statistical computing mice: multivariate imputation by chained equations in r point-of-care universal screening for meticillin-resistant staphylococcus aureus: a cluster-randomized cross-over trial routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial implementation of filmarray respiratory viral panel in a core laboratory improves testing turnaround time and patient care comparison of turnaround time and time to oseltamivir discontinuation between two respiratory viral panel testing methodologies role of a respiratory viral panel in the clinical management of pediatric inpatients impact of a rapid respiratory panel test on patient outcomes impact of early detection of respiratory viruses by multiplex pcr assay on clinical outcomes in adult patients comprehensive molecular testing for respiratory pathogens in communityacquired pneumonia the potential of molecular diagnostics and serum procalcitonin levels to change the antibiotic management of community-acquired pneumonia the clinical impact of the detection of potential etiologic pathogens of community-acquired pneumonia department of health: improving emergency care in england. sixteenth report of session together with formal minutes, oral and written evidence ann-louise caress, james beresford-davies, biofire and biomérieux (nadia albrecht, ryan anderson, sam bozette, christine ginocchio, marie-francoise gros, karen jordan, alasdair macmillan, wade stevenson), nergish desai, british lung foundation central manchester breathe easy patient/carer support group, chandan kainth, staff and patients of the study wards, pei jun wu. the theory behind the study and the turn-around times were an oral presentation at the biomérieux integrated symposium entitled 'from single to syndromic testing: the new paradigm in microbiology' at the th eccmid, amsterdam, netherlands, amus: acute medical units; cap: community acquired pneumonia; cft: complement fixation test; crp: c-reactive protein; ed: emergency department; epma: electronic patient medicines administration system; epr: electronic patient record; ews: early warning score; mac: medical assessment centre; pcr: polymerase chain reaction; poc: point of care; rp: respiratory pathogen; rsv: respiratory syncytial virus; rti: respiratory tract infection; tat: turn-around time; wcc: peripheral white cell count all authors contributed to the execution of the study and/or study analysis, and/or writing of the manuscript and all approved the manuscript.ethics approval and consent to participate written informed consent was obtained from each patient prior to participation in the study. ethical approval for the study to proceed was obtained from the nres committee london -westminster. rec reference /lo/ . competing interests dj has received speaker's fees from biomérieux. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord- -u f kvg authors: broeck, wouter van den; gioannini, corrado; gonçalves, bruno; quaggiotto, marco; colizza, vittoria; vespignani, alessandro title: the gleamviz computational tool, a publicly available software to explore realistic epidemic spreading scenarios at the global scale date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: u f kvg background: computational models play an increasingly important role in the assessment and control of public health crises, as demonstrated during the h n influenza pandemic. much research has been done in recent years in the development of sophisticated data-driven models for realistic computer-based simulations of infectious disease spreading. however, only a few computational tools are presently available for assessing scenarios, predicting epidemic evolutions, and managing health emergencies that can benefit a broad audience of users including policy makers and health institutions. results: we present "gleamviz", a publicly available software system that simulates the spread of emerging human-to-human infectious diseases across the world. the gleamviz tool comprises three components: the client application, the proxy middleware, and the simulation engine. the latter two components constitute the gleamviz server. the simulation engine leverages on the global epidemic and mobility (gleam) framework, a stochastic computational scheme that integrates worldwide high-resolution demographic and mobility data to simulate disease spread on the global scale. the gleamviz design aims at maximizing flexibility in defining the disease compartmental model and configuring the simulation scenario; it allows the user to set a variety of parameters including: compartment-specific features, transition values, and environmental effects. the output is a dynamic map and a corresponding set of charts that quantitatively describe the geo-temporal evolution of the disease. the software is designed as a client-server system. the multi-platform client, which can be installed on the user's local machine, is used to set up simulations that will be executed on the server, thus avoiding specific requirements for large computational capabilities on the user side. conclusions: the user-friendly graphical interface of the gleamviz tool, along with its high level of detail and the realism of its embedded modeling approach, opens up the platform to simulate realistic epidemic scenarios. these features make the gleamviz computational tool a convenient teaching/training tool as well as a first step toward the development of a computational tool aimed at facilitating the use and exploitation of computational models for the policy making and scenario analysis of infectious disease outbreaks. the h n influenza pandemic highlighted the importance of computational epidemic models for the real-time analysis of the health emergency related to the global spreading of new emerging infectious diseases [ ] [ ] [ ] . realistic computational models are highly complex and sophisticated, integrating substantial amounts of data that characterize the population and geographical context in order to attain superior accuracy, resolution, and predictive power [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the challenge consists in developing models that are able to capture the complexity of the real world at various levels by taking advantage of current information technology to provide an in silico framework for testing control scenarios that can anticipate the unfolding of an epidemic. at the same time, these computational approaches should be translated into tools accessible by a broader set of users who are the main actors in the decision-making process of health policy, especially during an emergency like an influenza pandemic. the tradeoff between realistic and accurate descriptions of large-scale dynamics, flexibility, computational feasibility, ease of use, and accessibility of these tools creates a major challenge from both the theoretical and the computational points of view [ , , , , , ] . gleamviz is a client-server software system that can model the world-wide spread of epidemics for human transmissible diseases like influenzalike illnesses (ili), offering extensive flexibility in the design of the compartmental model and scenario setup, including computationally-optimized numerical simulations based on high-resolution global demographic and mobility data. gleamviz makes use of a stochastic and discrete computational scheme to model epidemic spread called "gleam" -global epidemic and mobility model, presented in previously published work [ , , ] which is based on a geo-referenced metapopulation approach that considers , subpopulations in countries of the world, as well as air travel flow connections and short-range commuting data. the software includes a client application with a graphical user interface (gui) for setting up and executing simulations, and retrieving and visualizing the results; the client application is publicly downloadable. the server application can be requested by public institutions and research centers; conditions of use and possible restrictions will be evaluated specifically. the tool is currently not suitable for the simulation of vector-borne diseases, infection transmission depending on local contact patterns such as sexually transmitted diseases and diseases with a time scale that would make demographic effects relevant. the tool, however, allows the introduction of mitigation policies at the global level. localized intervention in space or time can be implemented in the gleam model and their introduction in the gleamviz computational tool are planned for future releases. only a few computational tools are currently available to the public for the analysis and modeling of epidemics. these range from very simple spreadsheet-based models aimed at providing quick estimates for the number of patients and hospitalizations during a pandemic (see e.g. flusurge [ ] ) to more complicated tools based on increasingly sophisticated simulation approaches [ , , , , , ] . these tools differ in their underlying modeling approaches and in the implementation, flexibility, and accessibility of the software itself. influsim is a tool that provides a visual interface to simulate an epidemic with a deterministic compartmental model in a single population [ ] . the model includes age structure and explicit sojourn times with different stages in each compartment, extending an seir compartmentalization to include hospitalizations and intervention measures. the software provides the infectious disease dynamics and the user can set parameter values and add or remove interventions. however, no spatial structure or other forms of heterogeneity and stochasticity are considered in the model. on the other hand agent-based models describe the stochastic propagation of a disease at the individual level, thus taking into account the explicit social and spatial structure of the population under consideration. communityflu is a software tool that simulates the spread of influenza in a structured population of approximately , households with , persons [ ] . user interaction with the software is limited to the spreadsheet portion of the program, where one can choose the type of intervention and other parameters describing the disease and the population. a larger population is considered in flute, a publicly available tool for the stochastic simulation of an epidemic in the united states at the level of individuals [ ] . the model is based on a synthetic population, structured in a hierarchy of mixing social groups including households, household clusters, neighborhoods, and nation-wide communities. flute comes with a configuration file in text format that can be modified by an expert user to set various parameters such as the initiation of the epidemic, the reproductive number, and the interventions considered. no gui is provided, and the output of the simulations is given in the form of text files that must be analyzed through additional software. epifast involves a parallel algorithm implemented using a master-slave approach which allows for scalability on distributed memory systems, from the generation of synthetic population aggregated in mixing groups to the explicit representation of the contact patterns between individuals as they evolve in time [ ] . the epi-fast tool allows for the detailed representation and simulation of the disease on social contact networks among individuals that dynamically evolve in time and adapt to actions taken by individuals and public health interventions. the algorithm is coupled with a webbased gui and the middleware system didactic, which allows users to specify the simulation setup, execute the simulation, and visualize the results via plots. epidemic models and interventions are pre-configured, and the system can scale up to simulate a population of a large metropolitan area on the order of tens of millions of inhabitants. another class of models focuses on the global scale, by using a metapopulation approach in which the population is spatially structured into patches or subpopulations (e.g. cities) where individuals mix. these patches are connected by mobility patterns of individuals. in this vein two tools are currently available. the global epidemic model (gem) uses a metapopulation approach based on an airline network comprised of major metropolitan areas in the world for the stochastic simulation of an influenza-like illness [ ] . the tool consists of a java applet in which the user can simulate a hypothetical h n outbreak and test pre-configured intervention strategies. the compartmentalization is set to an seir model, and the parameterization can be modified in the full or stand-alone mode, but not currently in the java applet. the spatiotemporal epidemiological modeler (stem) is a modeling system for the simulation of the spread of an infectious disease in a spatially structured population [ ] . contrary to other approaches, stem is based on an extensible software platform, which promotes the contribution of data and algorithms by users. the resulting framework therefore merges datasets and approaches and its detail and realism depend on continuous developments and contributions. however, these are obtained from a variety of sources and are provided in different formats and standards, thus resulting in possible problems related to the integration and merging of datasets. such issues are left to the user to resolve. the existing tools described above thus offer the opportunity to use highly sophisticated data-driven approaches at the expense of flexibility and ease of use by non-experts on the one hand, or very simplified models with user-friendly guis and no specific computational requirements on the other. our approach aims at optimizing the balance of complex and sophisticated data-driven epidemic modeling at the global scale while maintaining an accessible computational speed and overall flexibility in the description of the simulation scenario, including the compartmental model, transition rates, intervention measures, and outbreak conditions by means of a user-friendly gui. in the gleamviz tool the setup of the simulations is highly flexible in that the user can design arbitrary disease compartmental models, thus allowing an extensive range of human-to-human infectious diseases and intervention strategies to be considered. the user interface has been designed in order to easily define both features specific to each compartment, such as the mobility of classes of individuals, and general environmental effects, such as seasonality for diseases like influenza. in addition, the user can define the initial settings that characterize the initial geographical and temporal conditions, the immunity profile of the population, and other parameters including but not limited to: the definition of an outbreak condition in a given country; the number of stochastic runs to be performed; and the total duration of each simulation. the tool allows the production of global spreading scenarios with geographical high resolution by just interacting with the graphic user interface. while an expert input would be required to interpret and discuss the results obtained with the software, the present computational platform facilitates the generation and analysis of scenarios from intensive data-driven simulations. the tool can be deployed both in training activities as well as to facilitate the use of large-scale computational modeling of infectious diseases in the discussion between modelers and public health stakeholders. the paper is organized as follows. the "implementation" section describes the software application architecture and its major components, including the computational model gleam. the "results and discussion" section presents in detail the gleamviz client and its components that allow for software-user interaction, including an application of the simulator to an influenza-like-illness scenario. the top-level architecture of the gleamviz tool comprises three components: the gleamviz client application, the gleamviz proxy middleware, and the simulation engine. the latter two components constitute the gleamviz server, as shown in figure . users interact with the gleamviz system by means of the client application, which provides graphical userinterfaces for designing and managing the simulations, as well as visualizing the results. the clients, however, do not themselves run the simulations. instead they establish a connection with the gleamviz proxy middleware to request the execution of a simulation by the server. multiple clients can use the same server concurrently. upon receipt of requests to run a simulation, the middleware starts the simulation engine instances required to execute the requests and monitors their status. once the simulations are completed, the gleamviz proxy middleware collects and manages the resulting simulation data to be served back to the clients. a schematic diagram of the workflow between client and server is shown in figure . this client-server model allows for full flexibility in its deployment; the client and server can be installed on the same machine, or on different machines connected by a local area network or the internet. the two-part decomposition of the server in terms of middleware and engines additionally allows for advanced high-volume setups in which the middleware server distributes the engine instances over a number of machines, such as those in a cluster or cloud. this architecture thus ensures high speed in large-scale simulations and does not rely on the cpu-specific availability accessible by the user. the gleamviz simulation engine uses a stochastic metapopulation approach [ ] [ ] [ ] , [ ] [ ] [ ] ] that considers data-driven schemes for the short-range and design the compartmental model of the infectious disease in the model builder. configure the simulation of the world-wide epidemic spreading in the simulation wizard. submit the simulation for execution by the engine on the server. inspect the results of a simulation in the interactive visualization. inspect all simulations and retrieve results in the simulations history. long-range mobility of individuals at the inter-population level, coupled with coarse-grained techniques to describe the infection dynamics within each subpopulation [ , ] . the basic mechanism for epidemic propagation occurs at multiple scales. individuals interact within each subpopulation and may contract the disease if an outbreak is taking place in that subpopulation. by travelling while infected, individuals can carry the pathogen to a non-infected region of the world, thus starting a new outbreak and shaping the spatial spread of the disease. the basic structure of gleam consists of three distinct layers -the population layer, the mobility layer, and the epidemic layer (see figure ) [ , ] . the population layer is based on the high-resolution population database of the gridded population of the world project by the socio-economic data and applications center (sedac) [ ] that estimates population with a granularity given by a lattice of cells covering the whole planet at a resolution of × minutes of arc. the mobility layer integrates short-range and longrange transportation data. long-range air travel mobility is based on travel flow data obtained from the international air transport association (iata [ ]) and the official airline guide (oag [ ] ) databases, which contain the list of worldwide airport pairs connected by direct flights and the number of available seats on any given connection [ ] . the combination of the population and mobility layers allows for the subdivision of the world into geo-referenced census areas obtained by a voronoi tessellation procedure around transportation hubs. these census areas define the subpopulations of the metapopulation modeling structure, identifying , subpopulations centered on iata airports in different countries. the model simulates the mobility of individuals between these subpopulations using a stochastic procedure defined by the airline transportation data [ ] . short-range mobility considers commuting patterns between adjacent subpopulations based on data collected and analyzed from more than countries in continents across the world [ ] . it is modeled with a time-scale separation approach that defines the effective force of infections in connected subpopulations [ , , ] . on top of the population and mobility layers lies the epidemic layer, which defines the disease and population dynamics. the infection dynamics takes place within each subpopulation and assumes a compartmentalization [ ] that the user can define according to the infectious disease under study and the intervention measures being considered. all transitions between compartments are modeled through binomial and multinomial processes to preserve the discrete and stochastic nature of the processes. the user can also specify the initial outbreak conditions that characterize the spreading scenario under study, enabling the seeding of the epidemic in any geographical census area in the world and defining the immunity profile of the population at initiation. seasonality effects are still an open problem in the transmission of ili diseases. in order to include the effect of seasonality on the observed pattern of ili diseases, we use a standard empirical approach in which population layer short-range mobility layer long-range mobility layer the short-range mobility layer covers commuting patterns between adjacent subpopulations based on data collected and analyzed from more than countries on continents across the world, modeled with a time-scale separation approach that defines the effective force of infections in connected subpopulations. the long-range mobility layer covers the air travel flow, measured in available seats between worldwide airport pairs connected by direct flights. seasonality is modeled by a forcing that reduces the basic reproductive number by a factor α min ranging from . to (no seasonality) [ ] . the forcing is described by a sinusoidal function of months-period that reaches its peak during winter time and its minimum during summer time in each hemisphere, with the two hemispheres with opposite phases. given the population and mobility data, infection dynamics parameters, and initial conditions, gleam performs the simulation of stochastic realizations of the worldwide unfolding of the epidemic. from these in silico epidemics a variety of information can be gathered, such as prevalence, morbidity, number of secondary cases, number of imported cases, hospitalized patients, amounts of drugs used, and other quantities for each subpopulation with a time resolution of day. gleam has been under continuous development since and during these years it has been used: to assess the role of short-range and long-range mobility in epidemic spread [ , , ] ; to retrospectively analyze the sars outbreak of - in order to investigate the predictive power of the model [ ] ; to explore global health strategies for controlling an emerging influenza pandemic with pharmaceutical interventions under logistical constraints [ ] ; and more recently to estimate the seasonal transmission potential of the h n influenza pandemic during the early phase of the outbreak to provide predictions for the activity peaks in the northern hemisphere [ , ] . the gleamviz simulation engine consists of a core that executes the simulations and a wrapper that prepares the execution based on the configuration relayed from the client by the gleamviz proxy middleware. the engine can perform either single-run or multi-run simulations. the single-run involves only a single stochastic realization for a given configuration setup and a random seed. the multi-run simulation involves a number of stochastic realizations as set by the user and performed by the core (see the following section), each with the same configuration but with a different random seed. the results of the multi-run simulation are then aggregated and statistically analyzed by the wrapper code. the simulation engine writes the results to files and uses lock files to signal its status to the middleware component. the core is written in c++, resulting in a fast and efficient engine that allows the execution of a single stochastic simulation of a -year epidemic with a standard seir model in a couple of minutes on a high-end desktop computer. the wrapper code is written in python [ ] . the server components can be installed on most unix-like operating systems such as linux, bsd, mac os x, etc. the gleamviz proxy middleware is the server component that mediates between clients and simulation engines. it accepts tcp connections from clients and handles requests relayed over these connections, providing client authorization management. a basic access control mechanism is implemented that associates a specific client with the simulations it launches by issuing a private simulation identifier key upon submission. users can only retrieve the results of the simulations they launched, or simulations for which they have obtained the simulation definition file -containing the private simulation identifier key-from the original submitter. upon receipt of a request to execute a simulation, the middleware sets up the proper system environment and then launches an instance of the simulation engine with the appropriate configuration and parameters according to the instructions received from the client. for singlerun simulations, the daily results are incrementally served back to the client while the simulation is being executed. this allows for the immediate visualization of the spreading pattern, as described in "visualization interface" subsection. for multi-run simulations the results are statistically analyzed after all runs are finished, and the client has to explicitly request the retrieval of the results once they become available. the gleamviz proxy server component can be configured to keep the simulation data indefinitely or to schedule the cleanup of old simulations after a certain period of time. multi-run metadata is stored in an internal object that is serialized on a system file, ensuring that authorization information is safely kept after a server shutdown or failure. the gleamviz proxy component additionally provides control features such as accepting administrative requests at runtime in order to manage stored simulations or to modify several configuration parameters like the number of simultaneous connections allowed, the number of simultaneous simulations per client, the session timeout, etc. the middleware server is written in python [ ] and uses the twisted matrix library suite [ ] for its networking functionality. client and server communicate using a special purpose protocol, which provides commands for session handling and simulation management. commands and data are binary encoded using adobe action message format (amf ) in order to minimize bandwidth needs. the gleamviz client is a desktop application by which users interact with the gleamviz tool. it provides guis for its four main functions: ) the design of compartmental models that define the infection dynamics; ) the configuration of the simulation parameters; ) the visualization of the simulation results; and ) the management of the user's collection of simulations. in the following section we describe these components in detail. the client was developed using the adobe air platform [ ] and the flex framework [ ] and can thus be deployed on diverse operating systems, including several windows versions, mac os x, and several common linux distributions. the gleamviz client has a built-in updating mechanism to check for the latest updates and developments and prompts the user to automatically download them. it also offers a menu of configuration options of the interface that allows the user to customize preferences about data storage, visualization options, the server connection, and others. the software system presented above is operated through the gleamviz client, which provides the user interface: the part of the tool actually experienced on the user side. the gleamviz client integrates different modules that allow the management of the entire process flow from the definition of the model to the visualization of the results. in the following we will describe the various components and provide the reader with a user study example. the model builder provides a visual modeling tool for designing arbitrary compartmental models, ranging from simple sir models to complex compartmentalization in which multiple interventions can be considered along with disease-associated complications and other effects. (an example can be found in previous work [ ] .) a snapshot of the model builder window is shown in figure . the models are represented as flow diagrams with stylized box shapes that represent compartments and directed edges that represent transitions, which is consistent with standard representations of compartmental models in the literature. through simple operations like 'click and drag' it is possible to create any structure with full flexibility in the design of the compartmentalization; the user is not restricted to a given set of pre-loaded compartments or transition dynamics. the interactive interface provided by the model builder enables the user to define the compartment label, the mobility constraints that apply (e.g. allowed/not allowed to travel by air or by ground), whether the compartment refers to clinical cases, as well as the color and position of their representation in the diagram (see figure ). this allows the user to model many kinds of human-to-human infectious diseases, in particular respiratory and influenza-like diseases. transitions individuals is equal to  si n , where n is the total size of the subpopulation. the gleam simulation engine considers discrete individuals. all its transition processes are both stochastic and discrete, and are modeled through binomial and multinomial processes. transitions can be visually added by dragging a marker from the source to the target compartment. spontaneous transitions are annotated with their rates, which can be modified interactively. infection transitions are accompanied with a representation of the infection's source compartment and the applicable rate (i.e. b in the example above), which can also be modified in an interactive way. the rates can be expressed in terms of a constant value or in terms of a variable whose value needs to be specified in the variables table, as shown in figure . the value can also be expressed by simple algebraic expressions. the client automatically checks for and reports inconsistencies in the model in order to assist the user in the design process (see bottom right window in figure ). models can be exported to xml files and stored locally, allowing the user to load a model later, modify it, and share it with other users. the diagram representation can be exported as a pdf or svg file for use in documentation or publications. a few examples of compartmental models are available for download from the simulator website. the simulation wizard provides a sequence of panels that leads the user through the definition of several configuration parameters that characterize the simulation. figure shows some of these panels. the consecutive steps of the configuration are as follows: •choice of the type of the simulation (panel a) the user is prompted with three options: create a new single-run simulation or a new multi-run simulation from scratch, or a new one based on a saved simulation previously stored in a file. •compartmental model selection and editing the user can design a new compartmental model, modify the current compartmental model (when deriving it from an existing simulation), or load a model compartmentalization from a file. •definition of the simulation parameters (panel c) the user is asked to specify various settings and parameter values for the simulation, including, e.g., the number of runs to perform (only accessible in the case of a multi-run), the initial date of the simulation, the length of the simulation (in terms of days), whether or not seasonality effects should be considered, the airplane occupancy rate, the commuting time, the conditions for the definition of an outbreak, and others. •initial assignment of the simulation (panel d) here the user assigns the initial distribution of the population amongst compartments, defining the immunity profile of the global population on the starting date. •definition of the outbreak start (panel e) this panel allows the user to define the initial conditions of the epidemic by selecting the city (or cities) seeded with the infection. •selection of output results (panel f) here the user selects the compartments that will constitute the output provided by the client at the end of the simulation. the corresponding data will be shown in the visualization window and made available for download. when all the above configuration settings are defined, the user can submit the simulation to the gleamviz server for execution. this will automatically add the simulation to the user's simulations history. it is furthermore possible to save the definition of the simulation setup to a local file, which can be imported again later or shared with other users. the simulations history is the main window of the client and provides an overview of the simulations that the user has designed and/or submitted, in addition to providing access to the model builder, the simulation wizard, and the visualization component. the overview panel shown in figure lists the simulation identifier, the submission date and time, the simulation type (i.e., single or multi-run), the execution status (i.e., initialized, start pending, started, aborted, complete, failed, or stop pending) and the results status (i.e., none, retrieve pending, retrieving, stop retrieve pending, complete, or stored locally). additional file provides a detailed explanation of all these values. a number of context-dependent command buttons are available once a simulation from the list is selected. those buttons allow the user to control the simulation execution, retrieve the results from the server and visualize them, clone and edit the simulation to perform a new execution, save the simulation definition or the output data to the local machine (in order to analyze the obtained data with other tools, for example), and remove the simulation. in addition to exporting the compartmental model (see the "model builder" subsection) the user can export a complete configuration of a simulation that includes the compartmental model and the entire simulation setup to a local file, which can be imported again later or shared with other users. once the execution of a simulation is finished and the results have been retrieved from the server, the client can display the results in the form of an interactive visualization of the geo-temporal evolution of the epidemic. this visualization consists of a temporal and geographic mapping of the results accompanied by a set of graphs (see figure ). the geographic mapping involves a zoomable multi-scale map on which the cells of the population layer are colored according to the number of new cases of the quantity that is being displayed. several visualization features can be customized by clicking on the gear icon and opening the settings widget. it is possible to zoom in and out and pan by means of the interface at the top left of the map. dragging the map with the mouse (on a location where there are no basin marks) can also pan the visualization. all the widgets and the graphs displayed over the map can be re-positioned according to the user's preferences by clicking and dragging the unused space in the title bar. the color coding of the map represents the number of cases on a particular day. the time evolution of the epidemic can be shown as a movie, or in the form of daily states by moving forward or backward by one day at a time. for single-run simulations it is also possible to show the airline transportation of the 'seeding' individuals by drawing the traveling edge between the origin and destination cities. in the case where the output quantity is a subset of the infectious compartments, the edges show the actual seeding of the infection. note that the evolution of the epidemic depends strongly on the model definition. for example, it is possible that some basins are infected by a latent individual that later develops the disease. in this case no seeding flight will be shown if only infectious compartments are selected as output. beside the geographical map, the visualization window displays two charts. one chart shows the number of new cases per , over time (incidence), and the other shows the cumulative number of new cases per , over time (size). for multi-run simulations, median values and corresponding % confidence intervals are shown. the menu above each chart combo lets the user choose the context for which the corresponding charts show incidence and size data. this context is either: global, one of three hemispheres, one continent, one region, one country, or one city. the currently selected day is marked by a vertical line in these plots, and the day number, counted from the initial date selected for the simulation, is shown by side of the time slider. here we present an example application of the gleamviz tool to study a realistic scenario for the mitigation of an emerging influenza pandemic. disease-control programs foresee the use of antiviral drugs for treatment and shortterm prophylaxis until a vaccine becomes available [ ] . the implementation of these interventions rely both on logistical constraints [ , ] -related, e.g., to the availability of drugs -and on the characteristics of the infection, including the severity of the disease and the virus's potential to develop resistance to the drugs [ ] . here we focus on the mitigation effects of systematic antiviral (av) treatment in delaying the activity peak and reducing attack rate [ ] [ ] [ ] , , , , ] , and assume that all countries have access to av stockpiles. we consider a scenario based on the h n influenza pandemic outbreak and feed the simulator with the set of parameters and initial conditions that have been estimated for that outbreak through a maximum likelihood estimate by using the gleam model [ ] . the results provided by the present example are not meant to be compared with those contained in the full analysis carried out with gleam [ ] due to the fact that in the figure the simulation results can be inspected in an interactive visualization of the geo-temporal evolution of the epidemic. the map shows the state of the epidemic on a particular day with infected population cells color-coded according to the number of new cases of the quantity that is being displayed. pop-ups provide more details upon request for each city basin. the zoomable multi-scale map allows the user to get a global overview, or to focus on a part of the world. the media-player-like interface at the bottom is used to select the day of interest, or show the evolution of the epidemic like a movie. two sets of charts on the right show the incidence curve and the cumulative size of the epidemics for selectable areas of interest. present example we do not consider additional mitigation strategies that were put in place during the early phase of the outbreak, such as the sanitary control measures implemented in mexico [ , ] , or the observed reduction in international travel to/from mexico [ ] . indeed, the current version of gleamviz does not allow for interventions that are geographically and/or temporally dependent. however, these features are currently under development and will be available in the next software release. for this reason the simulation scenario that we study in this application of the simulator does not aim to realistically reproduce the timing of the spreading pattern of the h n pandemic. the results reported here ought to be considered as an assessment of the mitigating impact of av treatment alone, based on the initial conditions estimated for the h n outbreak, and assuming the implementation of the same av protocol in all countries of the world. we adopt a seir-like compartmentalization to model influenza-like illnesses [ ] in which we include the systematic successful treatment of % of the symptomatic infectious individuals (see figure ). the efficacy of the figure compartmental structure in each subpopulation in the intervention scenario. a modified susceptible-latent-infectious-recovered model is considered, to take into account asymptomatic infections, traveling behavior while ill, and use of antiviral drugs as a pharmaceutical measure. in particular, infectious individuals are subdivided into: asymptomatic (infectious_a), symptomatic individuals who travel while ill (infectious_s_t), symptomatic individuals who restrict themselves from travel while ill (infectious_s_nt), symptomatic individuals who undergo the antiviral treatment (infectious_avt). a susceptible individual interacting with an infectious person may contract the illness with rate beta and enter the latent compartment where he/she is infected but not yet infectious. the infection rate is rescaled by a factor ra in case of asymptomatic infection [ , ] , and by a factor ravt in case of a treated infection. at the end of the latency period, of average duration equal to eps - , each latent individual becomes infectious, showing symptoms with probability -p a , whereas becoming asymptomatic with probability p a [ , ] . change in travelling behavior after the onset of symptoms is modeled with probability p t set to % that individuals would stop travelling when ill [ ] . infectious individuals recover permanently after an average infectious period mu - equal to . days. we assume the antiviral treatment regimen to be administered to a % fraction (i.e. pavt = . ) of the symptomatic infectious individuals within one day from the onset of symptoms, reducing the infectiousness and shortening the infectious period of day. [ , ] . av treatment is accounted for in the model by a % reduction in the transmissibility of the disease by an infected person under av treatment when av drugs are administered in a timely fashion [ , ] . we assume that the drugs are administered within day of the onset of symptoms and that the av treatment reduces the infectious period by day [ , ] . the scenario with av treatment is compared to the baseline case in which no intervention is considered, i.e. the probability of treatment is set equal to in all countries. the gleamviz simulation results are shown in figure where the incidence profiles in two different regions of the world, north america and western europe, are shown for both the baseline case and the intervention scenario with av treatment. the results refer to the median (solid line) and % reference range (shaded area) obtained from stochastic realizations of each scenario starting from the same initial conditions. the resulting incidence profiles of the baseline case peak at around mid-november and the end of november in the us and western europe, respectively. these results show an anticipated peak of activity for the northern hemisphere with respect to the expected peak time of seasonal influenza. in order to make a more accurate comparison with the surveillance data in these regions, we should rely on the predictions provided by models that can take into account the full spectrum of strategies that were put in place during the h n outbreak, viz. the predictions obtained by gleam [ ] . in the case of a rapid and efficient implementation of the av treatment protocol at the worldwide level, a delay of about weeks would be obtained in the regions under study, a result that could be essential in gaining time to deploy vaccination campaigns targeting high-risk groups and essential services. in addition, the gleamviz tool provides simulated results for the number of av drugs used during the evolution of the outbreak. if we assume treatment delivery and successful administration of the drugs to % of the symptomatic cases per day, the number of av drugs required at the activity peak in western europe would be . courses per , persons, and the size of the stockpile needed after the first year since the start of the pandemic would be about % of the population. again, we assume a homogeneous treatment protocol for all countries in the world; results may vary from country to country depending on the specific evolution of the pandemic at the national level. computer-based simulations provide an additional instrument for emerging infectious-disease preparedness and control, allowing the exploration of diverse scenarios and the evaluation of the impact and efficacy of various intervention strategies. here we have presented a computational tool for the simulation of emerging ili infectious diseases at the global scale based on a datadriven spatial epidemic and mobility model that offers an innovative solution in terms of flexibility, realism, and computational efficiency, and provides access to sophisticated computational models in teaching/training settings and in the use and exploitation of large-scale simulations in public health scenario analysis. project name: gleamviz simulator v . project homepage: http://www.gleamviz.org/simulator/ operating systems (client application): windows (xp, vista, ), mac os x, linux. programming language: c++ (gleamsim core), python (gleamproxy, gleamsim wrapper), action-script (gleamviz) other requirements (client application): adobe air runtime, at least mb of free disk space. license: saas baseline scenario scenario with av figure simulated incidence profiles for north america and western europe in the baseline case (left panels) and in the av treatment scenario (right panels). the plots are extracted from the gleamviz tool visualization. in the upper plots of each pair the curves and shaded areas correspond to the median and % reference range of stochastic runs, respectively. the lower curves show the cumulative size of the infection. the dashed vertical line marks the same date for each scenario, clearly showing the shift in the epidemic spreading due to the av treatment. any restrictions to use by non-academics: none. the server application can be requested by public institutions and research centers; conditions of use and possible restrictions will be evaluated specifically. additional file : the gleamviz computational tool: additional file. this file includes information for installing the gleamviz client and details of the features of its various components. the transmissibility and control of pandemic influenza a (h n ) virus potential for a global dynamic of influenza a (h n ) seasonal transmission potential and activity peaks of the new influenza a(h n ): a monte carlo likelihood analysis based on human mobility modelling disease outbreaks in realistic urban social networks epifast: a fast algorithm for large scale realistic epidemic simulations on distributed memory systems multiscale mobility networks and the spatial spreading of infectious diseases strategies for containing an emerging influenza pandemic in southeast asia mitigation strategies for pandemic influenza in the united states mitigation measures for pandemic influenza in italy: an individual based model considering different scenarios flute, a publicly available stochastic influenza epidemic simulation model the influenza pandemic preparedness planning tool influsim an extensible spatial and temporal epidemiological modelling system centers for disease control and prevention (cdc) modeling the spatial spread of infectious diseases: the global epidemic and mobility computational model centers for disease control and prevention (cdc) controlling pandemic flu: the value of international air travel restrictions a mathematical model for the global spread of influenza assessing the impact of airline travel on the geographic spread of pandemic influenza forecast and control of epidemics in a globalized world delaying the international spread of pandemic influenza modeling the worldwide spread of pandemic influenza: baseline case and containment interventions predictability and epidemic pathways in global outbreaks of infectious diseases: the sars case study socioeconomic data and applications center (sedac). columbia university the architecture of complex weighted networks estimating spatial coupling in epidemiological systems: a mechanistic approach a structured epidemic model incorporating geographic mobility among regions infectious diseases in humans the role of airline transportation network in the prediction and predictability of global epidemics the modeling of global epidemics: stochastic dynamics and predictability modeling vaccination campaigns and the fall/winter activity of the new a (h n ) influenza in the northern hemisphere python programming language twisted matrix networking engine adobe flex framework modeling the critical care demand and antibiotics resources needed during the fall wave of influenza a (h n ) pandemic world health organization: pandemic preparedness antiviral treatment for the control of pandemic influenza: some logistical constraints hedging against antiviral resistance during the next influenza pandemic using small stockpiles of an alternative chemotherapy containing pandemic influenza with antiviral agents containing pandemic influenza at the source potential impact of antiviral drug use during influenza pandemic modelling of the influenza a(h n )v outbreak in mexico city secretaría de comunicaciones y transportes the who rapid pandemic assessment collaboration: pandemic potential of a strain of influenza a(h n ): early findings we are grateful to the international air transport association for making the airline commercial flight database available to us. this work has been partially funded by the nih r -da award, the lilly endowment grant - and the dtra- - award to av; the ec-ict contract no. (epiwork) to av, vc, and wvdb; the ec-fet contract no. (dynanets) to av and vc; the erc ideas contract n.erc- -stg (epifor) to vc, cg, and mq. authors' contributions cg, wvdb and bg designed the software architecture. wvdb and mq developed the client application. bg implemented the gleam engine. cg developed the proxy middleware. cg, vwdb, vc and av drafted the manuscript. mq and bg helped draft the manuscript. av and vc conceived and coordinated the software project, designed and coordinated the application study. all authors read and approved the final manuscript.competing interests av is consulting and has a research agreement with abbott for the modeling of h n diffusion. the other authors have declared that no competing interests exist. key: cord- - b a zc authors: liu, wen-kuan; liu, qian; chen, de-hui; liang, huan-xi; chen, xiao-kai; huang, wen-bo; qin, sheng; yang, zi-feng; zhou, rong title: epidemiology and clinical presentation of the four human parainfluenza virus types date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: b a zc background: human parainfluenza viruses (hpivs) are important causes of upper respiratory tract illness (urti) and lower respiratory tract illness (lrti). to analyse epidemiologic and clinical characteristics of the four types of human parainfluenza viruses (hpivs), patients with acute respiratory tract illness (arti) were studied in guangzhou, southern china. methods: throat swabs (n= ) were collected and tested from children and adults with arti over a -month period, and of ( . %) patients’ clinical presentations were recorded for further analysis. results: of patients tested, ( . %) were positive for hpiv. ninety-nine ( . %) samples were positive for hpiv- , ( . %) for hpiv- , ( . %) for hpiv- and ( . %) for hpiv- . / ( . %) hpiv-positive samples were from paediatric patients younger than years old, but no infant under one month of age was hpiv positive. seasonal peaks of hpiv- and hpiv- occurred as autumn turned to winter and summer turned to autumn. hpiv- and hpiv- were detected less frequently, and their frequency of isolation increased when the frequency of hpiv- and hpiv- declined. hpiv infection led to a wide spectrum of symptoms, and more “hoarseness” (p= . ), “abnormal pulmonary breathing sound” (p< . ), “dyspnoea” (p< . ), “pneumonia” (p= . ), and “diarrhoea” (p< . ) presented in hpiv-positive patients than hpiv-negative patients. / ( %) hpiv-positive adult patients (≥ years old) presented with systemic influenza-like symptoms, while / ( . %) hpiv-positive paediatric patients (< years old) presented with these symptoms (p= . ). the only significant difference in clinical presentation between hpiv types was “expectoration” (p< . ). co-infections were common, with . %– . % of samples positive for the four hpiv types also testing positive for other respiratory pathogens. however, no significant differences were seen in clinical presentation between patients solely infected with hpiv and patients co-infected with hpiv and other respiratory pathogens. conclusions: hpiv infection led to a wide spectrum of symptoms, and similar clinical manifestations were found in the patients with four different types of hpivs. the study suggested pathogenic activity of hpiv in gastrointestinal illness. the clinical presentation of hpiv infection may differ by patient age. human parainfluenza viruses (hpivs) are rna viruses in the genus paramyxoviridae. four hpiv types have been identified [ , ] . hpivs are important causes of upper respiratory tract illness (urti) and lower respiratory tract illness (lrti), especially in children [ , ] . an estimated five million lrti occur each year in the united states in children under years old, and hpivs have been isolated in up to one third of these infections [ ] [ ] [ ] . the hpiv- , hpiv- and hpiv- are second only to respiratory syncytial virus (rsv) as a cause of hospitalizations ( %- %) for acute respiratory infection among children aged younger than years in the united states [ , [ ] [ ] [ ] . compared with studies of hpiv infection in children, less is known about infections in adults. most hpiv infections in adults cause mild upper respiratory tract symptoms, but the elderly or those with compromised immune systems are at increased risk for severe hpiv * correspondence: zhou @yahoo.com † equal contributors infection [ , , [ ] [ ] [ ] . compared with types − , only a small number of reports have studied hpiv- [ ] [ ] [ ] [ ] [ ] , and the lack of epidemiologic data on hpiv- prevents a clear understanding of the full clinical pattern of hpivs. in addition, any differences in the clinical presentation of the four hpiv types are still largely unknown. the aims of this study were to explore the epidemiologic features and clinical manifestations of hpivs and other common respiratory pathogens in children and adults with acute respiratory tract illness (arti) in guangzhou, southern china, and to uncover clues that might help to establish clinical distinctions between different hpiv types. samples in this study were taken as part of standard care. the first affiliated hospital of guangzhou medical university ethics committee approved the experimental design and patient involvement in this study. written informed consent was obtained from the patient for publication of this report and any accompanying images. throat swab samples were collected from patients with arti (presenting with at least two of the following symptoms: cough, pharyngeal discomfort, nasal obstruction, coryza, sneeze, dyspnoea) at three hospitals in guangzhou, southern china between july and august . the samples were refrigerated at to °c and transported on ice to state key laboratory of respiratory diseases and analysed every working day or stored at − °c before testing. over samples were collected and tested during each month in our study. clinical presentations were collected and categorized retrospectively into the following six groups from the patients' medical records using designed presentation cards: urti, lrti, systemic influenza-like symptoms, gastrointestinal illness, neurologic symptom and others. patients with nasal obstruction, coryza, sneeze, cough, pharyngeal discomfort, or hoarseness were categorized as having urti. patients with pneumonia, bronchopneumonia, increasing lung markings, dyspnoea, or abnormal pulmonary breath sound were categorized as having lrti. patients with high fever (≥ °c), chills, dizziness, headache, myalgia or debilitation were categorized as having systemic influenza-like symptoms. patients with vomiting, poor appetite, or diarrhoea were categorized as having gastrointestinal illness. patients with convulsion were categorized as having an neurologic symptom. patients with other symptoms, including but not limited to rash, were classified as "others". some patients were assigned to multiple clinical presentation groups. pneumonia and bronchopneumonia were diagnosed by chest radiography. pneumonia was defined as an acute illness with radiographic pulmonary shadowing which was at least segmental or present in one lobe (excluding the bronchi); bronchopneumonia was defined as inflammation of the walls of the smaller bronchial tubes, with varying amounts of pulmonary consolidation due to spread of the inflammation into peribronchiolar alveoli and the alveolar ducts. other clinical symptoms were identified by common medical examinations and clinical descriptions. real-time polymerase chain reaction (pcr) for detection of hpiv and other common respiratory pathogens dna or rna from respiratory samples was extracted using qiaamp dna mini kit or qiaamp viral rna mini kit (qiagen co. ltd., shanghai, china) in accordance with the manufacturer's protocols. the four types of hpiv were tested by taqman real-time pcr as previously reported [ ] , and other common respiratory pathogens were also selected for taqman real-time pcr testing, including influenza a virus (infa), influenza b virus (infb), respiratory syncytial virus (rsv), adenovirus (adv), enterovirus (ev), human metapneumovirus (hmpv), four strains of human coronavirus (hcov- e, oc , nl and hku ), human bocavirus (hbov), mycoplasma pneumoniae (mp), and chlamydophila pneumoniae (cp) [ ] . for comparisons of categorical data, χ test and fisher's exact test were used where appropriate. all tests were twotailed and p< . was considered statistically significant. we tested samples for hpiv and other respiratory pathogens between july and august in guangzhou, southern china. the median age was . (interquartile range, . to . ), and ranged from one day to years. pathogens were detected in / ( . %) samples, and were detected in a higher proportion of samples from children (< years old) ( / ; . %) than from adults (≥ years old) ( / ; . %) (p< . ). the pathogens identified most frequently were infa ( / ; . %), rsv ( / ; . %) and mp ( / ; . %) ( table ) . hpivs were identified in / ( . %) samples. ninety-nine ( . %) samples were positive for hpiv- , ( . %) for hpiv- , ( . %) for hpiv- and ( . %) for hpiv- (table ). some samples were positive for multiple hpiv types, therefore the sum of these segments is more than . groups. the large majority ( / ; . %) of hpivpositive samples were from paediatric patients younger than years old ( figure ), but no infant under one month of age was hpiv positive. hpiv- was isolated from patients aged one month to years. hpiv- was isolated predominantly from patients under years of age, and the highest frequency was found in those aged - months ( / ; . %). hpiv- was isolated from patients aged one month to years, and hpiv- was isolated from patients aged months to years old. hpiv- -positive patients were more evenly distributed across ages than hpiv- -positive patients, but like hpiv- the highest frequency of hpiv- was detected from patients aged - months ( / ; . %). hpiv- was not isolated as frequently as hpiv- or hpiv- , but the highest frequencies of hpiv- were found in patients' age groups of - months ( / ; . %) and - months ( / ; . %). only ( . %) patients were hpiv- positive in this study, and patient age ranged from months to years. of ( . %) hpivs-positive-patients were coinfected with other / concerned pathogens except hcov-hku and cp. pathogens with the highest frequency of co-infection with hpiv were rsv ( / ; . %) and mp ( / ; . %), followed by ev ( / ; . %) and hcov-oc ( / ; . %) ( table ). the vast majority of co-infections were in children ( / ; . %), especially in patients under years of age ( / ; . %) (figure ). the co-infection rate of hpiv- was . % ( / ) , the rate of hpiv- was % ( / ) the rate of hpiv- was . % ( / ) and the rate of hpiv- was . % ( / ) ( table ) . table ). significant differences were seen between hpiv-positive and hpiv-negative patients for pharyngeal discomfort (p< . ), hoarseness (p= . ), "abnormal pulmonary breathing sound" (p< . ), "dyspnoea" (p< . ), "pneumonia" (p= . ), and "diarrhoea" (p< . ) ( table ). in hpiv-positive samples, the proportion of patients with "bronchopneumonia" was . % ( / ) and the proportion with "increasing lung marking" was . % ( / ), although these proportions were not significantly different from hpiv-negative patients (p= . and . ). hpivpositive patients were less likely than hpiv-negative patients including phlegm rale, wheeze rale, bubbling rale, moist rale, and rhonchi. § two-tailed χ test, testing the distribution of each illness or diagnosis between hpiv-positive and hpiv-negative patients. ※ two-tailed χ test, testing the distribution of each illness or diagnosis between the four hpiv types. # two-tailed χ test, testing the distribution of each illness or diagnosis between patients solely infected with hpiv and those co-infected. to present with each of the six symptoms of "systemic influenza-like symptoms" (p< . ) ( table ) . the only significant difference in clinical characteristics between patients solely infected with hpiv and those coinfected was "hoarseness" (p= . ), present in patients with "single hpiv" but no co-infected patient ( table ) . the only significant difference in clinical presentation between hpiv types was "expectoration," in which / ( . %) hpiv- positive patients, / ( %) hpiv- positive patients, / ( . %) hpiv- positive patients and / ( . %) hpiv- positive patients presented with the symptom (p< . ) ( table ) . within hpiv-positive patients, / ( %) adult patients presented with systemic influenza-like symptoms, while / ( . %) paediatric patients presented with these symptoms (p= . ). hpivs are common respiratory pathogens and are important causes of urti and lrti [ , , , ] . previous studies have predominantly focused on hpiv- , hpiv- and hpiv- infection in children because of high positive rate and morbidity of three types of hpiv infection in children, therefore less is known about hpiv- infection and hpiv infection in adults [ ] . in this study, we analysed the characteristics of the four hpiv types in children and adults with arti in guangzhou, southern china over a month period. of the pathogens investigated in this study, hpivs were the sixth most frequently isolated. the predominant types were hpiv- and hpiv- , which is consistent with previous reports [ ] [ ] [ ] . hpivs were isolated with higher frequency from males than females, similar to the previous study [ ] . immunity to hpivs is incomplete, and infections occur throughout life [ ] . in this study, hpivs were detected in patients over a wide age distribution. however, many more children were infected than adults (p< . ), and the vast majority of hpiv infections occurred in patients under years of age. the four hpiv types differed in age distribution of patients infected. hpiv- was mainly detected in paediatric patients under years old, while hpiv- and hpiv- were isolated from a broader age distribution than hpiv- ( figure ). no infant under one month of age was hpiv positive. these results are in accordance with seroprevalence studies indicating that newborn infants have high levels of hpiv antibodies, and that these levels decrease substantially by to months of age [ ] . in this study, hpiv- was only detected in children ( months to years old). this result is different from previous studies in which fairly equal infection rates were reported among infants younger than one year old, preschool children, school age children and adults [ ] [ ] [ ] [ ] [ ] . the low hpiv- positivity in our study may have influenced our results. co-infection of hpiv with other respiratory pathogens was common for all four hpiv types, similar to previous reports [ ] [ ] [ ] . rsv, mp, ev and hcov-oc were the main co-detected pathogens in this study. co-infections were mostly found in children, especially in patients under years of age (figure ). this might indicate that immature immune systems of children leave them susceptive to potential pathogens. however, no significant differences in clinical presentation were seen between patients solely infected with hpiv and patients co-infected with hpiv and other respiratory pathogens ( table ) . biennial fall epidemics of hpiv- have been reported in previous studies [ , , , ] . hpiv- has been reported to cause infections biennially with hpiv- , to alternate years with hpiv- , or to cause yearly outbreaks [ , , ] . hpiv- is reported to have occurred annually during april to june in the united states [ ] . only a small number of studies have studied the epidemiology of hpiv- , and the numbers of infections were too low to clearly identify seasonal peaks in activity [ , [ ] [ ] [ ] [ ] [ ] . in this study, hpivs were isolated throughout the year. seasonal peaks of hpivs, driven mostly by hpiv- and hpiv- , occurred in the time when autumn turned to winter and summer turned to autumn (september to november ; september to october ; april to july ). these results differ from previous reports [ , , , ] . in addition, hpiv- and hpiv- did not show the competitive interaction described previously, where hpiv- activity appeared to be greater during years when hpiv- was not circulating [ ] . the different geographic location might lead to the different seasonal distributions of hpivs. hpiv- and hpiv- were not isolated as frequently as hpiv- and hpiv- , and the seasonal peak of hpiv- and hpiv- was in the turn of winter to spring (december to march ). the frequency of detection of these two hpiv types increased when detection of hpiv- and hpiv- declined. hpivs can cause a spectrum of respiratory illness, and more "hoarseness" (p= . ), "abnormal pulmonary breathing sound" (p< . ), "dyspnoea" (p< . ), and "pneumonia" (p= . ) presented in hpiv-positive patients than hpivnegative patients. in our study, more "diarrhoea" (p< . ) presented in hpiv-positive patients than hpiv-negative patients suggesting pathogenic activity of hpiv in gastrointestinal illness ( table ) . systemic influenza-like symptoms were not main presentations of hpiv-positive patients overall (table ), but hpiv-positive adult patients presented with significantly more systemic influenza-like symptoms than hpiv-positive paediatric patients (p= . ). this result suggests that the clinical presentation of hpiv infection may differ by patient age as previously shown for hbov [ ] . the relationship between hpiv infection and neurologic disease has been studied for many decades. in a previous report, children hospitalized with hpivs had serious febrile seizures [ ] . in contrast, we found no significant difference in convulsion between hpiv-positive and hpivnegative patients (p= . ). this study investigated presentations of the four hpiv types and attempted to distinguish between different types of hpivs by clinical presentation. however, because all four hpiv types caused a wide spectrum of symptoms, distinguishing hpiv types by clinical characteristics alone was not possible as the previous study [ ] . hpiv infection led to a wide spectrum of symptoms, and lrti was the significant presentation. similar clinical manifestations were found in the patients with four different types of hpivs. more diarrhoea found in hpiv-positive than hpiv-negative patients suggested pathogenic activity of hpiv in gastrointestinal illness. all adults hpivpositive patients suffered from systemic influenza-like symptoms suggested that the clinical presentation of hpiv infection may differ by patient age. co-infections were common for the four hpiv types. however, no significant differences were seen in clinical presentation between patients solely infected with hpiv and patients co-infected with hpiv and other respiratory pathogens. this study explored characteristics of the four hpiv types and provided novel insights into the epidemiology and clinical implications of hpivs. epidemiology and clinical impact of parainfluenza virus infections in otherwise healthy infants and young children < years old seasonal trends of human parainfluenza viral infections: united states respiratory syncytial virus and parainfluenza virus progress in the development of respiratory syncytial virus and parainfluenza virus vaccines acute lower respiratory infections in nonhospitalized children parainfluenza virus type : seasonality and risk of infection and reinfection in young children seasonal pattern in childhood viral lower respiratory tract infections in melbourne new vaccine surveillance network: population-based surveillance for hospitalizations associated with respiratory syncytial virus, influenza virus, and parainfluenza viruses among young children human parainfluenza virus-associated hospitalizations among children less than five years of age in the united states bronchiolitis-associated hospitalization among us children detection of parainfluenza virus in turbinate epithelial cells of postviral olfactory dysfunction patients long-term therapy with aerosolized ribavirin for parainfluenza virus respiratory tract infection in an infant with severe combined immunodeficiency pediatric hospitalizations for croup (laryngotracheobronchitis): biennial increases associated with human parainfluenza virus epidemics antigenic variation among newly isolated strains of parainfluenza type virus recovery and identification of human myxoviruses acute respiratory disease in infancy and childhood: present understanding and prospects for prevention the isolation of parainfluenza subtypes a and b in england and serological studies of their prevalence antigenic characterization of parainfluenza a and b by the hemagglutination-inhibition test and distribution of hi antibody in human sera detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china parainfluenza viruses new vaccine surveillance network. parainfluenza virus infection of young children: estimates of the population-based burden of hospitalization dual respiratory virus infections multicentered study of viral acute lower respiratory infections in children from four cities of argentina, - diagnosis and clinical significance of parainfluenza virus infections in children impact of viral respiratory diseases on infants and young children in a rural an urban area of southern west virginia hospitalized pediatric parainfluenza virus infections in a medical center epidemiology and clinical presentation of the four human parainfluenza virus types the authors declare that they have no competing interests.authors' contributions rz, w-kl and ql designed the study. w-kl, ql, h-xl, x-kc, w-bh performed the pathogens testing. d-hc, z-fy and sq collected clinical data. all authors participated in the data analysis. w-kl, ql and rz drafted the manuscript. all authors read and approved the final version of this manuscript. key: cord- - qh pblc authors: quah, jessica; jiang, boran; tan, poh choo; siau, chuin; tan, thean yen title: impact of microbial aetiology on mortality in severe community-acquired pneumonia date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: qh pblc background: the impact of different classes of microbial pathogens on mortality in severe community-acquired pneumonia is not well elucidated. previous studies have shown significant variation in the incidence of viral, bacterial and mixed infections, with conflicting risk associations for mortality. we aimed to determine the risk association of microbial aetiologies with hospital mortality in severe cap, utilising a diagnostic strategy incorporating molecular testing. our primary hypothesis was that respiratory viruses were important causative pathogens in severe cap and was associated with increased mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. methods: a retrospective cohort study from january to july was conducted in a tertiary hospital medical intensive care unit in eastern singapore, which has a tropical climate. all patients diagnosed with severe community-acquired pneumonia were included. results: a total of patients were in the study. microbial pathogens were identified in ( . %) patients. mixed viral-bacterial co-infections occurred in ( . %) of patients. isolated viral infections were present in patients ( . %); isolated bacterial infections were detected in patients ( . %). hospital mortality occurred in ( . %) patients. the most common bacteria isolated was streptococcus pneumoniae and the most common virus isolated was influenza a. univariate and multivariate logistic regression showed that serum procalcitonin, apache ii severity score and mixed viral-bacterial infection were associated with increased risk of hospital mortality. mixed viral-bacterial co-infections were associated with an adjusted odds ratio of . ( % ci . – . , p = . ) for hospital mortality. conclusions: respiratory viruses are common organisms isolated in severe community-acquired pneumonia. mixed viral-bacterial infections may be associated with an increased risk of mortality. the microbial aetiology of severe community-acquired pneumonia (cap) remains varied throughout the world, with influences due to seasonal climate change, outreach of vaccination programmes and pathogen endemicity [ , ] . two decades ago, it was thought that viruses played a minor role in the pathogenesis of severe cap, notwithstanding influenza epidemics [ ] . recent literature contradicts this and suggests that viruses are frequently found in severe cap [ , ] . advances in molecular techniques have improved the sensitivity, accuracy and turnaround time of microbial diagnostic tests [ , ] . the availability of highly multiplexed commercial tests kits for common viral and bacterial pathogens has enabled these tests to be performed in large numbers of patients simultaneously, and across a variety of clinical settings [ , ] . multiple respiratory viruses may be present concurrently, or co-exist with bacterial pathogens to cause disease [ ] [ ] [ ] . the reported incidence of viruses in severe cap resulting in respiratory failure ranges . % to % [ , ] . this variation in the detection rate of viruses reflects potential limited availability of test assays and heterogeneity of physician practices in viral microbial diagnostic tests performed [ , ] . postulated prohibitive factors against the routine performance of viral diagnostics tests in patients with severe cap may include a lack of clear clinical guidelines, perceived low cost-effectiveness and the paucity of effective anti-viral therapies for respiratory viruses other than influenza. the primary aim of our study was to determine the risk association of microbial aetiologies with hospital mortality in severe cap, utilising a diagnostic strategy that incorporated molecular testing. our primary hypothesis was that respiratory viruses were important causative pathogens in severe cap and was associated with increased mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. this was a retrospective cohort study performed in an -bed medical intensive care unit of a -bed tertiary teaching hospital, in singapore. ethics approval was obtained from the singhealth centralised institutional review board (cirb / /fp), with waiver of consent. adults above the age of admitted to the medical intensive care unit with severe cap from january to july were included. written and electronic medical records were reviewed. cap was defined as an acute infection of the lung parenchyma associated with acute chest radiographic infiltrates and or more of the following: fever of °c or higher or hypothermia of °c or less; a new cough; dyspnea not explained by other reasons; worsening cough or change in respiratory secretions in a patient with pre-existing chronic cough. these symptoms should have been present at the time of, or within h of, hospital admission. chest radiographs reported by the hospital radiologists were obtained to confirm the presence of radiographic pulmonary infiltrates. patients with shock requiring vasopressor support, mechanically ventilated or who have out of minor criteria for pneumonia severity as defined by the infectious diseases society of america/american thoracic society, are considered to have severe cap [ ] . the minor criteria include: respiratory rate of breaths per minute or greater; pao /fio ratio equal or less than ; multi-lobar pulmonary infiltrates; confusion or disorientation; blood urea nitrogen equal or greater than mg/dl; leukopenia with white blood cell count of less than , cells/mm ; hypothermia indicated by core body temperature less than °c; hypotension requiring aggressive fluid resuscitation. all patients with prior hospitalisation within days of study enrolment; on active chemotherapy for neoplastic diseases; receiving renal replacement therapy by haemodialysis and prisoners were excluded. immunocompromised patients were not excluded as they were likely to have cap microbial pathogens as well as opportunistic infections. the time of admission to the intensive care unit was verified with electronic records. all patients had at least set of aerobic and anaerobic blood cultures performed within h of admission. routine collection of endotracheal aspirates for gram stain and semi-quantitative bacterial cultures occurred within h of intubation. viral and atypical pathogen polymerase chain reaction (pcr) amplification tests were collected from endotracheal aspirates. sputum samples were sent for bacterial aerobic cultures, while nasopharyngeal swabs were performed for atypical pathogen and viral pcr amplification tests for subjects who did not require intubation. urinary samples were tested for the presence of urinary streptococcus pneumoniae antigen and legionella pneumophilia serogroup antigen in patients without anuria. where indicated, acid-fast staining and mycobacterial cultures of respiratory samples were performed. nucleic acid was extracted from swabs or respiratory samples using ez virus mini kit (cat no. , qiagen, germany) performed on a semi-automated system (ez , qiagen, germany), and stored at - °c for more detailed molecular testing. besides routine standard clinical testing, additional multiplex real-time pcr testing was performed for each extracted sample. anyplex written and electronic medical records were reviewed for clinical data and laboratory indices. the most severe value was recorded for analysis if any blood test was repeated more than once within h after intensive care admission. serum c-reactive protein was measured by particle enhanced immunoturbidimetry (cobas® crpl ) and serum procalcitonin was measured using sandwich immunoassay (cobas® elecsys brahms pct). variables such as the acute physiology and chronic health evaluation ii (apache ii) severity score, presence of shock, mechanical ventilation, intensive care unit length of stay, and mortality were captured prospectively as part of administrative clinical care audits. empirical antibiotics were deemed to be adequate if it adhered to the hospital antimicrobial guidelines. combination therapy with beta-lactams and macrolides were recommended. where melioidosis was suspected, carbapenems were preferred to other classes of beta-lactams. categorical variables were expressed as number (percentages) and normally distributed quantitative variables were expressed as mean (± standard deviation). categorical variables were compared with chi-square test or fisher's exact test, and quantitative variables were compared with t-test or mann-u whitney test. the primary outcome measure was all-cause hospital mortality. demographic and disease variables were included in the univariate and multivariable logistic regression model. pneumonia symptoms were excluded from regression analysis as there were no known associations with mortality in severe cap. the variable for number of co-existent pathogens was excluded from regression analysis due to significant overlap with mixed viral-bacterial co-infections, which was included in the multivariate model. the following severity indicators: shock, altered mentation and ventilator support were not included as these variables are incorporated in the apache ii severity score. mycobacterium tuberculosis was classified as a bacterium for the logistic regression. demographic and disease variables were then compared between patients with and without virus infections, for identification of potential risk factors associated with acquisition of respiratory viral infections. a p-value of < . was considered significant. missing data was excluded from univariate and multivariate analysis. stata special edition version . (statacorp llc, texas, usa) was used for statistical analysis. one hundred seventeen patients were admitted to the medical intensive care unit for severe cap within the study period. baseline characteristics of patients who suffered in-hospital mortality (n = , . %) were compared with patients who survived, in table . of note, the patients who did not survived were older compared to survivors ( . years vs . years, p = . ). other baseline demographic and co-morbidity variables were not significantly different. blood cultures, respiratory specimen cultures and respiratory specimen pcr testing for viruses and atypical bacteria were performed in all patients. results of microbiological tests are presented in table . causative microbial aetiologies were identified in . % of patients with specific organisms presented in table . majority of pathogens identified were respiratory viruses and bacteria. viruses were found in . % of patients (n = ) with the most common virus being influenza a. . % (n = ) of patients with influenza a received empirical oseltamivir on the basis on clinical suspicion. bacterial infections were found in . % of patients (n = ). . % of patients (n = ) had mixed virus and bacterial co-infections. patients ( . %) had only viral pathogens and patients ( . %) had only bacterial pathogens, found as the microbial aetiology for severe cap. using hospital mortality as the primary outcome, univariate logistic regression was performed (table ). patients who did not survive had a higher apache ii score and higher serum procalcitonin levels. the microbiological aetiology that was significantly associated with increased hospital mortality was the detection of mixed viral-bacterial co-infections. on multivariate analysis, apache ii severity score, serum procalcitonin and mixed viral-bacterial co-infections remained significantly associated with increased adjusted odd ratios for hospital mortality. while apache ii severity score is known to be predictive for mortality in severe cap, the study found that the presence of mixed viral-bacterial co-infections was associated with increased hospital mortality by an adjusted odds ratio of . ( % confidence interval . , . , p = . ). the patients with respiratory viruses detected (both isolated viral pathogens and mixed viral-bacterial co-infections) were compared with patients who did not have any respiratory virus infections, in table . the mean serum c-reactive protein was found to be greater ( . ± . mg/l vs. . ± . mg/l) in patients without respiratory viruses compared to patients with detection of respiratory viruses (table ). respiratory virus infection as a cause of severe acute respiratory distress syndrome is well-established in literature data was missing for patient [ ] [ ] [ ] . however, its significance as a contributory pathogen in the outcomes of severe cap is uncertain. in this study, we showed that respiratory viruses were as commonly found as bacteria ( . % vs . %), as an aetiological pathogen. mixed viral-bacterial co-infections occurred in . % of patients and was associated with an adjusted odds ratio of . for hospital mortality. the impact of respiratory viruses on the prognosis of severe cap remains unclear with recent studies demonstrating contradictory results. fisher et al., in a prospective -year microbiologic survey of nosocomial pneumonia and cap complicated by respiratory failure, showed that . % of patients had viral infections, which was associated with a hospital mortality of . % [ ] . however, siow et al., found that viral infections were independently associated with lower hospital mortality compared to other microbial aetiologies, with an adjusted odds ratio of . (ci . - . ; p = . ) [ ] . in light of these findings, accurate characterisation of the impact of microbial aetiology on the outcomes of severe cap is required to influence future development of rapid molecular diagnostics assays and novel antimicrobial therapies that would target both viruses and bacteria [ , ] . piralla et al. reviewed the microbiological data of severe cap in northern italy during winter-spring seasons over years and found that . % of patients had one or more respiratory viruses identified [ ] . the most common viruses isolated were influenza a and rhinovirus, similar with our findings. while our study was performed in a tropical country, local microbiological surveillance has shown that influenza epidemics occur twice annually [ ] . this would mean that influenza seasons occurred over the course of this study. the molecular mechanisms in the viral pathogenesis of severe pneumonia are most well studied in influenza a and streptococcus pneumoniae co-infections. viral infections alter host immune responses that increase susceptibility to bacterial infection through viral-induced interferons [ ] [ ] [ ] . on clinical suspicion alone, only . % (n = ) of patients with influenza a in this study received empirical oseltamivir. the authors postulate that incorporation of early influenza a diagnostic tests may decrease the time to effective anti-viral therapies. the second most common virus detected in our study was rhinovirus (n = ). the association of rhinovirus with severe pneumonia has previously been shown in a surveillance program for middle east respiratory syndrome coronavirus in saudi arabia [ ] . its genotypes a to c are associated with severe pneumonia, with in-hospital mortality rates from . to . % [ ] . patients who are immunocompromised or who have chronic lung disease are most at risk [ ] . there are several reasons why viral infections may have been less prominent as a cause of severe cap in prior decades. firstly, grève et al. performed a prospective observational study on physician practices in the use of respiratory virus diagnostics demonstrating that despite clinical guideline recommendations on testing of respiratory viruses during influenza season, less than half of patients admitted to the intensive care unit with pneumonia were tested for viral pathogens [ ] . this may have led to under-recognition of the true significance of viral pathogens and mixed viral-bacterial infections, on outcomes in severe pneumonia. other factors which may have contributed to underdiagnosis of viral pneumonias include the unavailability or cost of molecular diagnostic assays, and the lack of effective anti-viral therapies [ ] . however, the authors argue that in this age of globalisation, highly virulent respiratory viruses have the potential to spread rapidly. constant surveillance is required to detect outbreaks and for the implementation of isolation precautions in a timely manner [ , ] . understanding the significance of respiratory viruses in the pathogenesis of severe cap would guide administrators with resource allocation when implementing vaccination programs for at-risk populations. the high rates of compliance with performing respiratory aerobic cultures, blood aerobic and anaerobic cultures in this study, were in accordance with sepsis guidelines [ ] . the most common bacterial pathogen found was streptococcus pneumoniae ( . %), which is consistent with the known epidemiology of cap globally [ ] [ ] [ ] . gadsby et al., in a prior study, was able to demonstrate a bacterial yield of . % when respiratory specimens from patients with cap were tested with bacterial multiplex pcr [ ] . incorporating the use of bacterial multiplex pcr in future studies, may increase the rate of bacteria detection, and shed light on potential molecular synergisms between specific viruses and bacteria in the pathogenesis of severe cap. pulmonary tuberculosis is endemic in the region where this study was performed. in our study, patients had tuberculosis, one of whom had concomitant adenovirus infection while another had streptococcus pneumoniae. the initial presentation of pulmonary tuberculosis with clinical features consistent with severe cap has been described by tseng et al. [ ] , where % of patients with pulmonary tuberculosis presented with respiratory failure or septic shock. the authors postulate that pulmonary tuberculosis may play a role in increasing host susceptibility to severe infection with cap organisms. the authors recognise that there were several limitations to this study. firstly, while first-dose antibiotics would have been administered as soon as sepsis is identified, we are unable to accurately define the time between administration of antibiotics and collection of specimens for microbiological assessment. this may affect the yield [ , , ] . secondly, the inherent retrospective nature of this study increases the risk of bias in data collection. however, as part of pre-established intensive care unit clinical audits and with the availability of national health records, clinical data such as participant demographics, co-morbid illnesses and severity indicators such as apache ii were established at the time of intensive care admission and stored prospectively. thirdly, given the high population density of the country ( rd in the world) where this study was performed, the microbial epidemiology of severe cap may only be extrapolated to urban settings. the study is a single-centre survey conducted in of the acute general hospitals serving a population of . million in a land area of . km . hence, the epidemiology may lack generalisability when extrapolated to other tropical countries. the fourth limitation is that we included patients who were immunocompromised with typical cap organisms and survived. they may potentially have had opportunistic infections that were not detected, however, as they did not contribute to the mortality outcomes, we felt that the microbiological data contributed by these patients were useful in the understanding of the prevalence of various data are presented as number (%), mean ± standard deviation apache acute physiology and chronic health evaluation a data was missing for patients. b data was missing for patient classes of cap organisms. lastly, another potential limitation was that vaccination records could not be retrieved, and we were unable to ascertain its influences on microbial aetiologies of severe pneumonia in our study population. the main strength of the study is the characterisation of the epidemiology of microbial pathogens in severe cap. we were able to show that in a tropical environment, the viral and bacterial pathogens associated with severe cap were similar with regions with a seasonal climate. despite a lower-than-expected mortality rate for severe cap in our study ( . %) compared with international data [ ] [ ] [ ] [ ] , we were able to demonstrate that mixed viral-bacterial co-infections were independently associated with hospital mortality. respiratory viruses are important causative pathogens in severe cap and are associated with increased risk of mortality when present with bacterial pathogens in mixed viral-bacterial co-infections. abbreviations apache ii: acute physiology and chronic health evaluation ii; cap: community-acquired pneumonia; pcr: polymerase chain reaction etiology and outcome of severe community acquired pneumonia in immunocompetent adults etiology of community-acquired pneumonia and diagnostic yields of microbiological methods: a -year prospective study in norway epidemiology of community-acquired respiratory tract infections in adults the intensive care global study on severe acute respiratory infection (ic-glossari): a multicenter, multinational, -day inception cohort study viral-bacterial coinfection affects the presentation and alters the prognosis of severe community-acquired pneumonia molecular diagnosis of respiratory viruses the filmarray® respiratory panel: an automated, broadly multiplexed molecular test for the rapid and accurate detection of respiratory pathogens rapid viral diagnosis for acute febrile respiratory illness in children in the emergency department clinical utility of on-demand multiplex respiratory pathogen testing among adult outpatients burden of respiratory viruses in patients with acute respiratory failure community-acquired polymicrobial pneumonia in the intensive care unit: aetiology and prognosis clinical characteristics and outcomes in hospitalized patients with respiratory viral co-infection during the h n influenza pandemic management of severe communityacquired pneumonia: a survey on the attitudes of physicians in iberia and south america clinical practice of respiratory virus diagnostics in critically ill patients with a suspected pneumonia: a prospective observational study infectious diseases society of america/american thoracic society consensus guidelines on the management of community-acquired pneumonia in adults acute management and long-term survival among subjects with severe middle east respiratory syndrome coronavirus pneumonia and ards virus-induced acute respiratory distress syndrome: epidemiology, management and outcome influenza a (h n ) vs non-h n ards: analysis of clinical course a prospective one-year microbiologic survey of combined pneumonia and respiratory failure the use of polymerase chain reaction amplification for the detection of viruses and bacteria in severe communityacquired pneumonia the past decade in bench research into pulmonary infectious diseases: what do clinicians need to know? laboratory diagnosis of pneumonia in the molecular age frequency of respiratory viruses among patients admitted to intensive care units in seven consecutive winterspring seasons ( - ) in northern italy characterization of influenza activity based on virological surveillance of influenza-like illness in tropical singapore the immunology of influenza virusassociated bacterial pneumonia integrated clinical, pathologic, virologic, and transcriptomic analysis of h n influenza virus-induced viral pneumonia in the rhesus macaque kinetics of coinfection with influenza a virus and streptococcus pneumoniae etiology of severe communityacquired pneumonia during the hajj-part of the mers-cov surveillance program clinical and molecular characterization of rhinoviruses a, b, and c in adult patients with pneumonia human rhinoviruses and severe respiratory infections: is it possible to identify at-risk patients early? viral pneumonia and acute respiratory distress syndrome burden of acute respiratory disease of epidemic and pandemic potential in the who eastern mediterranean region: a literature review global threat of animal influenza viruses of zoonotic concern: then and now surviving sepsis campaign: international guidelines for management of severe sepsis and septic shock global and local epidemiology of communityacquired pneumonia: the experience of the capnetz network global changes in the epidemiology of community-acquired pneumonia community-acquired pneumonia requiring hospitalization among u.s adults comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia empirical use of fluoroquinolones improves the survival of critically ill patients with tuberculosis mimicking severe pneumonia impact of antibiotic therapy in severe community-acquired pneumonia: data from the infauci study predictive factors of mortality in severe community-acquired pneumonia: a model with data on the first h of icu admission viral infection in patients with severe pneumonia requiring intensive care unit admission etiology of severe pneumonia in the very elderly assessment of prognosis in patients with community-acquired pneumonia who require mechanical ventilation the authors would like to thank the following: ms. carmen kam, the resident biostatistician for verification of the study statistical analysis; nurses ms. wang xi qin, ms. goh yuan xuan, ms. lim qian ru for assistance in data collection; senior medical technologist ms. heng ying xuan, ms. lee hui zi for assistance in performance of pcr tests; dr. tay tunn ren for her tutelage in manuscript writing. the study was performed with a grant awarded from changi general hospital research grant in (grant reference number chf . ), for an amount of $ singapore dollars (equivalent to usd , conversion usd = sgd . ). the datasets analysed during this current study are available from the corresponding author on reasonable request. authors' contributions jlq contributed to the design, analysis, interpretation of the study; drafting and revision of the manuscript. bj contributed to the design, interpretation and microbiological analysis of the study. pct contributed to the design and data acquisition of the study. cs contributed to the design of the work, drafting and revision of the manuscript. tyt contributed to the conception, analysis and interpretation of the study, drafting and revision of the manuscript. all authors have given final approval of the version to be published and agree to be accountable for all aspects of the work in ensuring that questions related to the accuracy or integrity of any part of the work are appropriately investigated and resolved.ethics approval and consent to participate ethics approval was obtained from the singhealth centralised institutional review board, singapore. reference number: cirb / /fp. waiver of consent granted for this study. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- - krf yxz authors: li, xi; huang, yongbo; xu, zhiheng; zhang, rong; liu, xiaoqing; li, yimin; mao, pu title: cytomegalovirus infection and outcome in immunocompetent patients in the intensive care unit: a systematic review and meta-analysis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: krf yxz background: cytomegalovirus (cmv) infection is common in immunocompetent patients in intensive care units (icus). however, whether cmv infection or cmv reactivation contributes to mortality of immunocompetent patients remains unclear. methods: a literature search was conducted for relevant studies published before may , . studies reporting on cmv infection in immunocompetent patients in icus and containing × tables on cmv results and all-cause mortality were included. results: eighteen studies involving immunocompetent patients admitted to icus were included in the meta-analysis. the overall rate of cmv infection was % ( %ci – %, i( ) = %, n = ) and the cmv reactivation was % ( %ci – %, i( ) = %, n = ). the odds ratio (or) for all-cause mortality among patients with cmv infection, compared with those without infection, was . ( %ci . – . , i( ) = %, n = ). moreover, upon exclusion of studies in which antiviral treatment was possibly or definitely provided to some patients, the association of mortality rate with cmv infection was also statistically significant (or: . , %ci . – . , i( ) = %, n = ,). for cmv seropositive patients, the or for mortality in patients with cmv reactivation as compared with patients without cmv reactivation was . ( %ci . – . , i( ) = %, n = ). patients with cmv infection required significantly longer mechanical ventilation (mean difference (md): days ( % ci – , i( ) = %, n = )) and longer duration of icu stay (md: days ( % ci – , i( ) = %, n = )) than patients without cmv infection. when analysis was limited to detection in blood, cmv infection without antiviral drug treatment or reactivation was not significantly associated with higher mortality (or: . , %ci . – . , i( ) = %, n = ; or: . , i( ) = %, n = ). conclusion: critically ill patients without immunosuppression admitted to icus show a high rate of cmv infection. cmv infection during the natural unaltered course or reactivation in critically ill patients is associated with increased mortality, but have no effect on mortality when cmv in blood. more studies are needed to clarify the impact of cmv infection on clinical outcomes in those patients. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. human cytomegalovirus (cmv) is a prototypic member of the β herpes virus subfamily [ ] . the prevalence of cmv seropositivity in human populations is roughly - % [ ] [ ] [ ] and highest amongst older people [ ] . cytomegalovirus infection induces innate immune responses (eg. natural killer cells) and adaptive immunity (eg. cd +/cd + t cells). however, the virus can evade host detection by expressing genes that interfere with both the innate and adaptive immune systems. eventually, cmv is able to establish latency in which either the host fails to eliminate the virus or the virus cannot replicate. however, cmv can become reactivated during periods of host immune suppression [ ] . it is well known that cmv infection is common in canonical immunodeficiency patients, such as those with human immunodeficiency virus infection, solid organ or stem cell transplantation and patients undergoing chemoor radiotherapy [ ] [ ] [ ] . with the development of more sensitive detection method, the rate of cmv detection is high in intensive care units (icus) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . however, so far, there is no convincing research to support the use of antiviral treatment when critically ill but immunocompetent patients present with cmv infection. furthermore, whether cmv is a contributor or simply a bystander to the severity of illness remains under debate [ ] [ ] [ ] . whether cmv infection is associated with increased mortality in immunocompetent icu patients remains controversial [ ] [ ] [ ] [ ] . a previous meta-analysis published in demonstrated that cmv infection was associated with a higher mortality rate, nearly twice that observed in patients without cmv infection [ ] . however, this study did not consider the influence of antiviral drugs on clinical outcomes. moreover, many clinical studies about cmv have been reported in recent years. thus, to acquire a better understanding of the potential role of cmv infection in contributing to mortality in critically ill patients, especially those not receiving antiviral agents and cmv detected in blood, we performed a meta-analysis of data available in the literature, focusing on the outcome in immunocompetent icu patients with cmv infection. a literature search for relevant publications included within the electronic databases pubmed, embase and the cochrane library was performed using combinations of the keywords "cytomegaloviruses", "salivary gland viruses", "herpes virus", "cytomegaloviral infection", "hhv ", "intensive care", "critical care", "critical illness", "mechanical ventilation", and "pulmonary ventilator". all searches were updated on may , . no language restriction was enforced. we also consulted relevant reference articles and searched using google scholar. two researchers (lx and hyb) performed data extraction independently, and any discrepancies were addressed by discussion and reevaluation until consensus was achieved. observational studies were eligible if they reported on cmv infection in immunocompetent patients in the icu, and if a × table could be constructed based on cmv results and all-cause mortality. all patients were over years of age. the systematic review included only studies in which all patients were tested for cmv. an episode of cmv infection was defined by one of the examination cmv viral culture, polymerase chain reaction (pcr), cmv antigen (pp ) in blood, tracheal aspirates, urine, or a combination of these. a case was defined by the presence of reactivation, where the patient had cmv infection and was seropositive. immunocompetent patients were defined as those patients who did not receive a solid organ or hematopoietic stem cell transplant, did not receive immunosuppressive treatment, did not have human immunodeficiency virus infection, did not have primary immunodeficiency, and did not receive chemotherapy or radiotherapy before icu admission. we obtained information on basic study characteristics (author, year of publication, country of origin, study period, setting, and study design), characteristic population, the site and detection method of sample, cmv seropositivity, cmv infection incidence, all-cause mortality, length of icu/hospital stay, length of mechanical ventilation, and administration of antiviral drugs. the newcastle-ottawa scale, developed for evaluating the quality of observational studies (additional file : table s ) [ ] , was used to assess the validity of included studies. continuous variables are reported as mean or median values and categorical variables are reported as n (%). meta-analytic pooling was performed for outcome variables with a logit transformation approach, reporting results as summary point estimates ( % confidence interval, ci). we used the mantel-haenszel method to obtain odds ratios (ors) and % ci. when only the median, range, or interquartile range of length of mechanical ventilation and the length of icu stay were reported, we used simple formulas to estimate the mean and standard deviation [ ] . between-study heterogeneity was examined using the i measure of inconsistency and the chi-square test of heterogeneity. to evaluate publication bias, we constructed a funnel plot and used the egger test. sensitivity analyses of the begg's test were additionally conducted to ascertain the robustness of our findings. all meta-analyses were performed with r software (version . . for windows) and spss (ibm, armonk, ny, usa). the initial database search identified potentially relevant studies. following this, assessment of the full text yielded studies suitable for analysis. another publication was incorporated after examining references from the extracted articles [ ] . consequently, our meta-analysis consisted of articles (fig. ) , including one case-control [ ] and cohort studies [ - , - , ] . most studies were conducted in the united states and europe, except one cohort study in egypt [ ] , and were published between and (table ) . overall, the studies were well done, with a median score of (range - ) on the newcastle-ottawa scale for appraising the quality of observational studies. a total of patients were included, having been admitted to the icu for a variety of reasons, with a median age of years. the median period of prospective studies was months, ranging broadly from to months. all studies used cmv blood assays, and studies also assayed sputum samples. most studies indicated that the frequency of sample collection was once a week. in our analysis, the methods used to assess cmv infection were virus culture, pp antigen detection and pcr detection of cmv dna in ten, three and two studies, respectively, and combinations of two diagnostic methods in the remaining three studies. as shown in fig. , the overall detection rate of cmv was % ( % ci - %, i = %, n = ). as compared with patients without cmv infection, the all-cause mortality of patients with cmv infection was significantly higher (or: . ; % ci . - . , i = %, n = ) (fig. a) . when analysis was limited to cmv detection in blood, there was still statistical significance in mortality rate between patients with cmv infection (or: . , % ci . - . , i = %, n = ) compared with patients without infection (additional file : figure s ). to rule out the impact of antiviral drugs on patients with cmv infection, four studies in which patients received antiviral drugs during their icu stay and eight studies that did not specify the use of antiviral drugs were excluded. the remaining six studies of patients without antiviral treatment during the course of icu stay were analyzed [ , , , , , ] . the difference in mortality rates between patients with cmv infection remained significant (or: . , % ci . - . , i = %, n = ) compared with patients without infection (fig. b) . when analysis was limited to cmv detection in blood, there was no statistical significance in mortality rate between patients with cmv infection (or: . , % ci . - . , i = %, n = ) as compared with patients without infection (additional file : figure s ). the mean difference in mechanical ventilation days and duration of icu stay was an increase of days ( % ci - , i = %, n = ) and days ( % ci - , i = %, n = ), respectively, between patients with and without cmv infection ( fig. a and b) . when analysis was limited to cmv detection in blood, there was still a statistically significant difference in length of mechanical ventilation and icu stay between patients with cmv infection as compared with patients without infection (md: days ( % ci - , i = %, n = ) and md: days ( % ci - , i = %, n = )), respectively (additional file : figure s and additional file : figure s ). the cmv seropositivity rate, which represents previous infection, was % ( % ci - %, i = %, n = ) in immunocompetent icu patients (fig. a) . patients with cmv reactivation, which represents cmv detected among seropositive patients, was % ( % ci - %, i = %, n = ) (fig. b) . the or for mortality in patients with cmv reactivation as compared with patients without cmv reactivation was . ( % ci . - . , i = %, n = ) (fig. c) . but for patients of cmv infection in blood, the reactivation was not associated with higher mortality (or: . , % ci . - . , i = %, n = ) (additional file : figure s ). we also analyzed the rate of cmv and mortality thought categorized by the detection methods (additional file : figure s , additional file : figure s : additional file : figure s and additional file : figure s ). we used the egger test to detect publication bias. there was no publication bias either in the overall cmv prevalence analysis (t = . , p = . ) or in the all-cause cmv mortality analysis (t = − . , p = . ). we also used begg's test to detect sensitivity analysis, and the results showed that the analyses were robust. in this meta-analysis, we have demonstrated that cmv infection frequently present in critically ill immunocompetent patients at icu admission. the overall rate of cmv infection was %, which was higher than the % presented in a previous meta-analysis [ ] , because eight recent studies detecting cmv infection by pcr assay were included in our meta-analysis [ - , , ] . polymerase chain reaction has been demonstrated to be the most sensitive method of cmv detection [ ] , but even so, the cmv infection rate may still be underestimated because we chose only the studies containing × tables on cmv results and all-cause mortality. we excluded studies where either the rate of cmv infection or mortality was zero and we also excluded some studies with a % infection rate that used early monitoring of cmv, often fewer than days after admission to the icu [ , [ ] [ ] [ ] . we believe this could have led to underestimation of the cmv infection rate because the transition to cmv infection requires time for the complete lytic virus cycle to develop from the latent phase [ ] . we found that the detection rate of cmv by culture, pp and pcr was , and %, respectively. desachy for cmv infection were obtained in a median of days by pcr compared with days by pp antigen detection after onset of sepsis [ ] . therefore, pcr facilitates earlier diagnosis of an episode of cmv infection than any other method. we then analyzed the association between cmv positivity and mortality, stratified by detection method. we also found that patients with cmv infection detected by pcr had higher mortality than patients without cmv infection (or: . , % ci . - . , i = %, n = ). however, when compared with other methods, the association with mortality was marginally less strong using pcr. we may think that viral burden of cmv is determinant of pathogenesis, and higher cmv loads is correlated with progression of some cmv infection disease [ , ] . the presence of cmv seropositivity, representing previous infection, was found in % of immunocompetent icu patients and the incidence of cmv reactivation was high, observed in % of seropositive patients in our meta-analysis. there are several factors that might explain the high prevalence. first of all, the rate of cmv seropositivity increases with advancing age [ ] and in our analysis, the median age was years. second, to inhibit the reactivation of cmv, as many as % of all peripheral cd + and cd + t cells are constantly required for immune surveillance to maintain functional latency [ ] . sepsis is associated with immunoparalysis, as apoptosis of cd + and cd + t cells is increased [ , ] . furthermore, some patients in the icu may be immunosuppressed after trauma and major surgery [ ] . in addition, treatments commonly received in the icu, such as massive transfusion, corticosteroids, or catecholamines may transiently compromise host immunity [ ] . it has also been reported that the use of heart-lung machines can lead to temporary systemic immunosuppression [ ] . therefore, patients in the icu may show transient immunoparalysis [ ] , potentially resulting in the observed cmv reactivation. third, some inflammatory cytokines including tumor necrosis factor alpha and interleukin- β, can stimulate reactivation of latent cmv [ ] . thus, significant numbers of immunocompetent patients harboring latent virus are susceptible to cmv reactivation during critical illness. when the mortality analysis was limited to cmv detection in blood, cmv infection without antiviral drug treatment or reactivation was not significantly associated with higher mortality. this maybe explained that the presence of high peripheral levels of functional cmv-specific cd + and cd + t cells in immunocompetent patients, which can suppress cmv during episodes of reactivation [ ] . it was observed that cmv infection was not associated with mortality in cmv colitis. in steroid-refractory patients with ulcerative colitis, cmv was found in the colon by histopathology, which is also not associated with adverse clinical outcomes [ ] . indeed, there has been no research to demonstrate that immunocompetent critically patients with cmv infection could benefit from antivirus therapy. and there are a number of side effects of antiviral drugs, such as hematologic complications (neutropenia, anemia and thrombocytopenia), renal dysfunction, mental disorders [ ] . therefore, giving antiviral drugs to critically ill patients should be considered cautiously in terms of advantage-disadvantage ratio. to address this issue, there are two ongoing, blinded, randomized placebo-controlled clinical trials of an antiviral drug with activity against cmv in critically ill patients in the icu (nct , nct ). patients with sepsis have the highest incidence of cmv infection [ ] . early in 's, bacterial sepsis was considered to trigger cmv reactivation [ ] . the reactivation associated with sepsis was consequence of inflammatory stimulation, transient immune compromise, and maybe involving some component of epigenetic regulation of viral dna [ ] . there are five limitations in this study. first, we observed large heterogeneity in many of our analyses. however, little or no heterogeneity was observed in the meta-analysis of 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cardiac surgery with cardiopulmonary bypass: pathways contributing to transient systemic immune suppression sir isaac newton, sepsis, sirs, and cars lipopolysaccharide, tumor necrosis factor alpha, or interleukin- beta triggers reactivation of latent cytomegalovirus in immunocompetent mice cytomegalovirus in inflammatory bowel disease: pathogen or innocent bystander? treating hsv and cmv reactivations in critically ill patients who are not immunocompromised: con our findings suggests that there is a high incidence of cmv seropositivity and cmv infection in critically ill patients without immunosuppression. this study suggest that cmv infection without antiviral drug treatment or reactivation in critically ill patients is associated with increased mortality, and is not associated with mortality when cmv infection is detected in blood. further research is necessary to determine the full role of cmv in this vulnerable patient demographic. additional file : table s . the newcastle-ottawa scale (pdf kb) additional file : figure s availability of data and materials all data generated or analyzed during this study are included in this published article.authors' contributions lx conducted the literature search, extracted data, performed statistical analysis, and drafted the manuscript. hyb conducted the search, extracted the data, and revised the manuscript. xzh performed the statistical analysis and edited the manuscript. zr conducted the literature search and extracted data. lxq interpreted the data. mp designed the study, interpreted data, and revised the manuscript. lym conceived and designed the study and revised the manuscript. all authors have read and approved the final manuscript. key: cord- - zrmsw f authors: liu, ming-der; chan, ta-chien; wan, cho-hua; lin, hsiu-ping; tung, tsung-hua; hu, fu-chang; king, chwan-chuen title: changing risk awareness and personal protection measures for low to high pathogenic avian influenza in live-poultry markets in taiwan, to date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: zrmsw f background: outbreaks of low and high pathogenic avian influenza (lpai, hpai) h n in chickens have occurred in taiwan since and , respectively. fully understanding the different awareness, attitudes and protective behaviors adopted by workers in live-poultry markets (lpmws) and local community residents (crs) to face the challenges of lpai and hpai is very important to minimize viral adaptations to human populations. methods: a structural questionnaire containing information on respondents’ occupation, personal risk awareness, attitudes toward different policies, and preventative measures was administered. the two-stage survey (before and after hpai h n outbreaks) was conducted from to , including: ( ) lpmws and crs at lpms from different geographical areas of taiwan after the government announced outbreaks of lpai h n during – , and ( ) lpmws and crs at two lpms in central taiwan after the hpai h n outbreaks in . the chi-squared test and logistic regression were applied for univariate and multivariate analyses, respectively. results: before hpai-h n outbreaks, higher educated respondents demonstrated greater risk awareness and concerns regarding ai. however, lpm-workers protected themselves less from ai viruses (aivs) and had lower acceptance of human or avian influenza vaccines. most importantly, the participants who opposed (versus agreed with) the policy on banning live-poultry slaughtering at lpms reported lower awareness of government prevention and control policies [odds ratio (or): . , % confidence interval (ci): . – . ] or practiced preventive measures (or: . , % ci: . – . ). after hpai-h n outbreaks, the risk awareness about ai in central taiwan significantly increased [lpai to hpai lpmws: . to . %, p < . ; crs: . to . %, p < . ] and lpmws’ belief in the effectiveness of vaccination to prevent human or avian influenza virus infection strikingly decreased ( . to . %, p < . ). conclusions: risk awareness depends on high or low pathogenicity of aivs, working in lpms, levels of education, age, and proximity to the sites of severe ai outbreaks. regardless of novel lpai or hpai virus reassortants that pose public health risks, prompt and clear risk communication focusing on both correct information about aivs and the most appropriate preventive measures are important for effective prevention of human infection. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. since the first occurrence of hpai h n human cases in hong kong in , the public health threat of high pathogenic avian influenza (hpai) has been a major global issue [ ] . exposure to live poultry was significantly associated with symptomatic or fatal cases of h n [ ] . as a result, hong kong government officials rapidly closed live-poultry markets (lpms), and slaughtered more than . million chickens around the end of [ ] , thus successfully controlling the outbreak [ ] . however, hpai h n viruses reappeared in , spread across continents, and sickened patients from to march , [ ] . the overall case fatality rate was . % ( / ). close contact with poultry is an important risk factor in h n infection [ , ] . in southeast asia, infections have mostly occurred in lpms, where activities such as slaughtering, removal of feathers, customers touching chickens, transportation, and cleaning poultry waste occur very frequently [ , ] . importantly, most of the low pathogenic avian influenza (lpai) h n viruses, which caused human infections in different parts of china since february of [ ] , had high viral sequence identities to the h n viruses isolated from wet poultry markets [ ] . this was quite different from the avian influenza (ai) outbreaks in europe and africa, which occurred mostly in poultry farms where migratory birds played an important role [ ] . therefore, exposure to ai viruses (aivs) in lpms in asia has been highly risky [ ] . the increasing number of fatal cases due to h n infections prompted the government of hong kong to initiate policies forbidding the slaughtering of live chickens or other poultry in wet markets [ ] . the awareness of ai has been documented to affect a persons' self-protection behaviors [ ] and live poultry purchases [ ] . it is important for mass media (such as television channels) to provide correct information to enhance the receivers' knowledge and risk awareness [ ] . an individual's level of education [ ] , occupation (such as being poultry workers) [ , ] , and the residential area's experiences with ai outbreaks [ , ] may all affect a person's perception of the risk of ai and their subsequent use of adequate personal protective equipment [ ] . thus, understanding all possible factors associated with risk awareness, attitudes, and practice of prevention measures (rap), as well as differences in risk perception of outbreaks due to lpai versus hpai viruses between the live-poultry market workers (lpmws) and community residents (crs) are all important for providing further education and implementing public health policies on preventing ai infection. taiwan, with close proximity to these asian ai epidemic and endemic centers, has many lpms which could be potential sources of ai virus maintenance for emerging novel influenza reassortant viruses. the first ai outbreak of h n in taiwan started in december , and subsequently these lpai h n viruses spread island-wide [ ] . although a policy to stamp them out was implemented from through , this virus subtype remained in circulation for many years. in october , another outbreak of h n occurred in kaohsiung (located in southern taiwan), and a molecular analysis of the cleavage site of ha of the isolated virus indicated that it was high pathogenic. as the chicken pathogenicity index (intravenous pathogenicity index, ivpi) of the specimens collected for the second time was below . (ivpi = . ), the government officials announced and reported it as an outbreak of lpai to the world organization for animal health (oie) (http://www.oie.int/) [ , ] . in fact, both of these lpai and hpai h n viruses are particularly unique, as they consist of reassortants of six internal segments derived from local taiwan lpai h n viruses, but the ha and na segments had the highest viral sequence identities with the mexican-like h n viruses [ , ] . after the first hpai h n outbreak was officially announced on march , , about , and , chickens were culled in changhua and tainan counties in central taiwan, respectively during february-march [ ] . the elevation from lpai h n to hpai h n viruses in recent years in taiwan provides a great opportunity to investigate whether the rap of high-risk populations of those working in lpms versus local crs were different when facing the greater challenges of hpai h n viruses compared to the past lpai h n ones. therefore, the specific aims of this study were: ( ) to investigate the factors associated with high and low levels of rap among lpmws and crs in outbreak areas throughout taiwan immediately following the announcement by the government on outbreaks of lpai h n ; ( ) to compare the differences in the factors associated with rap after the outbreaks of these ai viruses with low versus high pathogenicity; and ( ) to identify the different sources of information regarding the outbreaks of hpai-h n in chickens in central taiwan among lpmws and crs as well as to compare their willingness to take preventive measures against lpai-h n and the other important emerging infectious diseases. to the best of our knowledge, this is the first study to investigate public health awareness of both lpai and hpai of the same subtype viruses. moreover, our findings could help public health administrators in areas or countries with lpai to better prepare for possible subsequent hpai outbreaks or minimize the numbers of human infections with either lpai or hpai viruses. to fully understand the differences in local responses after the outbreaks of lpai and hpai of h n viruses in taiwan, we conducted two-stage surveys including: ( ) stage i: after the outbreaks caused by lapi h n viruses from january to january , and ( ) stage ii: after the outbreaks caused by hpai h n viruses from february to march . in the first-stage survey (before hpai h n outbreaks), representative lpms were selected across taiwan, including two markets each in northern, central, and southern taiwan, and five markets in eastern taiwan as illustrated in fig. . to increase the sample size for the areas with lpai h n outbreaks, which is the most neglected and important issue to be addressed, we covered all the major lpms in the outbreak areas and asked as many people as possible to answer the questions at each study site. since the outbreaks of lapi h n occurred in different years in various geographical areas, surveys were initiated in the wet markets in different time periods, right after the occurrence of outbreaks, including northern taiwan (january to june ), central taiwan (april to august ), eastern taiwan (february to may ), and southern taiwan (january ). after the outbreaks of hpai h n , which were restricted to changhwa county in central taiwan between february and march , the second-stage survey was conducted in the two lpms situated in the outbreak county (shown in fig. ) with a smaller sample size from late june to early july . for better comparison of the outcome measures between the two studied populations -( ) live-poultry market workers (lpmws) as a "high-risk group" and ( ) community residents (crs) as a "low-risk group", we used convenience sampling among these two groups for each of the study areas. the crs, who were buyers but did not sell or touch any live poultry, were sampled from visitors who shopped at the same lpms or visited the closest convenience stores (such as - stores) at the same time as we asked lpmws. a structural questionnaire was designed to investigate: ( ) ai awareness, ( ) knowledge of government policies, and ( ) protection measures used. to achieve the study objective, the team members who designed and reviewed the questionnaire included infectious disease physicians, infectious disease epidemiologists, scholars experienced in knowledge, attitude and practice (kap) of diseases, field workers who frequently went to lpm to take poultry specimens, and administrators in lpms. the questionnaire included items such as demographic information, job duties, prevention measures, personal perceptions, the impact of china's ai outbreaks on taiwan, attitudes toward different policies such as killing poultry at lpms, and potential confounding variables (age, gender, educational level, and living area) [additional file : appendix , additional file : appendix ]. we did a pilot test on both study groups in different geographical areas to assure full understanding and reliability. after a comprehensive review by questionnaire design team members, the wording of the questionnaire was revised and simplified to maximize the response rates. there were five main questions measuring risk awareness, attitudes and personal protection measures (rap) [additional file : appendix ] . in addition to these five main questions, questions on the awareness of hpai h n outbreaks in and risk perception in lpai-h n , hpai-h n and other important infectious diseases in taiwan [such as severe acute respiratory syndrome (sars)] were also included in the questionnaire of the second surveys for better comparison. most questions were multiple choice, with a comprehensive range of choices or differential scales or rankings [additional file : appendix ] . however, the second main question on possible future outbreaks of human cases of infection with aivs in taiwan was measured by the likert scale. the questionnaire was administered by well-trained interviewers. for better assessment of the exposure levels, the lpm workers were further classified into three risk groups, based on their occupational exposures. the "high-risk group" included butchers and sellers of raw chicken or duck meat. the "moderate-risk group" covered sellers of cooked chicken or duck meet, beef, mutton, pork, and other raw meat sellers. the "low-risk group" included other workers. regarding the risk levels among the market workers, the results showed that workers in all these three risk groups were located significantly more in northern taiwan than in the rest of taiwan (p = . , table ). we then focused on the comparisons of all possible factors that may be associated with rap; in particular, the differences on each question between lpmws and crs based on their occupation were analyzed. only statistically significant differences between these two study groups are presented in tables and , with the covariate of "occupation" adjusted in multivariate analyses. demographic characteristics (including age, gender, living area and education level) were summarized as frequencies and percentages. in order to analyze the respondents' answers, we classified their responses on rap measures into a binary scale (positive and negative perception of the questions) and used a chi-square test and logistic regression for univariate and multivariate analyses, respectively. additional file : appendix is the summary for all the assigned "positives" as " s" and "negatives" as " s" as a binary scale. for univariate analysis, a chi-squared test was used to compare differences in categorical variables [such as age: - , - and≧ (elderly)] and outcome of kap measures between lpmws and crs. the outcome variables, explanatory variables, as well as the model performance for data analyses in this study are all summarized in additional file : appendix . the comparison of perception changes before and after chicken h n outbreaks in central taiwan was analyzed by two-proportion z-test (table ) . for multivariate analysis, we pooled lpmws and crs together and then analyzed the outcome measures for each question. then, logistic regression with stepwise selection of variables was used for estimating the adjusted odds ratios (or) of explanatory factors and their % confidence intervals ( % ci) after adjusting for important confounding variables such as age [ ] , gender, residential area (northern, southern, central and eastern taiwan), education level, and occupation (i.e., lpmworkers and community residents), plus the other outcome variables in addition to the asked rap questions for both lpai and hpai surveys in tables and , respectively. for example, the last question, on effectiveness of vaccines, and the variables from all other questions in the same rap table such as impact of china on taiwan were entered for better assessment. for the best statistical performance, both age and education were entered as continuous variables. only significant variables (p < . ) were included in our final model. therefore, we controlled for the regional differences in each analyses of both the st-and nd-stage surveys. to ensure the validity of our results, basic model-fitting techniques for ( ) variable selection, ( ) goodness-of-fit (gof) assessment, and ( ) regression diagnostics were all used in our regression analyses. the statistical analysis was performed using sas . . (sas institute, cary, nc, u.s.a.). variables with p-value less than . were considered statistically significant. cox & snell r-square and nagelkerke r-square were applied and the results listed in additional file : appendix . the study and its consent procedures were approved by the ethical committee of national taiwan university hospital (approval number: rc). respondents were informed of the purpose of the study, while oral consent was obtained before anonymous questionnaires were administered. due to concern for privacy of the chinese signatures of names, written informed consent was not collected. whenever the respondents did not agree to join the study, the interviewers respected their opinions and did not continue for those cases. for the respondents aged less than years, the interviewers first got the agreement of their parents or guardians. otherwise, the interviewers dropped these cases. in other words, all the successfully collected questionnaires were agreed to verbally by the adult respondents themselves or children's parents or guardians. in addition, our data were fully de-identified to protect the respondents' privacy, and only group data were used for further analyses and statistical tests. both surveys recruited lpmws and crs. the response rates for high-, moderate-, and low-risk groups of lpmws and crs in the stage i survey were %, %, %, and %, respectively. such rates for lpmws and crs in the stage ii survey were % and %, respectively. the response rates for lpm workers after the hpai h n outbreaks were lower than those after the lpai h n outbreaks. in the first-stage survey after the lpai h n outbreaks but before the hpai h n outbreaks, a total of questionnaires were administered, including to lpmws and to crs (table ). in stage i, there were significant differences in gender and education, but the results were comparable across age and geographical distributions, without statistical differences between these two groups [ tables and ]. however, workers in the wet markets had a significantly higher proportion of males [ . % ( / )] as compared to the crs [ . % ( / )] (p < . ). overall, the crs had higher levels of education than the lpmws (p < . ). in the second stage survey (after the outbreaks of hpai h n in central taiwan), respondents ( lpmws and crs) completed the questionnaires. in this subgroup (table ) , the lpmws were significantly less educated (p < . ) and older [mean ± standard deviation (s.d.) of age (by years): . ± . vs. . ± . , p < . )] than those crs of the same local areas with hpai outbreaks. factors associated with risk awareness, attitude and preventive measures of ai before the outbreaks of hpai h n firstly, we analyzed possible factors influencing the awareness of ai in stage i before the outbreaks of hpai h n in taiwan (table ). as to the impact of china on taiwan (question of table ), the respondents with higher levels of education thought that the external outbreaks of ai in poultry or in human cases in china would affect taiwan (or: . , % ci: . - . ), whereas those who opposed the ban on live poultry slaughter in taiwan's traditional markets did not believe in such an influence (or: . , % ci: . - . ). next, the risk awareness regarding the impact of domestic ai outbreaks was assessed (question of table ) . besides level of education, respondents' age and residential area also influenced their risk awareness of human infection of ai in taiwan after the local lpai outbreaks. the older participants (or, . ; % ci, . - . ) and those living in central taiwan (or, . ; % ci, . - . ), where the population density of chickens is the highest and outbreaks of lpai-h n frequently occurred, were less likely to think that taiwan residents would get infection with aivs. however, the respondents living in the h n epidemic site of kaohsiung county in southern taiwan in january with different awareness of ai (after the controversial judgment on the causing agents as lpai or hpai viruses in the outbreak) compared with those in other areas, perceived that people in taiwan would become more likely to be infected with aivs (or: . , % ci: . - . ). since the government announced the new policy of "ten no's, five needs" in (additional file : appendix ) after many outbreaks of lpai h n , we then investigated the factors associated with knowing this policy and a possible future ban on slaughtering live poultry in traditional markets (question of table ). the results showed that greater percentages of respondents with higher levels of education or living in central or eastern taiwan knew the contents of the new government policy on ai than those in other areas (central taiwan in stage i, the mean, median, and range of age for crs were . ± . , . , and - , respectively whereas those for live poultry market workers (lpmws) were . ± . , . , and - , respectively. we used a chi-square test for the statistical analyses in table . there was no significant difference between these two groups [ table ] the data within the percentages of community residents related to the different demographical variables in the stage i survey served as the reference group in this tables and §high risk: butcher, raw chicken/duck sellers. moderate risk: sellers of cooked chicken/duck, beef, pork, mutton, and/or other raw meat. low risk: market cleaners, administrative officers, and those selling flowers, dry goods, vegetables and fruits. *p-value < . . ; % ci, . - . ), the respondents from eastern taiwan, where fewer outbreaks were reported (or, . ; % ci, . - . ), and those without opinions on the banning of poultry slaughtering (or, . ; % ci, . - . ), were all less motivated than those in the comparison groups to implement self-protection measures against aivs. by contrast, both respondents who believed human cases of ai would appear in taiwan (or: . , % ci: . - . ) and those who were also more aware about the new policy"ten no's, five needs" (or: . , % ci: . - . ) had more willingness to engage in self-protection against aivs. in other words, the study subjects' awareness of risk on ai in taiwan and attitude in supporting or opposing government policies were associated with their taking subsequent personal prevention and control measures. on regarding the perceptions on effectiveness of influenza vaccines (question of table factors associated with risk awareness, attitude and preventive measures after the outbreaks of hpai h n the results of the risk awareness, attitudes about, and protective behaviors against ai after the outbreak of hpai h n in taiwan are summarized in table . the older respondents were less likely to believe that taiwan would be affected by the influenza outbreaks in mainland china (or: . , % ci: . - . , question ). again, the impact of outbreaks of ai abroad as well as in taiwan was further explored (questions and table ). the risk awareness of ai causing serious disease and even death was evaluated (question of table ). crs of areas with documented chicken hpai h n outbreaks had higher awareness of ai leading to severe human clinical cases or fatalities (or: . , % ci: . - . ) than crs of other areas. these respondents with greater alertness of the ai severity not only had better knowledge of the new "ten no's, five needs" policy (or, . ; % ci, . - . ) but also were more likely to take preventive measures against aivs (or, . ; % ci, . - . ). after the government declared the outbreaks of hpai h n in taiwan in , we found protective behaviors and shopping habits were different between lpmws and crs. among the respondents, % of them washed their hands frequently ( / ) and . % of them ( / , with missing values) reported the intention to wear facemasks to protect themselves once ai outbreaks occur (table ). in this study, we did not differentiate surgical masks from cloth masks in our questionnaire on "facemasks". however, most of the public can easily buy surgical masks in convenience stores or drug stores. even among the crs, high percentages of them intended to change their shopping behaviors such as avoiding both live-poultry markets ( / , . %) and poultry purchases ( / , . %). comparing the perception differences before and after the outbreaks of chicken hpai h n among the study participants only in central taiwan, our results revealed significant increases in the proportion of both lpmws and crs who perceived taiwanese will be infected by aivs (table ) . after the occurrence of domestic hpai h n outbreaks, the lpmws' risk perception on the possibility of ai epidemics in mainland china affecting taiwan significantly decreased ( . to . %, p < . ), but their risk awareness on the likelihood of people in central taiwan being infected with aivs strikingly increased (from . to . %, p < . ). however, the lpmws' belief that vaccines are capable of preventing human or avian influenza virus infection strikingly decreased ( . to . %, p < . ). after the incident of hpai-h n outbreaks in central taiwan, we asked participants whether they knew that hpai outbreaks had occurred there. among those who knew about the hpai h n outbreaks, lpmws had significantly paid more attention to the ai outbreaks than crs [ . % ( / ) vs. . % ( / ), p = . ] ( table ). detailed analysis of the sources of information on these hpai outbreaks (table ) showed that the major channel for receiving information on the outbreaks for both groups was television broadcasts (lpmws vs cr: . % vs . %, p = . ), followed by the internet and relatives or friends for crs ( %) and newspapers for lpmws ( . %). however, seeking information through newspapers, internet and radio broadcasts was statistically more common among the crs than the lpmws (newspapers: . % vs. . %, p < . ; internet: . % vs. . %, p < . ; radio: . % vs. . %, p < . ). comparing the respondents' risk perception of lpai h n , hpai h n and other important emerging infectious diseases (eids) versus the old disease of tuberculosis (table ), severe acute respiratory syndrome (sars) was perceived as the most risky infectious disease by the respondents, while hpai h n was thought much more important than lpai h n , particularly among market workers (hpai vs lpai for lpmws: . % vs . %, p < . ; for crs: . % vs . %, p < . ). global epidemiology of ai has focused mostly on human cases after the outbreaks of hpai [ , ] , with little attention to lpai. to our knowledge, this is the first study to compare the differences in risk awareness, attitude and personal protection practice (rap) right after the outbreaks of both lpai and hpai of the same virus subtype. we have the following five major findings that may help future global efforts to prevent novel ai viruses (aivs) with pandemic threat to human populations. first, risk awareness, positive attitudes and taking preventive measures depend on several factors, including high or low pathogenicity of aivs (hpaivs or lpaivs), working in lpms, level of education, age, proximity to the sites of severe ai outbreaks, knowledge of ai outbreaks in neighboring countries or areas (e.g., mainland china or hong kong), the level of understanding of important knowledge on aivs, and learning preventive measures through various channels of mass media. second, table . lpmws were significantly older than crs (p < . ) *p-value < . . the data within the percentages of community residents related to the different demographical variables in the stage ii survey served as the reference group in this table a our government officials initiated the pilot study of phase h n avian influenza vaccine trial for animal-related workers in . at that time, the acceptance rate was quite low. therefore, the data of the reported "acceptance of avian influenza vaccine between live-poultry market workers and community residents" were thus compared only after the nd survey in table respondents with higher risk perception (concerning human ai infections in taiwan) before hpai outbreaks had not only more awareness about the ai outbreaks in mainland china affecting taiwan, but also better attitudes toward meeting domestic needs (endorsing the government's new policy on ai, and supporting a ban on slaughtering live poultry in markets). third, participants' better attitudes towards ai prevention and control were associated with higher motivation to practice self-protection measures, even in preventing lpaivs. fourth, individuals with lower educational levels, the lpm workers with high exposure to aivs, and the respondents living in areas with low frequency of ai outbreaks had a lower risk awareness of aivs, particularly lpaivs that might be transmitted to humans. fifth, the respondents' risk awareness and protective behaviors during the periods of lpai h n outbreaks strikingly rose after experiencing the outbreaks of hpai h n . all these together suggest that neglecting health education and precautions in lpms might facilitate adaptation of the virus in human populations, particularly the silent spreading of lpaivs. among all the factors associated with rap related to human infection of aivs, the pathogenicity of ai virus is crucially important, particularly in those areas or countries with no prior experience of hpai outbreaks. however, most past studies have targeted poultry workers as the high risk population due to exposure to possible hpaivs of h and h in sick or dead poultry [ ] , neglecting the dynamic changes of aivs from lpaivs to hpaivs. our study showed that risk perceptions changed significantly for both market workers and the general population after hpai outbreaks in taiwan. the increased pathogenicity of h n aivs may have caused the study subjects to feel nervous, as they faced the outbreaks of sars in and the novel h n influenza pandemic in , thus raising risk perceptions. lower risk perception in these high-risk populations is a general problem in different parts of the world, including taiwan [ ] , italy, thailand and china [ ] . this shows that high-exposure workers need more appropriate information on aivs to complement the information through mass media, which is usually obtained after rather than before outbreaks. generally, lpmws before the outbreaks of hpai in this study also had a lower perception of taiwanese ai risk than local residents (question in table ), so they did not adopt any preventive measures to avoid aiv infection, and did not believe the seasonal influenza vaccination was effective for preventing human or avian influenza. live-poultry markets, the major interface areas between poultry and humans offering conditions for sustainability, amplification, reassortment and cross-species we used logistic regression for the statistical analyses in this live-poultry market workers as the control group transmission of aivs, from h n hpaivs in [ ] to the most recent h n lpaivs in - , have been involved in many human-acquired aiv infections [ ] . most importantly, chickens sold in traditional lpms can transmit aivs to humans through respiratory transmission [ ] . interestingly, the older participants, who had more traditional thinking, and those living in central taiwan, where the density of layer-chickens ranks the highest and outbreaks of lpai-h n occurred more frequently than other areas, had lower risk awareness of aivs. by contrast, the respondents who lived in the epidemic site of kaohsiung county in southern taiwan, where the cleavage site of hemagglutinin (ha) was identified as hpaivs in , had higher perceptions than residents in other areas (or: . ) that people in taiwan would become infected with aivs. after the outbreaks of chicken hpai h n , the lpmws still had a lower belief in the effectiveness of vaccination to prevent human or avian influenza virus infection, regardless of their job duties. furthermore, compliance with and understanding of the government policy raised the individual's risk perception from lpaivs to hpaivs, while other measures of risk awareness had fewer differences among these two surveys, except for the reduction in risk perception on possible ai outbreaks in mainland china affecting taiwan among lpmws in central taiwan. such a striking decrease can be explained by the occurrence of the local hpai outbreaks instead of the imported infections. in other words, attitudes became positive and preventive measures were reported to be taken when they faced the threat of hpaivs. this may have been influenced by we used chi-square test for the statistical analyses in table the data within the percentages of community residents related to the different preventive measures served as the reference group in this this survey was implemented during late june-july , after the outbreak of hpai h n data in the two columns of "before hpai for live-poultry market workers" (lpmws) and "before hpai for community residents" (crs) served as two reference groups of lpmws and crs, respectively n number of participants who answered that specific question *p-value < . *, using a two-proportion z-test mass media or the experience of getting a voluntary h n ai vaccine. therefore, our study demonstrated that the perception of ai risk was elevated as the pathogenicity of aivs changed from low to high. these results emphasize the public health significance of educating high-risk populations, starting from lpaivs with capability to donate viral gene segments for generating novel reassortant viruses that may increase infectivity to humans, like h n and h n in china [ , ] and h n [ , ] and h n [ ] in taiwan. three important policies for ai prevention and control measures are closure of lpms, practicing personal protective measures (ppm), and receiving influenza vaccines before flu seasons. in general, numerous lpms are widely distributed in urban areas that might facilitate avian-tohuman and subsequent human-to-human transmissions of novel influenza viruses, unlike poultry farms, which are more frequently located in rural areas. such geographical differences between the urban and rural areas affected the awareness of ai in turkey [ ] and in china [ ] . in the past, closure of lpms has been implemented after the confirmation of severe or fatal human cases of h n and h n [ ] . however, the virus may still reemerge after temporary closure. therefore, market shutdown is not the most effective long-term measure, especially as the stakeholders are not likely to support it. the most likely approaches are: ( ) weekly and monthly off-market days (such as mondays and chinese festivals in taiwan) for cleaning and interrupting viral transmission, ( ) banning the slaughter of live poultry, ( ) practicing ppm, and ( ) receiving influenza vaccines. in fact, the lpmws with lower education in this study did adopt the latter three prevention measures less frequently. on the other hand, more highly educated participants believed that the outbreaks of ai in china would affect taiwanese, and thus supported the ban on slaughtering live poultry in markets. a ban on slaughtering live poultry in the market, first proposed in hong kong [ ] , has been implemented by the revised food commerce law [ ] . in , a woman in beijing without prior contact with live birds was infected with aivs after purchasing live poultry in a traditional market [ ] , implying that lpms are one of the sources of aiv infection. furthermore, most of the severe human h n cases in - also acquired their infections through contact with poultry or visiting wet markets [ ] . although the taiwan government initiated the implementation of the policy to ban slaughtering live poultry in lpms on april st, (e.g., after the h n outbreak in kaohsiung with controversial answers on viral pathogenicity), this was postponed, then reinstated on may , due to the occurrence of the first imported h n case in taiwan. in this study, lesseducated, high-risk groups had lower rap. therefore, enhancing surveillance of aivs in avian hosts as well as humans in lpms [ ] , timely epidemiologic data analyses and prompt risk communication with evidencebased data support, focusing on changing the minds of lower-educated, high-risk groups, will be very helpful to quickly control novel influenza viruses, thus minimizing the occurrence of potential pandemics. the data within the percentages of community residents related to the sources or channels of information served as the reference group in this ppms are particularly useful for reducing the risk of acquiring or transmitting emerging respiratory infections before the availability of commercial vaccines [ ] . education is the most cost-effective approach to deliver the correct knowledge and ways to prevent aiv infections and future epidemics. highly educated persons who had better access to the information on ai from television, newspapers and the internet in this study had higher risk perception of ai, similar to the findings in china [ ] and afghanistan [ ] . most importantly, strict compliance with personal protective equipment (ppe) requirements must be reinforced to manage the outbreaks of ai, regardless of the pathogenicity of the virus [ , ] . incomplete use of ppe was also associated with conjunctivitis and influenza-like illness after the outbreak of lpai h n in norfolk, england [ ] . generally, compliance with most ppe requirements tends to be suboptimal for the highly exposed groups [ ] . similarly, taiwanese lpm-workers generally feel that wearing ppe is uncomfortable, and they have not gotten used to it. the low risk perception accompanying such poor ppe usage needs to be guided with solid examples to finally achieve behavioral change. our study subjects who had higher risk perception of ai and who were more aware about the government's new policy had more motivation to use self-protection for preventing aivs. therefore, it is necessary to improve public awareness about the government's prevention policy and also educate different target groups with various approaches, based on educational levels, job duties, and residential areas. identifying populations with low acceptance rates of influenza vaccine is important before developing and implementing vaccination programs, particularly as the acceptance of influenza vaccines has become quite low in recent years in many parts of the world [ , ] . we found that market workers and the respondents living in taiwan (except the hpai h n outbreak sites in southern taiwan) did not believe that any influenza vaccine provided effective protection against aivs. a significant drop was observed in the perception of the vaccine effectiveness in preventing "avian influenza" after the impact of hpai compared to lpai h n outbreaks for both lpmws ( . % vs . %) and crs ( . % vs %). in addition, respondents who did not believe in the external influence of outbreaks of ai in mainland china, as well as those who paid no attention to domestic policy in taiwan, and those who found no need to protect themselves against aiv infection did not trust the effectiveness of any influenza vaccine for humans or poultry. to solve this problem, risk communication on sources of the risk as well as scientific data supporting safety becomes very important. additionally, easy access of high-risk populations to ai vaccines (similar to the established system for seasonal influenza vaccination of schoolchildren and the elderly in taiwan, supported by a well-established public health infrastructure), a feasible plan on resources allocation, and the available ai vaccine inducing higher immunogenicity through better innate immunity [ , ] all together, with systematic approaches, will reduce human infections of aivs. there are three major limitations of this study. first, there is a possibility of selection bias caused by the willingness of respondents to reply to the questions in the survey, even though we covered most of the live poultry markets affected by the outbreaks, and the lpmws' response rates were - % and % after the outbreaks of lpai h n and hpai h n , respectively. in addition, the nd-stage survey was conducted only in central taiwan, where the scale of layer chickens was the largest. second, our results may show reduced rap because more study subjects of lpms in the st-stage survey came from northern taiwan, where large-scale wholesale broiler chickens and ducks coming from different parts of taiwan are sold with better management and hygienic standards, whereas high densities of poultry farms are located in central and southern taiwan. third, the study subjects of the st and nd surveys were different and not comparable, and those results indicate only association rather than causation, because of the crosssectional study design. the outbreaks of lpai h n were larger in scale, and occurred much more frequently and in more places than those of hpai h n . to protect participants' privacy, we did not collect personal identification data, and therefore could not follow up on the respondents in the initial survey. future research should focus on the most effective methods and contents for risk communication in order to target different risk groups. risk perception problems on lpaivs need to be explored in relation to the scale, breeding style, types, and sanitation of poultry farms and different kinds of lpms, particularly in areas with limited resources and expertise. in addition, behavioral research is worth doing to direct the best prevention and control policies, considering the acceptance of influenza vaccination and acceptable behavior change in high-risk groups versus the general public. based on our findings, we sincerely recommend that health agencies enhance additional routine two-way risk communication with friendly interpersonal guidance for live-poultry market workers, poultry butchers and farmers, and related high-risk groups, particularly before outbreaks of ai. in addition, to minimize political concerns, fatal human cases after infection with aiv, including lpaivs of h n and h n [ ] in other countries can serve as solid examples for education, using easily influenza a (h n ) in hong kong: an overview case-control study of risk factors for avian influenza a (h n ) disease, hong kong chickens killed in hong kong to combat flu outbreak of avian influenza a(h n ) virus infection in hong kong in cumulative number of confirmed human cases for avian influenza a(h n ) reported to who risk of influenza a (h n ) infection among poultry workers, hong kong, - epidemiology of cases of h n virus infection in indonesia genetic analysis of avian influenza a viruses isolated from domestic waterfowl in live-bird markets of hanoi, vietnam, preceding fatal h n human infections in predicting the global spread of h n avian influenza epidemiology of human infections with avian influenza a(h n ) virus in china human infections with the emerging avian influenza a h n virus from wet market poultry: clinical analysis and characterisation of viral genome human avian influenza a (h n ) virus infection in china avian influenza and ban on overnight poultry storage in live poultry markets, hong kong knowledge, attitudes, practices and emotional reactions among residents of avian influenza (h n ) hit communities in vietnam avian influenza risk perception and live poultry purchase in knowledge, attitudes and practices (kap) relating to avian influenza in urban and rural areas of china relationships among trust in messages, risk perception, and risk reduction preferences based upon avian influenza in taiwan knowledge, attitudes, and practices of avian influenza, poultry workers knowledge, attitudes and practices related to avian influenza among poultry workers in nepal: a cross sectional study an exploration of how perceptions of the risk of avian influenza in poultry relate to urbanization in vietnam personal protective equipment and risk for avian influenza (h n ) the virulence variation in a h n low pathogenic avian influenza virus after passage in -day-old chicken embryonic eggs isolation and characterization of potentially pathogenic h n influenza virus from a chicken in taiwan in a low pathogenic h n influenza virus isolated in taiwan acquired high pathogenicity by consecutive passages in chickens isolation and characterization of potentially pathogenic h n influenza virus from a chicken in taiwan emergence and evolution of avian h n influenza viruses in chickens in taiwan the china post news staff: h n avian flu strikes taiwan: coa biased odds ratios from dichotomization of age global epidemiology of human infections with highly pathogenic avian influenza a (h n ) viruses molecular epidemiology of influenza a (h n ) viruses, bangladesh protecting poultry workers from exposure to avian influenza viruses spatial and temporal analysis of human infection with avian influenza a(h n ) virus in china infectivity and transmissibility of h n avian influenza virus in chickens and wild terrestrial birds infectivity, transmission, and pathology of human-isolated h n influenza virus in ferrets and pigs emergence in china of human disease due to avian influenza a(h n )-cause for concern? human infection with avian influenza a h n virus: an epidemiological analysis knowledge and anticipated attitudes of the community about bird flu outbreak in turkey knowledge, attitudes and practices (kap) relating to avian influenza in urban and rural areas of china global alert to avian influenza virus infection: from h n to h n hong kong places curbs on market poultry physical interventions to interrupt or reduce the spread of respiratory viruses attitudes, and practices regarding avian influenza (h n ) avoiding the risk of infection when working with poultry that is suspected of having h or h notifiable avian influenza effectiveness of personal protective equipment and oseltamivir prophylaxis during avian influenza a (h n ) epidemic, the netherlands self-reported use of personal protective equipment among chinese critical care clinicians during h n influenza pandemic barriers to vaccinating the elderly with h n vaccine why do i need it? i am not at risk! public perceptions towards the pandemic (h n ) vaccine ns -truncated live attenuated virus vaccine provides robust protection to aged mice from viral challenge live attenuated influenza viruses containing ns truncations as vaccine candidates against h n highly pathogenic avian influenza human influenza: one health, one world submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution submit your manuscript at www we would like to thank ms. wei-ru chen and ms. ju-feng wang at the institute of epidemiology and preventive medicine, college of public health, national taiwan university and students at st. mary's junior college of medicine, nursing and management, central taiwan university of science and technology, taiwan shoufu university, and hungkuang university for their help on field interviews and collecting questionnaires. in addition, we like to express our sincere gratitude to dr. muh-yong yen at taipei city hospital, dr. john allen at the duke-national university of singapore (duke-nus), ms. wen-wen wang, and administrators in live-poultry markets for their sincere assistance in questionnaire design and evaluation, statistical consultation, administrative support, and coordination in live-poultry markets, respectively. understandable wordings and movies to demonstrate the danger of aerosol transmission of aivs. above all, the policy on banning the slaughter of live poultry at lpms supported by incentives of tax reduction or free health care or certification to win customers' trust, as well as active virological and serological surveillance with random sampling in poultry farms and markets, could be the most efficient way to reduce cross-species transmission. in conclusion, person-to-person risk communication to high-risk groups using more acceptable and attractive approaches and effective public policies on "one health" [ ] , and post-policy evaluation with international comparison will be helpful to promote global health. additional file : appendix . additional file : appendix .additional file : appendix . the authors declare that they have no competing interests.authors' contributions mdl, hpl, tht and cck carried out the survey and designed the questionnaires. mdl, ctc, chw, cck drafted the manuscript. ctc, fch performed the statistical analysis. chw helped interpret the findings from the perspective of veterinary epidemiology. cck conceived of the study, participated in its design and coordination, and helped to revise the manuscript. all authors read and approved the final manuscript. key: cord- -fc u mx authors: neupane, dinesh; khanal, vishnu; ghimire, kamal; aro, arja r; leppin, anja title: knowledge, attitudes and practices related to avian influenza among poultry workers in nepal: a cross sectional study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: fc u mx background: avian influenza is a considerable threat to global public health. prevention and control depend on awareness and protective behaviours of the general population as well as high risk-groups. this study aims to explore the knowledge, attitudes and practices related to avian influenza among poultry workers in nepal. methods: the study was based on a cross-sectional study design, using a structured questionnaire administered in face-to-face interviews with poultry workers age and above from the rupandehi district in nepal. results: the majority of respondents were male ( %), mean age was (sd = . ). nearly everybody was aware that ai cases had been detected in nepal and that poultry workers were at risk for infection. the major sources of ai information were radio, tv and newspapers. knowledge about preventive measures was high with regard to some behaviours (hand washing), but medium to low with regard to others (using cleaning and disinfecting procedures or protective clothing). poultry workers who got their information from tv and newspapers and those who were more afraid of contracting ai had higher knowledge than those who did not. being employed as compared to being an owner of a poultry farm as well as having a high level of knowledge was associated with practising more preventive behaviours. while on one hand many specific government control measures found a high degree of acceptance, a majority of study participants also thought that government control and compensation measures as a whole were insufficient. conclusions: the study provides information about knowledge and practices regarding avian influenza among poultry workers in nepal. it highlights the importance of targeting lack of knowledge as well as structural-material barriers to successfully build preparedness for a major outbreak situation. in january nepal faced the first localized outbreak of highly pathogenic avian influenza (ai) among poultry, followed by a second outbreak in another area in february of the same year [ , ] , but no human cases were registered [ ] . if nepal were hit by pandemic flu as a consequence of re-assortment or adaptive mutation of the virus and ensuing full human-to-human transmissibility, consequences could be severe. depending on the planning scenario, fatality numbers are estimated between , and , and large numbers of people in need of clinical care might be faced with a severe shortage of hospital beds [ ] . beside the expected toll on human life, a pandemic is bound to incur disastrous economic losses in a country where sectors like farming and tourism, which make a significant contribution to the overall economy, are expected to be particularly hard hit [ , ] . prevention and control planning have to take account of the whole population, but there are subgroups which are particularly critical, such as poultry or pig farmers, who are among those first in line when it comes to risk of contracting ai. also they are expected to be a "bridging population" in terms of cross-species sharing of viruses and of spreading the disease into their local communities [ , ] . in early the government of nepal established the avian influenza control project (aicp) and endorsed a joint health and agriculture national avian influenza and influenza pandemic preparedness and response plan (naiipprp) [ , ] which placed particular emphasis on precautionary behaviours of poultry workers as well as the knowledge and attitudes which drive such practices. a mass media campaign informing about risks and motivating for protective behaviours had been started already soon after hpai hit asia in and was intensified after it had reached nepal in early . based on health-behaviour models such as the health belief model [ ] or protection motivation theory [ ] crisis communication campaigns usually have a strong focus on providing information and knowledge about risks and protective behaviours. most evidence accumulated within the context of sars, h n and the h n outbreak in is consistent with these models' assumptions about the relevance of risk perceptions and beliefs in the efficacy of protective behaviours [ ] [ ] [ ] [ ] [ ] [ ] . evidence on the role of knowledge about pandemic influenza, however, has been less unequivocal. while some studies have found positive effects on protective behaviours [ ] [ ] [ ] , others have failed to do so [ , ] . the objectives of the present study were ) to identify levels of knowledge about preventive behaviours as well as actual preventive behaviours with regard to avian influenza in nepalese poultry workers, ) to investigate factors associated with knowing about and practising preventive behaviours against ai, among them sociodemographic characteristics, media use (health information from tv and newspapers), and experience of fear. additionally, ) for preventive behaviours the role of knowledge about such behaviours was investigated. the study, which took place in april , was based on a cross-sectional cluster survey design. after obtaining ethical approval from research ethics committee of institute of medicine at tribhuvan university in kathmandu, nepal, face-to-face interviews with the help of a standardized questionnaire were conducted with poultry workers age and above from the rupandehi district, nepal. this district was chosen because it is one of those with the highest number of poultry farms in the country (n = ) and was therefore considered of critical importance, but no actual cases of h n had appeared in this area at the time of the study. to prepare the sampling frame a list of poultry farms in rupandehi was obtained from the district livestock services office (dlso), which runs a registry of all farms which habitually house more than chickens. from this overall district list the three village development committees (vdcs) with the highest density of poultry farms ( % of all farms in the region) were chosen. at the second stage farms were randomly selected from each of the three vdcs. from each of these farms, proportional to their size, between and poultry workers were interviewed based on a snowballing principle. prior to the interviews verbal informed consent was obtained from participants. sample size estimation for the proportion estimates was based on the following assumption: for a desired width of a % confidence interval of w = . , an alpha-level of p = . , an intra-cluster correlation of r = . and a number of m = clusters, the required number in each cluster was n = . which corresponds to an estimated sample size of . the actual sample size obtained from the field was slightly below the estimation i.e. n = . the interview was based on a standardized questionnaire. interviewers read the questions to the study participants and recorded responses on an answering sheet. questions about avian influenza were developed on the basis of a published questionnaire from a study on italian poultry workers [ ] as well as the who fact sheet on ai [ ] . socio-demographic information was collected for age, gender, school education and occupational status (owner of poultry farm versus paid employee). awareness about avian influenza was assessed by asking whether cases of avian influenza among poultry had appeared in nepal (yes/no). perceptions of professional risk were measured by a question asking about whether particular professional groups such as poultry workers, butchers or health workers were at risk for contracting avian influenza (yes/no). further, participants were asked from which sources they had obtained information about avian influenza, among them radio, tv, and newspapers. also, they indicated how afraid they felt that they could contract avian influenza (five-pointanswering scale from "not at all" to "very much"). knowledge about protective behaviours was assessed by an open-format question without pre-formulated answering options. respondents were asked to name all protective measures they knew against the danger of being infected due to work with poultry, and the interviewers recorded the answers on the interview sheet. for each behaviour correctly identified one point was assigned. afterwards study participants were asked to indicate how often they were using the following preventive measures when dealing with poultry: washing hands with soap and water, donning gloves, face masks, boots/boots covers, putting on protective body garments, and washing and disinfecting utensils and surfaces (five-step-answering format from "always" to "never"). attitudes towards government actions were assessed by first describing current government policies and then asking whether respondents agreed with, disagreed with or felt uncertain about these approaches. finally, another open-format item asked about habitual actions taken when sick or dead poultry was found on the farm. descriptive analyses were applied by using means, standard deviations and percentages. multivariable analyses were performed using logistic regression. the variables included were chosen on the basis of the research questions, intending to test socio-demographic differences in knowledge and behaviour, as well as mass media use, levels of fear, and, for the model testing protective behaviours, the role of knowledge about such behaviours. for the model explaining knowledge age, gender, school education (primary level and lower versus higher than primary education) and occupational status (owner of poultry farm versus paid employee), use of visual mass media for health information (receiving versus not receiving health information from tv and newspapers) and degree of fear experience (scale from "not at all" to "very much") were entered into the equation. knowledge was dichotomized as low level (knowing to protective behaviours) versus high level (knowing more than behaviours). the same variables were entered into the regression for the second model testing associations with protective behaviours, which additionally included the variable "knowledge about behaviours" (continuous, - ). the maximal number of correct behaviours named was actually seven. however, as only extremely few respondents fell into this category the variable was recoded so that the last category included those who knew six and more behaviours. the number of protective behaviours used on a habitual basis (always and often) was dichotomized into low level of behaviours (maximally two behaviours practiced) versus high (more than two behaviours practiced). p-values smaller or equal to . were considered statistically significant. the analysis was performed with spss ibm statistics . for windows. the majority of respondents was male ( %) and fell in the age bracket between and years ( %; m = ; sd = . ). nearly two thirds ( %) were owners of poultry farms, the others ( %) were employed workers. the size of the farms varied between approximately and , poultry. twenty eight per cent of the respondents had completed higher secondary and % lower secondary education, while % were illiterate. as the questionnaire was administered via personal interviews complete datasets were obtained for all respondents on the variables used in the analyses. nearly everybody ( %) was aware that ai cases in poultry had been detected in nepal. the main sources of information about ai were radio ( %), followed by tv ( %) and newspapers ( %); only one per cent had received health information from health workers. all of the respondents knew that poultry workers were among the "at-risk-groups" for being infected with avian influenza, and the majority ( %) expressed some degree of fear about ai. % described themselves as very afraid, while % said they were "rather afraid" (scale - , m = . , sd = . ). as for knowledge about protective behaviours in dealing with poultry, slightly over half ( , %) of the respondents knew about up to two such measures and a further % were aware of three, while only small percentages of respondents could name four or more such practices. clearly the best known among all measures was hand washing. protective properties of gloves and face masks were less well known, but still were referred to by a majority. only few, in comparison, named the protective potential of body suits, boots/boot covers or washing and disinfecting utensils and surfaces (see table ). when it came to practicing these behaviours, hand washing with soap and water was the most prevalent practice as it was uniformly reported as being used "always" or "often". use of other personal protective actions, however, seemed to be less common practice. habitual washing and disinfecting of surfaces and utensils was reported by about %, customary use of gloves and face masks by slightly less than one third, and only very few stated that they did use special boots or protective body garments (see table ). about half ( %) of the group regularly practiced up to two such behaviours, about one third ( , %) reported using three to four, and only , % more than four of these protection measures. percentage of poultry workers naming the specific behaviour when asked to list all protective practices against ai percentage of poultry workers indicating they were always or often using this practice most government control measures found uniformly high acceptance. thus, nearly everybody agreed that in case of an outbreak movements should be restricted ( %), all poultry on the respective farms or in the respective areas be culled ( %) and approval of the dlso be required to restart a business ( %). however, there were also notable exceptions. only % said they thought that the government steps being taken to prevent avian influenza outbreaks were sufficient, and only % thought that the compensation plans were adequate. when asked how they habitually acted when they encountered sick or dead poultry, most respondents stated that they used treatment ( %) and burial of carcasses ( %). only very few said that when finding sick ( %) or dead poultry ( %) they were in the habit of notifying the district livestock services office. factors associated with knowledge about protective behaviours against avian influenza table depicts the findings from a logistic regression analysis testing associations between socio-demographic factors, source of ai information, fear experience and level of knowledge about protective behaviours. none of the socio-demographic factors showed any relationship with knowledge. as nearly everybody reported having received information via radio, the analysis for media use compared only those who had received ai information from tv and newspapers with those who had not. poultry workers who got their information from these visual mass media were significantly more likely to be in the group with high knowledge than those who did not. also there was an inverse relationship between knowledge and fear, indicating that being less afraid was associated with knowing more about preventive behaviours. when it came to protective behaviours against personal infection one major significant difference occurred for occupational status, indicating that paid employees were considerably more likely to practice a larger number of preventive behaviours than owners of poultry farms. mass media use, while associated with knowledge was not related with precautionary practices, while level of knowledge did make a difference for such behaviours (see table ). awareness about the specific risk faced by poultry workers was uniformly high, and a majority of over % among respondents felt afraid of contracting ai. these findings are unsurprising given that the study was conducted shortly after the first ai outbreak in nepal but might also to some extent reflect increased governmental campaign efforts at promoting awareness after the outbreak had occurred. also, almost everyone knew about the importance of washing hands with soap and water, which had been the main message in the campaign. this finding is in line with studies on poultry workers in other countries which similarly found hand washing to be by far the best known practice [ , ] . assessment of knowledge in other areas, however, unveiled distinct gaps and deficits. while still almost % knew about the protective capacity of gloves, only half of the sample mentioned face masks as an option and only few knew about special boots or boot covers and body suits. also, only about one fourth named a basic procedure such as washing and disinfecting surfaces and utensils. another study, on poultry workers in nigeria, also reported "low" levels of knowledge about preventive behaviours [ ] , others, however, found distinctly higher rates for knowledge about face masks, boots covers and cleaning procedures [ ] than the present study did. one reason for this discrepancy might be that open-format questions like those used in the present study generate lower knowledge scores than identification tasks. also, the finding about low knowledge about cleaning/disinfecting could be interpreted as a tendency to perceive such behaviours as routine everyday practices instead of as extraordinary precautionary measures against ai. yet, these gaps in knowledge raise concern and suggest that future campaigns should make additional efforts to specifically target poultry workers and -beyond hand washing -focus also on the more specific behaviours which are relevant for prevention and containment of the virus at the source, i.e. on the poultry farms. analysis of the factors which were associated with knowledge about protection showed that tv and newspapers, which carried a substantial part of the campaign messages in nepal, played an important role. those who received information about ai via tv and newspapers were able to name more preventive behaviours than those without that kind of exposure -an effect also reported by other studies [ , ] . this finding certainly suggests a beneficial effect of the nepali mass media campaign but at the same time highlights deficits in reaching groups without access to these types of media -something to be considered for future health education efforts. another relevant factor, which was negatively associated with level of knowledge, was fear. at first glance, this seems to suggest that a higher degree of fear leads to less knowledge due to defensive processes such as not wanting to deal with the threat and therefore searching for less information. while such an explanation cannot be excluded, another mechanism is more plausible. the focus in this case was specifically on knowledge about protective behaviours, not on knowledge about ai in general, its danger potential, transmission pathways etc. while the latter type of knowledge is likely to make people aware of risks and therefore also more concerned, knowledge about effective protective behaviours might rather reduce fear by creating expectancies about successful control. the data on protective behaviours showed that washing hands with soap and water were fairly standard practice. high-frequency cleaning and disinfecting, however, was not and neither was habitual use of personal protective equipment. low usage rates for protective clothing have recently also been reported by studies with nigerian poultry farmers [ , ] , while findings from an italian study registered considerably higher rates [ ] , which probably reflects different financial resources to fund such equipment on a regular basis. the relevance of economic constraints was also indicated by the findings from the multivariable models. there was a substantial difference in usage rates of protective equipment between poultry farm owners and employees. the latter had higher odds to use personal protective equipment than owners of farms. employed poultry workers in nepal tend to work more often in larger-scale, economically better-off poultry businesses whereas many owners operate small-scale family businesses. paid employees might thus more often have been provided with protective equipment by farm management while owners of small-scale family businesses were more likely to save on expenses, thereby trading off possible longer-term preventive gains against more immediate economic savings [ , ] . the finding that those who had more knowledge were also those who actually acted more preventively is consistent with some other studies from the field [ ] [ ] [ ] even if the overall evidence on this issue is still inconsistent [ ] . one possible explanation for such discrepancies is that effects might depend upon the specific type of knowledge measured. knowledge about effective behaviours, which was the focus of the present study, is particularly likely to enable perceptions about efficacy of behaviours which have consistently been linked to precautionary practices [ ] . nevertheless, knowledge about a threat and potential countermeasures alone will most often be insufficient to achieve behaviour change, as other factors such as economic concerns or social norms are essential for enabling or disabling such change. yet, the findings emphasize the role of awareness-building about the availability of preventive options as a first step in generating preventive habits. a result which raises concern is that despite a generally voiced agreement with governmental emergency control measures and bio-security regulations large parts of the respondents expressed doubts with regard to the sufficiency of such control measures and eventual compensation mechanisms. also, in response to a question about what they habitually did in case of sudden chicken deaths only four per cent reported actually having notified authorities in case of sick/dead poultry. similar findings have been published by other studies [ ] [ ] [ ] [ ] [ ] [ ] . anticipated financial losses due to culling without sufficient compensation, lack of knowledge about how to proceed in notifying authorities, but also social considerations, such as stigma and shame might play a role and need dealing with to overcome avoidance of timely reporting [ ] [ ] [ ] . if early notification is a key component of prevention and rapid response, trust in government actions, including compensation measures, is crucial in order to enable pervasive compliance with drastic and economically threatening actions like mass culling in an outbreak situation [ ] . a major limitation of the study lies in the small, nonrandom sample which restricts possibilities to generalize findings from the present data and also, due to lack of power, might have led to underestimation of potential effects. another clear weakness is the cross-sectional study design which prohibits drawing causal conclusions about the relationships between some of the variables, such as fear and knowledge or knowledge and practices. finally, self-report on practices are generally vulnerable to recall bias and social desirability tendencies. the face-to-face-interview situation, while enabling full response-rates on all variables as well as participation of poultry workers who lack reading or writing abilities, might have additionally heightened this type of bias in assessing attitudes and behaviours. as for attitudes, however, the very low percentage of respondents reporting compliance with notification procedures indicates that such tendencies were not pervasive. the study points to issues that warrant attention in future prevention and preparedness efforts against ai. while it corroborates the relevance of cognitive factors, such as providing knowledge about effective protection measures, it also particularly highlights the important role of material resources which enable poultry workers in a low-resource country such as nepal 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beliefs we wish to express our thanks to chitra kumar gurung, bandana pradhan, jørn attermann and the poultry farmer's association of rupandehi as well as the district livestock service office, rupandehi and all the poultry farmers who participated in the study. authors' contributions vk, kg and dn conceived of the study and participated in its design and the data collection. dn and al performed the data analysis. dn, al and ara drafted the manuscript. all authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. key: cord- -h xnr authors: wiegers, hanke m. g.; van nijen, lisa; van woensel, job b. m.; bem, reinout a.; de jong, menno d.; calis, job c. j. title: bacterial co-infection of the respiratory tract in ventilated children with bronchiolitis; a retrospective cohort study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: h xnr background: viral bronchiolitis is the most common cause of respiratory failure requiring invasive ventilation in young children. bacterial co-infections may complicate and prolong paediatric intensive care unit (picu) stay. data on prevalence, type of pathogens and its association with disease severity are limited though. these data are especially important as bacterial co-infections may be treated using antibiotics and could reduce disease severity and duration of picu stay. we investigated prevalence of bacterial co-infection and its association with disease severity and picu stay. methods: retrospective cohort study of the prevalence and type of bacterial co-infections in ventilated children performed in a -bed tertiary care picu in the netherlands. children less than years of age admitted between december and november with a diagnosis of bronchiolitis and requiring invasive mechanical ventilation were included. tracheal aspirates (ta) and broncho-alveolar lavages (bal) were cultured and scored based on the quantity of bacteria colony forming units (cfu) as: co-infection (ta > (^ )/bal > (^ ) cfu), low bacterial growth (ta < (^ )/bal < (^ ) cfu), or negative (no growth). duration of mechanical ventilation and picu stay were collected using medical records and compared against the presence of co-infection using univariate and multivariate analysis. results: of included children ( . %) had a bacterial co-infection and ( . %) low bacterial growth. co-infections occurred within h from intubation in out ( . %) co-infections. h.influenza ( . %), s.pneumoniae ( . %), m.catarrhalis ( . %), and s.aureus ( . %) were the most common pathogens. picu stay and mechanical ventilation lasted longer in children with co-infections than children with negative cultures ( . vs . days, p = . and . vs . days, p = . ). conclusions: in this large study, bacterial co-infections occurred in more than a third of children requiring invasive ventilation for bronchiolitis and were associated with longer picu stay and mechanical ventilation. these findings support a clinical trial of antibiotics to test whether antibiotics can reduce duration of picu stay. bronchiolitis is a common respiratory condition in young children associated with a high morbidity. it is caused by viral infections, respiratory syncytial virus (rsv) being the commonest cause in hospitalized children [ ] . the clinical picture of these infections ranges from mild symptoms to respiratory distress requiring hospitalization for supportive therapy [ ] . about % of hospital admissions for rsv infection are so severe that invasive ventilation in a pediatric intensive care unit (picu) is needed [ ] . although several risk factors for a more severe course of bronchiolitis have been identified, including cardiac disease, chronic lung disease, prematurity, this only partly explains why some children with bronchiolitis have severe respiratory failure and require mechanical ventilation [ ] . previous studies suggested that bacterial co-infections may be associated with a more severe course, though definitive evidence is lacking [ ] [ ] [ ] [ ] . this is of particular interest as current treatment strategies for severe bronchiolitis are only supportive, whereas bacterial coinfections can be treated with antibiotics and thus could reduce morbidity. the aim of this study is to investigate the prevalence and types of bacterial co-infection of the respiratory tract in children with bronchiolitis requiring invasive ventilation. we further investigated whether bacterial co-infection was associated with a prolonged duration of mechanical ventilation and picu stay. finally, we analysed whether bacterial co-infection can be predicted using clinical or laboratory markers such as c-reactive protein (crp). this is a retrospective cohort study of children requiring invasive ventilation in the picu of the emma children's hospital/academic medical center amsterdam, between december and november . the picu is a -bed, tertiary unit, serving the greater amsterdam area in the netherlands. patients less than years of age who were clinically diagnosed with bronchiolitis and required invasive mechanical ventilation were included. in the netherlands patients are referred to the picu if they (are expected to) require (non) invasive ventilation. diagnoses were scored on discharge by the attending picu consultant and stored in an automated database containing all picu admissions. a standardised questionnaire was completed using data from the electronic patient chart that automatically recorded vital signs, ventilator settings; and collected medication given (metavision®). an electronic patient chart was used to gather demographic data, previous medical history, physical examination on admission, laboratory results, chest x-ray on admission, microbiological results, and outcome parameters. the mechanical ventilation duration was defined as the cumulative period of invasive ventilation and non-invasive ventilation. if extubation failed the cumulative periods of (non-)invasive ventilation was used. nasal specimens were collected and used for dna extraction and multiplex pcr as previously described [ ] . the multiplex platform included influenza viruses (a and b), enterovirus, adenovirus, respiratory syncytial virus (a and b), rhinovirus (a-c), human metapneumovirus; parainfluenza viruses ( - ), parechovirus, human bocavirus and coronaviruses (hcov; hku , nl , e, and oc ). with every extraction and pcr, three controls were run. a minority of children had previously been tested in other hospitals for rsv using pcr or antigen tests. these data were used if multiplex results from our hospital were not available. viral testing was performed by the discretion of attending physicians. in some children no viral testing was performed at all. the cultures performed in this study were ordered by the attending physicians in case of clinical suspicion of a bacterial (super)infection. during the study period two sampling techniques were used: tracheal aspirates and mini-broncho-alveolar lavage [ ] . sampling was performed the endotracheal tube without previous installation of normal saline (tracheal aspirate) or by mini broncho-alveolar lavage. tracheal aspirate was the primary sampling method. the mini-bal was performed as part of an observative study on cytokine levels in bronchiolitis. bacterial cultures were performed according to standard operating procedures at our laboratory. in brief, μl of specimen were inoculated on standard media (cled, colombia, sheep blood and chocolate agars) and incubated for - days. bacterial colonies were counted and identified by vitek (biomerieux) or malditof (bruker). other diagnostics x-rays were scored and reported by attending radiologists. plasma levels of c-reactive protein, were analyzed on modular p and modular analytics e systems (roche). leukocytes were determined on an automated hematology cell counter. to distinguish between colonisation and infection we used quantitative culture results, represented by the number of cfu per ml. we distinguished categories based on the american thoracic society definitions in adults and thorburn et al. [ , ] . to distinguish between colonisation and infection we used the quantity of cfu/ml [ , ] to distinguish categories. no bacterial growth: no growth or growth of commensal bacteria only. low bacterial growth: presence of pathogens in the bal < cfu/ml or trachea aspirates < cfu/ml. bacterial co-infection: presence of pathogens in the bal ≥ cfu/ml or trachea aspirates ≥ cfu/ml. to distinguish between bacterial co-infections present on admission and those associated with ventilation we used the following definitions. early infections (non-ventilatorassociated) apply to cultures taken within h of intubation, whilst late infections (ventilator -associated) refer to samples taken more than h after intubation [ ] . data was entered and analysed in an anonymised database spss . (spss inc., chicago, illinois, usa). for comparison of continuous variables we used independent ttests and for comparison of categorical variables chisquare test and fisher exact test were used. all pvalue reported are two-sided and values less than . was considered significant. potential predictors of duration of ventilation and picu stay were assessed using univariate and linear regression analysis. logistic regression was used to assess potential predictors of bacterial co-infection. variables were included in multivariate analyses if p-values of univariate associations were < . . multivariate models included potential confounding factors (e.g. previous antibiotic use). the study adhered to the strobe guidelines. medical ethical approval was waived as it concerned retrospective analysis of anonymised patient data. of picu admissions for bronchiolitis, ( . %) required invasive ventilation and were included in the study. the mean age was . months, patients ( . %) were less than months of age and ( . %) were boys. twenty-five patients had an underlying medical condition ( . %) and ( . %) were prematurely born (table ) . data on antibiotic use prior to picu referral was available in children, of these received antibiotics ( . %). one patient died due to pulmonary hypertension. rsv testing was performed in ( . %) patients and was detected in ( . % table ). in ( . %) children a full multiplex platform was performed and children ( . %) had presence of two of more viral pathogens (table ) . in of ( . %) patients one or more cultures (bal or tracheal aspirates) were performed, whereas in patients there was no culture performed ( . %). demographic data or underlying conditions were not different in these groups (data not shown). among the cultured children, the overall occurrence of bacterial co-infections was ( . % of children cultured, or . % of all children). low bacterial growth was found in children ( . %). in the children with a bacterial co-infection ( . %) coinfections were detected within h from intubation and were typed as early infection (table ) . thirty-four co-infections were detected by tracheal aspirate ( . %), and ( . %) by mini-bal. patients who received antibiotics prior to picu admission had more often negative cultures as compared to patients who did not receive antibiotics ( . % versus . %, p = . ). bacterial co-infections were found in . % of children with a proven rsv infection as compared to . % in the rsv-negative group ( / ) and . % in those who had no viral test performed ( / ; p = . ). in children with a bacterial co-infection a total of pathogens were isolated including h. influenza ( . %), s. pneumoniae ( . %), m. catarrhalis ( . %), and s. aureus ( . %) ( table ) . enterobacteriaceae were only seen as late co-infection. in patients with low bacterial growth the distribution of pathogens was comparable to the co-infection group (data not displayed). only s. aureus was more common in low bacterial growth as compared to the coinfection group ( . % vs . %, p = . ). s. pneumoniae was found in . % of rsv-positive patients ( / ), . % in rsv-negative patients ( / ) and in . % of patients who had no viral test performed group ( / , p = . ). children with a bacterial co-infection required a mean duration of ventilation of . days compared to . days in children with a negative culture result (p = . ; table ). picu length of stay was . and . days in children with and without a co-infection respectively (p = . ). no difference was noted in duration of ventilation nor picu stay between the groups with coinfection and low bacterial growth. early co-infections were not associated with a longer duration of ventilation or picu stay as compared to children with negative cultures ( . vs . , p = . and . vs . , p = . respectively). late co-infections were associated with a longer duration of ventilation and picu stay ( . vs . , p < . and . vs . , p < . ). a high crp was associated with bacterial co-infection in a multivariate analysis including antibiotic use prior to picu admission (p = . , table ). a crp level of mg/l or more had a sensitivity of . % and a specificity of . % to detect a bacterial co-infection. in this large and concise study assessing the prevalence and relevance bacterial co-infections of the respiratory tract in children with bronchiolitis requiring invasive ventilation, bacterial co-infections were identified in at least a third of patients. in our population bacterial coinfections were associated with a longer duration of ventilation and picu stay. crp was associated with bacterial co-infection and may be used to identify children at increased risk. in our study more than a third ( %) of the children with bronchiolitis requiring invasive ventilation had a bacterial co-infection. potentially this number may have been higher as we used: a) a very strict definition of bacterial coinfection, ignoring % of children with low bacterial growth; b) % of children received antibiotics prior to sampling, which may have decreased detection of bacterial co-infections. we may also have overestimated the prevalence of bacterial co-infections as we used a clinical diagnosis of bronchiolitis whilst some children may have had a primary bacterial infection. this hypothesis is less likely though as the prevalence of bacterial co-infections in the rsv-positive group was comparable to the overall prevalence ( and % respectively). the prevalence of bacterial co-infection is in line with previous studies that reported bacterial co-infections occurred in - % of children with bronchiolitis admitted to picu's [ , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . like our study, most data were retrospective and had similar limitations such as previous antibiotic use. a prospective study reported that % had a co-infection and % had low bacterial growth/possible co-infection [ ] . also, in this study more than % of children had received antibiotics prior to airway sampling. most co-infections were detected within h, a cut off that is used to distinguish pre-existing infections from ventilator acquired pneumonias (vap) [ ] . although it is not possible to distinguish pre-existing co-infections from infections acquired by our invasive techniques the fact that most co-infections occurred within these h suggest that these infections are not likely introduced by intubation or colonization of endotracheal tubes. in our setting most bacterial isolates were common airway pathogens such as h. influenza, s. pneumoniae, m. catarrhalis and to a lesser extent s. aureus. this corroborates with data from bronchiolitis studies in liverpool [ ] and zurich [ ] , however in our setting s. pneumoniae was more and s. aureus less common than in other studies. enterobacteriaceae were solely identified as late coinfections; which is in line with previous data on vap [ ] . in our setting empirical treatment with amoxicillin (with or without clavulanic acid) would be appropriate for early infections, however in late co-infections an antibiotic with a broader coverage should be considered. in this study we identified an association between bacterial co-infection and duration of picu stay and mechanical ventilation. the difference was days which can be considered clinically relevant. three other studies reported on duration of ventilation and the presence of bacterial co-infections in children with bronchiolitis requiring ventilation. kneyber et al. found a trend towards longer duration of mechanical ventilation in children with bacterial co-infections as compared to those without ( . vs. . days, p = . ) [ ] ; thorburn et al. reported that bacterial infection contributed to longer mechanical support [ ] and hennus et al. confirmed that mechanical ventilation and bacterial co-infection were positively correlated [ ] . the association between bacterial growth and a prolonged duration of mechanical ventilation suggests that bacterial co-infection could be of clinical importance. alternatively, prolonged ventilation may predispose to increased prevalence of bacterial infections (ventilator associated pneumonia) [ ] . in our study the difference in duration of ventilation was only significant in the late co-infections which corroborates with the latter theory. a similar trend was observed in the early co-infections which could suggest that both hypotheses are correct. the association between co-infections and length of ventilation in our and other studies justify a trial to assess the role of antibiotics in reducing duration of ventilation and picu stay in children with a bronchiolitis requiring invasive ventilation. ideally the design of such a study should include a curative and preventive arm to evaluate both the role of pre-existing and ventilator associated bacterial pneumonia. except for a study assessing the role of antibiotics to prevent picu admissions [ ] , such a study has not been performed so far. the use of prophylactic antibiotics in ventilated patients has been studied in adults using selective digestive decontamination (sdd). d'amico et al. showed that sdd given to adults admitted to icu reduced vap and overall mortality [ ] . the data on the overall effect of all ventilated children admitted to picu is conflicting however, but sdd appears to be effective in controlling respiratory infections [ ] . no studies have been performed in the current subgroup of bronchiolitis patients admitted to picu. in this study crp was a useful predictor of bacterial coinfection, which corroborates with previous data [ ] . that study used a much lower cut-off of mg/l, which in practice does apply to most children admitted with a bronchiolitis and may not be very discriminative. other studies did not find an association between crp and bacterial co-infection in bronchiolitis patients [ , ] . unlike these studies we used the maximum crp during admission, which may explain this discrepancy. crp is known to have a twofold increase every h, and therefor crp dynamics could be used to guide antibiotic use in this population awaiting culture results. several limitations apply to our study, firstly we may have underestimated the prevalence of bacterial co-infections as we used a) a strict definition and b) the majority of the patients received antibiotics prior to sampling. these factors potentially may have contributed to an underestimation of the actual prevalence and underline that the number of bacterial co-infections in children with viral bronchiolitis in picu is high. alternatively we may have overestimated the role of bacterial co-infections in children with bronchiolitis as we recruited children based on the clinical discharge diagnosis of the attending picu consultant. although of children had a proven viral infection some children may have had a primary bacterial lower airway infection. this is less likely though as also in the group with proven rsv infection the co-infection rate was % as compared to % in the overall study. secondly, not every patient who was admitted to the picu had a culture and cultures were performed based on clinically suspicion. this may mean that selection bias applied. however, the culture rate was nearly % and even if all children that were not cultured would have negative cultures still a third would have had a co-infection. thirdly, this was a retrospective and observational study, therefore we can only detect associations and not prove causal relationships. however, it justifies a prospective randomized controlled trial which could test the role of (curative or preventive) antibiotics to reduce duration of ventilation. fourthly, we have used different sampling techniques for bacterial cultures. however, we have applied strict definitions and corrected for this using the definition from thorburn [ ] et al. and international ats guidelines [ ] . until prophylactic or curative use of antibiotics have been tested in a double blind, randomized controlled trial, the use of antibiotics may be restricted to children with a suspicion of bacterial co-infection. especially in young children and children with an increased crp (> mg/l) cultures should be taken and antibiotics covering normal airway pathogens should be considered. in case late co-infection is suspected empiric antibiotics should cover gram-negative bacteria and s. aureus. future studies should prove if prophylactic or curative use of antibiotics could reduce duration of ventilation and picu stay in bronchiolitis patients. in this large and concise study on bacterial co-infections in children with bronchiolitis we have found that bacterial infections are common, associated with prolonged ventilation and a raised crp. bacterial co-infections of the respiratory tract occurred in at least . % of children with a bronchiolitis requiring invasive ventilation. in most cases bacterial co-infection was present within h after intubation and was caused by common airway pathogens: h. influenza, s. pneumoniae, m. catarrhalis. future studies should focus on the potential beneficial role of preventive or curative antibiotics to reduce duration of ventilation and picu stay. risk of bacterial infection in previously healthy respiratory syncytial virus-infected young children admitted to the intensive care unit the use of c-reactive protein in predicting bacterial co-infection in children with bronchiolitis bacteraemia and antibiotic use in respiratory syncytial virus infections pediatric investigators collaborative network on infections in canada (picnic) prospective study of risk factors and outcomes in patients hospitalized with respiratory syncytial viral lower respiratory tract infection high incidence of pulmonary bacterial co-infection in children with severe respiratory syncytial virus (rsv) bronchiolitis pulmonary and systemic bacteria co-infections in severe rsv bronchiolitis concurrent bacterial infection and prolonged mechanical ventilation in children with respiratory syncytial virus lower respiratory tract disease empiric antibiotics are justified for children with respiratory syncytial virus lower respiratory tract infection presenting with respiratory failure: a prospective study and evidence review development and evaluation of a four-tube real time multiplex pcr assay covering fourteen respiratory viruses, and comparison to its corresponding single target counterparts azithromycine does not improve disease course in hospitilized children with respiratory syncytial virus (rsv) lower respiratory tract disease: a randomized equivalence trial infectious diseases society of america. guidelines for the management of adults with hospital-acquired, ventilator associated, and healthcare-associated pneumonia risk of concurrent bacterial infection in preterm children hospitalized due to respiratory syncytial virus infection respiratory syncytial virus morbidity, premorbid factors, seasonality, and implications for profhylaxis survey of severe respiratory syncytial virus infection in kyoto prefecture from mechanical ventilation drives inflammation in severe viral bronchiolitis ventilator-associated pneumonia: diagnosis, treatment, and prevention antibiotic prophylaxis to reduce respiratory tract infections and mortality in adults receiving intensive care prevention of nosocomial infection in a pediatric intensive care unit (picu) through the use of selective digestive decontamination publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable.authors' contributions hw and ln performed data extraction. jc was an independent reviewer. jc, rb, and jw conceived the study. lab work and interpretation was supervised by mj. the article was written by all (hw, ln, jw, rb, mj and jc). all authors read and approved the final manuscript. not applicable. the anonymized datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate ethical approval was waived by the medical ethics review committee of the amc as it was retrospective analysis. not applicable. the authors declare that they have no competing interests. key: cord- -w kl c authors: li, jin; tao, yue; tang, mingyu; du, bailu; xia, yijun; mo, xi; cao, qing title: rapid detection of respiratory organisms with the filmarray respiratory panel in a large children’s hospital in china date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: w kl c background: respiratory tract infections (rtis) are the most common illness in children, and rapid diagnosis is required for the optimal management of rtis, especially severe infections. methods: nasopharyngeal swab or sputum specimens were collected from children aged days to years who were admitted to a hospital in shanghai and diagnosed with rtis. the specimens were tested with the filmarray respiratory panel, a multiplex pcr assay that detects viruses, mycoplasma pneumoniae (m. pneumoniae), bordetella pertussis (b. pertussis) and chlamydophila pneumoniae (c. pneumoniae). results: among the children studied, ( . %, / ) tested positive for at least one organism, and multiple organisms were detected in ( . %). rhinoviruses/enteroviruses ( . %, / ) were detected most often, followed by respiratory syncytial virus ( . %, / ), parainfluenza virus ( . %, / ), influenza a or b ( . %), adenovirus ( . %), m. pneumoniae ( . %) and b. pertussis ( . %). the prevalence of organisms differed by age, and most of the viruses were more common in winter. of the children suspected of having pertussis, . % ( / ) tested positive for b. pertussis. conclusions: filmarray rp allows the rapid simultaneous detection of a wide number of respiratory organisms, with limited hands-on time, in chinese pediatric patients with rtis. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory tract infections (rtis) are the leading causes of outpatient visits and hospitalizations in all age groups, especially during winter and spring. for children under years of age, rtis are the second leading cause of death [ ] . most acute rtis in children are caused by respiratory viruses, such as respiratory syncytial virus (rsv), adenovirus (adv), rhinovirus (rv) and influenza viruses. in addition to viruses, atypical pathogens are major causes of pediatric rtis. one of the most common atypical pathogens is mycoplasma pneumoniae (m. pneumoniae), accounting for - % of hospitalized children with community-acquired pneumonia [ , ] . in addition to m. pneumoniae, the incidence of pertussis in china has significantly increased since . nevertheless, multiple epidemiological studies have suggested that the incidence of pertussis in china has been significantly underestimated [ , ] . the early diagnosis of the pathogen is beneficial for the precise selection of medication, which can largely avoid the overuse or even abuse of the antibiotics and improve the clinical care of patients. more importantly, the early diagnosis of contagious pathogens, such as bordetella pertussis (b. pertussis) and influenza viruses, can enable early isolation of patients, thus reducing the spread of pathogens. at present, the routine detection methods for respiratory pathogens in china are mostly based on immunological methods, which include the detection of m. pneumoniae and several major viruses, such as rsv, adv, rv, parainfluenza virus (para), influenza a virus (flua) and influenza b virus (flub). other respiratory viruses and atypical bacteria, such as chlamydophila pneumoniae (c. pneumoniae) and b. pertussis, are typically not routinely detected. given their poor sensitivity and long turn-around time (tat), immunological methods usually lead to broad-spectrum therapy and have been gradually replaced by molecular-based methods, such as conventional and real-time polymerase chain reaction (pcr), in developed countries [ , ] . however, most of these molecular tests are technically challenging and require independent spaces, such as pre-pcr and post-pcr rooms, to eliminate the potential risk of cross-contamination, and such requirement limits their applications in china. therefore, faster, more sensitive and easy-to-use assays for multiplex respiratory pathogen detection are urgently needed. filmarray (biofire diagnostics, utah, usa, owned by biomérieux) is a small, desktop, fully automated multiplex pcr device. the molecular system includes automated nucleic acid extraction, an initial reverse transcription step and multiplex nested pcr, followed by a melting curve analysis [ ] . the filmarray respiratory panel (filmarray rp) is both fda-approved and ce ivd-marked. the current version of filmarray rp (v . ) is able to detect viral and atypical respiratory organisms. the test is performed in a closed system that requires min of hands-on time and min of instrumentation time. several comparison studies between fil-marray and other tests for respiratory organisms showed comparable results [ ] [ ] [ ] . the aim of this study was to evaluate the application of filmarray rp for the detection of respiratory organisms, and to provide information about the seasonality and prevalence of these organisms in pediatric patients with rtis in a large children's hospital in china. the study population was enrolled according to protocol definitions and inclusion criteria. patients with respiratory infections, with or without fever (defined as body temperature ≥ . °c), were included if they had at least one of the following symptoms: ( ) cough; ( ) nasal obstruction; ( ) tachypnoea; ( ) nasal flaring; or ( ) hypoxia. patients admitted to the hospital had at least one of the following conditions: ( ) unabating high fever; ( ) dyspnea, tachypnea or hypoxemia; ( ) anorexia or dehydration; ( ) radiological confirmation of lung infection; or ( ) respiratory infection with underlying diseases, such as congenital heart disease, bronchopulmonary dysplasia, airway malformations, severe malnutrition. according to the chinese center for disease control and prevention (cdc), patients suspected of having pertussis should have a cough for more than weeks and have at least one of the following symptoms: ( ) paroxysmal cough; ( ) inspiratory whoop; or ( ) post-tussive vomiting. in the present study, patients suspected of having pertussis were diagnosed with pertussis when b. pertussis was positive by filmarray rp detection and were otherwise diagnosed with pertussis-like syndrome. nasopharyngeal swab (nps) or sputum specimens were obtained from patients with symptoms of rtis on the day of hospitalization at shanghai children's medical center (scmc) from december , to november , . demographic data and clinical features, as well as laboratory test and imaging results, were obtained for each enrolled patient. the study was approved by the institutional review board and the ethics committee of shanghai children's medical center (scmcirb-k ), and written informed consent was obtained from the parents of each patient. the filmarray rp v . targets organisms, including adv, influenza a viruses h , h , h (flua-h , flua- h , flua-h ) and flub, parainfluenza virus types to (para - ), coronaviruses e, hku , oc , and nl (cov-hku , nl , e, oc ), human metapneumovirus (hmpv), rsv, human rhinovirus/ enterovirus (rhino/entero), c. pneumoniae, m. pneumoniae and b. pertussis. the filmarray rp assay was performed according to the manufacturer's instructions. the principle of the assay has been previously described [ , ] . each pouch included internal run controls for every step, and results for the assay were only provided by the software if the quality control reactions showed appropriate results. spss software package v . was used for all statistical analyses. categorical variables were expressed as frequencies and percentages. the chi-square and fisher's exact tests were used to compare groups. continuous variables are expressed as the mean and standard deviation. student's t-test was used to assess the statistical significance between groups. p < . was considered to be statistically significant. a total of patients diagnosed with upper or lower respiratory tract infections, aged days to years, were enrolled in the present study between december , , and november , . congenital heart disease, congenital biliary atresia, malignancy and congenital immunodeficiency were the most frequently observed underlying diseases in these patients and contributed to % of the deaths observed in this study. the general characteristics of the patients enrolled are presented in table . overall detection rate of filmarray rp v . analysis of the positive rates and prevalence in different age groups all the patients were grouped by age as follows: infants (age: < year), toddlers (age: - years), preschoolers (age: - years) and school-aged children (age: - years) ( table ). the highest specimen positivity rate, at . % ( / ), was in the < -year age group, followed by . % ( / ), . % ( / ) and . % ( / ) in the - -year, - -year and - -year groups, respectively. there were no significant differences in the positivity rate of the different age groups. in contrast, the prevalence of organisms were different between the different age groups ( table ) . rhino/entero, para , rsv and b. pertussis showed the highest prevalence in the < -year age group, while adv, hmpv and flua showed the highest prevalence in the - -year age group. the most prevalent organism in the - -year age group was m. pneumoniae. no organism showed a notably high prevalence in the - -year age group. there was only one c. pneumoniae-positive patient during the study period, and this patient was in the - -year age group. among the specimens, ( . %, / ) were positive for more than one organism. the largest proportion ( . %, / ) of multi-organism-positive specimens had combinations with rhino/entero. rhino/entero plus para was the most common combination, making up . % ( / ) of all multi-organism-positive specimens, while the combination of rhino/entero plus adv was the second most common type ( . %, / ), followed by rhino/ entero plus rsv ( . %, / ). the multi-organism combinations are listed in additional file : table s . seasonal prevalence of respiratory organisms from december , to november , the number of positive specimens was determined during different months of the year to demonstrate the epidemiology of the respiratory organisms. regarding the atypical bacteria, m. pneumoniae was detected throughout the year, with the highest incidence occurring in september and three minor peaks in december, january and june (fig. a) . the highest incidences of b. pertussis were observed in march and may. only one case of c. pneumoniae was detected in july. the seasonal prevalence of viruses with high detection rates were as follows. both flua and hmpv had two peaks that occurred in january and march, and adv showed a peak in january (fig. b) . the prevalence of para remained high from february-august. the peaks in our study, specimens were collected from pediatric patients with rtis over a period of one year and analyzed with filmarray rp v . . the overall results yielded a positivity rate of . %, with multiple organisms detected in . % of specimens, which is in accordance with litwin and piralla's reports [ , ] . as in other studies, a notable variation in the pathogen prevalence with season and age was observed. most viruses had their highest positivity rates in winter, except that para positivity rate was well distributed through the spring and summer, and the epidemiologic peaks for hmpv occurred to months later than those for rsv [ , ] . the majority of respiratory viruses were observed in children younger than years old. notably, rsv was the most prevalent virus in the < -year age group, and the prevalence decreased with age; while the incidence of m. pneumoniae increased with age [ ] . multiple respiratory organisms were detected in . % of the specimens in our study, the largest proportion of which included rhino/entero. other studies in adults reported lower multi-pathogen detection rates of approximately . - . % [ , [ ] [ ] [ ] , suggesting that pediatric patients with rtis are more likely to be infected by multiple pathogens than adults. however, the clinical significance, including disease severity and hospitalization time, of multi-pathogen infection, especially rhino/entero combination infections, is not clear. a previous report indicated that dual-positive results with rsv and rhino/entero specimens might be due to viral shedding from a previous rhino/entero infection [ ] . nokso-koivisto et al. also found that rhinovirus was the most prevalent virus in asymptomatic carriers [ ] . the most unexpected result in our study is the high detection rate of b. pertussis, with an overall detection rate of . % in the group of patients, further demonstrating the value of filmarray rp in clinical application. at present, the diagnosis of pertussis in china is based on culture and serology results. however, both the cdc and world health organization (who) use positive pcr results as the criteria for diagnosis, suggesting that filmarray rp testing, in addition to culture, can be considered for patients with suspected pertussis in order to better monitor disease outbreaks. additionally, the early diagnosis of patients with b. pertussis, which is typically difficult to distinguish from pertussis-like syndrome, can also help to reduce unnecessary macrolide treatment. the limitation of the panel is the lack of b. parapertussis, which contributes to more than % of pertussis cases [ ] . however, it has been added to the second-generation panel, filmarray rp v . [ ] , and the prevalence of b. parapertussis in our patients is currently under investigation. as stated in the manufacturer's instructions, "filmarray respiratory panel (rp) is a multiplexed nucleic acid test intended for use with filmarray systems for the simultaneous qualitative detection and identification of multiple respiratory viral and bacterial nucleic acids in nasopharyngeal swabs (nps) obtained from individuals suspected of respiratory tract infections". therefore, nps samples are recommended for filmarray rp, but there are also studies demonstrating a comparable or even higher detection rate in sputum [ , ] . however, the detection rate in sputum in our study was lower than that in nps samples. this might partially be attributed to the fact that most of the sputum samples ( . %, / ) were from icu patients, and the sputum-providing patients showed a higher positivity rate in their sputum culture than the nps-providing patients ( . % vs . %). in addition to sputum, bronchoalveolar lavage fluid (balf) is a common type of respiratory sample, and azadah et al. showed that detection in balf by filmarray rp can provide new and useful microbiological information within days after a negative nps result is obtained [ ] . therefore, the choice of the most appropriate sample type and time-point for each patient, particularly in specific clinical contexts, such as undergoing fiberoptic bronchoscopy or ventilator use, may require further investigation. as with other molecular methods, distinguishing whether the microbes detected in the filmarray analysis, especially those that are also detected in asymptomatic children, such as human rhinovirus, are causative pathogens or colonizers is not feasible [ ] [ ] [ ] . therefore, the clinicians should take caution when judging pathogens because the results are sometimes "false positive". on the other hand, despite the high detection rate of fil-marray rp, a negative result does not mean the patient is not infected; moreover, a positive result does not mean there is no other co-infecting agent, especially in critically ill patients, in whom a bacterial co-infection often occurs. for this "false-negative" limitation, biofire has a new pneumonia panel that also targets balf/sputum and covers common viruses, as well as bacteria, including klebsiella pneumonia, haemophilus influenza, streptococcus pneumonia and staphylococcus aureus. nevertheless, the filmarray panel only aims to rapidly provide results for potential pathogens as a reference. a more appropriate method is to comprehensively consider the results from other examinations, such as routine blood testing, c-reactive protein (crp), procalcitonin (pct), the erythrocyte sedimentation rate (esr), culture and radiography, as well as the patients' symptoms, including body temperature, breathing, blood oxygen, heart rate, and mental condition. our study also has several limitations. first, our study was performed in a single center and may not be representative of the entire chinese pediatric population. second, we did not have data from a more appropriate assay to evaluate the specificity of filmarray rp. additionally, we do not provide detailed information on the effects of filmarray rp on the use of antibiotics, clinical outcomes and health economics, which require further investigation. in conclusion, the filmarray rp assay significantly expands our ability to diagnose multiple respiratory infections caused by viruses and atypical bacteria. the array can detect respiratory organisms simultaneously, with a high detection rate, in min. our study provided the age groups and seasonal distributions of different organisms for pediatric rti patients. this study also provides new insights into the current status of pertussis infection in china. whether filmarray rp can enhance clinical decision-making and limit the unnecessary use of antibiotics in china as in other countries still requires further investigation. additional file : table s . combinations of multiple organisms detected with filmarray rp. (doc kb) additional file : table s . overall detection rates for the nasopharyngeal swab and sputum samples from the different age groups. epidemiology of viral respiratory infections community-acquired pneumonia requiring hospitalization among u.s. children epidemiology of acute respiratory infections in children in guangzhou: a three-year study seroprevalence of pertussis in china: need to improve vaccination strategies the seroepidemiology of immunoglobulin g antibodies against pertussis toxin in china: a cross sectional study routine molecular point-of-care testing for respiratory viruses in adults presenting to hospital with acute respiratory illness (respoc): a pragmatic, open-label, randomised controlled trial multiplex pcr and emerging technologies for the detection of respiratory pathogens an automated nested multiplex pcr system for multi-pathogen detection: development and application to respiratory tract infection charnot-katsikas a. comparison of cepheid xpert flu/rsv xc and biofire filmarray for detection of influenza a, influenza b, and respiratory syncytial virus comparison of the filmarray assay and in-house real-time pcr for detection of respiratory infection comparison of filmarray respiratory panel and laboratory-developed real-time reverse transcriptionpolymerase chain reaction assays for respiratory virus detection comparison of the filmarray respiratory panel and prodesse real-time pcr assays for detection of respiratory pathogens filmarray(r) respiratory panel performance in respiratory samples from neonatal care units seasonality and prevalence of respiratory pathogens detected by multiplex pcr at a tertiary care medical center human metapneumovirus subgroup changes and seasonality during epidemics seasonality, incidence, and repeat human metapneumovirus lower respiratory tract infections in an area with a high prevalence of human immunodeficiency virus type- infection etiologic spectrum and occurrence of coinfections in children hospitalized with community-acquired pneumonia performance of a novel microarray multiplex pcr for the detection of respiratory pathogens (symp-ari study) pcr for detection of respiratory viruses: seasonal variations of virus infections detection of respiratory viruses by molecular methods prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms update on pertussis and pertussis immunization multicenter evaluation of biofire filmarray respiratory panel for detection of viruses and bacteria in nasopharyngeal swab samples detection of respiratory viruses in sputum from adults by use of automated multiplex pcr evaluation and implementation of filmarray version . for improved detection of adenovirus respiratory tract infection filmarray respiratory panel assay: comparison of nasopharyngeal swabs and bronchoalveolar lavage samples respiratory viral detection in children and adults: comparing asymptomatic controls and patients with community-acquired pneumonia interactions of respiratory viruses and the nasal microbiota during the first year of life in healthy infants etiology of severe pneumonia in children in developing countries we would like to thank the patients and their parents for the support and cooperation in publishing this work. availability of data and materials all data described in this manuscript is available upon request via email. authors' contributions qc and xm initiated the study. jl, yt, myt and bld performed the detection of respiratory organisms. jl, yt, myt, qc and xm wrote the manuscript and analyzed the data. yjx provided technical support and assisted in the data analysis. all authors read and approved the final manuscript.ethics approval and consent to participate the study was approved by the institutional review board and the ethics committee of shanghai children's medical center (scmcirb-k ), and written informed consent was obtained from the parents of each patient. not applicable. yijun xia is an employee of biomérieux. he was involved in the technical support and data analysis. all the other authors declare they have no conflict of interests to disclose. all the other authors declare that they have no competing interests. key: cord- - wao a authors: dia, ndongo; richard, vincent; kiori, davy; cisse, el hadj abdoul khadir; sarr, fatoumata diène; faye, abdourahmane; goudiaby, déborah g; diop, ousmane m; niang, mbayame n title: respiratory viruses associated with patients older than years presenting with ili in senegal, to date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: wao a background: in africa, especially in west africa, studies about the prevalence and diversity of respiratory viruses (influenza and others) in elderly people are largely lacking. in studies done elsewhere, it is well established that older people, when compared with younger adults, are at greater risk of significant morbidity and mortality from complications arising from influenza. the main aim of this study was to determine the prevalence and the diversity of respiratory viruses associated with ili cases in adults over years old in senegal. methods: the recruitment period of this study was from january to december . patients aged years and above presenting ili cases were enrolled. nasal-pharyngeal and/or oral pharyngeal swabs were collected from patients. rna was extracted from μl of each sample followed by a two-step real-time rt-pcr. the anyplex™ ii rv detection kit was used for viral detection. the kit enabled the simultaneous detection of the presence of respiratory viruses. results: viruses were detected: influenza viruses ( . %) and rhinoviruses ( . %) were the most prevalent. we detected human parainfluenza viruses ( . %), human respiratory syncytial viruses ( . %), coronaviruses ( %), human metapneumoviruses ( . %), human adenoviruses ( . %) and human bocavirus ( . %). cases ( %) of dual virus infections and one triple viral detection case were encountered. ( . %) viruses detected were found in the - year old age group, ( . %; p < . ) from – year old age group and ( . %) were detected in the ≥ year old age group. the viral co-infections were more frequent in the - age group ( / ). conclusions: this pilot study demonstrates a variety of respiratory viruses in the elderly. it also highlights a high prevalence of these viruses in this age group. we speculate from these results that the impact of respiratory viruses other than influenza on the elderly has been considerably underestimated. a more exhaustive study seems necessary in order to provide a more complete picture of the burden of respiratory viruses on morbidity among adults over years old in the sub-saharan context. viral aetiology, prevalence and diversity data in people with influenza like illness (ili) and/or acute respiratory illness (ari) in africa, (especially in west africa), are scarce and often limited to the influenza viruses' infection. following the last influenza pandemic episode [ ] , few global and pediatric studies were conducted in some countries of the sub-region [ ] [ ] [ ] , and only a limited number of studies have described the etiology of ili due to viruses including non-influenza respiratory virus [ ] [ ] [ ] . however, no study has been conducted to describe the prevalence and the diversity of respiratory viruses (influenza and others) in west african elderly people. in studies done elsewhere, it is well established that older people, when compared with younger adults, are at greater risk of significant morbidity and mortality from complications arising from influenza [ , ] . for example in the united states alone, up to % of non-pneumonic lower respiratory illnesses in the elderly have been associated with respiratory viral infection [ ] , and an estimated , deaths annually have been attributed to the influenza and respiratory syncytial viruses (rsv) [ ] . it should be highlighted that in senegal the number of elderly people in consultation in healthcare centers for influenza like illness (ili) is very low. indeed, routine influenza monitoring in senegal showed that samples from people above years old represent only . % of the total, over a year surveillance period [ ] . some practices such as auto-medication and the use of traditional medicine to treat ili largely explain this situation with the socio-economic situation being another contributing factor. thus the main aim of this study was to determine the prevalence and the diversity of respiratory viruses associated with ili cases in adults over years old. the recruitment period of this prospective observational study was from january to december inclusive. all patients aged years and above presenting with ili during this period were enrolled in the study. it should be noted that samples were collected in the context of flu monitoring. an influenza sentinel surveillance system for outpatients with ili was established in in senegal and became part of the who global influenza surveillance and response system (gisrs). it is coordinated locally by the national influenza center (nic) at the institut pasteur de dakar. trained medical personnel were asked to screen all outpatients who were attended at the sentinel sites for signs and symptoms of ili. the symptoms of influenza are similar to those arising from other viral respiratory pathogens. the inclusion criteria, according to the cdc case definition, were sudden onset of fever (≥ °c) with cough or sore throat fewer than days in duration. nasal-pharyngeal and/or oral-pharyngeal swabs were collected from each enrolled ili case, placed in cryovials containing ml of viral transport medium (universal transport medium, copan diagnostics inc., murrieta, ca, usa) and stored at °c on site. if nasal-pharyngeal and oral-pharyngeal swab specimens were collected from the same patient, both swabs were placed in the same cryovial. upon arrival at the laboratory the specimens were separated into aliquots for analyses. the first aliquot was used for molecular analysis for the detection of influenza viruses (real-time reverse transcription polymerase chain reaction or rrt-pcr detection), the second was used for influenza virus isolation, and the third was stored at − °c for further analysis. the latter was used in the present study. for each patient who met the case definition criteria, a form collecting demographic and clinical data was completed.the questions included information on date of enrollment and symptom onset, sex, age, clinical symptoms, previous treatments, vaccination status for influenza, and whether or not the patient was hospitalized. ribonucleic acid (rna) extraction was performed from μl of each sample using the qiaamp viral rna kit (qiagen, valencia, ca, usa) according to the manufacturer's instructions. each rna sample was eluted with μl nuclease-free water before rna quantification with a nanodrop apparatus (nanodrop lite, thermo scientific). a two-step real-time rt-pcr was performed using the cfx real-time pcr detection system (bio-rad). the revertaid first strand cdna synthesis kit (thermo scientific) was used. first ng of rna was mixed with μl of random hexamer primer and nuclease free water for a final volume of μl. it was then incubated at °c for minutes and immediately put on ice in order to remove the secondary structures in gc-rich rna. for the cdna synthesis step, μl of x reaction buffer, μl of rnase inhibitor ( u/μl), μl of dntp mix ( mm) and μl of revertaid m-mulv reverse transcriptase ( u/μl) were added and incubated for minutes at °c followed by minutes at °c and °c for minutes. the cdna product could be used directly for the next step (pcr amplification) or stored at − °c until use. for viral detection, the anyplex™ ii rv detection kit (seegene) was used. the kit enabled simultaneous detection of influenza a virus, influenza b virus, human respiratory syncytial virus a, human respiratory syncytial virus b, human adenovirus, human metapneumovirus, human coronavirus e, human coronavirus nl , human coronavirus oc , human parainfluenza virus − , − , − , − , human rhinovirus a/b/c, human enterovirus and human bocavirus. reactions are duplicated in two panels (a and b) for detection of the viruses. the total reaction volume was μl for each sample (for each panel), containing μl x rv a (or x rv b), μl of -mop solution, μl of x anyplex pcr master mix (mix well by inverting times) and μl of cdna product. pcr was assessed after °c for minutes for transcriptase reverse enzyme inactivation, cycles of °c for seconds, °c for seconds and °c for seconds. additional cycle of °c for seconds was added for completion. the fluorescence is detected with a melting curve step, °c- °c ( seconds/ . °c). fisher's exact test was used to verify whether the associated proportions were statistically supported and a p-value < . was considered statistically significant. we used the - year' old group as the reference. the r. . tool was used to perform the analyses. the senegalese national ethical committee of the ministry of health approved the surveillance protocol as less than minimal risk research, and written consent forms were not required. throughout the study, the database was shared with the epidemiology department at the senegalese ministry of health and prevention for appropriate public health action. a total of patients above years old were enrolled into the study, ( . %) were women and ( . %) were men (table ). patients' ages ranged from to years, with a mean age of years. ninety-nine ( . %) enrolled patients were between and years old, ( . %) between and years old and ( . %) were years old or older. fever was the most reported clinical symptom, in ( . %; / ) of the enrolled patients, followed by cough ( . %; / ), rhinitis ( . %; / ), myalgia ( . %; / ) and pharyngitis ( %; / ). in all, ( . %) out of the patients were found to be infected with at least one of the viruses of interest. a total of ( . % of patients) viruses were detected. of these viruses, influenza viruses ( . %; / ) and rhinoviruses ( . %; / ) were the most prevalent viruses detected (table ) . we detected human parainfluenza viruses ( piv , piv , piv ) ( . %), human respiratory syncytial viruses ( rsv a and rsv b) ( . %), coronaviruses ( coronaviruses nl , one coronaviruses e and one coronavirus oc ) ( %), human metapneumoviruses ( . %), human adenoviruses ( . %) and one human bocavirus ( . %). a total of cases ( %) of dual virus infections and one triple viral detection case were encountered. influenza viruses ( cases) and rhinoviruses ( cases) were the most common type of virus found in samples with coinfections. regarding the number of viruses detected per age group, ( . %; / ) were from the - age group, ( . %; / ; p < . ) from the - year old age group and ( . %; / ) were detected in the older than year old age group ( table ) . the viral coinfections are more frequent in the - year old age group ( / ) followed by the ≥ years group ( / ). taking into account the clinical symptoms and viral detection, cough, rhinitis, pharyngitis or headache were in similar proportion in viral positive and non-positive patients: cough was observed in . % of positive patients and % in the negative patients group (p = . ), rhinitis . % and % (p = . ), pharyngitis in . % and % (p = . ) respectively. in contrast myalgia symptoms are significantly higher among viral-positive patients: . % versus % (p < . ). the pattern of the virus detection throughout the study period is showed in the figure . influenza viruses (a and b) were mostly detected from july to august (between weeks and ), which corresponds to the rainy season in senegal. a minor detection peak is also registered at the beginning of the year. rhinoviruses and parainfluenza viruses showed homogeneous detection levels throughout the study period. grouped, the remaining viruses seemed to have a similar temporal pattern to that of influenza viruses. the gap observed between weeks and correspond with a lack of samples from patients from our targeted age group. the present study is the first description of the etiology of respiratory viruses associated with patients with ili in a cohort of elderly people in the west african context. the results obtained showed that samples of the study population out of contained at least one of the targeted respiratory viruses. the frequency of virus detection ( . %) among the elderly with ili in our study is consistent with that of several studies already conducted. huo et al. ( ) [ ] , in a similar study in china detected at least one respiratory virus in % of patients years old or older, and munoz et al. ( ) [ ] % in elders in a long term care facilities in ontario during the - period. hasman et al. ( ) [ ] detected at least one respiratory virus in % patients in a study conducted in usa, without any precision about the ages of the adults. in others studies frequencies are lower. for example nicholson et al. ( ) [ ] % ( / ) of viral detection among the elderly between and years of old, % ( / ) detected in the elderly over years old in a study in china [ ] and . % in a recent study in japan [ ] . it is important to note that the technical approach used explains some discrepancies in rates of detection: primarily in their sensitivity and secondly in the number of targeted viruses. alternatively differences in rates of detection could be due to true geographical differences in overall burden, differences in study populations (outpatients or hospitalized patients) and to the studies sample collection periods. overall, the viral detection rate in the present study is very high as elderly people are often protected by pre-existing antibodies from previous illnesses, maybe illnesses suffered even decades back [ ] [ ] [ ] . indeed, because of pre-existing systemic and mucosal antibodies, elderly adults have been observed to have lower amounts of respiratory secretions and lower viral loads compared to children [ ] . consistent [ ] noted that the age distribution differed significantly between positive and negative patients, with positive patients being younger than negative patients (or = . , ic . - . ; p = . ). of the viruses detected in the elderly, influenza a virus was the most common viral pathogen. combined with influenza b viruses, influenza viruses represented % ( / ) of viruses detected. this influenza detection rate was expected as the enrollment of patients was directed towards patients with ili. these results are in agreement with previous findings in the elderly [ , , ] . our study revealed a high detection rate of rhinoviruses ( / ; . %). rhinovirus is the most common respiratory pathogen in all age groups [ ] . in a previous study, nicholson et al. ( ) [ ] showed that rhinoviruses were responsible for a greater disease burden (activities restriction, duration of illness) than that of influenza in elderly subjects representing % of detected viruses. in another study published by greenberg ( ) [ ] , rhinovirus was the most prevalent pathogen ( isolates; %) of the identified in upper respiratory episodes. these findings are in concordance with the high rhinovirus detection rate in the present study. with lower prevalence, piv, rsv, hcov, hmpv, enteroviruses, adenoviruses and bocaviruses were identified from elderly patient' specimens and contributed collectively to . % of all ili cases in our study. these results show the high diversity of respiratory viruses circulating in the elderly population. this viral diversity supports previous results [ , , ] and often in similar distributions with those of the present study. co-infections were relatively common in this study especially in the - years old age group ( . %; / ). the rate found in this age group was in line with the findings of hasman et al. ( ) [ ] ( %) and huo et al. ( ) [ ] , . %. huo and colleagues, in agreement with our results noted that co-infections were found most commonly in adults older than years of age. focusing on clinical symptoms, with the exception of myalgia, our study showed no significant differences between viral-positive and viral-negative patients with ili. viral circulation observed during the study period showed different patterns depending on the viral types. if we consider influenza viruses, we observed a circulation peak during the period starting in week and ending in week . this period corresponds to the middle of the rainy season in senegal. this result is further supported by a recent study conducted by mbayame and colleagues [ ] . these authors established clearly the seasonality of influenza viruses in senegal after many years of surveillance with a regular circulation during the year and a peak in the middle of the rainy season (july-august-september). the slight peak of influenza observed at the beginning of the year (february) is the result of the shift caused by the recent pandemic episode. the pandemic occurred in early in senegal with a peak in february [ ] . rhinoviruses showed a regular yearly circulation with peaks along the year corresponding to any rain season influence. the remaining respiratory viruses (piv, rsv, hcov, hmpv, enterovirus, adenovirus and bocavirus) were more likely associated with ili peak during the rainy season. this co-circulation with influenza viruses was also seen in a previous pediatric study in senegal [ ] . further studies (multiple year surveillance) are needed in order to properly define the temporal patterns of non-influenza virus circulation in senegal. our study did have several limitations. the first weakness is the small number of samples treated in this study. a more exhaustive sampling would give a better representation of the different targeted viruses in the ili cases among the elderly population in senegal. unfortunately after years of influenza sentinel monitoring we noted that the number of elderly presenting at healthcare centers for ili consultation is rather low compared to other age groups (children and young adults). the absence of nursing home services as in industrial countries, the use of traditional medicine (especially among the elderly) and economic constraints do not facilitate such studies in the west african context. it is worth noting that this was a retrospective study, the database contained limited information on disease outcome and atypical clinical symptoms in ili patients which were not reported. thus the association between viral infections (or co-infections) and severe signs could not be established. as in previous studies it appears that co-infections were associated with more severe signs than mono-infections [ , ] . without such data we could not measure the burden of targeted respiratory viruses in older patients with ili. another limitation is that our study is only focused on outpatient' cases; it would be interesting to investigate hospitalized patient cases (severe cases). a final limitation was that the study included mainly one geographic location, dakar, the capital city of senegal. despite the small number of samples included, the present pilot study demonstrates a variety of respiratory viruses in the elderly. it also highlights a high prevalence of these viruses in this cohort. from these results, it appears that the impact of respiratory viruses other than influenza was considerably underestimated. a more exhaustive study (increasing the number of elderly patients, with a better clinical picture and better documentation including disease outcomes, illness duration, hospitalizations etc.), relying on the new sentinel surveillance system (extension of sentinel sites in others geographical areas), seems necessary in order to provide a more complete picture of the burden of respiratory viruses on morbidity among adults over years old in the sub-saharan context. influenza-like illness; rrt-pcr: real-time reverse transcription polymerase chain reaction; who: world health organization; cdna: complementary deoxyribonucleic acid; rna: ribonuleic acid; gisn: global influenza surveillance network; nic: national influenza center rsv: respiratory syncytial virus; hcov: human coronavirus; hmpv: human metapneumovirus emergence of a novel swine-origin influenza a (h n ) virus in humans virological surveillance of influenza-like illness among children in ghana influenza viruses in nigeria, - : results from the first months of a national influenza sentinel surveillance system sentinel surveillance for influenza in senegal viral etiology of influenza-like illnesses in antananarivo viral etiology of respiratory infections in children under years old living in tropical rural areas of senegal: the evira project viral etiology of influenza-like illnesses in cameroon the impact of influenza on the health and health care utilisation of elderly people influenza-related hospitalisation and death in australians aged years and older new respiratory viruses and the elderly mortality associated with influenza and respiratory syncytial virus in the united states surveillance of respiratory viruses in patients with influenza-like illness in nanjing current research on influenza and other respiratory viruses: ii international symposium aetiology of influenza-like illness in adults includes parainfluenzavirus type acute viral infections of upper respiratory tract in elderly people living in the community: comparative, prospective, population based study of disease burden influenza a/h n pandemic and respiratory virus infections the post-infection outcomes of influenza and acute respiratory infection in patients above years of age in japan: an observational study characterization of an avian influenza a (h n ) virus isolated from a child with a fatal respiratory illness epidemiology of pandemic influenza a (h n ) deaths in the united states outbreaks of pandemic influenza a (h n ) among long-term-care facility residents-three states nationwide surveillance of respiratory viruses in patients with influenza-like illnesses: a pilot feasibility study in the french sentinel network rates of hospitalizations for respiratory syncytial virus, human metapneumovirus, and influenza virus in older adults fields virology viral respiratory infections in elderly patients and patients with chronic obstructive pulmonary disease a subregional analysis of epidemiologic and genetic characteristics of influenza a(h n )pdm in africa respiratory viruses in children admitted to hospital intensive care units: evaluating the clart pneumovir dna array comparison of human metapneumovirus, respiratory syncytial virus and influenza a virus lower respiratory tract infections in hospitalized young children respiratory viruses associated with patients older than years presenting with ili in senegal the authors thank dr abdou salam gueye, associate director for science at the us centers for disease control (cdc) in ivory coast, for his great help in the revisions of this paper, especially for the english language. many thanks to oumy niass (phd student, immunology unit, ipd) for her help in the statistical analyses. this work was funded by institut pasteur de dakar, senegal. the authors declare that they have no competing interests. the work presented here was carried out in collaboration between all authors. mnn and omd, defined the research and revised the manuscript; nd performed and coordinated technical work, wrote the draft and revisions of the paper; dk performed the main technical part of this work; eakc and af participated in the technical work; dg and eakc participated in data management and analysis; vr revised the manuscript and participated in the monitoring of the surveillance sites; fds participated in the monitoring of the surveillance sites. all authors have contributed to, seen and approved the manuscript. key: cord- - iecsho authors: wen, xiaohong; huang, qiuling; tao, hong; zou, weihua; gao, min; guo, huihui; yao, xing; cui, dawei; wang, xiang title: clinical characteristics and viral etiologies of outpatients with acute respiratory infections in huzhou of china: a retrospective study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: iecsho background: viruses are commonly found in patients with acute respiratory infections (aris). however, the viral etiologies and clinical characteristics of outpatients with aris are poorly understood in china. here, we identified the viral etiologies in outpatients with aris in huzhou, china. results: our results indicated that of outpatients, were positive for viruses. of them, were positive for a single virus, including influenza a, which comprised h n ( . %) and pandemic h n ( ) ( . %), enterovirus ( . %), and influenza b ( . %). other single viruses were detected at less than . %. twenty-five patients were positively coinfected with two viruses. the prevalent viruses in coinfections were rhinovirus and h n virus ( . %). viruses were major pathogens in young children (< years) ( . %). coinfections were prevalent in older adults ( . %) and young children ( . %). virus-positive outpatients presented higher temperatures and more sore throat, fatigue and shortness of breath than virus-negative outpatients. aris and most virus detections peaked during the winter, but enteroviruses emerged between april and september. conclusion: viruses are major agents of aris among outpatients in huzhou, china. there was a variation in the distribution of viruses across different age groups and seasons. these findings are beneficial for planning prevention and treatment services for outpatients with aris. acute respiratory infections (aris) are common and major public health threats, causing high morbidity and mortality worldwide, particularly in developing countries [ , ] . many pathogens can result in aris, and viruses have been identified as major causes in aris among various populations; the most common viruses of aris include influenza a and b virus (flua and flub), rhinovirus (rhv), respiratory syncytial virus (rsv), parainfluenza virus (piv) type - , enterovirus (ev), adenovirus (adv), human metapneumovirus (hmpv), human bocavirus (bov), and coronavirus (cov)- e, nl , oc and hku [ ] [ ] [ ] [ ] [ ] [ ] . currently, there are few available vaccines to prevent respiratory virus infections [ , ] . it is important to investigate the epidemic viral etiologies of aris to efficiently prevent and control viral epidemics in the future. it is well known that the early and rapid molecular detection of respiratory viruses is valuable to prevent and control aris [ , ] . however, only a portion of patients with aris have their viral etiologies detected because of the expensive testing costs in china and other developing countries [ , , , ] . moreover, the spectrum of viral etiologies is closely correlated with various factors, such as age, season, geographical region, medical condition, and immune status [ , , [ ] [ ] [ ] [ ] . in this study, we collected clinical and demographic data from outpatients including children and adults with aris, and their specimens were tested for viruses. this study aims to provide basic data to direct local disease prevention and control measures for aris in huzhou, china. this study was conducted from january to april in two general hospitals in huzhou city located beside tai lake of southeast china. demographic and clinical data from all enrolled aris outpatients of any age were collected, and clinical specimens from the upper respiratory tract of these outpatients were tested for viruses by the multiplex rt-pcr method using a seeplex® rv ace detection kit (seegene, korea). furthermore, a positive sample with flua virus was discriminated for seasonal h n (sh n ), seasonal h n (sh n ) and pandemic h n ( ) viruses by a one-step real-time rt-pcr assay from shanghai zj bio-tech co., ltd. (shanghai, china). outpatients of any age were enrolled from january to april at the department of fever outpatient clinic of the first people's hospital of huzhou and at the huzhou central hospital, huzhou, china. outpatients suffering from fever now or in recent days, such as influenza-like illness (ili) and aris patients, were seen at the fever outpatient clinic. a case definition of ari was described in a previous report [ ] . briefly, patients with aris presenting with at least one of the following symptoms: cough, sore throat, shortness of breath or coryza as an acute onset of symptoms within days were judged by a clinician for an infection. clinical specimens from the upper respiratory tract of patients, including throat swabs, nasal aspirates and washes, or sputum specimens, were collected and kept in ml of viral transport medium stored at − °c until testing for respiratory viruses. clinical data including age, oral body temperature, clinical symptoms and other information were recorded in case report forms during face-to-face interviews. statistical analysis was conducted with spss software (v . , spss, chicago, il, usa). descriptive statistics were used to analyze the seasonal and age distribution and infection rates of different respiratory viruses. chi-squared tests were used to compare different age groups, gender, and clinical characteristics between age virus-positive and virus-negative outpatients with aris. p-values < . were considered to be statistically significant. a total of outpatients with aris were enrolled from january to april in this study. of them, specimens ( . %, / ) were positive for at least one virus, and single infections accounted for . % ( / ) of cases. coinfections were observed in . % ( / ) of cases (fig. a) . of the single virus infections, flua virus was the most frequent virus, identified in . % ( / ) of the cases, comprising ( . %) cases of sh n virus and ( . %) cases of pandemic h n ( ) virus patients, followed by ev ( . %, / ), flub ( . %, / ), adv ( . %, / ), rhv ( . %, / ), hmpv ( . %, / ), and other viruses that were identified under . %, respectively (fig. b) . of the coinfections, rhv + sh n viruses were predominantly identified and accounted for . % ( / ) of cases. rhv + ev, rhv + adv and rhv + piv- viruses equally accounted for . % ( / ) of cases. adv + hmpv, adv + rsv-b, and flub+piv- also equally accounted for . % ( / ) of cases. other coinfections were identified in . % ( / ) of cases (fig. c) . sh n virus was not detected in this study. demographic and clinical characteristics of outpatients with aris are shown in table . of outpatients with aris, ( . %) were males, and ( . %) were females. the distribution of viruses did not significantly differ between males and females (χ = . , p = . ). however, the distribution of viruses notably differed among the different age groups (χ = . , p < . ). in addition, . % ( / ) of aris outpatients suffered from high fever (body temperature ≥ °c), followed by cough ( . %), sore throat ( . %), fatigue ( . %), and other respiratory symptoms. moreover, a significant difference was observed in clinical symptoms including fever (χ = . , p = . ), sore throat (χ = . , p < . ), fatigue (χ = . , p < . ), and shortness of breath (χ = . , p = . ) between virus-positive and virus-negative cases, and the proportion of patients with abdominal pain was greater among virus-negative cases (χ = . , p = . ). additionally, physical examinations showed abnormal lung auscultation ( . %) and x-rays ( . %). the distribution of the viral etiologies of the four age groups is shown in table . the highest proportion was observed in young children ( . %), and lowest proportion was observed in adults - years of age ( . %). similarly, the positive rate of cases with a single virus infection was highest in the young children ( . %) and lowest in adults of - years of age ( . %). moreover, the positive rate of the cases with coinfections was highest in older adults ( . %), followed by young children ( . %). the predominant viruses among four age groups differed. flua virus and subtypes were the most prevalent viruses in the older adults (≥ years), and the converse was true in the young children (< years). ev, rsv and piv viruses predominated in the young children (< years), and flub, rhv and adv viruses were more prevalent in the young adults ( ~ years). the virus detection rates for different seasons are shown in fig. . the proportion of positive viruses exhibited two waves corresponding to winter and spring, including jan to mar , and nov to feb . similarly, the flua virus also occurred more frequently in winter and spring. conversely, ev infections were predominant between april and september. other viruses occurred almost sporadically throughout the year without obvious seasonal trends, and a small number of ari outpatients with virus infection were observed between june and september, excluding ev infections. viruses are major agents contributing to the high morbidity and mortality of patients with aris, particularly in children under years of age [ , , ] . many reports describe the etiology and epidemiology of hospitalized aris patients, including children and/or adults worldwide [ , [ ] [ ] [ ] , although the study of outpatient aris in children and adults is more limited [ , ] . in this study, some clinical characteristics significantly differed between virus-positive and virus-negative outpatient aris, such as fever, cough, sore throat, fatigue, and other respiratory symptoms. these findings implied that viruses were the most common causes of aris, easily eliciting severe clinical symptoms. similar results were found in previous reports [ , [ ] [ ] [ ] [ ] . laboratory diagnosis of viruses is commonly conducted by conventional methods (such as culture or antigen detections), and real-time and multiplex rt-pcr assays have been considered to be important tools for identifying the etiologies of aris [ , , , ] . recently, a commercial multiplex pcr assay with a seeplex® rv ace detection kit was used to simultaneously and precisely identify the viruses of aris in many laboratories [ ] [ ] [ ] [ ] . the methods used in this study expanded and improved the capacity for testing viruses ( viruses and subtypes of flua virus). in this study, . % were positive for at least one virus, which was similar to the morbidity rates reported in previous studies in pittsburgh ( . %) and vitória of southeast brazil ( . %) [ , ] , but was different in china and compared with other reports [ , - , , ] . the single infection ( . %) was predominant in our study, particularly the flua virus ( . %), which was consistent with previous reports in shandong province, beijing, of china and other countries [ , [ ] [ ] [ ] [ ] [ ] . sh n and rhv coinfections were predominant among viral coinfections, which was different from previous reports [ , , , , ] . the discrepancy of the predominant coinfections might be closely associated with principal epidemical viruses in the local region. the proportion of respiratory viruses notably differed across different age groups; the virus positive rate was the highest in young children under years but was lowest in adults ( ~ years) in this study. these findings indicated that the viruses were the predominant pathogen found in young children with aris, and similar morbidity rates have been reported in previous studies [ , ] , although the morbidity rate in this study also differed from those in other studies [ ] . the flu a virus was the predominant virus among the three age groups, excluding young children, and was highest among older adults (≥ years) and lowest among young children; however, ev was the highest in young children among all age groups. some studies show that evs and rhvs can be difficult to discriminate with rt-pcr primers unless accompanied by amplicon sequencing, and flua virus and rhv might fail to detect rhv due to competition between the amplification reactions [ ] [ ] [ ] . therefore, all positive rhv and/or ev specimens and flua virus specimens with random selection were identified table age distribution of viruses from outpatients with aris aris, acute respiratory infections by sequencing assay, respectively, and among them, four rhv positive and ev positive specimens were not sequenced due to low viral load in the specimens. additionally, viral coinfections predominantly occurred in young children (< years) and older adults (≥ years), which was consistent with previous reports in china and other countries [ , , , , , ] . these results indicated that the detection rate of viruses in aris was closely associated with the age of outpatients because age affects immune status, exposure opportunities to viruses, and other lifestyle behaviors of people. generally, young children and older adults have weak immune systems against viruses, which might contribute to their higher susceptibility to viruses than young adults and adults who have strong immune status against viruses [ , ] . moreover, young children have more opportunities for exposure to evs than young adults and adults, which might cause higher incidences of ev infection than in other age groups [ , , ] . many studies have indicated that viral aris are affected by seasonal distributions and occur commonly in spring, autumn, and winter [ , , , , , ] . in this study, viruses from outpatient aris were detected throughout the whole year and commonly occurred in spring and winter, with peaks occurring in february and december . similar results were reported in other studies [ , , ] . moreover, our results demonstrated that ev infections occurred between april and september , with the peak detection rate in august , which was in accordance with previous reports [ ] . many studies have shown that evs are often detected in summer and autumn [ , [ ] [ ] [ ] [ ] . we speculate that climate conditions might be an important factor for ev detection in temperate regions. these findings indicated that the geographical diversity of surveillance areas with warm and wet climate conditions beside tai lake might contribute to the variability in seasonal trends and viral etiologies of aris. our study had some limitations. first, there were only outpatients with aris enrolled in the two local hospitals; because this was a small sample size, it may be difficult to estimate the disease severity of outpatient aris in detail. more large-scale surveillance for aris will performed, and analyses over additional years will provide a more accurate picture of seasonal variation in respiratory virus circulation in this community in the future. second, we detected only viruses, but did not test for bacterial pathogens in the respiratory tracts of outpatients with aris; therefore, our data regarding pathogens causing aris were not comprehensive, which also affected our ability to determine the relationships between pathogens and disease severity in outpatients with aris. taken together, our results were valuable to a certain degree for assessing outpatients and clinical treatments. in summary, this study provides important epidemiologic data regarding the clinical characteristics, viral spectrum, age distribution and seasonality of viruses in outpatients with aris in huzhou, china. these findings contribute to evaluating the burden of virus infections in outpatients, including young children and adults. timely and accurate diagnosis of pathogens in outpatients with aris is required to reduce the burden caused by these diseases. viral etiology of acute respiratory infection in gansu province prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen, china viral etiology of acute respiratory infections among children and associated meteorological factors in southern china estimates of worldwide distribution of child deaths from acute respiratory infections viral etiology of acute respiratory tract 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seeplex rv for simultaneous detection of respiratory viruses high incidence of multiple viral infections identified in upper respiratory tract infected children under three years of age in detection of respiratory viruses by real-time polymerase chain reaction in outpatients with acute respiratory infection etiology and incidence of viral and bacterial acute respiratory illness among older children and adults in rural western kenya prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing viruses associated with acute respiratory infections and influenza-like illness among outpatients from the influenza incidence surveillance project viral infections in outpatients with medically attended acute respiratory illness during the - influenza season rapid detection of respiratory tract viral infections and coinfections in patients with influenza-like illnesses by use of reverse transcription-pcr dna microarray systems enteroviruses as major cause of microbiologically unexplained acute respiratory tract infections in hospitalized pediatric patients new epidemiological and clinical signatures of pathogens from respiratory tract infections based on a -year study hand, foot, and mouth disease in china, - : an epidemiological study burden, seasonal pattern and symptomatology of acute respiratory illnesses with different viral aetiologies in children presenting at outpatient clinics in hong kong we thank the outpatients, nurses and clinicians of the participating hospitals for their participation and cooperation in this study. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -l voruz authors: tewara, marlvin anemey; mbah-fongkimeh, prisca ngetemalah; dayimu, alimu; kang, fengling; xue, fuzhong title: small-area spatial statistical analysis of malaria clusters and hotspots in cameroon; – date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: l voruz background: malaria prevalence in cameroon is a major public health problem both at the regional and urban-rural geographic scale. in , an estimated . million confirmed cases, and , cases were reported in health facilities and communities respectively, with about estimated deaths. several studies have estimated malaria prevalence in cameroon using the analytical techniques at the regional scale. we aimed at identifying malaria clusters and hotspots at the urban-rural geographic scale from the demographic and health survey (dhs) data for households between and using arcgis for intervention programs. methods: to identify malaria hotspots and analyze the pattern of distribution, we used the optimized hotspots toolset and spatial autocorrelation respectively in arcgis . for desktop. we also used pearson’s correlation analysis to identify associative environmental factors using the r-software . . . results: the spatial distribution of malaria showed statistically significant clustered pattern for the year and with moran’s indexes . (p < . ) and . (p < . ) respectively. meanwhile, the years and with moran’s indexes . (p = . ) and . (p = . ) respectively, had a random malaria distribution pattern. there exist varying degrees of malaria clusters and statistically significant hotspots in the urban-rural areas of the administrative regions. malaria cases were associated with population density and some environmental covariates; rainfall, enhanced vegetation index and composite lights (p < . ). conclusion: this study identified urban-rural areas with high and low malaria clusters and hotspots. our maps can be used as supportive tools for effective malaria control and elimination, and investments in malaria programs and research, malaria prevention, diagnosis and treatment, surveillance, should pay more attention to urban-rural geographic scale. malaria remains an international public health challenge as there has been an increase in the number of estimated malaria cases; million malaria cases from ( million) to ( million) [ ] . worldwide, countries are now malaria-free, whereas malaria is still an endemic disease in about countries [ ] . about and % of malaria cases and deaths respectively, reported in occurred in the who africa region with about counties all in sub-saharan africa (ssa) [ ] . the most prevalent malaria parasite in ssa is the plasmodium falciparum, accounting for % of malaria cases and most occurring in children under the age of five [ ] . in cameroon, the epidemiological transmission of malaria is high (> case per population) in about % ( . million people) and low ( - cases per population) in about % ( . million) in people of all sexes and age groups. malaria prevalence in cameroon is a major public health problem at both the regional (larger) and urban-rural (smaller) geographic scales, with an estimated . million confirmed cases reported in health facilities and , cases at the community level and ( - , ) estimated deaths in [ ] . generally, malaria intervention policies and control strategies in both the regional and urban-rural scales in cameroon, have been reported to focus on the use of insecticide treated bed-nets (itns), indoor residual spray (irs),larval control, diagnostic testing, treatments, disease surveillance, and national campaigns [ ] [ ] [ ] [ ] . the who and the roll back malaria global action plan [ ] anticipate having a malaria-free world by through its set milestones and targets pillars with a major focus to ensure universal access to malaria prevention, diagnosis, and treatment. malaria prevention strategies based on the use of itns and or irs in cameroon, has been a great method in the reduction of incident cases of the disease as about . million itns deliveries of the % itns deliveries in ssa, was made in cameroon between and [ ] . malaria risk maps and the applications of spatial malaria epidemiology in the fight against malaria in africa has been limited. a review by omumbo [ ] , examining the most recent national malaria strategies, monitoring and evaluation plans, as well as the types of maps presented and how they have been used to define priorities for investments in malaria control in countries in africa, found that about % of the countries did not present malaria maps within their national malaria prevention strategies. small-area statistical analysis and spatial epidemiology have emerged to solve issues of where disease clusters and hotspots are located. spatial epidemiology deals with the analysis and description of geographic health data with respect to demographic, environmental, behavioral, socioeconomic, genetic and other infectious agents or risk factors [ ] . a study by elliot [ ] on the current approaches and future challenges of spatial epidemiology reported that, recent advancements in data availability and analytical methods have created new openings for studies to improve on the local reporting of diseases at national or regional scale by observing changes in disease prevalence rates at a smaller scale. although, they reported on the absence of a satisfying definition of the term small-area in studying the variations in disease incidence and mortality, [ ] suggested a working definition as a rough guide which we will apply in our study; any region containing fewer than about cases of a disease can be considered a small area. for example, a disease with an annual incidence rate of about per , for a period of years, a small area constitutes a population size of around , or fewer in clusters of disease occurrence in a remote area or small village. they also identified four types of spatial analysis at a small-area scale: disease mapping, geographic correlation studies, disease clusters, and surveillance. some of the main techniques of spatial methods reviewed by robertson [ ] , used in emerging infectious disease research include; spatial autocorrelation, space-time interactions, hotspots and clusters. the global spatial autocorrelation technique is used to characterize a full map in one quantitative value. this method measures the total joint counts of nearby regions, attributes or locations against a null hypothesis of no spatial autocorrelation [ ] . moran's i and geary's c statistics are common methods of spatial autocorrelation. positive spatial autocorrelation indicates the existence of clustered patterns of a disease, negative autocorrelation can suggest a dispersion in the transmission pattern or surveillance among given regions. hotspot mapping and cluster detection are analyses executed through local spatial analysis methods. the basic technique is to calculate a test statistic for each location and then evaluate the distribution of these test statistics against a theoretical or random reference distribution. this technique is important in infectious diseases surveillance in that, it helps to identify geographic areas where and to what extent an observed spatial pattern of a disease is anticipated relative to a null hypothesis [ , ] . smith [ ] , conducted a systematic review of published reports of outbreak investigations worldwide to estimate the prevalence of infectious diseases using spatial methods such as dot maps, moran's i, rate maps, gestis-ord gi* on different diseases; hepatitis, influenza, malaria, rabies and many others. bhatt [ ] , found that, plasmodium falciparum infection in endemic africa has reduced and incidence of the clinical disease fell by % between and ; the authors used the geographical information systems (gis) applications. the gis computer system can describe, analyze, and predict disease patterns using feature (cartographic) and attribute data. gis has been used in many epidemiologic applications, including disease mapping, rate smoothing, cluster or hotspot analysis, and spatial modeling and have been reported and applied in small area units such as urban-rural and lower administrative scales [ ] [ ] [ ] [ ] [ ] . dot maps and geographic profiling have been used both in the united kingdom and egypt as spatial methods to identify locations of sources of cholera and malaria infections respectively [ ] . the moran's i spatial method has also been used to identify cholera clusters in areas with lower coverage of latrines in a peri-urban area of lusaka, zambia and advise for effective drainage systems [ ] . moreover, during the severe acute respiratory syndrome (sars) outbreak in hongkong, the moran's i technique was used to identify sars cluster patterns at the community level [ ] . findings from a study carried out in the small-area rural highlands of western kenya, identified significant spatial clusters of malaria in school children during an outbreak [ ] . the authors used household survey data and their analyses used the spatial scan statistic software. most studies focusing on malaria prevalence and incidence, or the use of itns / irs, in cameroon have applied the analytical statistics methods, tools evaluation, vector control and molecular techniques at both the higher and lower administrative levels [ , , ] . understanding the distribution of malaria cases in cameroon with the use of spatial statistical analysis approach, will help inform malaria control programs at a smaller scale. thus, we aim to identify malaria clusters and hotspots in cameroon at the urban-rural scale using the dhs global positioning system (gps) data for households. our objectives are to; i) use the spatial autocorrelation technique to analyze malaria spatial patterns in arcgis for desktop, ii) map the distribution of malaria cluster points, iii) identify urban-rural clusters with statistically significant hotspots of the disease, and iv) identify environmental factors associated with the distribution of malaria cases. data for this study was obtained after a granted request for registered users from the dhs program website https://dhsprogram.com funded by the united states agency for international developments (usaids) measure dhs project in collaboration with the national institute of statistics (institute nationale de la statistique) and the ministry of public health cameroon. the dhs are nationally-representative, probabilistic, household surveys that include a wide range of key demographic and health indicators used to monitor and evaluate population, health, and nutrition programs [ ] . the data contains the cameroon dhs malaria data for a five-year ( , , , and ) interval and some environmental covariates; enhanced vegetation index, rainfall, composite lights, and population density at the urban-rural scale. the gps point data for each sampled urban-rural cluster residence was linked to all the households and individual level attributes such as survey information for malaria indicator survey (mis) and aids indicator survey to be analyzed with arcgis. for reasons of confidentiality, the gps urban-rural location points were masked [ , ] and a python script in arcgis was used to displace the data within the appropriate administrative boundaries. for small-area administrative units, urban residence clusters were displaced a distance up to km and rural clusters up to km, with a further randomly selected % of the rural clusters displaced up to km [ ] . the dhs urban-rural residence clusters ( fig. ) as defined by the country's census bureau, is usually part of the sampling domain for lower levels of administrative units. census enumeration areas can be a city block or apartment building for urban areas while in rural areas is typically a village or group of villages. the population and size of sample clusters vary between and within countries (table ) . generally, clusters contain to households, of which to households are randomly selected for survey participation [ , ] . a malaria year was described as the average number of people per year who show clinical symptoms of plasmodium falciparum malaria within the cells whose centroid falls within a radius of km (for rural points) or km (for urban points). in surveys that collected specimens for malaria testing (primarily mis), indicators of the prevalence of malaria are provided, based on both rapid diagnostic tests (rdt) and on laboratory analysis [ ] . a clinical case was defined as a malaria-attributable febrile episode (body temperature in excess of . °c), typically accompanied by headaches, nausea, excess sweating and or fatigue censored by a -day window [ , ] . the global spatial autocorrelation (moran's i) statistical toolsets in arcgis . was used to identify statistically significant malaria clusters over the study area for the different regions in cameroon between and . the moran's index (m.i) statistical technique, evaluates the spatial autocorrelation of malaria cases at the urban-rural cluster locations where a moran's i value close to zero signifies spatial randomness of the disease, a positive value indicates spatial clustering [ , ] . to evaluate whether the spatial pattern is clustered, dispersed or random, a statistically significant estimate of moran's i (p < . , z score ≥ . ) indicates neighboring urban-rural areas have similar malaria cases under the null hypothesis that the distribution of malaria cases at the urban-rural scale is completely spatially random. the spatial autocorrelation tool runs through an input feature class, a conceptualization of spatial relationship (which include inverse distance, travel time, fixed distance, k nearest neighbors, and contiguity), and a distance band or threshold distance (cases have at least one neighbor). the tool returns five values: the moran's i index, expected index, variance, z-score, and p-value. this tool calculates a z-score and p-value which are measures of statistical significance to indicate whether or not the null hypotheses can be rejected. the average number of people per year who showed clinical symptoms for malaria in the urban-rural residence between and was symbolized using graduated symbols with five classes natural breaks jenks; which is a symbology technique in arcgis that shows quantitative differences in data values with varying symbol sizes. the this approach displays the relative density of points using a color scheme, ranging from low density to high density of points. moran's i have well established statistical properties to describe global spatial autocorrelations but has not been effective in identifying clustered spatial patterns and hotspots [ ] . for hotspots identification, we employed the optimized hotspot analysis tool in arcgis . to identify the malaria incident hotspots at the urban-rural clusters. the optimized hotspots analysis creates a map of statistically significant hotspots (areas with high malaria cases) and coldspots (areas with low malaria cases) using the getis-ord gi * statistic; which uses a default count incident points within fishnet polygons as the incident data aggregation method. the software generates polygons and aggregates these points into the polygons. the fishnet polygon technique produces a map of malaria incident cases with similar attribute values and automatically classifies them as coldspots (blue areas) or hotspots (red areas). malariological measures such as those of the environment, have been reported to be associated with malaria prevalence [ , , ] . to understand the association between malaria case distribution and environmental factors in our study, we measure the associations between the five-year interval malaria cases and environmental covariates such as rainfall, enhanced vegetation, and nightlights composite, by applying the pearson's product -moment correlation coefficient (pcc) denoted by r. the pcc was analyzed using the r-statistic software (version . . ). the mathematical computations and the applications of pcc are documented in [ ] and was applied in the spatiotemporal distribution and hotspots of hand, foot and mouth disease in northern thailand, where the authors found rainfall to be associated with the spread of the disease [ ] . the statistically significant result r, measuring the strength of the associations from − (perfectly inverse association) to + (perfectly strong association) were obtained. the global mi was greater than zero (more (table ) . in this study, we looked at the our main goal in this study was to identify malaria hotspots locations for future intervention using the spatial autocorrelation statistical techniques. in the year , statistically significant malaria hotspots ( % confidence) were identified in urban-rural clusters of the west, southwest, douala, yaoundé, littoral, center and the south regions (fig. ) . in , there were no statistically significant malaria hotspots in all the dhs administrative regions. the far north had areas with statistically significant coldspots; areas with low malaria cases (fig. ) . in , some urban-rural areas of the southwest and south regions had statistically significant malaria hotspots (fig. ). in , most urban-rural areas in the west, southwest, douala, center, and east had statistically significant malaria hotspots ( % confidence). some communities in the north had statistically significant malaria coldspots as illustrated in fig. . the map of the un population density shows high population densities in the urban-rural clusters of weights are adjustment factors applied to each case in the dhs survey to adjust for differences in probability of selection and interview between cases in a sample, due to different study designs [ ] yaoundé, douala and west regions and low population densities in some parts of the southwest, northwest, north and far north regions (fig. ) . table demonstrates the strength (r) of the association between the distribution of malaria cases and environmental factors. the application of spatial analytical techniques focusing on malaria is not new. however, very limited studies have focused on smaller administrative levels [ , , ] . given the z scores: . and . for the year and respectively, indicate there is a less than % likelihood for the observed clustered pattern to be due to chance (fig. ) . the null hypothesis of complete randomness is rejected, and the presence of cluster patterns indicate neighboring locations have high malaria cases at a given urban-rural area. the z scores: . and . for the year and respectively, illustrate that the malaria pattern does not appear to be significantly different than random (fig. ) , and the null hypothesis of complete randomness is accepted. this suggests that malaria cases are randomly spread across the urbanrural areas. knowing the hotspot locations of areas with clustered malaria patterns can inform for national malaria prevention programs and surveillance. the distribution of urban-rural malaria cases observed as graduated symbols in figs. , , and call for prevention programs as some urban-rural areas in yaoundé, douala, center, south west, north west, littoral, west, and south region had high malaria cases, and low cases in adamawa, east, north, and far north region. our finding is consistent with that reported by gemperli [ ] where they found high malaria prevalence in the west and low prevalence in the north and far north. contrary to our study that focused on the distribution of malaria cases at a smaller scale, the author focused on malaria prevalence at a regional scale. understanding the distribution of malaria cases and prevalence in these areas will advise for investments and prevention programs. the hotspots analysis identified varying intensities of malaria hotspots in the urban-rural areas of the west, southwest, northwest, douala, yaoundé, littoral, central, and south regions ( % confidence) between and (figs. and ) . in addition, there was a shift in the malaria hotspot location paradigm as some urbanrural areas in the east region recorded new incident malaria hotspots for which was not seen in the previous years. in a study ( ) , which focused on the mapping of plasmodium falciparum mortality in africa between to , the authors reported that several malaria hotspots areas in cameroon, niger, central africa republic and ivory coast, were associated with high mortality rate and low coverage of antimicrobial treatment(> malaria deaths per , ) [ ] . this study did not locate in detail, the various regions or urban-rural areas in cameroon with such hotspots. our hotspots maps are an affirming tool at the regional and even urban-rural scale for malaria prevention programs. furthermore, [ ] identified regions of adamawa, north, and east of having high mortality (> per , ) and low drug treatment < %. our study reported these regions of having low malaria cases and no statically significant malaria hotspots except for the east region. however, our study focused on malaria cases and advise for continues preventive measures in the urban-rural areas or regions of low malaria cases and high mortality. this study reported on the use of itns and irs as one of the most effective preventive strategy for malaria control in cameroon and though an effective method, spatial autocorrelation pattern for the malaria year (z-score: . ) and (z-score: . ) , ) were associated with low coverage of itns ( - %) for most regions in cameroon, nigeria, angola and parts of congo, central african republic, guinea and equatorial guinea [ ] . furthermore, an observational study that assesed itns possesion and their protective effects on malaria infection in semi-urban and rural communities in the south west region of cameroon, found that itns ownership was lower in rural settings compared to semi-urban settings [ ] . this also calls for malaria prevention and control campaigns such as those on itns distributions in urban-rural areas and particularly hotspots locations. the population density map (fig. ) at the urban-rural areas showed that malaria cases and hotspots locations were higher in regions of higher population density and lower in regions of lower population density. this corroborated with the findings of kabaria [ ] who reported the relationship between human population densities and malaria infection risk in children aged < in africa using the dhs data. they identified the correlation between high malaria risk prevalence in urban areas and argues for the decrease in transmission in rural areas due to urbanization. yaoundé (central region) and douala (littoral region) are the capital and economic capital of cameroon respectively and are full of more human activities than the other regions. we could not evidently support the reasons for the association between high malaria cases and high population densities and call for more research at a smaller scale in the future. the pearson's coefficient, r (table ) shows a positive association with some environmental factors such as rainfall, vegetation, and nightlights. again, this is not a new finding as a similar report on malaria prevalence on climatic factors have been demonstrated in cameroon, where the authors derived spatial distribution maps for malaria transmission under different climatic and intervention scenarios. their predictive study showed that temperature and rainfall were associated with malaria transmission [ ] . the association between malaria cases and rainfall (p < . and r = . ) examined in for example, highlights the necessity for malaria surveillance and response systems during the rainy seasons in cameroon since standing water provides breeding grounds for anopheles mosquitos responsible for transmission of the parasites. in the northern part of the country, the rainy seasons are from may to september (little rainfall) and from march to august (major rainfall) in the southern part. moreover, the nightlights composite (p < . and r = . ) in which indicates the number of human activities at night shows that cities in cameroon such as douala and yaoundé with the highest population densities have more night time activities due to increasing urbanization. the government should carry out more malaria preventive measures and campaigns in the urban-rural areas of these regions. vegetation indices are spectral shift of two or more bands designed to heighten the contribution of vegetation properties and allow reliable spatial and temporal inter-comparisons of terrestrial photosynthetic activity and leaf canopy structural changes [ ] . vegetations near human settlements increase the population of malaria vectors and thus transmission of malaria. kar [ ] in their study; a review of malaria transmission dynamics in forest ecosystem illustrated that forests serve as beds for malaria transmission as they provide favorable conditions such as vegetation cover, temperature, rainfall, and humidity for malaria transmission. in cameroon, most rural settlements and villages are located within forest areas and prevention campaigns should be extended to such areas with malaria clusters and hotspots. our study has the following limitations; i) the malaria prevalence clusters and hotspots at the various urban-rural areas, could be misinforming as the gps clusters data for these areas were displaced for confidentiality, though the clusters were maintained within the dhs administrative unit. ii) our study did not use socio-demographic factors that could find the association between malaria prevalence and social determinants of health and some related environmental data were missing, iii) the dhs project samples collection are subjected to bias due to disparities in the different urban-rural settings and various forms of bias such as the interviewee response bias. iv) the correlation analyses may be confounded by other factors and spatial techniques such as the geographically weighted regression may be considered to analyze the association between environmental variables and malaria distribution. we did not apply this technique because of missing gps urban-rural data points in some of the malaria years. the strength of this study includes; the application of spatial statistics and the use of arcgis in malaria research at a smaller geographic scale for public health interventions, the design of this study demonstrated the importance of using spatial data in dhs research. also, our study, unlike others will provide a new insight to the prevention of malaria in cameroon at the small-area scale and the techniques used can be applied to other disease phenomena. this research focused on malaria distribution at a smaller scale (urban-rural) and we identified urban-rural areas with high and low malaria cases and hotspots. global spatial demographic health datasets have been used to estimate the population at risk of malaria, which forms a fundamental system of measurement for decisionmakers at national and international levels [ ] [ ] [ ] . our maps are supporting tools for effective malaria control at the urban-rural scale and can be used to inform malaria prevention and control programs. despite the current advances to prevent malaria, more work is required particularly in targeting the population at the urban-rural geographic scale on spatial data collection and surveys, wide coverage and distribution of itns, campaigns, screening, and provision of treatment that will progressively eliminate the disease. we thank dhs, icf international for data access permission. we express our sincere gratitude to the chinese government and the teaching staff of the department of epidemiology and biostatistics,school of public health, cheeloo college of medicine, shandong university for study support and funding. we also thank mr alec lasko raymond for his advice and support throughout this study. geneva: world health organization call to action: priorities for malaria elimination who, world malaria country report the effect of insecticide treated nets (itns) on plasmodium falciparum infection in rural and semi-urban communities in the south west region of cameroon coverage and system efficiencies of insecticide-treated nets in africa from improving estimates of insecticides-treated mosquito net coverage from household surveys: using geographic coordinates to account for endemicity and seasonality. dhs analytical studies no. . calverton: icf international rbm, action and investment to defeat malaria how well are malaria maps used to design and finance malaria control in africa? spatial epidemiology: current approaches and future challenges geographic patterns of disease -wiley statsref: statistics reference online an overview of spatial analysis of emerging infectious diseases spatial methods for infectious disease outbreak investigations: systematic literature review global mapping of infectious disease geographic profiling as a novel spatial tool for targeting infectious disease control spatial analysis of risk factor of cholera outbreak for - in a peri-urban area of lusaka, zambia understanding the spatial clustering of severe acute respiratory syndrome (sars) in hong kong spatial clustering of malaria and associated risk factors during an epidemic in a highland area of western kenya evaluation of new tools for malaria vector control in cameroon: focus on long lasting insecticidal nets malaria risk factors in women on intermittent preventive treatment at delivery and their effects on pregnancy demographic and health surveys (dhs). icf international incorporating geographic information into demographic and health surveys: a field guide to gps data collection creating spatial interpolation surfaces with dhs data geographic displacement procedure and georeferenced data release policy for the demographic and health surveys. dhs spatial analysis reports no . calverton: icf international the effect of malaria control on plasmodium falciparum in africa between the malaria atlas project spatio-temporal distribution and hotspots of hand, foot and mouth disease (hfmd) in northern thailand climate variation and malaria prevalence in warri metropolis mapping malaria transmission in west and central africa effective use of pearson's product-moment correlation coefficient malaria prevalence, risk factors and spatial distribution in a hilly forest area of bangladesh analysing malaria incidence at the small area level for developing a spatial decision support system: a case study in kalaburagi mapping plasmodium falciparum mortality in africa between and the impact of urbanization and population density on childhood plasmodium falciparum parasite prevalence rates in africa spatial panorama of malaria prevalence in africa under climate change and interventions scenarios nasa measures vegetation index and phenology (vip) phenology evi yearly global . deg cmg a review of malaria transmission dynamics in forest ecosystems mapping malaria in municipalities of the coffee triangle region of colombia using geographic information systems (gis) spatial models for the rational allocation of routinely distributed bed nets to public health facilities in western kenya mapping populations at risk: improving spatial demographic data for infectious disease modeling and metric derivation funding and support for this study was provided by shandong university, people's republic of china. the datasets analyzed during the current study are publicly available from dhs website https://dhsprogram.com [ ] , and public access to the database is closed. administrative permission to access the data was obtained. mat; acquisition of data, analysis, and interpretation of data. fzx; conception and design, interpretation of results. mfpn, mat; drafting, writing and revising the article. ad, fk; assisted with data analysis, interpretation of results and revising the article. all the authors have read and approved the final manuscript. the procedures and questionnaires for dhs data collection have been reviewed and approved by the icf international institution review board (irb) [ ] . interviews are conducted only if the respondent provides voluntary informed consent. a written informed consent was obtained from all participants. the icf international irb ensures that survey complies with the u. s department of health and human services regulations for the protection of human subjects. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -c jdp jv authors: fu, yangxi; tang, zhengzhen; ye, zhixu; mo, shi; tian, xingui; ni, ke; ren, luo; liu, enmei; zang, na title: human adenovirus type infection causes a more severe disease than type date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: c jdp jv background: human adenovirus type (hadv- ) and (hadv- ) cause significant morbidity and develop severe complications and long-term pulmonary sequelae in children. however, epidemiologic reports have suggested that nearly all highly severe or fatal adenoviral diseases in children are associated with hadv- rather than hadv- . here, we conduct in-depth investigations to confirm and extend these findings through a comprehensive series of assays in vitro and in vivo as well as clinical correlates. methods: a total of nasopharyngeal aspirate (npa) samples were collected from hospitalized children with acute respiratory infections in children’s hospital of chongqing medical university from june to may . among samples that tested positive for hadvs, clinical data of cases of hadv- ( ) and hadv- ( ) infections were analyzed. all hadv-positive samples were classified by sequencing the hexon and fiber genes, and compared with clinical data and virological assays. we also performed in vitro assays of virus quantification, viral growth kinetics, competitive fitness, cytotoxicity and c a assay of the two strains. mouse adenovirus model was used to evaluate acute inflammatory responses. results: clinical characteristics revealed that hadv- infection caused more severe pneumonia, toxic encephalopathy, respiratory failure, longer mean hospitalization, significantly lower white blood cell (wbc) and platelet counts, compared to those of hadv- . in cell culture, hadv- replicated at a higher level than hadv- , and viral fitness showed significant differences as well. hadv- also exhibited higher c a production and cytotoxic effects, and hadv- -infected mice showed aggravated pathology and higher pulmonary virus loads, compared to hadv- -infected mice. macrophages in balf remained markedly high during infection, with concomitant increase in pro-inflammatory cytokines (tnf-α, il- β, ifn-γ, and il- ), compared hadv- infection. conclusions: these results document that hadv- replicates more robustly than hadv- , and promotes an exacerbated cytokine response, causing a more severe airway inflammation. the findings merit further mechanistic studies that offer the pediatricians an informed decision to proceed with early diagnosis and treatment of hadv- infection. human adenovirus is well-recognized as an important pathogen of respiratory tract infection in childhood [ ] [ ] [ ] . more than hadv serotypes are further subdivided into seven species (a-g) [ , ] ; however, species b (hadv- , , and ), c (hadv- , , and ), and e (hadv- ) are the ones most associated with respiratory infections [ ] . epidemiological studies have reported an approximately - % hadv-positive rate among acute respiratory tract infections in children [ , ] . adenovirus infections may in fact result in high morbidity and mortality in children, and fatality rates for untreated severe hadv pneumonia or disseminated disease may exceed % [ ] . severe adenovirus infection in children can be complicated with pleural effusions [ ] , acute respiratory distress syndrome (ards) [ ] , respiratory failure [ ] , myocarditis [ ] , and central nervous system dysfunction [ ] , leading to either mechanical ventilation or extracorporeal life support, even death. unfortunately, effective adenoviral vaccine for children and specific antiviral drugs against human adenoviruses are currently not available. hadv- and hadv- , belonging to subgenus b , cause infections that are usually mild and self-limiting in immunocompetent individuals; however, severe, and even life-threatening infections and outbreaks, associated with both type and in children, are increasingly reported [ , , ] . epidemiology of the disease suggests that hadv- and hadv- are the major types responsible for lower respiratory diseases in children less than years old worldwide [ , , , ] . in beijing, china, hadv- , and hadv- were the most common serotypes causing pneumonia from to [ ] . in chongqing, china, . % of the hadv- -infected patients were diagnosed as having severe pneumonia, significantly higher than the . % of the hadv- -infected patients from to [ ] . in taiwan, hadv- and hadv- , causing complication with respiratory failure, accounted for % of infected children; seven cases due to hadv- were among ten deaths reported in a single year, between and [ ] . in korea, both hadv- and hadv- cause epidemics of severe lower respiratory tract infections in young children. overall, and %, of the children infected with hadv- and hadv- , respectively, required mechanical ventilation [ ] . certain adenovirus serotypes are associated with particular clinical features and severe illness; however, no study has offered persuasive evidence for in the different degrees of disease severity caused by hadv- and hadv- infection because it still limits on the basis of clinical observation. in the present study, therefore, we undertook a comprehensive analysis of the comparative clinical features of hadv- and hadv- infection, as well as a serial of experiments, were performed to better understand the association between severity of the disease and the serotypes of hadvs. participants, demographic data, clinical data analysis patients ranging in age from month to years and requiring inpatient treatment due to acute respiratory tract infections (arti) at the department of respiratory medicine, children's hospital of chongqing medical university between june, and may, , were enrolled in this study. parents/guardians provided demographic data and medical history during an interview. severe disease was defined as respiratory failure confirmed by an abnormal blood gas analysis result (an oxygen saturation level of approximately % or less) or toxic encephalopathy. a total of nasopharyngeal aspirates (npas) were collected on the day of hospital admission after obtaining permission from the patients or their guardians. imaging and laboratory data on admission and during hospitalization were collected. white blood cell (wbc) count > , / μl was defined as leukocytosis, whereas that < /μl was defined as leukopenia. the study procedure was approved by the ethics committee of the children's hospital of chongqing medical university. informed consent was obtained from parent or guardian of all participants. hadv isolation, identification, and molecular typing npas from hadv-positive patients were inoculated into hep- cell monolayers and cytopathic effect (cpe) was monitored for days. viral dna and rna were extracted from μl aliquots of the npa samples by a qiaampminelute virus spin kit (qiagen, hilden, germany). hadv-positive samples were used to amplify the hexon hypervariable regions (hvrs) - and fiber genes for subsequent sequencing [ , ] . the hadvs strains were molecularly typed by pcr amplification and sequencing of all seven hypervariable regions in the hexon gene, described previously [ ] . the hadv- (cq ) and hadv- strains (cq ) used in this study were originally isolated from npas of children infected with hadv. the virus was purified using a standard cesium chloride gradient centrifugation procedure, as described [ ] . the concentration of viral particles (vps) was determined by spectrophotometry according to the a value. the virus concentration was calculated as vps/ml = a *dilution* . three cell lines (a , hbe, and hek- ) at % confluency in -well plates were infected with either hadv- or hadv- at a multiplicity of infection (moi) of vp/cell. viruses were cultured as previously described [ , ] . the culture supernatants and cells were sampled at h post-infection. viral loads were quantified by quantitative pcr (qpcr). briefly, μl reaction mixtures were prepared by adding μl of sample nucleic acid extract to μl of iq supermix (bio-rad, hercules, ca), primers and probes. the sequences of primers and probes for qpcr and the amplification conditions have been detailed [ , ] . the real-time hadv primer sequences were as follows: hadv- forward, ′-ggga gacaatattactaaagaaggtttgc- ′, hadv- reverse, '-caacttgaggctctggctgata- ′. the sequence of the hadv- probe was '-cactac"t"-gaaggagaagaaaagcccatttatgcc, which was labeled with -carboxyfluorescein (fam) and internally quenched at "t" with black hole quencher- on the ′end and phosphate on the ′-end. the forward primer sequences of hadv- were '-gaggagccag atat tgatatggaatt- ′, the reverse primer sequences of hadv- were: '-aattgacattttccgtgtaaagc a- ′, and probe, '-aagctgctgacgctttttcgcc tga- ′. the hadv- probe was labeled with -carboxy fluorescein (fam) on the ′-end and ′-terminally quenched with black hole quencher- . the thermal cycling condition was as follows: one cycle of min at °c; cycles of s at °c; and min at °c. a cells at % confluency in -well plates were infected with either hadv- or hadv- (moi = vps/ cell). after h adsorption with rocking every min, the inoculum was removed, ml of fresh dmem-f with % fbs was added to each well, and the plates were incubated at °c in % co . the culture media were harvested at , , , , , , , , and h post-infection. tcid was used to titer the virus at each time point. a cells were infected with a mixture hadv- and hadv- at a ratio of : and moi of vps/cell in standard -well cell culture plates, and incubated as described under 'viral growth kinetics'. media supernatants and cells were harvested at , , , , , , , h post-infection. virus copies were quantified at each indicated time point by qpcr described earlier [ , ] . a cells ( . × ) were seeded in each well of -well flat bottom plates and incubated at °c in % co overnight. thereafter, the media were removed, and cells were infected with hadv- or hadv- (low moi = vps/cell, or high moi = vps/cell). infected and uninfected cell viability were assayed at , , , , and h post-infection and the absorbance was determined as described [ , ] . cell viability was expressed as the ratio, and the mean (mean ± sem) of replicates of two strains was presented. all samples were collected as described previously, with minor modifications [ ] . purified hadvs ( . g) in pbs were mixed with a : dilution of normal human serum (nhs) in an assay volume of μl, and incubated for , and min at °c. the samples were further diluted : and tested using an opteia human c a elisa kit (bd biosciences), according to the standard procedure provided by the manufacturer. female balb/c mice, - -week old, were purchased from the animal laboratory of chongqing medical university and housed in a dedicated pathogen-free facility at chongqing medical university. the use of all experimental animals strictly met the ethical requirements of the animal committee of chongqing medical university (license numbers: syxk(yu) - ). the mice under anesthesia were infected intranasally (i.n.) with × vps/ml of hadv- or hadv- in μl. mock-infected mice were instilled with μl of phosphate-buffered saline (pbs) under the same conditions. mice were processed after , , and days of infection. for studies of viral load, total viral nucleic acid was directly extracted from the right lung lobe, using a qiaamp mini-viral dna extraction kit (qiagen, hilden, germany) and following the manufacturer's recommendations. quantification of hadv- and hadv- copies were performed as described earlier [ , ] . after , , and days of infection ( mice in each time point in every group), the animals were euthanatized by intraperitoneal injection of pentobarbital ( mg/kg). the left pulmonary hilum was closed by clamping, in order to collect balf from the right side. the specific steps were as described previously [ ] . briefly, bronchoalveolar lavage was performed using . ml of ice-cold sterile pbs for a total of times. balf was centrifuged at rpm for min in cold ( °c). the supernatant was aliquoted and stored at °c for subsequent cytokine measurements. the precipitate was resuspended in ml of pbs, and the total number of cells was counted under a microscope. the cells were centrifuged, smeared onto slides, fixed, and stained with diff quik (baxter healthcare corp, deerfleld, miami, fl) to analyze and classify white blood cells. cell counting was performed using a microscope. a total of random cells was counted from each slide and included macrophages, lymphocytes, and neutrophils. mice were sacrificed at , , and d after infection for pathological analyses. the left lung lobe was collected, fixed in % paraformaldehyde for h, dehydrated, and then embedded in paraffin. lung tissue blocks were cut into μm thick sections that were then stained with hematoxylin and eosin to evaluate lung tissue pathology associated with hadv- and hadv- infection. the levels of interferon-γ (ifn-γ), tumor necrosis factor-α (tnf-α), interleukin- β (il- β), and interleukin- (il- ) in balf were all measured using commercial murine elisa reagent kits (neobioscience, shenzhen, china) according to the manufacturer's instructions. descriptive statistics were performed for all variables; the continuous variables were summarized as means and standard deviations (sd) or as medians and ranges, and the categorical variables were summarized as frequencies and proportions. the graph pad prism . software was used for data analysis. statistical significance was assessed by one-or two-way analysis of variance (anova). one-way anova was used to analyze significant differences between three or more groups. two-way anova was used to analyze significant differences between two variables. if an overall test was significant, the tukey's test was utilized for specific comparisons between individual groups. differences were considered significant at p < . . more severe disease in patients infected with hadv- compared to hadv- among the npas, specimens were identified as positive for adenovirus, of which hadv- ( ) and hadv- ( ) were the most common types. as shown in table , age and gender distribution, underlying disease, and coinfection were comparable between hadv- and hadv- . co-infection with other respiratory viruses were observed in . and . % of patients infected with hadv- and hadv- , respectively. the most common clinical manifestations of hadv- and hadv- infections, such as cough, wheezing, croup, dry rales, moist rales and fever, including the peak of febrile body temperature (> °c), were not significantly different. in contrast, pneumonia was the most common diagnoses of the patients. among the hadv- -infected patients, . % were diagnosed as having severe pneumonia, significantly higher than that of the hadv- -infected patients ( . % vs . %, p = . ). when the median duration of hospitalization was analyzed, we found that the hadv- inpatients had a longer mean duration of hospitalization, . ± . days (p = . ). several severe outcomes, toxic encephalopathy, and respiratory failure were significantly higher in patients with hadv- infection compared with those with hadv- (p < . ), but no statistically significant differences were found in icu admission, pleural effusion, suspicious bo and intracranial infection. notably, one child, infected by hadv- , exhibited complications of acute respiratory distress syndrome (ards). laboratory findings for the hadv- -positive inpatients were also significantly different from those infected by hadv- (table ) . specifically, the hadv- -positive inpatients had lower white blood cell count ( . ± . vs. . ± . × cells/l; p = . ), platelet count ( . ± . vs. . ± . × cells/l; p = . ). in contrast, hemoglobin and c-reactive protein levels, and the percentages of lymphocytes, neutrophils and positive sputum culture were found to be statistically similar. in the chest radiographs (table ) , alveolar infiltration, consolidation and pleural effusion were more frequently observed with the hadv- patients compared to hadv- patients but they did not differ significantly. increased lung texture and interstitial inflammation showed a similar proportion. two patients infected with type , and four patients infected with type , had atelectasis. notably, two patients infected with adenovirus type had complications with pneumothorax. since type-specific adenovirus infection is known to cause different tissue tropisms and clinical manifestations as indicated before, viral loads and fitness of hadv- and hadv- were evaluated in several human epithelial cells to determine if there were differences. as shown (fig. a) , at h post-infection, hadv- replicated to a higher number of viral copies than hadv- in all three cell lines tested, namely, a , hek- and hbe cells. hadv- also exhibited stronger growth than hadv- in a cells at , and h post-infection (fig. b) . in comparing the competitive fitness between hadv- and hadv- , however, no significant difference was observed. lastly, hadv- loads were significantly higher than those of hadv- at , , and h post-infection (p < . ) (fig. c) . cytotoxicity assay may serve as a measure of cellular distress induced by viral infection. as shown (fig. ) , at a low moi of vps/cell, viability of hadv- -infected cells was slightly higher than that of the hadv- -infected cells, but there was no statistically significant difference. in contrast, at high moi of vps/cell, hadv- induced a remarkably greater decrease in cell viability at both and h post-infection ( . and . %, respectively; p < . ) (fig. ) . to compare complement activations by hadv- and hadv- , the anaphylatoxin c a, a split fragment of c (the step where classical, lectin, and alternative pathways of complement activation merge), was measured in vitro. as shown (fig. ), hadv incubation with serum, compared with nhs incubation alone, resulted in a marked increase of c a at , and mins. importantly, elevation of c a by hadv- , compared with hadv- , was statistically higher at mins (p < . ). to better understand the differences in adenovirus-induced airway inflammation, we established a mouse respiratory infection model for human adenovirus. in this model, assays of viral load in the lung tissues revealed that virus copies of hadv- -infected groups were higher than those of hadv- -infected groups at , and days of infection, particularly on day , when virus copies in the hadv- group were . -fold higher than the hadv- group (p < . ) (fig. a) . pulmonary inflammation in mice was also observed following hadv- and hadv- infection at each time point and found to gradually increase from day to day , decreasing on day . compared with hadv- -infected mice, lung tissue damage of hadv- -infected mice was also higher on all days of infection (fig. b) . consistent with the morphological changes, the total number of cells in balf was higher, compared with those in the control group on all days (p < . ) (fig. c-e) ; importantly, the hadv- group showed a significantly greater increase in these cells, in comparison with the hadv- group (p < . ) (fig. d-e) . specifically, abundant macrophages and smaller number of lymphocytes, neutrophils infiltrated into the balf at all days post-infection. whereas the neutrophils registered a slight rise, only on day , followed by dramatic decrease, the lymphocyte and macrophage numbers remained persistently elevated through days and . in addition, macrophages in balf of the hadv- group were significantly higher than the hadv- group after and days of note: data are shown as medians (interquartile range) or number (%) abbreviations: wbc white blood cell, crp c-reactive protein " a ": a significant difference between two groups infection ( fig. d-e) , while the lymphocytes exhibited significant difference only on day (p < . ) (fig. d) . neutrophils in balf of the hadv- group was not significantly different compared with that of the hadv- group at all time points (fig. c-e) . together, these results showed that hadv- infection induced a relatively more severe acute airway inflammation. the levels of inflammatory mediators associated with airway inflammation, including il- β, tnf-α, ifn-γ, and il- , were also clearly elevated in balf at , , and days post hadv- and hadv- infection. nevertheless, the levels of il- β, il- , and tnf-α in the hadv- group were higher than the hadv- group post , , and days of infection (p < . ) (fig. f-i ). hadv- and hadv- have been prevalent throughout the world for decades, and both serotypes frequently play an important role in the etiology of lower respiratory infection in children. in the present study, coinfection with hadv- and hadv- with other respiratory viruses accounted for half of the pediatric cases, but coinfection per se did not result in a difference in clinical manifestation or severe clinical outcomes. these results and the severe respiratory diseases and complications, which we identified with hadv- infection in hospitalized children, were all consistent with previous reports [ , , , ] . remarkably, we also found a higher rate ( . %) of toxic encephalopathy associated with hadv- infection. previous studies in japan showed that mild encephalitis/encephalopathy with a reversible splenial lesion (mers) could be triggered by adenovirus infection [ , ] , which was proposed to be caused by neurotoxins, likely acting as an antigen that induced a strong immune response, leading to a series of symptoms. recently, silkworm chrysalis ingestion was shown to induce toxic encephalopathy [ ] ; however, our results constitute the first reported case of toxic encephalopathy associated with adenoviral infection. earlier studies reported that several viruses, including adenoviruses, influenza a virus, parainfluenza virus, and japanese encephalitis virus, can use the olfactory nerve as a shortcut into the central nervous system, causing infection of nerve cells and induction of immune response [ ] ; however, the exact mechanisms of such neurotropic infections remain to be elucidated. in the current study, we have compared and correlated the biological characteristics of hadv- and hadv- with their properties ex vivo and in vitro. first of all, the results of cellular infectivity (fig. a) indicated that a , hbe, and hek- are susceptible cell lines for the growth of adenovirus ex vivo. furthermore, a difference of viral loads recorded in a and hbe cell lines between adenovirus and indicated differential inflammatory reactions were produced from infection of host respiratory epithelial cells by hadv stimulated the innate immune system that might partially explain the role of hadv- infection in the pathogenesis of a more severe pneumonia. this is likely because the airway epithelial cells not only serve as a passive barrier to infectious particles but also actively participate in the innate immune response to foreign antigens. the immune response of epithelia to infection and antigen exposure involves the release of chemokines and cytokines into the submucosa, which initiates an inflammatory reaction [ ] . the results of co-infection of the two viruses indicated that distinctive fitness is type-specific and not interaction. the result of cell viability also provided useful information that was important for understanding type-specific viral virulence (fig. ) . taken together, we conclude that the differences observed in the replication, fitness, and virulence of the two adenoviruses are dependent on virus type. the complement system plays important roles in innate immunity and is vital in the protection against invading pathogens. the complement cascade can be initiated through three main pathways: classical, lectin, and alternative. the classical pathway is mainly antibodydependent. it is activated by interaction of c q (a subunit of the first complement protein c ) with the fc portion of the igm or igg immune complex, although activation can also be achieved in an antibody-independent manner by some membrane components of viruses, bacteria and fungi. the lectin pathway is first activated when the mannose-binding lectin, a structural homologue of c q, binds to carbohydrate moieties on the surface of pathogens, including yeast, bacteria and viruses. the alternative pathway is antibody-independent; it is spontaneously activated on biological surfaces, as well as in plasma and other body fluids, when the level of c hydrolysis remains consistently low. this spontaneous cleavage readily initiates amplification of the activation cascades [ ] . although every pathway is triggered independently, all of the complement cascades culminate in the central cleavage of c and the generation of its active fragments c a and c b, which can bind covalently to viral components to aid in opsonization and phagocytosis. furthermore, complement activation also mediates the inflammatory reaction via the generation of anaphylatoxins (c a and c a) and recruitment of inflammatory cells to the site of infection [ , ] . therefore, activation of the complement system leads to inflammation, opsonization, and membrane perturbation. previous studies by johnson et al. and others [ , [ ] [ ] [ ] have shown that the complement is strongly activated by a wide range of negative-strand rna viruses, including parainfluenza virus (piv ), nipah virus (niv), mumps virus ; c-e the number of total cells and the differential counting of cells in balf. f-i concentration of cytokine il- β, tnf-α, and il- in balf; n = ; *,**,*** indicate p < . , . , and . , respectively, compared with the mock group; #, ##, ### indicate p < . , . , and . , respectively, compared with hadv- group implicate the plasma level of c a as a marker of the early innate response to adenovirus [ ] . in previous studies, our group successfully established a hadv- pneumonia model in the laboratory mouse, and studies in this in vivo model suggested that hmgb is a mediator of hadv- -induced pulmonary inflammation [ ] . our current study has addressed whether different types of human adenovirus infection result in distinct inflammatory reactions in vivo. in this study, we clearly observed robust virus growth, and also documented significantly higher viral load in hadv- infected mice at days post-infection. these results are different from those reported by kajon et al. in which only low levels of hadv- and hadv- replication were seen at the early stage of infection in mice [ ] . we speculate that the apparent discrepancy is due to the effect of different experimental animal models and detection methods used. adenovirus infection is characterized pathologically by a time-dependent progression in the type of inflammatory cells present. the initial inflammation is a neutrophilic interstitial infiltration with neutrophilic alveolitis. subsequently, monocytes become evident and, finally, predominantly lymphocytic infiltrates appear. during the acute stage of hadv- and hadv- infection, we also found that the total number and classes of cells in balf increased, primarily accounted for by macrophages. neutrophils only transiently increased at days post-infection. a mixture of mononuclear cells (macrophages and lymphocytes) exclusively became evident at and days postinfection. macrophages were most prominent than neutrophils and lymphocytes at all time points. these results are fully consistent with several studies, including those of kajon et al., which showed that hadv- and hadv- -induced pneumonia mainly produces neutrophil infiltration at and days post-infection, and affects macrophages [ ] . apart from phagocytosing and killing the pathogens, macrophages also secrete chemokines to recruit cells to the sites of infection. yoon et al. found that adenovirus type produced a more robust il- and il- response than adenovirus in human bronchial epithelial cells [ , ] . diaz et al. compared cytokine responses between type and type , and documented significantly higher production of interferon-γ from type [ ] . similarly, subsequent production of il- β, induced by macrophages, also reached the highest levels after , , and days of hadv- and hadv- infection. asgari et al. have reported that engagement of the c a receptor triggers il- β processing and release via caspase- activation [ ] . the results are consistent with the immune response of the epithelia to adenovirus infection and antigen exposure, which involves the release of chemokines and cytokines into the submucosa and initiates an inflammatory reaction. lastly, complement activation mediates the inflammatory reaction via anaphylatoxins (c a and c a) and recruitment of inflammatory cells to the site of infection. the inflammation then leads to the recruitment of phagocytes that help clear the invaders. collectively, we conclude that these innate immune responses play an important role in the initial defense against adenovirus infection. although our results implicate a novel type-specific relationship between adenoviral infection, it has potential limitations. the data presented herein only interrogated virological differences; however, adenovirus infection is a very complex process that involves not only the structural proteins of the virus, such as fiber, hexon, and penton, but also includes host and environmental factors. nonetheless, our data confirm that severe adenovirus infection was associated with hadv- rather than hadv- . the significance of the study lies in that the pediatrician should be aware of the importance of early diagnosis and treatment of hadv- infection in children in clinical practice. the findings should also stimulate further studies of mechanisms of the different pathogenicity among human adenovirus serotypes. the severity of adenoviral infection, as studied in chongqing, china, may be correlated to human adenovirus type (hadv- ) instead of type (hadv- ). overall, strain of the hadv- type caused a more severe pneumonia and an exacerbated cytokine response, which also paralleled their more robust replication in cell culture, as compared to hadv- . while the exact mechanism of the type-specific pathogenicity merits further investigation, these findings may eventually contribute to better control and treatment of adenoviral infection. serum inflammatory markers in patients with adenovirus respiratory infection adenovirus serotype and infection with acute respiratory failure in children in taiwan epidemical features of hadv- and hadv- in pediatric pneumonia in chongqing human mastadenovirus type : a novel, multiple recombinant species d mastadenovirus isolated from diarrhoeal faeces of a haematopoietic stem cell transplantation recipient computational analysis of a species d human adenovirus provides evidence 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infections: sequencing of the hexon hypervariable region reveals high sequence variability phylogenetic analysis of the main neutralization and hemagglutination determinants of all human adenovirus prototypes as a basis for molecular classification and taxonomy identification and typing of respiratory adenoviruses in guangzhou, southern china using a rapid and simple method a simple and efficient method for purification of infectious recombinant adenovirus a comparison of viral fitness and virulence between emergent adenovirus p and prototype adenovirus p strains in vitro characterization of human adenovirus type in comparison with its parental adenoviruses, types and pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr quantitative real-time pcr assay panel for detection and type-specific identification of epidemic respiratory human adenoviruses a novel factor i activity in nipah virus inhibits human complement pathways through cleavage of c b mmp- -mediated by sarm-trif signaling pathway contributes to ifngamma-independent airway inflammation and ahr post rsv infection in nude mice epidemiology of acute encephalopathy in japan, with emphasis on the association of viruses and syndromes transient hemiparesis and hemianesthesia in an atypical case of adult-onset clinically mild encephalitis/ encephalopathy with a reversible splenial lesion associated with adenovirus infection the effect of hemoperfusion on patients with toxic encephalopathy induced by silkworm chrysalis ingestion the olfactory nerve: a shortcut for influenza and other viral diseases into the central nervous system cytokine induction by respiratory syncytial virus and adenovirus in bronchial epithelial cells complement: a unique innate immune sensor for danger signals complement. first of two parts second of two parts virion-associated complement regulator cd is more potent than cd in mediating resistance of mumps virus and vesicular stomatitis virus to neutralization incorporation of host complement regulatory proteins into newcastle disease virus enhances complement evasion differential mechanisms of complementmediated neutralization of the closely related paramyxoviruses simian virus and mumps virus adenovirus activates complement by distinctly different mechanisms in vitro and in vivo: indirect complement activation by virions in vivo hmgb mediates hadv- infection-induced pulmonary inflammation in mice acute inflammatory response and remodeling of airway epithelium after subspecies b human adenovirus infection of the mouse lower respiratory tract differential effects of respiratory syncytial virus and adenovirus on mononuclear cell cytokine responses c a modulates il- beta secretion in human monocytes by regulating atp efflux and subsequent nlrp inflammasome activation we thank professor rong zhou, dr. xingui tian, and colleagues (state key lab of respiratory disease, guangzhou medical university, china) for kind assistance in various areas of the study. we would like to acknowledge the assistance of the patients and their caregivers involved in the study, the staff in the department of respiratory medicine and the department of intensive care unit of children's hospital of chongqing medical university help for nasopharyngeal aspirates collection. this study was supported by the national natural science foundation of china for young scholars ( ). the funders had no role in the study design, data collection, analysis and interpretation of data and writing the manuscript. the datasets used and analyzed during the current study are included within the article and its tables. additional data may be available from the corresponding author on reasonable request.authors' contributions yxf performed the experiments, analyzed and/or interpreted the data, and wrote the manuscript. zzt, zxy, sm helped to perform the experiments. xgt contributed to the design of the paper and revised the manuscript. kn contributed to the design of the study. lr assisted revision of the manuscript. n z contributed to the design of the study and assisted in the analysis and/or interpretation of data, revised the manuscript. em l contributed to conception, collected clinical information, and revision of the manuscript. all authors read and approved the final manuscript. the study procedure was approved by the ethics committee of the children's hospital of chongqing medical university, chongqing, china. written informed consent was obtained from parent or guardian of all participants. not applicable. the authors declare that they have no competing interests. key: cord- -u u k hj authors: ding, hua; chen, yin; yu, zhao; horby, peter w; wang, fenjuan; hu, jingfeng; yang, xuhui; mao, haiyan; qin, shuwen; chai, chengliang; liu, shelan; chen, enfu; yu, hongjie title: a family cluster of three confirmed cases infected with avian influenza a (h n ) virus in zhejiang province of china date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: u u k hj background: a total of laboratory-confirmed cases infected with avian influenza a (h n ) virus (including deaths) have been reported till october , , of which . % ( / ) of the cases were identified from zhejiang province. we describe the largest reported cluster of virologically confirmed h n cases, comprised by a fatal index case and two mild secondary cases. methods: a retrospective investigation was conducted in january of . three confirmed cases, their close contacts, and relevant environments samples were tested by real-time reverse transcriptase-polymerase chain reaction (rt-pcr), viral culture, and sequencing. serum samples were tested by haemagglutination inhibition (hi) assay. results: the index case, a -year-old farmer with type ii diabetes, who lived with his daughter (case , aged ) and wife (case , aged ) and his son-in-law (h n negative). the index case and case worked daily in a live bird market. onset of illness in index case occurred in january , and subsequently, he died of multi-organ failure on january . case presented with mild symptoms on january following frequent unprotected bed-side care of the index case between january to , and exposed to live bird market on january . case became unwell on january after providing bedside care to the index case on january to , and following the contact with case during january to at the funeral of the index case. the two secondary cases were discharged on february and separately after early treatment with antiviral medication. four virus strains were isolated and genome analyses showed . ~ % genetic homology, with two amino mutations (v i in ns and v a in np). % ( / ) of environmental samples collected in january were h n positive. twenty-five close contacts remained well and were negative for h n infection by rt-pcr and hi assay. conclusions: in the present study, the index case was infected from a live bird market while the two secondary cases were infected by the index case during unprotected exposure. this family cluster is, therefore, compatible with non-sustained person-to-person transmission of avian influenza a/h n . electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. human infection with avian influenza a/h n virus was first identified in march of , in china, a total of confirmed cases were found in the world up to date [ ] . the seasonal epidemiology is characterized to occur from november through april in china, coinciding well with both seasonal human influenza and h n in birds [ ] . almost all cases were hospitalized, and / of cases died. the fatality is much higher than that for seasonal influenza in the china ( . %), but it is lower than for cases of h n ( %) [ , ] . current evidence suggests that human infection appears to be associated with exposure to infected live poultry or contaminated environments, including markets where live poultry are sold [ ] [ ] [ ] . in the light of this opinion, the closure of live bird markets (lbm) has been associated with a reduction in the incidence of human infections [ ] . despite the fact that h n remains to be a zoonotic infection of avian origin, there are concerns that the virus show genotypic and phenotypic evidence of partial adaptation to mammals [ ] . compared to other subtypes of avian influenza virus, h n virus show increased binding affinity to mammalian-type receptors, and their amount grow up rapidly at the temperatures that are close to the normal body temperature in mammals (although it is lower than that of birds). in addition, they possess pb gene mutations that are associated with adaptation to mammals [ ] [ ] [ ] . whilst sequence analyses had shown that the haemagglutinin (ha) and neuraminidase (na) genes of h n virus detected in china show very high homology, whereas the genes for coding internal proteins are diversified [ ] . ferret and mouse models confirm that strains isolated from humans could replicate efficiently in both mammalian and human airway cells, with efficient transmissibility by direct contact and modest transmissibility by respiratory droplets [ , ] . given these signatures of partial adaptation to mammals, it is imperative to closely monitor and investigate all clusters of human h n virus to determine the transmissibility and severity of virus infection, as well as its potential host and pathogen determinants. a few of family clusters of h n infections (in shanghai, jiangsu, shandong, guangdong and beijing) have been described involving two family members. it was concluded that limited person-to-person transmission may occur following close, prolonged, and unprotected contact with the symptomatic index case, while sustained transmission was not found [ ] [ ] [ ] . here we describe an additional cluster, comprised of three laboratory-confirmed cases of human infection with h n virus reported in zhejiang province in january . this is the largest reported cluster of virological confirmed h n cases, and the full genome data of the virus were isolated from all cases and associated with clinical and epidemiological data and their close contacts. all three h n confirmed cases and adult contacts and surveillance cases had provided written consent for the participation in this study and the publication of their individual details. data collection for h n cases was determined by the national for h n cases was determined y f man of china, as a part of the continuing public health outbreak investigation; therefore, it was exempt from assessment by institutional review board. the protocol for collecting epidemiological data and conducting serological test of close contacts were approved by the institutional review board of the china cdc. suspected cases of human infection with h n virus are identified through the chinese surveillance systems for influenza-like illness, severe acute respiratory illness (sari), pneumonia of unexplained origin, and clinical diagnostics of cases of pneumonia. based on the chinese guidance, an individual could be considered as a confirmed case of h n virus infection if the presence of the h n virus is verified by real-time reverse transcriptase polymerase chain reaction assay (rt-pcr), virus isolation, or serologic testing [ ] . epidemiological and clinical data were collected through interviews and reviews of medical records between january and , . all three cases and their relatives were interviewed by public health staff to record their exposure history during the two weeks before the onset of symptoms, to validate the timeline of events and to identify close contacts. respiratory tract samples were collected from the index case, case , and case , on january , , and , respectively. environmental samples were collected from the lbm (a market) and the secondary wholesale markets (b , c , and d markets) and from a neighboring household where several chickens were bred. all samples were placed in sterile viral transport medium and shipped within hours to the laboratory of zhejiang cdc at °c for h n testing. viral rna was extracted using qiagen rneasy mini kit. real-time rt-pcr was used to detect influenza type a, subtype h and n using the protocol, specific primer and probe sets provided by china cdc [ ] . specimens were also tested by rt-pcr for the presence of seasonal influenza virus (h , h , and b) and h n virus. complete genomic fragments of the h n virus were amplified directly from clinical samples, and sequencing was performed using an abi xl automatic dna analyzer. the nucleotide sequences were determined by dideoxy sequencing using an abi prism bigdye terminator cycle sequencing kit as previously described [ ] . nucleotide sequences were analyzed with the dnastar package (lasergene, madison, wi, usa). phylogenetic analysis was done by neighbor-joining method with mega (version . ). close contacts were placed under daily active surveillance for fever and respiratory symptoms, which was last for seven days after their last exposure to the h n infected case. close contacts were defined as individuals who had close contact (< meter) with any case without the use of personal protective equipment at any time before illnesses onset to the time of isolation of the case in hospital. antiviral chemoprophylaxis was neither recommended nor provided to contacts. following written informed consent, a structured questionnaire was used to gather demographic information and data on use of personal protective equipment, antiviral chemoprophylaxis, symptoms, and potential risk factors for h n infection during the two weeks starting from their last exposure to h n -infected cases. respiratory specimens for h n testing were taken from close contacts with a febrile respiratory illness occurred during the -day observation period. contacts were asked to provide a single convalescent serum collected ≥ - weeks after their last exposure to a case with h n . h n serological testing was done by hi assay using a modified horse red-blood-cell assay, recommended by the who. the antigen used for the hi assays was the a/zhejiang/ / (h n ) strain. a hi titer ≥ : in single serum sample and a four-fold or greater rise in titer in paired sera was defined as seropositive. the index case, a -year-old farmer with type ii diabetes, taking antidiabetic drugs for one year, had been unwell since january , , with fever ( . °c) and flank pain. after consulting a health care clinic (a clinic) on january and , he was treated as an outpatient with ciprofloxacin and intravenous amoxicillin/ clavulanate potassium. on january , he made a further consultation at a local hospital (b hospital) owing to persistent fever. chest radiography showed a leftlower-lobe pneumonia; meanwhile, treatment with ciprofloxacin was continued. peripheral blood cell count was normal. on january , index case's condition was worsened and again medical advice was sought; therefore, index case was admitted to a different hospital (hospital c ). upon admission in hospital c , he had severe leucopenia, lymphopenia and thrombocytopenia (table ) . he was diagnosed with community acquired pneumonia with a left pleural effusion. on january , he consulted at hospital d (a more advanced hospital) where a sputum sample was collected and sent to zhejiang cdc for microbiologic testing. on january , h n virus-specific rna was detected by rt-pcr (ct value ) in the sputum sample. once the h n virus infection was confirmed, the patient was transferred from hospital c to d immediately. at hospital d , he was isolated in a single room, where he was intubated, mechanically ventilated and commenced on oseltamivir ( mg, twice daily by nasogastric tube) and peramivir ( mg, once daily, intravenously). on january , the patient died of acute respiratory distress syndrome (ards) and multi-organ failure (table , figure , figure , and additional : figure s ). case (index case's daughter, figure ), a -year-old female with no underlying diseases, developed a throat sore and cough on january , the day her father died. she initially consulted the healthcare clinic in hospital a due to constant fever on january , where she was treated with antibiotics (amoxicillin) and then transferred to hospital d for further examination, where sputum and throat swab samples were taken. rt-pcr was conducted on the samples of sputum and throat swabs on the january , and the influenza a/h n specific rna (ct value ) was positive. afterwards, case was admitted directly to an isolation room at the hospital d and commenced on oral oseltamivir ( mg, twice daily), and intravenous peramivir ( mg, once daily). on admission her peripheral blood count, serum blood biochemistry, and chest ct scan were normal (table ) . she was given supplemental oxygen via nasal cannula with a flow rate of - l/min, whereas her oxygen saturation was %. her condition remained stable, and symptoms were improved during hospitalization. later, she was completely recovered and was discharged on february after sputum samples tested negative for h n rna by rt-pcr on january and february (table , figure ). case (index case's wife and case 's mother), a year-old female farmer, with no underlying diseases, developed an acute cough with expectoration on january . she attended the hospital d where a throat swab was collected and an rt-pcr assessment was conducted on the throat swab sample, which was positive for h n (ct value of ). she was admitted to the hospital d on january . although chest radiography was normal, she was treated empirically with oral oseltamivir ( mg, twice daily) and intravenous peramivir ( mg, once daily). results of peripheral blood cell count, serum electrolytes, renal and liver function, and coagulation profiles were normal. arterial blood gas results were normal while the patient was breathing room air. the case remained stable during her admission and then she was discharged on february after sputum tested h n negative by rt-pcr on february (table , figure ). the husband of case , who had been in close contact with the index case and case , had no respiratory symptoms, and throat swabs and paired serum samples were negative ( figure ). rt-pcr-positive throats swabs or sputum samples were obtained on days, days and day of illness for the index case, case , and case , respectively. from these samples, four complete full genome sequences were amplified. sequence analyses indicated that the four isolates were highly homologous the other h n strains previously identified in shanghai, jiangsu, anhui province, and with candidate vaccine strains (sharing . % identity in amino acid sequences of all segments). the four sequences from the three confirmed h n case shared . ~ . % homology with the animal isolates (a/chicken/zhejiang/sd / ), and phylogenetic analysis showed that the four isolates were almost genetically identical to other h n virus isolated from the other provinces and chickens. furthermore, amino acid analyses showed that the ha gene of all four strains possessed the mutation l, indicating high affinity to human receptor alpha - sialic acid receptors. it showed that the four isolates were entirely of human origin, and na protein possessed amino acid sequences associated with susceptibility to neuraminidase inhibitor antiviral drugs (h and e and h in na). the fragments isolated from the index case, and two secondary cases were identical except for three non-synonymous amino substitutions identified in the index case. these were g a nucleic acid substitution (aa mutation v i) in the ns gene, g a nucleic acid substitution in the pb gene (nonsense amino mutation) and c t nucleic acid substitution (v a) in the np gene. (figure , additional file : figure s , table and table ). the index case, his daughter (case ), his wife (case ), and his son-in-law (the husband of case ) lived in separate rooms of one large house with three floors. there were no domestic animals and birds within the home or in the immediate vicinity of the home. however, two neighboring families located meters and meters respectively from the cases' homebred ducks and chickens, and there were a several free-range domestic poultry in the village. the index case and case worked in the lbm (a market), selling vegetables and bird eggs between am and pm during the two weeks prior to the illness onset in the index case. furthermore, two weeks before the illness onset, the index case had visited a wholesale lbm (d market) to buy vegetables twice per week (each time he stayed there for hours). the last known exposure date of the index case in a market was january ( hours), and the last exposure date of case to a market was on january for around three hours. in total. case had been exposed to the live bird market on three occasions for a total of hours from january - as follows: case visited d market for a total of three hours between january - and she worked in a market for , and, hours on january , , and , respectively. the index case became ill on january and was admitted to hospital on january . between january and , cases and lived together with the index case in one house. furthermore, case and index case had very close contact between january and , sleeping together in one room. case had three hours faceto-face contact with the index case on january . on january , case provided bedside care in the hospital for the index case for approximately hours. between january and , cases and provided bedside care to the index case in hospital without any personal protective equipment for approximately hours and hours, respectively, including washing, cleaning his body, change his clothes and disposing urine and feces of the index case. during this period, the index case had high fevers ( . °c), frequent coughing, and extensive sputum production. after the index case had been confirmed h n infection on january , he moved to icu for treatment and isolation. cases and visited the index case for four hours on january , wearing facemasks. case had frequent close contact with case during the funeral ceremony of the index case on january - . case visited case on the january when case was hospitalized with mild symptoms; case wore a facemask during this visit. a summary of the cases' exposure to each other was shown in table . the results of rt-pcr assay of environment samples were listed as follows: swabs of chicken and duck eggs from the index case working site (a market) were h n negative; of environmental samples from secondary live wet market (b market, a wholesale for a market) were positive for h n ; of sewage samples from c market (located nearby b market) were positive for h n ; of environmental samples taken from d market through routine avian influenza surveillance were h n positive; environmental samples from the area where neighbors were breeding poultry were all h n negative; of environmental samples from different live birds markets under routine surveillance in xiaoshan district were h n positive during january (source: unpublished data from the zhejiang avian surveillance system, additional file : figure s ). none of the close contacts developed acute respiratory symptoms during the seven days surveillance period. throat swabs collected from all twenty-five close contacts on january were negative for influenza a/ h n virus by rrt-pcr, and all serum samples tested negative for h n antibodies (titer < : ) by microneutralization and horse red-blood-cell hi assays (see table ). no close contacts were reported taking oseltamivir chemoprophylaxis. here we describe a family cluster of three confirmed cases of h n virus infection, involving a fatal index case, his wife and daughter (both survived). the index case presented with severe pneumonia and died of ards and multi-organ failure. the presence of chronic diseases has been associated with an increased risk of hospitalization with h n virus infection [ ] , and the index case had pre-existing diabetes, which requires oral anti-diabetic medication. another factor that may have played a role in the severity of disease was the late diagnosis of h n virus infection and the late commencement of anti-viral therapy. the efficacy of neuraminidase inhibitors (nais) in reducing the risk of mild influenza infection progressed to severe illness has not been fully assessed in randomized controlled trials; however, observational data suggest that early treatment with nais of hospitalized patients with influenza infection is associated with better outcomes [ ] . the other two cases i v i i i v i i i i i t k t t t t t t t t t single letters refer to the amino acid (aa) found in the noted protein at a specific site. *the numbering starts with the first condon of methionine for these proteins. were previously reported healthy, and presented with lower viral loads and mild symptoms that did not progress. both patients received early antiviral treatment, but it is not possible to determine whether the lack of clinical progression was result from antiviral treatment or as a consequence of a naturally indolent course [ ] [ ] [ ] . since there were no functionally important differences in the genotype of the virus infecting the three cases, viral virulence is not likely to contribute the differential severity. who evaluates all clusters of human cases of nonseasonal influenza virus to determine whether humanto-human transmission or common exposure to infected animals or contaminated environments may have occurred [ ] . the homology of all eight gene segments was between . ~ %, suggesting it was either a common source exposure or a person-to-person transmission. whilst all three individuals were exposed to potentially contaminated market environments within a putative maximum incubation period of days, case and case had extensive unprotected exposure to the index cases when he was ill. we believe that most likely explanation for this family cluster is that the index case was infected from the live bird market, and the virus was transmitted directly from the index case to his daughter and his wife. several reasons could explain for this conclusion, as follows: ( ) days prior to illness onset in the index case, he had not been in contact with any people with a febrile illness and other confirmed cases, but was frequently exposed to the a live bird market for hours daily and to the d secondary live bird market. although the a market was h n negative based on the environments samples collected on january , , the samples from wholesale market b that supplied a market were h n positive. furthermore, . % ( / ) of environments samples from different live bird markets under routine surveillance in xiaoshan district during the same period were h n positive (source: unpublished data from the zhejiang avian surveillance system); ( ) case stayed with the index case and provided beside bed medical care frequently on the january , , and - . she had close unprotected contact with the index case for cleaning and washing his body on january without any personal protection when the index case had severe symptoms such as high fever and cough. although case had visited the a live bird market for three hours in three days prior to her illness onset, she reported no direct contact with live birds or poultry products. ( ) there were multiple potential sources of infection for case , including the index case, the live market a , and case . however, the index case and case shared the same room every day and worked closely together after the illness onset in the index case. most importantly, case provided beside bed care to the index case including washing his body, dealing with his secretions, and changing his clothes for him, without any personal protective equipment. the day numbers between the onset of illness in the index case and the onset of illness in the secondary cases (the serial interval) was and days, [ , ] . furthermore, sequence analysis showed that four strains isolated from the three cases were genetically similar to each other. all four isolates possessing amino acids q l and g s in the ha segment were associated with increased affinity for human receptors (α- , linked sialo-saccharides) [ ] . virus from all three cases possessed p s in ns and e k and d n in pb (which were associated with increased virulence in mice) and i v and h y in pb (which was associated with aerosol transmission of avian virus between ferrets) [ , ] . there were only two amino differences (v i in ns and v a in np) between the virus infecting the index case and the secondary cases. those two mutations are not associated with any known functional change. therefore, field investigation and h n full genomics analyses supported the secondary cases acquired infection most likely from the index case. person-toperson transmission of h n has been reported [ , ] . previous animal experiments (ferrets, mice, and pigs) also indicate that h n virus possess the capability to bind to both avian and human receptors and it might be transmissible by respiratory droplets under certain conditions [ , ] . our findings indicate that the virus has not gained the ability for efficient sustained transmission from person to person [ ] . in this study, four close contacts and frequent contacts were negative for h n infection by hi testing and rt-pcr. although the husband of case had close contact with the index case, case , and case without any personal protective equipment, he showed no evidence of infection with the h n virus. there were several limitations in this paper. firstly, h n positive samples in environmental or bird samples were not found from a live bird market where the index case and case were working. secondly, the full genetic sequence of h n virus detected in the environment and live birds could not obtained. thus it is not able to compare human, avian, and environmental strains. on the basis of experiences of controlling of h n and h n virus, continued risk assessment, surveillance, and vigilance are required. a high degree of clinical awareness is necessary for people with possibility of h n infection, especially for health workers who are occupationally exposed to poultry and for people with respiratory illness following recent contact with live poultry or live bird markets [ , ] . here we report a largest size of the family cluster with confirmed h n in china, in which the index case was fatal while the secondary cases were mild. in the term of an infectious source, the index case was infected from a live bird market while the index case infected the two secondary cases during unprotected frequent exposure. this family cluster supported that the transmission of avian influenza a/h n was limited and not sustained. all of h n referred isolates in additional materinals were download from genbank (http://www.ncbi.nlm.nih. gov/nuccore/?term=h n ++and+china) and the global initiative on sharing all influenza data (gisaid) (http://platform.gisaid.org/epi /frontend# dda ). additional file : figure s . family pedigree showing three h n affected individuals and their close contacts. figure s . phylogenetic analysis of six segments (mp, np, ns, pa, pb , and pb ) from the four h n isolates in three confirmed cases of a family cluster in hangzhou, zhejiang province, china, in january of . figure s . all authors have declared: no support from any organization for the submitted work; no financial relationships with any organizations that might have an interest in the submitted work in the previous three years; no other relationships or activities that could appear to have influenced the submitted work. authors' contributions hd, yc, zy, pwh, ec, and hy designed the study. fw, jh, and xy conducted the field investigation and analyses. hm, sq and cc collected and tested the samples, performed and sequence analyses. sl and pwh wrote the first draft and all authors contributed to review and revision of the report. ec and hy are guarantors. all authors read and approved the final manuscript. human infections with avian influenza a(h n ) virus h n : preparing for the unexpected in influenza writing committee of the second world health organization consultation on clinical aspects of human infection with avian influenza av pandemic characteristics and controlling experiences of influenza h n virus year after the inception in hangzhou amino acid substitutions in polymerase basic protein gene contribute to the pathogenicity of the novel a/h n influenza virus in mammalian hosts dynamic reassortments and genetic heterogeneity of the human-infecting influenza a (h n ) virus human infection with avian influenza a(h n ) virus re-emerges in china in winter live-animal markets and influenza a (h n ) virus infection pathogenesis and transmission of avian influenza a (h n ) virus in ferrets and mice limited airborne transmission of h n influenza a virus between ferrets novel avian-origin human influenza a(h n ) can be transmitted between ferrets via respiratory droplets the k r substitution in viral protein pb enhances the effects of e k on influenza virus replication environmental connections of novel avian-origin h n influenza virus infection and virus adaptation to the human epidemiology of human infections with avian influenza a(h n ) virus in china family outbreak of severe pneumonia induced by h n infection surveillance of the first case of human avian influenza a (h n ) virus in beijing probable person to person transmission of novel avian influenza a (h n ) virus in eastern china, : epidemiological investigation one family cluster of avian influenza a(h n ) virus infection in characterization of h n influenza a viruses isolated from humans infectivity, transmission, and pathology of human-isolated h n influenza virus in ferrets and pigs human infection with a novel avian-origin influenza a (h n ) virus comparison of patients hospitalized with influenza a subtypes h n , h n , and pandemic h n determinants of antiviral effectiveness in influenza virus a subtype h n risk assessment on the epidemics of human infection with a novel avian influenza a (h n ) virus in jiangsu province global concerns regarding novel influenza a (h n ) virus infections analysis of the clinical characteristics and treatment of two patients with avian influenza virus (h n ) comparative epidemiology of human infections with avian influenza a h n and h n viruses in china: a population-based study of laboratory-confirmed cases probable longer incubation period for human infection with avian influenza a(h n ) virus in jiangsu province receptor binding by an h n influenza virus from humans probable person-to-person transmission of avian influenza a (h n ) three indonesian clusters of h n virus infection in genomic signature and protein sequence analysis of a novel influenza a (h n ) virus that causes an outbreak in humans in china human infection with avian influenza a h n virus: an assessment of clinical severity we thank all of staff at zhejiang provincial and hangzhou municipal cdc, xiaoshan district cdc, for their help in field investigation and collection of environmental samples. the views expressed are those of the authors and do not necessarily represent the policy of the china cdc. note: data are median (iqr) or n (%). *including direct contact (touching), preparation, cooking, and consumption of well-appearing poultry. key: cord- -pbahviaz authors: garg, shikha; jain, seema; dawood, fatimah s.; jhung, michael; pérez, alejandro; d’mello, tiffany; reingold, arthur; gershman, ken; meek, james; arnold, kathryn e.; farley, monica m.; ryan, patricia; lynfield, ruth; morin, craig; baumbach, joan; hancock, emily b.; zansky, shelley; bennett, nancy; thomas, ann; schaffner, william; finelli, lyn title: pneumonia among adults hospitalized with laboratory-confirmed seasonal influenza virus infection—united states, – date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: pbahviaz background: influenza and pneumonia combined are the leading causes of death due to infectious diseases in the united states. we describe factors associated with pneumonia among adults hospitalized with influenza. methods: through the emerging infections program, we identified adults ≥ years, who were hospitalized with laboratory-confirmed influenza during october through april , and had a chest radiograph (cxr) performed. pneumonia was defined as the presence of a cxr infiltrate and either an icd- -cm code or discharge summary diagnosis of pneumonia. results: among , adults hospitalized with influenza, ( %) had pneumonia. in multivariable analysis, factors associated with pneumonia included: age ≥ years, adjusted odds ratio (aor) . ( % confidence interval . – . ), white race aor . ( . – . ), nursing home residence aor . ( . – . ), chronic lung disease aor . ( . – . ), immunosuppression aor . ( . – . ), and asthma aor . ( . – . ). patients with pneumonia were significantly more likely to require intensive care unit (icu) admission ( % vs. %), mechanical ventilation ( % vs. %), and to die ( % vs. %). conclusions: pneumonia was present in nearly one-third of adults hospitalized with influenza and was associated with icu admission and death. among patients hospitalized with influenza, older patients and those with certain underlying conditions are more likely to have pneumonia. pneumonia is common among adults hospitalized with influenza and should be evaluated and treated promptly. electronic supplementary material: the online version of this article (doi: . /s - - -y) contains supplementary material, which is available to authorized users. influenza illness is generally characterized by acute onset of fever, mylagias, and respiratory symptoms, and while disease usually resolves without complications in healthy indiviudals, influenza is associated with an annual increase in hospital admissions for pulmonary, cardiovascular and neuromuscular compliations [ ] [ ] [ ] . the etiology of influenza-associated pneumonia may include primary influenza pneumonia, secondary bacterial pneumonia, or concomitant viral and bacterial pneumonia [ , , ] . pulmonary complications of influenza, including pneumonia and exacerbations of chronic pulmonary disease, are common and result in significant morbidity and mortality. oliveira and colleagues found that among all patients admitted to a large metropolitan hospital with influenza during the - season, % had pneumonia [ ] . further, in a study conducted over influenza seasons ( ) ( ) ( ) ( ) ( ) , murata and colleagues found that among patients hospitalized with influenza a, % had some type of acute findings on chest radiograph and % had definitive pneumonic infiltrates [ ] . although there is evidence that adult patients with underlying cardiac or pulmonary disease are more likely to develop influenza-associated pneumonia than those without underlying medical conditions [ , ] , much of the data describing factors associated with influenzaassociated pneumonia among adults comes from case series conducted at single sites and during a limited number of seasons. using data from a large multi-center, geographically diverse, population-based surveillance system, we describe factors associated with pneumonia among adults hospitalized with influenza over three consecutive years in which seasonal influenza viruses circulated. the emerging infections program (eip) network conducts active population-based surveillance for laboratory-confirmed influenza-associated hospitalizations. the network began adult surveillance in and covers over counties in states (california, colorado, connecticut, georgia, maryland, minnesota, new mexico, new york, oregon, and tennessee), representing approximately % of the adult u.s. population [ ] . patients were included in eip influenza surveillance if they resided and were hospitalized in an eip catchment area and were hospitalized within days of a positive influenza diagnostic test result. patients were excluded if the first positive influenza specimen was obtained > days after hospital admission because these patients might have had healthcare-associated influenza infection. influenza testing was performed at the discretion of health care providers. medical charts of hospitalized patients with laboratory-confirmed influenza were retrospectively reviewed [ , ] . the study period comprised influenza seasons, - to - . patients were included in this analysis if they were ≥ years of age, were hospitalized with laboratory-confirmed influenza during the - through - influenza seasons, and had a chest radiograph (cxr) performed during hospitalization. the following data were collected on patients: demographics, results of laboratory tests for influenza, influenza vaccination status for the current season, underlying medical conditions, bacterial coinfections, cxr data, antiviral treatment, clinical outcomes, and discharge diagnoses. laboratory confirmation of influenza was based on viral culture, direct or indirect immunoflourescence antibody staining, reverse-transcription polymerase chain reaction, or a rapid antigen test. surveillance staff completed medical record abstractions using check boxes to indicate whether or not a new infiltrate or consolidation was recorded on the official cxr transcript. discharge diagnoses were captured in two ways: ) the first nine international classification of diseases (icd- -cm) codes for each case were abstracted from the medical record; ) check boxes were marked for certain diagnoses, including pneumonia, if they were recorded by clinicians on the discharge summary. pneumonia was defined as the presence of a new infiltrate on cxr and either an icd- -cm discharge diagnosis code for pneumonia ( - . ) or a diagnosis of pneumonia recorded on discharge summary. information on the presence of selected bacterial infections was available only for patients who had a positive culture. a bacterial infection was recorded if bacteria other than those that are commonly considered to be contaminants grew from a sterile body site or a non-sterile respiratory site culture obtained within calendar days of hospital admission. sterile body sites for bacterial infections included blood, pleural fluid, cerebrospinal fluid, bronchoalveolar lavage fluid, and deep tissue biopsy. non-sterile respiratory sites included sputum and endotracheal aspirates. use of influenza antiviral therapy was examined for all individuals. among those who were treated with antiviral agents, timing of treatment was assessed in relation to hospitalization date. early antiviral treatment was defined as initiation of antiviral treatment within days of hospital admission. we used bivariate analysis to compare adults hospitalized with influenza with and without pneumonia. we used χ and fisher exact tests for categorical variables and t-tests and wilcoxon-rank sum tests for continuous and ordinal variables. all variables significant in bivariate analysis, as well as biologically plausible variables, and potential confounders were included in a multivariable logistic regression model to identify factors independently associated with influenza-associated pneumonia. we used the breslow-day test for homogeneity to assess for effect modification of select variables. all tests were two-tailed and a p-value of . was considered significant. analyses were conducted using sas version . (sas institute inc., cary, nc). ethics statement eip adult influenza hospitalization surveillance activities during the - influenza seasons were determined by the centers for disease control and prevention (cdc) institutional review board (irb) not to involve research in accordance with the federal regulations for the protection of human subjects in research. starting with the - season, research questions were added to evaluate factors associated with severe outcomes during hospitalizations, and irb review was conducted at all surveillance sites and the cdc. the protocol was approved by the cdc irb and was either approved or received exempt status by all surveillance site irbs. because all surveillance data was analyzed anonymously, neither verbal nor written informed consent was obtained from participants. during the study period, of adults hospitalized with laboratory-confirmed influenza, ( . %) had an available cxr report and discharge diagnosis information and were therefore included in our study. of the adults, ( %) had pneumonia. the prevalence of pneumonia did not vary significantly over the influenza seasons included in the analysis. adults ≥ years of age represented the age group with the highest proportion of patients hospitalized with and without influenza-associated pneumonia (fig. ) . the median age of patients with pneumonia compared with patients without pneumonia was years versus years (p < . ) ( table ). the majority of patients hospitalized with and without influenza-associated pneumonia were white. white patients were older (median age years) than black patients ( years), hispanic patients ( years), and patients of other races including asian, pacific islander, american indian, alaskan native, and multi-race ( years) (p < . ). patients aged years and above had a higher proportion of underlying conditions ( %) compared to patients aged < years ( %) (p < . ). influenza was diagnosed by rapid test only in / ( %) patients with pneumonia and in / ( %) patients without pneumonia (p < . ). the median number of days from symptom onset to hospital admission was days for patients with and without pneumonia (table ) . patients with pneumonia were significantly more likely than patients without pneumonia to reside in a nursing home prior to hospital admission, to have received influenza vaccine, and to have the following underlying medical conditions: chronic lung disease, cardiovascular disease, and immunosuppression. patients with pneumonia were significantly less likely than patients without pneumonia to have asthma (table ) . a description of the most frequent discharge diagnoses (based on first listed icd- diagnosis code) among patients with and without pneumonia can be found in additional file : table s . except for influenza vaccination and cardiovascular disease, all factors included in a multivariable model remained independently associated with pneumonia including age ≥ years [adjusted odds ration (aor) . ], white race (aor . ), nursing home residence (aor . ) chronic lung disease (aor . ), immunosuppression (aor . ) and asthma (aor . ) ( table ) . sixty-one patients with pneumonia and patients without pneumonia had sterile site bacterial infections, % of which were cultured from the blood ( table ). the most common pathogens cultured from sterile sites in patients with pneumonia were staphylococcous aureus (s. aureus) and streptococcus pneumonia (s. pneumonia). patients with pneumonia had a longer median length of hospital stay than patients without pneumonia ( days versus days; p < . ). patients with pneumonia were also significantly more likely to have a hospital length of stay greater than one week (aor . ), require intensive care unit (icu) admission (aor . ), require mechanical ventilation (aor . ), and die (aor . ) ( table ) . among patients with pneumonia, factors independently associated with a poor outcome, defined as icu admission, need for mechanical ventilation or death, included nursing home residence (aor . ), chronic lung disease (aor . ), cardiovascular disease (aor . ), (table ). of note, older age was inversely associated with a poor outcome (aor . ) among patients hospitalized with pneumonia (table ) . patients with pneumonia [ / ( %)] were significantly more likely to receive influenza antiviral therapy than patients without pneumonia [ / ( %); p < . ]. through this large, population-based surveillance system, we found that pneumonia was present in almost one-third of u.s. adults hospitalized with laboratoryconfirmed influenza over three consecutive years in which seasonal influenza viruses circulated. patients with pneumonia were older and were more likely to have certain underlying medical conditions than patients without pneumonia. patients with pneumonia were also more likely to have a prolonged hospital stay, be admitted to an icu, require mechanical ventilation for respiratory failure, and die. while patients with pneumonia were more likely to receive antiviral therapy than those without pneumonia, treatment was more often delayed among patients with pneumonia. similar to findings from smaller inter-pandemic studies [ , ] pneumonia was common among adults hospitalized with influenza in this study. among those hospitalized with influenza, older adults and nursing home residents were at significantly increased risk for having influenza-associated pneumonia. respiratory viruses including influenza are a common etiology of pneumonia in older adults, and several factors may contribute to the development of severe lower respiratory tract disease in these individuals, including decreased respiratory muscle strength and lung compliance, and waning humoral and cell-mediated immunity [ ] [ ] [ ] . additional risk factors for lower respiratory tract disease among older nursing home residents include immobility and swallowing difficulties leading to aspiration [ ] . within closed settings such as nursing homes, large outbreaks of influenza and its subsequent complications, including severe pneumonia, may rapidly evolve and lead to significant morbidity and mortality [ , ] . influenza virus infection should thus be considered a potential cause of pneumonia in older individuals and nursing home residents during fall and winter months ( ) other streptococci c ( ) ( ) other pathogens d ( ) ( ) unknown pathogens ( ) ( ) [ ] and should be diagnosed and treated promptly. influenza vaccination is the most effective method to prevent influenza and its complications, and older adults, residents of nursing homes and other long-term-care facilities, and adults with underlying medical conditions should be considered high priority groups for receipt of annual influenza vaccination [ ] . similar to earlier studies conducted during periods of seasonal influenza virus circulation, patients with pneumonia in this study were more likely to have underlying medical conditions including chronic lung disease and heart disease [ , ] . an unexpected finding was that patients with asthma in our analysis were less likely to have a diagnosis of pneumonia than patients without pneumonia. our study results contrast with eip surveillance data in hospitalized children < years of age which has shown that children with influenza-associated pneumonia were more likely to have asthma than those without pneumonia [ ] . studies of the association between asthma and seasonal influenza-associated pneumonia among adults are lacking. a possible explanation for our finding is that respiratory distress caused by influenza-associated asthma exacerbation provided an alternate reason for hospitalization in adult patients in the absence of pneumonia. biases in hospital admission practices based on the presence of underlying conditions may have also contributed to admission of asthmatic patients with a less severe respiratory presentation compared to patients without underlying medical conditions. invasive bacterial infections, especially due to s. aureus and s. pneumoniae, were observed among patients with influenza-associated pneumonia in this study as well as other studies conducted during inter-pandemic [ ] and pandemic periods [ ] . among patients with pneumonia, s. aureus was the most common organism cultured from specimens collected from sterile sites. influenza virus and s. aureus co-infections are increasing [ ] [ ] [ ] and have been associated with particularly severe cases of community-acquired pneumonia during periods of seasonal influenza virus circulation [ ] . in patients hospitalized with influenza, sterile site cultures should be collected as early as possible for detection of bacterial infection and empiric antimicrobial coverage of the most likely bacterial organisms should be considered [ , ] . in our study, s. pneumoniae was the only organism to be cultured from a sterile site more frequently in patients with pneumonia that in patients without pneumonia. in addition to annual influenza vaccination, pneumococcal vaccine should be administered to adults aged - years with certain health conditions and to all persons aged ≥ years [ ] . patients with influenza-associated pneumonia had a significantly increased risk of icu admission, respiratory failure requiring mechanical ventilation, and death compared with patients without pneumonia. while case series conducted during the h n pandemic demonstrated elevated frequencies of icu admission ( - %) [ , ] , respiratory failure ( - %) [ , ] and death ( - %) [ ] [ ] [ ] [ ] among patients hospitalized with pandemic h n influenza-associated pneumonia, limited data is available on the association between seasonal influenzaassociated pneumonia and severe outcomes. in a small case series of patients hospitalized with influenza during the - season, ( %) of patients with pneumonia were admitted to the icu and ( %) patients died [ ] . in another observational study of patients hospitalized with influenza during - , ( %) of patients with acute pulmonary disease were admitted to the icu, ( %) required mechanical ventilation, and ( %) died [ ] . while pneumonia and acute respiratory distress syndrome (ards) have been shown to account for a majority of deaths associated with influenza virus infection during pandemics [ ] , data is limited on the association between seasonal influenza virus infection and death from pneumonia or ards. in our analysis, only % of patients hospitalized with laboratory-confirmed influenza received influenza antiviral treatment. when limiting the analysis to patients who presented to the hospital within days of symptom onset, only % of all patients received antiviral treatment; the majority received antiviral treatment within day of hospital admission. multiple studies have found early antiviral treatment to be associated with a reduction in serious influenza-associated outcomes including the development of lower respiratory tract infections [ ] [ ] [ ] . the advisory committee on immunization practices recommends empiric influenza antiviral treatment for all adults with suspected or confirmed influenza who are hospitalized, have severe, complicated, or progressive illness, or are at high risk for influenza-associated complications [ ] . several limitations to this study should be noted. influenza diagnostic testing was performed at the discretion of treating clinicians at the various eip hospital sites. while all hospitalized patients who tested positive for influenza were included in surveillance, data is unavailable for hospitalized patients who tested negative for influenza or who were not tested. thus, these data may not be representative of all individuals hospitalized with influenza who may not have been tested or have laboratory confirmation of influenza virus infection. it is possible that patients included in surveillance were more likely to be tested for influenza because they were more severely ill; thus a higher proportion of patients exhibiting pneumonia-like symptoms may have been tested for influenza than patients presenting with other symptoms. furthermore, in our analysis, patients with pneumonia were compared to patients without pneumonia but with a wide array of other diagnoses. clinican influenza testing practices based on patient diagnoses at presentation may have biased our findings. in one study conducted in an emergency department in australia, patients presenting with fever and respiratory diagnoses were more likely to be tested for influenza than patients presenting with cardiac or other diagnoses [ ] . this study assessed pneumonia specifically among adults hospitalized with laboratory-confirmed influenza, including those whose influenza virus infection preceded hospitalization by more than a few days, and findings are not generalizable to all hospitalized individuals with pneumonia of other etiologies or to non-hospitalized individuals. several of the findings in this study may have been biased by hospital admission practices. for example, the finding of an inverse association between asthma and pneumonia may have been due to more aggressive admission of asthmatic patients presenting with respiratory distress despite the absence of pneumonia, compared with patients without asthma. biases related to hospital admission practices were likely reduced by including patients from multiple hospital sites in geographically diverse settings. for certain underlying conditions such as chronic lung disease and cardiovascular disease, disease type and severity were not captured by the case report form. availability of detailed data on type and severity of underlying conditions may have helped to better identify factors more strongly associated with development of influenza-associated pneumonia. radiographic data were based on review of cxr reports by surveillance officers and not by actual review of radiographs by a designated study radiologist. as a result, some individuals may have been misclassified as having pneumonia based upon a report of infiltrates or opacities, when in fact they had a more chronic pulmonary condition or a transient episode of pulmonary edema or effusion. there was no requirement regarding timing of identification of radiologic abnormalities during the hospitalization, and the timing of chest radiographs during the hospitalization was not collected as part of eip surveillance; thus, some misclassification of communityacquired pneumonia versus nosocomial pneumonia may have occurred. using icd- -cm code data may also have led to misclassification if a diagnosis code was listed incorrectly or not listed at all. a joint case definition for pneumonia which used both radiographic data and discharge diagnosis data from icd- -cm codes or discharge summaries was utilized to minimize some of these biases. bacterial culture data was only available for patients with a positive culture result rather than for all specimens spent, thus limiting the interpretation of the culture data. pneumonia is common among adults hospitalized with seasonal influenza virus infection. among patients hospitalized with influenza, older adults and those with underlying medical conditions may be more likely to have pneumonia. further studies are needed to explore the association between influenza-associated pneumonia and asthma in adults. influenza-associated pneumonia can lead to severe outcomes including icu admission and death. adults hospitalized with suspected or confirmed influenza should receive early antiviral therapy, prompt evaluation for pneumonia, and appropriate management upon diagnosis of pneumonia. additional file : table s . the most frequent icd- diagnosis categories based on first icd- code listed among adults hospitalized with laboratory-confirmed influenza with and without pneumonia (n= ). abbreviations cxr: chest radiograph; aor: adjusted odds ratio; eip: emerging infections program; icd- -cm: international classification of diseases; cdc: centers for disease control and prevention; irb: institutional review board; icu: intensive care unit; s. aureus: staphylococcus aureus; s. pneumonia: streptococcus pneumonia. the authors declare that they have no competing interests. authors' contributions sg: contributed to conception and design of study, analysis of data and interpretation of results, and drafting of the manuscript; sj: contributed to conception and design of study, interpretation of data, and criticial review and revision of manuscript; fd: contributed to conception and design of study, analysis of data, and critical review of manuscript; mj: contributed to conception and design of study, interpretation of data, and criticial review of the manuscript; ap: contributed to analysis and cleaning of data, interpretation of results, and critical review of the manuscript; td: contributed to analysis and cleaning of data, interpretation of results, and critical review of the manuscript; ar: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; kg: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; jm: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; ke: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; mf: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; pr: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; rl: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; cm: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; jb: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; eh: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; sz: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; nb: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; at: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; ws: contributed to conception and design of study, acquisition of data, interpretation of results, and critical review of the manuscript; lf: contributed to conception and design of study, interpretation of data, and criticial review and revision of manuscript; all authors 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-recommendations of the advisory committee on immunization practices (acip) van hal sj. influenza a testing and detection in patients admitted through emergency departments in sydney during winter ; implications for rational testing we wish to thank the following individuals for their help with surveillance efforts: deborah submit your next manuscript to biomed central and take full advantage of: key: cord- -bd zswo authors: lin, sheng; pan, hao; wu, huanyu; yu, xiao; cui, peng; han, ruobing; jiang, chenyan; kong, dechuan; zheng, yaxu; gong, xiaohuan; xiao, wenjia; mao, shenghua; jin, bihong; zhu, yiyi; sun, xiaodong title: epidemiological and clinical characteristics of discharged cases with coronavirus disease in shanghai, china date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: bd zswo background: in december , the outbreak of coronavirus disease (covid- ) began in wuhan, china, and rapidly spread to other regions. we aimed to further describe the epidemiological and clinical characteristics of discharged covid- cases and evaluate the public health interventions. methods: we collected epidemiological and clinical data of all discharged covid- cases as of february in shanghai. the key epidemiological distributions were estimated and outcomes were also compared between patients whose illness were before january and those whose illness were after january. results: of discharged covid- cases, the median age was years, and ( . %) cases were male. all of the cases were categorized as clinical moderate type. the most common initial symptoms were fever ( . %), cough ( . %), fatigue ( . %), muscle ache ( . %), sputum production ( . %), and there were six asymptomatic cases. ( . %) cases got infected in shanghai, and three of them were second-generation cases of shanghai native cases. the estimated median of the time from onset to first medical visit, admission, disease confirmation, and discharge for cases was . day ( % ci, . – . ), . days ( % ci, . – . ), . days ( % ci, . – . ), . days ( % ci, . – . ), respectively. the estimated median of the time from admission to discharge was . days ( % ci, . – . ). the time from onset to first medical visit, admission and disease confirmation were all shortened after the shanghai’s first-level public health emergency response. in cox regression model, the significant independent covariates for the duration of hospitalization were age, the time from onset to admission and the first-level public health emergency response. conclusions: local transmission had occurred in shanghai in late january . the estimated median of the time from onset to discharge of moderate covid- was . days in shanghai. time intervals from onset to first medical visit, admission and disease confirmation were all shortened after the shanghai’s first-level public health emergency response. age, the first-level public health emergency response and the time from onset to admission were the impact factors for the duration of hospitalization. coronavirus disease (covid- ) is an infectious disease caused by novel coronavirus ( -ncov). the most common signs of infection include fever, respiratory symptoms (such as cough and sputum production) and fatigue [ ] . the first covid- case was identified in wuhan, china in late december [ ] . the covid- has rapidly spread from wuhan to other areas [ , ] . as of february , a total of , covid- cases in china have been confirmed and cases have been reported in countries and continents internationally [ ] . to curb the spread of covid- , the shanghai authorities have declared the first-level public health emergency response on january [ ] . the measures included: travelers from wuhan and other epidemic areas were advised to report their travel records and to conduct selfquarantine for weeks to prevent community transmission; comprehensive implementation of sanitary quarantine at the entrance of shanghai; cancellation various large public events; masks were recommended to be worn in public places; strengthened publicity of health knowledge, etc. public health interventions played an important role in controlling the epidemic. as of february, there were a total of confirmed covid- cases in shanghai and of them had been cured to discharge [ ] . epidemiological and clinical characteristics of confirmed covid- cases in shanghai have been reported [ ] . however, at present, the impact of first-level public health emergency response on the epidemic of covid- was not estimated and information regarding the epidemiology and clinical features of discharged covid- cases is scarce [ ] [ ] [ ] [ ] . therefore, we provided an analysis of key epidemiological determinants and clinical characteristics of discharged covid- cases in shanghai. moreover, we described and estimated the time interval from onset to discharge, which might helpful to understanding the progression of the disease. we performed a comprehensive study of all the discharge covid- cases reported in shanghai in the case reporting system as of february . all cases were tested covid- positive in laboratory and diagnosed by clinical experts according to covid- prevention and control program ( nd ed.) [ ] . the symptom severity of covid- was classified into moderate, severe and critical. moderate cases refer to those cases who have symptoms such as fever and respiratory tract symptoms, etc. and pneumonia manifestations can be seen in imaging. severe cases refer to any of the following criteria: (i) respiratory rate ≥ breaths/ min, (ii) oxygen saturation ≤ % at a rest state, (iii) arterial partial pressure of oxygen (pao )/oxygen concentration (fio ) ≤ mmhg. critical cases refer to those cases that meeting any of the following criteria: (i) occurrence of respiratory failure requiring mechanical ventilation, (ii) presence of shock, (iii) other organ failure that requires monitoring and treatment in the icu. the criteria of discharge included: (i) body temperature returned to normal (< . °c) for more than days, (ii) respiratory symptoms improved significantly, (iii) rrt-pcr of -ncov was negative for two consecutive times (sampling interval at least day). after cases were reported to shanghai municipal centers for disease control and prevention (cdc), epidemiological investigations were conducted within h. demographic data, clinical symptoms or signs, laboratory tests during hospital admission, comorbidities, exposure history in days and prevention and control measures were all collected. when cases were discharged, clinical records of discharge were also collected. the specific information in epidemiological investigation was entered into a computerized database of epidata software (epidata association) in duplicate. the data were analyzed anonymously. the -ncov laboratory test assays were based on the technical guidelines for laboratory testing of novel coronavirus pneumonia [ ] . upper or lower respiratory specimens of suspected covid- cases were collected and tested for -ncov by real-time reverse-transcriptase polymerase chain reaction (rrt-pcr) assay. tests were carried out in biosafety level two facilities at district cdcs or municipal cdc. the case was considered as laboratory tested positive only when two targets, open reading frame a or b (orf ab) and nucleocapsid protein (n), were both positive. orf ab: forward primer ccctgtgggttttacacttaa; reverse primer acgattgtgcatcagctga; probe ′-vic-ccgtctgcggtatgtggaaagg ttatgg-bhq - ′. forward primer ggggaacttctcctgctagaat; reverse primer cagacattttgctctcaagctg; probe ′-fam-ttgctgctgcttgacagatt-tamra- ′. the shanghai authorities have activated the first-level public health emergency response to curb the spread of covid- on january. so cases were divided into two groups (illness onset during jan to jan, illness onset during jan to feb). estimated median intervals of onset to first medical visit, onset to admission, onset to disease confirmation, admission to discharge, and onset to discharge were obtained for the two groups, assuming that the times were γ distributed. we present continuous variables as medians (interquartile ranges, iqr) and compared using wilcoxon rank-sum tests between different groups. categorical variables were described as counts and percentages in each category, and compared using chi-square or fisher's exact tests between different groups. time-delay distributions (onset to first medical visit, onset to admission, onset to disease confirmation, admission to discharge, and onset to discharge) were fitted to γ distributions by maximum likelihood estimation methods. cox regression model was used to identify factors significantly associated with the duration of covid- hospitalization. these factors included: age, gender, hightest temperature, place of infection, smoking, drinking, body mass index, white blood cell count, neutrophil count, lymphocyte count, comorbidies, time from onset to admission and first-level public health emergency response. analyses of the time-delay distributions were performed with r software (r foundation for statistical computing). other analyses were performed with spss (statistical package for the social sciences) version . software (spss inc). as of february , confirmed covid- cases had been discharged in shanghai. the first cured case was discharged on january ( fig. ). the median age was years (iqr, - ; range, - ), and four ( . %) were younger than years. ( . %) cases were male. ( . %) cases got infected in shanghai, and three of them were second-generation cases of shanghai native cases (table ) . on admission, were all categorized as moderate severity. the most common reported initial symptoms at illness onset were fever ( table ) . ( . %) cases reported fever plus any one other symptom, and ( . %) cases reported fever plus two other symptoms. ( . %) cases had one or more basic diseases, ( . %) cases had hypertension, ( . %) cases had cardiovascular diseases, ( . %) cases had diabetes. of cases, the median white blood cell counts was . × /l (iqr, . - . ), the median neutrophil cell counts was . × /l (iqr, . - . ), the median lymphocyte cell counts was . × /l (iqr, . - . ). the time from onset to discharge for cases ranged from to days. the estimated median of the time from onset to discharge was . days ( % ci, . - . ) ( table ). the estimated median of the time from onset to discharge for cases who had onset symptoms before january was . days ( % ci, . - . ), which was significantly longer than cases with illness onset after january, having a median of . days ( % ci, . - . ) (p < . ) (fig. ) . symptom severity - the estimated median of the time from onset to first medical visit for cases was . day ( % ci, . - . ). the estimated median of the time from onset to first medical visit for the cases was . days ( % ci, . - . ), which was significantly longer than the cases with a median of . days ( % ci, . - . ) (p < . ). the estimated median of the time from onset to admission for cases was . days ( % ci, . - . ). the estimated median of the time from onset to admission for the cases was . days ( % ci, . - . ), which was significantly longer than the cases with a median of . days ( % ci, . - . ) (p < . ). the estimated median of the time from onset to disease confirmation for cases was . days ( % ci, . - . ). the estimated median of the time from onset to disease confirmation for the cases was . days ( % ci, . - . ), which was significantly longer than the cases with a median of . days ( % ci, . - . ) (p < . ). the estimated median of the time from admission to discharge for cases was . days ( % ci, . - in cox regression model, we used discharge as the outcome variable (tables and ). for all cases, the significant independent covariates for the duration of onset to discharge were age, the time from onset to admission and the first-level public health emergency response. the significant independent covariates for the duration of hospitalization were age, the time from onset to admission and the first-level public health emergency response (fig. ) . potential influence which did not apparently impact duration of hospitalization was gender. as far as we know, this research includes the largest discharged covid- case series and report an initial evaluation of the epidemiological characteristics, clinical characteristics, laboratory results, and disease course of covid- cases. as of february , a total of covid- cases in shanghai had been confirmed, of which ( . %) cases had been cured and discharged. among them, one ( . %) case died, and case fatality rate is consistent with national (except hubei) [ , ] . among discharged cases, . % cases were imported to shanghai after infection in other provinces, mainly in hubei ( . %). three of them were secondgeneration cases of shanghai native cases, the onset of which was late january, indicating that local transmission had occurred in shanghai in late january. the range age table the duration of onset to discharge analyzed by multivariate cox regression of the cases was to years, indicating that all age groups are susceptible to the -ncov. common symptoms at onset of illness were fever, dry cough and fatigue. however, a significant proportion of cases presented initially with atypical symptoms, such as vomit, diarrhea and dyspnea. there were a certain proportion ( . %) of cases without fever, if screening is focused on the detection of fever, some cases may be missed. the study found that there were six asymptomatic cases of covid- , which indicates that asymptomatic infections or pre-symptomatic infections is possible. the asymptomatic infections make it difficult to recognize illness and difficult to quickly and effectively isolate asymptomatic and pre-symptomatic cases, increasing the effective infectious period and the risk for transmission. in nanshan chen et al. study [ ] , mainly in moderate patients infected with -ncov, % of patients had lymphocytopenia, and lymphocytopenia occurred in more than % of critically ill patients in xiaobo yang et al. study [ ] , indicating that the severity of lymphocytopenia reflects the severity of -ncov infection. lymphocytopenia occurred in more than % of cases in our study. in the cox regression model, the lower the lymphocyte count, the longer the duration of hospitalization, but there is no statistical significance, our sample size may be limited to finding a statistical significance. the estimated median of the time from onset to the first medical visit, admission, disease confirmation was day, days and . days, respectively. after first-level public health emergency response, the time were reduced to . days, . days, and . days, respectively, which was significantly shorter than that before first-level public health emergency response ( . days, . days, and . days, respectively). this indicates that the early identification, isolation and confirmation of cases with covid- have been accelerated after the first-level public health emergency response. shortening the duration of onset to admission does not seem to impact clinical outcomes [ , ] . however, shortening the duration of onset to admission facilitates quarantine and reduces the risk of transmission, and the effective communicable period. and any additional shortening of the duration that symptomatic cases are in the community will bring about further benefits at the whole crowd level. quarantine is a traditional and yet the most effective measure to control an epidemic. because there is no specific vaccine or cure against -ncov infections, standard public health emergency measures usually prove most efficient, including isolating the sources of infection, interrupting or cutting off transmission routes, and special care for the most susceptible people. and the covid- epidemic has shown that the essential for risk disclosure that will warn and inform the citizens, in such a way that will enhance personal protection, without triggering raised fear and anxiety, as an essential part of covid- epidemic control. a change in disease risk awareness would potentially bring about an increase in early reporting of covid- . cox regression analysis showed that the elder the case, the longer the duration of hospitalization. the possible explanations were that the younger case has higher recovery ability after infection with -ncov, and the elder case has a higher proportion of comorbidities. we review previous studies that found a greater number of male than female [ , , ], but our research shows that there was no significant difference in the course of disease between male and female. after first-level public health emergency response, the duration of hospitalization was shorter. shortening the time from initial symptoms to admission does not decrease the duration of hospitalization for moderate covid- cases. more generally, the average time from onset to discharge was days. one reason may be that there is no specific cure or vaccine against -ncov infections except for meticulous supportive. another reason may be that it may indicates that moderate covid- is self-limited disease. this study has several limitations. first, only three of cases had short and defined periods of exposure to known covid- cases, so we did not estimate the distribution of the incubation period, the time from infection to the onset of symptoms of covid- . second, the symptom severity of the discharged cases was moderate pneumonia, so we are unable to estimate severe or critical pneumonia. in conclusion, local transmission had occurred in shanghai in late january . the estimated median of the time from onset to discharge of moderate covid- was . days in shanghai. time intervals from onset to first medical visit, admission and disease confirmation were all shortened after shanghai's first-level public health emergency response. age, first-level public health emergency response and the time from onset to admission were the impact factors for the duration of hospitalization. male and female have the same course of disease. world health organization. question and answer on coronaviruses (covid- ) outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan the continuing -ncov epidemic threat of novel coronaviruses to global health -the latest novel coronavirus outbreak in wuhan, china world health organization. coronavirus disease (covid- ) situation reports- shanghai launched a first-level response to resolutely curb the spread of the epidemic shanghai municipal people's government, one new confirmed case of covid- in shanghai epidemiological and clinical characteristics of confirmed cases with coronavirus disease clinical course and outcomes of critically ill patients with sars-cov- pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical findings in a group of patients infected with the novel coronavirus (sars-cov- ) outside of wuhan, china: retrospective case series epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia national health commission of the people's republic of china novel coronavirus pneumonia emergency response epidemiology t. the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) in china characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong the epidemiology of severe acute respiratory syndrome in the hong kong epidemic: an analysis of all patients clinical features of patients infected with novel coronavirus in wuhan clinical characteristics of coronavirus disease in china we thank all medical workers taking part in investigation and treatment of covid- patients in shanghai. authors' contributions sxd, why, ph conceived the study. ls designed the study, analyzed data and wrote manuscript. yx, cp, kdc, hrb, jcy, zyx, gxh, xwj, msh, jbh and zyy were involved in collecting data and data cleaning. all authors have read and approved the final manuscript. this work was support by a grant from science and technology commission shanghai municipality grant/award number: jc . the datasets used and/or analysed during the current study are available from the corresponding author (xiaodong sun, sunxiaodong_scdc@ .com) on reasonable request. this study was reviewed and approved by shanghai municipal center for disease control and prevention ethics review committee, which waived the written informed consent for emerging infectious diseases. the data were analyzed anonymously. not applicable. the authors declare that they have no competing interests.received: march accepted: october publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -iht zndl authors: de angelis, giulia; posteraro, brunella; biscetti, federico; ianiro, gianluca; zileri dal verme, lorenzo; cattani, paola; franceschi, francesco; sanguinetti, maurizio; gasbarrini, antonio title: confirmed or unconfirmed cases of novel coronavirus pneumonia in italian patients: a retrospective analysis of clinical features date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: iht zndl background: since december , the severe acute respiratory syndrome coronavirus (sars-cov- ) emerged as a novel etiologic agent of viral pneumonia. we aimed to compare clinical features of italian patients with laboratory confirmed or unconfirmed -ncov pneumonia. methods: on march , , hospitalized patients who presented with fever and/or respiratory symptoms, exposures, and presence of lung imaging features consistent with -ncov pneumonia were included. before admission to a hospital ward, patients underwent rt-pcr based sars-cov- rna detection in their nasopharyngeal swab samples. results: of patients studied, had positive rt-pcr results and were rt-pcr negative for days or longer (i.e., when the last swab sample was obtained). the median age was years (iqr, – ), and ( . %) of patients had at least one comorbidity. the majority of patients ( / , . %) had a mild pneumonia, and the remaining patients ( / , . %) a severe/critical pneumonia. we did not find any substantial difference in symptoms, incubation periods, and radiographic/ct abnormalities as well as in many of the biological abnormalities recorded. however, at multivariable analysis, higher concentrations of hemoglobin (or, . ; % ci, . – . ; p = . ) and lower counts of leukocytes (or, . ; % ci, . – . ; p < . ) were statistically associated with confirmed covid- diagnosis. while mortality rates were similar, patients with confirmed diagnosis were more likely to receive antivirals ( % vs . %, p < . ) and to develop ards ( % vs %, p = . ) than those with unconfirmed covid- diagnosis. conclusions: our findings suggest that unconfirmed -ncov pneumonia cases may be actually covid- cases and that clinicians should be cautious when managing patients with presentations compatible with covid- . the novel coronavirus ( -ncov), named severe acute respiratory syndrome coronavirus (sars-cov- ), became notorious since december as a new etiologic agent of viral pneumonia [ ] . in early illness stages, patients with coronavirus disease (covid- ) present with symptoms of acute respiratory infection, which can progress to acute respiratory distress syndrome (ards) and other serious complications [ ] . because of substantial pneumonia-related morbidity and mortality [ ] , testing for sars-cov- infection of patients who meet the suspected-case definition for covid- [ ] is central for their management. accordingly, provision of supportive care (e.g., oxygenation, ventilation, and fluid therapy) and/or administration of antiviral agents may be decisive [ ] . real-time reverse-transcriptase-polymerase-chain-reaction (rt-pcr) based sars-cov- rna detection in respiratory samples (e.g., nasopharyngeal swabs) is the reference diagnostic method to confirm covid- [ ] . however, one or more negative results do not exclude the likelihood of sars-cov- infection [ ] . currently published studies suggest lung imaging, biomarkers, and other non-microbiological tests as ancillary diagnostic methods [ ] , encouraging further investigation to understand the value of radiological or laboratory findings to diagnose covid- . we comparatively explored the clinical features of patients with laboratory confirmed or unconfirmed -ncov pneumonia admitted to covid- wards of the fondazione policlinico a. gemelli irccs, which is a tertiary care university hospital in rome, italy. thus, we investigated the prospect that cases with a negative rt-pcr test result are actually cases of -ncov pneumonia or, in other words, are not to distinguish from those without confirmation test performed. this retrospective, single-center observational study was conducted in accordance with the declaration of helsinki and was approved by the ethics committee of the fondazione policlinico a. gemelli irccs (reference number / ), and written informed consent was obtained from each enrolled patient. all patients who were hospitalized for suspected -ncov pneumonia [ ] on march , were considered for recruitment. inclusion criteria were fever and/or respiratory symptoms, exposures, and presence of lung imaging features consistent with -ncov pneumonia [ ] . at the emergency room (before admission to a hospital ward), all patients had undergone nasal and oropharyngeal swabs for detection of one or more sars-cov- specific nucleic acid targets [ ] , using the korean ministry of food and drug safety approved allplex -ncov assay (arrow diagnostics s.r.l., genova, italy). samples resulted negative had been repeated after - h. to rule out the presence of infections due to common viral (adenovirus, coronaviruses e, hku , nl , oc , influenza viruses, rhinovirus/enterovirus, etc.) or bacterial (bordetella pertussis, chlamydophila pneumoniae, legionella pneumophila, mycoplasma pneumoniae) pathogens, patients' samples had also been tested with the genmark's eplex respiratory pathogen panel assay. eleven patients yielding positive results for the etiological agents above mentioned were not included. we retrieved demographic, clinical, laboratory, imaging, treatment, and outcome data from the patients' medical chart records. we classified cases as mild (see above specification), severe (i.e., dyspnea, respiratory rate ≥ breaths/min, blood oxygen saturation ≤ %, and partial pressure of arterial oxygen [pao ]/fraction of inspired oxygen [fio ] ≤ mmhg), or critical (i.e., respiratory failure, septic shock, and/or multiple organ failure) pneumonia. we recorded chest x-ray or computed tomography (ct) features using the fleischner society terminology [ ] , we defined ards based on timing, lung imaging, origin of edema, and oxygenation as specified in the berlin definition [ ] , and we defined liver injury as transaminase elevation two-to three-times the upper limit of normal (e.g., u/l for alanine aminotransferase). according to the italian society of infectious diseases guidelines for covid- treatment [ ] , we administered antiviral agents (e.g., lopinavir/ritonavir) to all patients with severe/critical disease or to patients with mild disease who had comorbidities including age > . categorical variables were expressed as number with percentage and were compared using the χ test, and continuous variables were expressed as median with interquartile range (iqr) and were compared using the mann-whitney u-test. a two-sided p value of < . was considered statistically significant. we performed univariate analysis using the aforementioned tests, and all significant variables (among demographics and baseline characteristics) were included in a multivariable logistic regression model to identify those variables that were statistically associated with confirmed covid- diagnosis. odds ratio (or) values with % confidence intervals (cis) were calculated. all analyses were performed with stata software version . (stata-corp, college station, tx, usa). on march , , patients were hospitalized at our center with a suspicion of -ncov pneumonia, and patients were finally included. of them, were confirmed covid- cases based on positive rt-pcr results on nasopharyngeal swabs [ ] , and were rt-pcr negative for days or longer (i.e., when the last swab sample was obtained). table shows demographic and clinical characteristics of patients at baseline. the median age was years (iqr, - ), and patients were males. one hundred and twenty-three ( . %) patients had at least one comorbidity. the most common symptoms at admission were fever (n = , . %), dyspnea (n = , . %) and cough (n = , . %), and the median time from symptom onset to covid- diagnosis was days (iqr, - ). overall, lactate dehydrogenase levels (median value, u/l; iqr, - ) and c-reactive protein levels (median value, . mg/l; iqr, . - . ) were elevated. one hundred and twenty-seven patients ( %) presented with x-ray signs of ground-glass opacity, and ( . %) with signs of consolidation. at admission, ( . %) presented with a mild pneumonia, and the remaining patients ( . %) with a severe/critical pneumonia. treatments and outcomes of patients are detailed in table . overall, patients ( . %) developed ards, and of them ( . %) septic shock, which needed transfer to icu. most patients (n = , . %) were treated with oxygen support, antivirals (n = , . %), and antibiotics (n = , . %). forty-six patients ( . %) received therapy with interleukin- receptor inhibitors. overall, ( . %) of patients died at the follow-up end (n = because ards, n = because of septic shock, n = because of multiple comorbidities). at univariate analysis, fever was significantly more frequent in patients with confirmed diagnosis ( . % vs . %, p = . ). this group presented also with significantly lower levels of leucocytes (median value, . × /l vs . × /l; p < . ), neutrophils (median value, . × /l vs . × /l; p < . ), platelets (median value, × /l vs × /l; p = . ), and procalcitonin (median value, . ng/ml vs . ng/ml, p = . ), and higher levels of hemoglobin (median value, . g/dl vs . g/dl; p < . ), alanine aminotransferase (median value, u/l vs u/l, p = . ), and lactate dehydrogenase (median value, u/l vs u/l; p < . ). patients with confirmed diagnosis were also more likely to receive antivirals ( % vs . %, p < . ) and to develop ards ( % vs %, p = . ) than those without confirmed diagnosis. there were no other significant differences between the two groups. at multivariable analysis, higher concentrations of hemoglobin (or, . ; % ci, . - . ; p = . ) and lower counts of leukocytes (or, . ; % ci, . - . ; p < . ) were found to be statistically associated with confirmed diagnosis in the overall cohort. we tested the hypothesis that negative patients did not differ from sars-cov- rna positive patients by comparing features of cases with clinically diagnosed -ncov pneumonia in our hospital. we did not find any substantial difference in symptoms, incubation periods, and radiographic/ct abnormalities as well as in many of the biological abnormalities recorded. however, blood/serum test results showed that patients with laboratory-confirmed diagnosis of -ncov pneumonia were more likely to have higher levels of hemoglobin and lower levels of leukocytes. additionally, the proportion of ards in the group of covid- confirmed patients was significantly higher than in the group of covid- unconfirmed patients. large or small descriptive studies published in mainly focused on patients with laboratory-confirmed -ncov pneumonia [ , , ] . nonetheless, among , cases (as of february , ) from the covid- outbreak in china, , ( %) and , ( %) of them could not receive laboratory confirmation for covid- and, then, were classified as suspected cases or clinically diagnosed cases, respectively [ ] . although testing for sars-cov- in our laboratory was not restricted [ ] , ( . %) of patients with -ncov pneumonia did not have a laboratory-confirmed diagnosis in our study. despite a well-documented active virus replication in the upper respiratory tract [ ] , swab samples may have a limited sensitivity to identify cases. sampling or testing related factors may be responsible for false-negative rt-pcr results, necessitating additional sample collection from the lower respiratory tract specimens including sputum [ ] . it is plausible that our sars-cov- negative patients were outside the window of peak shedding in the upper respiratory tract samples or did not have symptoms highly suggestive for upper respiratory tract infection [ ] . accordingly, eight of patients with positive rt-pcr results became positive only with swabs taken on subsequent days after the first (negative) sampled swab, and their median time from symptom onset did not differ from that of remaining patients ( days vs days, p = . ). furthermore, the sensitivity of the allplex -ncov assay might have limited by its requirement that three genes were all detectable for a positive result. in our study, all patients had a radiological evidence of pneumonia not attributed to any typical respiratory viral infection agents, including the human coronaviruses hku , oc , nl , e, the influenza virus a and b, and others (data not shown). it is worthy to note that the finding of typical ground glass opacities in chest cts of clinically diagnosed patients [ ] prompted the chinese authorities, at one point in early february , to count these patients as confirmed cases [ ] . this allowed identifying and quarantining patients as early as possible. in conclusion, using the clinical diagnosis as the reference standard, the rt-pcr testing allowed to correctly identify two thirds of our patients as covid- while one third was not correctly laboratory-based confirmation of -ncov pneumonia was done by sars-cov- rna detection using a well-established rt-pcr assay [ ] b according to multivariable logistic regression model analysis (see text for details) c includes anemia, endocrine disorders, inflammatory bowel disease, and obesity d ct findings in all patients were assessed according to imaging features described elsewhere [ ] e not calculated because the relative variable was not entered into multivariable logistic regression model (see text for details) f patients were admitted between / / and / / , with follow-up through / / identified. however, as undocumented sars-cov- infections may be a relevant source of transmission among hospitalized patients [ ] , we believe that clinicians should exceed on the side of caution when managing patients with presentations compatible with covid- . a novel coronavirus from patients with pneumonia in china clinical features of patients infected with novel coronavirus in wuhan global epidemiology of coronavirus disease (covid- ): disease incidence, daily cumulative index, mortality, and their association with country healthcare resources and economic status laboratory testing for coronavirus disease (covid- ) in suspected human cases treatment of covid- : old tricks for new challenges diagnostic testing for severe acute respiratory syndrome-related coronavirus- : a narrative review global surveillance for covid- caused by human infection with covid- virus characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention detection of novel coronavirus ( -ncov) by real-time rt-pcr fleischner society: glossary of terms for thoracic imaging acute respiratory distress syndrome: the berlin definition vademecum per la cura delle persone con malattia da covi- . version . (in italian) epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus of the lombardy region clinical microbiology laboratory adaptation to covid- emergency: experience at a large teaching hospital in virological assessment of hospitalized patients with covid- correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases sars-cov- and covid- : the most important research questions substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (sars-cov ) publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors are grateful to franziska lohmeyer phd for her english language assistance and to clinical staff members for collection of samples. authors' contributions gda, bp, ms, and ag conceived and designed the study. gda, fb, gi, lzdv acquired, analyzed, and interpreted the data. gda and fb performed statistical analyses. bp, ms, and ag supervised the work. bp was the major contributor in writing the manuscript. pc and ff critically revised the manuscript for important intellectual concept. all authors read and approved the final manuscript. the reale group and the fondazione valentino garavani & giancarlo giammetti, to support the covid- research in our institution, financed this work. the funders had no role in the design and conduct of the study; collection, management, analysis, and interpretation of the data; preparation, review, or approval of the manuscript; and decision to submit the manuscript for publication. the datasets generated and analyzed during the current study are not publicly available as the data also forms part of an ongoing study but are available from the corresponding author on reasonable request. this retrospective, single-center observational study was conducted in accordance with the declaration of helsinki and was approved by the ethics committee of the fondazione policlinico a. gemelli irccs (reference number / ), and written informed consent was obtained from each patient. not applicable. the authors declare that they have no competing interests. key: cord- - dli emd authors: o’grady, kerry-ann f.; hall, kerry k.; sloots, theo p.; anderson, jennie; chang, anne b. title: upper airway viruses and bacteria in urban aboriginal and torres strait islander children in brisbane, australia: a cross-sectional study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: dli emd background: respiratory morbidity in australian indigenous children is higher than their non-indigenous counterparts, irrespective of urban or remote residence. there are limited studies addressing acute respiratory illness (ari) in urban indigenous children, particularly those that address the upper airway microbiome and its relationship to disease. we aimed to describe the prevalence of upper airway viruses and bacteria in symptomatic and asymptomatic urban-based australian indigenous children aged less than years. methods: a cross-sectional analysis of data collected at baseline in an ongoing prospective cohort study of urban aboriginal and torres strait islander children registered with a primary health care service in the northern suburbs of brisbane, australia. clinical, demographic and epidemiological data and bilateral anterior nasal swabs were collected on enrolment. polymerase chain reaction was performed on nasal swabs to detect respiratory viruses and bacteria. the primary outcome was the prevalence of these microbes at enrolment. logistic regression was performed to investigate differences in microbe prevalence between children with and without acute respiratory illness with cough as a symptom (ariwc) at time of specimen collection. results: between february and october , children were enrolled. the median age at enrolment was . months (iqr . – . ), . % were boys and children ( . %) had ariwc. overall, / ( %) nasal swabs were positive for at least one organism; ( . %) for any bacteria, ( . %) for any virus and ( . %) for both viruses and bacteria. co-detection of viruses and bacteria was more common in females than males ( . % vs . %, p = . ). no microbes, alone or in combination, were significantly associated with the presence of ariwc. conclusions: the prevalence of upper airways microbes in asymptomatic children is similar to non-indigenous children with ariwc from the same region. determining the aetiology of ariwc in this community is complicated by the high prevalence of multiple respiratory pathogens in the upper airways. study registration: australia new zealand clinical trial registry registration number: , , , , . retrospectively registered. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. acute and chronic respiratory illnesses are predominant causes of morbidity and mortality in aboriginal and torres strait islander (hereforth respectfully called indigenous) children in australia [ ] . in some remote regions of australia, indigenous infants present on average at least once a fortnight to community clinics and acute respiratory illnesses (ari) are the most common reason for attendance [ ] . indigenous children are . times more likely to present to emergency departments (ed) with ari [ ] and twice as likely to be hospitalised for an ari than non-indigenous children [ ] . however, respiratory research in indigenous children has predominantly focused on remote-based children [ , ] , although the majority of indigenous children live in urban or inner regional areas of australia, with brisbane having the largest of those communities [ ] . there is a conspicuous lack of current data on ari and related respiratory microbes in urban indigenous children at the community level and it is unknown whether it is similar to remote children or non-indigenous children in urban settings. further, cough is the most common symptom associated with health care utilisation amongst australian children [ ] , including indigenous children and, if present in ari, is likely indicative of a lower ari particularly if wet. establishing the microbiological aetiology of ari with cough (ariwc) in children is complex, particularly if upper airway specimens are used [ ] , given many organisms are also detected in the nasopharynx of healthy children. however upper airway microbial data obtained from the nasopharynx are still considered important because they can provide important epidemiological information on the prevalence of, and temporal trends in, organisms within and between different populations. knowing upper airway microbial epidemiology is particularly important to informing public health strategies such as vaccination. increasingly co-detection of viruses with bacteria is considered important and a recent south african study reported different types of organisms per episode were found in the upper airways of young children [ ] . yet, there are limited data on factors that are associated with virus and bacteria co-detection in indigenous children, particularly those in urban settings. thus, in urban-based indigenous children presenting to an urban primary health care service, we described the prevalence of upper airway respiratory viruses and bacteria. we also sought to identify factors associated with virus-bacteria codetection. we hypothesised that virusbacteria codetection was more likely in children with ariwc than those without. we analysed data from a cohort of urban aboriginal and torres strait islander children aged less than years collected at time of enrolment into a prospective study of ariwc. the full protocol of the prospective study has been previously published [ ] . the study was conducted in a large primary healthcare clinic in the northern suburbs of subtropical brisbane which has a patient population of approximately , people. fifty-nine percent of the patients identify as being indigenous. an aboriginal research officer approached all children aged less than five years and their parent or guardian at time of presentation to the clinic for any reason (including well child checks and accompanying another person presenting for health care). children were eligible for inclusion in the primary cohort study [ ] if they were: a) identified by the parent/guardian as being indigenous; b) a regular patient of the clinic; c) aged less than five years at time of enrolment, and; d) parents/guardians were willing and able to complete the study requirements. the reason for presentation and the presence of ariwc were not determined until after the child had been enrolled. for the analysis presented in this manuscript, only children who had a nasal swab performed were included. there were no exclusion criteria. at enrolment, detailed demographic, environmental, clinical and socio-economic data were collected, and an anterior bilateral nasal swab was performed. nasal swabs were collected using the virocult™ system (medical wire and equipment, corsham, uk) by inserting the tip at least cm into each nare and turning the swab four times against the nasal mucosa. a child was considered symptomatic of ariwc if any of the following symptoms occurred within seven days prior to and including the day of enrolment: cough and other local or systemic symptoms suggestive of a respiratory illness (eg. runny nose, wheeze, dyspnoea and tachypnoea). children did not meet the case definition if cough was not present during that time. nasal swabs were stored refrigerated until they were transferred within one week to - °c freezers. multiplex polymerase chain reaction (pcr) was used to test for adenovirus, respiratory syncytial virus (rsv) groups a and b, influenza virus types a and b, parainfluenza virus types - , human metapneumovirus, human rhinoviruses, human coronaviruses (oc , e,nl + hku ), human bocavirus, human polyomaviruses ki and wu, m. pneumoniae, c. pneumoniae, b. pertussis, s. pneumoniae, s. aureus, non-typeable haemophilus influenzae (nthi) and m. catarrhalis using previous established methods [ , ] . descriptive analyses were performed with data expressed as proportions and/or means of the selected characteristics. where continuous data were not normally distributed, medians with accompanying interquartile ranges are presented. univariate analyses were performed to evaluate potential differences in child characteristics between swabs in which codetection of virus and bacteria did and did not occur. chi statistics were used to assess differences in proportions and the wilcoxon rank sum test was used to compare differences in medians. given the lack of sufficient significant findings on univariate analyses, regression analyses were not performed. all analyses were performed in stata v se (statacorp, college station, tx, usa). between february and november , children were screened and indigenous children were enrolled. reasons for non-enrolment were ( . %) were non-indigenous, ( . %) declined, ( . %) were ineligible and ( . %) were not enrolled for other reasons. there were no differences in age and gender between children who were and were not enrolled. one child was withdrawn as a screen failure, children/parents refused specimen collection and thus nasal swabs were collected from children. of the children, the median age at enrolment was . months (interquartile range (iqr) . - . ) and . % were male; respiratory symptoms at time of enrolment were present in children ( . %). there were no differences in the median ages of children with and without ariwc (p = . ) nor any differences within and between age groupings (ie. < months, -< months, -< months and + months; p = . ). overall / ( %) nasal swabs were positive for at least one organism, ( . %) for any bacteria, ( . %) for any virus and ( . %) for both viruses and bacteria ( table ). all but two swabs that were positive for one or more viruses also had bacteria detected. three or more organisms were detected in . % of swabs. c. pneumoniae and m. pneumoniae were not detected in any specimens and are not considered further. there were no statistical differences in the prevalences of any virus or bacterium, alone or in combination, between children with and without ariwc (table ) . even in children without ariwc, a virus was detected in . % of children. virus-bacteria co-detection was more frequent in females than males ( . % vs . %, p = . ) and this was not age-dependent. no other characteristics were significantly different between children with and without codetection of viruses and bacteria ( table ). the prevalences of specific bacteriumbacterium, virusvirus and virus-bacterium codetections are presented in the additional file : table s . the seasonal distribution of organisms detected is presented in fig. ; influenza and b. pertussis are not included given each was only detected in one swab over the entire study. all other bacteria were detected across all seasons of the year however this did not occur for the parainfluenza viruses, adenovirus, polyomaviruses and coronaviruses. given the lack of microbiological data in the upper airways of urban-based indigenous children, we investigated this in a cohort of children aged < years attending a primary health care service with and without ariwc. irrespective of the reason for presentation to the clinic, a third of the children had ariwc symptoms at enrolment. the prevalence of upper airway respiratory viruses and bacteria were very high; at least one virus or bacteria was detected in % of children and ≥ organisms were detected in a third. the detection of any upper airway viruses and/or bacteria, alone or in combination, was similar between children with and without ariwc and was not associated with age. the prevalence of any organism in children in this study of % is similar to children aged < years presenting acutely to a tertiary paediatric emergency department (ed) in the same geographical location with ariwc ( %), the majority of whom were non-indigenous [ ] . the prevalence of co-detection of viruses and bacteria in the ed study was . % [ ] , higher than the . % in this cohort. however, children in the ed study were all symptomatic for ariwc whilst the majority of children in this current study were asymptomatic. the two studies utilised the same specimen collection and laboratory methods and tested for the same organisms at the research laboratory. in both studies, s. pneumoniae followed by m.catarrhalis and nthi were the dominant bacteria and rhinoviruses were the most common virus. b. pertussis, m. pneumoniae and c. pneumoniae were rare and influenza was uncommon. respiratory syncytial virus was only observed in % of children in this study but was detected in % of children in the ed study [ ] . in that study, rsv was weakly associated with children being hospitalised [ ] and its higher prevalence amongst ed children compared to community children possibly reflects the severity of illness if infected rather than community prevalence. the seasonal distribution of organisms was also similar to that identified in the ed study [ ] , including a predominance of the autumn months for nthi and rsv and that virus-bacteria codetection occurred predominantly in autumn and winter. in a study of upper airway viruses and bacteria in central australian aboriginal children hospitalised for pneumonia [ ] , a population with high rates of hospitalised lower ari [ ] and nasal colonisation [ ] , the [ ] . that study differed from the two brisbane studies in that the nt study focused on children hospitalised with pneumonia rather than non-severe ariwc, different specimen collection techniques were used, the pcr for bacteria was performed at a different laboratory and the central australian study was undertaken prior to widespread implementation of pneumococcal conjugate vaccines. however, more recent community based studies of nasopharyngeal carriage of these bacteria in the northern territory in the -valent pneumococcal conjugate vaccine era identified prevalences of % for s. pneumoniae, % for m. catarrhalis and % for nthi [ ] . viruses were not reported in that study. in a western australian study of asymptomatic rural aboriginal children that included testing for the same viruses as our study with the exception of bocavirus and the polyomaviruses [ ] , viruses were detected in % of children (most commonly rhinoviruses: . %). thus our data suggest that viral infection may be comparable between urban and remote indigenous children however bacterial carriage is likely to be higher in remote children. we identified only two swabs that were positive for viruses only (i.e. most had virus with bacteria codetection); one was a single isolation of a polyomavirus and the other was a co-detection of rhinovirus and bocavirus. the reasons why we found so few virus-only detections are uncertain, particularly given few data in the literature that have tested for the same spectrum of organisms by pcr that was undertaken in this study. both swabs were from children with ariwc at the time of testing but the clinical significance of virus only detections is unknown. a recent study of respiratory viruses (n = ) detected by pcr in paediatric episodes of ari reported episodes were virus positive, of which were single infections and were multiple infections; testing was undertaken for only two bacteria (c. pneumoniae and m. pneumoniae) [ ] . there was no difference in clinical severity and management between children with single infections and those with multiple infections. we found no relationship between the child characteristics and virus-bacteria codetection other than gender. notably there were no differences in codetection between children with and without ariwc, although the lack of difference may be attributable to a secondary analysis of data and hence lack of power to identify [ ] . m. catarrhalis in the presence of viruses was also associated with rhinitis, nasal congestion and cough [ ] . in norwegian children attending two daycare centres over a -year period, nasopharyngeal swabs (nps) were collected over time points and analysed by pcr for b. pertussis, m. pneumoniae, c. pneumoniae and viruses [ ] . overall % of specimens were positive for at least one virus and none were positive for the three bacteria. in swabs collected from children who underwent a clinical examination, % of children with clear signs of respiratory tract infection were virus positive, compared to % with mild findings and % in those who were asymptomatic (p < . ), with rhinovirus the most common virus detected in all groups [ ] . in a study of respiratory viruses in alaska native children hospitalised with acute lower respiratory infections and age-matched community controls, viruses were detected by pcr in nps in % of hospitalized children and % of asymptomatic community controls, with rhinoviruses the most common in both groups [ ] . bacteria were not reported in that study. while comparisons between children across studies are problematic given differences in demographics, geography and study methods, our study and those above emphasise the complexity in assigning ariwc causality based on nasal specimens in children given the high prevalence of multiple organisms in asymptomatic children. the probable exceptions are rsv, influenza virus and human metapneumovirus given their relatively strong association with severe ari in children and a low prevalence in asymptomatic children in several studies [ ] . as these viruses were uncommon in our study it was not possible to examine their role in symptomatic respiratory infections. our study has limitations given the cross-sectional nature of the analyses, the relatively small number of children enrolled and that given this was single centre study, the children who were enrolled may differ to the general population of urban indigenous children in australia posing a risk of selection bias. our study children differed from national indigenous statistics with respect to the high prevalence of exposure to environmental tobacco smoke and other household characteristics such as the high number of single parent households, low total annual household income and low levels of attendance at childcare [ ] . further, pcr detection of viruses and bacteria does not necessarily equate to active infection at the time of testing and simply provides an indication of recent exposure to the organism. next generation sequencing holds promise for the improved detection and differentiation of respiratory pathogens [ ] . however the tests are costly which currently limits the use of the technology in population-based studies. our study is the first to report upper airway microbial in urban-based indigenous children with and without ariwc that includes the range of microbes we tested for. with the exception of rsv, the prevalence of upper airway respiratory viruses and bacteria in urban indigenous children is comparable to acutely unwell non-indigenous children from the same urban area but differs from remote indigenous children with respect to the latter having a higher prevalence of respiratory bacteria. given the high prevalence ( %) of organisms detected in children without ari, upper airway microbiology in urban-based indigenous children should be interpreted with caution. additional file : qld , australia. child health research centre, centre for children's health research australian institute of health & welfare. aboriginal and torres strait islander health performance framework report: detailed analyses. cat. no. ihw . canberra: australian government disease burden and health-care clinic attendances for young children in remote aboriginal communities of northern australia can linked emergency department data help assess the out-of-hospital burden of acute lower respiratory infections? a population-based cohort study diverging trends for lower respiratory infections in non-aboriginal and aboriginal children lower respiratory infections in australian indigenous children estimates of aboriginal and torres strait islander australians general practice activity in australia upper airway viruses and bacteria detection in clinical pneumonia in a population with high nasal colonisation do not relate to clinical signs aetiology of childhood pneumonia in a well vaccinated south african birth cohort: a nested case-control study of the drakenstein child health study the respiratory health of urban indigenous children aged less than years: study protocol for a prospective cohort study successful application of a simple specimen transport method for the conduct of respiratory virus surveillance in remote indigenous communities in australia mailed versus frozen transport of nasal swabs for surveillance of respiratory bacteria in remote indigenous communities in australia prevalence, codetection and seasonal distribution of upper airway viruses and bacteria in children with acute respiratory illnesses with cough as a symptom upper airway viruses and bacteria and clinical outcomes in children with cough hospitalisation of indigenous children in the northern territory for lower respiratory illness in the first year of life general health, otitis media, nasopharyngeal carriage and middle ear microbiology in northern territory aboriginal children vaccinated during consecutive periods of -valent or -valent pneumococcal conjugate vaccines the interaction between respiratory viruses and pathogenic bacteria in the upper respiratory tract of asymptomatic aboriginal and non-aboriginal children single-and multiple viral respiratory infections in children: disease and management cannot be related to a specific pathogen role of nasopharyngeal bacteria and respiratory viruses in acute symptoms of young children respiratory virus detection and clinical diagnosis in children attending day care viral respiratory infections in hospitalized and community control children in alaska aetiological role of common respiratory viruses in acute lower respiratory infections in children under five years: a systematic review and meta-analysis australian institute of health & welfare. the health and welfare of australia's aboriginal and torres strait islander peoples: . canberra: australian government unbiased detection of respiratory viruses by use of rna sequencing-based metagenomics: a systematic comparison to a commercial pcr panel the authors would like to thank the children and families who participated in this study and the staff of caboolture community medical for supporting the implementation of the study in their practice. we thank our indigenous research reference group for cultural oversight of the study throughout its duration. at the queensland paediatric infectious diseases laboratory, jane gaydon was instrumental in the processing and reporting of respiratory specimens. study data and materials may be made available on request with appropriate human research ethics committee approval and with the consent of the participating community as required by australian criteria for research with indigenous communities.authors' contributions kfo conceptualized the study, analysed the data and lead the production of the manuscript. kkh contributed to study design, had primary responsibility for recruitment and data collection and contributed to the manuscript. tps was responsible for the laboratory components of the study and interpretation of laboratory data. ja contributed to study design, managed the study at ccm and contributed to the manuscript. abc contributed to study design and implementation and provided significant input to the drafting of the manuscript.competing interests ja is the director of the clinic in which this study was conducted. she had no role in the recruitment and consent of participants and did not receive financial support for the study. not applicable. the study was approved by the human research ethics committees of the queensland children's hospital and health services (hrec/ /qrch/ ), and the queensland university of technology ( ). informed consent was obtained from parents or guardians. an indigenous research reference group provided cultural oversight of the study. written informed consent was obtained from parents/guardians following provision of a plain language statement explaining the study. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord- - esrg jw authors: tam, clarence c.; offeddu, vittoria; anderson, kathryn b.; weg, alden l.; macareo, louis r.; ellison, damon w.; rangsin, ram; fernandez, stefan; gibbons, robert v.; yoon, in-kyu; simasathien, sriluck title: association between semi-quantitative microbial load and respiratory symptoms among thai military recruits: a prospective cohort study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: esrg jw background: multiplex real-time polymerase chain reaction assays have improved diagnostic sensitivity for a wide range of pathogens. however, co-detection of multiple agents and bacterial colonization make it difficult to distinguish between asymptomatic infection or illness aetiology. we assessed whether semi-quantitative microbial load data can differentiate between symptomatic and asymptomatic states for common respiratory pathogens. methods: we obtained throat and nasal swab samples from military trainees at two thai army barracks. specimens were collected at the start and end of -week training periods (non-acute samples), and from individuals who developed upper respiratory tract infection during training (acute samples). we analysed the samples using a commercial multiplex respiratory panel comprising bacterial, viral and fungal targets. we used random effects tobit models to compare cycle threshold (ct) value distributions from non-acute and acute samples. results: we analysed non-acute and acute swab samples from participants. haemophilus influenzae type b was the most commonly detected microbe ( . % of non-acute and . % of acute samples). in acute samples, nine specific microbe pairs were detected more frequently than expected by chance. regression models indicated significantly lower microbial load in non-acute relative to acute samples for h. influenzae non-type b, streptococcus pneumoniae and rhinovirus, although it was not possible to identify a ct-value threshold indicating causal etiology for any of these organisms. conclusions: semi-quantitative measures of microbial concentration did not reliably differentiate between illness and asymptomatic colonization, suggesting that clinical symptoms may not always be directly related to microbial load for common respiratory infections. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. multiplex polymerase chain reaction (pcr)-based diagnostic techniques allow rapid, simultaneous identification of a broad range of respiratory pathogens [ ] . compared to classical microbiological diagnostic methods, pcr-based assays offer higher sensitivity, specificity, and reproducibility [ ] . however, the high sensitivity of multiplex pcr diagnostics does not directly translate into clinical utility, because such assays do not distinguish between viable and dead organisms, or acute infection and asymptomatic colonisation [ ] . in the clinical setting, the etiological agent is seldom identified and unspecific respiratory symptoms are often treated empirically [ ] . although the quantification of microbial load may vary depending on the presence of co-infections, specimen type, sampling technique, or timing of sampling, quantitative or semi-quantitative microbial load data from real-time pcr assays may help define organism densities that are consistent with colonization or infection and distinguish between symptomatic and asymptomatic states [ ] . in this study, we assessed whether semi-quantitative microbial load availab from real-time pcr assays can differentiate between symptomatic and asymptomatic states for common respiratory agents in a cohort of basic military trainees at two royal thai army barracks. details of the study setting and procedures have been described previously [ ] . briefly, participants were recruited from six consecutive cohorts of basic military trainees at two royal thai army barracks between may and july . trainees entered the camps for a -week training period at the start of may and november each year. individuals aged ≥ years entering one of the two army barracks involved in the study were eligible for enrolment. suspected tuberculosis cases or individuals with immune deficiencies, such as acquired immune deficiency syndrome, leukemia or lymphoma, were excluded. throat and anterior nasal swab samples were collected using stiff synthetic swabs by trained study staff at the start and end of each training period (non-acute samples) and were placed in viral transport media (universal transport medium c ; copan diagnostics) and stored at − °c until time of transfer to the armed forces research institute of medical sciences for further testing. in addition, enrolled participants were asked to consult the camp's medical unit if they experienced respiratory symptoms during the training period. medical staff took a history, conducted a medical exam, and recorded symptoms of upper respiratory illness (uri) or influenza-like illness (ili). uri was defined as an illness with at least two of the following: (i) runny nose or sneezing; (ii) nasal congestion; (iii) sore throat, hoarseness or difficulty swallowing; (iv) cough; (v) swollen or tender glands in the neck; and (vi) fever (oral temperature > °c). ili was defined as a respiratory illness with acute onset presenting with fever and cough or sore throat. throat and nasal swab samples were collected on average . days after symptom onset from individuals who developed uri or ili during the -week follow-up (acute samples). specimens from two of the six cohorts (total number of individuals = ) were tested using a commercial multiplex real-time pcr assay comprising bacterial, viral and fungal targets according to the manufacturer's instructions (ftd kit, fast track diagnostics, esch-sur-alzette, luxembourg). these two cohorts were selected because they underwent concurrent routine environmental sampling of air and surfaces within the barracks, which were then similarly tested using the ftd kit (data not shown). multiplex testing of specimens from the remaining cohorts was not done due to resource constraints. a cycle threshold (ct) value below the detection limit of the assay (< ) was considered a positive result. non-acute samples collected at the end of the training period from participants who experienced an acute episode during follow-up were excluded from the analysis, as the ct-value might reflect post-infectious shedding. we used the mcnemar test to determine whether target-specific frequencies were significantly different in non-acute baseline samples and acute samples. in addition, we computed the chi-square (χ ) or fisher's exact test (for expected values < ) to assess whether co-detection of specific microbe pairs occurred more frequently than expected by chance in non-acute baseline or acute samples. to account for data censoring at ct-value = , random effects tobit regression models were used to compare ct-value distributions from non-acute and acute samples, or ct-value distributions from samples containing a single or multiple organisms. in addition, we used the kruskal-wallis test to compare the median delay between illness onset and sample collection between samples containing one or multiple organisms. all analyses were conducted using stata software (stata corporation). the study was approved by the institutional review boards of the royal thai army in bangkok, thailand, the walter reed army institute of research and the london school of hygiene & tropical medicine. all participants provided written informed consent. the investigators have adhered to the policies for protection of human subjects as prescribed in army regulation - . we analyzed a total of non-acute swab samples collected from recruits at the start (n = ) or end (n = ) of the training period, and acute specimens from individuals who developed one or more uri episodes during follow-up. of targets contained in the respiratory panel, were detected in at least one specimen (table ) . viruses were detected in . % ( / ) and bacteria in . % ( / ) of non-acute samples. among acute samples, viruses were detected in . % ( / ) and bacteria in . % ( / ) of specimens. haemophilus influenzae type b (hi-b) was the most commonly detected microbe ( . % of non-acute and . % of acute samples). other frequently detected bacteria included non-type b haemophilus influenzae (hi-nonb), streptococcus pneumoniae, and klebsiella pneumoniae (table ) . rhinovirus was the most prevalent virus, detected in . % of non-acute and . % of acute samples. all other viruses were detected in < % of collected specimens (table ) . hi-nonb, rhinovirus, and coronavirus were detected significantly less frequently in non-acute samples collected at the start of the training period than acute samples (p-values < . ) ( table ) . influenza b was identified in none of the non-acute, but . % of acute specimens. multiple microbes were detected in . % ( / ) of non-acute samples collected at the start of the training period. co-detection of multiple organisms was significantly higher in both non-acute samples taken at the end of the training period ( . %) and acute specimens ( . %) (p-values < . ; table ). among acute samples, specific organism pairs were co-detected more frequently than expected by chance (p-values < . ) ( were found in < % of acute specimens (table ) . no microbe pair occurred more frequently than expected by chance among non-acute baseline samples. overall, there was a substantial overlap in ct-value distributions from non-acute samples collected at the start or end of the training period and acute samples collected from symptomatic individuals during follow-up (fig. ) . this was the case even when considering only samples where a single organism was identified (fig. ) . for hi-nonb and s. pneumoniae, our tobit regression models indicated significantly lower microbial load in non-acute baseline compared to acute samples (p-values < . ) ( table ). for hi-nonb, a coefficient of . represents a . higher average ct-value in non-acute baseline samples compared to acute specimens, which corresponds to an approximately -fold lower microbial load in non-acute compared to acute samples. for s. pneumoniae, the average microbial load was . -fold lower in non-acute baseline samples compared to acute specimens. our analysis also indicated a significantly lower average rhinovirus load in non-acute samples collected either at the start or at the end of the training period compared to acute samples (p-values < . ) ( table ). this was in contrast with hi-b, for which regression analysis indicated a . -fold higher average microbial load in non-acute baseline samples compared to acute samples (p-value < . ) ( table ). for hi-non b and s. pneumoniae, there was a . -fold or . -fold increase in average microbial load in non-acute samples collected at the end of follow-up compared to acute samples collected during an uri episode, respectively (p-values ≤ . ). there was no significant difference in delay between symptom onset and specimen collection in acute samples containing one (median delay: days; interquartile range (iqr): - ) or more (median delay: days; iqr: - ) organisms (p-value = . ). six acute specimens were negative for all agents tested (median delay: . days; iqr: - ). thus, sampling delay is unlikely to account for any observed differences in ct-value distributions. we analyzed the patterns of infection with common respiratory agents in a well-defined population of military recruits. the use of highly sensitive multiplex pcr diagnostics allowed an accurate characterization of the spectrum of organisms contained in non-acute and acute samples. the data indicate co-circulation of several different viral agents, and high frequency of bacterial colonization in both non-acute and acute samples. up to one third of respiratory illness cases among army personnel are reportedly caused by viral or bacterial infections [ ] . the gathering of individuals from diverse geographic locations and the crowded living conditions increase the risk of microbe transmission in these settings [ ] . illnesses are usually self-limiting, although the emergence of highly virulent strains can lead to high morbidity and mortality [ ] . streptococcus bacteria, adenoviruses, coronaviruses and influenza are among the most widely distributed microbes in the military environment, and are implicated in > % of febrile illness cases reported at military medical facilities [ ] . we identified each of these organisms in one or more samples. for most of these microbes, overall detection frequencies were comparable in non-acute and acute samples, although influenza b and coronavirus were more commonly identified among acute specimens. other infectious agents commonly circulating among military personnel include h. influenzae, rhinovirus, and, to a lesser extent, parainfluenza, rsv, and l. pneumophila, although their presence does not necessarily imply the occurrence of clinical symptoms [ ] [ ] [ ] . h. influenzae and rhinoviruses were the most frequently detected organisms in our population in both non-acute and acute samples. we detected parainfluenza and l. pneumophila, but we did not find rsv in any of our samples. for individuals developing uri during follow-up, illness etiology could not be unequivocally determined. among acute samples, hi-b was the most frequently detected organism. it was the sole agent identified in % of acute specimens, while it was co-detected with other microbes in > % of acute samples. however, colonisation with hi-b was also common among non-acute baseline samples, where it was detected alone or in combination with other microbes in . % and . % of specimens, respectively. for organisms rarely detected among asymptomatic individuals but frequently found in acute samples, a causal association may be more likely. for instance, influenza b was detected in none of the non-acute, but . % of acute samples. similarly, the proportion of both hi-nonb-and rhinovirus-positive samples was significantly lower among non-acute specimens collected at baseline compared to acute samples. however, > % of acute samples positive for hi-non b, rhinovirus or influenza b were also positive for one or more additional microbe, so that a causal table ) association could not be determined. some agents, such as hi-non b or adenovirus, were most frequently detected in non-acute samples collected at the end of follow-up, possibly indicating post-infectious shedding or persistent infection at sub-clinical levels. in the clinical setting, overlapping clinical presentations and poor capabilities to determine the etiology of respiratory illnesses often lead to inappropriate treatment with broad-spectrum antibiotics [ ] . this might occur even more frequently in the military setting, where molecular diagnostic tools are usually inaccessible [ ] . since a considerable fraction of respiratory illnesses is caused by viruses, the unsubstantiated use of antibiotics is particularly problematic, because it can lead to negative health outcomes and promote the development of antimicrobial resistance [ ] . studies evaluating the impact of multiplex diagnostic procedures on patient management report inconsistent results. in the outpatient setting, access to rapid molecular diagnostic tools for respiratory pathogens significantly reduced antibiotic a b fig. cycle threshold value distribution in non-acute and acute samples. ct-value distribution for selected a bacteria and b viruses detected in non-acute samples collected at the start or end of the training period (orange bars) or acute samples from individuals experiencing an upper respiratory tract infection during follow-up (blue bars). a ct-value of < was considered a positive result prescription rates for patients presenting with respiratory illness [ ] . however, these findings were not confirmed in the hospital setting. pcr-based testing failed to reduce hospital admissions and duration of hospital stay in patients with acute respiratory infection [ , ] . although molecular diagnostic tools may help to differentiate bacterial and viral respiratory agents, it is unlikely that antibacterial treatment would be terminated based on the mere presence of viral agents in an acute respiratory sample, especially considering the high rates of bacterial co-infection [ ] . quantitative or semi-quantitative diagnostic tools can potentially help define clinically significant pathogen densities, and have proven highly valuable to understand the dynamics of diarrheal disease [ ] and to improve the management of gastrointestinal illnesses [ ] . among acute diarrhea patients, quantitative amplification of norovirus rna from fecal samples can help determine pathogen load thresholds that effectively distinguish between causal association and sub-pathogenic carriage [ ] . similarly, rotavirus load correlates with disease severity among children with gastroenteritis [ ] . because of the crucial role of microbial replication in viral pathogenesis, the value of pathogen load quantitation could be most clearly established for gastrointestinal illnesses of viral etiology, although some evidence is available for bacterial infections as well. for instance, microbial load of enteropathogenic e. coli is significantly higher among children with diarrhea compared to control subjects, especially when enteropathogenic e. coli is the sole agent identified [ ] . in this study, tobit regression indicated significantly lower microbial load in non-acute relative to acute samples for rhinovirus, hi-nonb, and s. pneumoniae. however, due to a substantial overlap in ct-value distributions, it was not possible to identify a ct-value threshold indicating causality for any of these organisms. previous studies assessing the association of viral load with clinical symptoms of respiratory infections reported similar findings. mean viral load for rhinovirus and six additional viruses was significantly higher in upper respiratory tract aspirates from children with pneumonia compared to healthy controls, but the overlap in viral load distribution was substantial [ ] . in pediatric patients, high rhinovirus load was associated with the presence of lower respiratory tract symptoms [ , ] , but a threshold for clinical relevance could only be determined if rhinovirus was the sole agent identified [ ] . additional studies reported a correlation between microbial load and occurrence or severity of respiratory symptoms for rsv [ ] , bocavirus [ ] , and human metapneumovirus (hmpv) [ , ] , although these findings were inconsistent [ , ] or conditional on the presence of the virus as a single microbe [ ] . we did not detect any significant association between microbial load and clinical manifestations for viruses other than rhinovirus. for both h. influenzae and streptococcus species, previous studies reported a significant correlation of bacterial densities with clinical manifestations of disease [ ] . in young patients with acute respiratory tract infection, s. pneumoniae load fluctuated with symptom incidence and resolution [ ] . among children hospitalized with pneumonia, median nasopharyngeal s. pneumoniae load was substantially higher compared to healthy controls [ ] . pneumococcal density was also associated with severity of symptoms [ ] and increased duration of children's hospital stay [ ] . similar associations were observed in pneumonic adults, although the correlation was not significant in this population [ ] . the association between microbial load and clinical manifestations may depend on specific pathogen-host interactions. if pathogenesis is primarily related to microbial replication, a stronger correlation between microbial load and illness magnitude may be observed [ ] . if clinical manifestations are largely attributable to host immune defences or bacterial toxins, the correlation with microbial load may not be obvious [ ] . temporal variations in microbial load may also play an important role if the quantity of nucleic acid is significantly more abundant at the time and location of pathology [ , ] . in acute respiratory illness patients, high bacterial colonization densities are often associated with the presence of viral co-infections [ ] , and clinical manifestations may vary depending on specific co-infection patterns [ ] . the ecology of respiratory pathogens is also likely to be influenced by the living conditions in military settings. mixing of individuals from diverse backgrounds living in close-quarters with high levels of inter-personal contact increases the potential for introduction and spread of multiple microbes in this population, which could account for the broad range of organisms and co-detections in this study. we analysed both non-acute and acute samples from a closely monitored population in a semi-closed, longitudinal setting. the study population was well-defined and relatively homogeneous with regards to demographics and living conditions. however, our findings may not be applicable to populations with different socio-demographic characteristics and populations outside the military environment, such as cohorts of children among whom the impact of respiratory infections may be greater. the frequent co-detection of multiple respiratory agents and the failure to distinguish between viable and dead organisms, or microbes that colonize the host at sub-pathogenic levels, may prevent the unambiguous interpretation of test results [ ] . a positive result may indicate illness aetiology, asymptomatic colonisation, post-infectious shedding, or an incipient infection. therefore, ct-values may not always be a reliable surrogate for infectious load. samples from only two out of six cohorts were tested by real-time pcr. although there might be bias from seasonal effects, these are usually less pronounced in the tropics. given the relatively low frequencies of viral detection, a larger sample size and a longer follow-up may have captured a more precise picture of infection patterns in this population. this study was also limited to the detection of organisms contained in the respiratory panel. we cannot exclude the presence of additional organisms in our specimens. in addition, the data were obtained from throat and nasal swab samples, but our findings may not apply to nasopharyngeal or sputum specimens. finally, the quality and quantity of material obtained through nose and throat swabs may differ significantly among subjects, and the success of pcr-based methods also depends on the availability of intact genome sequences and the absence of random mutations. overall, the multiplex respiratory panel provided a comprehensive characterization of the microbe spectrum contained in non-acute and acute respiratory samples collected among recruits. however, semi-quantitative assessment of microbial load could not reliably distinguish between symptomatic and asymptomatic samples. more research is warranted to compare new multiplex diagnostic techniques with traditional methods and evaluate their potential with regards to diagnostic accuracy [ ] and clinical utility [ , ] in the context of respiratory infections. additional file : dataset. title of data: semi-quantitative microbial load in throat and nasal swab samples from thai army recruits. description of data: semi-quantitative microbial load in non-acute and acute throat and nasal swab samples from thai army recruits, determined using a commercial multiplex real-time pcr assay comprising bacterial, viral and fungal targets; includes names, labels, and coding for individual variables. (xls kb) abbreviations pcr: polymerase chain reactionuriupper respiratory illnessiliinfluenza-like illnessftdfast track diagnosticsctcycle thresholdhi-bhaemophilus influenzae type bhi-nonbnon-type b haemophilus influenzaeiqrinterquartile rangehmpvhuman metapneumovirus we are grateful to the participants of this study, the royal thai army, and the clinical, laboratory and administrative personnel at afrims. material has been reviewed by the walter reed army institute of research. there is no objection to its presentation and/or publication. the opinions or assertions contained herein are the private views of the authors, and are not to be construed as official, or as reflecting true views of the department of the army or the department of defense. this work was supported by the united states department of defense -global emerging infectious disease surveillance (dod -geis), protocol a. the datasets analysed for the current study are available as additional file in this publication. author's contributions cct conceived the idea for this paper, vo conducted the analysis and wrote the manuscript. ka, aw, lm, de, rr, and ss participated in project oversight. sf, rg, rr, ss, and iy participated in the design of the study. all authors contributed to drafting the manuscript and approved the final submission. the study was approved by the institutional review boards of the royal thai army in bangkok, thailand, the walter reed army institute of research and the london school of hygiene & tropical medicine. all participants provided written informed consent. the investigators have adhered to the policies for protection of human subjects as prescribed in army regulation - . not applicable. cct is associate editor for bmc infectious diseases, research area viral diseases. all other authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. author details saw swee hock school of public health, national university of singapore and national university health system, singapore , singapore. advances in multiparametric molecular diagnostics technologies for respiratory tract infections the increasing application of multiplex nucleic acid detection tests to the diagnosis of syndromic infections infectious diseases society of a. an unmet medical need: rapid molecular diagnostics tests for respiratory tract infections frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values elevated transmission of upper respiratory illness among new recruits in military barracks in thailand. influenza other respir viruses respiratory tract infections in the military environment environmental factors, immune changes and respiratory diseases in troops during military activities overcrowding and mortality during the influenza pandemic of broad spectrum respiratory pathogen analysis of throat swabs from military 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different virus load of human bocavirus in patients with lower respiratory tract infection association between high nasopharyngeal viral load and disease severity in children with human metapneumovirus infection clinical disease and viral load in children infected with respiratory syncytial virus or human metapneumovirus clinical characteristics and viral load of respiratory syncytial virus and human metapneumovirus in children hospitaled for acute lower respiratory tract infection analysis of viral load in children infected with human metapneumovirus human bocavirus in children: mono-detection, high viral load and viraemia are associated with respiratory tract infection association between nasopharyngeal load of streptococcus pneumoniae, viral coinfection, and radiologically confirmed pneumonia in vietnamese children nasopharyngeal pneumococcal density and evolution of acute respiratory illnesses in young children dna bacterial load in children with bacteremic pneumococcal community-acquired pneumonia dna bacterial load in children and adolescents with pneumococcal pneumonia and empyema comprehensive molecular testing for respiratory pathogens in community-acquired pneumonia new concepts in diagnostics for infectious diarrhea high nasopharyngeal pneumococcal density, increased by viral coinfection, is associated with invasive pneumococcal pneumonia clinical characteristics of children with lower respiratory tract infections are dependent on the carriage of specific pathogens in the nasopharynx development of two real-time multiplex pcr assays for the detection and quantification of eight key bacterial pathogens in lower respiratory tract infections key: cord- -dc xbxha authors: cowling, benjamin j; lau, lincoln lh; wu, peng; wong, helen wc; fang, vicky j; riley, steven; nishiura, hiroshi title: entry screening to delay local transmission of pandemic influenza a (h n ) date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: dc xbxha background: after the who issued the global alert for pandemic influenza a (h n ), many national health agencies began to screen travelers on entry in airports, ports and border crossings to try to delay local transmission. methods: we reviewed entry screening policies adopted by different nations and ascertained dates of official report of the first laboratory-confirmed imported h n case and the first laboratory-confirmed untraceable or 'local' h n case. results: implementation of entry screening policies was associated with on average additional - day delays in local transmission compared to nations that did not implement entry screening, with lower bounds of % confidence intervals consistent with no additional delays and upper bounds extending to - day additional delays. conclusions: entry screening may lead to short-term delays in local transmission of a novel strain of influenza virus. the resources required for implementation should be balanced against the expected benefits of entry screening. pandemic influenza a (h n ) virus emerged in mexico in early . rapid global spread was primarily associated with air travel [ ] . as the world health organization (who) raised their pandemic alert level to and then in april, national health agencies across the world activated their pandemic plans. following guidance by who, many authorities began to screen travelers on entry in airports, ports and border crossings, isolate suspected or confirmed cases, and quarantine their close contacts [ ] . exit screening was not implemented by source nations. modeling studies suggested that entry screening could not prevent introduction but might be able to delay local epidemics by a few weeks [ ] [ ] [ ] [ ] . entry screening and quarantine did not substantially delay introductions in previous pandemics [ ] . we reviewed entry screening policies adopted by different nations and estimated the range of delays in local epidemics associated with entry screening. to explore potential delays in local h n transmission associated with entry screening, we ascertained dates of official report of the first laboratory-confirmed imported h n case and the first laboratory-confirmed untraceable or 'local' h n case (i.e. a case not otherwise epidemiologically linked with international travel, contact with an imported case or their secondary infectees) and the interval between these two events. we calculated the additional delays associated with entry screening tools versus the observed delays in nations that did not screen. since the data did not follow a normal distribution we estimated % confidence intervals for these differences using bootstrapping, which is a resampling technique suitable for statistical inference in small sample sizes with non-normal distributions [ ] . we based each bootstrap confidence interval on , resamples. statistical analyses were conducted using r (r development core team, vienna, austria) [ ] . the study was conducted between july and august , . the methods of entry screening employed were identified by review of official national health ministry websites and the media, and google searches in english using queries of the form ("<country name>" and ("influenza" or "h n " or "swine flu" or "pandemic" or "mexican flu")). we included each nation that had notified more than confirmed h n cases to the world health organization by july , , except mexico and the united states where local transmission occurred prior to the who global alert. to determine the date of first imported case and first local case the search queries were extended accordingly. queries were translated by google language tools http:// www.google.com/language_tools and babelfish http:// babelfish.yahoo.com to local official languages and searches were repeated to further increase our scope. we searched for websites in languages including chinese, dutch, french, german, greek, hebrew, japanese, korean, portuguese, spanish and thai. we identified nations that had reported more than confirmed h n cases to the world health organization by july (we included hong kong separately from mainland china as it has separate administration) (additional file ). the date of the first untraceable local case could not be determined for / of the nations. further details and web links to relevant reports and original data sources are available from the corresponding author on request. we identified four broad approaches to entry screening. first, temperature checks were performed onboard aircraft prior to disembarkation. second, health declaration forms were collected from every traveler or all travelers from countries identified with confirmed h n cases. third, arriving travelers were observed by alert staff for influenza symptoms (e.g. cough). fourth, travelers were scanned for elevated body temperature by thermal scanners. in the majority of countries screening was implemented by may , although we were unable to determine whether there were any substantial changes in screening protocols after commencement of screening but before confirmation of the first local case. because of stochasticity (i.e. chance variations in the occurrence of secondary transmission due to small number of cases initially), the single observed interval between the confirmation of the first imported case and the first local case in a given country is not easily interpretable. we examined patterns in aggregated data expecting that errors due to stochasticity should tend to average out in comparisons between groups of countries using similar tools. two nations (china and japan) implemented all four tools. five nations did not implement any of the four. table shows the intervals between confirmations of first imported cases and first local cases, categorized by entry screening tools. overall, implementation of the four tools alone or in combination were associated with on average additional - day delays in local transmission compared to nations that did not implement entry screening, with lower bounds of % confidence intervals consistent with no additional delays and upper bounds extending to - day additional delays ( table ) . dates of illness onset were available for the first imported cases in / nations and the first local cases in / nations, and mean delays were similar in that subset (data not shown). our results suggest that entry screening did not lead to substantial delays in local h n transmission (table ) . this empirical study is consistent with theoretical results from previous modeling studies [ ] [ ] [ ] [ ] and findings from previous pandemics [ ] . while longer delays in local transmission to the summer in countries in the northern hemisphere could have substantially aided pandemic mitigation, due to seasonal factors [ ] and school vacations [ , ] leading to lower peak attack rates [ ] , the observed delays in the present pandemic suggest entry screening provided around - weeks of additional time for preparation and planning. while our study focused on the impact of entry screening, some nations also implemented other containment and mitigation measures, such as isolation of suspected or confirmed cases, quarantine of their contacts with or without antiviral chemoprophylaxis, school closures or other social distancing measures, and public health campaigns to improve hygiene. most nations enhanced their influenza surveillance. if countries that expended greater effort into entry screening also had more effective containment and mitigation measures in the general population, these might have led us to overestimate the effect of entry screening. conversely, if countries that expanded greater effort into entry screening also tended to have better influenza surveillance and were able to identify local transmission earlier, we may have underestimated the effect of entry screening. other differences between countries in laboratory capacity and availability of public health resources may also have confounded our evaluation, and all of these factors are limitations of our study. previous mathematical modeling studies have questioned the value of entry screening, since it could only delay rather than prevent local epidemics [ ] [ ] [ ] [ ] . however, most models assumed that source countries would conduct exit screening and infectious cases would not travel [ ] [ ] [ ] [ ] . in such a scenario it is not surprising that entry screening would add little benefit, since most journeys are shorter than the average . - day incubation period for influenza a virus infections [ , ] . screening is unlikely to identify % of ill travelers, while some might use antipyretics to reduce a fever prior to passing through thermal scanners, or fail to report symptoms on declaration forms. many individuals with subclinical or asymptomatic illness would not be identified, and could initiate outbreaks after arrival [ ] . in hong kong, only one third of confirmed imported h n cases were identified through screening on entry to hong kong, the majority of imported cases were identified through the local health care system after arrival (t. tsang, personal communication). a similar experience has been reported in singapore [ ] . nevertheless, entry screening could act as a deterrent to traveling when ill or lead to other indirect benefits such as improving public awareness of the pandemic. for entry screening to be successfully employed, substantial resources are required to identify the small fraction of travelers who may have h n infection [ ] . further resources may be needed to isolate identified cases, and trace and quarantine close contacts. an important caveat is that a delay in inevitable local transmission of a pandemic virus may not be desirable if it would defer local transmission into a season associated with higher transmissibility such as the winter in temperate regions [ ] , or if it led to importation and local transmission of antiviral resistant strains [ ] . in addition to the caveats on potential confounding by resource availability, competing interventions, and other differences discussed above, there are a number of further limitations to our study. first, identification, confirmation and notification of h n cases is unlikely to have been perfect given the mild and self-limiting nature of most infections, and dates of importation and local transmission that we report may lag behind the true events of interest. nations that devoted greater resources to entry screening may have identified imported cases earlier. secondly, we have not considered the size of local epidemics, or how the degree of connectivity with source regions (for example the number of travelers per day) might relate to time delays between imported and local cases. thirdly, by focusing on nations with at least confirmed cases by july , we may have excluded nations where entry screening was more effective in delaying local transmission, or excluded some nations with fewer resources available for surveillance and confirmation of local cases. fourthly, while we searched for the dates of reporting of the first imported case and first local case, these dates may not have corresponded exactly to the dates of identification and confirmation of those cases, since in some cases delays may have occurred for various reasons including political considerations. finally, we collected data from online sources including official government websites, and we have included the hyperlinks in additional file , but information available on the internet could be inaccurate. in conclusion, our results suggest that entry screening could delay local transmission for an additional - weeks. the uncertainty bound of the delay estimates ranged from no delay to - days delay. a delay of - weeks could be useful if the additional time permits more comprehensive planning and preparation for a local epidemic, or shortens the time required for other pandemic mitigation measures such as school closures to be sustained. however the benefits of local screening should be balanced against the considerable resources required to implement screening [ ] . our empirical results are consistent with the modeling literature, and support the guidance from the world health organization that entry screening can only prevent local spread for a short period of time [ ] . additional file : use of entry screening* and interval between confirmation of first imported pandemic influenza a (h n ) case and confirmation of first untraceable local case spread of a novel influenza a (h n ) virus via global airline transportation world health organization: pandemic influenza preparedness and response delaying the international spread of pandemic influenza entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation the waiting time for inter-country spread of pandemic influenza airport entry screening in response to pandemic influenza: modeling and analysis world health organization (who) writing group: nonpharmaceutical interventions for pandemic influenza, international measures the jackknife and bootstrap team: r: a language and environment for statistical computing estimating the impact of school closure on influenza transmission from sentinel data reduced transmissibility of pandemic influenza a (h n ) associated with school closures and summer vacation controlling pandemic flu: the value of international air travel restrictions incubation periods of acute respiratory viral infections: a systematic review world health organization: new influenza a (h n ) virus: who guidance on public health measures epidemiology of travel-associated pandemic (h n ) infection in patients screening for influenza infection in international airline travelers hedging against antiviral resistance during the next influenza pandemic using small stockpiles of an alternative chemotherapy pre-publication history the pre-publication history for this paper can be accessed here entry screening to delay local transmission of pandemic influenza a (h n ) authors' contributions bjc conceived of the study and drafted the manuscript. bjc, llh, pw and hwcw participated in data collection. vjf conducted the statistical analyses. sr participated in interpreting the results. hn participated in planning the study and interpreting the results. all authors were involved in critical review and editing of the first draft, and subsequent revisions to the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -dk snw r authors: yang, lin; chan, king pan; wong, chit ming; chiu, susan shui seng; magalhaes, ricardo j. soares; thach, thuan quoc; peiris, joseph syrial malik; clements, archie c. a.; hu, wenbiao title: comparison of influenza disease burden in older populations of hong kong and brisbane: the impact of influenza and pneumococcal vaccination date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: dk snw r background: influenza and pneumococcal vaccine uptake in the older population aged years or over of hong kong dramatically increased since the sars outbreak. this study is aimed to evaluate the impact of increased coverage of influenza and pneumococcal vaccines by comparing the change of disease burden in the older population of hong kong, with the burden in the older population of brisbane with relatively high vaccine coverage in the past fifteen years. methods: time series segmented regression models were applied to weekly numbers of cause-specific mortality or hospitalization of hong kong and brisbane. annual excess rates of mortality or hospitalization associated with influenza in the older population were estimated for the pre-sars (reference period), post-sars and post-pandemic period, respectively. the rate ratios (rrs) between these periods were also calculated to assess the relative change of disease burden. results: compared to the pre-sars period, excess rates of mortality associated with influenza during the post-sars period in hong kong decreased for cardiorespiratory diseases (rr = . , % ci . , . ), stroke (rr = . , % ci . , . ), and ischemic heart diseases (rr = . , % ci . , . ). the corresponding rrs in brisbane were . ( % ci . , . ), . ( . , . ), and . ( . , . ), respectively. only the mortality of ischemic heart diseases showed a greater reduction in hong kong than in brisbane. during the post-pandemic period, excess rates of all-cause mortality increased in hong kong, but to a lesser extent than in brisbane (rr = . vs . ). conclusion: a relative decrease (or less of an increase) of influenza disease burden was observed in the older population of hong kong after increased coverage of influenza and pneumococcal vaccines in this population, as compared to those of brisbane where vaccination rates remained stable. the lack of significant findings in some disease categories highlights the challenges of evaluating the benefits of vaccination at the population level. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. globally, influenza has been associated with a heavy burden of mortality and morbidity [ ] . vaccination remains an important strategy to reduce disease severity and virus transmission within the community [ ] . although numerous clinical trials have demonstrated the effectiveness of influenza vaccines in children [ ] , adults [ ] , and healthy elderly people [ ] , few studies have included high-risk groups particularly the elderly with underlying chronic conditions. a recent systematic review also concluded that influenza vaccine only had a modest effect in preventing influenza infections among community-dwelling elderly people [ ] . previous cohort or case-control studies reported that vaccine effectiveness was % in preventing all-cause mortality [ ] . however, according to a study in the us, < % of all-cause mortality was specifically associated with influenza, suggesting that the estimates from the observational studies could have been seriously overestimated [ , ] . another ecological study conducted in ontario, canada also found significant relative reductions in influenza-associated mortality and health care utilization after the introduction of universal vaccination since in those aged < yrs., but not in those aged ≥ yrs. [ ] . taken together, available evidence suggests that there is a need to assess the effect of influenza vaccination at the population level, especially for those aged ≥ years. previous studies in hong kong have shown that annual vaccination rates for community-dwelling elderly people were less than % during the period - [ ] , but increased to more than % in - [ ] . since october , a subsidy of hk$ (us$ . ) for annual influenza vaccine and hk$ (us$ . ) for pneumococcal vaccine has been provided to those aged ≥ years under the elderly vaccination subsidy scheme. the vaccination rate remained nearly % in the elderly in the / season [ ] . in australia, the federal government has been providing free influenza vaccinations for people aged ≥ years since , and the coverage rates in the older population remained between and % during the period of - [ ] . unlike hong kong, where the sars outbreak and a new subsidy program greatly increased influenza and pneumococcal vaccine coverage among the older population, brisbane has had a relatively stable vaccination rate for both vaccines since . here we hypothesize that if influenza vaccine was effective in older people, the dramatically increased uptake among the older population of hong kong since sars could have resulted in a reduced influenza disease burden. we expect such a reduction to be larger than in brisbane, where uptake of the vaccine has remained stable among the community dwelling elderly people. further decrease in disease burden of influenza could have occurred after , as the increased uptake of pneumococcal vaccines in the older population could have reduced the risk of secondary bacterial pneumonia after influenza infections. hong kong is located at a latitude of °n, with a population of . million in residing in an area of km . brisbane is located at a latitude of °s, with a population of . million in and a territory of km . both hong kong and brisbane have a subtropical climate, with average temperatures of °c and °c, and relative humidity of and %, respectively. in terms of socioeconomic conditions, both are developed cities with a comparable gross domestic product per capita ($ , in hong kong vs. $ , in australia in ) [ ] . during the study period, the percentage of the population aged ≥ years was . % in hong kong and . % in brisbane. virology data, death registry data, hospital admission, and meteorological data during the study period of to were obtained from difference data sources of hong kong and brisbane, respectively. the detailed information is provided in additional file : appendix . in hong kong, seasonal influenza peaks during january -march and june -july, whereas in australia the peak usually occurs in august -october (fig. ) . given that seasonal influenza peaks at different times in these two cites, we defined annual study period as january -december in hong kong and may -april of next year in brisbane. these periods begin three months after the usual launch dates for the annual seasonal influenza vaccination campaigns (march in brisbane and september in hong kong), which shall allow for a valid assessment of the vaccination effectiveness. the whole study period was divided into the pre-sars, sars, post-sars, influenza pandemic and post-pandemic periods. the pre-sars period in hong kong was featured with a much lower vaccination rate in the older population compared to the post-sars and post-pandemic periods, whereas the vaccination rate in the older population of brisbane was stable across these periods. the burden during the sars and pandemic periods was not presented, as this was highly affected by different control measures adopted by the health authorities of hong kong and brisbane. the cut-off dates for these periods in hong kong and brisbane are listed in additional file : appendix . we constructed time series segmented regression models to estimate cause-specific mortality or hospitalization risks associated with influenza in the older population during the pre-sars, post-sars, and post-pandemic periods for hong kong and brisbane. the proxy variable for influenza in the model was the percentage of specimens that tested positive for influenza each week out of annual total number in each city. the reason why we decided to use annual percentage instead of weekly proportion used in our previous studies is that total numbers of specimens were not available in brisbane during the whole study period. we added seasonal trends, temperature, humidity, and other respiratory viruses to the model as covariates to estimate influenza-associated excess risks. dummy variables for the pre-sars, post-sars, and post-pandemic periods, together with the interaction terms between these period dummies and the virus activity variables, were also added to test the statistical differences in risk estimates between the different periods, respectively for hong kong and brisbane. the best-fit models were chosen by the minimal generalized cross-validation (gcv), according to our previous study [ ] . baseline rates of cause-specific mortality and hospitalizations associated with influenza were calculated for different periods by setting the virus proxy to zero and the corresponding period dummy to one (other dummies were simultaneously set to zero). we first estimated excess numbers by subtracting baseline rates from the observed data, and calculated excess rates (er) by dividing the excess numbers with age-specific population size. we calculated the % confidence interval (ci) of er by bootstrapping times. because the periods were of different lengths, annual excess rates (aer) of mortality (or hospitalizations) were calculated to facilitate comparisons between different periods. for each disease category, the rate ratios (rrs) of post-sars (or post-pandemic) versus pre-sars were derived by dividing annual excess rates during the post-sars (or post-pandemic) period with those of the pre-sars period (as reference): rr = aer (post-sars) / aer (pre-sars). since the pre-sars period was treated as the reference period in this study, hereafter the post-sars rr refers to the risk ratio of mortality or hospitalization in the post-sars period relative to those in the pre-sars period. similarly, the post-pandemic rr refers to the risk ratio of mortality or hospitalization in the post-pandemic period relative to those in the pre-sars period. the % ci and p-value of rr were derived from a normal approximation of their logarithmic transformations [ ] . we also conducted a subset analysis by using the data of influenza peak seasons only. the influenza season was defined as january to july in hong kong, and may to november in brisbane. we conducted another subset analysis by excluding the data for the mismatched years ( , , and in this study). all of the analyses were conducted in r software version . . . the significance level was set to . for all analyses. during the study period, there were around , and , people older than years living in hong kong and brisbane, respectively (tables and ). compared to hong kong, during the study period brisbane had higher mortality rates for all-cause ( . vs . per , population), cardiorespiratory diseases (crd, . vs . ), stroke ( . vs . ) and ischemic heart diseases (ihd, . vs . ), but a lower rate for pneumonia and influenza (p&i, . vs . ), and a comparable rate for chronic obstructive pulmonary disease (copd, . vs . ) (additional file : appendix ). all the cause-specific hospitalization rates were much lower in brisbane than in hong kong ( . vs . per , population for crd, . vs . for p&i, . vs . for copd, . vs . for stroke), with the only exception of ihd ( . vs . ) (additional file : appendix ). hong kong and brisbane have opposite seasonal climate patterns; the former is located in the northern hemisphere and the latter in the southern hemisphere. mean temperature and relative humidity of brisbane were lower than those of hong kong (additional file : appendix ). compared to the pre-sars period, average number of hospitalizations in hong kong was lower in the post-sars period for most disease categories, with the exception of a three-fold increase in p&i hospitalizations. this significant increase is likely due to the change in coding practice after the sars outbreak (table ). average number of hospitalizations was higher in the post-pandemic period for all the disease categories. weekly figures for mortality and hospitalization in brisbane did not obviously differ across the pre-sars, post-sars, and post-pandemic periods ( table ) . due to negative estimates in the annual excess rates for mortality, the post-sars rr could not be estimated for ihd hospitalizations in hong kong, and p&i mortality, stroke, and ihd hospitalizations in brisbane. similarly, the post-pandemic rr could not be estimated for p&i and ihd hospitalization in hong kong, and stroke and copd hospitalization in brisbane (additional file : appendix ). influenza-associated all-cause mortality rates were found increased after sars in hong kong (post-sars vs pre-sars rr = . ) but decreased in brisbane (rr = . ). the post-sars copd mortality increased in both cities ( . and . in hong kong and brisbane, table ). decreased excess rates of mortality in hong kong were observed for crd, stroke, and ihd mortality (rr = . and . ), while the corresponding rrs in brisbane were . and . , respectively. only ihd mortality had a greater reduction observed in hong kong than in note. weekly positive percentage (%) is defined as the percentage of positive specimens among annual total specimens brisbane (rr = . vs . ). none of rr estimates for the control disease injuries were significant (data not shown). compared to the pre-sars period, excess rates of crd hospitalizations in the post-sars period decreased in hong kong, but increased markedly in brisbane (rr = . vs . ). influenza-associated hospitalizations for p&i and copd increased more in brisbane than in hong kong (rr = . vs . and . vs . ). compared to the pre-sars period, excess mortality rates increased in hong kong for all the disease categories except for ihd, but only all-cause and copd mortality increased in brisbane. difference between hong kong and brisbane was found statistically significant for all-cause and stroke mortality. annual excess rates of all-cause mortality increased in hong kong to a lesser extent than in brisbane (rr = . vs . ), whereas an opposite trend was observed for stroke mortality (rr = . vs . ). subset analysis with peaks seasons only, or vaccine matched years only, generally derived larger rr estimates ( table ). the estimates for crd became significantly higher than one, and the brisbane estimates were much greater than the hong kong ones. many outcomes could not be estimated due to negative values of excess rates. in this study, we estimated excess rates of mortality or hospitalizations attributable to influenza in different periods (pre-sars, post-sars, and post-pandemic) for two subtropical cities hong kong and brisbane. we hypothesized that the influenza disease burden decreased more, or increased less, in hong kong than in brisbane since , because the uptake rate of influenza and pneumococcal vaccines increased more markedly in hong kong than in brisbane during the same period. kwong et al. [ ] compared the relative change of disease burden in ontario, where a universal influenza vaccination program was launched, to that in other canadian provinces without such a policy. they found that influenza-associated mortality fell in ontario and other provinces, but a larger reduction occurred in ontario. in this study, we found that excess rates of ihd mortality decreased more from the pre-sars to the post-sars period in hong kong than in brisbane, but with regard to the other mortality outcomes, excess rates increased more in hong kong. with respect to hospitalization in the post-sars period, significantly lower excess rates were only found for crd in hong kong. p&i and copd hospitalization rates increased in both cities, but to a lesser extent in hong kong. many countries have recommended annual influenza vaccination or providing subsidy programs to the older note. weekly positive percentage (%) is defined as the percentage of positive specimens among annual total specimens population [ ] . however, due to ethical concerns, a large body of knowledge on the effectiveness of influenza vaccine in the older population has been derived from observational studies, as few randomized controlled trials have been conducted in this high-risk population. a review by goodwin et al. found that seroprotection and seroconversion achieved in the older population after vaccination was only - % of vaccine response in younger adults [ ] . a cochrane review concluded that influenza vaccines were of limited effects, which could probably be explained by weak antibody response in the older population [ ] ; however, in a recent reanalysis using the same data, walter et al. made the opposite conclusion [ ] . they estimated that influenza vaccine resulted in a % reduction in complications after influenza infections, % in influenza-like illnesses, and % in laboratory confirmed influenza infections, specifically during influenza epidemics. however, there is still an on-going debate on vaccine effectiveness in the older population. our findings add some evidence of a decrease, or a slow increase, in influenza-associated disease burden among the older population, following a marked increase in influenza vaccine coverage at the population level. however, it should be noted that this effect could have been partially caused by an increase of pneumococcal vaccination at the same time. given that the effectiveness of influenza vaccine is affected by many factors including pre-existing immunity, antigenic shift, and underlying condition, it is not surprising that we failed to find consistent and significant estimates. this also highlights the challenges of evaluating the benefits of vaccination at the population level, even in the most susceptible older populations. a higher disease burden was found in both hong kong and brisbane after , which was consistent with the findings of our previous studies and others [ , ] . the potential explanation could be that h n was more often predominant after and this subtype has been found associated with higher disease burden than h n and b. the point estimates of rrs were sensitive to modeling parameters, and most had wide confidence intervals. this could be due to only a relatively small proportion of deaths or hospitalizations attributable to influenza. according to our previous studies, each year influenza is associated with nearly deaths and , hospitalizations in hong kong, accounting for only and % of annual total deaths and hospitalizations, respectively [ ] [ ] [ ] [ ] . negative estimates of excess rates were occasionally derived from some disease outcomes, making it difficult to assess the relative increase/decrease between two cities. relatively small counts in brisbane could be the reason why we obtained extremely large or small point estimates for the post-sars rr of crd hospitalizations and the post-pandemic rr of stroke mortality in brisbane; hence, these rrs need to be interpreted with caution. unfortunately, good quality mortality, hospitalization, influenza surveillance and vaccination data are available in few subtropical countries/regions. nevertheless, this study is the first to investigate the effectiveness of influenza and pneumococcal vaccination at the population level in warm climates, to our best knowledge. there are several limitations in this study. first, ecological fallacy is unavoidable given the ecological study design. individual vaccination status of those who have died or been hospitalized is unknown and the outcome variables are not specific to influenza. nevertheless, we have used a previously validated modeling approach to estimate disease burden associated with influenza. second, we assume that circulating influenza strains and pre-exisitng immunity at the population level are similar between hong kong and brisbane. therefore, a relative decrease (or less of an increase) in influenza-associated disease burden could reflect the effectiveness of influenza vaccination in terms of reducing adverse outcomes after influenza infections. this assumption may not hold, but there is also no strong evidence against it. third, only two to five years of data were included in each study period, because influenza virology and hospitalization data prior to were not available in hong kong or brisbane. our model obtained some unstable points estimates, especially in the pre-sars period, which could be due to the short time series and low counts. last but not least, although we have carefully adjusted for seasonal trends, temperatures, and humidity in our models, there are many confounding factors that remain unadjusted for in this study, such as the prevalence of underlying condition, and difference in health-seeking behaviors between two older populations. in conclusion, we found some but limited evidence that markedly increased rates of influenza and pneumococcal vaccination among the hong kong older people did lead to a reduction in their influenza disease burden. however, furture cohort studies with individual data are warranted to provide stronger evidence to support the promotion of influenza vaccination among the older population. additional file : appendix . additional information on data sources and statistical analysis. appendix . study periods defined for hong kong and brisbane. appendix . time series plots of weekly numbers of cause-specific mortality data of hong kong (black line) and brisbane (gray line). emerging infections: pandemic influenza influenza vaccines assessment of the efficacy and effectiveness of influenza vaccines in healthy children: systematic review vaccines for preventing influenza in healthy adults efficacy and effectiveness of influenza vaccines in elderly people: a systematic review vaccines for preventing influenza in the elderly effectiveness of influenza vaccine in the community-dwelling elderly mortality benefits of influenza vaccination in elderly people: an ongoing controversy impact of influenza vaccination on seasonal mortality in the us elderly population the effect of universal influenza immunization on mortality and health care use immune response to influenza vaccination in community-dwelling chinese elderly persons preliminary findings of a randomized trial of non-pharmaceutical interventions to prevent influenza transmission in households seasonal influenza vaccination coverage survey for the / season australian institute of health and welfare international monetary fund: world economic outlook database model selection in time series studies of influenza-associated mortality modern epidemiology influenza vaccination in : recommendations and vaccine use in developed and rapidly developing countries antibody response to influenza vaccination in the elderly: a quantitative review cochrane re-arranged: support for policies to vaccinate elderly people against influenza excess mortality associated with influenza a and b virus in hong kong excess mortality associated with the pandemic of influenza a(h n ) in hong kong age and sex differences in rates of influenza-associated hospitalizations in hong kong dietary habits and the short-term effects of air pollution on mortality in the chinese population in hong kong influenza-associated hospitalization in a subtropical city we thank the census and statistics department, hospital authority of hong kong, hong kong observatory, australian bureau of meteorology, australian institute of health and welfare, department of health of the australian government, and queensland health australia for providing the datasets used in this study. this study was supported by health and medical research fund (grant number: ), from the research fund secretariat, food and health bureau, the government of the hong kong special administrative region. the study sponsor was not involved in study design, data collection, analysis, and interpretation of data; nor in the writing of the report and in the decision to submit the paper for publication. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. administrative permissions from the data providers were required to access the raw data provided by the census and statistics department, hospital authority of hong kong, australian institute of health and welfare, department of health of the australian government, and queensland health australia). meteorological data are available at the websites of the hong kong observatory and australian bureau of meteorology. the r scripts used in this study are available from the corresponding author on request.authors' contributions ly, cmw, sssc, jsm and wbh designed the study and collected data. ly and kpc conducted data analysis and drafted the manuscript. sssc, rjsm, tqt, acac and wbh interpreted the results and finalized manuscript. all the authors gave final approval of the version to be published.ethics approval and consent to participate because no personal data was collected, individual consent was not required. the ethical approval was obtained from the institutional review board of the university of hong kong/hospital authority hong kong west cluster (reference number uw - ) and from the departmental research committee of the school of nursing, the hong kong polytechnic university (reference number hsears ). not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -e y l a authors: liu, ye; zhang, shoufeng; wu, xianfu; zhao, jinghui; hou, yanli; zhang, fei; velasco-villa, andres; rupprecht, charles e; hu, rongliang title: ferret badger rabies origin and its revisited importance as potential source of rabies transmission in southeast china date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: e y l a background: the frequent occurrence of ferret badger-associated human rabies cases in southeast china highlights the lack of laboratory-based surveillance and urges revisiting the potential importance of this animal in rabies transmission. to determine if the ferret badgers actually contribute to human and dog rabies cases, and the possible origin of the ferret badger-associated rabies in the region, an active rabies survey was conducted to determine the frequency of rabies infection and seroprevalence in dogs and ferret badgers. methods: a retrospective survey on rabies epidemics was performed in zhejiang, jiangxi and anhui provinces in southeast china. the brain tissues from ferret badgers and dogs were assayed by fluorescent antibody test. rabies virus was isolated and sequenced for phylogenetic analysis. the sera from ferret badgers and dogs were titrated using rabies virus neutralizing antibodies (vna) test. results: the ferret badgers presented a higher percentage of rabies seroconversion than dogs did in the endemic region, reaching a maximum of % in the collected samples. nine ferret badger-associated rabies viruses were isolated, sequenced, and were phylogenetically clustered as a separate group. nucleotide sequence revealed . - . % homology within the ferret badger isolates, and - % homology to the dog isolates in the nucleoprotein and glycoprotein genes in the same rabies endemic regions. conclusions: our data suggest ferret badger-associated rabies has likely formed as an independent enzootic originating from dogs during the long-term rabies infestation in southeast china. the eventual role of fb rabies in public health remains unclear. however, management of ferret badger bites, rabies awareness and control in the related regions should be an immediate need. rabies is caused by neurotropic viruses in the genus lyssavirus, family rhabdoviridae, and is transmissible to all mammals [ ] . dogs are the main hosts responsible for human rabies in africa, latin americas and asia, especially in china [ , ] , where rabies is re-emerging as a major public health threat, and its severity is only second to hiv and tuberculosis (tb) among all reportable infectious diseases. from the annual~ human deaths, southeast china counts for most cases, with more than % attributed to rabid dog bites [ ] . notably, both human population and dog density are high in the region with low rabies vaccination coverage in dogs. given that the program of dog rabies elimination has not been listed in the priority of governmental agenda, it is possible that long term dog rabies enzootics will lead to spillover events of dog-associated rabies into wildlife species. in addition to rabies transmitted by rabid dogs, other sources of rabies exposure to humans, such as cats, ferret badgers (fb), and pigs, have been continuously reported in china [ ] [ ] [ ] [ ] [ ] . interestingly, in provinces like zhejiang, jiangxi and anhui, the percentage of dog-associated human rabies is relatively low. meanwhile, up to % of the reported human rabies cases were inferred to be caused by fb bites in some districts in zhejiang province from to [ ] . although rabies in badgers was previously recorded in other countries [ , ] , fb-associated human rabies has never been reported except in china [ , ] . the frequent occurrence of fb-associated human rabies cases in southeast china highlights the lack of laboratorybased surveillance and urges revisiting the potential importance of this animal in rabies transmission. nevertheless, management of such animal bites in humans needs a clear guideline on post-exposure prophylaxis (pep) for rabies. currently, fb trading and its meat consumption are common in the related areas, resulting in a frequent source of fb bite to humans. similar to severe acute respiratory syndrome (sars) outbreaks through consumption of civet in south china, the close and frequent contact of fb by humans could be an important factor in human rabies cases in southeast china. to determine if the fb actually contributes to human and dog rabies cases, and the possible origin of the fbassociated rabies in the region, we conducted an expanded retrospective/prospective epidemiological survey, which encompassed both descriptive and molecular epidemiological approaches. a retrospective survey on rabies epidemics was carried out in zhejiang, jiangxi and anhui provinces in southeast china. the data were collected and summarized from the provincial cdc surveillance system and epidemiological records. some information was obtained verbally after interviewing animal traders or hunters in the endemic areas. in human rabies cases and its potential association with fb transmission, we conducted a preliminary investigation of fb population density, exposure frequency of sick fb to humans, and management of rabies pep after a fb bite. dead or live fb collected in fields and houses, and dogs that had bitten people were sent by selected farmers to the designated laboratories for diagnoses. the surveyed mountainous areas were huzhou, hangzhou, jinhua, quzhou, lishui in zhejiang province; nanchang, jingdezhen, shangrao, wuyuan in jiangxi province; and huangshan, xuancheng, anqing, jingxian in anhui province. the animal heads were packed appropriately and shipped to our laboratory under cold conditions. the brains were removed by opening the skull under sterile conditions. animal experiments related to this study were approved by the committee of animal welfare and ethics of veterinary research institute, academy of military medical sciences. humane endpoints were used throughout this study in accordance with the ethical principles for in vivo studies. all animals including ferret badgers, dogs and mice that showed clinical signs of infection were killed humanely. this study did not refer to any issue of human ethics as only the epidemiological data were collected and analyzed, and no sample was collected from any healthy and contracted humans. the fat on brain specimens was performed according to methods described elsewhere [ ] . briefly, the brain tissue impressions were made on slides. after air drying, the slides were fixed with % acetone for min. the fitc-conjugated anti-rabies virus nucleoprotein monoclonal antibodies (made in laboratory of epidemiology, veterinary institute, changchun, for routine rabies diagnosis) was added and incubated for min at ºc. after three-times' wash using pbs buffer ( . m, ph . ) -tween- ( . %), the slides were left to dry in the air at ambient temperature, and observed under a uv fluorescent microscope (zeiss corporation, germany) for typical rabies virus staining. for fat positive specimens, the suspension of the sample was injected intracerebrally to -day-old suckling mice (kunming mice, animal core laboratory, changchun institute of biological products) according to protocols described elsewhere [ ] . the mice were observed for~ days. the brain smear was made to re-check for the presence of rabies virus antigen using the fat if the inoculated mice died from day to . total rna of the infected fb brains were extracted using trizol reagent (invitrogen, carlsbad, ca). the nucleoprotein (n) and glycoprotein (g) genes were amplified by rt-pcr following the protocol described by nadin-davis et al [ ] using the total rna with the following primers. the positions of the primers were referred against strain fj (genbank acc. no. the takara (takara corp ltd, dalian, china) was the contracted company responsible for sequencing the amplicons. we generated a phylogenetic tree using the neighbor-joining (nj) method in mega (megalign, dnastar software suite, version . . , copyright - , dnastar inc.). the bootstrap values were calculated from , repeats using % as the cut-off. serum samples were collected from the captured fbs and dogs in our expanded surveyed areas. all the fbs were alive and appeared healthy when the serum was collected. dog sera were from watchdogs belonging to residents of different villages in zhejiang, jiangxi and anhui provinces. the method of titration of virus neutralizing antibodies (vna) by fluorescent antibody virus neutralization (favn) test was described elsewhere [ ] . briefly, -fold serial dilutions of standard serum ( . international units, iu/ml) and test serum samples were prepared in microplates in quadruplicate. fifty _μl of challenge rabies virus (cvs- ) containing tcid was also added to each well. after min incubation at °c in a humidified % co incubator, μl cell suspension containing × cells was added to each well and the plates were incubated for h at °c. after fixation at room temperature for min in % acetone, the cell monolayers were stained by addition of fitc-conjugated anti-rabies nucleoprotein monoclonal antibodies (laboratory of epidemiology, veterinary institute, changchun, china) to each well. staining was carried out in an incubator at °c for min and fluorescence was observed by uv microscope (zeiss, germany). wells exhibiting no specific fluorescence were considered antibody positive. neutralizing antibody titers were calculated using the spearman-kärber formula and expressed in iu/ml by comparison with a reference serum ( . iu/ml, product of afssa, france), . iu/ml is considered as the cut-off of seroconversion after rabies virus infection in both ferret badgers and dogs. human rabies cases potentially associated with fb transmission were inferred retrospectively and prospectively on the basis of exposure records ( table ). the patients included fb-hunters who capture and sell fbs, farmers with occasional exposure to sick fbs, and residents who were exposed to sick fbs in their yard or house. healthy fbs do not actively attack humans because of their nocturnal behavior, but rabid fbs become excited, run into residential areas, and will bite. in the case of hunters or occasional hunters, exposure to fb happened almost every day. the fb bites in humans were usually on the hands, feet, or occasionally on the arms, legs, and very rarely upon the upper body. the population density of fbs is unknown. empirically, hunters lay their traps according to the number of fb tracks. on average, - tracks could be found within chinese acres ( chinese acre = m ). the number of fb is about - per km . however, this rough estimation needs ecological support. in our investigation, there was at least one hunter in almost every village. seasonally, more than fb was caught by a hunter per day. despite frequent contact with rabies-susceptible animals, no hunters are aware of the potential danger of rabies exposure. no pep occurred after fb bites. furthermore, the hunters and farmers live in remote rural areas and mountainous regions, and their income ( rmb or less per month) cannot afford the high cost of pep ( - rmb) in china. therefore, the recommendation of pep is not only ignored, but also intentionally neglected due to the associated costs. in total, we collected dead fb from our surveyed regions, and of were partially decomposed when the brains were removed for diagnosis. eight of brain samples were confirmed rabies positive using fat ( . %), and rv was successfully isolated by mit from the samples. the rabies incubation period in -day-old suckling mice was - days. from the brain samples we collected from live fbs in jiangxi, anhui and zhejiang provinces, only sample was rabies positive by fat ( . %, / ). since captured and injured by the hunter, this positive animal did not present obvious clinical signs of rabies before euthanized for diagnosis. from the dog brain samples collected in the same regions, were rabies positive by fat ( %, / ). all rvs from fat positive samples were successfully isolated by mit. fifty six serum samples were collected from live fbs in the selected provinces. the average rabies seroconversion rate was . %, ranging from (no neutralizing antibody in all individuals) to % in different collections, and rabies vna ranged from to . iu/ml ( table ). in the dog serum samples, the detectable vna was . % ( / ), and the overall percentage of vna positive was relatively lower from dogs than the samples from fbs (table ). since no rabies vaccination campaign has been performed in the fbs and dogs could be vaccinated occasionally due to the disease awareness by the owners, the higher seroconversion in the fbs is an interesting phenomenon. we compared rv n sequences from rabid fb isolates with those in dogs from china, foxes from europe/middle east, dogs in africa, and rabies vaccine strains from asia, europe and the us (figure ). dog rvs in china were categorized into two groups. group compromises rvs all across south and southeast china, and group is formed by chinese isolates distributed in guizhou, jiangsu, henan, jilin provinces, and is closely related to asian and occidental vaccine strains, and cosmopolitan dog rvs (figure ). in general, chinese dog-associated lineages shared nucleotide identity from to % (figure ). same phylogenetic pattern was reconstructed using the g gene sequences (data not shown). the fb rvs were segregated in an independent lineage (figures ) . the nucleotide identity of fb isolates with current south and southeast chinese dog-related lineages (from zhejiang, jiangxi, fujian, jiangsu, hunan, guangxi, guizhou, hubei, yunnan, henan) ranged from to % ( . - . % homology by amino acid sequence comparison). however, the intra-group identity in the fb isolates only varied from . to . % ( . - % homology by amino acid sequences). a notable difference was detected on the g protein motif at residual position - with serine-valine-arginine (s-v-r) in fb isolates. this motif has not been found in any isolates from dogs in china and the corresponding motif in dog isolates was serine-isoleucine-arginine (s-i-r), which does not exist in the fb isolates (using pv as the reference sequence, genbank acc. no. m ). the current reporting system of human rabies in china covers two aspects. one is the statistical distribution status of human rabies cases, which can be reviewed on governmental websites by authorized personnel only. the other is the documented epidemiological data of each case available at local provincial cdcs. the establishment of this system occurred after the lessons learned from the sars outbreaks. although annual epidemiological data represented about , human rabies deaths during the last five consecutive years, rabies is still not considered as a priority in the china public health system. although fb-associated human rabies cases have been reported in china [ , , ] , the actual number of human cases remain unknown. in africa, about . % rabies seroconversion was recorded in badger populations [ ] . rabies in such animals was detected in africa [ ] and europe [ ] , but was simulated in great britain [ ] . however, fb-associated human rabies has never been reported except in southeast china. in our surveyed counties, where - % of human rabies cases were inferred to be caused by this animal species, such epidemiological data lack laboratory-based diagnosis, and may confuse the actual role of fb in the transmission of rabies to domestic dogs and humans. therefore, in this study, we conducted an active laboratory-based conversely, apparently healthy fbs were found with a high percentage of rabies seroconversion ( . %), whereas dogs were only . %. however, no rabies vaccination program has been performed in wildlife in china. the high percentage of rabies seroconversion in fbs is an interesting discovery, and could be due to abortive rabies infections in the population. domestic dogs still are the most affected species, and are the most likely source for rabies transmission to humans and other animals in the endemic region. our data also suggest that fbs may not be as susceptible to rabies as other carnivores. in the dog serum samples, the detectable vna was . % ( / ), and the overall percentage of vna positive was relatively lower than the samples from fbs. however, some of the vna positive dogs had high levels of antibodies with two reached . and . iu/ ml, respectively. we think the randomly collected serum samples include those from vaccinated dogs due to rabies awareness by the owners. the consistent amino acid signatures along with the phylogenetic findings in this report suggest that the fb rv variant may be maintained as an independent enzootic by local fb populations. nonetheless, the origin of fb rv has its root in south/southeast china dog rv variants. our phylogenetic analysis indicates a broad cocirculation of at least two major dog-related groups throughout south/southeast china. group is relatively distant to the fb lineage, and seems to act as a new emergence. group is closely related to the fb lineage, and connects to historical dog rvs circulating in the late s in china. as human populations in southeastern china are condensed and widely distributed in hills and mountainous area, their contact with rabid fbs could become a greater potential source of exposure to rv. in some rabies endemic regions, illegal fb trade for meat consumption increased the risks among hunters, farmers, traders, slaughterers, and chefs. the awareness of rabies should be a priority in the southeast populations at risk. clearly pep should be initiated after fb bite, provided that timely post-mortem rabies diagnosis of the biting animal is not feasible. for eventual rabies control, china should implement more effective rabies surveillance programs structured by animal control units (where suspicious animals are seized, euthanized and sampled) and a highly proficient rabies diagnostics laboratory network at the governmental and local levels to detect and characterize any rabies outbreaks in any susceptible hosts. the circulation of a dog-related rv variant in wildlife populations (in this case of fb) may pose a severe delay and complicate the elimination of dog and human rabies. with a low vaccination coverage and high dog population density, the fb rv may potentially return to the dog population, or vice versa. thus, an integral rabies control program should be implemented, targeting both dog and fb populations, by using novel vaccination strategies. otherwise, the goal of controlling animal rabies and eliminating human rabies by may not be achievable (the aim set by asean plus rabies conference). in conclusion, we demonstrated that fb rabies is likely occurring as an independent enzootic that became established in the fb populations from a dog rv variant distributed in southeast/south china. the actual role of fb rabies in public health remains unclear. however, since potential rabies transmission from fb to humans and dogs cannot be excluded, immediate rabies awareness should be altered. standard pep should be recommended once an exposure is confirmed by reliable laboratory diagnostics. to meet the goal of elimination of rabies in humans by , china must strengthen its rabies surveillance system and develop feasible strategies and programs for vaccination of dogs and wildlife. the neighbor-joining phylogenetic tree of chinese fb rabies virus isolates, using the full length n gene ( bp) for reconstruction. rabies as a traveler's risk, especially in high-endemicity areas slate d: control and prevention of rabies in animals: paradigm shifts inferior rabies vaccine quality and low immunization coverage in dogs (canis familiaris) in china analysis on the epidemiologic characteristics of human rabies in all the provinces in china human rabies cases caused by dogs and cats human rabies cluster following badger bites, people's republic of china molecular epidemiology of rabies in guangxi province, south of china overview and preventive measures of human rabies recent years in zhejiang province a retrospective study of wildlife rabies in zimbabwe. trop anim health prod rabies in wild carnivores in central europe i. epidemiological studies epidemic characteristics and preventive measures of human rabies of zhejiang province in - rabies in ferret badgers: an emerging public health threat in south-eastern china the use of fluorescent antibody staining in the diagnosis of rabies comparative field evaluation of the fluorescent-antibody test, virus isolation from tissue culture, and enzyme immunodiagnosis for rapid laboratory diagnosis of rabies a molecular epidemiological study of rabies virus in central ontario and western quebec development of a fluorescent antibody virus neutralisation test (favn test) for the quantitation of rabiesneutralising antibody epidemic characteristics and preventive measures of human rabies of zhejiang province from to seven cases of human rabies caused by ferret badgers modelling disease spread in a novel host: rabies in the european badger meles meles pre-publication history the pre-publication history for this paper can be accessed here ferret badger rabies origin and its revisited importance as potential source of rabies transmission in southeast china the research was funded by the key project of national science foundation of china (approval no. ) and the china national " " program (approval no. cb ). authors' contributions yl carried out the epidemiological survey on ferret badger-associated human rabies. sz and xw carried out the sequence alignments and analysis on phylogeny. jz participated in specimen collection of both ferret badgers and dogs. yh participated in the sequence analysis using mega /megalign softare, avv analyze the results of molecular epidemiology, fz assayed the viral neutralizing antibody, cr and rh designed the study, wrote the manuscript and coordinated the research. yl, sz, xw and jz contribute equally to this manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -jvnwivrg authors: wang, jian; chen, chong; li, qilin; cai, pengcheng; wang, zheng; wang, lin title: covid- confirmed patients with negative antibodies results date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: jvnwivrg background: a new coronavirus disease (covid- ) has escalated to a pandemic since its first outbreak in wuhan, china. a small proportion of patients may have difficulty in generating igm or igg antibodies against sars-cov- , and little attention has been paid to them. case presentations: we present two cases of confirmed covid- patients and characterize their initial symptoms, chest ct results, medication, and laboratory test results in detail (including rt-pcr, igm/ igg, cytokine and blood cell counts). conclusion: both of patients with confirmed covid- pneumonia failed to produce either igm or igg even to days after their symptoms onset. this work provides evidence demonstrating that at least a small proportion of patients may have difficulty in rapidly gaining immunity against sars-cov- . during the outbreak of coronavirus (covid- ) [ ] [ ] [ ] , a small proportion of confirmed covid- patients fail to produce igm or igg antibodies against sars-cov- even days or longer periods of time after onset of their initial symptoms. however, most of the current studies so far are focused on the general population but for these patients. from january to march , of covid- patients who were positive for sars-cov- real time reverse-transcription pcr (rt-pcr) testing and received igm and igg detection at wuhan union hospital (wuhan, china) were enrolled. rt-pcr was performed through amplifying orf ab gene and n gene of sars-cov- (biogerm, shanghai, china) using oropharyngeal swab specimens of all patients. from march to , igm and igg of sars-cov- were tested using blood samples for all these patients. two different kits were used to detect antibodies through immune colloidal gold (icg) technique (yingnuote, tangshan, china) and chemiluminescence immunoassay (clia) technique (yahuilong, shenzhen, china). laboratory test results were collected and analyzed. among covid- -confirmed patients, of them were tested positive for igm and/ or igg, but only two patients were negative for igm and igg detection. case patient (fig. ) , a -year-old man, developed a cough and a sore throat with no fever on january . groundglass opacities in chest ct and positive rt-pcr test results were obtained on february and , respectively. four days later (february ), this patient developed mild pneumonia and was hospitalized for treatment ( mg of antiviral arbidol, orally, every h). from february to , four consecutive rt-pcr test results (every day) using his throat swab specimens were all negative. with remission of pneumonia symptoms and absorption of ground-glass opacities, the patient was discharged on february . however, on march , his rt-pcr test result was tested positive again during his follow-up, and he was hospitalized again in the next day. after two negative rt-pcr test results on march and , the patient was discharged on march . no evidence showed that the patient's immune function was compromised ( table ) . analyses of a large range of laboratory results revealed that most of tests were normal, including the immunoglobulin g, m, a (igg, igm, iga) and complement , (c , c ) (table s ). igm and igg were repeatedly tested using his serum samples by two different detection methods (see methods for details) on march and , which were all negative. patient (fig. ) , a -year-old man, developed a fever and a cough on january and were admitted to wuhan central hospital on january . multifocal ground-glass opacities were observed on chest ct images and the rt-pcr test for sars-cov- was positive on february . despite anti-infection treatment and oxygen support, his symptoms worsened over the next few days, leading to severe pneumonia. on february , the patient was transferred to wuhan union hospital for further treatment ( mg of moxiflxacin, daily; mg of tienam, every h; mg of arbidol, every h; mg of methylprednisolone, every h). the patient with glucocorticoid therapy showed reduced lymphocyte numbers and low ratios of cd , cd and cd t cells (table ) , suggesting his compromised immune functions. elevated il- level was related to his severe pneumonia as previously reported [ ] . moreover, of laboratory tests of this patient were abnormal, such as elevated levels of creactive protein (crp), neutrophils and lactate dehydrogenase (ldh), and decreased levels of albumin, and hemoglobin (table s ). on february and march , he received two consecutive rt-pcr tests, which were positive. however, igm and igg in his serum samples remained undetectable on march , , and . serological tests have been widely utilized in the diagnosis of covid- . igm could be detected as early as day post-symptom onset (pso) and was detectable in % of covid- confirmed patients days pso [ ] . as for igg, over % of covid- confirmed patients produced this type of antibody days after illness [ ] . in this study, two patients with confirmed covid- failed to produce either igm or igg even to days after their symptoms onset. given that all of covid- patients reportedly had sero-positive for igm and igg approximately days after symptoms onset [ ] , the window period for antibody production in these two covid- patients fig. chronology of symptom onset, hospital admission/ discharge, chest ct test, rt-pcr test and igm/ igg test may be much longer, possibly up to days or even longer (if they eventually produce antibodies). these finding suggests that at least a small proportion of patients may have difficulty in rapidly gaining immunity against sars-cov- . the cross-reactivity among different coronaviruses may lead to false-positive results in igm and igg detection [ ] , but few falsenegative outcomes were yielded, especially jointly using clia and icg techniques. thus, the fact that two or three times of negative results in the two patients using two different kits suggests that the results are unlikely to be false-negative. analyses of the laboratory results showed that the -year-old patient (case ) had compromised immune functions, which might contribute to the negative igm/ igg results. however, the young patient (case ), who possessed normal immune functions and did not have any underlying disease, yet fails to produce igm/ igg days after symptom onset. the reason remains unclear. a further long-term follow-up should be carried out for these patients to determine whether they produce igm/ igg or persistently remain with no antibodies. additionally, recurrence of covid- was observed in the -year-old patient. if our finding of pneumonia recurrence in covid- patients without antibodies is replicated, the management of these no-antibody patients needs to be more cautious as they may be prone to recurrence or re-infection. in summary, this work presents two typical cases of covid- patients without producing igm/ igg and comprehensively characterizes their initial symptoms, chest ct results, medication, and laboratory test results, hoping to draw more attentions to this type of patients. coronavirus infections-more than just the common cold early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study the role of antibody testing for sars-cov- : is there one? antibody responsed to sarscov- in patients of novel coronavirus disease springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank xueping wang and colleagues from immunoassay team of clinical laboratory for generously providing antibodies results of covid- patients. we thank fenghua chen, zhi geng, yang liu, qianwen zhang and colleagues from nucleic acid testing team of clinical laboratory for generously providing rt-pcr test results of covid- patients. not applicable. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file : table s . laboratory examination results of patient . table s . laboratory examination results of patient . authors' contributions jw, cc and pc contributed to data collection. jw, zw, lw and ql contributed to data analysis. all authors contributed to data interpretation. jw, zw and lw drafted the manuscript. all authors critically commented on the manuscript and approved the final version. no funding was received for this publication. the data-sets used during the current study are available from the corresponding author on reasonable request. this study was approved by the wuhan union hospital of huazhong university of science and technology institutional review board and informed consent were waived. written informed consent was obtained from the individuals for the publication of any potentially identifiable images or data included in this article. the authors declare that they have no conflicts of interest. key: cord- -b sm y authors: murillo-zamora, efrén; trujillo, xóchitl; huerta, miguel; ríos-silva, mónica; mendoza-cano, oliver title: male gender and kidney illness are associated with an increased risk of severe laboratory-confirmed coronavirus disease date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: b sm y background: to identify factors predicting severe coronavirus disease (covid- ) in adolescent and adult patients with laboratory-positive (quantitative reverse-transcription polymerase chain reaction) infection. method: a retrospective cohort study took place, and data from subjects, from all states of mexico, were analyzed. the association between the studied factors and severe (dyspnea requiring hospital admission) covid- was evaluated through risk ratios (rrs) and % confidence intervals (cis). results: severe illness was documented in % of participants. in multiple analysis, male gender (rr = . , % ci . – . ), advanced age ([reference: – years old] – , rr = . , % ci . – . ; – , rr = . , % ci . – . ; years or older, rr = . , % ci . – . ), chronic kidney disease (rr = . , % ci . – . ) and thoracic pain (rr = . , % ci . – . ) were associated with an increased risk of severe disease. conclusions: to the best of our knowledge, this is the first study evaluating predictors of covid- severity in a large subset of the latin-american population. male gender and kidney illness were independently associated with the risk of severe covid- . these results may be useful for health care protocols for the early detection and management of patients that may benefit from opportune and specialized supportive medical treatment. severity in cases is fundamental to improve the survival rate from covid- [ ] . we aimed to identify what factors are associated with the risk of severe laboratoryconfirmed covid- among adolescent and adult patients in mexico. a retrospective cohort study was conducted in april . potential eligible subjects (laboratory-confirmed cases of covid- , quantitative reverse-transcription polymerase chain reaction, rt-qpcr) were identified from nominal records found in a national normative online system for respiratory viruses surveillance (rvss). eligible cases were registered at any of more than medical units (three levels of care) that the mexican institute of social security (imss, the spanish acronym) has all across mexico. the imss provides health care services to more than a third of the total population of mexico [ ] . individuals aged years or older, with symptoms onset from february to march , , and with conclusive test results (sars-cov- infection confirmed) were eligible. individuals with missing clinical or epidemiologic data of interest were excluded. in mexico, phase of the covid- pandemic started on march and, before that, laboratory testing was performed in all suspected ambulatory and no ambulatory cases [ ] . demographic characteristics (sex, age), tobacco use (current), personal history of chronic communicable disease (hiv infection, no/yes) and noncommunicable disease (no/yes: obesity [body mass index of or higher], arterial hypertension, type diabetes mellitus, asthma, chronic kidney disease, immunosuppression, chronic obstructive pulmonary disease, or cardiovascular illness) were collected from the surveillance system. date of healthcare-seeking and, when applicable, dates of hospital admission and discharge were also extracted from the audited database. additional clinical and epidemiologic data, such as if the influenza vaccine was applied during the same season as the onset of the acute illness (no/yes), and if acute symptoms were reported (cough, fever, headache, myalgia, arthralgia, odynophagia, chills, rhinorrhea, thoracic pain, diarrhea, polypnea, no/yes) were also extracted from the database. the primary data sources were the medical records of the enrolled patients; these records were obtained from the employed surveillance resource system. laboratory-confirmed covid- patients, and for epidemiological purposes, were classified as severe if they reported dyspnea [ ] that resulted in hospital admission. patients with severe manifestation were the main binary outcome (no/yes). patients without dyspnea, despite being admitted to hospital, were considered as non-severe covid- cases. according to normative standards [ ] , clinical specimens (nasopharyngeal or deep nasal swab) are analyzed (superscript™ iii platinum™ one-step qrt-pcr kits) at any of four specialized regional laboratories integrated with the imss network for epidemiologic surveillance. the laboratory methods employed in the imss network follow strict quality assurance standards in the diagnosis of viral respiratory pathogens [ ] . summary statistics were calculated, and the significance level was set at %. risk ratios (rrs) and % confidence intervals (cis), estimated by using generalized linear regression models, were employed to evaluate the association between the exposures to the analyzed risk, and the risk of severe covid- . all analyses were conducted using stata version . (statacorp). the local research ethics committee approved this study at the mexican institute of social security (imss, the spanish acronym). with the research ethics approval number/id: r- - - . data from participants registered by medical units located all across the country was analyzed. the study profile is shown in fig. . dyspnea was registered in individuals, and of them required hospital admission. therefore, they were classified as severe covid- cases ( . % from enrolled subjects). table shows the characteristics of the study sample for the variables selected. most of the participants were male ( . %), and the overall mean age (± standard deviation) was . ± . years old. no gender-related differences were observed in terms of age (p = . ). two-thirds of enrolled patients were aged - years old. antipyretic drugs were prescribed to most ( . %) subjects before they sought healthcare and, in out of them, acetaminophen was consumed. the mean length of hospital stay in severe cases was . ± . days and ranged from to days. severe covid- cases were older ( . ± . vs. . ± . , p < . ) when compared with non-severe patients (table ) , and particularly in the eldest age group (≥ years old; . % vs. . %). severe cases were also more likely to seek healthcare after four or more days after symptoms onset ( . % vs. . %, p < . ). regarding the acute symptoms profile, severe cases had a higher frequency of fever ( . % vs. . %, p = . ), thoracic pain ( . % vs. . %, p < . ) and polypnea ( . % vs. . %, p < . ). significant and higher prevalences of pain-related symptoms (headache, myalgia, arthralgia, odynophagia) were documented in non-severe cases (table ). in the multiple regression analysis (table ) , male gender (rr = . , % ci . - . ) and older patients ([reference: - years old] - , rr = . , % ci . - . ; - , rr = . , % ci . - . ; years or older, rr = . , % ci . - . ), subjects to thoracic pain (rr = . , % ci . - . ) or chronic kidney disease (rr = . , % ci . - . ) were also more likely to present severe covid- . on the other hand, in multiple analysis the history of obesity, tobacco use, cardiovascular or metabolic diseases, and pulmonary illness (namely asthma or chronic obstructive pulmonary disease) were not associated with the evaluated outcome. our findings suggest that more than of analyzed patients developed severe covid- and its predictors were identified. to the best of our knowledge, this is the first study evaluating factors associated with the illness severity in a large subset of latin-american subjects. severe disease, according to our results, was presented by % ( / ) of enrolled laboratory-confirmed cases. this proportion is similar to government estimates ( %) at the national level (all healthcare institutions, including private facilities) by the end of the first week of april and when rt-qpcr-confirmed cases had been registered [ ] . after adjusting by factors such as age, obesity and personal history of chronic noncommunicable diseases, males from our study sample were more likely (rr = . , % ci . - . ) to develop dyspnea and to require hospital admission. similar findings were previously described among chinese adults, where male patients were more likely to develop severe covid- [ ] . further research has to determine the pathogenic underlying mechanism between gender and the severity of sars-cov- infection. published data suggests that gender differences in susceptibility to sars-cov (severe acute respiratory syndrome coronavirus) in mice were secondary to estrogen receptor signaling, which seemed to be critical for protection in females [ ] . however, the role of sex hormones in the regulation of innate immune cells in the lung, as a response to viral respiratory pathogens, is poorly understood [ ] . in our study, a positive gradient between age and the risk of severe illness was observed (per additional year of age, rr = . , % ci . , % ci . ) in multiple regression analysis. aging has been consistently associated with disease severity [ ] [ ] [ ] and its determining factors have not been described but may be related to viral load. a recently published analysis showed a positive and strong correlation (spearman's p = . , % ci . - . ) between older age and rt-qpcr quantified viral load [ ] . teenagers from our study sample (aged - years old, n = ) seemed to have mild symptoms of sars-cov- infection and all of them presented a non-severe form of disease. patients with previous medical diagnosis of chronic kidney disease had a % increase in the risk of severe covid- (rr = . , % ci . - . ). interestingly, in our multiple regression study, neither type diabetes mellitus (rr = . , % ci . - . ) nor arterial hypertension (rr = . , % ci . - . ), which are leading causes of renal impairment worldwide, were associated with the risk of severe covid- . the association of previously diagnosed chronic kidney disease with the severity of covid- was recently described in a subset of chinese patients [ ] when compared with national estimates (type diabetes mellitus, . %; arterial hypertension, . %) [ ], higher prevalence of chronic noncommunicable diseases were observed in the study sample, particularly in participants with severe manifestations (type diabetes mellitus, . %; arterial hypertension, . %). mexico lacks of a national registry of chronic kidney disease patients and its precise prevalence remains unknown [ ] . a previously published study where randomly selected adults residing in urban areas of mexico were analyzed, estimated that the population prevalence of chronic renal disease (creatinine clearance, cockcroft-gault formula) was % (< ml/minute) and . % (< ml/minute) respectively [ ] . thoracic pain was a common acute symptom and it was registered in medical records from . % of enrolled subjects. it is a subjective symptom that seemed to be related to dyspnea, since it was presented by . and . % of dyspneic and non-dyspneic patients respectively (p < . ). the pathogenic mechanism of sars-cov- in severe outcomes, including viral pneumonia, remains unclear; however, immunological changes seem to be crucial in the development of severe illness [ ] . increased levels of proinflammatory cytokines, particularly interleukin , have been described among patients with severe covid- [ ] . current tobacco use, obesity, and personal history of chronic obstructive pulmonary disease were only associated with the risk of severe disease in bivariate analysis. the overall prevalence of tobacco use (current) in the study sample was similar to the national mean ( . and . %, respectively) [ ] and it was % higher among severe covid- cases ( . %). in china, there was a higher frequency for severe patients to be current smokers ( . % vs. . %), however, no association analysis was performed [ ] . obesity is assessed by the analyzed surveillance system as a dichotomous variable (body mass index (bmi) equal or higher than , no/yes) and the current consensus is step qrt-pcr kits were used to confirm the covid- ; ) the absolute and relative (%) frequencies are presented, unless otherwise specified. ) p-value from chi-square o t-tests as corresponding a the arithmetic mean ± standard deviation is presented b among subjects (non-severe disease, n = ; severe disease, n = ) in whom the use of antipyretic drugs was documented before healthcare seeking c during the same influenza season than acute covid- onset that morbidly obese individuals (bmi ≥ ) are at increased risk of severe covid- [ ] . since the patients' height and usual weight are not collected by the surveillance system, we were unable to compute de bmi and therefore to estimate the prevalence of severe obesity in the study sample. recently published data suggest the rapid decay of anti-sars-cov- immunoglobulin g (igg) in early infection [ ] , and this decay seems to be faster among asymptomatic patients and those with mild covid- [ ] . these findings may be highly relevant to immunity strategies and other pandemic control strategies. the potential limitations of our study must be cited. first, only users from a healthcare institution were enrolled (imss) and their characteristics may not be entirely representative of the source population. however, the profile of its users remains heterogeneous and sars-cov- testing is provided for free to suspected covid- cases. second, the system that served as a source of data focuses on epidemiological surveillance and clinical data (i.e. blood oxygen saturation, the ratio of the partial pressure of arterial oxygen to the fraction of inspired oxygen (pao :fio ), or lung radiographic reports) are not systematically collected. other relevant information, such as the personal history of malignant tumors, is neither obtained by this system. most of the collected data is dichotomous (no/yes) in order to simplify its operation and we were unable to obtain other clinical and epidemiological data of interest. besides, we analyzed a relatively small sample size and this may have an impact on the significance of multiple regression coefficients. only cases occurring prior to march (phase ) were enrolled and this allowed us to reduce the risk of bias in analyzing only severe cases that were more likely to seek healthcare. and third, the covid- severity from an undetermined fraction of participants from our study may be misclassified given that we only used dyspnea requiring hospital admission to identify it. however, this standalone marker seemed to have an acceptable predictive value since invasive ventilator support was required in . and . % of non-severe and severe cases (p < . ), respectively. a fatal outcome was documented in almost half ( . %) of analyzed subjects with severe illness (vs. . %; p < . ). the covid- pandemic is a major public health issue across the world. an effective response of healthcare systems is needed, which must include early identification of patients who are at increased risk of severe symptoms and poorer disease outcomes. in our analysis, male gender and kidney disease were independently associated with an increased risk of severe respiratory illness. to the best of our knowledge, this is the first study evaluating predictors of covid- severity in a large subset of the latin-american population. these results may also be useful in medical decision making related to the start of sars-cov- antiviral therapy, when available. chain reaction; bmi: body mass index pao : pressure of arterial oxygen; fio : fraction of inspired oxygen references a novel coronavirus from patients with pneumonia in china covid- en méxico: información general viral and host factors related to the clinical outcome of covid- predictors of covid- severity: a literature review kidney disease is associated with in-hospital death of patients with covid- datos abiertos: información en salud. dosis, antiinfluenza, aplicados, delegación lineamiento estandarizado para la vigilancia epidemiológica y por laboratorio de covid- characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention analysis of influenza data generated by four epidemiological surveillance laboratories in mexico comunicado técnico diario nuevo coronavirus en el mundo (covid- ) sex-specific clinical characteristics and prognosis of coronavirus disease- infection in wuhan, china: a retrospective study of severe patients sex-based differences in susceptibility to severe acute respiratory syndrome coronavirus infection sex hormones regulate innate immune cells and promote sex differences in respiratory virus infection clinical characteristics of patients infected with sars-cov- in wuhan severe outcomes among patients with coronavirus disease (covid- ) -united states clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov- : an observational cohort study prevalence of chronic kidney disease in an urban mexican population hypothesis for potential pathogenesis of sars-cov- infection-a review of immune changes in patients with viral pneumonia characteristics of lymphocyte subsets and cytokines in peripheral blood of hospitalized patients with novel coronavirus pneumonia (ncp) clinical characteristics of coronavirus disease in china groups at higher risk for severe illness clinical and immunological assessment of asymptomatic sars-cov- infections rapid decay of anti-sars-cov- antibodies in persons with mild covid- springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions emz conceived and designed the experiments also wrote the first draft of the manuscript; xt made data analysis and data collection, mh contributed with the methodology and writing-review and editing; mrs contributed with revisions and data analysis and omc performed the experiments, analyzed the data, and is responsible for the final version of the manuscript, that has been read and approved by all authors. this research received no external funding. all data generated or analyzed during this study are included in this published article and its supplementary information files.ethics approval and consent to participate local imss health research ethics committee, with approval code: r- - - . not applicable. none declared under financial, general, and institutional competing interests.author details key: cord- - yxmaihl authors: katsurada, naoko; suzuki, motoi; aoshima, masahiro; yaegashi, makito; ishifuji, tomoko; asoh, norichika; hamashige, naohisa; abe, masahiko; ariyoshi, koya; morimoto, konosuke title: the impact of virus infections on pneumonia mortality is complex in adults: a prospective multicentre observational study date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: yxmaihl background: various viruses are known to be associated with pneumonia. however, the impact of viral infections on adult pneumonia mortality remains unclear. this study aimed to clarify the effect of virus infection on pneumonia mortality among adults stratified by virus type and patient comorbidities. methods: this multicentre prospective study enrolled pneumonia patients aged ≥ years from september to august . sputum samples were tested by in-house multiplex polymerase chain reaction assays to identify respiratory viruses. viral infection status and its effect on in-hospital mortality were examined by age group and comorbidity status. results: a total of patients were enrolled in the study and . % was aged ≥ years. ( . %) did not have comorbidities, ( . %) had chronic respiratory disease, and ( . %) had other comorbidities. viruses were detected in ( . %) patients. human rhinovirus ( . %) was the most frequently identified virus, followed by influenza a ( . %) and respiratory syncytial virus ( . %). respiratory syncytial virus was more frequently identified in patients with chronic respiratory disease ( . %) than those with other comorbidities ( . %) and without comorbidities ( . %) (p = . ). the frequencies of other viruses were almost identical between the three groups. virus detection overall was not associated with increased mortality (adjusted risk ratio (arr) . , % ci . – . ). however, influenza virus a and b were associated with three-fold higher mortality in patients with chronic respiratory disease but not with other comorbidities (arr . , % ci . – . ). intriguingly, paramyxoviruses were associated with dramatically lower mortality in patients with other comorbidities (arr . , % ci . – . ) but not with chronic respiratory disease. these effects were not affected by age group. conclusions: the impact of virus infections on pneumonia mortality varies by virus type and comorbidity status in adults. electronic supplementary material: the online version of this article ( . /s - - -y) contains supplementary material, which is available to authorized users. pneumonia is the major cause of morbidity and mortality among adults, especially in the elderly. management of pneumonia is a critical problem in an ageing society like japan. streptococcus pneumoniae and haemophilus influenzae are the leading bacterial causes of adult pneumonia, while viruses also play important roles in disease development. recent advances in molecular diagnostic techniques have enabled us to detect multiple viruses simultaneously [ ] . studies have shown that viral infection is common in pneumonia patients [ , ] . according to a recent systematic review and meta-analysis, viruses were detected in . % of respiratory samples from community-acquired pneumonia (cap) patients [ ] . various viruses are known to be associated with respiratory infections, including pneumonia. according to the systematic review, influenza is the most commonly detected virus in cap, followed by human rhinovirus (hrv), respiratory syncytial virus (rsv), and human coronavirus (hcov) [ ] . in addition to these endemic respiratory viruses, emerging respiratory viruses, such as severe acute respiratory syndrome coronavirus, middle east respiratory syndrome coronavirus, and avian influenza, are posing a particularly serious threat to global health security [ ] . studies have suggested that these emerging viral infections are associated with an increased risk of severe conditions and mortality among pneumonia patients [ , ] . it must be noted that pneumonia mortality varies substantially according to patient characteristics, such as comorbidities, aspiration risk factors, and physical functional status [ , ] . to establish effective control measures, high-priority viruses and patient groups must be identified. however, the prevalence of viruses in adult pneumonia and their virus-specific effects on clinical outcome remain largely unknown. to the best of our knowledge, no large-scale study has investigated the different effects of viruses on pneumonia mortality by patient characteristics. we conducted this prospective multicentre study to determine the distribution of viruses associated with pneumonia in adults and to establish their virus-specific effects on pneumonia mortality stratified by age group and comorbidity status. the adult pneumonia study group-japan (apsg-j) conducted multicentre prospective hospital-based surveillance for community-onset pneumonia at four community-based hospitals in japan. in our previous paper, the burden and aetiology of adult pneumonia were reported based on the data and clinical samples collected during the st phase of the study (september to january ) [ ] . the current study included all data and samples collected during the whole study period (september to august ). details of the study settings and enrolment criteria were described previously [ ] . in brief, all outpatients and inpatients were screened by hospital physicians, and eligible patients were identified using a standardized case definition: patients aged ≥ years with respiratory symptoms compatible with pneumonia and new infiltrative shadows on chest xrays or computed tomography scans. clinical information was collected from patients and medical charts using a standardized data collection form. sputum, blood, and urine samples were collected at the time of diagnosis. gram staining, sputum culture, and blood culture were performed on site. sputum samples were further tested by in-house multiplex polymerase chain reaction (pcr) assays to identify viral and bacterial pathogens at the institute of tropical medicine, nagasaki university. and legionella pneumophila) were tested using multiplex pcr assays. details about the primers and pcr methods used have been described previously [ , ] . urinary antigen testing was performed for the detection of s. pneumoniae and l. pneumophila (binax now streptococcus pneumoniae, binax now legionella; alere inc., waltham, ma, usa). diagnosis of viral infection was made according to pcr results. bacterial infection was diagnosed when any of the following criteria were fulfilled: ) culture yielded pathogenic bacteria from microscopically purulent sputum samples (i.e., geckler's classification groups and ) or normally sterile site samples; ) pcr assays were positive for bacterial dna in microscopically purulent sputum samples; or ) urinary antigen tests showed a positive result. patients were categorized into four age groups: - years, - years, - years, and ≥ years. patients' disability status was evaluated using the eastern cooperative oncology group performance status (ps) score [ ] . pneumonia severity was assessed using the curb scoring system [ ] . to estimate the effect on pneumonia mortality, viruses were categorized into four groups: ) hrv; ) influenza a and b viruses; ) paramyxoviruses (rsv, hmpv, and piv type - ); and ) other viruses (hadv, hbov, and hcov). we divided patients into three groups according to comorbidity status: ) patients without comorbidity; ) patients with chronic respiratory disease; and ) patients with comorbidities other than chronic respiratory disease (i.e., other comorbidities). chronic respiratory disease included bronchial asthma, chronic obstructive pulmonary disease (copd), interstitial pneumonia, pneumoconiosis, and bronchiectasis. other comorbidities included diabetes mellitus, cerebrovascular disease, dementia, neuromuscular disease, cardiac failure, ischaemic heart disease, collagen disease, malignancy, renal disease, and liver disease. patients were considered to have aspiration risk factors when they had any of the following factors: episodes of aspiration, the presence of dysphagia, consciousness disturbances, neuromuscular diseases, cerebrovascular diseases, tube feeding, and bedridden status [ ] . the in-hospital death was defined as any death occurred during the hospitalization. during the first year of study, we followed up our patients after the enrolment and confirmed that no outpatient had died within days of enrolment. we therefore considered the in-hospital death as a good marker of short-term mortality in pneumonia patients regardless of their hospitalization status. patients were categorized according to their comorbidity status (i.e., patients without comorbidity, with chronic respiratory disease, or with other comorbidities) and compared using chi-squared tests. viral and bacterial infection status were compared by age group and comorbidity status using chi-squared tests, fisher's exact tests, and chi-squared tests for trend. in-hospital mortality rates were calculated by viral and bacterial infection status and compared with those of the virus-negative group. the effects of viral infection on in-hospital mortality were expressed as risk ratios with % confidence intervals (ci) and estimated using poisson regression models with robust standard errors. age, study site, comorbidity status, duration of symptoms, month of diagnosis, antibiotic use, and presence of bacteria were considered potential confounders based on prior knowledge and were included in the multiple regression models. for patients whose onset of symptoms were unknown (< %), we coded those missing values as "unknown" and included all patients in our analysis. the data were analysed using stata version (stata corp., college station, tx, usa). this study was approved by the institutional review boards (irbs) of the institute of tropical medicine, nagasaki university, ebetsu city hospital, kameda medical center, chikamori hospital, and juzenkai hospital. the requirement for obtaining written consent from all participants was waived by all irbs because of the study's observational nature without any deviation from the current medical practice. anonymized data were used for the analyses. during the study period, patients were enrolled in the study. of these, were excluded because of refusal to participate in the study (n = ), absence of pulmonary infiltrates (n = ), and non-pneumonia diagnosis (n = ). after excluding ( . %) patients whose sputum samples for pcr assays were unavailable, patients were eligible for analysis ( fig. ). table shows the clinical characteristics of pneumonia patients by comorbidity status. approximately % of patients were male, and the median age was . years (interquartile range, to years). the proportions of patients aged ≥ years and ≥ years were . and . %, respectively; . % of patients had aspiration risk factors. of all patients, ( . %) did not have comorbidities, ( . %) had chronic respiratory disease, and ( . %) had other comorbidities. patients with comorbidities were more likely to be male, older, more frequently required hospitalization, more frequently developed severe disease, more frequently had aspiration risk factors, had higher ps scores, and visited the hospital earlier than those without comorbidities. the proportion of patients with aspiration risk factors was particularly high ( . %) among patients with other comorbidities. multiple symptoms were most frequently observed in patients with chronic respiratory disease ( . %), followed by those without comorbidities ( . %) and those with other comorbidities ( . %). in total, ( . %) patients tested positive for at least one virus (table ) . hrv was the most common virus identified (n = [ . %]), followed by influenza a (n = [ . %]) and rsv (n = [ . %]). two or more viruses were detected in patients ( . %). the most frequent combinations of viruses were hrv plus influenza a (n = ), followed by hrv plus rsv (n = ), and hrv plus hmpv (n = ). three viruses (hrv, hmpv, and piv type ) were detected in one patient. bacterial pathogens were detected in ( . %) patients, and both viral and bacterial pathogens were detected in ( . %) patients (i.e., viral-bacterial co-infection). viral and bacterial infection status were compared by age group (table ) and comorbidity status ( table ). the proportion of overall virus-positive pneumonia did not differ by age group. rsv was more frequently identified in older age groups, while hcov was more frequently identified in younger age groups. the proportion of influenza- other comorbidities include diabetes mellitus, cerebrovascular disease, dementia, neuromuscular disease, cardiac failure, ischaemic heart disease, collagen disease, malignancy, renal disease, and liver disease positive pneumonia was similar across all age groups. bacterial pathogens were more frequently identified in younger patients. for patients' comorbidity status, rsv was most frequently identified in patients with chronic respiratory disease ( . %), followed by those with other comorbidities ( . %) and without comorbidities ( . %) (p = . ); the frequencies of other viruses were almost identical between the three groups (table ) . bacterial pathogens were more frequently identified in patients without comorbidities than in those with comorbidities. we explored symptoms of patients with each respiratory virus groups (additional file : table s ). the proportion of patients with multiple symptoms (i.e., the number of symptoms ≥ ) was higher in patients with paramyxovirus infection than those without viral infection ( . % vs . %, p < . ). in the group of patients with aspiration risk factors, those with paramyxovirus were more likely to have a cough than patients without virus ( . % vs . %, p < . ). among patients, patients died before discharge, with an overall in-hospital mortality of . %. the mortalities among virus-positive and -negative groups were . and . %, respectively, and the overall effect (adjusted risk ratio [arr]) of viruses on mortality was . ( % ci . - . , p = . ). intriguingly, when the effect of specific virus type was analysed, paramyxoviruses, including rsv, hmpv, and piv type - , were associated with a dramatically lower mortality (arr . , % ci . - . , p = . ). among paramyxovirus-positive pneumonia, five died: three were rsv-positive, one was hmpvpositive, and one was hmpv-positive at the enrolment. rsv alone was also associated with a lower mortality (arr . , % ci . - . , p = . ), but the association did not reach a statistically significant level. none of the other virus types were associated with mortality. the virus type-specific effects were further investigated after patients were stratified by age group and comorbidity status (tables and ). similar effects of viruses were seen across all age groups. however, influenza virus a and b were strongly associated with higher mortality in patients with chronic respiratory disease (arr . , % ci . - . , p = . ), while no influenza-related death was observed in those without comorbidity. intriguingly, paramyxoviruses were associated with markedly lower mortality in patients with other comorbidities (arr . , % ci . - . , p = . ), but this association was not observed in other groups. hrv was not associated with mortality in the three groups. virus only, bacteria only, and both virusand bacteria-positive pneumonia demonstrated higher mortality than virus-and bacteria-negative pneumonia in patients with chronic respiratory disease, but these associations did not reach statistically significant levels. we explored the association between viruses and inhospital mortality in patients with aspiration risk factors (additional file : table s ). paramyxovirus was the only virus type significantly associated with reduced mortality in this category of patients (arr . , % ci . - . , p = . ). influenza a and b were not associated with mortality (arr . , % ci . - . , p = . ). the mortality was higher among patients with cough than those without cough ( . % vs . %, p < . ). in this multicentre prospective study, . % of adult pneumonias were associated with viruses. hrv was the leading virus identified, followed by influenza a and rsv. this pattern was almost identical across all age groups. influenza was strongly associated with higher mortality in patients with chronic respiratory disease but not in other groups. paramyxoviruses, including rsv, hmpv, and piv type - , were associated with improved survival in patients with other comorbidities, especially in those with aspiration risk factors. to the best of our knowledge, this study is the first to systematically investigate virus-specific effects on pneumonia mortality by age group and comorbidity status among adults. viruses are frequently observed in pneumonia patients. according to previous studies, viruses were positive in , , and % of cap patents in the us [ ] , norway [ ] , uk [ ] , and china [ ] , respectively, and hrv, influenza a, and rsv were the leading viruses identified; these findings were confirmed in our study. however, the role of viruses in pneumonia development and progression has not been fully established. a systematic review showed that the risk of death was higher in patients with viral infection, although the association did not reach a statistically significant level (odds ratio . , % ci . - . ) [ ] . the major limitation of previous studies is that all viruses and patient groups were pooled, which may have overlooked their intergroup differences. in fact, in the current study, viruses overall were not associated with increased mortality among all pneumonia patients (arr . , % ci . - . ), but the effects were different by viruses and patient characteristics. influenza increased pneumonia mortality by . -fold ( % ci . - . ) in our patients with chronic respiratory disease but did not change the mortality in other patients. although influenza is known to be an important cause of pneumonia and death, only a few studies have formally compared the mortality of influenza pneumonia with that of non-influenza pneumonia, and the findings have been inconsistent [ , ] . on the other hand, previous studies have demonstrated that chronic respiratory disease increases the risk of severe outcome among influenza patients [ , ] . bronchial epithelial cells of copd are susceptible to replication of influenza virus because of their impaired antiviral immunity [ ] ; thus, the effect of influenza on disease progression may be stronger in patients with this condition. according to the cochrane review, influenza vaccination reduces exacerbations in patients with copd [ ] . seasonal influenza vaccination campaigns must therefore pay special attention to this patient group. interestingly, paramyxoviruses including rsv were associated with improved survival in our patients with other comorbidities. inconsistent findings have been reported about the effect of paramyxoviruses on pneumonia severity. a multinational study showed that older patients who had been infected with rsv were more likely to be hospitalized than those with other respiratory viruses [ ] , while a study conducted in the us demonstrated that patients with rsv infection were less frequently hospitalized than those with influenza infection [ ] . a retrospective cohort study conducted in hong kong showed that the -day and -day mortality rates were similar between adult patients hospitalized with rsv and those with seasonal influenza [ ] . these inconsistent findings suggest that the effects of paramyxovirus infection substantially vary by patients' conditions. in fact, in the current study, compared with virus-negative pneumonia, the mortality of paramyxovirus-associated pneumonia was substantially lower among patients with other comorbidities but this finding was not observed among patients without comorbidities and patients with chronic respiratory disease. the low mortality of paramyxovirus-associated pneumonia in this groups may be associated with its high prevalence of multiple symptoms. in our study, the proportion of patients with multiple symptoms (i.e., the number of symptoms ≥ ) was higher in patients with paramyxovirus infection than those without viral infection. patients with paramyxovirus-associated pneumonia are more likely to develop symptoms and are probably more likely to visit hospitals, and this benefit may be observed in patients with comorbidities. in the group of patients with aspiration risk factors, those with paramyxovirus-associated pneumonia were more likely to have a cough than patients without virus ( . % vs . %, p < . ), and the mortality was higher among patients with cough than those without cough ( . % vs . %, p < . ). the low mortality of paramyxovirus-associated pneumonia in patients with aspiration risk factors also suggests that these viruses may stimulate the cough reflex and improve patients' survival; however, our study does not provide conclusive evidence. further studies are needed to unveil the mechanisms of potential benefits of paramyxovirus infection on pneumonia mortality. in our study, multiple viruses were identified in . % of virus-associated pneumonia and were associated with higher mortality than single viral infection in patients with chronic respiratory disease and other comorbidities. the association between multiple viral infections and pneumonia mortality remains uncertain [ ] . systematic reviews have shown that multiple viral infections in patients with respiratory disease are not associated with disease severity [ , ] ; however, the majority of previous studies included young children but not adults. the effect of multiple viruses on disease progression may be different in children and adults. consistent with previous studies [ , , ] , half of the viral pneumonia patients were co-infected with bacterial pathogens. a systematic review showed that viral-bacterial co-infection increased mortality [ ] ; however, this association was not observed in our study. viral infections of the lower respiratory tract may increase the risk of secondary bacterial infection, which may increase the risk of pneumonia and mortality [ ] . however, sputum samples were collected once in our study; thus, we were unable to determine the time course of viral and bacterial infections. a cohort study with sequential respiratory sampling may be a preferable design to establish a causal association between viral-bacterial co-infection and pneumonia mortality. advances in molecular diagnostic techniques have enabled virus detection in clinical settings. our findings raise the question of whether all pneumonia patients should be tested for viruses. the increased mortality of influenzaassociated pneumonia in patients with chronic respiratory disease suggests the importance of early diagnosis of influenza and initiation of antivirals for this patient group. on the other hand, no substantial increase of mortality was found for other viruses. screening for viruses in all pneumonia patients may be unnecessary in clinical settings. our study has limitations. first, sputum samples were not available for % of the enrolled patients. however, the clinical characteristics of patients without sputum samples did not differ from those of patients with sputum samples. exclusion of this group did not affect our findings. second, we used pcr to detect viruses. the detection of viral rna and dna in respiratory samples does not always indicate the presence of a causal pathogen; particularly, the detection of viruses in nasopharyngeal swabs may not be reflecting lower respiratory infections in pneumonia patients. we, therefore, used sputum samples from pneumonia patients, and the presence of viruses in the lower respiratory tract must be very likely causative [ ] . third, due to the nature of an observational study design, unmeasured confounding factors may have remained in our risk factor analyses for pneumonia mortality. viral infections are common in adult pneumonia, and their impact on pneumonia mortality varies by viruses and comorbidities. variable impacts of viruses by population characteristics must be considered in the development of antiviral drugs and vaccines. division of respiratory medicine - - sakamoto, nagasaki - , japan. department of general internal medicine etiology of community-acquired pneumonia: increased microbiological yield with new diagnostic methods community-acquired pneumonia requiring hospitalization among u.s. adults etiology of community-acquired pneumonia and diagnostic yields of microbiological methods: a -year prospective study in norway viral infection in community-acquired pneumonia: a systematic review and meta-analysis identification of new respiratory viruses in the new millennium middle east respiratory syndrome predictors of in-hospital mortality of older patients admitted for community-acquired pneumonia prognostic implications of aspiration pneumonia in patients with community acquired pneumonia: 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distinguishing pandemic h n influenza-related pneumonia from interpandemic community-acquired pneumonia in adults comparison of clinical features and outcomes of hospitalized adult patients with novel influenza a (h n ) pneumonia and other pneumonia factors associated with poor outcomes among adults hospitalized for influenza in france: a three-year prospective multicenter study pneumonia among adults hospitalized with laboratory-confirmed seasonal influenza virus infection-united states targeting pi k-p alpha suppresses influenza virus infection in chronic obstructive pulmonary disease influenza vaccine for patients with chronic obstructive pulmonary disease respiratory syncytial virus and other respiratory viral infections in older adults with moderate to severe influenza-like illness medically attended respiratory syncytial virus infections in adults aged >/= years: clinical characteristics and outcomes high morbidity and mortality in adults hospitalized for respiratory syncytial virus infections viral pneumonia single and multiple respiratory virus infections and severity of respiratory disease: a systematic review clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis incidence and characteristics of viral community-acquired pneumonia in adults high incidence of community-acquired pneumonia among rapidly aging population in japan: a prospective hospital-based surveillance yield of sputum for viral detection by reverse transcriptase pcr in adults hospitalized with respiratory illness we are grateful to all the laboratory staff at the participating hospitals. we would like to thank rina shiramizu (institute of tropical medicine, nagasaki university, nagasaki, japan) and kyoko uchibori (institute of tropical medicine, nagasaki university, nagasaki, japan) for performing pcr and yumi araki (institute of tropical medicine, nagasaki university, nagasaki, japan) for administrative work. adult pneumonia study group-japan (apsg-j) are: takao wakabayashi , naoto hosokawa , norihiro kaneko , kei nakashima , yoshihito otsuka , eiichiro sando , kaori shibui , daisuke suzuki , kenzo tanaka , kentaro tochitani , masayuki chikamori , masayuki ishida , hiroshi nakaoka , hiroyuki ito , kei matsuki , yoshiko tsuchihashi , bhim g dhoubhadel , akitsugu furumoto , sugihiro hamaguchi , , shungo katoh , , satoshi kakiuchi , emi kitashoji , takaharu shimazaki , masahiro takaki , additional file : table s . proportion of patients with multiple symptoms (number of symptoms ≥ ) by virus. ethics approval and consent to participate this study was approved by the institutional review boards of the institute of tropical medicine, nagasaki university, ebetsu city hospital, kameda medical center, chikamori hospital, and juzenkai hospital. the requirement for obtaining written consent from all participants was waived by all irbs because of the study's observational nature without any deviation from the current medical practice. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. submit your next manuscript to biomed central and we will help you at every step: key: cord- -fn gzsw authors: nishiura, hiroshi; kamiya, kazuko title: fever screening during the influenza (h n - ) pandemic at narita international airport, japan date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: fn gzsw background: entry screening tends to start with a search for febrile international passengers, and infrared thermoscanners have been employed for fever screening in japan. we aimed to retrospectively assess the feasibility of detecting influenza cases based on fever screening as a sole measure. methods: two datasets were collected at narita international airport during the pandemic. the first contained confirmed influenza cases (n = ) whose diagnosis took place at the airport during the early stages of the pandemic, and the second contained a selected and suspected fraction of passengers (self-reported or detected by an infrared thermoscanner; n = , ) screened from september to january . the sensitivity of fever ( . °c) for detecting h n - was estimated, and the diagnostic performances of the infrared thermoscanners in detecting hyperthermia at cut-off levels of . °c, . °c and . °c were also estimated. results: the sensitivity of fever for detecting h n - cases upon arrival was estimated to be . % ( % confidence interval: , . ) among nine confirmed h n - cases, and . % of the h n - cases were under antipyretic medications upon arrival. the sensitivity and specificity of the infrared thermoscanners in detecting hyperthermia ranged from . - . % and . - . %, respectively. the positive predictive value appeared to be as low as . - . %. conclusions: the sensitivity of entry screening is a product of the sensitivity of fever for detecting influenza cases and the sensitivity of the infrared thermoscanners in detecting fever. given the additional presence of confounding factors and unrestricted medications among passengers, reliance on fever alone is unlikely to be feasible as an entry screening measure. the rapid international spread of severe acute respiratory syndrome (sars) from to led countries around the world to extensively assess the entry screening measures at their international borders as one of the countermeasures to prevent the global spread of infectious diseases [ , ] . pandemic influenza has been one of the most important subjects for entry screening [ ] . including an analysis of the historical records of maritime quarantine during the - influenza pandemic [ ] , many scientific discussions concerning the scientific value and public health performance of entry screening took place prior to the pandemic. although the efficacy of entry screening in correctly detecting and diagnosing influenza cases is likely to be small, mainly because of the impossibility of detecting incubating individuals at the border [ , ] and the presence of asymptomatic cases [ ] [ ] [ ] , many countries adopted entry screening measures to some extent during the early stages of the pandemic [ ] . japan followed its original guideline [ ] to enforce entry screening at international airports as well as other border control measures during the very early stages of the pandemic, with the aims of detecting influenza cases at the border and preventing secondary transmissions arising from potentially exposed individuals through strict quarantine (e.g. at hotels close to airports) or voluntary home quarantine. since the diagnostic criteria and definitions of both sars and influenza-like illness include fever, entry screening tends to start with a search for febrile international passengers, and such fever screening has tended to largely rely on the use of infrared thermoscanners because of their non-invasive nature and the need to screen massive numbers of travelers at the border [ ] [ ] [ ] . because of the relatively high sensitivity and specificity, the negative predictive value (npv) of infrared thermoscanners in excluding non-febrile passengers is believed to be high [ ] [ ] [ ] [ ] [ ] , which supports the use of infrared thermoscanners for releasing negative individuals (i.e. a strict screening measure through diagnosis by exclusion), under an important assumption that the prevalence of infected individuals is small among the total number of passengers and with the expectation that "cases" are represented as febrile passengers. although border control does not fully rely on infrared thermoscanners to detect febrile passengers, narita international airport (also known as tokyo-narita airport or new tokyo international airport), comprising the largest international airport in japan and dealing with % of arriving international passengers, has placed stationary infrared thermoscanners as an aid to monitor and screen for fever among arriving international passengers since . however, despite the high diagnostic accuracy and npv under the above-mentioned assumption and expectation, the readings of infrared thermoscanners are known to be influenced by several confounding factors including age and outdoor temperature, and the small positive predictive value (ppv) with the small prevalence of febrile passengers is not believed to realistically permit less strict entry screening (e.g. use of infrared thermoscanners to actively detect hyperthermia) [ ] [ ] [ ] . the validity of fever screening in relation to its theoretical rationale (e.g. the above-mentioned assumption and expectation) should be assessed in practical settings. japan is one of the countries that implemented the most strict entry screening during the early stages of the pandemic [ ] . this allowed us to retrospectively analyze epidemiological datasets of confirmed cases whose diagnosis took place at an international border during the early entry screening practice and of a portion of passengers screened by the infrared thermoscanners. the datasets of the influenza cases and passengers provide us with a unique opportunity to critically investigate the public health performance of fever screening in correctly detecting and diagnosing influenza (h n - ) at international borders. the purpose of the present study was to retrospectively assess the feasibility of detecting influenza cases based on fever screening as a sole measure through the analysis of actual entry screening data, thereby identifying practical issues surrounding fever screening of passengers including influenza cases. in the present study, we analyzed two different datasets collected at narita international airport, which receives approximately , international flights per year (i.e. flights per day) and through which approximately million passengers per year enter japan (i.e. , - , passengers per day) including japanese passengers returning from abroad. the first dataset contained the limited number of confirmed cases infected with h n - or other influenza viruses whose diagnosis took place at the airport during the very early stages of the pandemic, and the second dataset contained non-randomly sampled passengers, comprising a selected and suspected fraction of passengers (selfreported or detected by an infrared thermoscanner) arriving at narita international airport from september to january ( figure ). the first dataset was based on strict entry screening measures implemented from april to june , which targeted passengers arriving from canada, mexico and the united states. the strictest border control measures (i.e. those involving fever screening of passengers on board before disembarkation from an arriving aircraft) were performed until may. from may to june, a clinical examination and laboratory testing were performed for self-reporting passengers and those detected by the stationary infrared thermoscanners. passengers with a travel history to the above-mentioned three countries with fever greater than . °c (directly measured by the tympanic or axillary temperature, for example) or with two or more acute upper respiratory tract symptoms (e.g. cough, sputum or breathlessness) underwent rapid diagnostic testing for influenza. briefly, nasal swab specimens were taken for the rapid diagnostic testing and, if positive for influenza type a virus, a confirmatory diagnosis was made by rt-pcr. the primers for real-time rt-pcr for h n - detection were developed by the national institute of infectious diseases and became available on april [ ] . during the -day screening period, a total of , commercial aircrafts landed at narita international airport from the three countries bringing , passengers and , airline crew members [ ] . among these, persons underwent rapid diagnostic testing, and tested positive. including those who tested negative but were strongly suspected of having the disease (e.g. those with a history of apparent contact with a known case), a total of cases were confirmed as having influenza (figure ). among these cases, had h n - , had other influenza type a virus subtypes (four with h and three with h other than h n - ) and one had influenza type b virus. one of the h n - cases was healthy upon arrival, but had a history of contact with other symptomatic cases. since this case developed the illness during quarantine at a hotel, the case was excluded from our analysis. the temperature of one h case upon arrival was unknown. accordingly, a total of cases with h n - and cases with other influenza viruses with data regarding age, gender, history of medications prior to arrival and axillary temperature upon arrival were evaluated. the other dataset included data for axillary temperature, surface temperature measured by an infrared thermoscanner and other variables among a total of , passengers arriving at narita international airport from september to january . during the month study period, a total of , , passengers entered japan through narita international airport, and all were screened by infrared thermoscanners. a total of eight tvs- infrared thermoscanners (nec/avio infrared technologies co. ltd., tokyo, japan) were placed near the quarantine station before immigration. the infrared sensors optically scanned the surface of each passenger, and the temperature distributions were recorded as two-dimensional thermal images. our subjects comprised a selected and suspected fraction of passengers among the total passengers, who fulfilled one of the following selection criteria: (a) those who selfreported some symptom or actively visited the health consultation room of the quarantine station; (b) relatives or friends of self-reporting individuals; or (c) those who were detected by an infrared thermoscanner (based on a predefined threshold reading of . °c) and were asked by quarantine officers to undergo further examinations. hereafter, we refer to these , passengers as the "selected and suspected fraction" of passengers, because the passengers were selected based on the above-mentioned criteria and were more likely to be suspected of fever than the remaining passengers. figure shows a map of narita international airport, which employs a satellite terminal design (i.e. an airport building detached from other airport buildings so that aircraft can park around its entire circumference). there are two terminals, namely terminal with four satellites and terminal with two satellites. these satellites can be crudely classified into three areas, and each is utilized by a single alliance of airline companies. four infrared thermoscanners were set up in each terminal. the distances between the infrared thermoscanners and the passengers varied slightly in the satellites, being - m in terminal and - m in terminal . guided by quarantine officers, all the subjects voluntarily entered the health consultation room. upon entrance, the axillary temperature of the subjects was measured once using a c axillary thermometer (termo corporation, tokyo, japan). the sensor was directly inserted into the axilla, and the temperature was measured by a thermistor. the temperature was displayed at approximately seconds after the insertion. figure flow chart of participants in the study. two datasets were collected at narita international airport. the first contained confirmed influenza cases whose diagnosis took place at the airport during the early stages of the pandemic (study a). the second contained a selected and suspected fraction of passengers (self-reported or detected by an infrared thermoscanner) screened from september to january (study b). in addition to the temperature, we collected information regarding age, gender, aircraft (i.e. place of embarkation), self-reporting (i.e. presence or absence of voluntary reporting of any symptoms) and information of the satellite where the surface temperature was measured by an infrared thermoscanner (i.e. alliances a, b or c). since air-conditioning greatly influences room temperature variations within the airport, and because the room temperature also varies with arrival gates and satellites (e.g. depending on individual routes of entry), we were not able to measure the room temperature for each subject. history of medications prior to the screening was not collected systematically. for both datasets, we used the axillary temperature as a reference variable, and axillary temperatures above defined thresholds were considered to be hyperthermia (our outcome variable). first, we documented the summary statistics of the demographic variables and axillary temperatures for both datasets. second, using the first dataset, we examined the sensitivity of fever for detecting influenza among the sample of confirmed cases, by using three different cut-off levels for defining hyperthermia ( . °c, . °c and . °c) based on the axillary temperature upon arrival. because the sample size was small, we computed the exact % confidence interval (ci) of the sensitivity, using the quantile function of the binomial distribution. we also examined the associations between hyperthermia and types of influenza virus (h n - or not), age, gender and history of medications. third, among the , screened passengers, we measured the associations of hyperthermia with age, gender, place of embarkation (categorized into six regions of the world health organization, but grouping southeast-asia and western pacific regions into one region owing to their geographic closeness), self-reporting (dichotomous) and satellite of measurement (categorized by three areas as shown in figure ). except for the axillary temperature and the surface temperature measured by an infrared thermoscanner, only age was a continuous variable. we employed the welch test to examine the association between hyperthermia and age. for all the remaining variables, we used fisher's exact test or the c test. fourth, we assessed the univariate correlation and association between the axillary temperature (outcome variable) and the surface temperature measured by an infrared thermoscanner. pearson's product-moment correlation was employed to examine the correlation between two continuous variables. subsequently, the diagnostic performances (including sensitivity, specificity and area under the receiver operating characteristic curve (auc)) of the infrared thermoscanners were estimated along with the ppv and npv. we employed the youden index (i.e. sensitivity plus specificity minus ) to identify the sensitivity and specificity of the infrared thermoscanners at an optimal threshold of the surface temperature. the % cis of the sensitivity and specificity were computed using normal approximation to the binomial distribution, while the calculations of the % cis of the ppv and npv were based on the wald method with the ppv and npv variances determined by the delta method [ ] . for calculation of the % ci of the auc, we employed the wald method using logit transformation of the auc [ ] . lastly, we measured an adjusted auc by incorporating a demographic variable that appeared to be a potential confounding factor of hyperthermia (i.e. age) by employing a multiple logistic regression. since our selection criterion (c) for the , passengers already included those detected by the thermoscanners, we also assessed the above-mentioned diagnostic performances of the thermoscanners in identifying fever among the sample of self-reporting passengers only. the study conformed to the principles of the helsinki declaration. eligible subjects were voluntarily enrolled, and informed consent was obtained before the enrollment. simplified map of narita international airport. the airport has two discrete terminals. a total of four infrared thermoscanners were placed in each terminal. terminal is mostly used by alliance a, while terminal is roughly divided into two groups of satellites used by alliances b and c, respectively. the stationary infrared thermoscanners were set up near the quarantine station before immigration. the survey was conducted during the entry screening practice following the guideline of the japan pandemic influenza plan issued by the government of japan [ ] . the fever screening, health examination and laboratory testing were conducted according to the quarantine act (articles and ), and the use of the infrared thermoscanners and examination of axillary temperature adhered to the health service bureau notice issued by the tuberculosis and infectious disease control division of the ministry of health, labour and welfare of japan. the analysis of the data and its publication are permitted by article - of the quarantine act. no names (only id numbers) were assigned to each study participant and the data were anonymously analyzed. the mean (standard deviation (sd)) age of all the confirmed cases was . ( . ) years. the ages did not differ significantly between patients with h n - and the other influenza viruses (p = . ). males accounted for cases ( . %), and gender was not significantly associated with h n - (p = . ). a total of cases ( . %) were under medications upon arrival. five of the h n - cases ( . %) had taken commercially available cold/cough medications containing antipyretic substances, and one child case among the remaining four cases took an antibiotic (azithromycin) prior to arrival. these medications were started at hours to days before arrival. all cases with the other influenza viruses were under medications: five with commercially available cold/cough medications containing antipyretic substances, one with oseltamivir and one with an antibiotic (cefcapene pivoxil hydrochloride). medications were not significantly associated with h n - , when the antibiotics were both included and excluded (p = . and p = . , respectively). among the confirmed cases with h n - , the axillary temperature upon arrival ranged from . - . °c with a mean (sd) of . °c ( . °c). the axillary temperature of the cases with the other influenza viruses ranged from . - . °c with a mean (sd) of . °c ( . °c). the axillary temperature did not differ significantly between the two groups (p = . ; figure ) , and the proportions of hyperthermia also did not differ significantly between the two groups for the cut-off levels of . °c, . °c and . °c (p > . for all cut-off levels). for the cut-off levels of both . °c and . °c, the sensitivities of hyperthermia for detecting influenza were estimated to be . % ( % ci: , . ) for h n - and . % ( % ci: . , . ) for the other influenza viruses. using . °c as the cut-off level, the sensitivities were estimated to be . % ( % ci: , . ) for h n - and . % ( % ci: , . ) for the other influenza viruses. age and gender were not significantly associated with the proportion of hyperthermia cases among the total of confirmed influenza cases using all three cut-off levels (p > . for all cut-off levels). medications were also not associated with hyperthermia among the cases, when the antibiotics were both included and excluded (p > . for all cut-off levels). among the cases with h n - , medications were not significantly associated with hyperthermia (p > . for all cut-off levels), but the proportion of hyperthermia cases was smaller among those with medications for the cut-off levels of . °c and . °c. for both cut-off levels, the sensitivities of fever for detecting influenza were . % ( % ci: . , . ) and . % ( % ci: . , . ) among those with and without medications (including antibiotics), respectively. the age distribution of the , subjects is shown in figure a . the mean (sd) and median (lower to upper quartiles) ages were . ( . ) and ( - ) years, respectively. males accounted for persons ( . %). regarding the place of embarkation, cases ( . %) were from countries belonging to western pacific or southeast asian regions, ( . %) were from the americas, ( . %) were from europe and ( . %) were from the eastern mediterranean region, africa or the mean (sd) axillary temperature was . °c ( . °c ). the proportions of cases with hyperthermia using the cut-off levels of . °c, . °c and . °c were . % ( % ci: . , . ), . % ( % ci: . , . ) and . % ( % ci: . , . ), respectively. the mean (sd) temperature measured by the infrared thermoscanners was . °c ( . °c). during the period of observation, no confirmatory diagnoses of h n - were made among the total screened passengers (i.e. including passengers who were not included in our study). overall, persons were diagnosed with malaria, and and were diagnosed with dengue virus infection and chikungunya virus infection, respectively. the axillary temperature tended to be higher among younger passengers (pearson's correlation coefficient = - . , p < . ; figure b ). using the cut-off levels of . °c, . °c and . °c, the ages of the passengers with hyperthermia appeared to be significantly younger than those without fever (p < . for all cut-off levels). there was no gender-specificity in the proportions of hyperthermia for the cut-off levels of . °c and . °c (p = . and p = . , respectively), whereas genderspecificity was observed for the cut-off level of . °c (p = . ; odds ratio of being male with hyperthermia = . ( % ci: . , . )). place of embarkation was not significantly associated with hyperthermia (p > . for all cut-off levels). self-reporting was not significantly associated with hyperthermia for the cut-off levels of . °c and . °c (p > . for both), but was significantly associated for the cut-off level of . °c (p = . ; odds ratio of being a self-reporting passenger with hyperthermia = . ( % ci: . , . )), perhaps reflecting the fact that passengers without self-reporting were more likely to be febrile owing to our selection by employing infrared thermoscanners. satellite was associated with the proportion of hyperthermia (p < . for all cut-off levels), but the significant association disappeared after adjustment for age using a multiple logistic regression (data not shown). in a comparison of the axillary temperatures and the surface temperatures measured by the infrared thermoscanners, the pearson's correlation coefficient was estimated to be . (p < . ). as shown in the scatter plot in figure a , the variances of both measurements were large. using the three cut-off levels for hyperthermia, the surface temperatures measured by the infrared thermoscanners were significantly higher among those defined as having hyperthermia (p < . for all cut-off levels). table shows the diagnostic performances of the infrared thermoscanners in identifying fever at each cut-off level. using the cut-off levels of . °c, . °c and . °c, the sensitivities were estimated to be . %, . % and . % and the specificities were estimated to be . %, . % and . %, respectively. the ppv and npv ranged from . - . % and . - . %, respectively. the ppv was smallest ( . %) for the cut-off level of . °c, while the npv was smallest ( . %) for the cut-off level of . °c. the receiver operating characteristic (roc) curves for the , subjects with the three different cut-off levels are shown in figure b . the expected auc values ranged from . - . %, and were much smaller than those in previously published studies [ , ] . table also summarizes the estimated auc values after adjustment for age. the adjustment offered only slight improvements, and the age-adjusted auc ranged from . - . %. we also measured the diagnostic performances of the infrared thermoscanners in correctly detecting fever among only the self-reporting passengers (n = ). a scatter plot is shown in figure c , and the pearson's correlation coefficient was estimated to be . (p < . ), which was slightly greater than that for the total screened subjects. the estimated sensitivity and ppv were also higher than those of the total screened subjects (ranging from . - . % and . - . %, respectively), whereas the specificity and npv were only partially significantly different compared with those for the total screened passengers ( table ). the roc curves for the self-reporting passengers are shown in figure d . despite small improvements, the expected auc values were as low as . - . %. although the passengers defined as having hyperthermia were significantly younger among the self-reporting passengers (p = . , p = . and p = . for the cut-off levels of . °c, . °c and . °c, respectively), adjustment for age did not result in apparent improvement of the estimated auc ( . - . %). the present study analyzed epidemiological datasets of confirmed influenza cases whose diagnosis took place at narita international airport during the early stages of the pandemic and of a selected and suspected fraction of passengers screened from september to january . in our retrospective assessments of the diagnostic performances of fever screening in detecting and diagnosing influenza at the main entrance airport to japan, three key findings appeared to be notable. first, despite the small sample size, the sensitivity of fever (e. g. . °c) for detecting h n - upon arrival was estimated to be as low as . % among the confirmed cases with h n - . in addition, of the confirmed cases with h n - ( . %) were under antipyretic medications upon arrival. second, the estimates of the diagnostic performances of the infrared thermoscanners in identifying fever among the selected and suspected fraction of passengers were smaller than those in previously published studies, in which the samples were mostly general populations based on prospective study designs and/or under ideal study conditions [ , , , , ] . for example, the sensitivity and auc for the cut-off level of . °c in the present study were as low as . % and . %, respectively. third, even though we examined a suspected fraction of passengers as our subjects (i.e. those who were theoretically more likely to be febrile than the remaining passengers), the ppv still appeared to be as low as . - . %. considering the total passengers arriving at narita international airport, the actual ppv will be smaller than our estimates (owing to the smaller prevalence of hyperthermia), implying more false-positive passengers during mass screening if one relies on infrared thermoscanners for active detection of hyperthermia [ ] . in summary, our retrospective study demonstrates that reliance on fever alone is unlikely to be feasible as an entry screening measure. the most important caveat of the present study is that there are two independent processes when considering the diagnostic performances of fever screening at an international border [ ] . the first is the sensitivity of fever for detecting influenza cases. although influenzalike illness (e.g. defined as a temperature higher than . °c plus either cough or sore throat) can be accurately found by clinical examinations, it is known that the clinical findings do not permit the confirmation or exclusion of the diagnosis of influenza [ , ] . whereas the sensitivity of fever alone is undoubtedly higher than that of influenza-like illness and fever screening may be useful for avoiding a substantial number of false-negatives [ ] , more critical studies on influenza-like illness have indicated that a high temperature ( . °c or higher) is not the prime indicator of influenza [ , ] . thus, even with these facts alone, it is evident that active identification of influenza cases by fever screening alone is unlikely to be feasible. in addition, our all the values are expressed as percentages. † proportion of subjects whose axillary temperature was above the specified cut-off level; ‡ auc unadj , area under the receiver operating characteristic curve without adjustment for age; auc adj , age-adjusted estimate of the area under the receiver operating characteristic curve. values in parentheses are the % confidence intervals. experience at narita international airport led us to realize that the axillary temperature tends to be readily modified by commercial medications (e.g. antipyretics) in practical settings. although the proportion of febrile cases among confirmed h n - cases was reported to be % in the united states [ ] , no direct comparison can strictly be made because the fraction of febrile cases at an international border is different from that among a total number of confirmed cases in a community. however, that figure of % and the figure of . % obtained in our study indicate that the antipyretic medications taken by our study participants potentially reduced the risk of fever by . %. second, even though the diagnostic performances of the infrared thermoscanners in detecting fever were not sufficiently high, the prevalence of hyperthermia would be very small among the total number of international passengers, and thus the ppv would be considerably lowered [ , ] . the finding our study adds to the literature on this point is that the ppv of infrared thermoscanners was still insufficient for actively detecting febrile passengers, even when our interest was restricted to a suspected fraction of passengers. the sensitivity of entry screening in correctly detecting and diagnosing symptomatic influenza is measured by the product of the above-mentioned two different sensitivities [ ] , i.e. the sensitivity of fever for detecting influenza cases and the sensitivity of a non-invasive device for detecting febrile passengers. the ppv of entry screening is therefore smaller than that of the infrared thermoscanners alone. of course, a confirmatory diagnosis of influenza is further required to account for the limited sensitivity of the rapid diagnostic testing. the present study does not criticize the use of infrared thermoscanners, but does emphasize that reliance on its use during the entry screening of influenza is unlikely to be feasible. such devices could be used for other purposes (e.g. estimation of true prevalence based on known estimates of sensitivity and specificity among the total passengers) or in other settings (e.g. screening of fever in a setting with a far greater prevalence of hyperthermia), because infrared thermoscanners improve the detection of fever and are especially useful in settings where the ppv and npv do not matter [ ] . our estimates of the diagnostic performances must be interpreted with caution ( table ). the analyses of our second dataset were based on a retrospective non-random sample that was considered to represent a suspected fraction of passengers. in other words, the estimated sensitivity and specificity are not applicable to other passengers owing to the imposed selection criteria, and instead are only useful for the sample population that we examined. nevertheless, given the previous reports of the sensitivity and specificity among a wider spectrum of the population [ , , ] , this point should not be regarded as a negative aspect. the scientific value of our retrospective study was to demonstrate that the diagnostic performances of infrared thermoscanners in detecting febrile passengers, especially the sensitivity, can be even worse among the suspected fraction of passengers than among all the passengers. in addition to previous studies indicating that the use of infrared thermoscanners for fever screening prior to voluntary selfreporting was not fully justified [ ] [ ] [ ] , our study has demonstrated that infrared thermoscanners were not useful for actively detecting fever, even among a selected and suspected fraction of passengers. our investigation of a selected and suspected fraction of passengers only, especially with the inclusion of those detected by the infrared thermoscanners, could partly provide a reason for the small estimates of the specificity. for example, owing to the representation of the suspected fraction of passengers, there were not many subjects with low axillary temperatures among our subjects, thereby leading to small estimates of the specificity compared with all arriving passengers. since the inclusion of cases detected by the infrared thermoscanners in our samples complicates an explicit interpretation of our estimates, we also examined the diagnostic performances only among the self-reported cases. the estimates of ppv and npv among the self-reporting passengers did not differ significantly from those among our total subjects. in addition to the limited diagnostic performance of fever screening in identifying febrile influenza cases, it should be remembered that the readings of infrared thermoscanners are known to be influenced by other confounding factors, most notably by age and outdoor temperature [ , [ ] [ ] [ ] ]. although we were not able to adjust for room temperature owing to its variation depending on air-conditioning and individual routes (e. g. gate and satellite combinations), age was shown to be a confounding factor, even among the suspected fraction of passengers. there are two plausible explanations for these findings: (a) physiological reasons including agedependent vascular reactivity (e.g. the temperature varies more easily among children than among elderly persons) [ ] and (b) influenza h n - has mainly been observed in younger individuals, most notably among school-age children [ ] [ ] [ ] [ ] . although no confirmatory diagnoses of h n - were made during the screening from september to january , it is likely that substantial numbers of undetected cases were allowed into japan during the study period [ ] . the above-mentioned point (b) poses a technical challenge, because the real-time dependence of age on the epidemiology of influenza introduces a time-dependency in its influence on the readings of the infrared thermoscanners (i.e. a simple statistical adjustment does not hold in such instances). as an additional complication but perhaps one of the most important features among international passengers, our experience at narita international airport led us to realize that the use of antipyretics and antivirals is very likely among febrile passengers in practical settings, thereby greatly complicating the detection owing to masked symptoms. among those with any suspicious symptoms, it is natural that medications with commercially available antipyretics are widely used without any restrictions, and the different timings, doses and medicines do not permit us to adjust for the influence by statistical modeling. except for cases of imminent public health risk, the revised international health regulations (ihr) in were intended to minimize interference with world travel, permitting only non-invasive and least intrusive medical examinations that could achieve a "public health objective" [ ] . although infrared thermoscanners are non-invasive and may detect a small portion of febrile influenza cases among the total passengers, our study has demonstrated fundamental problems in the reliance on fever in detecting and diagnosing influenza in international passengers. in addition to the issue of screening, the effectiveness of entry screening involves the presence of incubating individuals [ , ] and asymptomatic cases [ , ] . given the limited information that we can gain from fever alone, one could further examine other vital signs to improve the detection during mass screening [ ] , along with efforts to promote self-reporting and improve its coverage. in addition to such devices, it is vital to reconsider the public health objectives of entry screening measures with a specific disease in mind (e.g. influenza) [ ] , and the way forward requires us to explicitly define the roles and purposes of international border control in the event of the next pandemic [ ] . to retrospectively assess the feasibility of detecting the cases of influenza (h n - ) based on fever screening as a sole measure in a practical setting, we analyzed epidemiological datasets of confirmed influenza cases whose diagnosis took place at narita international airport during the early stages of the pandemic and of a selected and suspected fraction of passengers screened from september to january . among the confirmed h n - cases (n = ), the sensitivity of fever for detecting influenza upon arrival appeared to be as low as . %, and of the cases ( . %) were under antipyretic medications. the ppv of the infrared thermoscanners for detecting fever among the suspected fraction of passengers (n = , ) was shown to be insufficient to actively detect febrile influenza cases among passengers. given the additional presence of confounding factors and unrestricted medications among passengers, the reliance on fever alone is unlikely to be feasible as an entry screening measure against influenza. world health organization working group on international and community transmission of sars: public health interventions and sars spread border screening for sars. emerg infect dis world health organization writing group: non-pharmaceutical interventions for pandemic influenza, international measures protective effect of maritime quarantine in south pacific jurisdictions, - influenza pandemic entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation evaluation of measures to reduce international spread of sars time lines of infection and disease in human influenza: a review of volunteer challenge studies quarantine for pandemic influenza control at the borders of small island nations the state-reproduction number for a multistate class age structured epidemic system and its application to the asymptomatic transmission model entry screening to delay local transmission of pandemic influenza a (h n ) governmental committee of action plans against influenza pandemic: action plans against 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thermal environment epidemiology of influenza a(h n )v virus infection in japan narita airport quarantine station: report of responses to pandemic influenza at the narita airport quarantine station chiba, narita airport quarantine station confidence intervals for predictive values with an emphasis to case-control studies comparison of non-parametric confidence intervals for the area under the roc curve of a continuous-scale diagnostic test comparison of infrared thermal detection systems and self-report for mass fever screening joint quantification of transmission dynamics and diagnostic accuracy applied to influenza does this patient have influenza? real-time forecasting of an epidemic using a discrete time stochastic model: a case study of pandemic influenza evaluation of an influenza-like illness case definition in the diagnosis of influenza among patients with acute febrile illness in cambodia working towards a simple case definition for influenza surveillance is influenza an influenza-like illness? clinical presentation of influenza in hospitalized patients emergence of a novel swineorigin influenza a (h n ) virus in humans thermal image scanning for influenza border screening: results of an airport screening study cutaneous vascular reactivity is reduced in aging and in heart failure: association with inflammation travel and age of influenza a (h n ) virus infection does glycosylation as a modifier of original antigenic sin explain the case age distribution and unusual toxicity in pandemic novel h n influenza? an epidemiological analysis of severe cases of the influenza a (h n ) virus infection in japan assortativity and the probability of epidemic extinction: a case study of pandemic influenza a did modeling overestimate the transmission potential of pandemic (h n - )? sample size estimation for post-epidemic seroepidemiological studies world health organization: international health regulations ( ) geneva, world health organization estimation of the incubation period of influenza a (h n - ) among imported cases: addressing censoring using outbreak data at the origin of importation a novel screening method for influenza patients using a newly developed non-contact screening system theoretical basis to measure the impact of shortlasting control of an infectious disease on the epidemic peak delaying the international spread of pandemic influenza we thank the study participants for their time and involvement in the study. hn was supported by the japan science and technology agency presto program. authors' contributions kk and hn conceived the study. hn developed the methodological ideas and implemented the statistical analyses. hn drafted the manuscript and all the authors discussed and revised the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- - qq xsw authors: kramer, axel; schwebke, ingeborg; kampf, günter title: how long do nosocomial pathogens persist on inanimate surfaces? a systematic review date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: qq xsw background: inanimate surfaces have often been described as the source for outbreaks of nosocomial infections. the aim of this review is to summarize data on the persistence of different nosocomial pathogens on inanimate surfaces. methods: the literature was systematically reviewed in medline without language restrictions. in addition, cited articles in a report were assessed and standard textbooks on the topic were reviewed. all reports with experimental evidence on the duration of persistence of a nosocomial pathogen on any type of surface were included. results: most gram-positive bacteria, such as enterococcus spp. (including vre), staphylococcus aureus (including mrsa), or streptococcus pyogenes, survive for months on dry surfaces. many gram-negative species, such as acinetobacter spp., escherichia coli, klebsiella spp., pseudomonas aeruginosa, serratia marcescens, or shigella spp., can also survive for months. a few others, such as bordetella pertussis, haemophilus influenzae, proteus vulgaris, or vibrio cholerae, however, persist only for days. mycobacteria, including mycobacterium tuberculosis, and spore-forming bacteria, including clostridium difficile, can also survive for months on surfaces. candida albicans as the most important nosocomial fungal pathogen can survive up to months on surfaces. persistence of other yeasts, such as torulopsis glabrata, was described to be similar ( months) or shorter (candida parapsilosis, days). most viruses from the respiratory tract, such as corona, coxsackie, influenza, sars or rhino virus, can persist on surfaces for a few days. viruses from the gastrointestinal tract, such as astrovirus, hav, polio- or rota virus, persist for approximately months. blood-borne viruses, such as hbv or hiv, can persist for more than one week. herpes viruses, such as cmv or hsv type and , have been shown to persist from only a few hours up to days. conclusion: the most common nosocomial pathogens may well survive or persist on surfaces for months and can thereby be a continuous source of transmission if no regular preventive surface disinfection is performed. within the global infection control community, there is an ongoing controversy about the appropriate treatment of inanimate surfaces in hospitals in order to prevent transmission of nosocomial pathogens within an institution. based on a lack of epidemiological data that would provide evidence of a benefit for the patient from surface disinfection (e.g., from a significant reduction of nosocomial infection rates), some scientists postulate that cleaning of surfaces with non-antimicrobial detergents is generally sufficient [ ] . others prefer cleaning of surfaces with antimicrobial agents, based on data on the risk of infection due to microbial contamination and potential transmission of nosocomial pathogens, at least in the immediate vicinity of patients [ ] [ ] [ ] . new guidelines on treatment of surfaces in hospitals take into account more parameters which are considered to be relevant for preventing the transmission of nosocomial pathogens, such as the type of ward or the expected frequency of hand contact with a surface [ , ] . irrespective of the divergent opinions regarding the appropriate treatment of surfaces, an important parameter for a fair scientific assessment remains, that is, the persistence of nosocomial pathogens on surfaces. the longer a nosocomial pathogen persists on a surface, the longer it may be a source of transmission and thus endanger a susceptible patient or healthcare worker. the aim of this review was therefore to collect and assess the data that have been published in the last decades on persistence of all types of nosocomial pathogens on surfaces, both in the context of surface disinfection and the control of nosocomial outbreaks. the literature was systematically reviewed in medline on the internet homepage of the national library of medicine without language restrictions. the search was done on december and covered all years available in medline. the following search terms were applied: persistence, survival, surface, fomite, bacteria, virus, pathogen, transmission, and nosocomial. in addition, the citations in each study found during the main search were reviewed for potential relevance. finally, standard textbooks on infection control, bacteriology and virology were examined for information. all reports with experimental evidence on the duration of persistence of a nosocomial pathogen on any type of inanimate surface were included. information from textbooks was also included, even if the chapter itself did not contain experimental evidence. at least two of the investigators decided on the relevance of each report. reports were not blinded to the investigators so that they knew the names of the authors of all studies. for a clinically relevant summary, all nosocomial pathogens were grouped according to their importance in causing hospital-acquired hand-transmitted infections [ ] and according to their mode of nosocomial transmission [ ] . the range of the reported duration of persistence was used as the principle outcome of the search for each nosocomial pathogen. in addition, parameters with potential influence on persistence were evaluated in all experimental studies. most gram-positive bacteria, such as enterococcus spp. (including vre), staphylococcus aureus (including mrsa), or streptococcus pyogenes survive for months on dry surfaces (table ). in general, there was no obvious difference in survival between multiresistant and susceptible strains of staphylococcus aureus and enterococcus spp. [ ] . only in one study was such a difference suggested, but the susceptible strains revealed a very brief survival as such [ ] . many gram-negative species, such as acinetobacter spp., escherichia coli, klebsiella spp., pseudomonas aeruginosa, serratia marcescens, or shigella spp. can survive on inanimate surfaces even for months. these species are found among the most frequent isolates from patients with nosocomial infections [ ] . a few others, such as bordetella pertussis, haemophilus influenzae, proteus vulgaris, or vibrio cholerae, however, persist only for days (table ) . mycobacteria -including mycobacterium tuberculosis and spore-forming bacteria, including clostridium difficilecan also survive for many months on surfaces (table ) . overall, gram-negative bacteria have been described to persist longer than gram-positive bacteria [ , ] . humid conditions improved persistence for most types of bacteria, such as chlamydia trachomatis [ ] , listeria monocytogenes [ ] , salmonella typhimurium [ ] , pseudomonas aeruginosa [ ] , escherichia coli [ ] , or other relevant pathogens [ , ] . only staphylococcus aureus was found to persist longer at low humidity [ ] . low temperatures, e.g., °c or °c, also improved persistence of most types of bacteria, such listeria monocytogenes [ ] , salmonella typhimurium [ ] other factors were rarely investigated and hence provide inconsistent results. longer persistence has been described with higher inocula [ ], in the presence of protein [ ] , serum [ , ] , sputum [ ], or without dust [ ] . candida albicans as the most important nosocomial fungal pathogen can survive up to months on surfaces ( table ). persistence of other yeasts was described to be similar (torulopsis glabrata months) or shorter (candida parapsilosis days). the presence of serum or albumin, a low temperature, and high humidity have been described as leading to longer persistence [ ] . most viruses from the respiratory tract such as corona-, coxsackie-, influenzavirus, sars, or rhinovirus can persist on surfaces for a few days. viruses from the gastrointestinal tract, such as astrovirus, hav, polio-and rotavirus persist for approximately months. blood-borne viruses, such as hbv or hiv, can persist for more than one week. herpes viruses such as cmv or hsv type and have been shown to persist from only a few hours up to days. [ ] or norovirus [ ] . other investigators found that per- sistence was favored on non-porous surfaces for influenzavirus [ ] , on formica and gloves for rsv [ ] , and on a telephone receiver for fcv [ ] . other parameters for a longer persistence of viruses include the presence of fecal suspension [ ] and a higher inoculum [ ] . cryptosporidium species have been reported to survive on dry surfaces for only hours [ ] . the most relevant nosocomial pathogens can persist on dry inanimate surfaces for months. in addition to the duration of persistence, some studies have also identified factors influencing persistence. a low temperature, such as °c or °c, was associated with longer persistence for most bacteria, fungi and viruses. high humidity (e.g., > %) was also associated with longer persistence for most bacteria, fungi, and viruses, although for some viruses conflicting results were reported. a few studies also suggest that a higher inoculum is associated with longer per-sistence. the type of surface material and the type of suspension medium, however, reveal inconsistent data. overall, a high inoculum of the nosocomial pathogen in a cold room with high relative humidity will have the best chance for long persistence. in most reports with experimental evidence, persistence was studied on dry surfaces using artificial contamination of a standardized type of surface in a laboratory. in most studies, bacteria were prepared in broth, water or saline. viruses were usually prepared in a cell culture medium [ ] . the main advantage is that the environmental conditions are consistent regarding temperature and air humidity. in addition, the effect of temperature or relative humidity can only be determined under controlled conditions, which are much easier to ensure in the laboratory. however, this may not always reflect the clinical situation, in which surfaces can be simultaneously contaminated with various nosocomial pathogens and different types of body fluids, secretions etc. yet the question remains: what is the clinical evidence for the role of surfaces in nosocomial infections? in hospitals, surfaces with hand contact are often contaminated with nosocomial pathogens [ ] [ ] [ ] , and may serve as vectors for cross transmission. a single hand contact with a contaminated surface results in a variable degree of pathogen transfer. transmission to hands was most successful with escherichia coli, salmonella spp., staphylococcus aureus (all %) [ ] , candida albicans ( %) [ ] , rhino virus ( %) [ ] , hav ( % - %) [ ] , and rota virus ( %) [ , ] . common modes of transmission from inanimate surfaces to susceptible patients figure common modes of transmission from inanimate surfaces to susceptible patients. susceptible patient direct transmission compliance in hand hygiene: ~ % to more surfaces [ ] or other subjects [ ] . contaminated hands can also be the source of re-contaminating the surface, as shown with hav [ , ] . compliance rates of healthcare workers in hand hygiene are known to be around % [ ] . due to the overwhelming evidence of low compliance with hand hygiene, the risk from contaminated surfaces cannot be overlooked (figure ). the main route of transmission is via the transiently contaminated hands of the healthcare worker [ ] [ ] [ ] . an outbreak of nosocomial infections due to acinetobacter baumannii in a neurosurgical intensive care unit may serve as an example. a direct correlation was found between the number of environmental isolates obtained during screening and the number of patients who were colonized or infected with the same strain during the same calender month [ ] . during outbreaks, the environment may play a significant role for transmission of nosocomial pathogens, as suggested by observational evidence. this has been described for various types of microorganisms, such as acinetobacter baumannii [ ] [ ] [ ] , clostridium difficile [ ] [ ] [ ] , mrsa [ , ] , pseudomonas aeruginosa [ , ] , vre [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , sars [ , ] , rota- [ , ] , and norovirus [ ] . however, the evidence to support a role of environmental contamination is not equally strong for all types of nosocomial pathogens. for clostridium difficile, mrsa, and vre, data are stronger than for other pathogens, such as pseudomonas aeruginosa or acinetobacter baumannii, of which multiple types were detected in the environment, and which did not always correlate with the acquired strain [ ] . the role of surface disinfection for the control of nosocomial pathogens has been a contentious issue for some time [ ] . routine treatment of clean floors with various types of surface disinfectants (some of them had rather poor bactericidal activity) has been described to have no significant impact on the incidence of nosocomial infections [ ] . disinfection of surfaces in the immediate environment of patients, however, has been described to reduce acquisition of nosocomial pathogens such as vre [ ] or acinetobacter baumannii [ 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have no acknowledgements. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /pre pub key: cord- -w n o authors: wang, yan; yan, jiangwei; shi, yuling; li, ping; liu, chuanxuan; ma, qingjun; yang, ruifu; wang, xiaoyi; zhu, lina; yang, xiao; cao, cheng title: lack of association between polymorphisms of masp and susceptibility to sars coronavirus infection date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: w n o background: the pathogenesis of severe acute respiratory disease syndrome (sars) is not fully understood. one case-control study has reported an association between susceptibility to sars and mannan-binding lectin (mbl) in china. as the downstream protein of mbl, variants of the mbl-associated serine protease- (masp ) gene may be associated with sars coronavirus (sars-cov) infection in the same population. methods: thirty individuals with sars were chosen for analysis of masp polymorphisms by means of pcr direct sequencing. tag single nucleotide polymorphisms (tagsnps) were chosen using pairwise tagging algorithms. the frequencies of four tag snps (rs , rs , rs and rs ) were ascertained in sars patients and control subjects, using the beckman snpstream ultra high throughput genotyping platform. results: there is no significant association between alleles or genotypes of the masp tagsnp and susceptibility to sars-cov in both beijing and guangzhou populations. diplotype (rs and rs )were analyzed for association with susceptibility to sars, no statistically significant evidence of association was observed. the beijing and guangzhou sample groups were homogeneous regarding demographic and genetic parameters, a joined analysis also showed no statistically significant evidence of association. conclusion: our data do not suggest a role for masp polymorphisms in sars susceptibility in northern and southern china. severe acute respiratory disease syndrome (sars), a new and highly infectious disease that is caused by a previously undescribed coronavirus in humans, has created a major public health threat in many countries [ ] [ ] [ ] . progress has been made in understanding sars coronavirus (sars-cov) and the epidemiology, clinical manifestations, laboratory findings and radiological features of this disease have all been studied in detail [ , ] . however, its pathogenesis is still not fully understood. it has been reported that diabetes mellitus and heart disease are risk factors for adverse prognosis of sars [ ] , however, little is known about the contribution of genetic factors. sars has been found to have a profoundly adverse effect on the immune system [ ] . variation in host immunity may be one of the important factors that determine susceptibility to sars. a few case-control studies have reported an association between sars susceptibility and human leucocyte antigen (hla) and mbl [ ] [ ] [ ] [ ] . deficiency of mbl, a key component of the innate immune system, has been detected in sars patients. such a deficiency may increase susceptibility to sars infection. three papers reported the association of hla and susceptibility to sars [ ] [ ] [ ] . of these, two reported the association of hla with susceptibility and resistance to the development of sars [ , ] . in a more recent paper, no statistical significance was found with the susceptibility and severity of the disease [ ] . mbl is a member of the collectin family and plays an important role in innate immunity [ , ] . mbl and ficolins distinguish between self, non-self and altered-self by recognizing patterns of ligands on the surface of microorganisms or aberrant cells. when this happens, mbl-associated serine protease- (masp- ) is activated and cleaves complement factors to initiate the antibody-independent pathway of the complement system, thus starting inflammatory reactions. mbl-associated serine proteases interact with mbl via the collagenous region of larger mbl oligomers. four related proteins derived from two genes have been reported; namely masp , its alternative splicing variant masp , and masp with its alternative splicing variant map [ ] [ ] [ ] . masp activates the complement system by cleaving complement proteins c and c . masp is an essential component of the lectin pathway of complement activation. masp deficiency is observed because of genetic polymorphisms. it is known that a masp polymorphism, namely d g has a functional effect on the protein, and does not allow the formation of an active mbl-masp complex. this variant has been described for the first time in a patient with an inherited deficiency of masp , who was characterized by augmented susceptibility to infection and development of immunological disease. with regard to sars-cov infection, the codon variant of the mbl gene has been shown to be associated with infection susceptibility but not with disease severity [ ] . as the downstream protein of mbl, variants of the masp gene may be associated with sars-cov infection. to examine the hypothesis that polymorphisms of the masp gene in sars patients are genetic factors that influence infection susceptibility, we studied masp gene polymorphisms in dna from two groups of chinese sars patients, and compared these with normal blood donors from the same region. this study was performed with the approval of the ethical committee of the chinese human genome center. a total of sars patients, who were diagnosed based on who criteria, were recruited during the outbreak. all sars patients selected for study were unrelated and were shown to be seropositive by anti-sars-n protein elisa and immunofluorecent assay (ifa). the specificity of the elisa assay is more than % [ , ] . of these, were from the wards at xiaotangshan hospital, beijing, china, and the remaining were from the eighth people's hospital of guangzhou in southern china. a total of age, sex, and ethnicity-matched healthy, genetically unrelated and seronegative (confirmed by anti-sars n protein antibody elisa and sars antibody ifa) adults were recruited as control subjects. we extracted genomic dna from peripheral blood leukocytes of affected individuals and controls using the qiaamp dna mini kit (qiagen, valencia, ca). dna was quantified using standardized fluorometric reading on a du spectrophotometer (beckman coulter, fullerton, ca). each sample was diluted to a final concentration of ng/ ul. genomic dna from individuals with sars was chosen for analysis of masp gene polymorphisms. the sample included chromosomes, which provided a % confidence level to detect alleles with a frequency > % [ ] . with a candidate-gene strategy, we screened for polymorphisms in all exons (~ . kb), '-and '-flanking regions (lengths of . kb), untranslated regions (~ . kb), and about . kb intronic sequences. dna sequence spanning the masp gene was obtained from the national center for biotechnology information (ncbi) website (available from http://www.ncbi.nlm.nih.gov/; nt_ . ). primers were designed using the premier program (primer premier ). pcr primer sequences see additional file . dna samples from the individuals were amplified and purified. the pcr reaction volume was μl, containing - ng of dna, pmol of each forward and reverse primer, . mmol/l of each dntp, . u of dna polymerase(tiangen biotec co, beijing, china), mmol/l of tris-hcl, . mmol/l of mgcl , mmol/l of kcl, and . % triton x- . amplication was carried out in a geneamp pcr system (abi) with cycle parameters of min °c (initial denaturation), rounds of °c s, - °c s, and °c s, and a final extention for min at °c. pcr products were sequenced using an abi prism dye terminator sequencing kit with amplitaq gold dna polymerase (applied biosystems division/perkin-elmer, foster city, ca) and loaded onto an abi sequencer. polymorphism candidates were identified by the polyphred program. (p. green, personal communication) snp genotypes were verified by manual evaluation of the individual sequence traces. genotyping was completed using snpstream ultra high throughput genotyping system (beckman coulter, fullerton, ca) according to the manufacture's instructions. briefly, the method combines solution-phase multiplex single nucleotide extension (sne) with a solid-phase sorting of labeled sne primers by hybridization to a chip that contains × arrays of oligo-nucleotide tags and oligo-nucleotides for positive and negative controls. each sne primer contained of the oligonucleotide tags at its ' end, and the sne reactions were performed in -plex. the microarray plate was imaged by the snpscope reader (beckman coulter, fullerton, ca). the two-color system allowed the detection of the snp by comparing signals from the two fluorescent dyes. the image signals were then transferred to genotyping software that translated the images of the arrays into genotype calls. the error rate based upon sequencing for % of the snps examined in the present study was - . %. the exact test was calculated to evaluate if the population was in hardy-weinberg equilibrium by the markov chain method, taking p < . as the cutoff for assessing significance. the tagsnps were chosen on a pairwise basis and linkage disequilibrium (ld) calculation was performed on the confidence interval basis using haploview . software. genotype and alleles frequencies for each polymorphism were determined by gene counting. diplotypes of each individual were inferred using the algorithm developed by stephens et al. ( ) (phase). the chi square test was used to determine whether allele frequencies differed between sars cases and controls. binary logistic regression was used to analyze the association between a single locus and sars susceptibility, adjusted for sex and age status, and odds ratio and % ci were used to measure strength of association in a genetic risk association study. these statistical analyses were implemented in statistical package for the social sciences . software (spss inc, chicago, illinois, usa). demographic characteristics are shown in table . the age and sex distribution of the patients and controls were not significantly different, which indicated that the matching of controls to cases was adequate. screening masp for polymorphisms sequencing of the exons of masp , the ' and ' regions of the gene, and some intronic sequences in individuals with sars identified polymorphisms ( table ). eleven of the snps have been published in the dbsnp database http://www.ncbi.nlm.nih.gov/snp/ index.html. allele and genotype frequencies were consistent with those expected under hardy-weinberg equilibrium. nine snps (allele frequency > %) were chosen with haploview for assessment. the snps were contained in two blocks of ld (fig. ) , as defined by lewontin's| d'|. snps including rs , rs and rs were located outside of the defined ld blocks. four tag-snps were chosen with the pairwise tagging algorithm implemented in the tagger program of haploview: rs , rs , rs and rs (r threshold was . ). genotype frequencies of the tag-snp except rs in other populations that have been published in the hapmap database were shown in table genotype data and diplotype (rs and rs ) were analyzed for association with susceptibility to sars, in beijing and guangzhou populations, using binary logistic regression for overall genotypic association (tables and ). no statistically significant evidence of association was observed. the beijing and guangzhou sample groups were homogeneous regarding demographic and genetic parameters, so a joined analysis was done, no statistically significant evidence of association was observed (table ). possible contribution of host genetic factors to the susceptibility and outcome of sars-cov infection has been investigated through several association studies [ ] [ ] [ ] [ ] , mbl deficiency because of polymorphisms in the mbl gene has been shown to be involved in sars-cov infection. as the downstream protein of mbl, improper masp activity can interfere with complement functions [ ] . there is an association between the genetics of masp and its serum level [ ] [ ] [ ] [ ] . thiel et al. [ ] have analyzed the mutation of p. _ -dupchnh and snps p.r q, p.r c, p.d g, p.p l and p.v a in four popu- lations: africans from zambia, hong kong chinese, brazilian amerindians and danish caucasians. p. _ -dupchnh was only found in chinese (gene frequency . %), associated with low levels of masp , and p.d g was found only in caucasians. p.p l and p.r q were present in africans and amerindians only. masp levels were reduced in individuals with p.v a (rs ). therefore, we chose individuals with sars from beijing for analysis of masp gene polymorphisms. the sample included chromosomes, which provided a % confidence level to detect alleles with a frequency > %. however, we only observed the snp rs (p.v a) among those mentioned in the study of thiel et al. we analyzed four tagsnps in the masp gene in two different sars patients and controls, searching for a possible genotype-phenotype correction, but no statistically significant difference was found for any polymorphisms between the different groups genotyped, while the possible role of the rare variants is to be determined. our analysis indicates that masp polymorphisms is not directly related to sars-cov susceptibility in northern and southern chinese. the authors declare that they have no competing interests. a two snp diplotypes span the genomic region of exon (rs and rs ). a two snp diplotypes span the genomic region of exon (rs and rs ). zln have contributed to genotyping; cc, lcx and mqj performed the data analyses, prepared the manuscript and supervised this study. all authors contribute to writing of the final manuscript. all authors read and approved the final manuscript. coronavirus as a possible cause of severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome(sars) world health organization: consensus document on theepidemiology of severe acute respiratory syndrome (sars) clinical features and short-term outcomes of patients with sars in the greater toronto area pathogenetic mechanisms of severe acute respiratory syndrome association of hla class i with severe acute respiratory syndrome coronavirus infection association of human-leukocyteantigen class i (b* ) and class ii (drb * ) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome lack of association between hla-a, -b and-drb alleles and thedevelopment of sars: a cohort of sars-recovered individuals in a population of guangdong southern china influence of fcgammariia and mbl polymorphisms on severe acute respiratory syndrome. tissue antigens association between mannan-binding lectin genepolymorphisms and susceptibility to severe acute respiratory syndromecoronavirus infection a newcomplementdependent bactericidal factor found in nonimmune mouse sera: specific binding to polysaccharide of ra chemotype salmonella masp- and its association with distinct complexes of the mannan-binding lectin complement activation pathway a second serine protease associated with mannan-binding lectin that activates complement two constituents of the initiation complex of the mannan-binding lectin activation pathway of complement are encoded by a single structural gene profile of antibodies to the nucleocapsid protein of the severe acute respiratory syndrome (sars)-associated coronavirus in probable sars patients diagnosis of severe acute respiratory syndrome (sars) by detection of sars coronavirus nucleocapsid antibodies in an antigencapturing enzyme-linked immunosorbent assay variation is the spice of life ace polymorphism and progression of sars the association of rantes polymorphism with severe acute respiratory syndrome in hong kong and beijing chinese association of sars susceptibility with single nucleic acid polymorphisms of oas and mxa genes: a case-control study homozygous l-sign (clec m) plays a protective role in sars coronavirus infection deficiency of mannanbinding lectin associated serine protease- due to missense polymorphisms mannan-binding-lectin-associated serine proteases, characteristics and disease associations horcajada. novel masp variants detected among north african and sub-saharan individuals genetic influences on mannan-binding lectin (mbl) and mannan-binding lectin associated serine protease- (masp- ) activity primers used for discovery of masp polymorphisms. click here for file [http://www.biomedcentral.com/content/supplementary/ - - - -s .doc] the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -gf mgz x authors: zhang, xi; rao, huaxiang; wu, yuwan; huang, yubei; dai, hongji title: comparison of spatiotemporal characteristics of the covid- and sars outbreaks in mainland china date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: gf mgz x background: both coronavirus disease (covid- ) and severe acute respiratory syndrome (sars) are caused by coronaviruses and have infected people in china and worldwide. we aimed to investigate whether covid- and sars exhibited similar spatial and temporal features at provincial level in mainland china. methods: the number of people infected by covid- and sars were extracted from daily briefings on newly confirmed cases during the epidemics, as of mar. , and aug. , , respectively. we depicted spatiotemporal patterns of the covid- and sars epidemics using spatial statistics such as moran’s i and the local indicators of spatial association (lisa). results: compared to sars, covid- had a higher overall incidence. we identified clusters (predominantly located in south-central china; the highest rr = . , % ci: . – . ) for covid- and clusters (mainly in northern china; the highest rr = . , % ci: . – . ) for sars. fewer secondary clusters were identified after the “wuhan lockdown”. the lisa cluster map detected a significantly high-low (hubei) and low-high spatial clustering (anhui, hunan, and jiangxi, in central china) for covid- . two significant high-high (beijing and tianjin) and low-high (hebei) clusters were detected for sars. conclusions: covid- and sars outbreaks exhibited distinct spatiotemporal clustering patterns at the provincial levels in mainland china, which may be attributable to changes in social and demographic factors, local government containment strategies or differences in transmission mechanisms. supplementary information: the online version contains supplementary material available at . /s - - -y. since the world health organization (who) declared the outbreak of coronavirus disease (covid- ) a public health emergency of international concern (pheic) on january , , this emerging infectious disease has spread rapidly in china and to other countries beyond china. as of march , , the total number of confirmed cases of covid- climbed to approximately , , with more than reported deaths. approximately , people had been identified as close contacts of infected patients, and , people had been under medical observation or quarantine in china [ ] . compared to the severe acute respiratory syndrome (sars) outbreak in , which was also caused by a similar coronavirus, covid- has been much more transmissible and rapidly spread from a single city to the entire country in just days [ ] . the estimated basic reproductive numbers (r s) for covid- and sars were approximately . [ ] and . [ ] , respectively. the transmission mechanisms of covid- are currently poorly understood, although this disease is considered to be one of the most widespread and destructive infectious diseases. there is a need for a more integrated investigation and coordinated international response to the outbreak. spatiotemporal analyses, which integrate spatial and time-series analyses, can provide additional information on the persistence of patterns over time and illuminate any unusual patterns. therefore, in this study, by collecting the daily numbers of newly confirmed covid- and sars cases during the two epidemics, we aimed to determine the spatial behavior and temporal features of the covid- spread in mainland china and compared them with respective features from the sars epidemic using spatiotemporal analysis. the present study included the number of incident cases of covid- and sars in provinces (provincial-level regions on the chinese mainland). incident cases infected by covid- were extracted from the daily briefings on novel coronavirus cases from january to march , , provided on the official website of the national health commission of the people's republic of china [ ] . we confirmed the daily total numbers of reported cases with the surveillance data provided by the who [ ] . incident cases of sars were extracted from daily situation reports for mainland china from april to august , , which were posted by china.org.cn (in chinese) and were also provided by the national health commission. we confirmed the daily total numbers of reported cases of sars with the cumulative numbers of reported cases provided by the who [ ] . cases of covid- included diagnosed cases confirmed by at least one of the following three methods: isolation of covid- virus, at least two positive results for covid- virus by real-time reversetranscription polymerase chain reaction (rt-pcr) assay or a genetic sequence that matches covid- virus [ ] . a clinically diagnosed case was defined as a suspected case with imaging features of pneumonia, which has only been applicable in hubei province since february , [ ] . cases of sars were defined in accordance with the "national case definition of infectious atypical pneumonia (sars) in china, ," which was updated by the national health commission on april , . criteria for probable and suspected sars included a) travel to a sars epidemic area in the weeks before onset of symptoms or close contact with a probable sars patient; b) fever of > °c; c) chest x-ray abnormalities; d) normal or decreased leukocyte count; and no response to treatment with antimicrobial drugs [ ] . we used arcgis software v . . (esri inc., redlands, ca, usa) to depict the spatial distribution and perform global and local spatial autocorrelation analyses. we used kulldorff's space-time scan statistical analysis to detect the space-time clusters of sars and covid- and to examine whether and to what extent the geographic clustering was explained by random variation. considering the relatively low incidence rate, we used the discrete poisson probability model as the scanning statistical model. in kulldorff's space-time scanning, the radius of the population coverage was used, and the maximum spatial scanning area was set to cover % of the risk population. the maximum temporal scanning window was set to cover % of the total research time. the scan window was increased gradually from to the maximum, and the log-likelihood ratios (llrs) were calculated for each window. the window with the maximum likelihood was defined as the most likely cluster area. other clusters with statistically significant llrs were defined as the secondary potential clusters. the llr p-value was estimated through , monte carlo simulations. a p-value < . indicated a significantly high risk inside of the scan window and a potential high-risk cluster of the disease. the relative risk (rr) and its % confidence interval ( % ci) of the disease in each cluster was calculated to evaluate the risk of sars and covid in the detected cluster areas. the results of spatiotemporal scans are sensitive to various parameters, such as the maximum spatial and temporal cluster sizes. thus, the selection of the maximum radius of the spatial scanning window and the maximum length of the temporal scanning window were very important. in this study, we selected the maximum spatial cluster sizes as and % of the total population at risk and the maximum temporal cluster sizes as % of the total study period. based on the optimal spatiotemporal parameters, retrospective space-time scanning analysis was applied to identify the geographic areas and time periods of potential clusters with significantly higher covid- and sars incidence than those of nearby areas. the spatial autocorrelation analysis was conducted by using open geoda software v . . (geoda center for geospatial analysis and computation, arizona state university, az, usa). to identify the spatial clustering of the covid- and sars incidence at the provincial level, we used row standardized first-order contiguity rook neighbors as the criterion for identifying neighbors, as described in [ ] . we calculated moran's i value and the local indicators of spatial association (lisa) statistic to analyze the global and local clusters as well as spatial outliers. there were four categories of spatial patterns in the lisa map. the high-high and low-low locations (positive local spatial autocorrelation) were typically referred to as spatial clusters, while the highlow and low-high locations (negative local spatial autocorrelation) were termed spatial outliers. a cluster was computed as such when the value at a location (either high or low) was more similar to its neighbors than would be the case under spatial randomness. the highhigh locations referred to hot spot areas where the risk of case spreading was higher than average, whereas the low-low locations referred to cool spot areas where the risk of case spread was lower than average. in detail, high-high indicates a spatial relationship in which an area with high incidence is surrounded by an area with high incidence; low-low indicates a spatial relationship in which areas with low incidence are surrounded by areas with the same low incidence. both high-high and low-low denote positive spatial autocorrelation, implying a spatial homogeneity. high-low represents a spatial relationship between areas with high morbidity and areas with low morbidity; low-high represents a spatial relationship in which a low-incidence area is surrounded by a high-incidence area. low-high and high-low represents negative spatial autocorrelation, implying a spatial heterogeneity. considering the stringent measure of quarantining in wuhan (hubei) and neighboring cities introduced on january , , we further conducted subgroup analyses by dividing the covid- data into two stages: stage (january to february , , quarantine date plus a -day incubation period) and stage (february to march , ). we also performed spatiotemporal clustering analysis for covid- by excluding cases in hubei. as of march , , provinces ( % of mainland china) reported , covid- cases, with the number of incident cases ranging from to , per day. the average incidence rate was . infections per , persons (range: . - . ) during the selected period of the covid- epidemic. outside of the hubei province epicenter, beijing and shanghai were among the first case-reported provinces for covid- on january , . compared with covid- , sars had a less widely influential area but a longer epidemic duration, and only provinces ( % of mainland china) reported sars cases as of august , , with an average incidence rate of . per , (range: . - . ). (fig. ) . to illustrate the spread of the two diseases nationally, we plotted the temporal changes in covid- and sars in provinces in mainland china (fig. , ordered by administrative area code). in most provinces except hubei, the rate of increase in the number of cases for covid- was fast for the first two weeks and reached a peak at the end of january. on the other hand, the incidence trend for sars was mostly flat, except in beijing, tianjin, hebei, shanxi and inner mongolia. notably, compared to sars, there was an obvious increasing trend for covid- in terms of the number of new cases in provinces, such as heilongjiang, shanghai, jiangsu, zhejiang, anhui, jiangxi, shandong, henan, hubei, hunan, chongqing and sichuan. on the other hand, several provinces in western china, such as guangxi, yunnan, shaanxi, gansu, qinghai, ningxia, xinjiang and tibet, had a much lower prevalence for both covid- and sars. through spatiotemporal clustering analysis, we identified high-risk clusters for covid- within cluster time frames (fig. a) . the most likely cluster was the epicenter, hubei, with an rr of ( % ci: - ) compared with the neighboring provinces and the longest high-risk period of days (p < . ), indicating that the risk of covid- infection in this most likely cluster areas were times higher than those outside this area. two significant secondary clusters were identified in zhejiang (from jan. to jan. , , p < . ) and shandong (in feb. , , p < . ), with similar rrs of . ( % ci: . - . ) and . ( % ci: . - . ), respectively. another possible cluster was identified in jiangxi (from february to february , , p = . ). when considering the measure of quarantine in hubei, the rr of ( % ci: - ) in stage (from february to march , ) was remarkedly increased compared to the rr of ( % ci: - ) in stage (january to february , ) (additional file a and b). there were different spatial behaviors and temporal features between the two stages. when excluding cases in hubei, the high-risk clusters were centered on the areas around hubei and in beijing, shanghai, and heilongjiang in stage , whereas the high-risk clusters were only restricted within the neighborhood areas of hubei in stage . moreover, the rrs in both stages were significantly decreased for the most likely cluster, with rrs of . ( % ci: . - . ) in stage and . ( % ci: . - . ) in stage (additional file a and b). different from covid- , the most likely cluster of sars was centered on beijing (fig. b) , lasting from april to may , , with the highest rr of ( % ci: - ) and a longest period of days (p < . ). three significant secondary clusters were identified in shanxi and hebei (from april to may , , p < . ), guangdong (from april to may , , p < . ), and provinces of jilin, liaoning, heilongjiang and tianjin (from april to may , , p < . ), respectively. the global moran's i values for covid- and sars were − . and . , respectively (both p > . ), which indicated that the case distribution may be due to chance rather than global autocorrelation (fig. ) . the lisa cluster map showed the significant locations color coded by the type of spatial autocorrelation. for covid- , the high-low spatial clustering was in hubei province. in addition, we identified significant clusters at p < . and significant clusters at p < . . specifically, liaoning, inner mongolia, and most western provinces had significantly low-low spatial clustering, whereas anhui, hunan and jiangxi of central china had significantly low-high spatial clustering. for sars, two significant high-high (beijing and tianjin) and low-high (hebei) clusters were detected. sichuan, tibet and anhui showed significant low-low clustering (fig. ) . this study showed significantly distinct spatiotemporal clustering patterns between covid- and sars outbreaks in mainland china. compared to sars, covid- had a higher incidence as well as wider and faster transmission. the significant high-risk areas for covid- were predominantly clustered in south-central china, around hubei, from january to february , . additionally, our results suggest that the quarantine measure taken in hubei might have played a crucial role in restricting the infected areas, shortening the epidemic period, and reducing the national infected risk of the disease. the sars outbreaks represented one of the most serious public health challenges to china and the world [ ] . seventeen years later, the outbreak of covid- could encounter a similar situation but lead to a different outcome. different transmission mechanisms of these coronaviruses can also present different spatial and temporal distributions nationally and globally. for sars, we observed that the distance transmission chain started from guangdong to beijing and the nearby provinces. however, for covid- , we observed a shorter transmission chain around hubei but a wider infected region nationally. outside the epicenter, we identified more secondary clusters for sars, indicating that the transmission was wider for second generations. compared to sars, the secondary clusters of covid- were mainly clustered around hubei. this could be explained by the relatively high infection rate nationally, as well as different demographic factors and local government containment strategies regionally. another secondary cluster identified in shandong around february , was mainly due to the newly reported cases previously identified in jails [ ] . because the reporting system of the jails was independent from the national reporting system, these cases were reported with a time lag. when these cases were not considered, the incidence was generally low during the last two weeks in february. because the mandatory quarantine for hubei ("wuhan lockdown") has been in effect since january , , and because social-distancing measures, such as population movement restrictions, school closures and temperature monitoring at public locations, have also been in effect in most provinces in mainland china since this date, we distinguished the spatial patterns of the covid- epidemic before and after this date plus a -day incubation period. we found that covid- cases were clustered mainly in hubei, and other secondary clusters disappeared, except in shandong. this reinforced that quarantine and isolation can help to contain the virus, prevent transmission and effectively reduce the number of secondary clusters. although our study covered the peak period of the outbreaks in most provinces, transmission patterns of sars in beijing and guangdong were biased due to lack of the available data before april , . daily briefing on novel coronavirus cases in china characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention transmissibility of -ncov transmission dynamics of the etiological agent of sars in hong kong: impact of public health interventions national health commission of the people's republic of china covid- ) situation reports. world health organization cumulative number of reported probable cases of severe acute respiratory syndrome (sars). world health organization clinical characteristics of coronavirus disease in china national health commission of the people's republic of china superspreading sars events spatial transmission and meteorological determinants of tuberculosis incidence in qinghai province, china: a spatial clustering panel analysis evaluation of control measures implemented in the severe acute respiratory syndrome outbreak in beijing china finds spike in coronavirus cases in two jails, officials fired early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to show our respect and gratitude to all the health workers who are at the front line of the outbreak response and fighting against covid- in china. comparatively, reported cases for covid- could also be biased due to missing data before january , in this study. moreover, early infections with atypical presentations may have been missed [ ] . in this study, covid- and sars outbreaks exhibited distinct spatiotemporal clustering patterns at the provincial levels in mainland china. our results indicated that the different spatiotemporal clustering patterns may reflect changes in social and demographic factors, public health emergency preparedness and response capabilities, as well as differences in transmission patterns and mechanisms of these coronaviruses in china. nevertheless, the conclusions should be interpreted with caution with regard to the early stages of the two epidemic outbreaks before more data become available on transmission patterns and epidemiologic characteristics of covid- and sars. further investigation with a detailed comparison with the corresponding characteristics between the two diseases is warranted. the online version contains supplementary material available at https://doi. org/ . /s - - -y. authors' contributions xz and hd conceived and designed the study. xz, hr, yw, yh and hd collected the data. xz and hr performed the statistical analysis. xz and hd prepared the manuscript. all authors read and approved the final manuscript. there was no funding source for this research. the datasets supporting the conclusions of this article are available from daily notification of the outbreak provincial health commissions in china (http://wwsw.nhc.gov.cn/xcs/yqtb/list_gzbd.shtml) and who (https://www. who.int/emergencies/diseases/novel-coronavirus- /situation-reports/.). the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no conflicts of interest.author details key: cord- -r w jm authors: yi, hana; yong, dongeun; lee, kyungwon; cho, yong-joon; chun, jongsik title: profiling bacterial community in upper respiratory tracts date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: r w jm background: infection by pathogenic viruses results in rapid epithelial damage and significantly impacts on the condition of the upper respiratory tract, thus the effects of viral infection may induce changes in microbiota. thus, we aimed to define the healthy microbiota and the viral pathogen-affected microbiota in the upper respiratory tract. in addition, any association between the type of viral agent and the resultant microbiota profile was assessed. methods: we analyzed the upper respiratory tract bacterial content of healthy asymptomatic people ( health-care workers and community people) and patients acutely infected with influenza, parainfluenza, rhino, respiratory syncytial, corona, adeno, or metapneumo viruses using culture-independent pyrosequencing. results: the healthy subjects harbored primarily streptococcus, whereas the patients showed an enrichment of haemophilus or moraxella. quantifying the similarities between bacterial populations by using fast unifrac analysis indicated that bacterial profiles were apparently divisible into oropharyngeal types in the tested subjects. the oropharyngeal types were not associated with the type of viruses, but were rather linked to the age of the subjects. moraxella nonliquefaciens exhibited unprecedentedly high abundance in young subjects aged < years. the genome of m. nonliquefaciens was found to encode various proteins that may play roles in pathogenesis. conclusions: this study identified oropharyngeal microbiome types. no virus-specific bacterial profile was discovered, but comparative analysis of healthy adults and patients identified a bacterium specific to young patients, m. nonliquefaciens. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. recent culture-independent community analysis performed on the human microbiome has provided an overall picture of commensal microbial communities. studies have revealed that diverse microbes occupying body habitats with strong niche specialization both within and among individuals [ ] . in the case of the respiratory tract microbiome, a catalogue was initially established in [ ] and then respiratory microorganisms were extensively characterized [ ] [ ] [ ] [ ] [ ] [ ] . collectively, studies to date have revealed that the respiratory tract harbors a homogenous microbiota that decreases in biomass from the upper to the lower tract [ ] , and that the lung microbiome resembles the oral microbiome, although these microbiomes are distinguished by the overrepresentation of distinct bacterial species in the lung [ ] . as with other human-body habitats, the core microbiome of nasopharynx remains undefined because it varies substantially from person to person [ ] . however, existence of core microbiome was observed despite the significant inter-individual variation [ ] . one study reported that the microbial composition of the upper respiratory tract is typically unique to each person and it changes little over time [ ] . although the available evidence is not sufficiently strong, microbiome types are speculated to eventually affect a person's risk of disease or response to distinct drugs [ ] . the human microbiota is considered to benefit the host by promoting the differentiation of the mucosal structure and function, stimulating both the innate and adaptive immune systems, and providing "colonization resistance" against pathogen invasion [ ] . recently, the composition of the airway microbiota has been suggested to play roles in determining the presence and severity of diseases [ , ] . for example, the clinical outcomes of respiratory infections caused by pseudomonas aeruginosa vary depending on the diversity of the airway microbiota [ , ] , and a temporal loss of the diversity is linked to the development of ventilator-associated pneumonia and patient mortality [ , ] . the importance of intact commensal microbiota was also demonstrated in viral infection, with the commensal microbiota composition critically regulating host immune response following influenza virus infection [ ] . to reveal the links that exist between microbiome types and clinical traits, we have to first understand the diversity of the microbial community in target body sites. most respiratory tract infections are caused by viruses including rhinovirus, respiratory syncytial virus, parainfluenza virus, adenovirus, coronavirus, human metapneumovirus, and influenza virus. infection by pathogenic viruses significantly changes the condition of the respiratory tract as a result of the epithelial damage caused by viral invasion itself and/or by inflammatory mediators produced by the host immune response [ ] . given, the pathophysiology and mechanism of local immune responses are virus specific [ ] , a virus-specific bacterial profile in the respiratory tract could potentially be characterized. discovering any specific bacterial species that exhibits a tendency of opportunistic infection or coinfection in a viral species-dependent would benefit future preventive measures and current treatments. to date, no study has evaluated whether the composition of the respiratory microbiota changes in relation to the type of infectious virus. in this study, our aim was to determine whether a viral infection-related bacterial profile exists in the respiratory tract and evaluate any disparities in the microbiota structure that develops depending on the infectious virus species. we used culture-independent high-throughput sequencing to analyze the bacterial content in the upper respiratory tract of patients and healthy asymptomatic people. we also examined the presence or absence of dissimilarities in the microbiota of hospital staff and community people. this study was approved by the institutional review board of the severance hospital, yonsei university health system, seoul, korea (protocols - - , - - , and - - ). patients and healthy adults provided written informed consent to be enrolled. de-identified demographic data and clinical measures were taken from electronic medical record system. additional file : table s presents the list and features of samples used in this study. we selected patients with confirmed acute viral infections from yonsei university hospital during a month period (december to may ). the viral agents of the infections were confirmed using pcr by yonsei university hospital. the viruses included influenza (if, n = ), parainfluenza (pi, n = ), rhino (rh, n = ), respiratory syncytial (rs, n = ), corona (cr, n = ), adeno (ad, n = ), and metapneumo (mp, n = ) viruses. the upper respiratory tract samples were collected from patients' oropharynx by using swabs and suspended in ml of viral transport medium (vtm; becton dickinson universal viral transport, usa). sputum or nasopharyngeal aspirate was collected when available instead of swabs. sputum samples were diluted with an equal volume of suspension medium and homogenized as described [ ] . the upper respiratory tract samples were also obtained from healthy adults including health-care workers ( non-icu and icu staff ) and community people. the hospital staffs and community people were recruited over the same period in yonsei university hospital (june ) and community people were additionally recruited in the same hospital (june ). the oropharyngeal swabs were obtained using aseptic technique, suspended in vtm and transported to the laboratory for further processing. the samples were stored at − °c until dna extraction. dna extraction, pcr, and pyrosequencing dna was extracted from μl of samples by using a commercial microbial dna isolation kit (qiagen). the extracted dna was amplified using primers targeting the v to v regions of the prokaryotic s rrna gene by using methods described elsewhere [ ] . dna was sequenced by chunlab inc. (seoul, korea) by using a roche/ gs junior system according to the manufacturer's instructions. the processing of pyrosequencing data of s rrna gene sequences were performed as described elsewhere [ ] . chimeric sequences were detected using uchime [ ] and eztaxon-e database (http://eztaxon-e.ezbiocloud.net; [ ] ) was used to taxonomically assign each pyrosequencing read. phylogenetic analyses of s rrna gene sequences were performed using the neighbor-joining [ ] tree method implemented in mega program [ ] . an evolutionary distance matrix was generated for the neighbor-joining tree according to the model of jukes and cantor [ ] and the resultant tree topologies were evaluated using bootstrap analyses [ ] . the draft genome sequence of moraxella nonliquefaciens dsm t was determined through paired-end shotgun sequencing performed by using the miseq system (illumina) with × coverage. the sequencing reads were assembled using clc genomics wb (clcbio). annotation, comparative genomic analyses and average nucleotide identity (ani) calculation were performed as described [ ] . random subsampling was conducted to normalize the data size to , reads, because the total number of reads that remained after pre-processing varied depending on the samples. all statistical analyses were performed using this subset. the simpson diversity index [ ] was calculated using the rrna database project's pyrosequencing pipeline (http://pyro.cme.msu.edu/). the overall phylogenetic distance between each pair of communities was estimated using the fast unifrac web interface (http://unifrac.colorado.edu/) [ ] and visualized using principal coordinate analysis (pcoa) implemented in r program (http://www.r-project.org/). to compare microbiome structures based on categorical metadata, samples were pooled into binds (healthy/patient, male/female, vtm/aspirate/sputum, smoking/non-smoking, ages, causal viruses, etc.) and statistical significance tests were performed using r program. the significance of differences in bacterial profiles according to categorical metadata was determined using hotelling's t test. significant bacterial taxa based on categorical metadata were identified using q-values after multiple testing correction [ ] to eliminate false discovery rates. the difference in shannon diversity index among categorical metadata was evaluated using wilcox two-sample t test. the sequencing data supporting this article are available in the genbank repository, sub . the genome data of m. nonliquefacience is under submission to the ddbj/embl/genbank databases under accession no. prjna . we sequenced upper respiratory tract samples from healthy adults including health-care workers ( non-icu and icu staff ) and community people. the patients with confirmed acute viral infections with influenza (if, n = ), parainfluenza (pi, n = ), rhino (rh, n = ), respiratory syncytial (rs, n = ), corona (cr, n = ), adeno (ad, n = ), and metapneumo (mp, n = ) viruses were also successfully sequenced. the pyrosequencing of s rrna gene amplicons resulted in , quality-filtered reads for the samples. we observed an average of bacterial phylotypes ( % clustering) for each samples (range to , ). in the sample-size-normalized ( , nt) subsamples, the number of bacterial species ranged from to (average, ), depending on samples. the genus streptococcus was identified as the core microbiome of the healthy human respiratory tract. in all healthy subjects tested in this study, members of streptococcus dominated the bacterial community, exhibiting an average abundance ratio (percentage of the taxon in the total bacterial community) of . % (range . %- . %, depending on subjects) ( table ). the genera neisseria ( . %) and gemella ( . %) were also dominant in healthy subjects, but their abundance ratios were considerably less than that of streptococcus. the genera observed in all healthy subjects were streptococcus, prevotella, and veillonella. the genera haemophilus, gemella, rothia, and leptotrichia were detected in most subjects at abundance ratios of . %- . %. fast unifrac analyses for the bacterial profiles in healthy samples showed that hospital staff and community people were not discriminated based on their bacterial composition (additional file : figure s ), and age, sex, year and month of sample collection, and smoking status did not discriminate the bacterial profile (data not shown). analyzing the bacterial communities of healthy-adult and patient groups revealed clear differences. we used the shannon index in which higher values represent higher diversities; the average values calculated for the healthy-adult and patient groups were . ± . and . ± . , respectively ( figure ). this indicated that healthy adults harbored significantly more complex and diverse microbiota than did patients (p < . , wilcox test). the microbiota profiles of healthy-adult and patient groups also differed in the relative composition of the microbiome, which was highlighted in the graph showing the abundant bacterial genera observed in the tested samples ( figure and additional file : figure s ). to identify the bacterial taxa that were more abundant (in a statistically significant manner) in the patient group than in the healthy-adult group, p-values were calculated for all the taxa detected. the result demonstrated that distinct bacterial genera were overrepresented in the patient and healthy-adult groups. whereas haemophilus (p = . ) and moraxella (p = . ) were identified as patient group-specific genera, streptococcus (p = . ), neisseria (p = . ), gemella (p = . ), aggregatibacter (p = . ), and actinobacillus (p = . ) were determined to be bacteria specific to the healthy-adult group. to hierarchically visualize the bacterial profile similarities among the samples, a upgma dendrogram was generated from the fast unifrac distance matrix. based on bacterial composition, the samples analyzed in this study were divided into oropharyngeal microbiome types (additional file : figure s ), with the clusters being characterized by the dominance of several bacterial genera: type (dominated by streptococcus + prevotella + veillonella), type (streptococcus + haemophilus + neisseria), type (streptococcus), type (moraxella), type (haemophilus), and type (klebsiella). only samples were not grouped into any of the types. the healthy adults and a subset of patients harbored bacterial communities dominated by streptococcus, and to a lesser extent by haemophilus, neisseria, prevotella, veillonella, and/or gemella (types - in figure ). the remaining patients carried impaired microbiota dominated by moraxella (type in figure ), haemophilus (type ), or klebsiella (type ), and this was coupled with a massive reduction in the levels of streptococcus. types and were dominated by well-known pathogens like h. influenzae and k. pneumonia, but type was dominated by a previously unknown one, moraxella nonliquefaciens. we elucidated the differences in bacterial profiles in the context of causal agents of infections and demonstrated that virus type did not determine the structural differences in bacterial communities (additional file : figure s a ). moreover, sex, sample type (swab, aspirate, or sputum), and smoking status did not influence the bacterial community structure (additional file : figures s b, s c and s d), which was also unaffected by the year and month of sample collection (data not shown). by contrast, subjects' age was associated with the bacterial profile in a statistically significant manner (additional file : figure s e ), and the samples were categorized into age groups, - and - years (p < . , hotelling's test). in this study, we discovered a bacterial species that was dominant in young patients ( - years old): m. nonliquefaciens was detected in . % of the patients, with abundance ratios of . %- . % depending on the subject, but this species was not observed in any healthy subject. most of the patients ( out of cases) harboring m. nonliquefaciens were under years old. two rsv infected patients (rs and rs ) showed and % of abundance of m. nonliquefaciens, indicating that the upper respiratory tracts of these patients were overwhelmed by this bacterial species. in addition, a closely related pathogenic species, m. catarrhalis, was detected in . % of the patients, with abundance ratios of . %- . %. analyzing the s rrna gene sequence revealed that m. nonliquefaciens and m. catarrhalis were clearly distinct species that showed . % similarity between type strains (additional file : figure s ). the moraxella contigs recovered from patient samples were divided into clades based on phylogenetic analysis (figure ). clade i was closely related ( . %- . % s rrna gene similarity) to the type strain of m. nonliquefaciens. the branching pattern of the contigs within the radiation of clade i indicated that m. nonliquefaciens populations in the patients encompassed phylogenetic lineages. contigs belonging to clade ii were clustered together with m. catarrhalis ( . %- % s rrna gene similarity), and these contigs were further divided into subpopulations, type and type populations [ ] . to determine whether m. nonliquefaciens has a possibility to be an opportunistic pathogen, its potential pathogenicity was inferred using the genome sequence of the type strain. genome sequencing identified a . -mb-sized genome of m. nonliquefaciens dsm t , featuring a . % g + c ratio. the genomic relatedness calculated using ani showed that m. nonliquefaciens dsm t and m. catarrhalis rh shared low genomic relatedness, % ani, which is considerably less than the cut-off value used for species circumscription, %- % ani [ ] . the ani value further confirmed that the species were distinct. various proteins have been reported to play pivotal roles in m. catarrhalis pathogenesis [ ] . thus, we examined whether the virulence proteins in the m. catarrhalis rh genome were also encoded in the newly determined m. nonliquefaciens genome. the genes responsible for host-cell adhesion and invasion, evasion of host immune system, and biofilm formation were included as putative virulence factors. comparative genomic analysis revealed that most of the virulence genes identified in m. catarrhalis were encoded in the m. nonliquefaciens genome (additional file : table s ). moreover, resistance to β-lactam antibiotics was predicted based on the presence of the β-lactamase class c gene. however, bro- and bro- β-lactamases encoded by most of m. catarrhalis strains were not detected in m. nonliquefaciens. our results demonstrated that changes in bacterial profiles elicited by viral infection were not associated with the causal viral species: the microbiome compositions in samples obtained from various viral infections were not differentiated based on the causal infectious agents. regardless of the causal agents involved, the respiratory tract microbiota of patients differed substantially from the microbiota of healthy subjects in the kinds and diversities of prevalent bacteria. however, the heterogenicity of the patient group of this study (age, sampling type, and number of samples/virus type) hinders strong conclusions for this point. nevertheless, the current results from this study provide the first insight into microbiome alterations associated with viral infection in the upper respiratory tract. figure dependence of microbiome structure on several key genera. principal coordinate analysis (pcoa) of the bacterial communities isolated from healthy-adult and patient samples was performed using the weighted pairwise unifrac distance matrix. the unifrac distance represents the distance between samples in terms of the microbial community structure. samples are color-coded according to the clustering groups. the diminished bacterial diversity observed in patients agreed well with previous studies reporting that the diversity in commensal airway microbiota declined following infection by specific pathogens [ , ] . for example, in the nares of patients with s. aureus carriage, species diversity was half of that found in healthy adults' nares [ ] . these results indicate that the normal microflora is depleted in respiratory tract cells impaired due to viral infection and is replaced by a few opportunistic pathogens. moreover, the dominance of streptococcus in the respiratory tracts of healthy subjects (table ) agreed with previous culture-independent massive metagenomic sequencing studies [ , ] . streptococcus is also known to be abundant in the oral cavity [ , ] . thus, the oropharynx of healthy people could be characterized by high bacterial diversity and by an overwhelming abundance of the genus streptococcus. by analyzing the bacterial community, we defined oropharyngeal types of bacterial populations in the upper respiratory tract. we use the word "oropharyngeal type" here based on the "enterotype" concept, which was introduced by arumugam et al. and defined as the clusters of human gut microbiome determined based on bacterial composition [ ] . the concept suggests that people can be classified into several enterotypes according to the abundance of key bacterial taxa in gut microbial communities [ ] . in this study, the samples included were restricted to one ethnic group, and thus the suggested oropharyngeal types may be accepted only temporarily. however, because no other efforts to cluster respiratory tract microbiomes have been reported to date, our results may serve as a favorable starting point for future studies on this subject. our results revealed that haemophilus and moraxella were patient-specific genera. unlike h. influenzae, m. nonliquefaciens has not been studied for its possible role in pneumonia. although m. nonliquefaciens has been isolated from clinical cases including chronic bronchitis [ ] , bronchial infection [ ] , pneumonitis [ ] , endophthalmitis [ ] , septic arthritis [ ] , thyroiditis [ ] , discitis [ ] , botryomycosis [ ] , and endocarditis [ ] , this bacterium is widely considered to be a part of the normal flora in the human upper respiratory tract and to exhibit low pathogenicity [ , ] . by contrast, a closely related species, m. catarrhalis, is a well-studied respiratory tract pathogen that frequently colonizes the nasopharynx and is an exclusively human pathogen that displays an affinity for the human upper respiratory tract [ , ] . long considered to be a commensal bacterium of the upper respiratory tract, m. catarrhalis has now been established as an etiological cause of otitis media and the exacerbations of chronic obstructive pulmonary disease (copd) [ , ] . despite the distinctiveness of the species at the taxonomic level, m. nonliquefaciens and m. catarrhalis share several common features. first, the age-related incidence of m. nonliquefaciens infection determined here is concordant with that of m. catarrhalis. previously, m. catarrhalis was reported to be mostly associated with upper respiratory tract infections in children [ ] , and its carriage rate was shown to be high in children (up to %) and extremely low in healthy adults ( %- %) [ , [ ] [ ] [ ] [ ] . moreover, the phenotype and gene incidences of m. catarrhalis isolates of children and adults presenting with respiratory disease differ substantially, possibly as a result of immune evasion in adults [ ] . the age-related incidence of m. nonliquefaciens and m. catarrhalis may be indicative of the weak pathogenicity of moraxella species, which may be unable to evade the well-established immune system of adults. second, most strains of m. catarrhalis are known to produce β-lactamases and thus exhibit ampicillin resistance [ ] . this antibiotic resistance was also predicted in m. nonliquefaciens based on the presence of the class c β-lactamase gene. however, although both species possess class c β-lactamase genes, the species differ with respect to the possession of bro β-lactamase; bro is unique because it shows no substantial similarity to any β-lactamase genes identified so far [ ] . the absence of bro- and bro- in m. nonliquefaciens suggests that m. catarrhalis acquired the bro genes by means of lateral gene transfer after the species evolved into distinct lineages. third, all but one virulence factors reported for m. catarrhalis were found to be encoded by m. nonliquefaciens (additional file : table s ), which indicates that m. nonliquefaciens has a high potential to be pathogenic even though it is currently considered to be a commensal bacterium. several reasons may account for why the overabundance of m. nonliquefaciens has not been reported. growing this organism and distinguishing it from m. catarrhalis are challenging, which may have resulted in a poor recognition of m. nonliquefaciens as a respiratory pathogen. moreover, although m. catarrhalis is focused on by clinicians, the isolation of m. catarrhalis from clinical samples is complicated by the presence of neisseria strains because these organisms share morphological similarities [ ] . furthermore, m. nonliquefaciens may have been considerably underestimated because of being misidentified as m. catarrhalis or neisseria spp. [ ] . lastly, the current absence of clinical interest or familiarity with m. nonliquefaciens may have resulted in under-reporting or identification of this pathogen. indeed, m. catarrhalis was previously underreported since other better recognized pathogens were also recognized and growing in the same cultures [ ] . to clarify the incidence of this potential pathogen, future studies will need to differentiate between the true rates of incidence of m. catarrhalis and m. nonliquefaciens. the results of this study suggest that m. nonliquefaciens deserves considerable attention as a potential opportunistic pathogen in the respiratory tract. human microbiome project c: structure, function and diversity of the healthy human microbiome bacterial community variation in human body habitats across space and time variability and diversity of nasopharyngeal microbiota in children: a metagenomic analysis disordered microbial communities in the upper respiratory tract of cigarette smokers topographical continuity of bacterial populations in the healthy human respiratory tract the human nasal microbiota and staphylococcus aureus carriage comparison of the respiratory microbiome in healthy nonsmokers and smokers pyrosequencing analysis of the human microbiota of healthy chinese undergraduates gut microbial 'enterotypes' become less clear-cut what are the consequences of the disappearing human microbiota? airway microbiota and pathogen abundance in age-stratified cystic fibrosis patients from microbe to microbiota: considering microbial community composition in infections and airway diseases loss of bacterial diversity during antibiotic treatment of intubated patients colonized with pseudomonas aeruginosa a persistent and diverse airway microbiota present during chronic obstructive pulmonary disease exacerbations microbiota regulates immune defense against respiratory tract influenza a virus infection understanding the symptoms of the common cold and influenza metagenomic analysis of respiratory tract dna viral communities in cystic fibrosis and non-cystic fibrosis individuals effect of genetically modified poplars on soil microbial communities during the phytoremediation of waste mine tailings uchime improves sensitivity and speed of chimera detection introducing eztaxon-e: a prokaryotic s rrna gene sequence database with phylotypes that represent uncultured species the neighbor-joining method: a new method for reconstructing phylogenetic trees mega: a biologist-centric software for evolutionary analysis of dna and protein sequences evolution of protein molecules confidence-limits on phylogenies -an approach using the bootstrap comparative genomics of neisseria weaveri clarifies the taxonomy of this species and identifies genetic determinants that may be associated with virulence measurement of diversity fast unifrac: facilitating high-throughput phylogenetic analyses of microbial communities including analysis of pyrosequencing and phylochip data a direct approach to false discovery rates analysis of moraxella catarrhalis by dna typing: evidence for a distinct subpopulation associated with virulence traits shifting the genomic gold standard for the prokaryotic species definition molecular aspects of moraxella catarrhalis pathogenesis defining the normal bacterial flora of the oral cavity ecology of viridans streptococci in the oral cavity and pharynx enterotypes of the human gut microbiome a guide to enterotypes across the human body: meta-analysis of microbial community structures in human microbiome datasets association of mucoid encapsulated moraxella duplex var. nonliquefaciens with chronic bronchitis moraxella duplex var. nonliquefaciens as a cause of bronchial infection pneumonitis and pulmonary abscess associated with moraxella nonliquefaciens posttrabeculectomy endophthalmitis caused by moraxella nonliquefaciens moraxella nonliquefaciens septic arthritis in a patient undergoing hemodialysis acute thyroiditis caused by moraxella nonliquefaciens discitis due to moraxella nonliquefaciens botryomycosis caused by moraxella nonliquefaciens endocarditis caused by moraxella nonliquefaciens henriksen and bøvre , al . in bergey' s manual of systematic bacteriology the type- pilin of moraxella nonliquefaciens exhibits unique similarities with the pilins of neisseria gonorrhoeae and dichelobacter (bacteroides) nodosus moraxella catarrhalis, a human respiratory tract pathogen age-related genotypic and phenotypic differences in moraxella catarrhalis isolates from children and adults presenting with respiratory disease in a: moraxella catarrhalis: from emerging to established pathogen epidemiology of moraxella catarrhalis in children during the first years of life: relationship to otitis media comparison of the local immune response to nontypable haemophilus influenzae (nhi) and moraxella catarrhalis (mc) during otitis media acute otitis media caused by branhamella catarrhalis: biology and therapy impact of antimicrobial therapy on nasopharyngeal carriage of streptococcus pneumoniae, haemophilus influenzae, and branhamella catarrhalis in children with respiratory tract infections respiratory-tract infections due to branhamella catarrhalis -epidemiologic data from western-australia genesis of bro beta-lactamase-producing moraxella catarrhalis: evidence for transformation-mediated horizontal transfer genome analysis of moraxella catarrhalis strain bbh , a human respiratory tract pathogen profiling bacterial community in upper respiratory tracts we thank dr. s won for his advices on statistical analyses. this work was supported by research funds ( -e - , -e - , and -e - ) of the korea centers for disease control and prevention. the pyrosequencing of bacterial community identified oropharyngeal microbiome types in the upper respiratory tract, but the bacterial profile was not related to the type of causal infected viruses. the microbiota of patients differed substantially from that of healthy subjects in the kinds and diversities of prevalent bacteria. comparative analysis of healthy adults and patients identified a bacterium specific to young patients, m. nonliquefaciens. the results of whole-genome sequencing raised the possibility of m. nonliquefaciens being an opportunistic pathogen. additional file : table s . list and clinical characteristics of samples used in this study. nd, not determined.additional file : figure s . ordination diagram showing the relatedness of microbiomes in the upper respiratory tract of healthy people. principal coordinate analysis (pcoa) of bacterial communities isolated from healthy adults was performed using the weighted pairwise unifrac distance matrix. the unifrac distance represents the distance between samples in terms of the microbial community structure.additional file : figure s . dendrogram and circle map showing the clustering of samples into groups depending on bacterial population dynamics. the unweighted pair group method with arithmetic mean (upgma) dendrogram was generated from the fast unifrac distance matrix to hierarchically visualize the manner in which samples are grouped. the relative abundance of representative microbial genera is indicated as a circle map; circle sizes represent the percentage ratio within a sample.additional file : figure s . ordination diagram showing the relatedness of microbiomes in the upper respiratory tract of people afflicted with diverse viral infections. we performed principal coordinate analysis (pcoa) on the bacterial communities isolated from healthy-adult and patient samples by using the weighted pairwise unifrac distance matrix. the unifrac distance represents the distance between samples in terms of the microbial community structure. the structure of the bacterial community was not affected by (a) the type of virus or by (b) the sex, (c) sample type, and (d) smoking status of the subjects. however, (e) age was correlated with the microbiome structure.additional file : figure s . diagram showing the relationships among the type strains and the representative contigs obtained from patient samples. the average nucleotide identity (ani) value indicating genome relatedness was calculated using the complete genome sequence of m. catarrhalis rh strain (prjna ), which shows % s rrna gene sequence identity with the type strain of the species.additional file : table s . comparison of major virulence genes present in m. nonliquefaciens and m. catarrhalis. the list of virulent genes was obtained from previous reports [ , ] . the gene locus in each genome is presented together with gene size in amino acids (in parenthesis).abbreviations upgma: unweighted pair group method with arithmetic mean; pcoa: principal coordinate analysis; ani: average nucleotide identity; vtm: viral transport medium; icu: intensive care unit. the authors declare that they have no competing interests. key: cord- - jbf ik authors: alsaleh, asma n; whiley, david m; bialasiewicz, seweryn; lambert, stephen b; ware, robert s; nissen, michael d; sloots, theo p; grimwood, keith title: nasal swab samples and real-time polymerase chain reaction assays in community-based, longitudinal studies of respiratory viruses: the importance of sample integrity and quality control date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: jbf ik background: carefully conducted, community-based, longitudinal studies are required to gain further understanding of the nature and timing of respiratory viruses causing infections in the population. however, such studies pose unique challenges for field specimen collection, including as we have observed the appearance of mould in some nasal swab specimens. we therefore investigated the impact of sample collection quality and the presence of visible mould in samples upon respiratory virus detection by real-time polymerase chain reaction (pcr) assays. methods: anterior nasal swab samples were collected from infants participating in an ongoing community-based, longitudinal, dynamic birth cohort study. the samples were first collected from each infant shortly after birth and weekly thereafter. they were then mailed to the laboratory where they were catalogued, stored at - °c and later screened by pcr for respiratory viruses. the quality of specimen collection was assessed by screening for human deoxyribonucleic acid (dna) using endogenous retrovirus (erv ). the impact of erv load upon respiratory virus detection and the impact of visible mould observed in a subset of swabs reaching the laboratory upon both erv loads and respiratory virus detection was determined. results: in total, nasal swabs were received in the laboratory. erv load in nasal swabs was associated with respiratory virus detection. reduced respiratory virus detection (odds ratio . ; % confidence interval . - . ) was observed in samples where the erv could not be identified. mould was associated with increased time of samples reaching the laboratory and reduced erv loads and respiratory virus detection. conclusion: suboptimal sample collection and high levels of visible mould can impact negatively upon sample quality. quality control measures, including monitoring human dna loads using erv as a marker for epithelial cell components in samples should be undertaken to optimize the validity of real-time pcr results for respiratory virus investigations in community-based studies. acute respiratory infections (aris) caused by viruses are the most common illnesses experienced by all age groups. aris are particularly important during early life as infants have the highest infection rates and they can transmit infectious agents to other household members [ ] . recently introduced molecular-based diagnostic techniques have much improved sensitivity compared with previous classical culture and phenotypic-based methods and have led to the discovery of new respiratory viruses [ ] . however, contemporary studies employing these new techniques have often used convenience samples obtained from patients admitted to hospital or attending emergency department clinics [ ] [ ] [ ] . understanding more fully the ari disease burden in the community is important for developing public health interventions, such as vaccination programs [ ] , and for understanding the role respiratory viruses may play in the pathogenesis of certain chronic pulmonary disorders, such as asthma [ ] [ ] [ ] . this has led to the instigation of community-based studies. such studies do however have some logistical challenges, particularly concerning respiratory sample collection and transport. most studies have relied upon clinic or home visits by trained healthcare workers to collect specimens during an ari episode, which imposes restrictions upon busy families and may lead to biased disease estimates and specimen availability [ ] [ ] [ ] . cost and feasibility of using healthcare workers are also important when large longitudinal, community-based cohort studies, involving frequent specimen collections, are planned. to help address these limitations, we and others have begun testing parentcollected, anterior nasal swab specimens that have been transported to the research laboratory using the standard mail [ ] [ ] [ ] [ ] . this approach is considered to be safe, convenient and cost-effective [ ] . importantly, when using highly sensitive polymerase chain reaction (pcr) assays the detection rates for respiratory viruses are similar in both anterior nasal swab specimens and samples collected by the more traditional method of nasopharyngeal aspiration [ , ] . building on this information, later studies have also shown that pcr testing for respiratory viruses provided similar results for parent-collected anterior nasal swab specimens and either nasal swab or nasoparyngeal aspirates collected by healthcare professionals [ , ] . other studies examining sample transport have also shown that mailing swabs at ambient temperature has limited or no impact on respiratory virus detection by pcr [ , , ] , although investigating further the effects of transporting samples for extended periods and at higher temperatures was highlighted in one study [ ] . the observational research in childhood infectious diseases (orchid) project is a longitudinal, communitybased, dynamic birth cohort study, which seeks to describe the nature and timing of respiratory viruses detected in australian children during the first -years of life [ ] . the study commenced in late and involves parents collecting and mailing nasal swabs weekly to the research laboratory for pcr-based respiratory virus screening. during the first year mould was seen in some samples as they arrived in the laboratory and we became concerned about the impact of this contaminant upon sample integrity. therefore, as part of the orchid study, we undertook a broader investigation of sample quality, examining collection and transportation, and how these impact on respiratory virus detection. our objectives were first to determine the quality of specimen collection by testing for the presence of human dna (endogenous retrovirus ; erv ) and then to investigate the effects of sample quality and the presence of visible mould in samples reaching the laboratory upon pcr performance. briefly, as part of orchid, families expecting a healthy term baby were recruited antenatally at either the publically funded royal brisbane and women's hospital or the north west private hospital, in brisbane, australia, a subtropical city of more than million inhabitants [ ] . the human research ethics committees of the children's health queensland hospital and health service, the royal brisbane and women's hospital and the university of queensland approved the study. parents/caregivers of each baby provided written, informed consent at the time of enrolment into the study. parents were asked to record from birth a daily symptom diary and to collect anterior nasal swab samples every week until their infant's second birthday. instructions on sample collection were provided at the initial visit by research staff who also demonstrated the technique by undertaking the initial nasal swab specimen shortly after delivery of the newborn baby. in addition, parents were given written instructions on how to collect nasal swab specimens. they also received regular text messages, emails or telephone calls as means of research staff keeping in contact with participating families. regular supplies of sterile rayon swabs (virocult, mw , medical wire & equipment, england) were provided, which were rotated against the internal anterior walls of both nostrils and then placed in the provided transport tube that contained a viral transport media-soaked foam pad in the base. parents were instructed to squeeze the foam pad to release the fluid and bathe the top of the swab. ideally within hours of collection, the nasal swabs were then sent by regular postal mail (in accordance with australia post regulations [ ] ) at ambient temperature to our research laboratory where they were stored at − °c until analysis. nasal swabs were vortexed in ml of phosphate buffered saline from which μl was spiked with μl of equine herpes virus- (ehv ) culture supernatant, which served as an extraction and inhibition control agent, before nucleic acid was extracted using the cas xtractorgene automated system (qiagen-australia) according to the manufacturer's instructions. the final volumes of specimen extracts were μl/specimen eluted in well racks (matrix, thermo scientific, australia). for each run ( extracts/run), extracts were tested using a duplex real-time pcr assay for ehv and erv in the following reaction compositions; pmoles of each primer, pmoles of each probe (table ) , μl of sensimix ii probe pcr mix (bioline, australia) and μl of extract in a μl final reaction. cycling conditions used for amplification were: initial hold at min at °c; followed by cycles of sec at °c and sec at °c. the ehv component was performed as an extraction and inhibitor control as described previously [ ] , while erv was used as a marker to evaluate the quality of nasal swab sample collection [ ] . briefly, the samples were considered to have failed the ehv component (ie. failed extraction or possessed pcr inhibitors) if the ehv real-time pcr cycle threshold (ct) results for individual samples were more than two standard deviations from the mean value of all samples, which for this study was calculated to be approximately cycles [ ] . samples that passed ehv dna extraction quality control testing were screened for respiratory viruses using previously optimized and described pcr and reverse transcriptase pcr assays. virus testing assays included: rhinovirus (rv) [ ] , influenza viruses (a and b) [ ] , respiratory syncytial viruses (a and b) [ ] , parainfluenza viruses ( - ) [ ] , human adenoviruses [ ] , human metapneumovirus [ ] , human coronaviruses (oc , hku , e, and nl ) [ , ] , human bocavirus [ ] and human polyomaviruses (wupyv and kipyv) [ ] . for all viruses, except rv, samples were tested in a × pooled format. briefly, aliquots of the sample extracts were pooled using the cas- liquid handling system (qiagen-australia) and pools tested for the presence of respiratory viruses. for positive pools, individual sample extracts were then tested to confirm positivity. rv screening was performed on individual sample extracts, and not on the pooled extracts, as the number of expected positive samples was considered too high for there to be any benefits from pooling. during the initial phases of the study, mould was observed growing on a small number of nasal swabs at the time of their arrival at the laboratory. in light of this observation, before extraction all swabs were inspected visually for mould and were assigned a semi-qualitative score according to a sliding scale ( to ), whereby = no mould observed, = low, = medium, and = high levels of visible mould present. dna sequencing was used to identify the type of fungi present on a subset of swabs exhibiting varying degrees of visible mould growth ( swabs where no mould was seen, and each where low, medium and high levels, respectively, of mould contamination was present). pcr amplification of a fungal internal transcribed spacer (its) region was performed using pmoles of forward and reverse primers (its forward primer tccgtaggt gaacctgcgg and its -reverse primer tcctccgc tta ttgatatgc [ ] , μl of qiagen sybr master mix (qiagen, australia) and μl of template in a total μl reaction mix. cycling was performed using the following conditions: °c for min, cycles of °c for sec, °c for sec and °c for sec and a melting step of - °c at the end of the thermal cycling. pcr products were examined by gel electrophoresis using a % agarose gel and sent to the australian genome research facility (the university of queensland, brisbane) for automated sequencing. for this study, samples that failed ehv criteria or were not inspected for mould growth were excluded from the analysis (figure ). the association between variables of interest and binary outcomes was investigated using mixed effects logistic regression models, with participants included as a random intercept to account for the possibly correlated outcomes within each infant. the association with continuous outcomes was investigated using mixed effects linear regression. when examining the association of mould level with sample quality and respiratory virus detection we conducted both univariate and multivariate analyses, with multivariate analyses adjusting for the potential confounders of the child's age, gender, relationship of collector to participant (e.g. father, mother or others), season specimen collected, and time from specimen collection to being frozen in the laboratory. analyses were conducted using stata statistical software v. . (statacorp, college station, tx, usa). between september and july , infants were recruited into the study. all participants lived within the greater brisbane metropolitan area and none were from rural communities. one-hundred and twentyfive recruits remained active study participants up until the date of this analysis. of the withdrawals, four had moved out of the study area, two others were later deemed ineligible, ten withdrew for personal reasons and eleven were ineligible because they could not fulfill sampling requirements. for the active families, swab return rates were > % for almost , child-days of observation. in total, weekly nasal swab specimens (~ nasal swabs/ month) were batched in ( well) racks, extracted and tested. the median time from collection to swab arrival in the laboratory was (interquartile range - ) days; however . % of swabs were received more than -days after their collection. for ehv extraction and inhibition testing, ( . %) dna extracts failed the ehv criteria. the initial samples were not inspected for mould growth during the early stages of the study and therefore were excluded from further analysis. of (figure ). however, following a cluster of samples negative for erv (figure ; batches , , ) we contacted parents and reminded them of the optimal swab collection technique they had been shown at enrolment of their baby. after this feedback the numbers of erv negative samples declined. at least one respiratory virus was detected in ( . %) samples. dual or multiple virus detections were observed in ( . %) samples. rv was the most common virus detected, being present in almost % of specimens, followed by human bocavirus, human polyomavirus kipyv, respiratory syncytial viruses and human adenoviruses ( table ) . of (table ) . a diverse range of species was observed with epicoccum nigrum and cladosporium cladosporioides the most prevalent. of the samples that were erv positive, ( . %) had at least one respiratory virus detected by pcr. in contrast, the respiratory virus detection rate in erv negative samples was significantly lower ( / , . %; crude odds ratio (or) = . ; % ci . - . ) when erv was absent in swab specimens. we also observed that among erv positive swabs, the average erv ct value for samples positive for any respiratory virus ( . cycles) was significantly lower (indicating greater erv load) than the average ct value ( . ) in samples negative for all viruses (crude difference = . , % ci . - . ; figure ). moreover, there was a significant difference in erv ct values (p = . ) in samples that table examines the association between erv and respiratory virus detection and potential explanatory and confounding variables. erv positive sample rates increased with age, varied by season and declined with increasing mould levels and time taken for samples to reach the laboratory and to be frozen. similarly, respiratory virus detection rates increased with age, specimen collection outside the summer months, and time taken to reach the laboratory, while decreasing as visible mould levels in samples reaching the laboratory increased. the orchid project is an ongoing comprehensive community-based study using pcr assays to detect respiratory viruses in anterior nasal swab specimens taken weekly by parents from their infants throughout the first -years of life. this requires parents following a standardized protocol of obtaining swabs regularly and mailing them promptly to our laboratory. however, we have observed that suboptimal sample collection as determined by erv detection and presence of visible mould in swab samples reaching the laboratory can negatively affect sample quality and potentially respiratory virus detection. the data from the first -months of our longitudinal study indicate that respiratory virus detection is associated with the erv load in nasal swab specimens. swabs negative for erv , presumably from sub-optimal collection, had reduced respiratory virus detection rates compared with samples containing erv . furthermore, in those specimens positive for erv , a higher erv load was associated with a higher likelihood of respiratory virus detection. overall, this shows the importance of measuring human dna as a marker for epithelial cells in swab samples, which if tested and monitored in real time during the study, can identify problems associated with collection that can be addressed quickly. this is illustrated in the current study when a sudden increase in erv negative samples was observed. parents were contacted and reminded about sample collection protocols following which there was a decline in erv negative sample rates towards baseline levels. we were also concerned at finding mould on some samples, which occurred despite the commercial swab tubes containing antifungal agents. most fungal species identified in the swabs were saprophytic, and the most common fungus found, epicoccum nigrum, is a known contaminant of clinical specimens [ ] . the relationship between fungal airspora counts and meteorological conditions is complex and impacts at the species level [ ] . in brisbane, cladosporium and alternaria airspora are detected commonly throughout the year, but as with epicoccum,sp their levels peak during the warmer, humid months. other factors, such as rainfall and wind speed, table. can also influence fungal airspora composition [ , ] . in our study, mould was associated mainly with longer time intervals between taking swabs and their arrival at the laboratory. however, this was especially evident during the warm, humid spring and summer months, which leads us to speculate that fungal contamination occurred during sample collection and was influenced by the aforementioned environmental factors. unfortunately, we could not explore this further as it was beyond the scope of the present study. in addition, while mould growth proved to be an issue in the subtropical climate of brisbane, this may be less of a problem in more temperate climates with lower temperatures and humidity levels. we now remind parents regularly to mail swabs promptly after collection. of interest however, was that respiratory virus detection rates were not affected by prolonged transport times, but in fact appeared to increase with time taken to reach the laboratory. while the observed increase was unexpected and may have occurred simply by chance, it is plausible that viral nucleic acids were protected to some extent by being encapsulated within the viral capsid, and by using viral transport medium in the swabs. fungi were found to be associated with both reduced erv detection and, at high levels, reduced significantly respiratory virus detection. at least three points emerge from this study. first, although swabs may contain antimicrobial agents, the risk of fungal and potentially bacterial contamination may still arise. second, the times between swab collection and laboratory arrival should be monitored and feedback provided if delays occur. finally, if delays are expected swabs should be placed in the household refrigerator until mailed to the laboratory [ ] . we found that erv as a marker for human dna and epithelial cells was also an important indicator of sample quality for our study. for community-based investigations similar to our own, real-time sample processing and erv detection can facilitate rapid interventions to maintain sample quality and to optimize respiratory virus detection. indeed, this may have broader implications since nasal swabs are beginning to replace the traditional, but more invasive nasopharyngeal swab or aspirate sampling techniques in hospitals and clinics, especially following the influenza pandemic [ ] . thus, similar erv testing strategies could be used by diagnostic laboratories to improve or monitor sample collection quality for optimal respiratory virus detection. finally, the potential problem of visible mould contamination of swabs taken during community-based studies can be 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spora of brisbane submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the orchid study team: anne cook, hannah cox, jane gaydon, rebecca holding, kevin jacob, frances maguire, lebo mahango and clair wang, the study support volunteers: lynne grimwood and patricia sloots; and especially the families who participated in the study. the authors declare that they have no competing interest. key: cord- -d duepnb authors: wolfensberger, aline; clack, lauren; stefanie, von felten; kusejko, katharina; hesse, mirjam faes; jakob, werner; saleschus, dirk; meier, marie-theres; kouyos, roger; held, leonhard; sax, hugo title: implementation and evaluation of a care bundle for prevention of non-ventilator-associated hospital-acquired pneumonia (nvhap) – a mixed-methods study protocol for a hybrid type effectiveness-implementation trial date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: d duepnb background: hospital acquired pneumonia (hap) is divided in two distinct groups, ventilator-associated pneumonia (vap) and non-ventilator-associated hap (nvhap). although nvhap occurs more frequently than vap and results in similar mortality and costs, prevention guidelines and prevention focus almost exclusively on vap. scientific evidence about nvhap prevention and its implementation is scarce. therefore, we designed a mixed-methods hybrid type effectiveness-implementation study to investigate both the effectiveness and implementation of a newly developed nvhap prevention bundle. methods: this single-centre project at the -bed university hospital zurich (uhz) will engage the wards of nine departments with substantial nvhap rates. the nvhap bundle consists of five primary prevention measures: ) oral care, ) prevention of dysphagia-related aspiration, ) mobilization, ) stopping unnecessary proton pump inhibitors, and, ) respiratory therapy. implementation includes the engagement of department-level implementation teams, who sustain the ‘core’ intervention components of education, training, and environmental restructuring and tailor the implementation strategy to local needs. both effectiveness and implementation outcomes will be assessed using mixed-methods. as a primary outcome, nvhap incidence rates will be analysed by poisson regression models to compare incidence rates before, during, and after the implementation phases (on the hospital and department level). additionally, the association between process indicators and nvhap incidence rates will be analysed using longitudinal poisson regression models. a longitudinal, qualitative study and formative evaluation based on interviews, focus groups, and observations identifies supporting or hindering factors for implementation success in participating departments dynamically over time. this accumulating implementation experience will be constantly fed back to the implementation teams and thus, represents an active implementation element. discussion: this comprehensive hybrid mixed-methods study is designed to both, measure the effectiveness of a new nvhap prevention bundle and multifaceted implementation strategy, while also providing insights into how and why it worked or failed. the results of this study may contribute substantially to advancing knowledge and patient safety in the area of a rediscovered healthcare-associated infection - nvhap. trial registration: clinicaltrials.gov: nct . registered december . hospital acquired pneumonia (hap) is defined as pneumonia with first symptoms ≥ h after admission. it is divided into two distinct groups, ventilator-associated pneumonia (vap) and non-ventilator-associated hospital acquired pneumonia (nvhap). together, hap and lower respiratory tract infections were shown to be the most common healthcare-associated infections (hai) in both the european point prevalence study of / and the multistate u.s. point prevalence study in [ , ] . in these studies, more than half of hap - and % -were nvhap [ , ] . further, nvhap leads to substantial morbidity and was shown to have comparable mortality and similar costs as vap [ ] . however, current research and prevention efforts still focus almost exclusively on vap. scientific evidence about prevention of nvhap is scarce and of limited quality [ ] . there are no formal recommendations or evidence-based guidelines for nvhap, and the existing hap prevention guidelines focus almost exclusively on vap [ ] [ ] [ ] . in a narrative review, passaro et al. highlighted that oral care is the most studied measure and was commonly associated with a decreased hap rate, although a broad range of interventions are proposed [ ] . evidence is lacking for other measures such as dysphagia programs, early mobilization, and head of bed elevation [ ] . the estimated proportion of preventable hai in general ranges from to % [ , ] , and the preventable proportion of vap specifically was reported to be - % [ , ] . in a systematic literature review and metaanalysis about the proportion of hai that could be prevented with multifaceted interventions only two of included studies dealt with the prevention of nvhap [ ] . hiramatsu et al. found that an outpatient bundle of nvhap prevention measures, comprising three procedures of breathing exercises, two procedures of oral care, a procedure of nutritional control and smoking cessation prior to planned surgery, was effective to prevent postoperative pneumonia among patients with oesophageal cancer [ ] . kazaure et al. found that use of an incentive spirometer, oral hygiene with chlorhexidine, ambulation with good pain control and head-of-bed elevation to at least °and sitting up for all meals, accompanied by initial and ongoing education, progress reports, prevention measure documentation and order sets lead to a . % decrease of postoperative pneumonia in non-cardiac surgical patients [ ] . to our knowledge, there are no studies evaluating the effectiveness of an nvhap prevention bundle on a broad patient population. implementation science is the scientific study of methods to promote uptake of evidence-based best practices into routine healthcare practice [ ] . although quality improvement studies often report on the effectiveness of studied interventions to improve both, process indicators and patient outcomes, little is usually reported about the context of the intervention and what factors played a role in the successful implementation of practice measures. further, the implementation strategies used in such studies are often described in poor detail and lack theoretical justification, therefore hindering the development of an evidence base for their effectiveness [ ] [ ] [ ] . a detailed understanding of not only what works, but also how and why it works, is helpful to ensure that evidence-based practices of proven effectiveness can be successfully replicated and implemented in other settings. to simultaneously evaluate our multifaceted implementation strategy while also testing the effectiveness of the clinical nvhap prevention bundle, we undertake a type hybrid effectiveness-implementation study [ , ] . this comprehensive type hybrid effectivenessimplementation study aims to assess the effectiveness and success factors of both, a new prevention bundle against nvhap and a specifically designed departmentbased multifaceted implementation strategy in a medical and surgical patient population. with this mixed-methods study, we aim to investigate the impact of the implementation of a newly designed nvhap prevention bundle on the nvhap incidence rate among inpatients in our tertiary care hospital. we will quantify the adherence to the individual bundle elements and qualitatively identify the factors that influence successful implementation. to determine the nvhap bundle effectiveness on the nvhap incidence rate . to determine adherence to the nvhap bundle and each of the bundle elements . to relate adherence to nvhap bundle elements with nvhap incidence rate . to qualitatively monitor changes and identify trends in implementation outcomes throughout the study periods . to identify which factors in the implementation setting are associated with the actual degree of local implementation of the nvhap bundle the study is conducted at the university hospital zurich (uhz), switzerland, a -bed tertiary-care teaching hospital covering all medical specialties except paediatrics and orthopaedics. all patients hospitalized in nine predefined medical and surgical departments and their corresponding wards will be included in this study. the nine departments were chosen based on the following criteria; ) nvhap rate above the th percentile according to uhz nvhap data from the year ; ) high absolute number of patients with nvhap according to uhz nvhap data from the year ; ) organizational structure, e.g. departments sharing same nursing or medical personnel; ) representing both medical and surgical specialties. the university hospital zurich nvhap bundle was designed by an interprofessional and interdisciplinary group of experts. elements were chosen based on the evidence, although scarce, of their effectiveness and based on their anticipated feasibility and implementability. the bundle consists of five prevention measures (details see additional file nvhap bundle). . oral care, i.e. mechanical oral care with or without pharmacological products, once daily in all patients, and three times daily in patients with swallowing difficulties. . prevention of dysphagia-related aspiration, i.e. applying a 'modified swallowing assessment' (msa) adapted from the 'standardized swallowing assessment' by perry et al. [ ] (additional file 'msa perry') in a defined risk population, followed by further evaluation and treatment of dysphagia residing with the responsible physicians. . mobilization, i.e. mobilization at least once at the day of surgery and at least twice daily in all other patients without contraindications. . stopping unnecessary ppi and antacids, according to a list of indications in in-hospital guidelines. . respiratory therapy, i.e. referral to respiratory therapists advised for a defined patient population, with a final decision at the discretion of the responsible physician. all patients will be assessed regarding whether an active intervention of healthcare providers is indicated for each of the prevention measures at the following time points: after admission, after clinical deterioration, and after major surgery during. if yes, the prevention measure is executed according to the above description. the execution of the bundle element will be documented in the electronic medical record (emr). our multifaceted implementation strategy is designed to increase ownership and local adoption in each department by engaging local implementation teams, who establish department-specific actions tailored to local needs. this strategy is also intended to facilitate adaptability, i.e. the degree to which the intervention can be adapted to meet local needs [ , ] . based on an initial behavioural analysis informed by sensitizing frameworks (see below, "implementation frameworks") [ , , ] , we identified the following as promising intervention functions to increase adherence to the nvhap bundle: increasing knowledge and understanding about the nvhap bundle elements through education; imparting skills through technical training; and changing the physical context to increase awareness and support performance of nvhap measures through environmental restructuring. whereas these intervention functions to increase adherence to the nvhap bundle elements make up the foreseeable core intervention components, each department is encouraged to adapt the delivery of these components and to employ additional promotional components according to local context, making up the 'adaptable periphery' of the intervention [ ] . local implementation teams, composed of one nurse, one physician and one physiotherapist, will be established in each department. during recurrent "action plan" meetings, the local implementation team from each department, with support from the nvhap study team, will be responsible for assessing the current implementation status with respect to each bundle element and establishing an "action plan" with a list of planned actions aimed to increase adherence to bundle elements according to local needs. local implementation teams will be responsible for implementing the nvhap bundle in their respective departments. established "action plans" will be revisited to assess progress and refine necessary actions, as described below. the nvhap study team, based in the infection prevention department, will form a central coordinating team to provide local teams with support, example training materials, and feedback on process and outcome data. additionally, we will employ a formative approach, during which we aim to continuously identify influences on implementation efforts (e.g. barriers and facilitators) and feed these insights back to local implementation teams to optimize the potential for implementation success [ ] . this formative evaluation will occur in stages throughout the project, as described by stetler and colleagues [ ] and presented in table . the formative evaluation will rely primarily on "action plan" meetings as an opportunity to feed information back to local implementation teams regarding identified barriers and facilitators to implementation and to refine implementation action plans accordingly. this mixed methods study collects and analyses quantitative and qualitative data collected during the three study periods (baseline, implementation, and intervention period). the conceptual model of the study is depicted in fig. . effectiveness outcomes the primary outcome is nvhap incidence rate, defined as the number of patients suffering from nvhap per patient days per month. secondary outcomes are in-hospital mortality rate; length of stay; and adherence to individual bundle elements and the nvhap bundle as a whole. implementation outcomes we will use a qualitative definition of implementation success composed of the following four implementation outcomes [ ] : ) acceptability, how satisfied are study participants with the intervention; ) appropriateness, what is the perceived fit of the intervention and to what extent did participants succeed in adapting the intervention to meet the needs of their local context; ) implementation fidelity, how closely did participants succeed in implementing the core bundle components as described in the study protocol; and ) sustainability, to what extent did the intervention become institutionalised and anchored within ongoing operations. implementation outcomes will primarily be assessed qualitatively through semistructured interviews at multiple time points throughout the project, both, to assess implementation progress and to inform our formative evaluation (table ) . implementation fidelity will further be assessed through observation and artefact analysis by comparing planned and actual implementation activities. quantitative data on adherence to the five bundle measures, as described below, will also be considered in assessing implementation fidelity. sustainability will be particularly assessed by identifying examples of how the intervention has been integrated into local processes and structures such that it is likely to continue as a part of stable operations [ ] . in assessing implementation outcomes at multiple time points, we aim to identify what has been described by proctor and colleagues as "leading" and "lagging" indicators of implementation success [ ] where leading indicators are those that reflect the outcome of a change assess levels of current practices and their determinants prospectively identify potential barriers and facilitators to implementation during the baseline period "action plan" interview with local implementation teams, the current state of practice for each nvhap bundle measure will be assessed and determinants of current behaviour discussed. an "action plan" of promotional activities will be established, taking into account potential barriers and facilitators. assess discrepancies between established implementation plan and its operationalization continually identify barriers and facilitators to implementation refine implementation plan during the "action plan" interview with local implementation teams following the implementation period, the previously established "action plan" will be revisited and actual vs. planned interventions assessed. refinements to the action plan will be made as needed taking into account newly identified barriers and facilitators. during the intervention period, feedback about nvhap outcomes and process indicators will be fed back to local implementation teams. triangulate qualitative and quantitative data to enhance understanding of implementation results upon project completion, qualitative findings will be used to illuminate quantitative results and inform guidance about how the nvhap bundle can best be implemented in further settings. in practice early on or even predict it, and lagging indicators reflect the delay between a change in practice and the observable outcomes. baseline period will start at the same time for all departments and will be of different length (minimum months) as implementation of nvhap prevention measures will occur at the department level and the start of implementation activities is chosen by every department, primarily relying on availability of resources. we define three study periods on the department level, ) department baseline period, before implementation of nvhap bundle in the specific department; ) department implementation period, a two month time frame starting with the beginning of implementation activities in the respective department; ) department intervention period following the department implementation period. on the hospital level the three periods are defined as follows: ) hospital baseline period, before starting implementation in the first department; ) hospital implementation period, from the beginning of the implementation period of the first department until end of implementation period of the last included department; ) hospital intervention period following the hospital implementation period. figure depicts an anticipated study timetable. quantitative and qualitative data collection will continue throughout the project and follows the study periods on the department level (fig. ) . legend: nvhap = non-ventilator-associated healthcare-acquired pneumonia. this figure portrays the conceptual model of the nvhap implementation process, in which the entire implementation process is moderated by the context in which the process is set. the contextual influencers of implementation include the larger organizational setting (i.e. the hospital and wider national context), the inner setting (i.e. the departments in which the bundle is being implemented), as well as the characteristics of individuals directly and indirectly involved in the implementation process. the contextual influencers moderate the effectiveness of specific intervention components used to implement the nvhap bundle elements in participating departments, resulting in varying levels of implementation success, as reflected by levels of adherence to bundle components, and ultimately by the resulting outcome measures the first department (pilot department) is used to test quantitative and qualitative data collection tools and the feasibility of the implementation strategy. insights from this pilot department will help to improve the implementation strategy and study tools prior to the inclusion of further departments. our study is theoretically informed by the consolidated framework for implementation research (cfir) [ ] and the theoretical domains framework (tdf) [ ] . both the cfir and the tdf integrate findings from theoretical literature into synthesized frameworks consisting of constructs that may mediate behaviour change [ , ] . whereas the tdf domains represent a set of constructs related to individual behaviour change, the cfir domains include constructs relating to broader organizational behaviour change. for the current inquiry, we find the use of both frameworks useful to capture influencers of behaviour at the individual level, as well as the department, the overall hospital, and the wider environmental context. the cfir and the tdf will inform the intervention implementation strategy, as previously described, and guide the qualitative data collection (semi-structured interview guides) and analyses (use of tdf as deductive coding framework). in particular, use of these sensitising frameworks throughout our study will facilitate the timely identification of barriers and facilitators and will also provide insights as to which additional intervention components are most likely to be successful in addressing the identified barriers [ ] . in the study hospital, all patient data are charted electronically via an emr system. selected data are stored in a clinical data warehouse. nvhap surveillance we apply the european centre for disease prevention and control (ecdc) definition criteria for pneumonia that are used in the ecdc point prevalence studies [ ] (additional file 'ecdc nvhap definition'). in brief, the pneumonia definition comprises radiologic criteria, systemic symptoms (fever > °, leukopenia or leukocytosis) and pulmonary symptoms (e.g. cough, sputum production). pneumonia is defined as hospital-acquired, if symptoms start ≥ h after admission. if an invasive respiratory device was present in the h preceding symptom onset, the pneumonia is considered a ventilator-associated pneumonia and thus not subject of this study. a validated semi-automated surveillance system for nvhap is used [ ] . place of nvhap acquisition is defined as department, ward and room to which the patient was affiliated h before first symptoms of nvhap, unless shorter incubation period is evident from patient history. process indicators portraying adherence to the nvhap bundle elements will be monitored in two ways. first, for all five prevention measures, at least one surrogate parameter for adherence is continuously extracted from the emr of the total patient population (continuous process indicators; see additional file 'process indicators'). this parameter, e.g. tooth brushing provided by nurses, will be expressed per department, and month, and per hospital days. second, we will monitor process indicators on a sample basis with individual assessment of a subset of patients (denominator) at four different time points per department (intermittent process indicators; see additional file 'process indicators'). the latter allows a more detailed description of adherence, including non-documented prevention measures (e.g. oral care executed by patient) and takes into consideration the individual need of patients for the specific prevention measure (e.g. respiratory therapy is indicated only in a subset of patients). from the intermittent process indicators the 'nvhap adherence score' will assess patient based adherence per department and time point. the score is based on samples of patients, the 'nvhap adherence indicator' takes the value in the case the specific prevention measure was completed in the specific patient, if that was not the case, and "empty" in the case of missing values. the 'nvhap adherence score' is calculated by summing up the five proportions of patients with completed specific prevention measures (i.e. 'nvhap adherence indicator'= ) dividing it by factor five (additional file 'nvhap adherence score'). longitudinal qualitative data will be collected throughout the project as portrayed in fig. , including action plan interviews with local implementation teams, dropin interviews with frontline staff, and focus group interviews, as described in table . the researchers involved in qualitative data collection and analysis, who are also part of the implementation team, will seek to demonstrate empathic neutrality [ ] , for example by prefacing interviews with the fact that we are interested in learning about implementation experiences and that there are no right or wrong answers. in doing so, we hope to limit desirability bias in the information shared. having three data collection activities will also allow for rigorous triangulation of findings among data sources and will all inform the ongoing formative evaluation (see table ). for drop-in and focus groups interviews, participants will be purposefully sampled to include a representative mix of professions (nurses, physicians, and physiotherapists) from wards within the participating departments given inconsistencies in definition and application of 'saturation' as a measure of sufficient sampling, 'information power' has been proposed as a concept to guide adequate sample size [ ] . by having a clearly defined qualitative study aim, an information-rich sample of interview participants, guiding theoretical frameworks to inform structured data collection by skilled interviewers expert in the study topic, our study design and sampling strategy is designed to achieve high information power [ ] . analyses of nvhap bundle effectiveness to evaluate the effectiveness of the intervention bundle, two distinct analyses are performed. first, a change point model will be combined with piecewise constant rates with additional sine-cosine waves to account for seasonality. poisson regression (with log link function) is used to analyse the monthly overall nvhap incidence rate over all departments, using the monthly sum of the nvhap cases over all departments as "count" and the monthly sum of the number of patient days (in thousands) over all departments as offset. study period on the hospital level (hospital baseline, implementation, intervention period) will be used as explanatory factor (see additional file 'statistical analysis' for detailed statistical model). we may use a quasi-poisson model in case of overdispersion. second, a longitudinal poisson regression will be used. the monthly number of nvhap cases in each department will be modelled by a generalized estimating equation (gee) with departments as clusters. this allows to account for the non-independence of consecutive nvhap counts within departments, to model the temporal correlation structure (e.g. first order autoregressive) and to account for over-dispersion, if necessary. study periods and data collection in a single exemplary department legend: the baseline period of months or longer is followed by an implementation period defined to be months long, and an intervention period of again months or longer. the figure depicts the data collection time points, with squares indicating quantitative data collection and circles indicating qualitative data collection time points we will assume a poisson error distribution for the nvhap counts and use the log link function. as above, we may use a quasi-poisson model in case of overdispersion. we will use a time-dependent, department-specific binary indicator variable for department-level implementation of the intervention bundle (possibly with an intermediate level for the implementation phase) as explanatory variable. further, we will adjust for seasonality of nvhap incidences by inclusion of sine/cosine waves. because the baseline period includes nvhap rates from which served (inter alia) as basis for the choice of the nine departments, we will perform sensitivity analyses excluding data from for all analyses described above to assess a potential "regression to the mean" effect. to portray adherence to the single prevention measures and the nvhap bundle as a whole a descriptive analysis will be performed, summarizing continuous and intermittent process indicators and the 'nvhap overall adherence score' by department-level periods. further, we will evaluate whether the process indicators are associated with the nvhap incidence rate. we will use gees with poisson error and departments as clusters (as described above) to model the monthly nvhap rates as dependent on either single continuous process indicators or on all continuous process indicators combined. to model monthly nvhap as dependent on intermittent process indicators (either single process indicators, all process indicators combined, or the nvhap overall adherence score), we will use gees with poisson error and departments as clusters (as described above). because the intermittently collected process indicators are collected only at four time points, we will use linear interpolation to derive monthly values for these process indicators. longitudinal qualitative data from drop-in, action-plan and focus group interviews will be included in a crosscase analysis, where each participating department represents a case. in a first step, all interview transcripts and notes will be coded deductively using the theoretical domains framework (tdf) as a coding scheme as well as additional codes to capture our pre-defined implementation outcomes [ ] . inductive thematic analysis will then be conducted to identify themes relevant to the implementation within tdf domains. analyses will begin with at the case level to understand the local influencers of implementation at the department level, allowing us, for example, to assess how implementation outcomes shift over time in relation to the undertaken interventions and in light of local barriers and facilitators. then, cross-case matrices will be used to explore any trends across departments [ ] . this qualitative analysis will allow us to make a qualitative assessment about which local factors and interventions contributed to implementation success. our in-depth findings will also help to ultimately shine light on quantitative study results. the researchers involved in qualitative analysis will engage in an ongoing process of reflexivity [ ] , considering the role of our own preconceptions and close relation to the implementation process, while also aiming to provide an authentic account of the implementation process. with this mixed-methods study we will close critical knowledge gaps about the prevention of nvhap, a neglected but common hai. to date, literature about prevention measures against nvhap is scarce [ ] , and our study will provide further knowledge by assessing the effectiveness of a five element prevention bundle against nvhap on lowering nvhap incidence rates. to our knowledge, it is the first study testing an inpatient bundle of nvhap prevention measures on a broad semi-structured interviews of approximately one hour to assess the current implementation status of each nvhap bundle element throughout the study periods, as well as identify potential or actual barriers and facilitators to implementation, and plan a list of actions to be taken locally. interviews will be audio-recorded and transcribed where acceptable and structured notes will be taken systematically. these and the written action plan documents established after each interview will be included in qualitative analysis. short, semi-structured, drop-in interviews of - min to learn from frontline staff about their experience with the nvhap implementation and identify local barriers and enablers to implementation. detailed, structured notes will be taken during and after each drop-in interview and/or the interview will be audio-recorded and transcribed verbatim to be included in qualitative analysis. frontline clinicians semi-structured focus group interviews of approximately h to assess implementation outcomes among frontline staff. focus groups will be audio-recorded and transcribed verbatim for inclusion in qualitative analysis. patient population. moreover, as effective implementation is as important as choosing the right bundle elements [ , ] , we place focus on a theoreticallyinformed implementation strategy. the quantitative part of the study aims to not only measure the primary outcome parameter nvhap incidence rate over time, but to also measure process indicators. this will help us to better understand if the implementation process was successful and to evaluate direct association between prevention measures and nvhap incidence rate. as the nvhap bundle cannot be effective if it is not well implemented, it is important to also measure implementation outcomes (e.g. acceptability, appropriateness, fidelity, and sustainability) as necessary preconditions for achieving the desired changes in clinical outcomes. a major strength of this study is the mixed-methods approach, including an extensive formative qualitative study to provide insights about how and why departments succeeded, or faced challenges, in implementing the nvhap bundle. with some notable exceptions [ ] [ ] [ ] [ ] , many qualitative implementation evaluations are limited to inquiries conducted at a single point in time. such inquiries are prone to participant recall biases and may be insufficient to telling the whole implementation story [ ] . our longitudinal qualitative study aims to provide critical contextual insights to guide others hoping to implement the nvhap bundle. additionally, the participatory approach of our formative evaluation is intended to increase project commitment among stakeholders, particularly local implementation teams. the limitations of our study are the following: first, our study does not include a control group. we abstained from conducting a randomized controlled trial due to anticipated high contamination between departments/wards within the same hospital. second, the duration of the implementation period is determined to be months not accounting for possibly longer duration due to the formative approach of the implementation strategy. we aim to address this point by analysing the results both on the hospital and department level. third, by continuously collecting process indicators from emr, we cannot preclude reporting bias (e.g. increased documentation of oral care). we address this issue by additionally measuring process indicators on an individual basis. further, although we take efforts to demonstrate empathic neutrality during our qualitative data collection, we cannot entirely preclude the possibility that qualitative researchers may be perceived as being partial, leading to potential desirability bias in the qualitative data. finally, we acknowledge that our formative process evaluation does in itself lead to changes in implementation plans and that these changes must be documented with great care to keep track of the exact implementation activities. rather than purely a limitation, we view this as a strength of our study, and we anticipate that it should also be integrated into recommendations for those wishing to replicate results of our future nvhap study. in conclusion, with this innovative mixed-methods study design, we will assess the effectiveness of the nvhap bundle, but also measure process indicators of the nvhap bundle and contextual factors influencing implementation uptake. we will be able to triangulate our findings, i.e. correlate nvhap rates with adherence data of the prevention bundle and again with qualitative measures of implementation success. further, our mixed-method approach will be of great value to understanding the complex contextual interactions that influence implementation success, which are necessary to inform implementation guidance for other institutions planning to implement the nvhap bundle. addendum: due to the covid- pandemic, the study data collection had to be terminated earlier than planned (i.e. end of february ). additional file informs about the details of early study termination. european centre for disease prevention and control. point prevalence survey of healthcare associated infections and antimicrobial use in european acute care hospitals multistate point-prevalence survey of health care-associated infections the breadth of hospital-acquired pneumonia: nonventilated versus ventilated patients in pennsylvania prevention of hospital-acquired pneumonia in non-ventilated adult patients: a narrative review guidelines for preventing health-care--associated pneumonia, : recommendations of cdc and the healthcare infection control practices advisory committee management of adults with hospital-acquired and ventilatorassociated pneumonia: clinical practice guidelines by the infectious diseases society of america and the clinical practice 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investigate implementation problems the role of formative evaluation in implementation research and the queri experience outcomes for implementation research: conceptual distinctions, measurement challenges, and research agenda point prevalence survey of healthcareassociated infections and antimicrobial use in european acute care hospitals, protocol version . , ecdc pps development and validation of a semi-automated surveillance system-lowering the fruit for non-ventilator-associated hospital-acquired pneumonia (nvhap) prevention qualitative research and evaluation methods. ed. thousand oaks sample size in qualitative interview studies: guided by information power miles and huberman publication location is thousand oaks associations between ventilator bundle components and outcomes implementing infection prevention practices across european hospitals: an in-depth qualitative assessment practice based, longitudinal, qualitative interview study of computerised evidence based guidelines in primary care implementation and adoption of nationwide electronic health records in secondary care in england: final qualitative results from prospective national evaluation in "early adopter" hospitals implementation of infection control best practice in intensive care units throughout europe: a mixed-method evaluation study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors would like to thank the members of the interprofessional group of uhz healthcare workers who helped to design the uhz nvhap bundle, namely claudia barfuss, birgit david, brigitte eggenberger, and dragos ionescu. supplementary information accompanies this paper at https://doi.org/ . /s - - - .additional file . nvhap bundle.additional file . modified swallowing assessment (msa).additional file . ecdc definition for nvhap. additional file . nvhap adherence score.additional file . statistical analysis. additional file . addendum: early study termination. this study is supported by the swiss federal office of public health (nr. . ). development of the algorithm for semi-automatized surveillance of nvhap was supported by "innovationspool", a university hospital zurich funding program for developing new approaches in medical diagnostics and treatment. aw is supported by the academic career program "filling the gap" of the medical faculty of the university of zurich. the funding sources were not involved in the design of the study, nor in collection, analysis, and interpretation of data, nor in writing the manuscript. data sharing is not applicable to this article as no datasets were generated or analyzed during the current study. the necessity of a formal ethical evaluation was waived by the ethics commission of the kanton zürich, switzerland (kantonale ethikkommission zürich), based on the swiss law on research on humans (req- - ). no written informed consent will be obtained from hospitalized patients included in the study. written informed consent will be obtained from the healthcare workers participating in audio-recorded interviews. not applicable. none to declare for all authors.author details division of infectious diseases and hospital epidemiology, university hospital zurich, university of zurich, rämistrasse , ch- zurich, key: cord- -hrz bypr authors: omrani, ali s.; almaslamani, muna a.; daghfal, joanne; alattar, rand a.; elgara, mohamed; shaar, shahd h.; ibrahim, tawheeda b. h.; zaqout, ahmed; bakdach, dana; akkari, abdelrauof m.; baiou, anas; alhariri, bassem; elajez, reem; husain, ahmed a. m.; badawi, mohamed n.; abid, fatma ben; abu jarir, sulieman h.; abdalla, shiema; kaleeckal, anvar; choda, kris; chinta, venkateswara r.; sherbash, mohamed a.; al-ismail, khalil; abukhattab, mohammed; ait hssain, ali; coyle, peter v.; bertollini, roberto; frenneaux, michael p.; alkhal, abdullatif; al-kuwari, hanan m. title: the first consecutive patients with coronavirus disease from qatar; a nation-wide cohort study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: hrz bypr background: there are limited data on coronavirus disease (covid- ) outcomes at a national level, and none after days of follow up. the aim of this study was to describe national, -day all-cause mortality associated with covid- , and to identify risk factors associated with admission to an intensive care unit (icu). methods: this was a retrospective cohort study including the first consecutive patients with covid- in qatar who completed days of follow up by june , . the primary outcome was all-cause mortality at days after covid- diagnosis. in addition, we explored risk factors for admission to icu. results: included patients were diagnosed with covid- between february and april , . the majority ( , . %) were males and the median age was years [interquartile range (iqr) – ]. by days after covid- diagnosis, patients ( . %) had died, ( . %) were still in hospital, and two ( . %) were still in icu. fatal covid- cases had a median age of . years (iqr . – ), and were mostly males ( , . %). all included pregnant women ( , . %), children ( , . %), and healthcare workers ( , . %) were alive and not hospitalized at the end of follow up. a total of patients ( . %) required hospitalization, out of which ( . %) were admitted to icu. most frequent co-morbidities in hospitalized adults were diabetes ( . %), and hypertension ( . %). multivariable logistic regression showed that older age [adjusted odds ratio (aor) . , % confidence interval (ci) . – . per year increase; p < . ], male sex (aor . , % ci . – . ; p < . ), diabetes (aor . , % ci . – . ; p . ), chronic kidney disease (aor . , % ci . – . , p . ), and higher bmi (aor . , % ci . – . per unit increase; p . ), were all independently associated with increased risk of icu admission. conclusions: in a relatively younger national cohort with a low co-morbidity burden, covid- was associated with low all-cause mortality. independent risk factors for icu admission included older age, male sex, higher bmi, and co-existing diabetes or chronic kidney disease. supplementary information: supplementary information accompanies this paper at . /s - - - . conclusions: in a relatively younger national cohort with a low co-morbidity burden, covid- was associated with low all-cause mortality. independent risk factors for icu admission included older age, male sex, higher bmi, and co-existing diabetes or chronic kidney disease. keywords: coronavirus, covid- , sars-cov- , mortality, qatar background severe acute respiratory syndrome coronavirus (sars-cov- ), the cause of coronavirus disease , emerged in china in late . by july , , more than million confirmed sars-cov- infections were confirmed worldwide, with over thousand associated deaths [ ] . based on the number of deaths as a proportion of reported covid- cases, the overall estimated covid- -associated mortality rate is around . % [ ] . however, the accuracy of such a figure is uncertain given the variation in case finding policies from one healthcare setting to another [ , ] . furthermore, reported mortality has been mostly based on in-hospital outcomes or relatively short follow up [ ] [ ] [ ] [ ] [ ] . in their recently published recommendations for a minimal common outcome measure set for covid- research, the world health organization (who) favored that mortality outcomes are assessed at days [ ] . single and multi-center cohort studies suggested that risk factors for severe covid- include male sex, older age, and the presence of multiple comorbidities [ , , ] . the extent to which such risk factors are important at a population level in settings with ample healthcare resources, a covid- control program based on active case finding and isolation, and a low burden of comorbidities, is unknown. in this study, we describe -day outcomes of a nationwide covid- cohort from qatar, and explore patient characteristics associated with the need for admission to an intensive care unit (icu). hamad medical corporation (hmc) encompasses multiple hospital facilities and provides all covid- medical care for the . million population of qatar. in response to the covid- pandemic, existing clinical services were re-organized and two brand new hospital facilities were opened ahead of their originally planned dates. in total, non-icu bed capacity was increased from to ( . % increase), and icu beds from to ( . % increase). from a healthcare delivery perspective, hmc defines adults as those aged above years. sars-cov- infection was diagnosed by real-time polymerase chain reaction (rt-pcr) assays taqpath covid- combo kit (thermo fisher scientific, waltham, massachusetts) or cobas sars-cov- test (roche diagnostics, rotkreuz, switzerland) on respiratory tract specimens. severity of covid- was classified according to the who guidelines [ ] . sars-cov- testing was offered to all individuals presenting with symptoms suggestive of covid- , known close contacts of confirmed cases including healthcare workers, and all returning travelers. patients with asymptomatic sars-cov- infection or mild covid- without significant co-morbidities were isolated in dedicated community facilities until they had two consecutive negative sars-cov- rt-pcr results from upper airway samples taken more than h apart. covid- patients with significant co-morbidities or moderate to severe disease were hospitalized for inpatient management. standard care for hospitalized patients involved supportive care and investigational antiviral therapy. individual regimens were selected by the treating physicians based on severity of disease, the presence of contra-indications or potential drug-drug interactions, and the patients' preferences. twenty five individuals included in this study had been elsewhere reported [ ] . we used the hmc covid- database to identify the first consecutive patients with rt-pcr-confirmed covid- who would complete days of follow up from date of diagnosis by june , . during the period between may and june , , clinical and laboratory data were retrieved from hmc's electronic healthcare system. final status days after covid- diagnosis was ascertained against the electronic healthcare system and qatar's national deaths records. the report was prepared according the strengthening the reporting of observational studies in epidemiology (strobe) recommendations [ ] . the primary endpoint was all-cause mortality within days after rt-pcr confirmation of sars-cov- infection. for hospitalized patients, we also assessed risk factors for admission to icu. we summarized categorical data as numbers and percentages and compared them using pearson's chisquared or fisher's exact test, as appropriate. continuous data are presented as medians and interquartile ranges (iqr) and compared among groups using wilcoxon rank-sum test. the majority ( patients, . %) of admissions to icu occurred within of the first h from hospitalization. we therefore used logistic regression to explore predictors of admission to icu. baseline variables were included in the univariable logistic regression analysis if their between groups differences were associated with p values of < . . independent variables in the multivariable regression model were chosen based on their association with p values of < . in the univariable logistic regression, and on their ready availability before any covid- -related clinical evaluation. due to the number of events in the study, we limited the number of independent variables in the multivariable regression analysis to eight to avoid overfitting the model. the final multivariable logistic regression model included age, male sex, body mass index (bmi), defined as body weight in kilograms divided by squared height in meters, and co-existing diabetes mellitus, systemic hypertension, coronary artery disease, chronic liver disease, and chronic kidney disease. multiple imputations approach was applied for variables with > % missingness. all p values were two-sided with a threshold of < . for statistical significance. statistical analyses were performed using stata statistical software release . (statacorp llc, college station, texas). individuals included in this study were diagnosed with sars-cov- infection between february and april , . initial sars-cov- cases were diagnosed in travelers returning from iran and europe. sustained local transmission became established thereafter (fig. ) . of the rt-pcr-confirmed covid- cases included in this report, ( . %) were in males and the majority belonged to age groups - years ( , . %) and - years ( , . %) (fig. ) . the cohort included ( . %) individuals aged years or less, ( . %) pregnant women and ( . %) healthcare workers (table s , appendix). most individuals in this study did not require hospitalization ( , . %). those who were not hospitalized were significantly younger and had fewer co-existing chronic medical conditions (table s , appendix). of patients who required hospitalization, ( . %) were admitted to icu. overall, days after covid- diagnosis, patients ( . %) had died, patients ( . %) were still in hospital and two ( . %) were still in icu (fig. ) . out of individuals aged > included in this report, ( . %) were hospitalized. the majority ( , . %) were males and the median age was years (iqr - ). nationalities from who's south-east asia region ( , . %) and eastern mediterranean region ( , . %) were most frequent. diabetes ( , . %) and hypertension ( , . %) were the most common co-existing medical conditions. fever ( . %) and cough ( . %) were the most common presenting symptoms. median bmi was . kg/m (iqr . - . ). most patients ( , . %) did not require oxygen therapy within the first h of hospitalization. hydroxychloroquine ( , . %), azithromycin ( , . %), and lopinavir-ritonavir ( , %) were the most commonly used investigational antiviral agents. compared with those who did not require icu admission, icu patients were significantly more likely to be males (p . ), have higher median age (p < . ) and to have multiple co-morbidities (p < . ) ( table ) . they also had significantly higher median bmi ( . versus . , p < . ) and were more likely to present with fever, cough and dyspnea (p < . for each). within the first h of hospitalization, icu patients had significantly higher median heart rate ( versus per minute, p < . ), and respiratory rate ( . versus per minute, p < . ), significantly lower oxygen saturation ( % versus %, p < . ) ( table ). in addition, baseline blood investigations from icu patient were significantly more likely to show lower median peripheral lymphocyte count ( . versus . × /l, p < . ), and higher median serum creatinine ( versus μmol/ l, p < . ), and c-reactive protein (crp) ( . versus , p < . ). complications such as acute kidney injury ( . % versus . %), and myocardial injury ( . % versus . %, p < . ) were more common in icu compared with non-icu patients. other baseline characteristics, management, and complications variables in hospitalized covid- adults included in this study are shown in table . in univariable analysis, the odds of admission to icu were significantly higher in older patients, males compared with females, and in those with diabetes, hypertension, coronary artery disease, or chronic lung, liver, or kidney disease, and in those with higher bmi ( table ). the presence of cough, dyspnea, or fever, elevated baseline heart rate or respiratory rate, decreased oxygen saturation, lower lymphocyte count, and increased serum creatinine, crp, and alanine transaminase (alt) were also associated with admission to icu ( table ). in the multivariable logistic regression, we found that older age, male sex, co-existing diabetes or chronic kidney disease, and higher bmi were all independently associated with increased risk of need for icu admission ( table ) . a total of patients ( . %) died within days of follow up. the median age of fatal covid- cases was . years (iqr . - ). most deceased patients were males ( , . %) and most ( , . %) had two or more comorbidities (see table s in supplementary material). two patients died without hospitalization. the first was a -year-old man with a history of hypertension and heavy smoking. he had asymptomatic sars-cov- infection and was isolated in a community facility pending viral clearance. he developed severe chest pain and cardiopulmonary arrest days after covid- diagnosis. the second patient was a -year-old man with end-stage kidney disease, hypertension, diabetes and coronary artery disease. he developed cardiopulmonary arrest shortly after presenting to the emergency department in severe respiratory distress. a post-mortem examination to confirm the cause of death was not performed in either case. the remaining deaths all occurred in patients who were in icu with severe acute respiratory distress syndrome requiring prolonged invasive mechanical ventilation. deaths occurred after a median of days (iqr - ) from covid- diagnosis. ten ( . %) deaths occurred in patients aged or older. the remaining two were in patients aged years and years. the former had diabetes, hypertension, and obesity (bmi . ). the latter patient presented with fulminant hepatitis and his hepatitis b serology was positive for surface igm antibodies. he died within days with encephalopathy and multi-organ failure. the study included pregnant women with sars-cov- infection with median age of years (iqr . - ). nineteen ( . %) were hospitalized, including one ( . %) in icu, and all were discharged within the follow up period. ten ( . %) pregnant women with covid- gave birth during the follow up period; all resulting in healthy babies with negative sars-cov- tests (see table s in supplementary material). a total of patients in this cohort were healthcare workers. their median age was years ( - ) and the majority were males ( , . %). the most frequent professional background of affected healthcare workers was nursing ( , . %), and allied healthcare ( , %) (see table s in supplementary material). out of ( . %) who required hospitalization, three ( . %) required admission to icu. all healthcare workers in this table s in supplementary material). there were individuals aged years or less in the study, of which ( . %) were males. median age was years (iqr - ). children were mostly diagnosed in the context of contact screening ( / , %), and were not hospitalized ( , . %). the majority ( , . %) of children, including all seven infants, had family members with confirmed covid- (see table s in supplementary material). in this national covid- cohort, only ( . %) out of patients died within days of diagnosis, and ( . %) required ongoing hospitalization at the end of the -day follow up period. sars-cov- infection are generally slightly more common in males than females [ ] . however our report shows that . % of sars-cov- infections in qatar were in males. our findings reflect the country's demographic characteristics. male to female ratio in qatar's general population is . and the corresponding male to female sars-cov- incidence per , population in our report is . . notably, the population's male to female ratios are . - . in age groups between to ( . - . ), where . % of sars-cov- infections where reported (table s , supplement). our mortality rates are considerably lower than previously reported form large covid- cohorts from china, europe and united states [ ] [ ] [ ] [ ] [ ] . there are the other hand, nearly one third of patients reported in our study were identified through screening efforts. our lower mortality rates could therefore be in part due to higher detection of milder covid- cases. secondly, our cohort's demographic profile is consistent with the country's population being largely constituted of male migrants working in the country's numerous infrastructure projects (table s , supplement). older age and the presence of multiple co-morbidities have consistently been associated with increased risk of severe covid- , need for critical care support, and mortality [ , , ] . the majority ( %) of patients in our study did not have any pre-existing chronic medical conditions. moreover, with a median patient age of years (iqr - ), our patients were considerably younger than those reported in large cohorts from lombardy region in italy (median year, iqr - ), the united kingdom (median years, iqr - years) and new york city (median years, iqr - ) [ ] [ ] [ ] . note should also be taken of qatar's population being relatively younger than most countries reporting high covid- -associated mortality. for example, the median age in qatar is only . years, whereas the median population age is . years in italy, and . years in the united kingdom [ ] . in addition, the proportion of population aged over years is only . % in qatar, while it is % in italy and . % in the united kingdom [ ] . a third factor in explaining our low covid- associated mortality is the rapid escalation of the healthcare system's capacity to accommodate the expected hike in demand for hospital beds in general, and for icu support in particular. it has been suggested that some of the worst covid- -associated mortality rates have in part been the result of overwhelmed critical care resources that could not support a large influx of severely ill covid- patients [ , ] . this has stimulated discussions around rationing of critical care support for covid- patients, including potentially difficult decisions to withdraw resources from one patient to provide them to another [ ] . on the other hand, critical care support is rarely withheld in our setting, even in cases where the prognosis appears to be unfavorable. while diabetes mellitus, coronary artery disease, chronic liver disease, hypertension and chronic kidney diseases all appeared to be associated with risk of admission to icu in our univariate analysis, the association was statistically significant only for the latter wo in the adjusted logistic regression analysis ( table ). this is probably the result of interactions between our cohort's co-morbidities and their age. deaths observed in our study have largely occurred in older patients with multiple co-morbidities. though . % of deaths occurred in those aged years or above, this group constituted only . % of our entire cohort. our age group-specific mortality was . % in those aged - years, and . % in those aged years or more. these figures are comparable with mortality rates in similar age groups in china, italy, and the united states, but are considerably lower than those reported from the united kingdom [ , , , ] . one patient in our cohort died while in a community isolation facility with asymptomatic sars-cov- infection. his rapid demise after complaining of chest pain suggests that his death was caused by myocardial infarction or pulmonary embolism. both complications are increasingly recognized associations with covid- [ , ] . an increase in out-of-hospital cardiac arrests has been observed in association with sars-cov- pandemic, including in patients with symptoms compatible with covid- [ ] . moreover, . % of our icu patients and . % of our non-icu patients had evidence of myocardial injury during their hospitalization. the diagnosis of covid- in patients with known or increased risk of coronary artery disease should be an opportunity to review and optimize medical therapy to reduce the risk of acute coronary events. most hospitalized patients in our study received investigational antiviral therapies. however, recent reports from large cohort and randomized clinical trials do not support the use of hydroxychloroquine, alone or in combination with azithromycin, or lopinavir-ritonavir for patients with covid- [ , ] . it is likely that covid- management will continue to evolve as more results from ongoing clinical trials become available [ ] . our analysis showed that increasing age, male sex, higher bmi, and the presence of diabetes or chronic kidney disease are risk factors for admission to icu. remarkably, hypertension, chronic lung disease, and coronary artery disease, all of which are frequently reported as important predictors for severe covid- in previous studies, were not independently associated with icu admission in our setting [ ] . furthermore, our univariable analysis showed that presenting with dyspnea and cough as well as baseline blood abnormalities such as lower lymphocyte count, higher crp and serum creatinine are associated with increased risk of admission to icu [ , ] . higher bmi as a risk factor for severe covid- is particularly noteworthy [ ] . median bmi in our hospitalized patients was . kg/m (iqr . - . ), a reflection of the growing concern over the increasing prevalence of overweight and obesity in developing countries, along with its consequent health problems such as diabetes and cardiovascular disease [ ] . in the context of covid- , it is important to recognize the role of overweight and obesity as a driver of severe covid- . our findings help guide deployment of medical resources to better select patients for hospitalization, closer clinical monitoring, and early clinical support. healthcare workers represented . % of cases in our report. three ( . %) of our healthcare workers required admission to icu. unlike some unfortunate reports from elsewhere, all healthcare workers in our study fully recovered within the study follow up period [ ] . risk to healthcare personnel is highest in those with prolonged direct contact with symptomatic patients, especially where personal protective equipment are either in short supply or not used appropriately [ ] . also noteworthy is that . % of healthcare workers in this study were asymptomatic. single center healthcare worker screening studies reported asymptomatic rates ranging from . to % [ , ] . the most efficient healthcare worker screening strategy that combines practicality with patient protection is still unclear. like previous reports, children in our study had a largely uneventful sars-cov- infection [ ] . while only . % of the entire cohort were hospitalized, the majority ( . %) of pregnant women with covid- in our report were hospitalized. however, only one ( . %) out of pregnant women in this report required admission to icu, and none died within days of follow up. our findings are consistent with recent reports indicating that pregnancy may be independently associated with increased risk of hospitalization and severe covid- [ , ] . the limitations of this study include its observational nature and missing data for some variables. to address those limitations, we used multivariate analyses with multiple imputations to assess independent associations with the outcome. despite this, our study benefits from being, to the best of our knowledge, the first to report -day outcomes of sars-cov- , and to do so at a nationwide level. in a setting of proactive sars-cov- case finding, a younger population, and low co-morbidity burden, sars-cov- was associated with low all-cause mortality. independent risk factors for icu admission included older age, male sex, higher bmi, and co-existing diabetes or chronic kidney disease. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file table s . baseline characteristics and outcomes of individuals with coronavirus disease in qatar. table s . qatar population and corresponding sars-cov- infection incidence per , population by sex and age group. qatarpo box . division of critical care, department of medicine, hamad medical corporation, doha, qatarpo box . hazm mebaireek general hospital, hamad medical corporation, doha, qatar. hamad general hospital rumailah hospital qatarpo box . references . world health organization. coronavirus disease (covid- ) situation report - real estimates of mortality following covid- infection what other countries can learn from italy during the covid- pandemic covid- : investigation and initial clinical management of possible cases baseline characteristics and outcomes of patients infected with sars-cov- admitted to icus 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covid- and intensive care unit admission: a systematic review and meta-analysis high prevalence of obesity in severe acute respiratory syndrome coronavirus- (sars-cov- ) requiring invasive mechanical ventilation epidemic obesity and type diabetes in asia deaths from covid- in healthcare workers in italy-what can we learn? reasons for healthcare workers becoming infected with novel coronavirus disease (covid- ) in china covid- screening of health-care workers in a london maternity hospital characteristics of healthcare workers who underwent nasopharyngeal swab testing for sars-cov- in milan covid- in children, pregnancy and neonates: a review of epidemiologic and clinical features public health agency of sweden's brief report: pregnant and postpartum women with severe acute respiratory syndrome coronavirus infection in intensive care in sweden characteristics of women of reproductive age with laboratory-confirmed sars-cov- infection by pregnancy status -united states publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank all colleagues in hamad medical corporation and the ministry of public health for their outstanding service and dedication. the publication of this article was supported by the medical research center, hamad medical corporation. no other funding was required. the datasets used and analyzed during the current study are available from the corresponding author on reasonable request. the study was approved by hamad medical corporation's institutional review board (mrc ), with a waiver of informed consent. no additional administrative permissions were required to access the raw data. all data used in this study were anonymized before their use. not applicable. the authors declare that they have no competing interests. key: cord- -ltmu ncu authors: pfitscher, l. c.; cecatti, j. g.; pacagnella, r. c.; haddad, s. m.; parpinelli, m. a.; souza, j. p.; quintana, s. m.; surita, f. g.; sousa, m. h.; costa, m. l. title: severe maternal morbidity due to respiratory disease and impact of h n influenza a pandemic in brazil: results from a national multicenter cross-sectional study date: - - journal: bmc infect dis doi: . /s - - -z sha: doc_id: cord_uid: ltmu ncu background: the aim of this study was to assess the burden of respiratory disease, considering the influenza a pandemic season (h n pdm ), within the brazilian network for surveillance of severe maternal morbidity, and factors associated with worse maternal outcome. methods: a multicenter cross-sectional study, involving referral maternity hospitals in five brazilian regions. cases were identified in a prospective surveillance by using the who standardized criteria for potentially life-threatening conditions (pltc) and maternal near miss (mnm). women with severe complications from respiratory disease identified as suspected or confirmed cases of h n influenza or respiratory failure were compared to those with other causes of severe morbidity. a review of suspected h n influenza cases classified women as non-tested, tested positive and tested negative, comparing their outcomes. factors associated with severe maternal outcome (smo = mnm + md) were assessed in both groups, in comparison to pltc, using pr and % ci adjusted for design effect of cluster sampling. results: among cases of severe maternal morbidity, ( %) had respiratory disease. respiratory disease occurred in one-quarter of mnm cases and two-thirds of md. h n virus was suspected in cases with respiratory illness. around % of these women were tested, yielding confirmed cases. confirmed h n influenza cases had worse adverse outcomes (mnm:md ratio < ( . : ), compared to : in cases due to other causes), and a mortality index > %, in comparison to . % in other causes of severe maternal morbidity. delay in medical care was associated with smo in all cases considered, with a two-fold increased risk among respiratory disease patients. perinatal outcome was worse in cases complicated by respiratory disease, with increased prematurity, stillbirth, low birth weight and apgar score < . conclusions: respiratory disease, especially considering the influenza season, is a very severe cause of maternal near miss and death. increased awareness about this condition, preventive vaccination during pregnancy, early diagnosis and treatment are required to improve maternal health. background improvement in maternal health aiming a reduction in maternal mortality is a priority worldwide, in an attempt to accomplish the established millennium development goals set for [ ] [ ] [ ] . however, the expected reduction in maternal mortality is still far from ideal and varies widely across regions [ , ] . most recently, to better comprehend the burden of disease on female health and complement mortality inquiries, an alternative approach has been to study maternal morbidity. maternal morbidity can have an impact on both low-income and highincome settings. in , the world health organization (who) standardized the definitions of potentially life-threatening conditions (pltc) and maternal near miss (mnm) [ ] . pltc is defined by the number of maternal complications, including hemorrhagic (e.g., abruption placenta, ruptured uterus, atony and others), hypertensive disorders (e.g., severe preeclampsia, eclampsia, hellp syndrome), management indicators of severity (e.g., blood transfusion, intubation, intensive care unit admission) and other complications (e.g., pulmonary edema, cardiac disease and sepsis). maternal near miss (mnm) is any situation in which a woman survives a very severe complication with proven organ dysfunction, during pregnancy or puerperium ( days after childbirth), with at least one of the following criteria: clinical (e.g., shock or clotting disorder), laboratory (lactate > , pao /fio < mmhg) or management (hysterectomy due to infection or hemorrhage and blood transfusion ≥ units of packed red blood cells). severe maternal outcome (smo) accounts for cases of mnm plus maternal deaths (md) [ ] . recently, the concept of "obstetric transition" was incorporated into the study of maternal morbidity and mortality. the concept illustrates a global trend in which a pattern of high maternal mortality rates with predominantly direct obstetric causes (e.g., hemorrhage, preeclampsia or uterine infection) has been replaced by lower maternal mortality rates with an increasing proportion of indirect causes (preexisting disorders or those aggravated by pregnancy, such as cardiac disease, kidney disease or infection due to urinary or pulmonary foci), institutionalization and medicalization of childbirth and increased rate of obstetric interventions [ ] . obstetric transition is important to help understand the occurrence of severe maternal morbidity and provide patients with the appropriate treatment in different settings. among the indirect causes of maternal morbidity and mortality, respiratory disease plays a significant role, either due to the presence of severe infection or complications of the underlying conditions, such as asthma and heart disease. physiological and anatomical changes that occur during pregnancy to provide accommodation for the growing uterus, can affect the known clinical presentation of respiratory signs and symptoms. adequate diagnosis and treatment of respiratory disease may be delayed [ , ] . in addition, it is recognized that pregnancy may increase the risk of severe influenza-associated complications [ , ] . it became clear throughout the h n influenza pandemic [termed a(h n )pdm ] worldwide [ ] [ ] [ ] [ ] [ ] [ ] that pregnant women were a highly vulnerable group. from july to january , , , cases of the disease and deaths were reported in brazil, [ ] . however, the total number of cases and deaths were likely much higher than the notified number. we proposed a novel approach to analyzing the burden of h n influenza virus infection and other respiratory disease among patients with severe maternal morbidity. cases complicated by severe respiratory disease were compared to cases with morbid conditions due to other causes (such as hemorrhage and hypertension). in addition, factors possibly associated with a higher risk of smo were evaluated by using the who standardized definitions of morbidity in referral maternity hospitals. this study is a secondary analysis of the brazilian network for surveillance of severe maternal morbidity including referral maternity hospitals in brazil. the study evaluated severe maternal morbidity cases, from a prospective surveillance, according to the who newly publicized criteria for these conditions [ ] . the methodological details of the original study have already been published elsewhere [ , ] . briefly, this multicenter study included referral maternity hospitals distributed among the five brazilian geographical regions. from july to june , all women admitted to participating centers, who were identified as having any life-threatening condition, near miss or maternal death, according to the who definition, were included in the study. data collection, by the study team, was acquired through medical chart review after hospital discharge or death of the patient. if any doubt on diagnosis considered, the treating doctors were further contacted for clarifications. information was entered into the openclinica® electronic platform (version . . -waltham, ma, usa) through a structured form completed by the local coordinator from each participating center. this was not a population based study, however, there was a concern to reduce the impact of nonrandom sampling and an effort to consider representativeness of the national territory (with health facilities from all five macro-regions of the country) and of facilities from public and private sectors, university and non-university hospitals. all selected hospitals had to provide information concerning their characteristics, including location, complexity of level of care, population covered, number of maternity beds and availability of resources for severe cases. quality control was carried out during various phases of the study. initially, training was provided to the entire team participating in the study, using a detailed operations manual, with the definition of each variable. meetings were held between the local research team and the coordinating team of the study to standardize data. case review was conducted by the local investigator. subsequently, the coordinating team of the study performed random reviews of manual and electronic forms for data consistency in visits to monitor the centers' performance. periodically, review of the electronic system was carried out to check for data inconsistency, along with systematic case review. some reported conditions were delay or substandard care, which had been previously reported [ ] . reasons for the delay in treatment were the woman or family member (including delay in identifying the condition, seeking care and refusing to accept treatment), health service (difficulties in obtaining equipment or medical supplies) or health professional (delays in identifying the correct diagnosis and providing appropriate patient treatment). sample size was determined by the prevalence of about maternal near miss cases per births and a maternal mortality ratio of / , live-born infants ( % confidence interval). it was predicted that , births [ ] needed to be monitored. for the present analysis, we considered severe respiratory disease as a suspected or confirmed case of influenza or acute respiratory failure, defined as incapacity of the respiratory system to promote adequate gas exchange, with arterial blood gas parameters: pao < mmhg or peripheral saturation < %, associated or not with paco > mmhg. clinical parameters such as tachypnea (respiratory rate-rr > ) or bradypnea (rr < ), use of accessory respiratory muscles, nasal flaring, associated with torpor or agitation were also considered. for suspected or confirmed cases of a(h n )pdm , the definition of cases considered only those with severe morbidity, including acute respiratory insufficiency, sepsis, intensive care admission, intubation and others. cases of h n without severe complications were not included. a review of all h n influenza cases was necessary to confirm whether laboratory tests had been performed and to obtain the results of these tests, since data in the original study had not been collected in detail. case review was requested from each local center and new data were distributed into three groups: non-tested, positive and negative cases for h n influenza virus. initially, the prevalence of pltc, mnm and md was calculated per group, as well as the respective health indicators related to maternal morbidity and mortality: maternal near miss ratio, severe maternal outcome ratio, mortality index and maternal mortality ratio, according to the who definition [ ] . to evaluate the progression of severe maternal morbidity in cases complicated by respiratory disease throughout the study, maternal outcomes (pltc, mnm and md) were measured for each month studied. the risk of smo associated with procedures used to manage the severity of conditions was estimated for the group with severe respiratory disease and other causes of severe maternal morbidity, using prevalence ratios plus their respective % ci adjusted for the design effect of cluster sampling. subsequently, we performed an analysis considering the total number of cases with severe respiratory disease versus cases with other causes of severe maternal morbidity. in each group, pltc (less severe cases) and severe maternal outcome (smo: mnm + md) cases were compared to evaluate the factors potentially associated with more severe disease, including delay in obstetric care, also using the prevalence ratios plus their respective % ci adjusted for the design effect of cluster sampling. the prevalence of sociodemographic, obstetric and perinatal factors were evaluated between the two groups using chi-square tests. values statistically significant were considered those with a p-value under . . the statistical procedures for analysis were performed with spss and stata. during the -month study period, , women were screened. of these, had criteria for severe maternal morbidity. among these women, only ( %) had severe respiratory disease. however, in this group with respiratory illness, symptom severity progressed more rapidly, if compared to other causes of severe morbidity ( fig. ), such as bleeding or hypertensive disorders, and may be times more lethal. among the total number of women with respiratory disease, patients with suspected h n influenza a virus infection had more severe disease ( . % mnm and . % md) than those without suspected h n influenza a virus (prevalence of mnm: . %, md: . %) ( fig. ) . about % of cases of suspected h n influenza a were tested. women who tested positive ( cases) for h n had more severe disease, with a higher prevalence of smo. figure shows the distribution of cases with severe respiratory disease, according to progression of severity during the study period, based on date of admission in participating centers. there was a higher incidence of cases in the first months considered, especially july, august and september . national guidelines and availability of vaccination during pregnancy were instituted in march/ . considering health indicators, disease was more severe among cases tested and positive for h n (table ) . mortality rate was higher than % among positive cases for a(h n )pdm . the death rate was about % in cases testing negative for h n and . % in non-tested cases. in contrast, the mortality rate was only . % in morbid disorders due to other causes. the maternal near miss to mortality ratio was . : , . : and . : , among positive, negative and non-tested groups for a(h n )pdm , respectively, compared to a value of . : for other causes of severe maternal morbidity. more than % of patients with severe respiratory disease had three diagnostic criteria for near miss: laboratory, clinical and management, while for the remaining causes of severe maternal morbidity, around % of patients only had criteria for laboratory or management diagnosis ( table ). all procedures for management of severity were associated with a worse outcome in both groups, women with severe respiratory disease and those with severe maternal morbidity due to other causes ( table ) . analysis of sociodemographic and obstetric characteristics ( there was an association to smo and non-white color, history of diabetes, low weight and substance abuse (use of psychoactive substances, including alcohol and illicit drugs), in addition to delay in care. in contrast, the group of cases due to other causes of morbidity, low maternal age, first pregnancy, history of maternal obesity and lack of a partner were identified as having lower association to smo, while hospitalization in a non-public institution, parity, history of caesarian section, drug abuse, complication occurrence at an earlier gestational age and mainly in the postpartum period, in addition to any type of delay in obstetric care, were associated with smo. concerning characteristics of pregnancy and perinatal results (table ) , the group with severe respiratory disease had a higher rate of early preterm births, between and weeks of gestation, low birthweight (< g), apgar < at five minutes of life, stillborn and the need for hospital admission/transference of the newborn infant, compared to the group with severe maternal morbidity due to other causes. neonatal death increased threefold in women with severe respiratory disease. a statistically significant difference was observed in the groups compared, when the mode of delivery and onset of labor were taken into consideration (p < . ). the number of women who did not undergo pregnancy resolution and remained pregnant during the severe morbid event was much higher in the respiratory disease group. around % were "still pregnant" compared to % in the group with severe maternal morbidity due to other causes. our study presents the burden of severe respiratory diseases among cases of severe maternal morbidity and results of the h n influenza pandemic, considering referral maternity hospitals in brazil. overall, the prevalence of respiratory disease was rare ( %). nevertheless, respiratory disease accounted for one-quarter of mnm cases and two-thirds of md. worse adverse outcomes occurred among cases of confirmed a(h n )pdm , with an impressive mnm:md ratio below one, meaning that there were more deaths than near miss cases in this group. the mortality index (mi) was over % in the h n group, compared to . % for other causes of severe maternal morbidity. the mi is known to correlate to quality of care and when the index is above %, it represents substandard care [ ] . numbers of mi over % most likely reflect that poor outcomes were not only due to the severity of disease, but also to substandard care, including delays in diagnosis and management of the considered cases. our data further confirmed that the increased risk of smo was linked to delays in health care (delays due to women/family members, health services or health professionals). considering the impact of the a(h n )pdm on maternal health [ ] , a great effort towards prevention occurred worldwide, with strong recommendations for vaccination during pregnancy and empirical antiviral *adjusted for design effect of cluster sampling therapy, as soon as possible in case of suspected disease [ ] . brazil followed these recommendations and launched a national vaccination campaign before the winter of , targeted at high-risk groups, including pregnancy. the vaccine was available in all public health facilities, at no cost for the patient and reached very high coverage (around %), most likely due to the long term experience in the national immunization program for children and due to the awareness about the severity of the disease, among health professionals and among the society [ ] . we cannot evaluate the impact of those preventive measures in our study, since we lack information on the total number of cases and specific data on individual history of vaccination or treatment, however, from our fig. , we can see on the linear traces that there is a trend towards decrease in numbers of severe cases, through time, especially after vaccination. clinical evaluation should determine treatment, in order to ensure timely and effective interventions. in our study, around % of suspected cases of h n influenza a virus were tested. in accordance with previous reports, symptoms were more severe in positive cases [ ] . the majority of cases in brazil occurred during cold weather (july, august and september), period of increased infections by respiratory viruses and influenza outbreak in the country (brazil declared a pandemic in mid-july ). over half of the reported cases of severe respiratory disease were not due to suspected influenza infection. acute respiratory failure was the cause, including a broad number of conditions, as follows: pulmonary edema, cardiac disease community-acquired pneumonia, aspiration, pulmonary embolism, asthma exacerbation or venous embolism. unfortunately we do not have detailed information on each of the mentioned causes. nevertheless, these complications include mostly indirect causes of maternal morbidity and mortality, which represent novel or preexisting health problems unrelated to pregnancy, such as cardiac disease and asthma. asthma is the most common medical condition that may worsen during pregnancy and it is often underdiagnosed and under-treated [ ] . direct causes of maternal morbidity and mortality can also lead to respiratory failure, such as systemic consequences of sepsis due to uterine infection and severe preeclampsia and eclampsia, complicated by pulmonary edema [ , ] . it is very important to understand all differential diagnosis, since timely and adequate interventions can potentially improve maternal outcome. future studies focusing on the specific differences in diagnosis and management of causes of acute respiratory failure should consider the main aspects on diagnosis and management of these conditions. pneumonia in pregnancy and postpartum, for example, is the leading cause of fatal none obstetric infection and can be caused by bacteria, virus (at risk of secondary bacterial infection), fungus and mycobacteria and the clinical features include fever, cough, dyspnea and hypoxia [ ] . another important cause of severe complications is pulmonary edema, which can be consequence of left ventricular systolic or diastolic dysfunction, or due to the use of tocolytic agent, fluid overload, severe hypertension or severe renal disease. the clinical presentation of pulmonary edema is normally dyspnea, tachypnea, tachycardia, chest pain and diffuse crackles. there can be evidence of cardiac dysfunction, specific alterations in the electrocardiogram and radiographic abnormalities [ ] . during labor or immediate postpartum, a rare and feared complication is the aspiration of gastric contents, if needed intubation for general anesthesia, due to increased intraabdominal pressure and predisposing physiological changes of pregnancy such as relaxation of the lower esophageal sphincter and delayed gastric emptying. however, in the last decades, the incidence of aspiration significantly declined, even with food intake during labor [ ] . the definitions of severe respiratory complications that are usually reported can be rather confusing and sometimes difficult to incorporate [ ] . recent onset of fever and respiratory symptoms, including cough is the clinical definition of severe acute respiratory syndrome. in the setting of an epidemic, this definition is very useful to raise awareness and ensure prompt treatment, as soon as a suspected case is identified [ ] . ards is another acronym for acute respiratory distress syndrome, a different condition that represents hypoxemic respiratory failure and bilateral radiographic opacities, without congestive heart failure. this diagnosis depends on oxygenation deficit measurements and chest imaging [ , ] . in the current study, we couldn't accurately establish any of the above conditions, since we did not collect data on clinical symptoms (fever, cough) neither obtained the results of those specific laboratory findings or imaging. the diagnosis of h n influenza was also not standardized through all hospitals included. we understand that timing of sample collection, quality of sample and laboratory procedures are key for the accurate diagnosis and unfortunately we do not have data on details regarding these procedures. another limitation was the lack of data on the use of antiviral therapy or vaccination. for cases of smo, complicated by documented organ dysfunction, ards would probably be the diagnosis of respiratory disease. nevertheless, confirmation was lacking for all cases. in addition, we do not have a control group, with no underlying complication, what would be key to access risk factors. we only have data on severe maternal morbidity cases, comparing less severe (pltc) to more severe cases (smo). factors associated with smo, included non-white color, history of diabetes, low weight and substance abuse, along with delay in care, were reported for the majority of conditions under study. substance abuse associated with increased risk of severity in cases of respiratory disease, is in agreement with previous reports [ , ] . drug-related severe respiratory complications can occur, resulting from parenchymal (infectious and non-infectious pneumonitis, aspiration-related events, hemorrhage, pulmonary edema and pneumothorax), pulmonary vascular insults (endovascular infection, hemorrhage, and vasoconstriction) or airway (bronchospasm and hemorrhage) abnormalities. diabetes was also associated with an increased risk of smo among cases complicated by severe respiratory disease. previous studies had demonstrated this fact, even in the brazilian population. diabetes is one of the main risk factors for death from h n influenza a [ ] virus infection. medical history, including known factors related to worse outcomes should be highlighted and the awareness among patients and health professionals towards targeted cases could impact in the final outcome. pregnancy characteristics and perinatal outcomes according to the main cause of severe morbidity showed that pregnancies complicated by respiratory disease present an increased rate of preterm delivery and worse perinatal outcomes. this finding had already been demonstrated [ , ] . studies have shown that vaccination during the first trimester of pregnancy can improve those outcomes and decrease stillbirth rates without increasing the risk of malformations, which is a common concern among health practitioners and pregnant women [ ] . severe respiratory disease, especially considering the influenza season, is one of the most serious causes of maternal near miss and death. increased awareness of this condition, preventive vaccination during pregnancy, early diagnosis and treatment are required to improve maternal health. abbreviations smm: severe matenal morbidity cpr: cardiopulmonary resuscitation; pr: prevalence ratio a(h n )pdm : h n influenza pandemic; ards: acute respiratory distress syndrome; ci: confidential interval; md: maternal deaths; mi: mortality index mnm: maternal near miss; pltc: potentially life-threatening conditions rr: respiratory rate; smo: severe maternal outcome; who: world health organization hospital das clınicas da universidade federal de pernambuco united nations department of economic and social affairs maternal mortality for countries, - : a systematic analysis of progress towards millennium development goal targets and strategies for ending preventable maternal mortality: consensus statement. geneva: world health organization ending preventable maternal deaths: the time is now trends in maternal mortality: to . geneva: world health organization who working group on maternal mortality and maternal morbidity classifications. maternal near miss -towards a standard tool for monitoring quality of maternal health care obstetric transition: the pathway towards ending preventable maternal deaths surveillance for emerging respiratory viruses respiratory disease in pregnancy h n influenza and pregnancy- years later clinical aspects of pandemic influenza a (h n ) virus infection maternal mortality due to pandemic influenza a h n virus in colombia risk factors for severe illness with pandemic influenza a (h n ) virus infection in china risk factors for hospitalisation and poor outcome with pandemic a/h n influenza: united kingdom first wave maternal and neonatal outcomes among pregnant women with pandemic influenza a(h n ) illness in florida, - : a population-based cohort study infection and death from influenza a h n virus in mexico: a retrospective analysis impact on pregnancies in south brazil from the influenza a (h n ) pandemic: cohort study risk factors for death from pandemic (h n ) , southern brazil from planning to practice: building the national network for the surveillance of severe maternal morbidity network for surveillance of severe maternal morbidity: a powerful national collaboration generating data on maternal health outcomes and care brazilian network for the surveillance of severe maternal morbidity study group. delays in receiving obstetric care and poor maternal outcomes: results from a national multicenter cross-sectional study incidence and predictors of severe obstetric morbidity: case control study a(h n ) vaccination in pregnant women in brazil: identifying coverage and associated factors a cross-sectional analysis of symptom severity in adults with influenza and other acute respiratory illness in the outpatient setting the pulmonary edema preeclampsia evaluation (pepe) study pulmonary edema in severe preeclampsia-eclampsia: analysis of thirty-seven consecutive cases acute pulmonary edema in pregnancy effect of food intake during labour on obstetric outcome: randomised controlled trial personal view of sars: confusing definition, confusing diagnoses clinical management and infection control of sars: lessons learned critical illness in pregnancy: part ii: common medical conditions complicating pregnancy and puerperium ards in pregnancy the large spectrum of pulmonary complications following illicit drug use: features and mechanisms acute respiratory failure from abused substances risk factors for death from influenza a(h n )pdm , state of são paulo, brazil influenza and pregnancy in the united states: before, during, and after h n effect of influenza vaccination in the first trimester of pregnancy the authors declare that they have no competing interests.ethics and consent to participate the study complied with ethical principles guiding human research described in the declaration of helsinki and was approved by the institutional review board of each local center and research coordinating center (conep, brazilian ministry of helath: / ). the informed consent form was waived, since data were obtained from hospital records after patient discharge, without any contact with research subjects. the review boards of the following institutions reviewed and approved this study: maternidade cidade nova dona nazarina daou (manaus, am), consent to publish not applicable. all the data supporting the presented findings is contained within the manuscript. the idea for the study and this specific analytical approach arose in a group discussion among all the authors. analyses were planned by lcp, mlc and jgc. the first version of the manuscript was drafted by lcp and mlc. subsequently, all remaining authors complemented with suggestions. all authors contributed to the development of the study protocol and approved the final version of the manuscript. key: cord- -a jk w authors: ding, ji-guang; sun, qing-feng; li, ke-cheng; zheng, ming-hua; miao, xiao-hui; ni, wu; hong, liang; yang, jin-xian; ruan, zhan-wei; zhou, rui-wei; zhou, hai-jiao; he, wen-fei title: retrospective analysis of nosocomial infections in the intensive care unit of a tertiary hospital in china during and date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: a jk w background: nosocomial infections are a major threat to patients in the intensive care unit (icu). limited data exist on the epidemiology of icu-acquired infections in china. this retrospective study was carried out to determine the current status of nosocomial infection in china. methods: a retrospective review of nococomial infections in the icu of a tertiary hospital in east china between and was performed. nosocomial infections were defined according to the definitions of centers for disease control and prevention. the overall patient nosocomial infection rate, the incidence density rate of nosocomial infections, the excess length of stay, and distribution of nosocomial infection sites were determined. then, pathogen and antimicrobial susceptibility profiles were further investigated. results: among patients admitted over the period of time, the overall patient nosocomial infection rate was . % or . per patient days., lower respiratory tract infections (lrti) accounted for most of the infections ( . %), followed by urinary tract infections (uti, . %), bloodstream (bsi, . %), and gastrointestinal tract (gi, . %) infections. there was no significant change in lrti, uti and bsi infection rates during the years. however, gi rate was significantly decreased from . % in to . % in . in addition, a. baumannii, c. albicans and s. epidermidis were the most frequent pathogens isolated in patients with lrtis, utis and bsis, respectively. the rates of isolates resistant to commonly used antibiotics ranged from . % to . %. conclusion: there was a high and relatively stable rate of nosocomial infections in the icu of a tertiary hospital in china through year – , with some differences in the distribution of the infection sites, and pathogen and antibiotic susceptibility profiles from those reported from the western countries. guidelines for surveillance and prevention of nosocomial infections must be implemented in order to reduce the rate. nosocomial infections, also called healthcare acquired infections or health care-associated infections, is defined by the cdc as a localized or systemic condition resulting from an adverse reaction to the presence of an infectious agent(s) or its toxin(s), without any evidence that the infection was present or incubating at the time of admission to the acute care setting [ ] . nosocomial infections have become an important public health issue worldwide. nosocomial infections may result in an excess length of stay in hospital for up to days and an increase in the costs of hospitalization. [ , ] nosocomial infections pose a critical threat to patients, especially in the high-risk departments, such as the intensive care unit (icu). [ , ] in industrialized countries, nosocomial infections occurs in - % of hospitalized patients, with the rates being up to % in icu, while the rates are - % in hospitalized patients with the rates being up to % in icu. [ ] [ ] [ ] [ ] [ ] [ ] [ ] in china, there are more than , hospitals with significant differences among each other in the size, facilities, administration, teaching, research and academic levels. a few studies reported nosocomial infections in china, but these studies were limited in small sample sizes and short period of time, publication in chinese journals, with or without english abstracts. [ ] [ ] [ ] recently, we carried out a retrospective study to determine the current status of nosocomial infections in a tertiary hospital in east china. all data on nosocomial infections between year and were retrieved and reviewed. the overall patient nosocomial infection rate, the incidence density rate of nosocomial infections, the excess length of stay, and distribution of nosocomial infection sites were determined. then, pathogen and antimicrobial susceptibility profiles were further investigated. the study was performed in the affiliated hospital of wenzhou medical university, which situates in zhejiang province, east china, with over beds and one mixed icu of beds. since , the hospital started an infection control program, including collection of data on infections acquired in the hospital. in the present study, data from january to december for patients in the icu were retrieved by the infection control team since completed raw data of the patients in icu were available only after . this retrospective study was approved by the medical ethics committee of the third affiliated hospital, wenzhou medical college. all patients admitted to the icu for more than hours were monitored for nosocomial infections, which were defined according to the american cdc. [ ] infections developed within hours of discharge from the icu were also considered to be icu-acquired unless there was an identified cause after discharge. the major nosocomial infections, including lower respiratory tract infections (lrtis), urine tract infections (utis), bloodstream infections (bsis) and gastrointestinal tract infections (gis) were defined as followings. lrtis refer to lower respiratory tract infection, other than pneumonia, i.e. bronchitis, tracheobronchitis, bronchiolitis, tracheitis, without evidence of pneumonia. bsis refers to laboratoryconfirmed bloodstream infections, utis refers to symptomatic urinary tract infections and gis refers to gastrointestinal tract (esophagus, stomach, small and large bowel, and rectum) infections excluding gastroenteritis and appendicitis. the detailed criteria to diagnose these nosocomial infections were described in the cdc documents [ ] . data on the date and site of infection, patient demographic information and device use were collected for each infection. moreover, data on the isolated pathogens and their susceptibility testing to antimicrobial agents, if available, were also collected. the overall patient nosocomial infection rate was calculated by dividing the total number of patients with nosocomial infections by the total number of patients in the icu (× ) during the defined period of time (i.e. each year). the incidence density rate of nosocomial infections was calculated by dividing the total number of nosocomial infections by the total patient days (× ) during the defined period of time. the total patients days were calculated by summating the days of each patient in the icu. in the meantime, the length of stay, which was defined as the overall days of a patient spent in the hospital including the icu and another department to which the patient was transferred from icu after stabilization of the conditions. the excess length of stay was then calculated by subtracting the average length of stay for patients without nosocomial infection from that of patients with nosocomial infections. statistical analyses were performed using spss software version . (spss inc., chicago, ill., usa). chi-square test and spearman's rank-correlation coefficients were applied where appropriate. for all analyses, a p value of less than . was considered statistically significant. from to december , medical data of patients discharged from the hospital icu were colleted. the average length of stay was . days, giving patient-days. among these patients, patients acquired a total of nosocomial infections, including patients with two infections, patients with three infections, patients with four infections and one patient with five infections ( table ) . the overall patient nosocomial infection rate was . %, ranging from . % to . % among the years. there was a significant difference in the infection rates among the years (χ = . , p = . ). the incidence density rate of nosocomial infections was . per patient days, ranging from . to . among the years. the excess length of stay was . days, ranging from . lower respiratory tract infections (lrtis) including bronchitis, tracheobronchitis and pneumonia, were the most common infections, occurring in . % of the patients, followed by urine tract infections (utis) ( . %), and bloodstream infections (bsis) ( . %). among the nosocomial infections, lrtis accounted for . %, followed by utis ( . %), bsis ( . %) and gastrointestinal tract infections ( . %). most ( . %) patients with nosocomial lrtis had received mechanical ventilation or tracheotomy before the infections, whereas . % of nosocomial utis and . % of nosocomial bsi were catheter associated (table ) . there is no significant change in lrti, uti and bsi rates during the years. the gi infection rate was significantly decreased from . % in to . % in (χ = . , p = . ), whereas nosocomial infections in other sites was increased significantly (χ = . , p = . ). the nosocomial infection rates at the surgical sites and skin and soft tissues remained under % (table ) . pathogens were isolated and identified from ( . %) of nosocomial infections, or, in ( . %) of the patients. the isolated pathogens responsible for nosocomial infections differed among the infection sites ( table ). in patients with lrtis, acinetobacter baumannii and klebsiella pneumoniae were the most frequently isolated pathogens, followed by pseudomonas aeruginosa and staphylococcus aureus, accounting for more than half of the lrti related pathogen population. in patients with utis, the fungi, especially candida albicans, were the most com-mon pathogens, followed by escherichia coli. staphylococcus epidermidis, e. coli, and s. aureus were the first three most common pathogens for bsis. in addition, a. baumannii was commonly isolated in utis and bsis (table ) . data on susceptibility testing were available for isolates, including isolates of e. coli, isolates of s. aureus, and isolates of p. aeruginosa. overall, . %, . %, . % and . % of e. coli isolates were resistant to trimethoprim/sulfamethoxazole (tmp/smx) and ciprofloxacin, cefotaxime, and amoxicillin/clavulanic acid, respectively. all s. aureus isolates were sensitive to vancomycin, but . %, . %, . % and . % of isolates were resistant to nitrofurantoin, tmp/smx, rifampin and ciprofloxacin, respectively. in addition, . %, . % and . % of p. aeruginosa were resistant to tmp/smx, ciprofloxacin and levofloxacin, respectively [see additional file ]. all patients with nosocomial infections were treated with empirical antimicrobial therapies or according to the antimicrobial susceptibility test results, when available. the in the present study, the overall patient nosocomial infection rate in the icu was . % during and , which was higher than in the icus in many industrialized countries where the rates ranging from . % to . %, [ ] [ ] [ ] and even higher than the rate ( . %) observed in icus of developing countries. [ ] however, the rate is comparable with those reported in some latin american countries such as argentina and brazil [ ] , and slightly lower than that reported in india. [ ] over the years, the lowest rate was reported in ( . %), and the highest was reported in ( . %). one plausible explanation is that in the early of the country was suffering from the outbreak of highly infectious pneumonia, namely severe acute respiratory syndrome (sars). due to the massive campaign to prevent the spread of sars, nosocomial infections were indirectly reduced. being fatigued from the campaign over the previous year, disinfection and sterilization procedures might be loosen in , explaining the moderate rebound in . however, the incidence density rate of nosocomial infections was the lowest in , due to considerably longer stay of some patients in the icu ( table ). the average length of stay in the hospital varied from . to . between and , with the overall average being . days, which is generally in agreement with those ( . - . days) reported in european and the united states [ ] [ ] [ ] , but much less than that reported in taiwan [ ] . however, the it must be also mentioned that sars outbreak had some impact on the overall length of stay. although the average stay in the icu was only . days, the shortest among the years, the average stay in the hospital was the longest for both patients with and those without nosocomial infections due to the isolation policy imposed in the special period of time. the distribution of nosocomial infections in the present study differed from that reported in the united states. we found that the lrtis were the most common infections in the icu, accounting for . % of overall infections, whereas utis was the most frequently reported infections in the icus in the united states, with the rate of %, followed by pneumonia of %. [ ] the proportions of utis and bsis in the present study were relatively lower than the data reported in the united states ( . % vs. % and . % vs. %, respectively). [ ] although data from europe revealed same three most common infection sites as the present study did, the absolute proportion of lrtis was %, [ ] which was lower than the rate in the present study. the common reasons proposed by studies in many western countries have suggested that nosocomial lrtis are mainly due to mechanical ventilation. [ , ] in china, air pollution, high density of population, and improper health habits such as smoking may also account for the high rate of lrtis. the rate of lrtis slightly, but insignificantly, decreased from to . there was no change in utis and bsis rates during the years. notably, the gi rate significantly and stably decreased every year, suggesting an improvement in the environment and food sanitary in the region. since the lower respiratory tract and the urinary tract were the first two sites that nosocomial infections frequently occurred in the icu, constituting more than % of all nosocomial infections in and , more efforts were later made to control these two kinds of infections, leading to decreased rates of lower respiratory tract and the urinary tract infections, and correspondingly increased rates of nosocomial infections at other sites. the present study showed three quarters of lrti patients received mechanical ventilation or tracheotomy, more than half of nosocomial uti and bsi cases were catheter associated. these findings are consistent with previous studies, [ , , , , ] and indicate that the nosocomial infections are often associated with the use of invasive device. therefore, to effectively reduce nosocomial infections, the use of invasive device should be minimized and specific disinfection precautions taken during the device application. in the present study, pathogens were isolated from . % of overall nosocomial infections or . % of all patients with nosocomial infections. similar to the us report, gram-negative bacteria accounted for . % of the lrtis in the present study, but the most frequently pathogens were a. baumannii and k. pneurnoniae in the present study whereas p. aeruginosa and s. aureus were the most common pathogens in the us report. [ ] consistent with the us report, fungi was the most frequently pathogen for nosocomial utis; candida accounted for . % of utis in the present study, suggesting a relatively narrow profile of pathogens in nosocomial utis. s. epidermidis was the most common pathogen for bsis in both the present study and the us report; however, e. coli, instead of enterococci, was the second common pathogen in the present study. [ ] e. coli was the most common bacterial cause of nosocomial utis, and also frequently found in bsis and lrtis. it has been shown that the activity of beta-lactam antibiotics against e. coli is greatly reduced as a result of beta-lactamase production, but is restored by the addition of clavulanic acid. [ ] in the present study, only . % of e. coli isolates were sensitive to the formula amoxicillin combined with clavulanic acid, while . % of e. coli isolates exhibited susceptibility to the combination in an uk study. [ ] a high rate of resistance to tmp/smx ( . %) was observed in these e. coli isolates, in contrast to the rate of % reported in uk [ ] . in addition, there was a high proportion ( . %) of e. coli isolates resistant to ciprofloxacin, whereas the rate was less than % in uk and the united states. [ , ] these findings indicate that treatment with these antimicrobial agents for nosocomial infections caused by e. coli in china is likely to result in clinical failure in a substantial proportion of patients. we also found that a considerable number of p. aeruginosa isolates were resistant to fluoroquinolones, from . % to ciprofloxacin to . % to levofloxacin, which is comparable with the fluoroquinolone-resistant rates ( % to %) reported for patients in icus of eight developing countries, [ ] but much higher than that ( %) reported in the united states. [ ] all s. aureus isolates in the present study were sensitive to vancomycin, which was similar to the observation by tsuji et al in japan. [ ] in addition, we observed relatively low resistant rates to nitrofurantoin ( . %) and tmp/smx ( . %), which renders these antimicrobial agents suitable for empirical treatment for s. aureus infections. in china, the guidelines for surveillance and prevention of nosocomial infections was established in , and modified in . [ , ] however, surveillance systems and control measures for nosocomial infections described in the guidelines were not completely implemented and executed in all hospitals, due to the imbalanced development and health care resources within the countries, and less attention to nosocomial infections in some hospitals. therefore, it is believed that the nosocomial infection rates must be higher in some rural hospitals or even nontertiary hospitals. in addition, due to empirical use or abuse of antibiotics, the proportion of antibiotic resistant pathogens for nosocomial infections in many lower level hospitals would also be higher than that reported in the present study. it is noticed that although the mortality rates in patients nosocomial infections were between %- %, there was no mortality directly caused by nosocomial infections. the major reasons for this observation would be the fact that refractory nosocomial infections are relatively less encountered based on our susceptibility testing, which showed that most pathogens were sensitive to many most commonly used antibiotics, indicating that they can be effectively controlled. moreover, zhejiang is one of richest provinces in china where medical and healthcare systems are relatively well established and antiinfectious therapies are not a big problems. finally, it should be emphasized that our hospital is an tertiary infectious hospital with experience, methodologies and facilities to combat against various infections including nosocomial infections. the present study has some limitations, due to the retrospective nature. first, data on risk factors, except for the use of the medical device, that are potentially associated with nosocomial infections were not available. these factors may include the primary diseases for admission to the icu, patient resting posture (e.g. semirecumbent or supine body position), continuous prophylactic use of anti-peptic ulcer drugs, utilization of the alcohol-based handrubs and oral care, which need to be taken into consideration in the prospective studies. second, the data on the identification and isolation of the pathogens and their susceptibility were available only for half of the nosocomial infections. it would produce more accurate data if these numbers were increased. finally, the data on the clinical consequences were not available for most cases, making it impossible to compare the clinical outcomes between patients with and those without nosocomial infections. however, the present study was able to show that the length of stay in the hospital was significantly increased in patients with nosocomial infections, compared with those without the infections. in conclusion, there was a high and relatively stable rate of nosocomial infections in the icu of a tertiary hospital in china through year - , with some differences in the distribution of the infection sites, and pathogen and antibiotic susceptibility profiles from those reported in the western countries. the guidelines for surveillance and prevention of nosocomial infections must be implemented national wide in order to reduce the rate. cdc definitions for nosocomial infections effect of intensive care unit nosocomial pneumonia on duration of stay and mortality prolongation of hospital stay and extra costs due to ventilator-associated pneumonia in an intensive care unit nosocomial infection rates in adult and pediatric intensive care units in the united states prevalence and risk factors for nosocomial lower respiratory tract infections in german hospitals selected aspects of the socioeconomic impact of nosocomial infections: morbidity, mortality, cost, and prevention the prevalence of nosocomial infection in intensive care units in europe. results of the european prevalence of infection in intensive care (epic) study. epic international advisory committee mortality attributable to nosocomial infections in the icu national nosocomial infections surveillance (nnis) system report, data summary from nosocomial infections in medical-surgical intensive care units in argentina: attributable mortality and length of stay prevalence rate of nosocomial infection in a general hospital surveillance of risk factors for nosocomial infection in patients in intensive care units study on the changing trends in national nosocomial infection transaction investigation results nosocomial infections in medical intensive care units in the united states nosocomial infections: prospective survey of incidence in five french intensive care units nosocomial infections in a neurosurgery intensive care unit device-associated nosocomial infections in intensive care units of developing countries epidemiology, risk factors and outcome of nosocomial infections in a respiratory intensive care unit in north india the estimation of the cost of nosocomial infection in an intensive care unit the impact of adverse patient occurrences on hospital costs in the pediatric intensive care unit excess length of stay, charges, and mortality attributable to medical injuries during hospitalization impact of nosocomial infection on cost of illness and length of stay in intensive care units national healthcare safety network (nhsn) report, data summary for amoxycillin/clavulanic acid: a review of its antibacterial activity, pharmacokinetics and therapeutic use a uk multicentre study of the antimicrobial susceptibility of bacterial pathogens causing urinary tract infection antimicrobial resistance in community and nosocomial escherichia coli urinary tract isolates identification and pretherapy susceptibility of pathogens in patients with complicated urinary tract infection or acute pyelonephritis enrolled in a clinical study in the united states from november gram-negative antibiotic resistance: there is a price to pay communityand health care-associated methicillin-resistant staphylococcus aureus: a comparison of molecular epidemiology and antimicrobial activities of various agents anonymous: guidelines for nosocomial infections control and prevention (draft) anonymous: guidelines for nosocomial infections control and prevention (draft) the authors acknowledge that the manuscript was edited by a professional company, medjaden biomedical services. the authors declare that they have no competing interests. j-gd, q-fs and k-cl were the principal investigators who designed and conducted the study, analyzed the data, performed literature research and prepared the manuscript. m-hz, x-hm and wn participated in the design of the study, contributed to the data analysis and made constructive comments on the manuscript. lh, j-xy, z-wr, r-wz, h-jz and w-fh participated in the design of the study, collected the all required original data, and generated results and tables that were the basis of the manuscript. all authors have read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /pre pub key: cord- -zkrlm ds authors: cui, wei; zhao, hongwen; lu, xu; wen, ying; zhou, ying; deng, baocheng; wang, yu; wang, wen; kang, jian; liu, pei title: factors associated with death in hospitalized pneumonia patients with h n influenza in shenyang, china date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: zkrlm ds background: during the spring of , a pandemic influenza a (h n ) virus emerged and spread globally. we describe the clinical characteristics and factors associated with the death of patients who were hospitalized with h n influenza pneumonia in shenyang, china, from november to december . methods: we carried out a retrospective chart review of patients who were hospitalized with pneumonia and confirmed to have h n virus infection by a real time rt-pcr assay of respiratory specimens. results: of the patients we studied, ( %) were admitted to an intensive care unit and ( . %) died. the median age of patients was years (range, - ), and only one patient was over years of age. the male to female ratio was . : ( : ). of the patients, ( %) had at least one underlying medical condition, ( %) had a cigarette index ≥ and ( %) were obese. all patients underwent chest radiography on admission and the findings were consistent with pneumonia in all cases. all patients were treated with oseltamivir and treatment was initiated at a median time of seven days after the onset of illness. the laboratory test results indicated lymphopenia, hypoproteinemia and elevated lactic dehydrogenase and c reactive protein levels. of the patients, ( %) showed a reduction in cd t cell counts. of the patients who survived, ( %) had lymphopenia and recovered from this condition after five days. of the patients who died, nine ( %) had lymphopenia and only two patients recovered from this condition after five days. obesity and recovery from lymphopenia after five days were factors associated with death, as determined by multivariate logistic-regression analysis (obesity, odds ratio = . ; lymphocytopenia reversion, odds ration = . ). conclusions: during the evaluation period in shenyang, china, h n influenza caused severe illness requiring hospitalization in patients, ( . %) of which died. many of these patients were considered healthy adults and few were elderly ( years or older). obesity and lymphopenia, which was not restored after five days of treatment, were factors associated with poor outcomes of h n influenza infection. in early april , cases of human infection with pandemic influenza a (h n ) virus were identified in the united states [ , ] and mexico [ ] , and the virus then spread rapidly to other regions of the world [ , ] . on december , , more than countries and overseas territories or communities had reported laboratory confirmed cases of pandemic influenza a (h n ), and at least , deaths had occurred [ ] . the first case of pandemic influenza a (h n ) virus infection in china was documented on may . from may to november of , the number of reported cases in china was , [china centre for disease control, cdc]. the majority of the early cases reported mild, influenza-like illnesses [ ] [ ] [ ] [ ] [ ] , but more severe infections have been reported as the pandemic spreads and the number of infected individuals increases. the greatest number of cases of h n influenza infection resulting in severe clinical presentations and death have been reported in mexico and other western countries. the fatality rate of hospitalized patients with critical illness due to h n influenza was about % [ ] [ ] [ ] in united states and mexico. the clinical features included fever and respiratory symptoms and death most commonly resulted from severe acute respiratory distress syndrome (ards) and refractory hypoxemia [ , [ ] [ ] [ ] [ ] . obesity, underlying health conditions and delayed neuraminidase inhibitor treatment were the major risk factors for a poor outcome of infection [ , ] . however, the clinical outcomes and risk factors associated with h n influenza infection in china remain to be determined and variables that could help clinicians to identify patients at high risk of infection would be valuable. therefore, this report summarizes the clinical manifestations, clinical outcomes and the risk factors associated with death in hospitalized pneumonia patients associated with h n influenza infections in shenyang, china, during november to december . we carried out a retrospective chart review of patients who were hospitalized with pneumonia and h n influenza infection in the first affiliated hospital of china medical university from november , , to december , . during the h n influenza epidemic, patients in clinics or emergency departments who presented with an influenza-like illness were tested for h n influenza virus, and only those patients whose illness was complicated with pneumonia were admitted to our department. patients with mild symptoms were isolated for treatment at home. therefore, in total patients were treated in our department between november th and december st . we retrospectively studied these cases without any selection. the first affiliated hospital of china medical university is a tertiary care center that includes obstetric services, pediatric wards and oncology wards, and treats immunosuppressed patients and hiv-infected patients. it is also a tertiary referral center for adult patients infected with h n influenza, as appointed by the national government. however, this hospital is not a tertiary referral center for children and pregnant women infected with h n influenza, so no children and pregnant women were involved in our study. all patients were confirmed positive for h n influenza a by a real-time reverse-transcriptase-polymerase-chain-reaction assay on respiratory specimens carried out at the cdc. all tests used standard cdc-based primers. all patients underwent chest radiography on admission. the study was approved by the ethics committee of china medical university. medical-chart abstractions were performed by physicians from the infectious diseases department. they used a standardized form that included demographic data, seasonal influenza vaccination history for the previous year, smoking status, underlying medical conditions, clinical signs and symptoms, selected laboratory tests including c reactive protein (crp), white blood cell classification and count, lactic dehydrogenase (ldh), creatine kinase (ck), glutamic-oxaloacetic transaminase (ast), glutamic alanine aminotransferase (alt), albumin (alb), cd , cd and cd t cell counts, blood gas analyses, blood or sputum cultures, radiographic findings, intervals between symptom onset and initiation of oseltamivir therapy, treatment course and length of stay. the thresholds for each laboratory test are listed in annex and any elevation or reduction according to the threshold of normal was defined as an abnormality. the body mass index (bmi) (the weight in kilograms divided by the square of the height in meters) of each patient was calculated. patients were defined as overweight if their bmi was to . and obese if their bmi ≥ according to the criterion established by the working group on obesity in china (wgoc) in . lymphocytopenia was defined as an absolute lymphocyte count ≤ cells/μl. a cigarette index abnormality was defined as ≥ . blood gas analyses were tested by nova biomedical stat profile, and the oxygenation index (pao /fio ) was calculated and abnormality was defined as values ≤ . blood cultures were tested using the bactectm system (becton and dickinson company). continuous variables were summarized as the mean values (±sd). for categorical variables, the percentage of patients in each category was calculated. clinical characteristics were compared between subgroups of survivors and deceased and between patients who were admitted to an intensive care unit (icu) and those who were not, with the use of a non-parametric mann-whitney u test, chi-square test or fisher's exact test, as appropriate. a p value of less than . was considered to indicate statistical significance. we performed bivariate analysis to compare the clinical characteristics of patients who survived with those of patients who died. we used multivariate logistic-regression models to further investigate associations between illness and mortality. data with a p value < . was entered into the multivariate logistic regression model and data with a p value < . was kept in the model. all analyses were carried out with the use of spss software for windows (release . ) (from http:// www.bizinsight.com.cn). . symptoms at presentation included a fever, cough, difficulty breathing, hemosputum, diarrhea and myalgia. all patients presented with a fever. a cough was reported in ( %) patients, breathing difficulties were reported in ( %) patients, hemosputum in ( %) patients, diarrhea in ( %) patients and myalgia in ( %) patients. the median duration of symptoms before hospitalization was seven days (range, - days). five patients showed baseline chronic abnormalities in the results of laboratory tests as a result of their underlying medical conditions. three patients showed a reduced level of serum alb of , and . g/l, one patient showed a mild elevation of alt at u/l and one deceased patient showed an elevated level of cr at μmol/l. the majority of the laboratory test data was acquired within h of hospital admission, including crp, ldh, ck, ast, alt, alb, cd , cd and cd t cell count and blood gas analyses. as for white blood cell classification and counts, there were patients whose samples were taken in the clinics or emergency rooms of our hospital and nine patients whose samples were taken in other hospitals. the median interval from the onset of illness to the time of sample collection was five days (range, - ). ten patients ultimately died and the median interval from the time of sample collection to death was seven days (range, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the lymphocyte counts for all patients were monitored every other day until their lymphocyte counts recovered to normal levels. laboratory tests abnormalities included lymphopenia ( %), elevated serum ldh ( %), ck ( %), ast ( %), alt ( %) and crp ( %). all patients were tested for hiv infection and were negative. of the patients, ( %) had reduced cd t cell counts. blood cultures were performed for patients who displayed a high fever above . °c for at least three days or patients who had a repeated fever. sputum cultures were also performed for patients who showed symptoms of expectoration especially in cases where the sputum was yellowish and purulent. all but three patients had received antibiotics prior to sample collection. five of patients had positive blood cultures: two of which were acinetobacter baumannii, one was methicillin-resistant staphylococcus cohnii, one was methicillin-resistant staphylococcus saprophyticus and one was methicillinresistant staphylococcus aureus. nine of patients had positive sputum cultures: six of which were a. baumannii, two were pseudomonas aeruginosa and one was methicillin-resistant s. aureus. all patients who underwent chest radiography on admission displayed clinical signs that were consistent with pneumonia. radiographic findings included bilateral infiltrates (in patients), an infiltrate limited to one lobe and multi-lobe infiltrates limited to one lung. of patients who underwent blood gas analysis, ( %) had an oxygenation index below . all patients received oseltamivir treatment. the median time from the onset of illness to the initiation of antiviral therapy was seven days (range, - ). of patients, ( %) were treated with antiviral therapy within hours of hospital admission, and ( %) were treated after hours. all patients received antibiotic therapy, with % of the patients receiving more than one antibiotic. the median value for the number of antibiotics administered was two (range, - ) and the median duration of antibiotic treatment was seven days. commonly used antibiotics included cefminox and levofloxacin. of the patients, ( %) received corticosteroids by means of an injection. the average dosage of methylprednisolone was mg·kg - ·d - and the median time for initiation of methylprednisolone in hospital was day . of the patients, ( %) received extrasin alpha . of the patients we evaluated, ( %) were admitted to an icu and of these died. there were no significant differences in the average age, cigarette index ≥ and interval time from the onset of illness to the initiation of antiviral therapy between patients who were admitted to an icu and patients who were not (table ) . patients who were admitted to an icu were more likely than patients who were not admitted to an icu to have an underlying medical condition and a bmi ≥ (table ) . of the patients who were admitted to an icu, ( %) had at least one underlying medical condition, including tuberculosis ( ), asthma ( ), chronic obstructive pulmonary disease (copd) ( ), chronic renal failure ( ), chronic hepatitis b virus infection ( ), fatty liver disease ( ), hypertension ( ), congenital heart disease ( ), rheumatic heart disease ( ), guillain-barre syndrome ( ) and pituitary adenoma ( ) . seven of the ( %) patients who were not admitted to an icu had an underlying medical condition, including tuberculosis ( ), copd ( ), liver cirrosis ( ), diabetes ( ), hypertension ( ), nephrotic syndrome ( ) and a kidney transplant ( ) . laboratory test data showed that the levels of serum ldh and crp were higher in patients who were admitted to an icu than those who were not admitted (p < . ) ( table ) . patients who were admitted to an icu also showed a more significant reduction in serum alb, lymphocyte counts, cd , cd and cd t cell counts, compared with patients who were not admitted to an icu (p < . ) ( table ). in a multivariable model that included bmi ≥ , medical conditions, ldh, crp and cd t cell count, no variable was significantly associated with admission to an icu. of the patients who were admitted to an icu, required mechanical ventilation, had acute respiratory distress syndrome (ards), and had a clinical diagnosis of sepsis. all patients received antiviral drugs, antibiotics, corticosteroids and extrasin alpha . the median time from the onset of illness to the initiation of antiviral therapy was seven days (range, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . of the hospitalized patients, ( . %) were discharged and ( . %) died. the median time from the onset of illness to death was days (range, - ). the median time from hospital admission to death was six days (range, [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the median length of hospital stay for survivors was days (range, . there were no significant differences in median age, cigarette index ≥ and interval time from the onset of illness to the initiation of antiviral therapy, between patients who died and patients who survived (table ) . patients who died were more likely than patients who survived to have underlying medical conditions and a bmi value ≥ (table ) . of the patients who died, six had at least one underlying medical condition, including tuberculosis ( ), chronic renal failure ( ), chronic hepatitis b virus infection ( ), asthma ( ), copd ( ), hypertension ( ) and congenital heart disease ( ). of the patients who survived, had an underlying medical condition, including tuberculosis ( ), hypertension ( ), rheumatic heart disease ( ), guillain-barre syndrome ( ), fatty liver disease ( ), pituitary adenoma ( ), copd ( ), liver cirrosis ( ), diabetes ( ), nephrotic syndrome ( ) and a kidney transplant ( ). laboratory test results were compared between patients who survived and those who died. no differences were found in the levels of serum ck, ast and alt between patients who survived and those who died. however, patients who died were more likely than patients who survived to have elevated levels of serum ldh and crp and reduced levels of alb and cd , cd and cd t cell counts (p < . ) ( table ). in addition, we noticed that patients suffered from lymphocytopenia during the early stage of illness. among these patients, recovered and nine died. of the surviving patients, ( %) recovered from lymphocytopenia within five days, while just two of the patients who later died recovered from lymphocytopenia within five days (table ) . in a multivariate model that included bmi ≥ , medical conditions, crp, cd t cell counts and lymphocytopenia reversion, the variables that were significantly associated with death were bmi ≥ and delayed lymphocytopenia recovery ( table ). all patients who died had received [ , , , ] . in our study, almost % of the hospitalizations due to h n influenza infection involved individuals who were between the ages of and years. the male to female ratio was . : . these age and sex distributions were different from those reported nationally for the normal population in china (male to female ratio was . : ; age distributions were: - years, . %; years and over, . %; years and over, . %). we concluded that severe illness resulting from h n virus infection was more likely among male adults in shenyang, which was consistent with other reports [ , [ ] [ ] [ ] [ ] [ ] . persons aged years or older showed a low incidence of h n influenza infection, this may be due to "protection" by preexisting immunity resulting from previous exposure to h n influenza infection, along with reduced outdoor activity by these individuals because of the fear of h n infection. a recent study demonstrated that persons aged years or older who were hospitalized with pandemic influenza a (h n ) infection were among those most likely to die, despite having lower hospitalization rates [ ] . however, our findings did not support this conclusion, possibly due to the small sample size used in our study. nevertheless, it is appropriate that clinicians should closely monitor elderly patients with pandemic h n influenza infection and treat them accordingly. in our study, almost % of patients had pre-existing medical conditions, of which chronic pulmonary diseases, diabetes and hypertension were the most common diseases, as found in other studies [ , , ] . the patients included in our study who died had a higher rate of pre-existing diseases. asthma is also considered a high risk factor for h n influenza infection, however, our findings did not confirm this as only one patient in our study had asthma as a pre-existing condition. this discrepancy might be caused by the lower morbidity of asthma in china (approximately %) compared with western counties (approximately - %). copd and heart disease are also considered high risk factors for h n influenza infection, and in our study about two thirds of the patients with pre-existing medical conditions suffered from copd or heart disease. thus, it is appropriate that physicians should pay close attention to patients with copd or chronic heart disease during the h n influenza epidemic. in our study, % of patients were obese compared with % of adults in the normal population in china, which indicated severe illness from h n virus infection was more likely among obese individuals, as reported in other studies [ , , , , ] . almost % of patients who died from h n influenza infection were obese and using multivariate logistic-regression models, obesity was found to be a factor associated with death from h n influenza in our study, despite the wide % confidence interval (ci) for the odds ratio (or) for bmi due to the limitations of using a small sample size. the clinical features of patients who were hospitalized with h n influenza infection included fever, cough, myalgia and dyspnea, which were generally similar to other reports. whereas the incidence of gastrointestinal symptoms such as nausea, vomiting, and diarrhea was much lower than previously reported [ , [ ] [ ] [ ] [ ] , ] . the results of laboratory tests indicated lymphopenia, hypoproteinemia, elevated ldh and crp levels, which were consistent with other reports [ , , ] . abnormalities in the laboratory test results were more significant in patients who were admitted to an icu and/or died than in patients who were not admitted to an icu, but these abnormalities were not predictive factors in icu admission or death. our results were not consistent with the study in taiwan which found initial lymphocyte count less /microl was associated with the development of respiratory failure [ ] . although lymphopenia was not a risk factor for death in our study, we found lymphopenia was restored after about five days in most surviving patients, whereas this was not observed in patients who ultimately died. the multivariate logistic-regression model results indicated that lymphopenia that did not resolve after five days was a risk factor for death. furthermore, we determined the cd and cd t cell counts in most of the patients included in our study. the results indicated a reduction in cd and cd t cell counts in about half of the patients during the early stage of h n virus infection, which was similar to previous reports in china [ , ] . these findings indicated that lymphopenia was mostly caused by t cell reduction, in particular a reduction in cd t cells. we were unable to determine whether the low immunity was pre-existing or caused by h n infection because we did not know the basic values for each patient. during the severe acute respiratory syndromes (sars) epidemic, lymphopenia was considered to be caused by the virus infection but further studies are needed to investigate the precise host immune response to h n influenza virus. these results suggest the physicians should pay close attention to patients who are infected with h n influenza virus, who are also obese or have experienced long-term lymphopenia. in the - influenza a pandemic, most deaths were attributed to concurrent bacterial infection [ ] . a report into pandemic h n influenza also indicated that % of the patients displayed bacterial coinfection, which might have contributed to the death rate in the current pandemic [ ] . in our study, only % patients had positive blood or sputum cultures, and most of these pathogens could potentially be responsible ventilatorassociated pneumonia. the majority of bacterial coinfections were drug-resistant bacteria such as a. baumannii and methicillin-resistant s. aureus, whereas non drug-resistant bacteria were found predominantly in other reports [ , ] . this result may reflect the abuse of antibiotics in china, and chinese physicians should take measures to ensure protection against respiratory machine-related pneumonia, especially involving drugresistant bacteria. to achieve this, physicians, nurses and patients should obey the rules of segregation and sterilization strictly and pay attention to bacteria contamination and the rational use of antibiotics. few bacterial coinfections were detected in patients who did not have mechanical ventilation, which was consistent with most previous studies [ , ] . however, bacterial diagnostic tests were not performed for all patients, especially those who were not admitted to an icu, and most patients received antibiotics close to the time of culture collection, which could have reduced the diagnostic sensitivity. although antiviral therapy is most beneficial when treatment is initiated within hours after the onset of illness [ ] , a prospective cohort study of oseltamivir therapy in patients hospitalized with influenza infection indicated a reduction in mortality, even when such therapy was initiated more than hours after illness onset [ ] . recent data from thailand also showed that oseltamivir therapy was associated with survival in hospitalized patients with influenza pneumonia [ ] . under an emergency use authorization, the fda recently approved oseltamivir therapy for h n infection even if it is initiated more than hours after the onset of illness and also approved its use in children under the age of one year [ ] . in our study, antiviral drugs were administered to all patients, but such therapy was not initiated within hours of the onset of illness in all patients and there was no difference between surviving patients and those who died in the median number of days from the onset of illness to oseltamivir initiation. therefore, we were unable to conclude whether or not antiviral therapy in critically ill patients led to better clinical outcomes. no patients undertook the test of the isolated h n influenza a strains for oseltamivir resistance in our study, so whether oseltamivir resistance affected the outcomes of patients with h n infection was unclear. delayed initiation of antiviral therapy may have contributed to an increased severity of illness in our study. our study has several limitations. the patients we evaluated represented % of the total hospitalizations in shenyang for h n infection that were reported to the cdc during the surveillance period that ended in december, . no children or pregnant women were included in our study. participation in the study was voluntary and was therefore subject to reporting bias. we evaluated only patients with confirmed h n infection, so the group may not be representative of all hospitalized patients as some may have gone undetected. all diagnostic testing was clinically driven, and tests were not obtained in a standardized fashion. finally, despite the use of a standardized data-collection form, not all of the required information was collected for all of the patients and the sample size was small. during the evaluation period in shenyang, h n influenza caused severe illness requiring hospitalization. thirty percent of the patients need an intensive care unit and fifteen percent died. obesity and lymphopenia, which was not restored after five days of treatment, were factors associated with poor outcomes of h n influenza infection. early identification of pneumoniasusceptible patients at high risk of h n influenza infection may aid clinicians in carrying out effective clinical management of this disease. cdc: centre for disease control; crp: c reactive protein; ldh: lactic dehydrogenase; ck: creatine kinase; ast: glutamic-oxaloacetic transaminase; alt: glutamic ala- swine-origin influenza a (h n ) virus infections in a school swine influenza a (h n ) infection in two children --southern california outbreak of swine-origin influenza a (h n ) virus infection --mexico update: infections with a swine-origin influenza a (h n ) virus --united states and other countries an outbreak of swine-origin influenza a(h n ) virus with evidence for human-to-human transmission h n flu: international situation update national influenza a pandemic (h n ) clinical investigation group of china: clinical features of the initial cases of pandemic influenza 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clinical and organizational aspects: the experience in chile impact of pandemic h n influenza virus on critical care in australia: a single centre case series predictors and outcomes of respiratory failure among hospitalized pneumonia patients with h n influenza in taiwan clinical analysis of cases with the novel influenza a (h n ) virus infection in shanghai streptococcus pneumoniae coinfection is correlated with the severity of h n pandemic influenza bacterial coinfections in lung tissue specimens from fatal cases of pandemic influenza a (h n ) -united states updated interim recommendations for the use of antiviral medications in the treatment and prevention of influenza for the - season atlanta toronto invasive bacterial diseases network: antiviral therapy and outcomes of influenza requiring hospitalization in ontario severe human influenza infections in thailand: oseltamivir treatment and risk factors for fatal outcome we thank the staff members of the centre for disease control of liaoning province (particularly wei zhao and ling sun) for their assistance in virology laboratory analyses for h n influenza a. we thank the resident physician for their efforts on treatments of patients. we thank hongbo liu for her assistance in statistic analysis. we thank anonymous reviewers for their constructive comments on this manuscript. the authors wish to acknowledge financial support from the following sources: pei liu and hongwen zhao were supported by the natural science foundation of liaoning province. wei cui was supported by the china medical university. the authors declare that they have no competing interests.authors' contributions hz, wc, xl, yw, yz, bd, yw, ww and jk have made substantial contributions to acquisition of clinical data. pl and wc have made contribution to conception and design. wc has made contribution to analysis and interpretation of data. all authors read and approved the final manuscript. the pre-publication history for this paper can be accessed here: http://www.biomedcentral.com/ - / / /prepub key: cord- -aw rc authors: Österdahl, marc f.; lee, karla a.; lochlainn, mary ni; wilson, stuart; douthwaite, sam; horsfall, rachel; sheedy, alyce; goldenberg, simon d.; stanley, christopher j.; spector, tim d.; steves, claire j. title: detecting sars-cov- at point of care: preliminary data comparing loop-mediated isothermal amplification (lamp) to polymerase chain reaction (pcr) date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: aw rc background: a cost effective and efficient diagnostic tool for covid- as near to the point of care (poc) as possible would be a game changer in the current pandemic. we tested reverse transcription loop mediated isothermal amplification (rt-lamp), a method which can produce results in under min, alongside standard methods in a real-life clinical setting. methods: this prospective service improvement project piloted an rt-lamp method on nasal and pharyngeal swabs on residents of a high dependency care home, with two index covid- cases, and compared it to multiplex tandem reverse transcription polymerase chain reaction (rt-pcr). we recorded vital signs of patients to correlate clinical and laboratory information and calculated the sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) of a single swab using rt-lamp compared with the current standard, rt-pcr, as per standards for reporting diagnostic accuracy studies (stard) guidelines. results: the novel method accurately detected / rt-pcr positive cases and identified a further positive cases. eight further cases were negative using both methods. using repeated rt-pcr as a “gold standard”, the sensitivity and specificity of a single novel test were and % respectively. ppv was % and npv was %. incorporating retesting of low signal rt-lamp positives improved the specificity to %. we also speculate that hypothermia may be a significant early clinical sign of covid- . conclusions: rt-lamp testing for sars-cov- was found to be promising, fast and to work equivalently to rt-pcr methods. rt-lamp has the potential to transform covid- detection, bringing rapid and accurate testing to the poc. rt-lamp could be deployed in mobile community testing units, care homes and hospitals to detect disease early and prevent spread. current diagnosis of covid- relies on centralised laboratory-based rt-pcr (reverse transcription polymerase chain reaction) testing. although pcr provides a relatively rapid result, it is limited by the bottlenecks of transportation to the laboratory and the requirement to batch samples in a large run. moreover, alternative technologies to rt-pcr requiring different reagents, and dry swabs would reduce the strain on laboratory and clinical supplies, allowing greater numbers of tests to be performed [ ] . it is abundantly clear that urgent research is needed to enable health services globally to plan resources and this research must both move rapidly from bench to bedside and be scalable and rapidly available. in light of this urgency, we present a preliminary evaluation of a novel, quick test for covid- that can be implemented at the point of need. point-of-care (poc) testing may be critical to enable rapid detection of disease when an outbreak is suspected. this is particularly important in community settings like care homes, where multiple vulnerable patients reside together, and covid- can spread quickly if not identified early [ ] . older residents are at higher risk of mortality from covid- [ ] , and care homes have reported significant outbreaks both in the united kingdom (uk) and internationally [ ] . however, they have limited access to laboratory diagnostic services. a rapid, poc test would allow early case identification, and implementation of increased infection control measures to prevent further spread to residents and staff, as recommended by the world health organization (who) [ ] and british geriatric society [ ] . between th february and th april , six independent groups have posted preprints of submitted manuscripts evaluating novel rt-lamp testing methods against rt-pcr as gold standard (table ) . since then, a number [ ] of other groups have published high-quality studies demonstrating that rt-lamp has the potential to replace rt-pcr as a means for detecting sars-cov- (severe acute respiratory syndrome coronavirus ) within rna extracted from nose -throat swabs and endotracheal secretions/bronchoalveolar lavage fluid [ , , ] . to this end, we used a combination of magnetic bead viral genome capture and optimised rt-lamp (reverse transcriptase loop-mediated isothermal amplification) for amplification and detection of the sars-cov- genome; targeting the orf ab gene, to show proof of principle. the assay runs at °c allowing simpler and cheaper instrumentation to be used with rapid results (< min from swab to result). it can be used without a hospital laboratory and is suitable for a mobile testing unit model. compared to rt-pcr, the method has a high sensitivity and specificity in laboratory evaluation [ ] but is yet to be proven in clinical settings. the setting was a national health service (nhs) high dependency care home (category continuing care), where an outbreak was suspected. all residents were eligible for inclusion. on day (monday th march) two patients experienced fever and had other classical symptoms of covid- , arousing clinical suspicion. rt-pcr testing was performed on day and reported as positive to determine the extent of spread in the home, and protect patients and staff, on days & nasal and pharyngeal swabs were performed in all patients in the care home and analysed using multiplex tandem rt-pcr. on day a single rt-lamp swab was used to sample the throat, followed immediately by the nose. patients' vital signs (including temperature, heart rate, blood pressure, respiratory rate and oxygen saturations) were noted in the weeks before the known outbreak to determine whether the start of the outbreak may have occurred prior to the presumed day . standards for reporting diagnostic accuracy studies (stard) guidelines were used; stard guidelines aim to improve the completeness and transparency of reporting studies of diagnostic accuracy, to allow readers to assess the potential for bias and to evaluate its generalisability [ ] . in order to protect staff and patients, isolation and barrier nursing with full personal protective equipment were instituted for all patients. all patients were sampled on day and day using pharyngeal (day ) and deep nasal (day ) specimens (swabs) collected which were immediately placed into viral transport media (vtm) for rt-pcr or dry for the rt-lamp assay. staff taking the swabs were also swabbed and were negative for sars-cov- using rt-lamp. samples were urgently couriered to the hospital and microsensdx laboratory. the hospital performed multiplex tandem rt-pcr according to standard protocols with the rt-pcr test targeting the orf ab gene only; the limit of detection of the rt-pcr was not determined by the lab or manufacturer, but for this technology it is typically < copies per μl nucleic extract input [ ] . input volume for rt-pcr was μl of sample eluted to μl, with just μl of this used in the assay. if patients were positive on day , day samples were not analysed, but have been stored for later analysis. the rt-lamp method employed was the microsensdx rapiprep® sars-cov- research use test (see fig. ). this method used magnetic bead capture to maximise the yield of target nucleic acid during sample preparation from the dry swab, which is followed by rt-lamp to amplify and detect the sars-cov- genome, targeting the orf ab gene alone. the assay runs at °c allowing simpler and cheaper instrumentation which can yield results in min on average, often giving identification of positives in < min. results from this assay were compared to multiplex tandem pcr performed twice in the case of negative patients. input volume for rt-lamp was μl of the rna extract, which was the entire eluate from the magnetic bead extraction. the sensitivity, specificity, positive predictive value and negative predictive value were calculated using clopper-pearson confidence intervals (ci) by comparing our day rt-lamp result to rt-pcr. a patient was considered positive by rt-pcr if either a day / result or a day test result was positive. in view of the urgency of the covid- pandemic and the need to act quickly in the outbreak, formal ppi consultation for this clinical improvement study was not possible. the study was discussed extensively with the care team and virology department and senior management. this project was a clinical service improvement and the requirement for research ethics committee (rec) approval was therefore waived in line with nhs health research authority guidance (http://www.hradecisiontools.org.uk/research/). in the spirit of participant involvement, the study was discussed with all capacitous patients in the care home. all were enthusiastic to be involved and could see the value of rapid testing. in addition, relatives of all the patients who lacked capacity were appraised of the study and given a chance to comment, and for their relative to not take part. one family felt an additional swab might be intrusive, but all others were keen to be involved, gave some suggestions about swab technique (nasal vs. pharyngeal) and for the results to be shared for the benefit of others. twenty four residents were present in the care home on day . two patients lacked capacity and had no contactable next of kin to inform of the project. in one patient their informant did not agree to repeated testing as a service improvement. twenty one patients were included in the study (fig. ) . study participants were aged between and years (median years) and were predominantly female ( %). / died due to covid- , and / died from unrelated causes (for one patient, a progressive end-stage malignancy) within days of their positive test (table ) . testing results are shown in table . we defined cases as being rt-pcr positive on one of two tests at day or , and negative if negative on both tests. using this definition, / patients in the facility were covid- positive (rt-pcr ). of these cases, were identified with a single swab for rt-lamp, giving a sensitivity of % ( % ci - %) and positive predictive value of % ( %ci - %) ( table ) . this represented an improved rate of detection compared to single swab rt-pcr both in our sample and previous estimates. the specificity of the rt-lamp test was % on a single test and % on retesting lamp positives with low signal. three cases were initially identified as low positive using rt-lamp which were negative on rt-pcr, giving a total of patients testing positive on either rt-pcr or rt-lamp (table ). of these patients, patient had a high grade temperature of . °c on d of testing, patient had a temperature < . °c in the days prior to testing and patient had a temperature of . °c in the days prior to testing (table ). all three remained well at day with no other explanation for symptoms, such as upper respiratory or urinary tract infections. the routine test protocol now recommended by microsensdx includes retesting of low positive samples. repeat rt-lamp tests on samples from the three low positive patients were negative on repeat at day . it is possible that the rt-pcr results for one or more of these patients represent false negatives on day . of the two patients positive for rt-pcr and negative using rt-lamp one was contemporaneously symptomatic, and the other was well at the time of testing but had suffered a significant flu-like illness for the weeks prior to day . many patients in the home had altered vital signs in the week leading up to testing, with / negative cases, and / positive cases showing signs, e.g. fevers or reduced oxygen saturations. low temperatures (< °c were detected in a minority of covid pcr positive patients ( table ). the development of cases in the home and testing results are illustrated in fig. . no adverse events related to testing were reported. in a time of global crisis, it is critical that data are quickly shared on new testing methods, so that they can be scaled up more rapidly. to this end, we present data from patients in a care home tested within days of an outbreak in the home. in this patient group, a single rt-lamp test had a sensitivity of % and a specificity of % on single test compared to a "better than gold standard" of two consecutive rt-pcr swabs. the specificity of the rt-lamp improved to % when the new protocol of retesting low positive lamp tests is performed. we feel that this level of sensitivity is "clinically workable" in a time of crisis, particularly if repeated testing is utilised, and safeguards are put in place to guard against overconfidence in negative individuals, but this is somewhat subjective as no defined threshold of acceptability exists. it is comparable to other estimates of a single-swab rt-pcr test in our clinical experience and in posted pre-prints [ , ] . combined with the rapid result time, rt-lamp may have additional clinical utility to standard rt-pcr. the rt-pcr negative, rt-lamp initial low positive samples may indicate a lack of specificity of the low-level rt-lamp signals. given that some infected patients are assumed to be have been clinically asymptomatic and given that the rt-lamp assay used here tests more of the swab eluate than the pcr, these may be real positive results at day , that have missed by the rt-pcr. further testing and further studies will resolve this issue. in addition, we found fever > . °c, as expected was a common symptom, but hypothermia (t < . °c) and desaturation were also noted. the finding of hypothermia is important. it is a recognised symptom of sepsis and the systemic inflammatory response syndrome, particularly in older people [ ] . however, current phe and who covid- guidelines do not include hypothermia as a symptom. larger scale studies on prevalence of hypothermia, as well as other nonclassical symptoms, would shed more light on the presentation of covid- in institutionalised patients. loop-mediated isothermal amplification (lamp) was developed as a rapid and reliable, cheaper method to amplify from a small amount target sequence at a single reaction temperature, obviating the need for sophisticated thermal cycling equipment [ ] . two of these used only proven pcr-positive throat and nasal swabs and demonstrated sensitivity > % for rt-lamp methods targeting the orf ab gene when compared with gold standard rt-pcr [ , ] . only the studies by yang [ ] and yan [ ] included samples from both sars-cov- positive and negative patients and was thus able to produce both a sensitivity and a specificity. the remaining two groups, both based in the united states, lacked access to, or clearance to work with, sars-cov- samples and used either inactivated hiv with synthesised lamp sequences [ ] or other synthesised rt-lamp sequences [ ] . the majority of studies focused on the highlyconserved orf ab gene primer, also targeted by the rt-lamp method used by the microsensdx rapiprep® sars-cov- method. our study is the first "real world" study comparing the effectiveness of rt-pcr and rt-lamp testing in a group of patients at high risk for covid- and represents an important progression to clinical use for this novel sars-cov- testing method. we planned to perform rt-lamp testing just once due to a high degree of confidence that a single test would have satisfactory accuracy, allowing clinical decisions to be made immediately. however, the discrepant samples were fully concordant on re-test. as such, our standard for comparison was not a single rt-pcr, but two separate swabs for rt-pcr sent on consecutive days, thus representing what could be considered a "better-than gold standard" for comparison. however, as the pandemic has progressed, it has become apparent that there is no true "gold standard" for covid- testing with highly-anticipated antibody testing not always proving helpful; even in mild disease, antibodies in pcr positive patients may not be detected [ ] . we have been able to perform these tests quickly in a group at high risk for severe disease, and a setting where early identification of infected patients is key to preventing further spread. many other studies so far have used laboratory samples to estimate efficacy but have been unable to estimate the clinical utility. swabs were taken by the same clinician, minimising the risk of technical error or observer biases. all rt-lamp samples were tested in the microsensdx laboratory, and rt-pcr in the hospital laboratory, and there was no viral transport medium on the rt-lamp swabs. our samples were shipped to microsensdx because a level biosafety cabinet was available in the company's laboratory for initial sample handling and, due to the urgency of the study, there was not time to install a suitable cabinet in the care home. in the future a poc facility may still require a level cabinet, however recent developments in sample collection devices that inactivate the virus immediately after swabbing are expected to eliminate the need for operator protection and so a biosafety cabinet will not be required. actual poc testing, and or viral medium could be used to optimise performance further but use of dry swabs could ease issues with supply of viral transport media. we are limited by a small sample size, so our estimates have wide confidence intervals. however, they appear to be concordant with other (pre-print) studies on rt-lamp performed purely on laboratory samples. cost is a significant issue when large-scale testing within the setting of a pandemic is considered. the combined sample preparation and lamp assay kit at list price from microsensdx is equivalent in cost to a separate sample extraction kit and pcr test kit used in the reference laboratory. however, the lamp instrument is significantly cheaper than a pcr machine (by a factor of - x) providing a cost saving. subsequent technology developments in the lamp assay since this study was performed early in the pandemic include conversion to a colorimetric signal that can be read by eye, potentially negating the need for instrumentation altogether. additionally, lamp assays are currently being trialled with saliva. use of this rapid test could facilitate early identification of cases and enactment of infection control measures as required. we speculate that this could significantly reduce spread and subsequent mortality in care home residents, a speculation which could easily be tested if the method was more widely available. the test may also be suitable for use in other community settings such as pharmacies and care agencies, as well as emergency departments, and prisons or residential settings for homeless people where rapid diagnosis would be most useful. an area of global concern is covid- spread in developing countries, where reported cases are increasing. inexpensive poc testing that is not dependent on skilled and centralised technicians will be vital for less well-resourced countries and economies. however, evaluation in these settings would be advised to replicate its effectiveness. there is an urgent need for a rapid, robust and costefficient poc test that can be used in care homes, community settings and away from centralised large-scale laboratories, without the need for skilled technicians. magnetic bead capture and rt-lamp amplification and testing for sars-cov- was found to be promising, rapid, easy to use and to work equivalently to standard multiplex tandem pcr methods. definitive studies to evaluate this method in larger cohorts are underway. rt-lamp has the potential to transform covid- detection, bringing rapid and accurate testing to the poc. covid- : testing times a single and two-stage, closed-tube, molecular test for the novel coronavirus (covid- ) at home, clinic, and points of entry rapid detection of novel coronavirus/ severe acute respiratory syndrome coronavirus (sars-cov- ) by reverse transcription-loop-mediated isothermal amplification rapid molecular detection of sars-cov- (covid- ) virus rna using colorimetric lamp rapid detection of covid- coronavirus using a reverse transcriptional loop-mediated isothermal amplification (rt-lamp) diagnostic platform rapid detection of sars-cov- using reverse transcription rt-lamp method rapid and visual detection of novel coronavirus (sars-cov- ) by a reverse transcription loop-mediated isothermal amplification assay fermented barley and soybean (bs) mixture enhances intestinal barrier function in dextran sulfate sodium (dss)-induced colitis mouse model characteristics of and important lessons from the coronavirus disease (covid- ) outbreak in china: summary of a report of cases from the chinese center for disease control and prevention phenolic compounds from red wine and coffee are associated with specific intestinal microorganisms in allergic subjects prebiotic evaluation of cocoa-derived flavanols in healthy humans by using a randomized, controlled, double-blind, crossover intervention study probiotics, prebiotics and synbiotics-a review development and validation of a rapid, single-step reverse transcriptase loop-mediated isothermal amplification (rt-lamp) system potentially to be used for reliable and high-throughput screening of covid- standard operating procedures for sars-cov- detection by a clinical diagnostic rt-lamp assay development of reverse transcription loopmediated isothermal amplification assays targeting severe acute respiratory syndrome coronavirus (sars-cov- ) stard guidelines for reporting diagnostic accuracy studies: explanation and elaboration impact of fermented foods on human cognitive function-a review of outcome of clinical trials correlation of chest ct and rt-pcr testing in coronavirus disease (covid- ) in china: a report of cases evaluating the accuracy of different respiratory specimens in the laboratory diagnosis and monitoring the viral shedding of -ncov infections the impact of body temperature abnormalities on the disease severity and outcome in patients with severe sepsis: an analysis from a multicenter, prospective survey of severe sepsis loop-mediated isothermal amplification of dna covid- : two antibody tests are "highly specific" but vary in sensitivity, evaluations find publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank participants, their families and staff at the care home for their help. authors' contributions mfo, kal, mnl and cjs collected clinical data. mfo and kal contributed equally to this paper as joint first authors, and with cjs wrote the manuscript. cjs took all swabs. mfo performed the data analysis. rh, as, tds, cjs and sw were involved in project set-up and planning. sdg and sd provided lab and virology expertise. the authors reviewed and approved the final manuscript. this research received no specific grant from any funding agency in the public, commercial or not-for-profit sectors. testing was provided free of charge by microsensdx, who processed the rt-lamp tests. cw and sw both provided comments on the interpretation of the results. pcr tests were performed as part of routine clinical care. no other funding was sought for this study. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. in line with procedures relating to a clinical service improvement, all capacitous participants, and relatives in case of non-capacitous participants, were verbally appraised of the project and given the opportunity to not take part. written appraisal was not practical due to visiting restrictions. this approach was approved by the institutional review bodies. competing interests cjs is supported by hefce funding. cs and sw are employees of microsensdx ltd. testing was provided free of charge by microsensdx. other authors report no conflict of interest. key: cord- -p rtp m authors: weissbrich, benedikt; neske, florian; schubert, jörg; tollmann, franz; blath, katharina; blessing, kerstin; kreth, hans wolfgang title: frequent detection of bocavirus dna in german children with respiratory tract infections date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: p rtp m background: in a substantial proportion of respiratory tract diseases of suspected infectious origin, the etiology is unknown. some of these cases may be caused by the recently described human bocavirus (hbov). the aim of this study was to investigate the frequency and the potential clinical relevance of hbov in pediatric patients. methods: we tested nasopharyngeal aspirates (npa) obtained between and from pediatric in-patients with acute respiratory tract diseases at the university of würzburg, germany, for the presence of hbov dna. the specificity of positive pcr reactions was confirmed by sequencing. results: hbov dna was found in ( . %) of the npas. the median age of the infants and children with hbov infection was . years (mean age . years; range days – years). infections with hbov were found year-round, though most occurred in the winter months. coinfections were found in ( . %) of the hbov positive samples. rsv, influenza a, and adenoviruses were most frequently detected as coinfecting agents. sequence determination of the pcr products in the np- region revealed high identity ( %) between the nucleotide sequences obtained in different years and in comparison to the swedish viruses st and st . an association of hbov with a distinct respiratory tract manifestation was not apparent. conclusion: hbov is frequently found in npas of hospitalized infants and children with acute respiratory tract diseases. proving the clinical relevance of hbov is challenging, because application of some of koch's revised postulates is not possible. because of the high rate of coinfections with hbov and other respiratory tract pathogens, an association between hbov and respiratory tract diseases remains unproven. respiratory tract infections are a major cause of human morbidity and are caused by a broad spectrum of microbial agents. viruses account for the largest number of respiratory tract infections. the so-called respiratory viruses include influenza virus a and b, parainfluenzae viruses, adenoviruses, respiratory syncytial virus (rsv), rhinoviruses, and coronaviruses. in recent years, several novel viruses have been discovered in patients with respiratory infections using molecular biology methods. these novel viruses include the human metapneumovirus and several coronaviruses (sars, nl , hku ) [ , ] . the latest addition to this list was the human bocavirus (hbov) described by allander et al. [ ] . their screening method for unknown viral sequences in patient samples involved concentration of viral particles, nucleic acid isolation, random amplification of rna and dna, and finally sequencing and subsequent blasting of the amplified products. hbov is most closely related to the minute virus of canines (mvc) and the bovine parvovirus (bpv), which have been classified in the genus bocavirus within the parvoviridae [ ] . classically, the postulates of koch as modified by rivers have been used to establish a causal relationship between viruses and a disease [ ] . however, hbov has not been propagated in cell culture, and there is no animal model so far. therefore, proving the clinical relevance is challenging because application of some postulates of koch and rivers is not possible. in the first description of hbov, dna was detected in ( . %) of swedish children with lower respiratory tract disease. three of the children were coinfected with other viruses (two with rsv and one with adenovirus). because hbov-positive samples were negative for other respiratory viruses by standard screening, it was reasoned that hbov may cause respiratory tract disease. in a second report of hbov in australian children and adults, hbov was detected in ( . %) of respiratory samples. in ten of these ( . %), a coinfection with rsv (n = ), hmpv or adenoviruses was detected [ ] . in a third study from japan, hbov dna was found in ( . %) of respiratory specimens of children with lower respiratory tract disease [ ] . samples positive for other viruses (rsv, influenza virus a and b, hmpv) were excluded from the study. analysis of double infections was therefore not possible. in two more recent studies from canada and france, hbov dna was detected in of ( . %) and nine of ( . %) respiratory samples, respectively [ , ] . in the canadian study specimens of children and adults were tested. specimens positive for other respiratory viruses were excluded. the french study examined samples of children below five years of age. three of the nine hbov dna positive children ( . %) were coinfected with rsv. in order to better understand the epidemiological pattern of hbov infections and to analyze its clinical relevance, further studies on larger groups of patients are necessary. therefore, we retrospectively tested nasopharyngeal aspirates of patients from the university of würzburg children's hospital, germany for the presence of hbov dna. the samples tested for hbov infection consisted of stored nasopharyngeal aspirates (npa) that were sent by the university of würzburg children's hospital for screening of respiratory viruses from january to september . on arrival in the viral diagnostic laboratory, the samples were routinely tested for the presence of respiratory virus antigens with an immunofluorescence assay (respiratory panel ifa kit, chemicon). the screening reagent of the kit detects antigens of adenoviruses, influenza viruses a and b, parainfluenza viruses - , and rsv. npas showing positive reactions with the screening reagent were further studied by ifa using the seven single monoclonal antibodies contained in the screening reagent. remaining npa material was stored at - °c until further testing for hbov dna. in addition to the samples from - , a small number of samples (n = ) from - were also available for retrospective testing. the study was carried out in compliance with the helsinki declaration and was approved by the ethics committee of the medical faculty at the university of würzburg. hbov pcr and sequencing dna was extracted from μl of the npa samples using the high pure viral nucleic acid kit (roche, mannheim, germany) according to the instructions of the manufacturer. the elution volume was μl. amplification of hbov dna was performed with the np- primers bov f (gagctctgtaagtactattac) and bov r (ctctgtgttgactgaatacag) described by allander [ ] using the hotstartaq dna polymerase (qiagen, hilden, germany). pcr reactions were carried out in a μl volume consisting of μl extracted dna, × qiagen hotstar buffer, dntps at a final concentration of μm each, pmol of each primer, and . u of taq polymerase. the cycling conditions were cycles ( °c s, °c s and min at °c) after a preheating step of min at °c. after amplification, pcr products were visualized by staining with ethidium bromide on agarose gels. a pcr reaction was considered as positive when a band of the expected size ( base pairs) was visible. to confirm the sequence specificity, all pcr products from positive reactions were sequenced completely in both directions using big dye terminator chemistry and the abi prism (applied biosystems, darmstadt, germany). general laboratory procedures to prevent pcr contamination were strictly adhered to. one negative control was extracted and amplified for every five npa samples. all negative controls were found to be negative for hbov dna. a plasmid containing the pcr product cloned in the vector pcr ® . -topo ® (invitrogen) was used as positive control. the sensitivity of the hbov assay was approximately copies per reaction. from january to september , nasopharyngeal aspirates (npa) of hospitalized infants and children with febrile respiratory tract diseases were received for viral diagnostic evaluation. the median age of the patients was . years (mean age . years; range days - years), and % were boys. the seasonal distribution of all samples is shown in figure . because of insufficient volume of the stored material, samples had to be excluded from the retrospective testing of hbov dna. the median age and the seasonal distribution of the hospitalization were not significantly different between the patients with and without sufficient npa sample volume. by routine immunofluorescence testing for antigen of respiratory viruses, a positive diagnosis was obtained for ( . %) of the npa samples. rsv (n = ; . %) and influenza a virus (n = ; . %) were most frequently found. further details are shown in table . coinfections with two or more viruses were detected in of the cases using the antigen assay. of the npas tested for hbov dna, ( . %) samples were found to be positive by pcr and subsequent sequencing. the male to female ratio of the hbov positive infants and children ( . % boys) was similar to the ratio in the population tested. their median age was . years (mean . years; range days - years). the age distribution of the hbov positive patients and of the rsv, adenovirus and influenza a virus positive children for comparison is shown in figure . median ages were significantly different for these four infectious agents, except for the comparison between influenza a and adenovirus infections ( table ) . while infections with rsv peaked during the first six months of life (median . years), most hbov infections (n = ; . %) occurred at the age of - years. infections with influenza a virus were more evenly distributed over a wider age range (median . years). infections with hbov were found year-round, though most occurred in the winter months ( figure ). the shape of the curves of the total number of npa samples received and of the hbov positive samples appeared to be almost in parallel. there were no significant differences between the yearly frequencies of hbov positive results between and (table ). in samples that were available from before , hbov was retrospectively found in samples, two from and one from . in ( . %) of the hbov positive infants and children, coinfections with other respiratory viruses were present, most frequently with rsv (n = ) followed by influenza a (n = ). the percentage distribution of the coinfecting agents among the hbov positive samples was similar to the distribution of these agents in the total population (table ) . sequence determination of the hbov pcr products (np- region of the genome) revealed high identity ( %) between the nucleotide sequences obtained in different years and in comparison to the swedish viruses st and st . clinical data were available for of the hbov positive npas. the patients suffered from upper and/or lower respiratory tract diseases (table ) . associations between all hbov infections and distinct clinical manifestations were not apparent. because of the retrospective nature of the study and because clinical data for the hbov negative samples were not obtained, a statistical analysis of this aspect was not possible. when the clinical diagnoses of the children with and without coinfections with other respiratory pathogens were compared, pneumonia was found more often in the group of children without coinfections (table ). this association was borderline significant (p = . by fisher's exact test). we found hbov dna in . % of npa samples obtained from infants and children with respiratory tract diseases during the years to in the region of northern bavaria in germany. this is the highest frequency reported so far. to our knowledge, there have been five previous reports on hbov infections, the original report from sweden with a frequency of . %, and reports from australia, japan, canada and france with frequencies of . %, . %, . %, and . %, respectively. in our study, hbov infections were almost as frequently found as infections with influenza a virus, the second most common respiratory infection, and they were considerably more frequent than infections with influenza b, parainfluenzae, and adenoviruses. however, in contrast to the pcr method used for hbov detection, the other respiratory viruses in our study were examined by ifa. when comparing the detection frequencies, this difference in detection methods has to be taken into consideration. in general, pcr assays are more sensitive than antigen detection methods [ ] . therefore, it is likely that the true prevalence of the respiratory viruses that were analyzed by ifa is actually higher than here reported. there are several possible explanations for the higher frequency of hbov infections observed in our study compared to the previous reports. firstly, the difference may be due to an increased sensitivity of our pcr assay. in all previous studies as well as in ours, single round hot-start pcrs have been employed to detect hbov dna. however, the assays vary in the number of pcr cycles performed ( cycles [ , [ ] [ ] [ ] ; cycles [ ] ; cycles (present study)). depending on the assay optimization, cycles may not be sufficient to detect weakly positive samples. in addition, data on the assay sensitivity were not provided in either of the previous studies. using a plasmid control, we were able to show that our assay regularly detects approximately copies of hbov dna per reaction. in order to obtain information on the amount of hbov dna present in the npas and in other secretions, a real-time pcr assay is currently under development. a second potential reason for differing infection frequencies between studies may be due to regional and temporal differences in the incidence of hbov infection. in contrast to the previous reports, which have studied samples from only one or two winter seasons, the npas in our studies have been collected during four consecutive years. in general, seasonal differences of sample acquisition may account for varying incidence numbers. in our study, for example, proportions of influenza a, parainfluenza , and adenovirus infections were considerably different between winter seasons (data not shown). however, this was not the case for hbov infections. in four consecutive winter seasons, we observed a similar frequency of approximately %. therefore, seasonal variation is unlikely to account for the observed high frequency of hbov infections in our study population. so far, hbov has been detected in sweden, australia, japan, canada, france, and germany, and it appears that hbov has a worldwide distribution. it remains to be determined how incidence numbers are influenced by regional aspects. thirdly, the higher frequency observed in our study may be related to different patient populations. children hospitalized for respiratory tract diseases were included in all studies published so far [ , [ ] [ ] [ ] [ ] . two studies additionally examined adults and outpatients [ , ] . while three studies as well as ours included patients with upper and/or lower respiratory tract disease [ , , ] , two studies focused on patients with lower respiratory tract diseases [ , ] . thus, it is difficult to compare patient populations in the hbov studies. in agreement with the previous reports [ , [ ] [ ] [ ] [ ] , sequencing of the pcr products in the np- region revealed a nucleotide identity of more than % between different samples. this was also true for the two hbov dna positive samples from . although much more sequence information on hbov will be required, the data available so far indicate that hbov may be a highly conserved virus. the age distribution of hbov infections found in our study is similar to previous reports [ , [ ] [ ] [ ] [ ] . most hbov infections occurred between months and years of age. this distribution is compatible with protection from infection by maternal antibodies in the first year of life. future studies of the seroprevalence of hbov antibodies in different age groups will shed light on this issue. analysis of a potential association between hbov infection and clinical manifestations is limited by the retrospective nature of our study, by the high number of double infections, and by the fact that clinical information was obtained only for hbov positive patients. however, it seems that there is no obvious association between hbov infection and a distinct clinical manifestation. instead, a broad spectrum of both upper and lower respi- ratory tract diseases was observed. when clinical diagnoses of hbov dna positive patients with and without coinfections were compared, pneumonia was found more frequently in children without coinfection. however, this association of borderline significance (p = . ) should be regarded with caution because of small numbers. if pneumonia was caused by hbov infection, it is unclear how a coinfection could result in a less frequent manifestation of this disease. the assumption in the first description of hbov, that this virus might be an etiologic agent of respiratory tract disease, was based on the fact that hbov infections were found significantly more often in samples negative for other respiratory viruses. however, with coinfection rates ranging fom . % to . %, these findings were neither confirmed by the other studies that analyzed coinfections [ ] nor by us. the true number of coinfections in our study is probably even higher than the reported . %, because antigen-based methods were used for screening of respiratory viruses other than hbov, and because several respiratory pathogens such as coronaviruses, rhinoviruses, enteroviruses and the human metapneumovirus were not tested for. on the basis of the considerable number of coinfections, one might argue that hbov is an aggravating factor of respiratory diseases, an innocent bystander that is just detected by chance, or a persisting virus that is reactivated by the inflammatory process. thus, it is uncertain at present, if hbov is indeed an etiologic agent of respiratory tract (or other) diseases. several viruses detected by molecular biology methods in recent years are still in search for a relevant clinical disease [ , ] . if hbov has to be added to this list remains to be determined. hbov is frequently found in npas of hospitalized children with acute respiratory tract diseases. proving the clinical relevance of hbov is challenging, because application of some postulates of koch is not possible. because of the high rate of coinfections with hbov and other respiratory pathogens, an association between hbov and respiratory tract diseases remains unproven. newer respiratory virus infections: human metapneumovirus, avian influenza virus, and human coronaviruses characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia cloning of a human parvovirus by molecular screening of respiratory tract samples tijsses p: family parvoviridae viruses and koch's postulates evidence of human coronavirus hku and human bocavirus in australian children detection of human bocavirus in japanese children with lower respiratory tract infections human bocavirus infection human bocavirus in children simultaneous detection and typing of influenza viruses a and b by a nested reverse transcription-pcr: comparison to virus isolation and antigen detection by immunofluorescence and optical immunoassay (flu oia) ttv, a new human virus with single stranded circular dna genome gb virus type c/hepatitis g virus. semin liver dis we thank the technicians of the viral diagnostic lab for skillful and dedicated assistance and axel rethwilm and kirsty mcpherson for helpful comments on the manuscript. the author(s) declare that they have no competing interests. bw and hwk designed and coordinated the study. fn performed the hbov dna testing. hwk, kblessing and kblath collected clinical data. bw, js and ft collected virological data. all authors participated in the data anal-ysis. bw and fn drafted the manuscript. all authors read and approved the final version of the manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /pre pub key: cord- -ioemd ij authors: tellier, raymond; li, yuguo; cowling, benjamin j.; tang, julian w. title: recognition of aerosol transmission of infectious agents: a commentary date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: ioemd ij although short-range large-droplet transmission is possible for most respiratory infectious agents, deciding on whether the same agent is also airborne has a potentially huge impact on the types (and costs) of infection control interventions that are required. the concept and definition of aerosols is also discussed, as is the concept of large droplet transmission, and airborne transmission which is meant by most authors to be synonymous with aerosol transmission, although some use the term to mean either large droplet or aerosol transmission. however, these terms are often used confusingly when discussing specific infection control interventions for individual pathogens that are accepted to be mostly transmitted by the airborne (aerosol) route (e.g. tuberculosis, measles and chickenpox). it is therefore important to clarify such terminology, where a particular intervention, like the type of personal protective equipment (ppe) to be used, is deemed adequate to intervene for this potential mode of transmission, i.e. at an n rather than surgical mask level requirement. with this in mind, this review considers the commonly used term of ‘aerosol transmission’ in the context of some infectious agents that are well-recognized to be transmissible via the airborne route. it also discusses other agents, like influenza virus, where the potential for airborne transmission is much more dependent on various host, viral and environmental factors, and where its potential for aerosol transmission may be underestimated. the classification of an infectious agent as airborne and therefore 'aerosol-transmissible' has significant implications for how healthcare workers (hcws) need to manage patients infected with such agents and what sort of personal protective equipment (ppe) they will need to wear. such ppe is usually more costly for airborne agents (i.e. aerosol-transmissible) than for those that are only transmitted by large droplets or direct contact because of two key properties of aerosols: a) their propensity to follow air flows, which requires a tight seal of the ppe around the airways, and b) for bioaerosols, their small size, which calls for an enhanced filtering capacity. several recent articles and/or guidance, based on clinical and epidemiological data, have highlighted the potential for aerosol transmission for middle-east respiratory syndro me-associated coronavirus (mers-cov) [ , ] and ebola virus [ , ] . some responses to the latter have attempted to put these theoretical risks in a more practical light [ ] , and this nicely illustrates the quandary of how to classify such emerging or re-emerging pathogens into either the large droplet (short-range) versus airborne (short and possibly long-range) transmission categories. however, this delineation is not black and white, as there is also the potential for pathogens under both classifications to be potentially transmitted by aerosols between people at close range (i.e. within m). strictly speaking, 'aerosols' refer to particles in suspension in a gas, such as small droplets in air. there have been numerous publications classifying droplets using particle sizes over the years [ ] [ ] [ ] [ ] [ ] [ ] . for example it is generally accepted that: i) small particles of < - μm aerodynamic diameter that follow airflow streamlines are potentially capable of short and long range transmission; particles of < μm readily penetrates the airways all the way down to the alveolar space, and particles of < μm readily penetrates below the glottis ( ) ii) large droplets of diameters > μm refer to those that follow a more ballistic trajectory (i.e. falling mostly under the influence of gravity), where the droplets are too large to follow inhalation airflow streamlines. for these particle sizes, for example, surgical masks would be effective, as they will act as a direct physical barrier to droplets of this size that are too large to be inhaled into the respiratory tract around the sides of the mask (which are not close-fitting); iii) 'intermediate particles' of diameters - μm, will share some properties of both small and large droplets, to some extent, but settle more quickly than particles < μm and potentially carry a smaller infectious dose than large (> μm) droplets. ' aerosols' would also include 'droplet nuclei' which are small particles with an aerodynamic diameter of μm or less, typically produced through the process of rapid desiccation of exhaled respiratory droplets [ , ] . however, in some situations, such as where there are strong ambient air cross-flows, for example, larger droplets can behave like aerosols with the potential to transmit infection via this route (see next section below). several properties can be inferred from this, for example the penetration of the lower respiratory tract (lrt), as at greater than μm diameter, penetration below the glottis rapidly diminishes, as does any potential for initiating an infection at that site. similarly, any such potential for depositing and initiating an lrt infection is less likely above a droplet diameter of μm, as such large particles will probably impact onto respiratory epithelial mucosal surfaces or be trapped by cilia before reaching the lrt [ ] . the infectious diseases society of america (idsa) has proposed a scheme that is essentially equivalent [ ] , defining "respirable particles" as having a diameter of μm or less; and "inspirable particles" as having a diameter between μm and μm, nearly all of which are deposited in the upper airways. some authors have proposed the term "fine aerosols", consisting of particles of μm or less, but this has been in part dictated by constraints from measurement instruments [ ] . several authors lump together transmission by either large droplets or aerosol-sized particles as "airborne transmission" [ ] , or use "aerosol transmission" to describe pathogens that can cause disease via inspirable particles of any size [ ] . however, we think that it is important to maintain a distinction between particles of < μm and larger particles, because of their significant qualitative differences including suspension time, penetration of different regions of the airways and requirements for different ppe. in this commentary, we use the common convention of "airborne transmission" to mean transmission by aerosol-size particles of < μm. if the infected patients produce infectious droplets of varying sizes by breathing, coughing or sneezing, transmission between individuals by both short-range large droplets and airborne small droplet nuclei are both possible, depending on the distance from the patient source. figure illustrates these potential routes of short and long-range airborne transmission, as well as the downstream settling of such droplets onto surfaces (fomites). from such fomites, they may be touched and transported by hands to be self-inoculated into mucosal membranes e.g. in the eyes, nose and mouth) to cause infection, depending on the survival characteristics of individual pathogens on such surfaces, and the susceptibility (related to available, compatible cell receptors) of the different exposed tissues to infection by these pathogens. for example, when the infectious dose (the number of infectious agents required to cause disease) of an organism is low, and where large numbers of pathogen-laden droplets are produced in crowded conditions with poor ventilation (in hospital waiting rooms, in lecture theatres, on public transport, etc.), explosive outbreaks can still occur, even with pathogens whose airborne transmission capacity is controversial, e.g. the spread of influenza in a grounded plane where multiple secondary cases were observed in the absence of any ventilation [ ] . the more mechanistic approaches (i.e. arguing from the more fundamental physical and dynamic behavior of small versus larger particle and droplet sizes in the absence of any biological interactions) to classifying which pathogens are likely to transmit via the airborne route have been published in various ways over the years [ ] [ ] [ ] [ ] [ ] [ ] , but may have to be considered in combination with epidemiological and environmental data to make a convincing argument about the potential for the airborne transmissibility of any particular agentand the number of possible potential exposure scenarios is virtually unlimited). one should note that "aerosol" is essentially a relative and not an absolute term. a larger droplet can remain airborne for longer if ambient airflows can sustain this suspension for longer, e.g. in some strong cross-flow or natural ventilation environments, where ventilation-induced airflows can propagate suspended pathogens effectively enough to cause infection at a considerable distance away from the source. one of the standard rules (stoke's law) applied in engineering calculations to estimate the suspension times of droplets falling under gravity with air resistance, was derived assuming several conditions including that the ambient air is still [ ] [ ] [ ] [ ] [ ] . so actual suspension times will be far higher where there are significant cross-flows, which is often the case in healthcare environments, e.g. with doors opening, bed and equipment movement, and people walking back and forth, constantly. conversely, suspension times, even for smaller droplet nuclei, can be greatly reduced if they encounter a significant downdraft (e.g. if they pass under a ceiling supply vent). in addition, the degree of airway penetration, for different particle sizes, also depends on the flow rate. in the field of dentistry and orthopedics, where high-powered electric tools are used, even bloodborne viruses (such as human immunodeficiency virus -hiv, hepatitis b and hepatitis b viruses) can become airborne when they are contained in high velocity blood splatter generated by these instruments [ , ] . yet, whether they can cause efficient transmission via this route is more debatable. this illustrates another point, that although some pathogens can be airborne in certain situations, they may not necessarily transmit infection and cause disease via this route. here 'expiration' also includes normal breathing exhalation, as well as coughing and/or sneezing airflows. airborne droplets can then settle on surfaces (fomites) from where they can be touched and carried on hands leading to further self-inoculation routes of transmission outline over time, for a pathogen with a truly predominant airborne transmission route, eventually sufficient numbers of published studies will demonstrate its true nature [ ] . if there are ongoing contradictory findings in multiple studies (as with influenza virus), it may be more likely that the various transmission routes (direct/indirect contact, short-range droplet, long-, and even shortrange airborne droplet nuclei) may predominate in different settings [ , ] , making the airborne route for that particular pathogen more of an opportunistic pathway, rather than the norm [ ] . several examples may make this clearer. the selected pathogens and supporting literature summarized below are for illustrative purposes only, to demonstrate how specific studies have impacted the way we consider such infectious agents as potentially airborne and 'aerosol-transmissible'. it is not intended to be a systematic review, but rather to show how our thinking may change with additional studies on each pathogen, and how the acceptance of "aerosol transmission" for different pathogens did not always followed a consistent approach. chickenpox chickenpox is a febrile, vesicular rash illness caused by varicella zoster virus (vzv), a lipid-enveloped, double-stranded dna virus, and a member of the herpesviridae family. for chickenpox, the evidence appears to be mainly epidemiological and clinical, though this has appeared to be sufficient to classify varicella zoster virus (vzv) as an airborne agent. studies on vzv have shown that the virus is clearly able to travel long distances (i.e. up to tens of meters away from the index case, to spread between isolation rooms and other ward areas connected by corridors, or within a household) to cause secondary infections and/or settle elsewhere in the environment [ ] [ ] [ ] . in addition, tang et al. [ ] showed that airborne vzv could leak out of isolation rooms transported by induced environmental airflows to infect a susceptible hcw, most likely via the direct inhalation route. measles (also known as rubeola) is a febrile, rash illness caused by the measles virus, a lipid-enveloped, singlestranded, negative-sense rna virus, and a member of the paramyxoviridae family. for measles several studies examined a more mechanistic airflow dynamical explanation (i.e. based upon the fundamental physics and behaviour of airborne particles) for the main transmission route involved in several measles outbreaks [ ] , including that of riley and colleagues who used the concept of 'quanta' of infection [ ] . later, two other outbreaks in outpatient clinics included retrospective airflow dynamics analysis, providing more evidence for the transmissibility of measles via the airborne route [ , ] . tuberculosis is a localized or systemic, but most often respiratory bacterial illness caused by mycobacteria belonging to the mycobacterium tuberculosis complex. for tuberculosis (tb), definitive experimental evidence of airborne transmission being necessary and sufficient to cause disease was provided in a series of guinea-pig experiments [ , ] , which has been repeated more recently in a slightly different clinical context [ ] . numerous other outbreak reports have confirmed the transmissibility of tb via the airborne route [ ] [ ] [ ] , and interventions specifically targeting the airborne transmission route have proven effective in reducing tb transmission [ ] . smallpox is a now eradicated, febrile, vesicular rash and disseminated illness, caused by a complex, doublestranded dna orthopoxvirus (poxviridae family), which can present clinically in two forms, as variola major or variola minor. for smallpox, a recent comprehensive, retrospective analysis of the literature by milton has suggested an important contribution of the airborne transmission route for this infection [ ] . although various air-sampling and animal transmission studies were also reviewed, milton also emphasized clinical epidemiological studies where non-airborne transmission routes alone could not account for all the observed smallpox cases. at least one well-documented hospital outbreak, involving cases of smallpox, could only be explained by assuming the aerosol spread of the virus from the index case, over several floors. retrospective smoke tracer experiments further demonstrated that airborne virus could easily spread to patients on different floors via open windows and connecting corridors and stairwells in a pattern roughly replicating the location of cases [ ] . for sars-cov, several thorough epidemiological studies that include retrospective airflow tracer investigations are consistent with the hypothesis of an airborne transmission route [ ] [ ] [ ] . air-sampling studies have also demonstrated the presence of sars-cov nucleic acid (rna) in air, though they did not test viability using viral culture [ ] . although several studies compared and contrasted sars and mers from clinical and epidemiological angles [ ] [ ] [ ] , the predominant transmission mode was not discussed in detail, if at all. several other studies do mention the potential for airborne transmission, when comparing potential routes of infection, but mainly in relation to super-spreading events or "aerosolizing procedures"such as broncho-alveolar lavage, and/or a potential route to take into consideration for precautionary infection control measures [ ] [ ] [ ] . however, from the various published studies, for both mers and sars, it is arguable that a proportion of transmission occurs through the airborne route, although this may vary in different situations (e.g. depending on host, and environmental factors). the contribution from asymptomatic cases is also uncertain [ ] . for both sars and mers, lrt samples offer the best diagnostic yield, often in the absence of any detectable virus in upper respiratory tract (urt) samples [ ] [ ] [ ] . furthermore, infected, symptomatic patients tend to develop severe lrt infections rather than urt disease. both of these aspects indicate that this is an airborne agent that has to penetrate directly into the lrt to preferentially replicate there before causing disease. for mers-cov specifically, a recent study demonstrated the absence of expression of dipeptidyl peptidase (dpp ), the identified receptor used by the virus, in the cells of the human urt. the search for an alternate receptor was negative [ ] . thus, the human urt would seem little or non-permissive for mers-cov replication, indicating that successful infection can only result from the penetration into the lrt via direct inhalation of appropriately sized 'droplet nuclei'-like' particles. this makes any mers-cov transmission leading to mers disease conditional on the presence of virus-containing droplets small enough to be inhaled into the lrt where the virus can replicate. influenza is a seasonal, often febrile respiratory illness, caused by several species of influenza viruses. these are lipid-enveloped, single-stranded, negative-sense, segmented rna viruses belonging to the orthomyxoviridae family. currently, influenza is the only common seasonal respiratory virus for which licensed antiviral drugs and vaccines are available. for human influenza viruses, the question of airborne versus large droplet transmission is perhaps most controversial [ ] [ ] [ ] [ ] . in experimental inoculation experiments on human volunteers, aerosolized influenza viruses are infectious at a dose much lower than by nasal instillation [ ] . the likely answer is that both routes are possible and that the importance and significance of each route will vary in different situations [ , , ] . for example, tighter control of the environment may reduce or prevent airborne transmission by: ) isolating infectious patients in a single-bed, negative pressure isolation room [ ] ; ) controlling environmental relative humidity to reduce airborne influenza survival [ ] ; ) reducing exposure from aerosols produced by patients through coughing, sneezing or breathing with the use of personal protective equipment (wearing a mask) on the patient (to reduce source emission) and/or the healthcare worker (to reduce recipient exposure) [ ] ; ) carefully controlling the use and exposure to any respiratory assist devices (high-flow oxygen masks, nebulizers) by only allowing their use in designated, containment areas or rooms [ ] . the airflows being expelled from the side vents of oxygen masks and nebulisers will contain a mixture of patient exhaled air (which could be carrying airborne pathogens) and incoming high flow oxygen or air carrying nebulized drugs. these vented airflows could then act as potential sources of airborne pathogens. numerous studies have shown the emission of influenza rna from the exhaled breath of naturally influenzainfected human subjects [ ] [ ] [ ] [ ] [ ] and have detected influenza rna in environmental air [ ] [ ] [ ] . more recently, some of these studies have shown the absence of [ ] , or significantly reduced numbers of viable viruses in air-samples with high influenza rna levels (as tested by pcr) [ , , ] . the low number of infectious particles detected is currently difficult to interpret as culture methods are inherently less sensitive than molecular methods such as pcr, and the actual operation of airsampling itself, through shear-stress related damage to the virions, also causes a drop in infectivity in the collected samples. this may lead to underestimates of the amount of live virus in these environmental aerosols. an additional variable to consider is that some animal studies have reported that different strains of influenza virus may vary widely in their capacity for aerosol transmission [ ] . in some earlier articles that discuss the predominant mode of influenza virus transmission [ ] [ ] [ ] [ ] [ ] , these same questions are addressed with mixed conclusions. most of the evidence described to support their views was more clinical and epidemiological, and included some animal and human volunteer studies, rather than physical and mechanistic. yet, this mixed picture of transmission in different circumstances is probably the most realistic. it is noteworthy that several infections currently accepted as airborne-transmitted, such as measles, chickenpox or tb present, in their classical form, an unmistakable and pathognomonic clinical picture. in contrast the clinical picture of influenza virus infection has a large overlap with that of other respiratory viruses, and mixed outbreaks have been documented [ ] . thus, a prevalent misconception in the field has been to study 'respiratory viruses' as a group. however, given that these viruses belong to different genera and families, have different chemical and physical properties and differing viral characteristics, it is unwise and inaccurate to assume that any conclusions about one virus can be applied to another, e.g. in a cochrane review of published studies on interventions to reduce the spread of respiratory viruses, there were actually only two studies specifically about influenza viruses [ ] . as the authors themselves pointed out, no conclusion specific to influenza viruses was possible. while many airborne infections are highly contagious, this is not, strictly speaking, part of the definition. even so, the lower contagiousness of influenza compared to, say, measles has been invoked as an argument against a significant contribution of airborne transmission. yet, it should be noted that a feature of influenza virus infections is that the incubation time (typically - days) is much shorter than its duration of shedding. this allows for the possibility that a susceptible person will be exposed during an outbreak to several different infectious cases belonging to more than one generation in the outbreak. this multiple exposure and telescoping of generations may result in an underestimate of influenza virus transmissibility, as fewer secondary cases will be assigned to a known index case, when in fact the number of secondary cases per index could be much higher. for example, it is known that in some settings a single index case can infect a large number of people, e.g. in an outbreak on an alaska airlines flight [ ] . ebola is a viral hemorrhagic fever associated with a very high mortality, caused by the ebola viruses; these are enveloped single-strand, negative-sense rna viruses comprising five species within the family filoviridae. four ebola species have been implicated in human diseases; the most widespread outbreak, also the most recent, was caused by ebola zaire in west africa in - . the transmission of ebola viruses has been reviewed in depth by osterholm et al. ( ) . these authors noted the broad tissue tropism, as well as the high viral load reached during illness and the low infectious dose, from which it appears inescapable that more than one mode of transmission is possible. regarding aerosol transmission, concerns are raised by several documented instances of transmission of ebola zaire in laboratory settings between animals without direct contact [ , ] (also reviewed in [ ] ). experimental infections of rhesus monkeys by ebola zaire using aerosol infection has been shown to be highly effective [ , ] and this experimental procedure has in fact been used as infectious challenge in ebola vaccine studies [ , ] . rhesus monkeys infected by aerosol exposure reliably developed disseminated, fatal infection essentially similar to that caused by parenteral infection with the addition of involvement of the respiratory tract. autopsies showed pathological findings in the respiratory tract and respiratory lymphoid system in animals infected by the aerosol route that are not found in animals infected parenterally [ , ] . such respiratory pathological lesions have not been reported in human autopsies of ebola cases, but as noted by osterholm et al. [ ] , there have been few human autopsies of ebola cases, arguably too few to confidently rule out any possibility of disease acquired by the aerosol route. the precautionary principle would therefore dictate that aerosol precautions be used for the care of infected patients, and especially considering that infection of the respiratory tract in such patients is not necessary to create an aerosol hazard: ebola viruses reach a very high titer in blood or other bodily fluids during the illness [ , ] and aerosolization of blood or other fluids would create a significant airborne transmission hazard. in summary, despite the various mechanistic arguments about which organisms can be potentially airborne and therefore aerosol-transmissible, ultimately, the main deciding factor appears to be how many studies using various differing approaches: empirical (clinical, epidemiological), and/or experimental (e.g. using animal models), and/or mechanistic (using airflow tracers and air-sampling) methods, reach the same consensus opinion. over time, the scientific community will eventually form an impression of the predominant transmission route for that specific agent, even if the conclusion is one of mixed transmission routes, with different routes predominating depending on the specific situations. this is the case for influenza viruses, and is likely the most realistic. some bacterial and viral infections that have more than one mode of transmission are also anisotropic, like anthrax, plague, tularemia and smallpox: the severity of the disease varies depending on the mode of transmission [ , ] . older experimental infection experiments on volunteers suggest that this is the case for influenza, with transmission by aerosols being associated with a more severe illness [ , ] , and some more recent field observations are consistent with this concept [ ] . for anisotropic agents, even if a mode of transmission (e.g. aerosols) accounts for only a minority of cases, interruption 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authors critically reviewed the final version of the manuscript. all authors read and approved the final manuscript.ethics approval and consent to participate not required. no individual patient information is included. only previously published papers are discussed. not applicable. none of the authors have any competing interests to declare. key: cord- -b yl mh authors: lau, joseph tf; griffiths, sian; choi, kai chow; tsui, hi yi title: avoidance behaviors and negative psychological responses in the general population in the initial stage of the h n pandemic in hong kong date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: b yl mh background: during the sars pandemic in hong kong, panic and worry were prevalent in the community and the general public avoided staying in public areas. such avoidance behaviors could greatly impact daily routines of the community and the local economy. this study examined the prevalence of the avoidance behaviors (i.e. avoiding going out, visiting crowded places and visiting hospitals) and negative psychological responses of the general population in hong kong at the initial stage of the h n epidemic. methods: a sample of respondents was recruited in a population-based survey. using random telephone numbers, respondents completed a structured questionnaire by telephone interviews at the 'pre-community spread phase' of the h n epidemic in hong kong. results: this study found that . % of the respondents currently avoided going out or visiting crowded places or hospitals, whilst % felt much worried about contracting h n and % showed signs of emotional distress. females, older respondents, those having unconfirmed beliefs about modes of transmissions, and those feeling worried and emotionally distressed due to h n outbreak were more likely than others to adopt some avoidance behaviors. those who perceived high severity and susceptibility of getting h n and doubted the adequacy of governmental preparedness were more likely than others to feel emotionally distressed. conclusions: the prevalence of avoidance behaviors was very high. cognitions, including unconfirmed beliefs about modes of transmission, perceived severity and susceptibility were associated with some of the avoidance behaviors and emotional distress variables. public health education should therefore provide clear messages to rectify relevant perceptions. the who raised the influenza alert level to the highest pandemic 'phase ' level on june , . as of june , , , confirmed h n cases were detected in countries, territories and areas and deaths had been reported [ ] ; the number of death increased to , as of february , . a preliminary study showed that the new h n virus is more infectious than seasonal influenza [ ] . in hong kong, the first confirmed case, a traveler from mexico, was reported on may , , leading to the closure and isolation of the metropark hotel and to the quarantining of guests and staff from may l to may , . during the h n outbreak, the hong kong hospital authority raised the alert level to the highest 'emergency response level'. the government maintained its confinement strategy till june, , when it became obvious that cases were spread in the community, and the response thus became mitigation. as of june , , there are confirmed cases in hong kong and no h n -related death was recorded and as of february , , there were h n -related deaths. the lessons learned from the sars experience in hong kong [ ] and other countries demonstrated the importance of understanding community responses [ , ] . surveillance of community responses at the initial phase of an emerging epidemic is useful to inform both policy makers and the public about the state of preparedness. in hong kong, at the time of the sars epidemic, the perceptions and behaviors changed dramatically during the course of the outbreak [ , [ ] [ ] [ ] . panic and worry increased and became widespread during the epidemic and remained high in the post-sars period [ , ] . at the height of the epidemic, the general public avoided going out, traveling to other countries and gathering for social activities [ ] . scholars estimated that a loss of hk$ billion in spending on goods and services in hong kong domestic economy was attributable to the sars epidemic [ ] . similar studies were conducted to investigate community responsiveness to the threat of human-to-human h n avian flu transmissions in hong kong [ ] [ ] [ ] [ ] . previous studies on human avian flu or sars in different countries also suggested that widespread distress occurred in affected areas and nationwide populations even at the early phase of the outbreak, causing serious social and economic disruption [ , ] . a study was conducted to investigate community behavioral and emotional responses at the very initial phase after the identification of the first few h n cases in hong kong [ ] . a few other studies have investigated community's attitudinal and behavioral responses toward the early phase of the h n pandemic in countries including the u. k. [ ] , australia [ ] , malaysia and europe [ ] , france [ ] , japan [ ] . avoidance behaviors have been prevalent in a number of countries or cities, such as hong kong and malaysia but not in the u. k. mild emotional distress was observed in hong kong but the public in japan perceived overwhelming fear. the majority of the respondents in the hong kong study washed their hands more often than usual, but only around % of those in the u. k. did the same. variations in perceived susceptibility and perceived efficacy over preventive measures have also been reported in these studies. therefore, community responses to the h n pandemic are likely to be country-specific, possibly determined by previous experiences of epidemics such as sars, the health system, risk communication patterns and even culture [ ] . this study investigated whether the general population in hong kong avoided visiting different places (going out, visiting crowded places and visiting hospitals) and assessed some negative psychological responses to h n , including whether people were much worried about contracting h n and their level of emotional distress (panicking, depression or emotional disturbance) due to h n . factors in association with the outcome variables on avoidance behaviors and negative psychological responses were investigated, including variables such as socio-demographic characteristics, confirmed knowledge and unconfirmed beliefs about modes of h n transmission, evaluation towards governmental preparedness/ performance, perceived availability of treatment, and risk perception (perceived severity and susceptibility related to h n ). the study period of this report covers almost the entire pre-pandemic and pre-community outbreak phase of the h n epidemic in hong kong. the study population comprised all chinese adults who were years old or above in hong kong. anonymous telephone interviews were conducted by well-trained interviewers, using an identical structured questionnaire, from may to may (day - , n = ), from may to may (day - , n = ), and from june to june (day - , n = ), . there were respectively , and imported cases (and no community non-imported cases) detected at the beginning date of these surveys. preliminary data from the survey conducted from may to may have previously been reported [ ] . the first local community-infected case with an unknown source of infection was reported on june , , so that the surveys (may to june , ) therefore covered almost the entire 'pre-community outbreak phase' (may to june , ) of the local epidemic. random telephone numbers were selected from an up-to-date telephone directory and the last two digits of the selected telephone number were randomized to include some unlisted telephone numbers; over % of the households in hong kong have a fix-line telephone at home [ ] . the interviews were conducted from : to pm to avoid overrepresenting the non-working population. one member was selected by the last-birthday-rule from each of the contacted households. at least phone calls were made at different hours and days before an unanswered number is considered invalid. verbal consent was sought and the study was approved by the ethics committee of the chinese university of hong kong. a total of , phone numbers were made and being answered by someone ( , calls were unanswered with at least attempts made), out of which , were eligible households were identified and being invited to join the study. of these , eligible respondents, ( . %) could not be contacted after attempts, ( . %) refused to join or withdrew from the study, and ( . %) participated in the study. previously, preliminary data from the may to may survey have been reported elsewhere [ ] . dependent variables included current avoidance behaviors: ) 'avoided going out', ) 'avoided visiting crowded places' and ) 'avoided visiting hospitals', and exhibition of negative psychological responses: ) worried very much that oneself or one's family would contract h n and ) emotional distress ('panicking very much' or 'felt much depressed' or 'felt much emotionally disturbed' due to h n ). socio-demographic characteristics were recorded. correct knowledge and unconfirmed beliefs about modes of h n transmission were assessed. respondents were asked about perceived availability of treatment. risk perception questions include those related to perceived severity of h n (fatality and severe irreversible bodily damages), the relative chance for hong kong to have a large-scale h n outbreak as compared to other countries, and perceived susceptibility (oneself, one' family and the general public). questions were also asked to evaluate relevant actions taken by the government ( items), their ability to control the epidemic ( items), as well as the health system's preparedness toward the h n pandemic ( items: adequacy of medicine, vaccines and personal protection equipments). these items are listed in tables , and . they were modified from the questionnaires which had been used in some avian flu studies [ ] [ ] [ ] [ ] and sars studies [ , , , ] . they have also been used in the published baseline h n study [ ] . associations between the independent variables and the dependent variables (avoidance behaviors and negative psychological responses) were assessed by using univariate odds ratios (or) and their respective % confidence intervals (ci). variables that were significant in the univariate analysis were used as candidates for fitting logistic regression models. multivariate or and their % ci were reported. spss . was used for the data analyses with p < . as the level of statistical significance. of all respondents (n = ), . % were males; . % were of age years old or above; . % received some post-secondary education; % were currently married or were cohabitating with someone; % were currently employed full time; and . % were health care workers. the age and gender distributions did not vary across the surveys and were similar to those of the census data (footnote of table ). of all respondents, . % held at least one of the unconfirmed beliefs that h n could be transmitted through airborne spread across long distance (e.g. from a building to another building; . %), via water sources such as reservoirs ( . %), via insect bites ( . %) or via eating well cooked pork ( . %). respectively, . %, . % and . % of the respondents correctly knew that h n is transmittable via droplets, touching the body of infected person or contaminated objects; about . % were correct in all these three items ( table ) . around - % of all respondents believed that the local health system currently did not have enough medication ( . %), vaccine ( . %) or personal protection equipments ( . %) to deal with the h n epidemic, with . % holding at least one of such beliefs ( table ). the majority ( . %) of the respondents was confident in the public's or governmental ability to control the epidemic, i.e., either agreeing with the statements 'hong kong would be able to control the h n epidemic' ( . %) or with the statement that the 'hong kong government would be able to control a large-scale h n outbreak' ( . %). the majority ( . %) of the respondents gave a passing score > for the governmental performance in dealing with h n (range = to , with as the passing mark; table ). about % ( . %) of the respondents believed that there was so far no effective drug available to treat h n ( table ) . around % of the respondents believed that h n is highly fatal ( . %) or could cause severe irreversible bodily damages ( . %; table ). respectively, . % and . % believed that hong kong has a higher or a lower chance of having a large scale h n outbreak in the future year, as compared to other countries. close to % of the respondents perceived a high or very high chance for himself/herself ( . %), his/her family members ( . %) or the general public ( . %) to contract h n in the next year ( table ) . respectively . %, . % and . % of the respondents currently avoided going to crowded places, avoided going out or avoided visiting hospitals. around % ( . %) of the respondents were currently much worried that either they or their family members would contract h n ; . % showed signs of emotional distress (i.e. panicking very much or felt much depressed or were very much emotionally disturbed due to h n ). females, older respondents, those with >= unconfirmed beliefs about modes of h n transmission, those who knew that h n could be transmitted 'via droplets', 'bodily contact with infected person' or 'touching contaminated objects', those who were very worried that either they or their family members would contract h n , those expressing emotional distress (in panic or feeling very depressed or being highly emotionally disturbed due to h n ) were more likely than others to avoid visiting crowded places (multivariate or = . to . , p < . ; table ). females, older respondents, those with >= unconfirmed beliefs about modes of h n transmission, those who knew that h n could be transmitted 'via droplets', 'bodily contact with infected person' or 'touching contaminated objects', those who believed that h n would cause severe irreversible bodily damage, and those expressing emotional distress (in panic or feeling very depressed or being highly emotionally disturbed due to h n ) were more likely than others to avoid going out (multivariate or = . to . , p < . ). those who were full-time employed were less likely than others to avoid going out (multivariate or = . , p < . ; table ). the respondent . % family members . % the general public . % * governmental performance was assessed by items: timeliness of measures taken; effectiveness of implemented measures; clear explanations made to citizens; adequacy of implemented measures; coordination across governmental departments; overall performance of the government. (score range = to , with as the passing mark). an average was calculated for the item scores. # less than % missing cases exist for the listed variables. (score range = to , with as the passing mark). an average score was calculated for the item scores. variables that were not significantly associated with any of the dependent variables in the univariate analysis were not tabulated. these variables include being current health care practitioner, perceived availability of drugs, perceived high chances of contracting the disease for himself/herself, his/her family members and the general public. respondents who were married/cohabited, those with >= unconfirmed beliefs about modes of h n transmission, those who believed that h n would cause severe irreversible bodily damage, and those who were very worried that either they or their family members would contract h n were more likely than others to avoid visiting hospitals (multivariate or = . to . , p < . ; table ). the results of the multivariate analysis showed that those who believed h n would cause severe irreversible bodily damage and those who believed that they themselves had a high chance of contracting h n were more likely than others to be much worried that either they or their family members would contract h n (multivariate or = . and . respectively, p < . ; table ). those who believed either that the general public and/or the local government would be able to control a large scale local h n outbreak were less likely to show the worry (multivariate or = . ). in the multivariate analysis, females, those who doubted about adequacy of governmental preparedness (inadequate vaccine or medication or personal protection equipments in hong kong), those who associated h n with a high fatality, and those who believed that their family members had a high chance of contracting h n were more likely than others to indicate emotional distress (panicking or much depressed or much emotionally disturbed) due to h n . the significant multivariate or ranged from . to . (p < . ; table ). around % of the respondents showed some avoidance behaviors. the studies covered the entire early 'pre-community outbreak phase' of the h n epidemic in hong kong during which all confirmed cases were imported. during the study period, the local government had not given any public health advice about avoiding going to different places, though a previous analysis of our may to may data showed that . % of the public misconceived that such an advice was given [ ] . avoidance of visiting hospital may be due to the fear of getting infected in hospitals, which was prominent during the sars period [ ] . the government only started advising people to avoid crowded places at the 'community outbreak phase' of the epidemic. there seemed to be no serious immediate public health threat for going out or visiting different places. such avoidance behaviors were associated with negative psychological responses; emotional elements may therefore be strongly involved in making the decisions. experience from sars showed that such avoidance behaviors among large numbers in the popula-tion potentially damages the economy and disrupts daily lives. about half of the respondents believed that hong kong has a lower chance of having an h n outbreak as compared to other countries, whilst only less than % held the opposite belief. there were signs of underestimating the risk of having a community outbreak in hong kong [ ] . the shift into the pandemic phase as announced by the who and the explosion of non-imported community cases in hong kong ( as of june , ) may change the picture completely. the direction of change is however uncertain. a few international studies also documented strong levels of anticipated anxiety and avoidance behaviors at the early phase of human avian flu outbreaks or pandemic influenza [ , [ ] [ ] [ ] . the impact of pandemics and unknown emerging infections has not been widely studied. avoidance behaviors and emotional distress may have been under-emphasized in the preparedness plans. it is seen that females, older people and those who were not full-time employed were more likely than others to show avoidance behaviors or signs of emotional distress. the results are consistent with those reported during the sars period [ ] . a recent study exploring people's emotional and behavioral responses to an avian flu outbreak also showed that females and older people were, respectively, more likely to express negative emotional responses and exhibit avoidance behaviors (e.g., avoiding leaving their residence, avoiding crowds and avoiding visiting hospitals) in response to avian flu [ ] . attention should therefore be given to avoidance behaviors and psychological needs of these subpopulations at times of a pandemic. perceptions still count in this context. there were substantial unconfirmed beliefs about the mode of h n transmission ( . % had at least one unconfirmed belief ). around / of the respondents did not know that the virus could be spread by touching contaminated objects. the aforementioned unconfirmed beliefs about transmission mode were significantly associated with avoidance behaviors. unconfirmed beliefs about modes of transmission were also documented in h n studies [ ] , suggesting that similar unconfirmed beliefs exist in general for emerging respiratory infectious diseases. rectification of misconceptions is important -and may decrease and reduce unwarranted anxiety. around % of the respondents believed that h n would result in high fatality or severe irreversible bodily damages. such beliefs may be affected by the sars experience. perceived high fatality was associated with emotional distress (e.g. panic) due to h n and perceived severe irreversible bodily damage was associated with of the outcome variables on avoidance behaviors and negative psychological responses. up-to-date informa- tion about the clinical properties of h n should be disseminated to the public in layman terms. the actual fatality associated with h n , both local and international, remains low. the cost of assurance by hong kong government is however, high -with early summer closure of all primary schools and kindergarten and a number of secondary schools, billion hong kong dollars being spent ( . billion us$) to purchase h n vaccines and reorganization of the health services to accommodate escalating infection figures are not insubstantial. tourism may be adversely affected. a substantial proportion of the public may be overestimating its fatality and physical damages. since public understanding of risk and of these mitigation measures will help to reduce unnecessary concern and changes in lifestyle amongst the population, public education is important. as expected, perceived personal/family susceptibility for contracting h n was associated with negative psychological responses due to h n . the association between perceived personal/family susceptibility and avoiding going out was non-significant. the results suggest that the public did not avoid going out because of feeling susceptible. avoidance behaviors may involve an irrational element. it is speculated that the sars experience of avoiding going to different places [ ] might have a spill-over effect. the general public evaluated the government highly in the performance and ability to control the pandemic. they however, showed reservations about the availability of medicine and vaccine and protective equipments, possibly because h n was a new disease and it was not certain whether effective medicine, vaccine and equipments were then available. the positive evaluations of governmental performance and perceived ability for hong kong or the government to control the h n outbreak were significantly associated with the outcome variables in most of the univariate analyses. nonetheless, most of these associations were statistically non-significant in the multivariate analysis. the associations between such variables and the outcome variables (avoidance behaviors and negative psychological responses) were hence mediated by other variables, such as worry about contracting h n or perceived susceptibility. these potential mediators were multivariately associated with either the avoidance variables or the negative psychological response variables. the study has some limitations. first, this was a crosssectional baseline study. second, the response rate was comparable to those of other relevant published studies but some non-responder bias may still exist [ , , ] . some telephone numbers are unlisted and we randomized the last two digits to cover some of the unlisted numbers. moreover, the gender and age distributions were comparable to those of the census population data. third, results were self-reported and social desirability bias may exist. the study was however anonymous. fourth, hong kong went through unique sars experience, the results may not be comparable with those of other countries. fifth, the measures on negative psychological responses were based on those used in previous studies, rather than derived from some validated scales. finally, the study was not intended to track changes within the short study period of a month -interactions between time and various independent variables were not explored. similar data obtained from other countries are becoming available and can be compared with ours. in sum, the results of this study documented that a noticeable proportion of the public exhibited avoidance behaviors that had not been advised by the government and negative psychological responses at the early 'precommunity outbreak phase' of the h n outbreak. with the relatively mild nature of the h n , and with all the hygiene and public health measures continually reemphasized, an open debate on whether the public should avoid going out during the h n outbreak should be encouraged. it would facilitate appropriate responses and daily lives of people in hong kong, one of the most densely populated cities in the world, remain undisrupted. the study is part of an ongoing surveillance program, which is in place in hong kong. hong kong is now in the pandemic and 'community outbreak phase'. the public needs to be better informed about the modes of transmission and clinical consequences of the disease to make rational behavioral choices. early detection of mental health problems and primary preventions are warranted. comparisons with other parts of the world, such as mainland china, would be very informative. this study provides a better understanding of the factors associated with negative psychological responses due to h n , which would give useful insights to designing primary prevention of mental health distress at the initial phase of this and outbreaks of other emerging respiratory infectious diseases. the opinions expressed by authors contributing to this journal do not necessarily reflect the opinions of the centre for health behaviours research or the institutions with which the authors are affiliated. world health organization: influenza a(h n ) -update who rapid pandemic assessment collaboration: pandemic potential of a strain of influenza a (h n ): early findings severe acute respiratory syndrome(sars) expert committee: sars in hong kong: from experience to action monitoring community responses to the sars epidemic in hong kong: from day to day 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 impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong factors influencing the wearing of facemasks to prevent the severe acute respiratory syndrome among adult chinese in hong kong longitudinal assessment of community psychobehavioral responses during and after the outbreak of severe acute respiratory syndrome in hong kong sars-related perceptions in hong kong. emerging infectious diseases economic impact of sars: the case of hong kong a serial of surveillance surveys of anticipated behavioral and psychological responses to avian influenza pandemic in humans among the hong kong general public (sf- ). oral presentation at the th apacph annual conference perceptions about status and modes of h n transmission and associations with immediate behavioral responses in the hong kong general population anticipated and current preventive behaviors in response to an anticipated human-to-human h n epidemic in the hong kong chinese general population perceptions related to human avian influenza and their associations with anticipated psychological and behavioral responses at the onset of outbreak in the hong kong chinese general population differences in public emotions, interest, sense of knowledge and compliance between the affected area and the nationwide general population during the first phase of a bird flu outbreak in israel crisis prevention and management during sars outbreak widespread public misconception in the early phase of the h n influenza epidemic public perceptions, anxiety, and behaviour change in relation to the swine flu outbreak: cross sectional telephone survey the community's attitude towards swine flu and pandemic influenza swine flu") lay perceptions of the pandemic influenza threat responses to the outbreak of novel influenza a (h n ) in japan: risk communication and shimaguni konjo a population-based study of depression and three kinds of frequent pain conditions and depression in hong kong impacts of sars on health-seeking behaviors in general population in hong kong prevalence and factors of sexual problems in chinese males and females having sex with the same-sex partner in hong kong: a population-based study predicting the anticipated emotional and behavioral responses to an avian flu outbreak pandemic influenza in australia: using telephone surveys to measure perceptions of threat and willingness to comply realities and enigmas of human viral influenza: pathogenesis, epidemiology and control sars preventive and risk behaviours of hong kong air travellers an outbreak of the severe acute respiratory syndrome: predictors of health behaviors and effect of community prevention measures in hong kong the authors would like to thank all participants of this study. thanks are extended to mr. nelson yeung for his help in the early drafts of the manuscript, mr. tony yung and mr. johnson lau for their assistance in the preparation of the questionnaire, ms. mw chan, mr. mason lau, and ms. cheri tong for coordination of the telephone survey and all colleagues who served as telephone interviewers of this study. the study was supported by the research fund for the control of infectious diseases from the food and health bureau. the authors declare that they have no competing interests. jtfl designed and oversaw the study and wrote the manuscript. sg and hyt proposed suggestions to improve study and revised the manuscript intellectually. kcc performed the data analysis. all authors read and approved the manuscript. the pre-publication history for this paper can be accessed here: key: cord- - vcts w authors: chan, kc allen; tang, nelson ls; hui, david sc; chung, grace ty; wu, alan kl; chim, stephen sc; chiu, rossa wk; lee, nelson; choi, kw; sung, ym; chan, paul ks; tong, yk; lai, st; yu, wc; tsang, owen; lo, ym dennis title: absence of association between angiotensin converting enzyme polymorphism and development of adult respiratory distress syndrome in patients with severe acute respiratory syndrome: a case control study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: vcts w background: it has been postulated that genetic predisposition may influence the susceptibility to sars-coronavirus infection and disease outcomes. a recent study has suggested that the deletion allele (d allele) of the angiotensin converting enzyme (ace) gene is associated with hypoxemia in sars patients. moreover, the ace d allele has been shown to be more prevalent in patients suffering from adult respiratory distress syndrome (ards) in a previous study. thus, we have investigated the association between ace insertion/deletion (i/d) polymorphism and the progression to ards or requirement of intensive care in sars patients. method: one hundred and forty genetically unrelated chinese sars patients and healthy volunteers were recruited. the ace i/d genotypes were determined by polymerase chain reaction and agarose gel electrophoresis. results: there is no significant difference in the genotypic distributions and the allelic frequencies of the ace i/d polymorphism between the sars patients and the healthy control subjects. moreover, there is also no evidence that ace i/d polymorphism is associated with the progression to ards or the requirement of intensive care in the sars patients. in multivariate logistic analysis, age is the only factor associated with the development of ards while age and male sex are independent factors associated with the requirement of intensive care. conclusion: the ace i/d polymorphism is not directly related to increased susceptibility to sars-coronavirus infection and is not associated with poor outcomes after sars-coronavirus infection. the outbreak of the severe acute respiratory syndrome (sars) has made a great impact to the health care systems around the world. the pandemic affected over individuals and resulted in deaths worldwide [ ] . several clinical parameters, including male sex [ , ] , age of over years [ , ] , elevated lactate dehydrogenase activity [ ] [ ] [ ] , low platelet count [ ] and high viral load on presentation [ ] , have been identified to be predictive of the severity of the disease in affected individuals. moreover, it has been postulated that genetic variations of the host and the virus may account for the individual difference in the susceptibility to the infection and the severity of the disease. with regard to viral factors, it has been shown that there is no significant difference in the genetic sequences of viruses causing the two major outbreaks in hong kong, namely the prince of wales hospital and amoy gardens outbreaks, despite the significant difference in the mortality rates and diarrheal rates of the two cohorts [ ] . furthermore, several association studies have been conducted to investigate the possible contribution of host genetic factors in the determination of the susceptibility and prognosis of sars-coronavirus infection. thus, certain human leukocyte antigen subtypes have been shown to be more prevalent in sars patients [ ] and in those who had poorer outcomes [ ] . on the other hand, the polymorphism in the angiotensin converting enzyme ii gene, coding for a functional receptor of the sars-coronavirus, is not associated with the susceptibility or outcome of sars [ ] . recently, it has also been reported that the deletion of the bp alu repeat (d allele) in intron of the ace gene is associated with hypoxemia in sars patients [ ] . however, there are several limitations to this previous study. first, only sars patients were studied. second, hypoxemia was arbitrarily defined as requiring oxygen supplementation. moreover, patients who died were excluded from the study. these factors may be potential confounders to a genetic association study. therefore, in this study, we investigated the association of the ace insertion/deletion (i/d) polymorphism of the bp alu repeat to the susceptibility to sars and the development of adult respiratory distress syndrome (ards) with a larger population. this study was reviewed and approved by the ethical committee of the prince of wales hospital, hong kong. patients who were admitted to the hospitals of the new territories east cluster of hong kong for the treatment of sars were recruited retrospectively. the recruitment of patients depended on the availability of blood samples. all patients, including survivors and deceased patients, with available blood samples were recruited. for geneti-cally related sars patients, only the index case (the first individual who developed symptoms) was recruited. all patients were of chinese ethnicity and fulfilled the world health organisation case definition of probable sars [ ] . three hundred and twenty-six healthy individuals undergoing routine health check were recruited as controls. the control subjects were recruited before the sars epidemic and none of them had respiratory symptoms. all control subjects were ethnical chinese and were not genetically related. the association between genotype and disease outcome was studied in the sars patients. two categories of patients were considered as having a severe disease: ( ) patients who developed ards; and ( ) patients who required admission to the intensive care unit (icu). a patient was classified as having ards if he or she fulfilled all criteria of the joint american/european consensus for ards [ ] , including: ( ) acute onset of respiratory distress; ( ) presence of bilateral infiltrates on chest x-ray; ( ) having a ratio of arterial partial pressure of oxygen to inspired fractional oxygen concentration (pao /fio ) of less than . kpa and absence of clinical evidence of left heart failure. dna was extracted from whole blood using a qiaamp dna blood mini kit (qiagen) with the 'blood and body fluid spin protocol' as recommended by the manufacturer. ace i/d genotypes were determined by polymerase chain reaction amplification. the forward and reverse primers were '-ctggagaccactcccatcctttct- ' and '-gatgtggccatcacattcgtcagat- ', respectively. reactions were set up in a volume of µl containing . µm of each primer, x buffer ii (applied biosystems), mm mgcl , . mm of each dntp, . u taq polymerase (amplitaq gold dna polymerase, applied biosystems) and ng dna. after initial denaturation at °c for min, the reaction mixtures were subjected to cycles of °c for min, °c for min and °c for min, and a final extension at °c for min. this method yielded amplification products of bp for the i allele and bp for the d allele. the products were electrophoresed and visualized in % agarose gels with ethidium bromide. statistical analyses were performed using sigmastat, ver. . ; spss. disease associations were compared by chisquare tests. univariate and multivariate logistic regression analyses were performed to identify predictors of ards or the outcome of sars. one hundred and forty sars patients ( males, females) and healthy individuals ( males, females) were recruited. the mean ages of the sars patients and control subjects were . and . years, respectively (p = . ). seventeen of the sars patients developed ards during the course of their illness. the demographic data of the sars patients who had or had not developed ards are summarized in table . patients who developed ards were significantly older than those who did not develop ards (p < . ). there was no significant difference in gender, smoking habits, hepatitis b status and the presence of comorbidity between the two groups. thirty-five patients required intensive care and sixteen died. patients who required intensive care were significantly older than those with milder disease. the genotypic distributions and allelic frequencies of ace i/d polymorphism in the sars patients and control subjects are shown in table . the genotypic distributions of the sars patients and the healthy control subjects follow the hardy-weinberg equilibrium using chi-square analysis. there was no significant difference in the genotypic distributions (χ value = . , df = , p = . ) and allelic frequencies (χ value = . , df = , p = . ) of the two groups. among the sars patients, we further analyzed the genotypic distributions and allelic frequencies of ace i/d polymorphism in patients who developed ards and in those who did not develop ards in the course of their illness. the results are shown in table a. there was no significant difference in the genotypic distributions (χ value = . , df = , p = . ) and allelic frequencies (χ value = . , df = , p = . ) between the two groups. besides, there was also no significant difference in the genotypic distributions (χ value = . , df = , p = . ) and allelic frequencies (χ value = . , df = , p = . ) between patients who did or did not require intensive care. the results are shown in table b. in the univariate analysis, we did not detect any significant difference in the number of d alleles in the ace polymorphism between patients who did and did not develop ards (p = . , or = . ( % ci: . - . ). following multivariate logistic regression analysis, age was found to be the only significant factor that determined the development of ards in sars patients (table a). in the multivariate analysis for the requirement of intensive care, we have shown that age and male sex are associated with the requirement of intensive care (table b) . the possible contribution of host genetic factors to the susceptibility and outcome of sars-coronavirus infection has been investigated through several association studies [ ] [ ] [ ] [ ] . in contrast to a recent report showing an association between the presence of the d allele of the ace gene and hypoxemia in sars patients [ ] , we have shown that the i/d polymorphism of the ace gene is associated with neither increased susceptibility to sars-coronavirus infection nor progression to ards once infected. in multivariate logistic regression analysis, we have identified that age is the only significant factor associated with the development of ards while age and male sex are independently associated with the requirement of intensive care in sars there are several possible explanations for the discrepancies in our conclusion and that by itoyama et al [ ] concerning the association between ace polymorphism and the outcome of sars. first, the inclusion of subjects within the same family and exclusion of deceased patients by the previous study might cause potential bias, especially when the frequency of the dd genotype was reported to be as low as % in control subjects [ ] . in this study, we have only included the index patient if more than one member in a family developed sars. second, we have used a well defined endpoint of ards instead of the requirement of supplemental oxygen. sars infection commonly leads to respiratory distress and over % of patients were given supplemental oxygen during the course of their illness in our cohort. therefore, it seems to be more appropriate to use ards instead of the requirement of oxygen supplement to define the severity of sars. as ards is the more severe end of the spectrum of disease progression, any potential association between genotype and disease progression would become even more obvious when the most severe cases were considered. similarly, the disease outcome was not associated with ace i/ d genotype when we also used another broader definition for severe disease after sars infection (requiring intensive care or death). previous studies on caucasian populations have suggested that the presence of the d allele of the ace gene is associated with increased incidence of ards [ ] . this effect has been postulated to be related to the higher enzyme activity in individuals with dd genotype [ ] . however, it is unclear whether these observations can also be seen in chinese as the frequencies of dd genotype and d allele of the ace gene are much lower in chinese than in caucasian subjects [ , ] . furthermore, the sarscoronavirus characteristically affects the pneumocytes, and the formation of multinucleated pneumocytes and intrabronchial fibrogranulation (bronchiolitis obliterans organizing pneumonia-like lesions) are commonly observed in the lung biopsies of sars patients in addition to the typical pathological changes of ards [ ] . therefore, it is possible that the pathogenesis and genetic factors predisposing to sars-related ards may be different from ards resulted from other respiratory illnesses. previous reports have highlighted the inconsistency of the results of genetic association studies for complex diseases [ , ] . this inconsistency may be attributable to the difference in the genetic composition of the studied population and study design. here, we showed that both susceptibility and disease outcome of sars infection were not associated with ace i/d polymorphism among chinese patients in contrast to the recent report studying vietnamese patients [ ] . the sample size was definitively larger in our study. two different better-defined criteria were used as indicators of severe disease progression, yet no association was found between disease severity and ace i/d genotype. the d allele which was the hypothetical high risk allele [ ] , did not show any sign of over-representation in the subgroups of patients with severe disease. our analysis indicates that ace i/d polymorphism is not directly related to poor outcomes after sars-coronavirus infection in chinese. cumulative number of reported probable cases of sars severe acute respiratory syndrome: clinical outcome and prognostic correlates hong kong epidemic: an analysis of all patients serum ld isoenzyme and blood lymphocyte subsets as prognostic indicators for severe acute respiratory syndrome quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome genomic characterisation of the severe acute respiratory syndrome coronavirus of amoy gardens outbreak in hong kong association of human-leukocyte-antigen class i (b* ) and class ii (drb * ) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome association of hla class i with severe acute respiratory syndrome coronavirus infection ace gene polymorphisms do not affect outcome of severe acute respiratory syndrome ace polymorphism and progression of sars who: case definitions for surveillance of severe acute respiratory syndrome (sars) the american-european consensus conference on ards. definitions, mechanisms, relevant outcomes, and clinical trial coordination angiotensin converting enzyme insertion/deletion polymorphism is associated with susceptibility and outcome in acute respiratory distress syndrome relationship between angiotensin-converting enzyme id polymorphism and vo( max) of chinese males alhenc-gelas f: angiotensin iconverting enzyme in human circulating mononuclear cells: genetic polymorphism of expression in t-lymphocytes angiotensin i-converting enzyme (ace) gene polymorphism and breast cancer risk among chinese women in singapore pulmonary pathological features in coronavirus associated severe acute respiratory syndrome (sars) problems of reporting genetic associations with complex outcomes candidate gene case-control association studies: advantages and potential pitfalls the work is supported by the research fund for the control of infectious disease (rfcid) from the health, welfare and food bureau of the hong kong sar government. we thank coral lee, wb lui and katherine chow for technical support. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -zntwwqod authors: dabisch-ruthe, mareike; vollmer, tanja; adams, ortwin; knabbe, cornelius; dreier, jens title: comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder assay and respifinder smart assay date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: zntwwqod background: a broad spectrum of pathogens is causative for respiratory tract infections, but symptoms are mostly similar. therefore, the identification of the causative viruses and bacteria is only feasible using multiplex pcr or several monoplex pcr tests in parallel. methods: the analytical sensitivity of three multiplex pcr assays, respifinder- , respifinder-smart- and xtag-respiratory-virus-panel-fast-assay (rvp), were compared to monoplex real-time pcr with quantified standardized control material. all assays include the most common respiratory pathogens. results: to compare the analytical sensitivity of the multiplex assays, samples were inoculated with different quantified viruses in the range of ( ) to ( ) copies/ml. concordant results were received for rhinovirus, whereas the rvp detected influenzavirus, rsv and hmpv more frequently in low concentrations. the respifinder- and the respifinder-smart- showed a higher analytical sensitivity for adenoviruses and coronaviruses, whereas the rvp was incapable to detect adenovirus and coronavirus in concentrations of ( ) copies/ml. the respifinder- and respifinder-smart- a did not detect influenzaviruses ( ( ) copies/ml) and rsv ( ( ) copies/ml). the detection of all viruses in one sample was only achieved using monoplex pcr. to analyze possible competitive amplification reactions between the different viruses, samples were further inoculated with only different viruses in one sample. compared to the detection of viruses in parallel, only a few differences were found. the incidence of respiratory viruses was compared in tracheal secretion (ts) samples (n = ) of mechanically ventilated patients in winter (n = ) and summer (n = ). in winter, respiratory viruses were detected in ts samples ( %) by respifinder- , whereas the detection rate with rvp was only %. the most frequent viruses were adenovirus ( %) and piv- ( %). multiple infections were detected in ts samples ( %) by respifinder- . fewer infections were found in summer (respifinder- : %; rvp: %). all positive results were verified using monoplex pcr. conclusions: multiplex pcr tests have a broad spectrum of pathogens to test at a time. analysis of multiple inoculated samples revealed a different focus of the detected virus types by the three assays. analysis of clinical samples showed a high concordance of detected viruses by the respifinder- compared to monoplex tests. acute respiratory tract infections are the most widespread type of infection in adults and children and are responsible for a considerable morbidity and mortality worldwide. a high rate of respiratory tract infections is caused by viruses (approximately %) [ , ] . within the last ten years, diagnosis of respiratory viruses has become more important, because of the unexpected emer- however, an efficient pathogen-based prophylaxis or therapy has a significant effect on the disease progress in patients [ , ] . to overcome limitations concerning the use of several monoplex tests in parallel and the resulting shortage of sample volume, the development of multiplex tests for a fast and exact identification is necessary. today, several multiplex tests are commercially available [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . a number of studies have already compared the detection frequencies of multiplex assays with conventional monoplexpcr assays in clinical samples [ , [ ] [ ] [ ] , but a comparison of the analytical sensitivity of these multiplex assays with quantified standardized control material does not exist. this study presents the first comparison of the analytical sensitivity of three novel multiplex pcr methods, the respifinder- assay, respifinder-smart(single tube multiplex amplification in real-time)- assay (both pathofinder, maastricht, netherlands) and the xtag respiratory virus panel fast assay (abbott molecular, wiesbaden, germany), with quantified virus control material. the respifinder- assay, which is based on the multiplex ligation-dependent probe amplification analyzed by capillary electrophoresis, detects respiratory viruses and bacteria in one reaction [ ] . a further development of the respifinder- assay is the respifinder-smart- assay, which is also based on the multiplex ligation-dependent probe amplification. this assay differentiates respiratory viruses and bacteria in one reaction by melt curve analysis. the xtag respiratory virus panel fast assay (rvp) is a bead array-based system for the detection of different respiratory viruses [ , ] . previous studies with clinical samples showed that the sensitivity and specificity of the rvp assay was . % and . %, respectively, compared to real-time pcr-methods, that are currently declared as the gold standard [ ] . another study performed a comparison with clinical samples of the respifinder- with the precursor assay of the rvp [ ] . both assays in this study have an excellent specificity and the sensitivity was % and % for the rvp and the respifinder- assay, respectively. the aims of the present study were as follows: (a) the quantification of commercially available qualitative control material for (b) the evaluation of the performance and sensitivity of the three multiplex assays respifinder- , respifinder-smart- and rvp fast assay and (c) the applicability and performance of these multiplex pcr assays in a routine setting. furthermore, this is the first screening study determining the incidence of infections with respiratory pathogens in a mechanically ventilated patient cohort developing an atypical pneumonia during post-operative monitoring using multiplex pcr assays. tracheal secretion (ts) samples (n = ) were collected in february (winter, n = ) and july (summer, n = ) from mechanically ventilated non-immunocompromised patients (male: %, female: %, mean age . ± . years, range - ). patients were suspected of atypical pneumonia during postoperative monitoring after coronary artery bypass, heart or lung surgery. ts samples were initially analyzed for bacterial and viral pathogens with our routine diagnostic profile including cmv, legionella pneumophila, pneumocytisjirovecii, mycoplasma pneumoniae and chlamydophilapneumoniae. the residual material was used for the comparative analysis with the respifinder- and rvp assay. the results of the performance evaluation were received retrospectively and had no influence on patient's therapy. all patients provided informed consent. for our study we did not need an ethical approval, because in paragraph } of the german act on medical devices (in german: gesetzuebermedizinprodukte -mpg) an ethical approval is not required for clinical specimens without a separate invasive sampling. the qualitative respiratory validation panel global (natrvp- , zeptometrix corporation, buffalo, new york) was quantified by real-time qpcr. external plasmid standards were used to determine the concentration of the different viruses. plasmid standards of adenovirus (from position to , accession number ac_ ), influenza a virus (from position to , accession number cy ), rsv-a (from position to , accession number m ), enterovirus (from position to , accession number d ) and rhinovirus (from position to , accession number d ) were established [ ] . samples were analyzed in triplicate in three independent pcr assays. the viruses hmpv, piv- , piv- and piv- were quantified by lightmix assays (tib molbiol, berlin, germany). the virus controls of the natrvp- are purified intact virus particles that have been chemically modified to render them non-infectious and refrigerator stable. quantifications of viruses included in this panel were as follows: influenza a virus (h : . e + copies/ml, h : . e + copies/ml), rsv-a ( . e + copies/ml), adenovirus ( . e + copies/ml), rhinovirus ( . e + copies/ml), hmpv ( . e + copies/ml), piv- ( . e + copies/ ml), piv- ( . e+ copies/ml) and piv- ( . e+ copies/ml) ( table ) . for the undiluted samples with viruses, μl of each virus were mixed (total volume: μl). accordingly, for the undiluted samples inoculated with viruses, μl of each virus were mixed and filled up with pbs to achieve also a final volume of μl. subsequently, μl of the sample was extracted using the nuclisenseasymag automated system with an elution volume of μl. the total nucleic acid from inoculated samples and tracheal secretion samples (ts) was extracted using the nuclisenseasymag automated system (biomérieux, nürtingen, germany) according to the manufacturer's instructions. nucleic acids were extracted from μl of ts samples and μl of inoculated samples and were eluted in a final volume of μl (routine diagnostic) or μl elution buffer, respectively (biomérieux, nürtingen, germany). the manufacturer of the respi-finder- and smart- assays (pathofinder) recommend an elution volume of μl, whereas the manufacturer of the rvp assay recommend an elution volume of μl. to avoid false negative results due to a deviation from the manufacturer instructions, we extracted residual material and used an elution volume of μl. the nucleic acid extracts were tested using the rvp assay (abbott molecular, wiesbaden, germany) according to the manufacturer's instructions. the rvp employs a multiplex pcr with labelled primers and a single-step hybridization of pcr products to the fluorescent bead array. the detection was performed using the xmap is instrument (luminex molecular diagnostics inc., toronto, canada) and the analysis was performed using tdas rvp fast software (version . , abbott molecular). the rvp simultaneously detects influenza a virus (subtyped as h , h or h ), influenza b virus, rsv-a and -b, adenovirus, hmpv, piv- , - , - and − , coronaviruses e, nl , oc and hku , picornavirus (enterovirus and rhinovirus) and human bocavirus. the assay also includes an internal positive control added to each specimen at the extraction step (phage ms ) and a positive run control that is added to each plate (phage lambda dna) [ ] . the respifinder- and the respifinder-smart- (both pathofinder, maastricht, the netherlands) were used according to the manufacturer's instructions except for the nucleic acid extraction of patient samples, because we use μl of ts samples instead of μl as recommend. elution volumes were used as described previously. briefly, the assays comprised a preamplification step, which combines reverse transcriptase and multiplex target amplification pcr, followed by a probe hybridization step, a probe ligation step and a probe amplification step. the respifinder- analyzes the amplified pcr products by capillary electrophoresis using a dna analyzer (abi , applied biosystems, darmstadt, germany), whereas the respifinder-smart- analyzes by melt curve analysis on the rotorgene q (qiagen, hilden, germany). the respifinder- simultaneously detects respiratory viruses (adenovirus, coronaviruses e, nl , oc , hmpv, influenza a virus, influenza a virus h n , influenza b virus, piv- , - , - and − , rsv-a and -b, rhinovirus) and four bacteria (bordetella pertussis, chlamydophila pneumoniae, legionella pneumophila, mycoplasma pneumoniae). the respifinder-smart- additionally detects coronavirus hku , enterovirus and bocavirus. both assays also tests an internal positive control added to each specimen at the extraction stage (rna transcript of the polyprotein gene from encephalomyocarditis virus) [ ] . pcr primers and probes were adapted as previously described: adenovirus [ ] , coronavirus (types nl , hku- , oc , e) [ ] , cytomegalovirus (cmv) [ ] , enterovirus [ ] , influenza a virus [ ] , rsv-a [ ] , rhinovirus [ ] and legionella pneumophila [ ] . the bacteriophage ms was used as an internal control (ic) for the reverse transcription pcr as previously described [ ] . a bp fragment of the bacteriophage lambda (position to , accession number j ) was used as ic to avoid competitive co-amplification of dna viruses. rna amplification of coronavirus (types nl , hku- , oc , e), influenza a virus, rsv-a and rhinovirus was carried out in . ml tubes containing μl reaction mix and μl rna extract. the reaction mix consisted of × invitrogen rnx-reaction mix (including mm mgso ), nm of target primers, nm of the target probe, nm of ic primers, nm of the ic probe and u of invitrogen superscript platinum taq-enzym mix (superscript iii one-step rt-pcr with platinum-taq, invitrogen, darmstadt, germany). a -bp pcr product of the bacteriophage ms replicase gene was added to the reaction mixture as an exogenous ic. pcr was performed on the rotorgene q system (qiagen, hilden, germany) with a reverse transcription at °c for min, preliminary denaturation at °c for min, followed by cycles of denaturation of °c for s, annealing and extension at °c for s, with a single fluorescence acquisition step at the end of the annealing step. dna amplification of cmv, adenovirus and enterovirus was carried out in . ml tubes containing μl reaction mix and μl dna extract. the reaction mix consisted of × taq buffer (including mm mg), nm of each deoxynucleoside triphosphate, nm of target primers, nm of the target probe, nm of ic primers, nm of the ic probe, . u of uracil-dna glycosylase (ung, roche diagnostics, mannheim, germany) and u of taq dna polymerase ( prime, hamburg, germany). pcr was performed on the rotor-gene system (corbett life sciences, sydney, australia) with an ung activity step at °c for min, preliminary denaturation at °c for min, followed by cycles of denaturation of °c for s, annealing and extension at °c for s, with a single fluorescence acquisition step at the end of the annealing step. the legionella pneumophila pcr was carried out in glass capillaries containing μl reaction mix and μl dna extract. the reaction mix consisted of × faststart dna master plus hybridization probes (roche diagnostics, mannheim, germany), nm of each target primer, nm of the target probe, nm of each ic primer for lambda, nm of the ic probe and . u of ung (roche diagnostics, mannheim, germany). pcr was performed on the lightcycler . system (roche diagnostics, mannheim, germany) with an ung activity step at °c for min, preliminary denaturation at °c for min, followed by cycles of denaturation of °c for s, annealing at °c for s and extension at °c for s, with a single fluorescence acquisition step at the end of the annealing step, and a following melt analysis ( °c for s and °c for s). the real-time pcr for hmpv, piv- , - and − was performed using lightmix assays (tib molbiol, berlin, germany) on the lightcycler . system according to the manufacturer's instructions. the cut-off value for the decision positive/negative was adjusted to < cycles. the specificity of all monoplex-real-time pcr assays was determined by the exclusion of cross-amplification with different bacterial or viral dnas/rnas (eight bacteria, seven viruses). the analytical sensitivity was determined to be < copies/ ml. the reproducibility of the assay was demonstrated by analyzing the inter-assay variation for the crossing threshold (ct) values, determined from six independent pcr runs. key parameters for real-time pcr assays are shown in table . virus control material, natrvp (zeptometrix), was quantified by external plasmid standards for a comparison of the analytical sensitivity of the rvp, respifinder- and respifinder-smart- assays. subsequently, pbs-buffer was inoculated with different quantified viruses, or viruses in different combinations, in the range of - copies/ml. nucleic acid extracts were analyzed in parallel with the three multiplex methods and monoplex real-time pcr methods (elution volume μl). the rvp showed the detection of influenzavirus a (inf-a, . e + copies/ml), respiratory syntical virus a and b (rsv-a/b, . e + copies/ml), coronavirus oc (cov oc ) and human metapneumovirus (hmpv, . e + copies/ml) even in a high dilution ratio ( table ). rhinovirus (hrv) was also detected in low concentration by rvp; however this assay is not able to differentiate between human rhinovirus and enterovirus due to the high sequence similarity (manufacturer's information). inf-b, cov e, parainfluenzavirus - (piv- - ) and adenovirus (adv) were not detected. in contrast, the respifinder- and the respifinder-smart- detected adv and cov oc and e also at low concentrations (e.g. adv . e + ). however, both assay did not detect inf-a or -b, rsv-a or b, piv- and − as well as hmpv in concentrations in the range of copies/ml. only the respifinder-smart- detected piv- at a concentration of copies/ml). none of the three multiplex assays was capable of detecting all viruses in parallel. this was only achieved using monoplex real-time pcr assay. ct values were shown in table . in order to simulate clinical-relevant multiple infections as well as to analyse possible competitive amplification reactions between the different viruses, pbs-buffer was further inoculated with only different viruses in one assay ( table ) table . only a few differences were found using the same elution volume of μl. rsv-a and adv were now detected by the respifinder- and respifinder-smart- assays at concentrations ranging from copies/ml (panel ) to copies/ml (rsv panel , adv panel ). the rvp assay now detect adv ( copies/ml panel , copies/ml panel ) and piv- ( copies/ml, panel ). the rvp was still incapable of detecting cov e. however, more differences were found in the detection of the pathogens using different elution volumes for both respifinder assays. the company pathofinder suggests an elution volume of μl for the respifinder- and the respifinder-smart- assay, whereas abbott molecular advises for the rvp assay an elution volume of μl. the two assays of the company pathofinder did not detect inf-a in samples which were extracted in μl elution buffer, but detection was possible by using the advised extraction volume of μl. the respifinder- detect inf-a at a concentration of copies/ml (panel ), whereas both assays detect inf-a at concentrations ranging from - copies/ml (panel ). the same observation was made for the detection of adv at a concentration of copies/ ml (panel ) with the respifindersmart- by using the advised extraction volume of μl. in contrast, the detection frequency of rsv-a virus was reduced for both respifinder assays analysing samples with μl elution volume compared to μl elution volume (panel : undiluted sample; panel : respifinder all samples, respifindersmart- dilution : ). no differences in the detection frequency were found for the rvp assay ( table ) . additionally, we evaluated the applicability of the rvp and the respifinder- for routine diagnosis and the distribution of viruses in our patient cohort in ts samples during winter (february) and summer (july). the respifinder-smart- as a further development of the respifinder- was not participating in this study, because it started before the respifinder-smart- was commercially available. all positive results were verified with real-time pcr as monoplex analysis. ts samples were obtained routinely from mechanically ventilated patients after coronary artery bypass, heart or lung surgery who were suspected of atypical pneumonia during post-operative monitoring. respiratory viruses were detected by respifinder- in ts samples from winter ( %) (figure ). the most frequent viruses were adv, which was found in ts samples ( %), and piv- , which was found in ts samples ( %). other detected viruses were cov e ( %), piv- ( %), hrv ( %), inf-b ( %), rsv-a ( %), inf-a ( %) and hmpv ( %). in comparison, the rvp detected respiratory viruses only in ts samples ( %). rhinovirus, which was found in ts samples ( %), was the most frequent virus in addition to cov e ( %), cov hku ( %), adv ( %), piv- ( %), rsv-a ( %) and hmpv ( %). in summer fewer figure comparison of rvp, respifinder- , and real-time pcr results for ts samples in winter and summer. the rectangular boxes symbolize the different assays. * l. pneumophila is not in the spectrum of pathogens of the rvp and cov hku is not in the spectrum of the respifinder- . respiratory virus infections were found in the ts samples by the different assays. the respifinder- detected in ts samples ( %) respiratory viruses in contrast to the rvp, which found viruses only in ts samples ( %), respectively ( figure ) . furthermore, the spectrum of detected viruses was smaller in summer: adv ( %), hrv ( %) and piv- (both %). additionally, l. pneumophila was detected by the respifinder- ( %). in order to confirm positive results obtained by rvp and respifinder, samples were additionally tested by monoplex real-time pcr with % concordance (figure ). the analyses of three ts samples with rvp failed ( table ). the error message "sample failure in saline due to unexpected control call" appeared. the manufacturer provided the information that this is possible, if the sample is positive for a pathogen which is not in the spectrum of pathogens of the kit. in these three cases the samples contained cmv (detection only with monoplex real-time). in order to exclude cmv pneumonia due to reactivation processes, samples had already been tested for cmv in line with our standard diagnostic profile, and data was provided for completeness. in ts samples ( %) from february, multiple infections were detected (table ) . one triple infection with inf-b, piv- and cmv was found. double infections with adenovirus and piv- ( %) as well as adenovirus and cmv ( %) were the most frequent combination. in july, double infections were only detected in ts samples ( %). the detection of multiple infections was only possible with respifinder- . the highest detection rate of viruses in all samples was reached with the respifinder- . a confirmation reaction was performed with monoplex pcr assay, which found a total of viruses in ts samples. the viruses which were only detected with respifinder- showed high cycle threshold points in monoplex pcr methods due to low virus concentrations. the detection of respiratory viruses by multiplex pcr has been described as an important tool for the identification of the pathogens in respiratory tract infections. but until today no comparison of these three multiplex pcr methods with quantified standardized virus control material was performed. therefore, we quantified virus control material to compare the analytical sensitivity of three commercially available multiplex pcr methods, followed by evaluation of application for routine diagnosis with regard to hands-on-time, time-to-result, costs and accomplishment (table ) . we observed that the rvp, the respifinder- and the respifinder-smart- assay had a different focus of the detected virus types in the inoculated samples. the rvp showed an advanced detection of inf-a, rsv, and hmpv, whereas the respifinder- and the respifinder-smart- showed an improved detection of cov e and adv. the respifinder-smart- further showed an improved detection of piv- . viruses were detected also at low concentrations of - copies/ml. cov oc and hrv were detected equally also at low concentrations with the three assays, whereas piv- and piv- were not detected at all. the parallel analysis with monoplex real-time pcr assays showed as expected the highest analytical sensitivity: all viruses were detected in all concentrations and dilution factors. our results indicated a possible competition for nucleotides, primer or enzymes between the different viruses in the detection of a high number of multiple infections ( viruses in one assay). viruses in one sample was the maximum demand on the multiplex pcr tests. this highly artificial experiment emphasizes the general methodological limitation of multiplex pcr assay, because nearly all multiplex assays performed terribly analyzing samples with a high number of parallel pathogens. therefore, these results have to be interpreted with attention. in the routine clinical setting, a parallel infection with this high number of different viruses is uncommon up to impossible, and the clinical relevance of these results is initially arguable. the highest virus load in our patient cohort was a triple infection. this was also observed by other research groups [ ] . due to this fact we tested three different combinations of quadruple infections. surprisingly, only a few differences were found compared to the highly artificial experiment with viruses. potential competitive reactions were found for the detection of rsv and adv (respifinder) or inf-a, adv and piv- (rvp). however, some viruses were still not detected (e.g. rvp: cov e, respifinder: inf-a). but we observed an influence of the elution volumes for the detection with the respifinder- and the respifinder-smart- . an elution volume of μl showed the tendency of a higher analytical sensitivity. however, the elution volume seems to have differing influences depending on the contained viruses. for example, inf-a was detected more frequent in an elution volume of μl, whereas rsv showed a higher detection frequency using an elution volume of μl. no differences were found in the detection with the rvp assay, although we expected that the dilution of the dna/rna extract may result in a reduced analytical sensitivity. for routine testing using this different multiplex assays in parallel, we suggest an elution volume of μl, because the remaining extract is available for retesting or analysis for non-included pathogens. if only one multiplex assay will be implemented, elution should be performed according to the specifications of the manufacturer. the analysis of ts samples was done in parallel with the rvp and the respifinder- . at that time, the respifinder-smart- was not commercially available and later on, no residual extracted material was available for additional testing. for a comparison of the three multiplex assays with clinical samples further studies have to be done. this study indicated a higher clinical sensitivity of the respifinder- in the detection of virus infections in clinical samples with low concentrations in contrast to the rvp. respiratory tract infections were found in % of the ts samples by respifinder- . the rvp detected only % in the same samples, respectively. gadsby et al. [ ] also described problems in the detection of low virus concentrations with rvp; either the pathogens were not detected or they produced a false-positive for adenovirus. raymaekers et al. [ ] investigated clinical samples for a comparison of the respifinder- with the precursor assay of the rvp (xtag respiratory viral panel assay, abbott molecular, wiesbaden, germany). the precursor assay of the rvp included a target specific primer extension (tspe) in contrast to the new assay, therefore these results are not directly comparable to the present study. in raymaekers' study, respiratory viruses were detected by rvp in of clinical samples and by respifinder- in of . these results are comparable to our results and verified our results in present study. a comparison of the precursor assay of the rvp with the newer rvp assay was made by pabbaraju et al. [ ] . they showed that the older assay was more sensitive than the newer rvp fast assay ( . % and . % sensitivities, respectively) for all the viral targets combined. this corresponds to our observations concerning the respifinder- . however, the higher sensitivity of the respifinder- assay may also be due to the detection of incidental but non-causal viruses. in % of the february ts samples the respifinder- assay detected multiple infections, corresponding to results of fox [ ] , whereas the rvp assay detected no multiple infections. raymaekers et al. also described problems in the detection of co-infections table patient samples with positive pathogen detection from winter and summer with the rvp. samples which include adenovirus and coronavirus were false-negative with the rvp. we observed these problems in samples, which were positive for cmv and adenovirus. the importance of viral coinfections remains uncertain [ ] [ ] [ ] . in summary, we detected multiple infections in % of the ts samples analyzed and the detection of multiple infections was only possible using the respifinder- assay. however, other studies also demonstrated the detection of multiple infections using the rvp assay [ , ] . in this context, the question arises, whether all detected viruses in multiple infections are clinically relevant or not. in some cases the pathogens of the co-infections cause more serious illnesses, e.g. coronavirus, rsv or hmpv, than the pathogens of the primary infection, e.g. rhinovirus. with this study approach, none of the three assays can differentiate between pathogens of the primary infection and the co-infecting virus. therefore, the clinical relevance of additionally detected viruses has to be correlated in further studies. the interpretation of the generated data of the three assays has different levels of difficulty. abbott molecular supplies with the rvp a software tool (tdas rvp fast software), which analyze the generated data and provided automatically a positive or negative signal for the detection of the particular virus. the interpretation of prices for one sample. the personal training will be very low, if technicians are familiar with sequencing using an abi system. the results of the respifinder- is more difficult, because the manufacture did not provide helping data for the analysis (e.g. threshold). so it is possible that low positive samples were missed. the further developed respifinder-smart- , has an improved analysis of the generated data with given melting points. in general, the comparison of the spectrum of pathogens detected in our patient cohort of mechanically ventilated patients with other studies (e.g. [ ] ) is hardly realizable, because the comparability is limited by regional, seasonal and methodological differences. in this context, the development of a respiratory networks (e.g. www.medical-dpc.com/respvir) will allow a constant comparison of seasonal accumulations and chronological trends. for the application of the three multiplex pcr methods in a routine diagnostic setting, the hands-on-time, the time-to-result, the costs and the accomplishment are also very important parameters. table summarizes the specifications of respifinder- , respifinder-smart- and rvp. the time-to-result differs from three and a half hours with rvp to eight hours with respifinder- . the respifinder- also needs more hands-on-time steps than the rvp. the respifinder-smart- , as a further development of the respifinder- , delivers a result after six hours. this amounts to a time saving of one and a half hour. for a significant effect on disease progress, patients with respiratory tract infections need an efficient pathogen based prophylaxis or therapy. multiplex pcr methods allow a fast and exact identification of the causative pathogens. for routine application the analytical sensitivity must balance out the time-toresult. in our opinion, the turn-around-times of the respifinder-smart- and rvp assay did not have a significant impact on patient treatment, because both assays provided results within one working day. in this context, the respifinder- demands for a strict time management to achieve results within a day. study limitations: unless samples were not analyzed in an adequate number of replicates to perform statistical analysis, our approach allow the comparison of the performance and the range of sensitivity of the three multiplex assays by analysis of inoculated samples with quantified virus material. reported differences may be due to chance regarding a single consideration of individual results, however a tendency towards difference in lower or higher detection efficiencies by the different assays is observable. in conclusion, our study shows that before a multiplex pcr method is applied in routine diagnostics it has to balance out the analytical sensitivity between time-to-result, hands-on-time and the clinical relevance of the detected pathogens. the respifinder- has a higher analytical sensitivity than the rvp, but needs more than twice as long for a result compared to the rvp. the respifinder-smart- , as a further development provides a faster result, but has to been tested with routine samples in further studies. osterhaus adme: a newly discovered human pneumovirus isolated from young children with respiratory tract disease mimivirus in pneumonia patients b: identification of a new human coronavirus clinical and molecular epidemiological features of coronavirus hku -associated community-acquired pneumonia cloning of a human parvovirus by molecular screening of respiratory tract samples new dna viruses identified in patients with acute viral infection syndrome timely diagnosis of respiratory tract infections: evaluation of the performance of the respifinder assay compared to the xtag respiratory viral panel assay xtag rvp assay: analytical and clinical performance comparison of the luminex xtag respiratory viral panel with xtag respiratory viral panel fast for diagnosis of respiratory virus infections respiratory virus surveillance and outbreak investigation human bocavirus: prevalence and clinical spectrum at a children's hospital journal of clinical virology respiratory viral diagnostics -the pathogens, the challenges and the solutions -introducing the xtagtm respiratory viral panel comparison of the eragen multi-code respiratory virus panel with conventional viral testing and real-time multiplex pcr assays for detection of respiratory viruses epidemiology and clinical outcome of viruspositive respiratory samples in ventilated patients: a prospective cohort study tropheryma whipplei infection of an acellular porcine heart valve bioprosthesis in a patient who did not have intestinal whipple's disease principles of the xtag respiratory viral panel assay (rvp assay) quantitative real-time pcr assays for detection of human adenoviruses and identification of serotypes and development and implementation of a molecular diagnostic platform for daily rapid detection of respiratory viruses real-time monitoring of cytomegalovirus infections after stem cell transplantation using the taqman polymerase chain reaction assays respifinder: a new multiparameter test to differentially identify fifteen respiratory viruses comparison of the luminex respiratory virus panel fast assay with in-house real-time pcr for respiratory viral infection diagnosis lion t: real-time quantitative pcr assays for detection and monitoring of pathogenic human viruses in immunosuppressed pediatric patients increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms real-time reverse transcription-pcr assay for comprehensive detection of human rhinoviruses real-time pcr assay targets the s- s spacer for direct detection and differentiation of legionella spp. and legionella pneumophila use of bacteriophage ms as an internal control in viral reverse transcription-pcr assays comparison of three multiplex pcr assays for the detection of respiratory viral infections: evaluation of xtag respiratory virus panel fast assay, respifinder assay and respifinder smart assay the results of our analysis of ts samples were registered with the network of respiratory viruses (www.medical-dpc.com/respvir). this network was founded for the acquisition, documentation and statistical analysis of epidemiological data from respiratory tract infections caused by viruses. this study was fundedbythe german aif (arbeitsgemeinschaft industrieller forschungsvereinigungen "otto von guericke" e.v.). we thank sarah kirkby for her linguistic advice.author details institut für laboratoriums-und transfusionsmedizin, herz-und diabeteszentrum nordrhein-westfalen, universitätsklinik der ruhr-universität bochum, bad oeynhausen, germany. institut für virologie, universitätsklinikum, düsseldorf, germany. the authors declare that they have no competing interests. submit your manuscript at www.biomedcentral.com/submit key: cord- -lkrmg qr authors: xie, yewei; wang, zaisheng; liao, huipeng; marley, gifty; wu, dan; tang, weiming title: epidemiologic, clinical, and laboratory findings of the covid- in the current pandemic: systematic review and meta-analysis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: lkrmg qr background: the covid- pandemic has affected the world deeply, with more than , , people infected and nearly , deaths. this review aimed to summarize the epidemiologic traits, clinical spectrum, ct results and laboratory findings of the covid- pandemic. methods: we scoped for relevant literatures published during st december to th july based on three databases using english and chinese languages. we reviewed and analyzed the relevant outcomes. results: the covid- pandemic was found to have a higher transmission rate compared to sars and mers and involved stages of evolution. the basic reproduction number (r( )) is . ( % ci: . – . ), the incubation period was . days ( % ci: . – . , studies) on average, and the average time for symptoms onset varied by countries. common clinical spectrums identified included fever ( . – . °c), cough and fatigue, with acute respiratory distress syndrome (ards) being the most common complication reported. body temperatures above . °c, dyspnea, and anorexia were more common symptoms in severe patients. aged over years old, having co-morbidities, and developing complications were the commonest high-risk factors associated with severe conditions. leucopenia and lymphopenia were the most common signs of infection while liver and kidney damage were rare but may cause bad outcomes for patients. the bilateral, multifocal ground-glass opacification (ggo) on peripheral, and the consolidative pulmonary opacity were the most frequent ct results and the tendency of mortality rates differed by region. conclusions: we provided a bird’s-eye view of the covid- during the current pandemic, which will help better understanding the key traits of the disease. the findings could be used for disease’s future research, control and prevention. the emergence of covid- has made it the first infectious disease pandemic in the twenty-first century. as of th july , a total of , , people got infected, and , were confirmed dead in countries, territories, and areas globally [ ] . while more than countries had issued the highest level of response, the sars-cov- (pathogen of continues to spread in different regions around the world [ ] . however, the key information on the virus epidemiology, clinical spectrum, and on the pathogen was delayed in response during the early outbreaks in many countries. to fill the research gaps mentioned above, this review article systematically summarizes global findings on the natural history, clinical spectrum, transmission patterns, laboratory findings, ct results, and risk factors of the covid- . we searched for publications in epidemiology and clinic domains of the covid- broadly. the databases we searched were: chkd v . of the cnki [in chinese], pubmed, and medrxiv, by using such search terms as 'covid- , sars-cov- , and ncov' (see additional file ). the publication date was restricted from st dec to th jul . both english and chinese were applied for the search. only the full-text available human studies were eligible for selection. like the realtime data, other data were obtained from health departments of multiple countries, global ngos, and reputable media sources. the searched records were firstly screened by reading titles and abstracts. then, the rest records were screened again by full-text reading. if there were disagreements initially, the records then submitted to the whole team for further discussions. besides, a prisma diagram was conducted to illustrate the entire flows of the review (fig. ). the data for the quantitative analysis was extracted and managed by using microsoft excel (microsoft©, redmond, wa, usa). the meta-analysis was performed by the r version . and rstudio ( ) [ ] . the cochrane handbook for systematic reviews of interventions suggested review authors collect missing data from investigators. considered that using the imputation method to tackle the missing data problem could not reduce bias, we only analyse data available to us if we could not collect the missing data from the investigators [ ] . the heterogeneity of the included studies was assessed by using i . the p-value was generated by wald-type test and likelihood-ratio test. the overlapping confidence intervals (cis) were displayed by the forest plots (see additional file ). we categorized and combined the data about epidemiologic traits, clinical spectrums, laboratory, and imageology findings in a narrative. then we further analyze the data about common symptoms, reproduction number, and incubation period through meta-analysis. the quantitative outcomes were combined with the narrative of epidemiological and clinical findings. we collected , records after removing duplications. after three batches of screening, records were included in this review (see screening details in fig. ). in a china based study involving , covid- patients, the majority of patients were aged - ( . %) with only . % of the patients being years and below. the median age of the patients was (ranged days- years old) [ ] . similarly, in the united states, more than half of patients were aged between and years ( %), with only % of patients being under years old. older aged patients were more prone to getting infected compared to the young [ ] . by gender, the male to female ratio of confirmed cases was . : . in china [ ] . however, in south korea and iceland, the male population had a higher incidence rate than the female population [ , ] . males had twice the secondary attack rate than females [ ] . the covid- transmission stages could be categorized into four temporal stages according to the chronological order of case reports. the first stage: people with exposure histories to huanan seafood market (hsm) got infected [ ] . forty-one patients were found to be having sars-like symptoms in december , and the hsm was believed to be one of the origins of the virus. however, of the patients reported no prior exposure to the hsm thus indicating that the origin of the virus needed further investigation [ ] . the second temporal stage is the transition from community transmissions to the outbreak in wuhan [ ] . the virus was mainly spread to multiple communities directly and indirectly by people with hsm exposure histories. the interpersonal transmissions and clustered transmissions formed community transmissions [ ] . an earlier study showed that the proportion of patients with hsm exposure histories decreased from to . % within days, indicating when people who did not have exposure histories to the hsm became infected [ , ] . the third stage: the epidemic in china. at this stage, transmissions began to expand to communities outside wuhan and the hubei province as a whole [ ] . on th jan , a study involving covid- patients outside wuhan found that all the patients had been exposed to wuhan, which demonstrated an established local transmission outside wuhan [ ] . the fourth temporal stage is the global pandemic. on th jan , the first case outside china was reported in thailand [ ] . on th jan , the who declared a public health emergency of international concern (pheic) [ ] . it subsequently took about days for transmission to escalate from the first reported case to the , th reported case outside china. globally, it took days for the number of reported cases to increase from , th cases to , th cases, days from , th cases to , th cases, only days from , th cases to , , th cases and days from , , th cases to , , th cases [ ] . the main transmission route of this virus was by human-to-human spread, since only . % patients among confirmed patients had history of direct contact with wild animals [ ] . the vital transmission routes were through respiratory droplets and contact transmissions. there remains the possibility of aerosol transmission when exposed to high concentrations of aerosols for a long time in a relatively closed environment [ ] . mother-to-child transmission has been confirmed, whiles fecal-oral transmission was also considered possible but lacked direct evidence until now [ , ] . other suspected routes of transmission still needed further clarification. community transmission, nosocomial transmission, household transmission, and transmission in closed environments were four typical transmission patterns of the covid- . firstly, community transmission was considered to be an important pattern in covid- spread [ ] . in the netherlands, community transmissions were found in the noord-brabant regions [ , ] . in north america, community transmissions were reported in winnipeg, canada, and eastern idaho, united states [ , ] . secondly, the potential risk of transmission among medical personnel and through medical facilities was deemed high and thus extreme attention should be paid. transmissions between patients and health workers were in higher proportions during the sars outbreak, while transmission through medical facilities was higher in proportion during the mers outbreak [ ] . in wuhan, the proportion of severely infected medical workers was higher than the national average [ ] . in italy, health workers were reported infected with the covid- before th march and accounted for . % of the total number of cases nationwide. the number however increased to by th march and represented % of the country's total number of cases [ , ] . in spain, the number of diagnosed cases among medical workers increased to within days and more than % of the country's confirmed cases remained among medical workers until march th [ ] . update from another source reported an increase in the number of cases from to % among spain healthcare workers by st march and this was attributed to lack of medical supplies, such as masks and gowns. other reasons accounting for these high infection rates among medical personnel varied according to different country's circumstances. an italy study pointed out hospitals as a potential hotspot for infection. facilities and medical personnel turned into untested vectors and patients [ , ] . in the us for example, the reasons that turned hospitals into infection hotspots included the overload of covid- patients and inappropriate management against the pandemic in hospitals [ ] . similar to the us, medical workers got infected in a county hospital in romania due to inadequate hospital management. in egypt, a serious wave of emigration by physicians for years led to patient overload for remaining medical workers and placed them at higher risk of infection through continuous exposure. the emigration wave was purportedly caused by low salary, undesirable working conditions, lack of legal protection, and shortage of medical supplies and equipment [ ] . thirdly, household transmission contributed to cluster infections and was the major transmission pattern observed in china. for instance, among reported cases in guangdong and sichuan provinces, most cluster infections occurred in families ( - %) [ ] . the who in this regard issued a statement that household transmission highly occurred among medical workers' families than health facility infection in china. household transmission was also a significant pattern observed in south korea and the us [ , ] . the european centre for disease prevention and control (ecdc) had provided guidance for the control of household transmission in european countries [ , ] . what made household transmission worse was that some groups (age < and > ) had high risk got infection within households than the general population [ ] . so, children and elderly living with medical workers at a higher risk of getting than other populations. fourthly, transmissions in a closed environment besides the home should also be of a keen focus on the prevention and control of this outbreak. a japanese health department reported that a closed environment could promote super-spreading events because the transmission of the sars-cov- in a closed environment was the same as large-scale transmission, such as the ski chalet-cluster infection in france and the church-hospital infection clusters in south korea [ ] . for example, outbreaks of the covid- were observed in multiple prisons in china, the uk, and the us [ , , ] . cluster infections also happened on cruise ships, such as the diamond princess, grand princess, golden princess, ruby princess, phoenix reisen, ms westerdam, and punta arenas [ ] . further studies are however required to identify and assess other potential transmission patterns for further prevention, especially since some cases were asymptomatic [ , ] . in addition, patients who were considered cured and no longer needed quarantine still tested rt-pcr positive after to days [ ] . we systematically used the data of the incubation period and the reproduction numbers for meta-analysis (see details of selected studies on additional file ). the result suggested that the mean incubation period was . days ( % ci: . - . , studies), and ranged from to . days [ ] [ ] [ ] [ ] [ ] . however, the incubation period in some special cases could be as long as days [ ] . the result also illustrated that the basic reproduction number (r ) of sars-cov- was . ( % ci: . - . , studies) and varied between . - . [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this finding suggested that the transmission ability of sars-cov- was stronger than sars ( ) and mers (≤ ) [ , ] . moreover, the median time from the first symptom to first hospital admission was days with the median duration from illness development to severe symptoms development being: - days for dyspnea, - days for ards, . days for mechanical ventilation and icu admission [ , ] . for covid- related deaths, the duration from the onset of symptoms to death averaged days in china and in italy (median) [ ] , and days in south korea (median) [ ] . by th july , nations had reported over , covid- cases in each of the countries, together contributed to . % of the confirmed cases and . % of death in the world . the world case fatality rate (cfr) was . % on th july; however, it was apparently different by country. one third of these countries had a cfr of over . %. france ( . %), united kingdom ( . %), italy ( . %), and mexico ( . %) were the top four countries with over % cfr while qatar ( . %) and saudi arabia ( . %) were the two countries with no more than % cfr. most countries experienced an increase of cfr at first, and the number was then gradually becoming stable during the disease outbreak (fig. ) . however, the cfr was high in iran ( . % on th february) and the united states ( . % on th march) at first, experienced a sharp decrease to . % on th march and . % on th march, and rebounded to . and . % on th july, respectively. bangladesh was the only country that had high cfr of around % at the beginning and then continuously decreased until . % on th july. as the pandemic outbreak continued, more surveillance is needed for the cfr of covid- [ ] . the mortality is higher among elderly, patients requiring intensive care unit admission and male. however, mortality rate among younger age group and patients with mildly disease is less. the us's data indicated that patients younger than had milder covid- illness, with almost no hospitalizations or deaths reported [ ] . based on a worldwide data, the elderly (aged over ) were at a high risk of developing into death [ , , , ] . the mortality in icu was extremely higher than non-icu patients, varied from to % [ ] [ ] [ ] [ ] . about the gender ratio, there is a seemingly unquestionable pattern that covid- killed more men than women [ ] . unlike the less report in the research from china, south korea or other asia areas, the reports from europe and american reflect the male gender is the risk factor for heavy illness. to figure out the general situation around the world, here we analyzed the data from countries, compiled centrally and individually verified by authors against country-specific reports [ ], shown that the case-fatality rate among male is about % higher than female (ir = . based on the data collected from selected articles [ , , , , (details of selected articles were put in the additional file ), we conducted the meta-analysis using a random-effects model to identify the clinical feature of covid- . fever ( . , % ci: . - . %) and cough ( . , % ci: . - . %) were the most fig. case fatality rate of countries reported over , cases, *. *data was collected until july (i.e. the th day of year ). the cfr of a country was not included on those dates when the country reported less than cases, with the consideration that the cfr may not be reliable if the size of infected population was small common symptoms. other common symptoms included: olfactory ( . %), gustatory ( . %), dyspnea ( . %), fatigue ( . %), sputum production ( . %), sore throat ( . %) and headache ( . %). all the other data showed in table . besides, studies pointed out that most patients had more than one symptom [ , , ] . additionally, there were . % of patients without viral pneumonia symptoms [ ] , which was opposite to previous studies [ , ] . the asymptomatic cases varied from . - . % [ , , , ] . the top common symptoms among mild and severe patients are summarized and displayed in a figure (fig. ) [ , , , , [ ] [ ] [ ] [ ] . fever was found to be the most common symptom in all patients. in a study, . % of patients had fever initially and the proportion increased to . % following hospitalization [ ] . the body temperatures of - . % of patients ranged between . - . °c. the higher body temperatures (above . °c), dyspnea and anorexia were more frequent among patients in severe conditions [ , , ] . cough and fatigue were more widely reported among mild and severe patients. additionally, another study reported that dyspnea ( %) was the most common symptom among severe patients in the united states [ ] . the proportion of patients who needed icu care varied based on the local pandemic circumstances. for example, the who speculated that around . % of patients were in severe conditions in china [ ] . however, - % of patients needed icu care in wuhan [ , , ] . currently documented covid- related complications include ards, arrhythmia, septic shock, acute cardiac injury, myocarditis, acute coronary syndrome, cardiomyopathy, acute respiratory injury, and acute renal injury, etc. [ , , , , , ] . the ards was the most common complication, among both mild and severe patients [ , , , , ] . most icu patients had a higher risk of developing ards and having complications [ , ] . the progress of some patients with ards to septic shock was fast and quickly evolved into multiple organ failure finally [ ] . among covid - patients, a decrease in leukocytes such as eosinophil and lymphocyte were commonly reported. this might be because the cytokine storm caused by the novel virus changes the peripheral of white blood cells and immune cells [ , , , , , ] . severe lymphopenia was also common among the dead patients [ , ] . myocardial zymogram abnormality was found in many patients. for instance, % of patients had an increase in lactate dehydrogenase, while % of patients had increases in creatine kinase [ ] . the level of c-reactive protein was important to evaluate the infection [ ] . most patients were found to have a higher level of c-reactive protein ( %) and serum ferritin ( %) compared to the normal range [ ] . the biomarkers related to liver and renal damage were found to be abnormal among covid- patients. the abnormality of liver-related biomarkers was not widespread but yet still common in severe cases [ , , , ] . besides, although only % of patients showed renal biomarker abnormalities, renal damage might contribute to the final multi-organ failure and death outcome [ , ] . the icu patients showed higher levels of white blood cells, neutrophil counts, d-dimer, creatine kinase, and creatine with longer prothrombin times [ , , ] . compared to patients who survived, the patients who died had higher levels of d-dimer, high-sensitivity cardiac troponin i, serum ferritin, lactate dehydrogenase, il- , blood urea, creatinine, white blood cell counts and neutrophil counts. severe lymphopenia was also common among dead patients [ , ] . the computed tomography scan (ct scan) was widely used for disease diagnosis, prognosis, and management during the covid- [ ] . the ct was found more sensitive for identifying sars-cov- patients than the rt-pcr assay ( % vs. %) in a study [ ] . the ct evidence for confirming the highly suspected patients' positive may precede the rt-pcr results [ , ] . most patients had ggo and the bilateral lung involvement [ , , [ ] [ ] [ ] . one study found that bilateral lung involvement was more frequently shown in the intermediate course and late course, compared to the earlier clinical course [ ] . the clinical course could be divided into four stages based on ct scan findings [ ] . in the first stage (pre-symptom), ggo, unilateral and multifocal were observed among most patients in this stage [ , ] . in the second stage (symptoms ≤ week), lesions soon developed into bilateral and diffused except for ggo. this stage was considered a period from transition to consolidation. a mixed pattern of transition and consolidation develops during this stage. in the third stage (symptoms - weeks), the ggo was still common and the consolidation pattern showed. findings indicated an interstitial change, which was considered as the development of fibrosis. in the fourth stage (symptom - weeks), consolidation and mixed patterns were more common, and the ggo started to shrink [ ] , the consolidation was gradually absorbed among patients who recovered at last [ ] . among icu patients, the bilateral multiple lobular and sub segmental areas of consolidation were considered typical findings [ ] . patients in severe condition showed diffuse lesions, with density increasing in both lungs. ct scans showed 'white lung' appearances, indicating the serious influence the infection has on patients' lung functions [ ] . being old (≥ years old), male sex, having a higher bmi value (> kg/m ), having co-morbidities (e.g. hypertension, diabetes, cardiovascular and cerebrovascular diseases, etc.), and developing complications were vital risk factors for patients to develop severe conditions [ , , , , , , ] . the cytokine storm, raised inflammatory markers, elevated cardiac troponins, the requirement of mechanical ventilation, and the requirement of intensive care unit stay predict the bad outcome of admission patients [ ] . findings from multiple studies showed that patients who are more than years of age, with co-morbidities such as diabetes and heart diseases had a high mortality rate [ , , [ ] [ ] [ ] . late hospitalization and bacterial infections were also considered high risk factors for disease progression [ , , ] . smoking history could be a potential risk factor for developing severe conditions [ , ] . people with underlying disorders were considered to be at a high risk of getting infected [ ] . our review identified several research gaps. firstly, large amounts of data from african were missing from this review. as the number of people in african suffering from malnutrition, anemia, malaria, hiv/aids and tuberculosis is high, a large "low immunity population" has been created which has made the control and prevention of covid- in the region a challenge. the situation could be worsened by the limited health resources region [ ] and hence, more african focused research is required to support africa in fighting the epidemic. secondly, the proportion of asymptomatic patients is large but the current transmission ability by asymptomatic patients might be weak. however, further exploration of risks posed by the group is needed as limited studies exist on the subject matter [ ] . meanwhile, data on the distribution of asymptomatic patients in large-scale community groups is also lacking, prompting the need for large scale of active screening and testing to help identify them [ , ] . this approach is however difficult and expensive for most countries to undertake as accurate strategies to identify asymptomatic currently are non-existent. further research focus on asymptomatic patients is needed. third, a 'super-spreader' was defined as infected individuals who infected numerous others during the sars outbreak. for example, a nephrotic hospitalized patient who infected people was classified as a 'superspreader' during the sars in china. in those patients were medical workers who came in contact with the 'super-spreader'. the incidence rate among the medical workers was . % ( / ) in the nephrotic department [ ] . in the covid- era, the emergence of 'super-spreaders' were found in multiple places worldwide. a saudi arabian study linked the concept of 'super-spreaders' to 'super spreading' events noting that 'super-spreaders' might cause unexpected transmissions during the pilgrimage [ ] , as huge numbers of people gather. reasons causing the super-spreading events might include: immune suppression, increased disease severity and viral load, asymptomatic individuals, and extensive social interactions [ ] . however, the characteristics and features of how an individual becomes a super-spreader are still not clear [ ] . summarizing the features of the 'super spreader' concept, as well as their characteristics and role in transmissions, are needed in future disease control [ ] . fourth, it has been reported that some cured patients covid- retested positive by pcr after being discharged and quarantined at home in multiple places [ , ] . the reason for this phenomenon is still unclear and hence further investigations are required for future pandemic control [ ] . there existed some limitations in this review. firstly, this review was based on english and chinese resources only. as the covid- transformed from a regional outbreak to a global pandemic, comprehensive collection of the related information worldwide is needed. secondly, the clinical spectrum presented in this review is based on general population only, and thus a further subgroup analyzes in future may help to figure out more on the entire picture of the covid- . for instance, although kawasaki disease was found in children in the uk and europe countries, other places did not report the gathering kawasaki disease cases [ ] . the covid- had a stronger transmission ability than sars and mers, timely intervention should be conducted to reduce the spread of the disease. the common symptoms included in this study could assist in identifying the potential patients. the summary of the common complications, lab findings, ct features and risk factors could help medical personnel better manage patients who may develop into severe conditions or death. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . searching terms. additional file . detail of selected studies for meta-analysis (r &incubation period). additional file . detail of selected studies for clinical symptoms' metaanalysis. additional file . the forest plots of clinical symptoms' meta-analysis. who. situation 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-status of diagnostic test, significance of "super spreaders problems to be solved in sars research in china fu yang": there is no unified conclusion, and discharge management is being strengthened at present, it seems that fuyang patients are not infectious what is kawasaki disease and its possible link with covid- in children? publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank mr. peizhen zhao for technical support.authors' contributions wt designed the study protocol. yx, zw, and hl did the literature search. the titles, abstracts, and full texts were screened and selected by yx, zw, and hl. the data were extracted and analysed by yw, zw and hl. yx, zw and hl drafted the manuscript. yx, zw, gm, dw, and wt edited the draft. all authors read and approved the final manuscript. key: cord- -usfvulc authors: sharifipour, ehsan; shams, saeed; esmkhani, mohammad; khodadadi, javad; fotouhi-ardakani, reza; koohpaei, alireza; doosti, zahra; ej golzari, samad title: evaluation of bacterial co-infections of the respiratory tract in covid- patients admitted to icu date: - - journal: bmc infect dis doi: . /s - - -z sha: doc_id: cord_uid: usfvulc background: covid- is known as a new viral infection. viral-bacterial co-infections are one of the biggest medical concerns, resulting in increased mortality rates. to date, few studies have investigated bacterial superinfections in covid- patients. hence, we designed the current study on covid- patients admitted to icus. methods: nineteen patients admitted to our icus were enrolled in this study. to detect covid- , reverse transcription real-time polymerase chain reaction was performed. endotracheal aspirate samples were also collected and cultured on different media to support the growth of the bacteria. after incubation, formed colonies on the media were identified using gram staining and other biochemical tests. antimicrobial susceptibility testing was carried out based on the clsi recommendations. results: of nineteen covid- patients, ( %) patients were male and ( %) were female, with a mean age of ~ years old. the average icu length of stay was ~ days and at the end of the study, cases ( %) expired and only was case ( %) discharged. in total, all patients were found positive for bacterial infections, including seventeen acinetobacter baumannii ( %) and two staphylococcus aureus ( %) strains. there was no difference in the bacteria species detected in any of the sampling points. seventeen of strains of acinetobacter baumannii were resistant to the evaluated antibiotics. no metallo-beta-lactamases -producing acinetobacter baumannii strain was found. one of the staphylococcus aureus isolates was detected as methicillin-resistant staphylococcus aureus and isolated from the patient who died, while another staphylococcus aureus strain was susceptible to tested drugs and identified as methicillin-sensitive staphylococcus aureus. conclusions: our findings emphasize the concern of superinfection in covid- patients due to acinetobacter baumannii and staphylococcus aureus. consequently, it is important to pay attention to bacterial co-infections in critical patients positive for covid- . a novel coronavirus known as severe acute respiratory syndrome coronavirus (sars-cov- , also called covid- and -ncov) was first reported in wuhan, hubei province, china in december . ever since the virus has been spreading worldwide claiming thousands of lives. due to serious respiratory disease in humans, some patients need to be hospitalized and in severe cases intensive care with mechanical ventilation support is essential (~ - %) [ , ] . although covid- associated deaths have mainly occurred in the elderly with serious underlying diseases [ ] , nosocomial pneumonia (np) in intensive care units remains a major risk factor for the patients and the health of patients, especially when intubated, may deteriorate in the presence of lower respiratory tract infections. nosocomial infections (nis) are usually described as infections acquired during hospitalization within - h after admission and they mainly spread through person-to-person contact, devices, and instruments [ ] . among microorganisms, the bacteria including staphylococcus spp., enterococcus spp., klebsiella pneumoniae, enterobacter spp., escherichia coli, acinetobacter spp., and pseudomonas spp. are the most frequently detected causative agents of nis [ ] . these opportunistic pathogens can also cause superinfections, especially in combination with viral respiratory tract infections in hospitalized patients. however, even patients without underlying diseases and in all age groups may be at the risk of co-infections as well [ , ] . some studies have shown that viral agents such as influenza viruses can be associated with secondary bacterial pneumonia that might occur throughout hospitalization and lead to the death of individuals with or without preexisting respiratory diseases [ ] . the damage of ciliated cells can also be observed in association with respiratory syncytial virus infection; it can result in deterioration of mucociliary clearance, increased adhesion of bacteria to mucins and, enhanced colonization of the bacteria in the airway. moreover, new receptors for bacterial adherence can emerge following the virus-induced death of the airway epithelial cells [ ] . in addition, after an acute inflammatory reaction and pulmonary tissue damage induced by viral infections, a resolving/repair phase of the lung tissue takes place. due to varied immune responses in different individuals, this phase may cause an enhanced susceptibility to respiratory bacterial infections. thus, bacterial superinfection can occur after a viral infection, which in turn might lead to increased morbidity and mortality [ ] . nevertheless, any probable contribution of the bacteria to the development of the infectious diseases caused by the newly discovered coronavirus is still completely unknown. in a study by póvoa et al., the risk of ventilatorassociated bacterial pneumonia in covid- patients was studied [ ] . in addition, although there are a few recently published retrospective reports of co-infections in patients with covid- [ , ] , our study is adding to a growing evidence base of the role bacterial coinfections may have in covid- patients. therefore, our aim was to evaluate secondary bacterial infections and their antibiotic resistance in covid- positive patients admitted to icus in qom, the first city in iran to report covid- disease [ ] . nineteen critically ill patients admitted to the icu wards in two referral hospitals for coronavirus in qom, iran, were enrolled in the present study. patients were given antibiotics such as ceftriaxone and azithromycin before admission to the icus. inclusion criteria were being infected by covid- , hospitalized, intubated, and mechanically ventilated > h in icus. ventilator-associated pneumonia (vap) was identified based on the following criteria: a new and persistent (> h) or progressive infiltrate on the chest radiograph plus of the following minor criteria: fever > °c or hypothermia < °c, blood leukocyte count of > , cells/ml or < cells/ml, purulent tracheal secretions, or decrease in the pao /fio . in cases with clinically suspected pneumonia, vap diagnosis was established with a positive quantitative culture (cut-off point ≥ [colony-forming units (cfu)/ml]) [ , ] . all patients in our study were also neutropenic with elevated erythrocyte sedimentation rate and c-reactive protein (crp); and had a history of sore throat, cough, and shortness of breath. to determine the status of the patients, i.e. death or discharge, we waited until the end of the admission of the last patient in our icus. reverse transcription real-time polymerase chain reaction (rt-pcr) for the detection of covid- this step was carried out once for each patient. briefly, naso-pharyngeal samples were obtained using a specific swab (medical wire, uk) and then placed in a separate collection tube containing two ml of viral transport medium and immediately sent to the coronavirusreference laboratory of the university. first, the extraction of the viral rna was performed using a commercial kit according to the manufacturer's protocol (geneall, seoul, south korea). next, rt-pcr was performed using lightmix® modular sars and wuhan cov e-gene kit and using one-step rt-pcr polymerase mix (tib-molbiol, berlin, germany) [ ] . the collection of the samples for bacterial infections was repeated at four stages with an interval of~ days for each patient who still stayed in icus. endotracheal aspirate (eta) specimens were collected in sterile tubes based on a standard clinical protocol [ ] . the specimens were immediately transferred to the bacteriological laboratory and were evaluated by conventional methods. first, the samples were cultured on blood agar, chocolate agar, eosin methylene blue (emb), and macconkey agar and then incubated at °c for - h under standard conditions. the colonial growth of the bacteria was confirmed by gram staining and other media and standard biochemical testing including (e.g. . minimum inhibitor concentration (mic) was also performed for colistin and vancomycin according to clsi protocol [ ] . s. aureus atcc and e. coli atcc were used as standard strains. phenotypic detection of mbl-producing clinical isolates was evaluated by the combination disk diffusion test (cddt) and modified hodge test (mht) as previously described by lee et al. [ ] . all patients were positive for bacterial infections. out of patients, ( %) patients were male and ( %) were female, with a mean ± standard deviation (sd) age of . ± . years (range of age - years). of all patients, cases ( %) had underlying diseases such as kidney disease, diabetes, hypertension, or heart diseases. at the end of the study, cases ( %) were dead ( . ± . years old), % ( cases) of whom had underlying diseases, and case ( %) was discharged ( years old). at first collection, a total of clinical specimens, all patients ( %) were found positive for bacterial infections, cases ( %) for acinetobacter (a.) baumannii and cases ( %) for staphylococcus (s.) aureus. in the stage of the , , and of sampling, , , and patients were included, respectively. in all samplings, the bacterium isolated from each patient remained the same. more information is presented in table . overall, the mean ± sd length of pre-icus stay for all included patients was . ± . days, while the average in our icus was . ± . days ( . ± . and days for expired and discharged patients, respectively). the median icu length of stay for a. baumannii -positive and s. aureus -positive patients was . ± . and ± . days, respectively. results of the antimicrobial susceptibility testing showed a high-level resistance of a. baumannii isolates to all tested antibiotics, except colistin with a resistance rate of %. no isolated a. baumannii strain produced mbls and the resistance pattern of a. baumannii isolates was not different between expired and discharged patients. one of the s. aureus isolates was detected as methicillin-resistant staphylococcus aureus (mrsa) and resistant to all other evaluated agents i.e. penicillin, cefoxitin, azithromycin, erythromycin, gentamycin, cotrimoxazole, linezolid, and ciprofloxacin. no resistance was observed to vancomycin or tetracycline. the mrsa strain was isolated from a patient who expired on the th day of icu admission. another s. aureus strain, isolated from a discharged patient, was identified as methicillin-sensitive staphylococcus aureus (mssa) and susceptible to all the above-mentioned drugs. three of our patients ( %) had no underlying diseases. one of them was infected with the mssa and the other two cases were infected with the a. baumannii strains. among, only mssa-infected patient was discharged and other two a. baumannii-infected patients were expired. covid- , a viral pneumonia with an unusual outbreak, is considered as a new public health concern threatening us worldwide. recent studies show that -ncov or sars-cov- originated from an animal source and later adapted to other variants as it crossed the species barrier to ultimately infect humans [ , ] . in recent months, less attention has been paid to hospital-acquired infections and opportunistic microorganisms, which could be due to the outbreak of covid- and its consequent long-term hospitalization of patients, and high workload on the healthcare personnel. in this study, with a focus on secondary infection of the lower respiratory tract of patients, a. baumannii was the most common organism followed by s. aureus. in recent years, emerging strains of both species that have acquired additional genetic features have shown to be commonly associated with hypervirulence and resistant to many types of antibiotics [ , ] . according to our infection control committee and laboratory reports, these were associated with other bacteria including pseudomonas aeruginosa, escherichia coli, klebsiella pneumoniae, enterobacter spp., serratia marcescens, and citrobacter freundii, etc. that were previously isolated from the icu wards and non covid- patients admitted to our icus. in addition, both a. baumannii and s. aureus were among the most isolated bacteria from non covid- icu patients in iran and other countries [ ] . in a study conducted in tehran, iran, klebsiella pneumoniae and acinetobacter had the highest rates of incidence in icus [ ] . in a study, a. baumannii and klebsiella spp. were the most common organism isolated in mysuru, india [ ] . in , the most common icu-acquired strains were acinetobacter baumannii, pseudomonas aeruginosa, stenotrophomonas maltophilia, staphylococcus aureus, enterococcus spp., and klebsiella pneumonia in shanghai, china [ ] . in the present study, our first samplings were performed in the patients who were admitted to icus for ≥ days, except for case with days of admission. certainly, this duration was an excellent opportunity for bacteria to infect the patients, and thus all of our first cultures were positive with secondary infection ( / , %) . this incidence rate is higher than similar recently published articles. in fu et al. study, . % ( of ) of the patients in the icu were diagnosed with severe acute respiratory syndrome coronavirus and secondary bacterial infection. in another report that was published from a uk secondary care setting, amongst patients identified as sars-cov- , cases ( . %) had early confirmed bacterial isolates identified ( - days post admission) rising to cases ( . %) during the admission [ , ] . in addition, our result indicates a higher incidence than other published studies on non covid- patients. in a study conducted in shiraz, iran, in , hassanzadeh et al. suggested that icu-acquired infections were documented in . % of icu patients, with a mortality rate of . % ( patients) [ ] . one of the reasons for the increase in infection rate in our study can be due to the simultaneous infection of the virus and bacterium. as previously mentioned, viruses can facilitate the attachment and colonization of the bacteria in the respiratory tract, which is certainly no exception for covid- ; however, understanding the accurate mechanisms of interactions between novel coronavirus and other bacteria requires further research. nevertheless, other factors such as icu type, used equipment rate, admission/ discharge criteria, high workload/nurse ratio, etc. can also affect the quality of care and the rate of icu acquired nosocomial infection [ , ] , especially in pandemics. except for colistin, a. baumannii strains showed widespread resistance to all different classes of antibiotics and no inhibition zone was observed in the disk diffusion method. resistant isolates of the bacteria, especially a. baumannii, are not uncommon among admitted patients in the hospitals and hospital-acquired infections have become a major concern to health systems. wang et al. showed that the resistance rate of a. baumannii isolates was approximately > % to piperacillin, imipenem, ceftriaxone, ciprofloxacin, and ceftazidime [ ] . castilho et al. also reported that a. baumannii isolates from icus in goiânia, brazil, were classified as multidrug resistant (mdr) with a high incidence of resistance to carbapenems. the development of resistance to carbapenems and other β-lactams may be due to the production of the mbls. these are one of the most common participating in resistance mechanisms that can inactivate a wide range of β-lactam antibiotics [ ] . nevertheless, no mbl-producing a. baumannii strain was isolated. however, the bacteria may use other strategies to resist the effects of antibiotics [ , ] . in our study, one of the strains of s. aureus was identified as mrsa. this organism plays an important role in the severe complication of infections in icu environments. the probability of acquiring mrsa may increase (> . - times) in patients with longer stays in the ward, i.e. more than one week [ ] . different studies have also shown that lower respiratory tract infections caused by mrsa can be associated with a significant level of mortality in the patients admitted to icus [ , ] . due to the covid- crisis conditions, we were not able to carry out mic and other phenotypic confirmatory tests for evaluating extended-spectrum betalactamases or esbls, etc., as well as molecular assays for detecting resistance genes. nevertheless, these pathogens showed extremely high rates of resistance to the majority of the antibacterial agents tested. this could not only delay the process of treatment and recovery of covid- patients but also increase the mortality rate. based on our local strategies, all patients in the current study routinely received ceftriaxone and azithromycin (except for some contraindications or interactions) before admission to the icus. in the cases of the infection in icu, these were changed to extended-spectrum antibiotics such as meropenem and vancomycin, but no changes in the isolation of our resistant bacteria were observed at different stages of sampling. however, the treatment protocols have been changed by the icu medical team based on the obtained results of the cultures and the pattern of antibiotic resistance, e.g. in this study, combination therapy with meropenem, colistin, and ampicillin-sulbactam was used for the treatment of infections caused by the resistant strains of acinetobacter. among our patients, three cases had no underlying diseases. one patient, infected by a susceptible strain of staphylococci, was discharged, while two other patients, infected with multidrug-resistant a. baumannii, expired. due to some limitations, the sample size of the current study was not sufficient for comparing and accurate statistical evaluation. however, further work is required to investigate whether there are increased mortality rates associated with patients co-infected with covid- and antibiotic-resistant bacteria. the median length of icu stay among patients in our study was higher, days (interquartile range, to ), compared with zhou et al. study, which reported a length of stay of . days ( . - . ) of all patients with covid- admitted to their icu. moreover, no bacterial pathogens were detected in their patients on admission [ ] . it seems that the length of icu stay can be prolonged, if patients become co-infected. a study on respiratory co-infection in patients with pandemic influenza a (h n ) virus infection showed that icu length of stay was days longer among patients who had co-infection [ ] . in addition, infections and antibiotic resistance in icu patients can also result in higher cost of treatment, and increased mortality [ ] . in a study conducted by toufen and colleagues on icu patients in brazil, the rate of mortality was . %, while the patients with infection had a mortality rate of . % and the most frequently reported infections were related to respiratory infections ( . %) [ ] . chastre et al. study also suggested that the mortality rate of vap in icu patients varies from to %, and even higher when caused by high-risk pathogens [ ] . according to previous studies, viral-bacterial synergistic interactions are reviewed and the mortality rate can be further increased when there is simultaneous an acute respiratory viral infection and a bacterial infection. a multicenter retrospective cohort study conducted by arabi et al. on mers sari (middle east respiratory syndrome severe acute respiratory infection) patients who were admitted to the participating icus showed that % ( cases) and % ( cases) of them had bacterial and viral co-infections, respectively [ ] . it has also been estimated that one-third of the world's population (~ million people) may have been clinically infected during the - influenza pandemic, which resulted in the death of at least million people worldwide (https://www.cdc.gov/flu/pandemic-resources/ -pandemic-h n .html). the findings suggest that the vast majority of individuals who died during the pandemic were infected by a bacterial infection [ ] . the co-infection of the influenza virus with staphylococcus aureus, especially mrsa, has been previously documented. in a study performed by bhat et al. during the - influenza season, bacterial co-infections were identified in of cases. accordingly, s. aureus was the most common etiology ( cases); six of these cases were detected as methicillin-resistant strains [ ] . randolph et al. also reported that among children with influenza a (h n ) virus admitted to a pediatric intensive care unit during the influenza a (h n ) pandemic, ( . %) had a presumed diagnosis of early s. aureus co-infection of the lung with % positive for mrsa [ ] . moreover, jia et al. project on mouse model findings also showed that secondary infection with methicillin-resistant staphylococcus aureus after infection with influenza virus was associated with high mortality rates [ ] . another study by liu et al. also confirmed that the co-infection of avian influenza a (h n ) virus and extensively antibiotic-resistant a. baumannii in the patients with invasive mechanical ventilation is a key factor for the severity of the disease and high mortality [ ] . our report is one of the first to demonstrate the presence of superinfections in the lower respiratory tract of patients with covid- . our findings emphasize the concern of bacterial infections in the patients due to a. baumannii and s. aureus that are resistant to the extended-spectrum antibiotics commonly used for the treatment of life-threatening bacterial diseases, especially in icu patients. secondary bacterial infections may develop during or following covid- and thus they are an undeniable fact. due to severe pandemic conditions, it was not possible to have a negative control group without covid- in our icus simultaneously. therefore, we could not certainly state what percentages of deaths in our patients were caused by bacterial coinfections. however, when mortality rates compared to other non covid- studies, e.g. . % in shiraz [ ] and . % in mysuru [ ] , it seems that mortality is increased in covid- patients and may be attributed to bacterial co-infections. thus, further studies are recommended to confirm this finding. mortality in covid- positive patients with no underlying diseases may be due to bacterial infections that this concern also requires more investigations. overall, it is important to limit the risk of 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teaching hospital ventilator-associated pneumonia critically ill patients with the middle east respiratory syndrome: a multicenter retrospective cohort study deaths from bacterial pneumonia during - influenza pandemic influenza-associated deaths among children in the united states critically ill children during the - influenza pandemic in the united states severe pneumonia caused by coinfection with influenza virus followed by methicillin-resistant staphylococcus aureus induces higher mortality in mice clinical, immunological and bacteriological characteristics of h n patients nosocomially co-infected by acinetobacter baumannii: a case control study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we wish to thank the research council of qom university of medical sciences and the personnel of our hospitals for supporting the study. the authors declare that they have no competing interests. authors' contributions ss, es, and jk developed and supervised the work. me and rfa performed the experiments. ss, ak, and sejg drafted the manuscript. zd contributed to data interpretation. all authors reviewed the manuscript. all authors read and approved the final manuscript. the study was supported by research council of qom university of medical sciences. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. the study was reviewed and approved by medical ethics committee of qom university of medical sciences (code: ir.muq.rec. . ). participants provided written informed consent to participate in this study. in unconscious patients or those under mechanical ventilation, consent was obtained from the relatives of them. not applicable. key: cord- -xm ae authors: liu, wen-kuan; chen, de-hui; liu, qian; liang, huan-xi; yang, zi-feng; qin, sheng; zhou, rong title: detection of human bocavirus from children and adults with acute respiratory tract illness in guangzhou, southern china date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: xm ae background: human bocavirus (hbov) is a newly discovered parvovirus associated with acute respiratory tract illness (arti) and gastrointestinal illness. our study is the first to analyze the characteristics of hbov-positive samples from arti patients with a wide age distribution from guangzhou, southern china. methods: throat swabs (n= ) were collected and analyzed from children and adults with arti over a -month period. the hbov complete genome from a year-old female patient isolate was also determined. results: hbov dna was detected in / ( . %) samples, of which / were from children (< years old) and / from adults (≥ years old). seasonal peaks of . % and . % were detected in may and june, respectively. of ( . %) hbov-positive samples were co-detected with / other potential pathogens. mycoplasma pneumoniae had the highest frequency of . % ( / ). upper and lower respiratory tract illness were common symptoms, with / ( . %) patients diagnosed with pneumonia by chest radiography. all four adult patients had systemic influenza-like symptoms. phylogenetic analysis of the complete genome revealed a close relationship with other hbovs, and a more distant relationship with hbov and hbov . conclusions: hbov was detected from children and adults with arti from guangzhou, southern china. elderly people were also susceptive to hbov. a single lineage of hbov was detected among a wide age distribution of patients with arti. respiratory tract infection etiology is complex and diverse, and new pathogens are continuously being reported. over the past few years, several novel respiratory viruses including human metapneumovirus (hmpv) [ ], severe acute respiratory syndrome (sars) coronavirus [ ] , human coronavirus nl (hcov-nl ) [ , ] , and coronavirus hku (hcov-hku ) [ ] [ ] [ ] have been identified. in , allander et al. [ ] reported a previously undescribed human parvovirus, human bocavirus (hbov) that belongs to the genus bocavirus, in respiratory secretions of children with respiratory tract disease in sweden. hbov is a single-stranded deoxyribonucleic acid (dna) virus with a small genome size of approximately . kilo-bases (kb), which has three open reading frames (orf) encoding two non-structural proteins ns and np , and the two structural proteins vp and vp . vp and vp are located within the same orf but have different initiator codon positions [ ] . subsequently, hbov was reported in respiratory samples from different countries and regions worldwide [ ] [ ] [ ] [ ] [ ] [ ] , where hbov was detected in . %- . % of respiratory samples from individuals with acute respiratory tract illness (arti), especially young children and infants. the virus was also found in stool samples from patients with gastrointestinal illness [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these reports suggest that hbov might be associated with upper and lower respiratory disease and gastrointestinal illness throughout the world. in , two viruses closely related to hbov, named hbov [ ] and hbov [ ] , were found in stool samples, and suggested hbov diversity. hbov infection has recently attracted increasing attention all over the world. however, the incidence and clinical presentation of this infection varies widely, and often involves co-infection with other potential pathogens [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . such characteristics have led to debate over the role of hbov as a true pathogen. therefore, additional evidence and studies are needed throughout the world to gain a better understanding of this virus. in this study, respiratory samples were collected from patients (with an age range of days to years) with arti in guangzhou, southern china, from november to november to analyze the characteristics of hbovpositive patients. samples in this study were taken as part of standard care. the first affiliated hospital of guangzhou medical university ethics committee approved the experimental design and patient involvement in this study. throat swab samples (n = ) were collected from patients with arti (presented at least two of the following symptoms: cough, pharyngeal discomfort, rhinobyon, snivel, sneeze, dyspnea) at three hospitals in guangzhou, southern china between november and november . patients' ages ranged from nine days to years, and included children (< -years-old) and adults (≥ -years-old). clinical characteristics of the patients were recorded for further analysis. real-time polymerase chain reaction (pcr) for hbov detection dna from respiratory samples was extracted using a qiaamp dna mini kit (qiagen), in accordance with the manufacturer's protocol. taqman real-time pcr primers and probe were designed based on the conserved region of the np gene. sequences were as follows: forward primer, '-gag aga ggc tcg ggc tca ta- ' ( - nt); reverse primer, '-tcg aag cag tgc aag acg at- ' ( - nt); and probe, '-fam-cat cag gaa cac cca atc agc cac c-bhq - ' ( - nt). primers and the probe were synthesized by takara. premix ex taq (perfect real time) real-time pcr reaction buffer was also purchased from takara. amplification was conducted using pmol of primers, pmol of probe and μl dna in a final volume of μl. cycling conditions included an initial incubation at °c for min, followed by cycles of °c for sec and °c for sec (abi- real-time pcr instrument, applied biosystems). the amplified np gene target sequence ( - nt) was inserted into the pmd -t vector (takara) and used as a positive control for quantification analysis. sensitivity of the pcr assay was calculated to be copies of plasmid dna using positive control plasmid diluted gradients. hbov dna positive samples were tested for other potential pathogens, including influenza a virus, influenza b virus, parainfluenza virus ( , , , ), respiratory syncytial virus, adenovirus, enterovirus, human metapneumovirus, human coronavirus ( e, oc , nl , hku ), mycoplasma pneumoniae, and chlamydia pneumoniae by taqman real-time pcr, in accordance with the manufacturer's protocol (guangzhou huyansuo medical technology co., ltd). the complete genome of hbov from a -year-old female patient isolate was sequenced and analyzed. sequencing primer sets were designed according to hbov genome sequences available in the genbank database (table ) . dna template ( μl) was added to the pfu dna polymerase reaction mixture (fermentas) in a final volume of μl and pcr amplified. products were purified after % agarose gel electrophoresis using a qiaquick gel extraction kit (qiagen). the purified dna was then sequenced (abi genetic analyzer xl) and assembled using dnastar-seqman software (dnastar, http://www. dnastar.com/t-products-lasergene.aspx). pcr amplification and sequencing were conducted at least twice to ensure sequence accuracy. the hbov complete genome (gu ) was aligned to other hbov genomes available in the genbank database using the basic local alignment search tool (blast, http://blast.ncbi.nlm.nih.gov/blast.cgi). phylogenetic analysis of complete genomes of hbov, hbov and hbov from different countries and regions, including usa, sweden, thailand, japan, australia, china, hong kong, and taiwan was conducted using molecular evolutionary genetics analysis version . (mega . , http://www. megasoftware.net/). phylogenetic trees were inferred from vp /vp ( - nt), ns ( - nt), np gene ( - nt) and complete genome data ( - nt) using the neighbor-joining method, and bootstrap values were calculated from replicates. for comparison of categorical data, χ test and fisher's exact test where appropriate. all tests were two-tailed and p < . was considered statistical significant. the positive patients were aged between days and years, comprising . % ( / ) that were ≤ years-old, . % ( / ) that were - years-old, and . % ( / ) adult patients. four hbov-positive adults were , , and years-old, respectively. during our -month study period, more than samples were collected for detection each month, and the ratio of ≤ year-old patients ranged from . hbov dna positive samples were co-detected with of upper referred pathogens in / ( . %) of the patients. mycoplasma pneumoniae had the highest frequency of . % ( / ), followed by rsv with . % ( / ) ( table ). the male:female ratio was : in the hbov-positive patients which did not differ significantly from the hbov-negative patients (p = . ). the clinical characteristics of the patients are listed in table . most patients presented with symptoms of upper respiratory tract illness (urti), including cough ( . %) and expectoration ( . %); ( . %) patients presented with (table ) . nineteen ( . %) were diagnosed as pneumonia by chest radiography, and / ( . %) pneumonia patients were co-detected with one or two other pathogens, and a statistic difference was found for the symptom of "pneumonia" (p = . ) between the two groups "single hbov" and "co-pathogens". two of seven patients with systemic influenza-like symptom samples were co-detected with influenza a virus-enterovirus (triple pathogens) and influenza b virus (dual pathogens), respectively, while the remaining five patients were detected with single hbov. all four adult patients (≥ years old) presented with systemic influenza-like symptoms; three had only hbov detection and the other year-old female patient was co-detected with influenza b virus. complete hbov genome sequences for isolates from a year-old patient were sequenced and submitted to the genebank (accession number:gu ). the full length of the genome was bases and the distribution of a/g/c/t was . %/ . %/ . %/ . %, respectively. compared to the other hbov genomes available in the genebank database, gu showed a % similarity with the hbov previously described by allander et al. [ ] . phylogenetic trees were inferred from vp /vp , ns , and np gene data, in addition to the complete genome sequence (figure ) . gu was similar to other hbovs, although it displayed obviously sequence variations from hbov and hbov . three hbov lineages were illustrated in all four phylogenetic trees (figure ). hbov is a novel parvovirus first described in by allander and colleagues [ ] . since that time, it has been associated with upper and lower-respiratory tract disease and gastrointestinal illness throughout the world. however, most studies were focused on young children and infants [ ] [ ] [ ] [ ] [ ] [ ] , and only a few papers have described the characteristics of hbov infection in adult patients [ , , ] . our study successfully analyzed the characteristics of hbov-positive samples from arti-infected patients with a wide age distribution from guangzhou, southern china for the first reported time. similar to previous study [ ] , the detection rate in pediatric patients (< years old) was significantly higher than that in adults (≥ years old) (p < . ), and most hbov-positive patients were ≤ years old. hbov was detected in four adult patients, including and year-old patients. this suggested that older people were also susceptive to hbov infection, although at much lower positive rates. four adult patients also presented with systemic influenza-like symptoms, which might suggest that hbov infection in adults is a more complex and serious problem than in children. while seasonal peaks of hbov infection vary among different counties and regions because of climate and other factors, many previous studies suggest a higher detection rate in winter [ , ] . in our study, a higher frequency of hbov was observed between may and june during the month testing period ( figure ). this result was similar to the report of choi and colleagues, in which a seasonal peak was observed between may and june [ ] . characteristics of hbov-positive patients in our study were also similar to previous reports [ ] [ ] [ ] [ ] [ ] [ ] . the male: female ratio in the hbov-positive patients did not differ significantly from the hbov-negative patients. in all clinical characteristics, seven major symptoms (cough, fever (≥ °c), abnormal pulmonary breath sound, dyspnea, expectoration, snivel and pneumonia) had the highest ratio (ratio > %) ( table ) and were common as urti and lrti. as previous studies [ , [ ] [ ] [ ] [ ] [ ] [ ] , co-detection with other potential pathogens was common in hbov-positive patients. in this work, . % ( / ) patients were codetected with other potential pathogens, and there would be higher ratio if human rhinovirus were concerned [ ] . furthermore, not only co-pathogens but also single hbov groups had a high ratio of major symptoms (table ) , which might suggest hbov is an important pathogen in urti and lrti. a statistic difference was found for the symptom of "pneumonia" between the two groups "single hbov" and "co-pathogens" (table ) , which might further suggest hbov is an important pathogen. further studies were required to determine whether hbov played a causative role in these co-infections or acted as an exacerbation factor. before , little variation was found in the surface protein of hbov (vp -vp ), and some researchers predicted that hbov infection might only occur after the subsequently development of life-long immunity via the neutralization of the target antibody [ ] . however, novel types of hbov and hbov were described successively in [ , ] , suggested diversity within this group of viruses. furthermore, we successfully sequenced the complete hbov genome (gu ) for an isolate from an adult patient and phylogenetic analysis revealed the existence of three lineages: group i, hbov; group ii, hbov ; group iii, hbov , and gu was located in group i. the frequency and clinical presentations of hbov infection vary widely [ ] [ ] [ ] [ ] [ ] [ ] , , ] and are typically associated with other pathogens [ , [ ] [ ] [ ] [ ] [ ] [ ] . such characteristics have subsequently led many scientists to question the role of hbov as a potential pathogen. this lack of information is largely because of the inability of hbov to grow in standard cell lines [ ] . to confirm the effects of hbov, more studies are required throughout the world, focusing on various aspects of this infection, including epidemiology, serology, molecular biology, in vitro culture and animal models. hbov was detected from children and adults with arti from guangzhou, southern china, and the features were described in this study. hbov was confirmed in elderly patients ( and years old), suggesting that older people were also susceptive to hbov. all four adult patients with hbov positive in this study presented systemic influenza-like symptoms, which potentially suggest that hbov infection in adults may develop more serious symptoms than those in children. phylogenetic analysis suggested that hbov-gu from an elderly patient is in a single 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a newly identified bocavirus species in human stool a novel bocavirus associated with acute gastroenteritis in australian children human bocavirus infection severe pneumonia and human bocavirus in adult. emerg infect dis evidence of human bocavirus circulating in children and adults detection of human bocavirus in canadian children in a -year study the association of newly identified respiratory viruses with lower respiratory tract infections in korean children evidence of human coronavirus hku and human bocavirus in australian children isolation of human bocavirus from swiss infants with respiratory infections. swiss paediatric respiratory research group human bocavirus infection among children human bocavirus in french children frequent detection of bocavirus dna in german children with respiratory tract infections the human bocaviurses: a review and discussion of their role in infection pre-publication history the pre-publication history for this paper can be accessed here submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution the state major infectious disease research program (china central government, zx - ) provided financial support for this work. authors' contributions rz and w-kl designed the study. w-kl, q l, h-xl performed the hbov dna testing. d-hc, z-fy and sq collected clinical data. all authors participated in the data analysis. w-k l and r z drafted the manuscript. all authors read and approved the final version of this manuscript. the authors declare that they have no competing interests. key: cord- -vip jtlv authors: ng, lisa fp; barr, ian; nguyen, tung; noor, suriani mohd; tan, rosemary sok-pin; agathe, lora v; gupta, sanjay; khalil, hassuzana; to, thanh long; hassan, sharifah syed; ren, ee-chee title: specific detection of h n avian influenza a virus in field specimens by a one-step rt-pcr assay date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: vip jtlv background: continuous outbreaks of the highly pathogenic h n avian influenza a in asia has resulted in an urgent effort to improve current diagnostics to aid containment of the virus and lower the threat of a influenza pandemic. we report here the development of a pcr-based assay that is highly specific for the h n avian influenza a virus. methods: a one-step reverse-transcription pcr assay was developed to detect the h n avian influenza a virus. the specificity of the assay was shown by testing sub-types of influenza a virus and other viral and bacterial pathogens; and on field samples. results: validation on field specimens from vietnam and malaysia showed that the assay was specific without cross reactivity to a number of other infuenza strains as well as human respiratory related pathogens. detection was % from allantoic fluid in h n positive samples, suggesting it to be a reliable sampling source for accurate detection. conclusion: the assay developed from this study indicates that the primers are specific for the h n influenza virus. as shown by the field tested results, this assay would be highly useful as a diagnostic tool to help identify and control influenza epidemics. influenza a virus infects many animals such as humans, pigs, horses, marine mammals, and birds [ ] . in avian species, most influenza virus infections cause mild local-ized infections of the respiratory and intestinal tract, but highly pathogenic strains such as h n cause system infections in which mortality may reach % [ ] . in humans, influenza viruses cause a highly contagious acute respiratory disease that resulted in epidemic and pandemic disease in humans [ ] . three types of influenza viruses, types a, b, and c are known and they belong to a family of single-stranded negative-sense enveloped rna viruses called orthomyxoviridae [ ] . the viral genome is comprised of eight rna segments (seven in type c). influenza a viruses can be classified into subtypes based on antigenic differences in the two surface glycoproteins, namely, hemagglutinin (ha) and neuraminidase (na) which are required for viral attachment and cellular release. other major viral proteins include the nucleoprotein (np) which is the main structural protein, membrane proteins (m and m ), polymerase proteins (pa, pb and pb ), and nonstructural proteins (ns and ns ). currently, sixteen subtypes of ha (h -h ) and nine na (n -n ) antigenic variants are known in influenza a virus mostly related with veterinary significance, with only three subtypes circulating in humans (h n , h n , and h n ). however, in recent years, the pathogenic h n subtype of avian influenza a has been reported to cross the species barrier and infect humans as documented in hong kong in and [ ] [ ] [ ] . since late , the h n avian a influenza in poultry reached epidemic proportions with reports of serious outbreaks in several asian countries including vietnam, thailand, south korea, laos, cambodia, indonesia, japan and malaysia [ , ] that resulted in massive culling of millions of poultry which had severe economic repercussions. as a result, h n avian influenza a virus represents a potential danger to human health not only in asia but to the world. therefore, in addition to containment procedures, sensitive detection assays for early diagnosis are vital to lower the chances of spread and reduce the risk of development into an epidemic. current methods employed to detect h n subtypes include various polymerase chain reaction (pcr) assays [ ] [ ] [ ] ] and antigen tests using various fluorescence and enzymelinked immunoassays [ ] . however, these assays are reported to be low in specificity and sensitivity, and clinically, the low sensitivity of these diagnostics may limit the usefulness for reliable detection of influenza a (h n ) virus in humans [ ] . therefore, there is an urgent need for improved, validated, sensitive diagnostic tests for rapid and early diagnosis. in this study, we describe the development of a nucleic acid detection test that is rapid, specific and sensitive, thus allowing greatly improved detection of the h n avian influenza a virus. extraction of total rna was performed following manufacturers' protocol from qiaamp viral rna mini kit (qia-gen, germany) and trizol (invitrogen, usa) using all necessary safety precautions. the resultant rna was dissolved in μl of rnase-free water. μl of rna was used in μl reaction mixtures using the one-step reverse transcription (rt)-pcr system (qiagen, germany) with h n specific primers (forward primer: '-actatgaagaattgaaacacct- ' and reverse primer: '-gcaatgaaatttccattactctc- '). the pcr program was set as: °c for min, °c for min, °c for min, and °c for min followed by cycles of °c for sec, °c for sec, and °c for min and lastly followed by °c for min. the size of this pcr product was bp and was resolved in . % agarose gels. pcr products were sequenced directly to confirm the identity of the products. primers were designed at the conserved regions of the viral ha gene which may be less likely to be affected by mutational changes. this allows the detection of a broad range of isolates and variants of the h n subtype. the performance of the primers was first assessed in gel-based assays using in vitro-transcribed rna generated by the t ribomax express in vitro transcription system (promega, usa). the concentration of purified transcribed rna was measured by ribogreen rna quantitation reagent (invitrogen, usa) and serial dilutions of in vitro-transcribed rna were prepared in duplicate. a single one-step rt-pcr was done using μl of rna in a thermal cycler as described in methods, and products were analyzed on agarose gels. non-template controls were included. the sensitivity of the assay was found to be less than × copies and was able to specifically detect h n rna (fig. a , lanes to ). to establish the specificity of the assays for h n subtype detection, we then tested the primers on several known strains of influenza a viruses derived from avian sources (h n , h n , h n and h n ). non-influenza viruses such as newcastle disease virus (ndv) were also included as controls (fig. b) . thirteen of other known human respiratory diseases caused by viruses and bacteria were also included ( fig. a , lanes to , and table ) as controls. results showed that detection was specific to h n only ( fig. a and b) . as the number of actual human avian influenza virus infected cases are extremely sparse during this study, the inclusion of a panel of known human samples that do not contain h n virus derived from patients exhibiting "flu"-like symptoms are very helpful in demonstrating the non cross-reactive nature of these primers. in an effort to further investigate the specificity of this set of primers, a panel of avian and human subtypes (h to h ) of influenza a virus was screened. results showed that there was reactivity only against the h subtypes with this primer set and no reactivity against the other subtypes ( fig. a, lanes to ) . to ensure that the rna from these subtypes were not degraded, the matrix gene from these samples were amplified in parallel. as expected, the bp product was amplified for all the subtypes tested ( fig. b , lanes to ). a total of field samples comprising of known and suspect cases from chickens, ducks and muscovies isolated from vietnam and malaysia during the to outbreak were tested for h n rna (table detection of h n avian influenza a virus by one-step rt-pcr ). samples ranged from homogenized pooled organs and tissues, allantoic fluid, cloacal and tracheal swabs. all yielded positive results with % positive detection for allantoic fluid, % for cloacal and tracheal swab, and % for homogenized pooled tissue and organs (table ) verifying the sensitivity of this rt-pcr assay. viral culture isolation methods performed in eggs were used as confirmatory tests for all positive samples ( table ). this variation in detection could be due to the different efficiencies in viral rna recovery from the different samples, with allantoic fluid fractions having the highest efficiency for h n rna recovery and detection among the samples ( table ) . optimizing the extraction protocols may also improve the rna recovery from other tissues. in a separate study, the sensitivity of this h n primer set was also evaluated against the currently recommended world health organization (who) h primer set. three different strains were used -one human archived rna (human , from the vietnam outbreak) and two freshly extracted avian strains (avian and avian b). a rna dilution series was done on the three samples from - to - (fig. a, lanes to and lanes to ) , and in all the three strains, the h n primer set described here gave to logs better performance when compared to the recommended who primer set (fig. a , table ), indicating that this primer set performs well and consistent. in addition, five positive samples from the vietnam set were also then tested in parallel with another current h primer set and results showed that the h n primers described here detected five out of five positive h n samples while the other primer set only managed to detect three out of five (fig. b) , indicating that the h n primers described here are more sensitive and could detect weak positive samples with lower viral load. in conclusion, we have reported an efficient, specific and sensitive assay that has been evaluated on field specimens to be able to detect a wide variety of h n influenza virus isolates. accurate and sensitive detection of viral rna is also strongly influenced by the sample type. the rapid sensitivity test against human and avian strains figure sensitivity test against human and avian strains. a. the h n primers were tested for sensitivity against the currently recommended who h primer set using three h n strains, one human strain from stored archive rna, and two freshly extracted avian strains. one-step single tube reaction described here not only reduce the detection time but also lowers the risk of crosscontamination which has a higher probability in twosteps rt-pcr methods. this cost effective gel-based system has a lower limit of detection in the picogram range which is equivalent to × copies, and is designed to cater for use in the field in regions where real-time pcr platform and equipments may not be available. clearly, the results would be further strengthened with the inclusion of more known h n influenza in archived samples from humans, but the availability of human samples are difficult due to the low number of human infections at this point. however, observations from this study strongly suggest that the primers are specific for h n which can be very useful for the early detection and monitoring of avian influenza outbreaks. avian influenza highly pathogenic avian influenza outbreak of avian influenza a (h n ) virus infection in hong kong in re-emergence of fatal human influenza a subtype h n disease clinical features and rapid viral diagnosis of human disease associated with avian influenza a h n virus cases of influenza a (h n ) -thailand (h n ) in patients in vietnam nucleic acid sequence-based amplification methods to detect avian influenza virus identification and subtyping of avian influenza viruses by reverse transcription-pcr singlestep multiplex reverse transcription-polymerase chain reaction (rt-pcr) for influenza a virus subtype h n detection we thank jer-ming chia, martin l. hibberd, christopher w. wong and jian-jun liu (genome institute of singapore), no-na yeoh, geok-huai ong and rafidah ahmad johari (veterinary research institute, malaysia), fook-kheong ng, chee-wee lim and wai-kwan wong (central veterinary laboratory, agri-food and veterinary authority of singapore), and kian-leong ong and woon-hsi chin (veredus laboratories singapore) for their contributions. positive control human human h h h h h h h h h h h h h h h ntc h h h h h h h h h h h h h h ntc h dna marker the author(s) declare that they have no competing interests. lfpn, ib, rspt, ecr conceived the study, its design and coordination, and results analysis. tn, smn, lva, sg, hk, tlt and ssh carried out the experiments and analysis. lfpn and ecr drafted the manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -hptjqmrl authors: xiang, nijuan; song, ying; wang, yu; wu, jiabing; millman, alexander j.; greene, carolyn m.; ding, zhentao; sun, jie; yang, wei; guo, guoxia; wang, ruirui; guo, ping; ren, zhixing; gong, lei; xu, pengpeng; zhou, suizan; lin, dan; ni, daxin; feng, zijian; li, qun title: lessons from an active surveillance pilot to assess the pneumonia of unknown etiology surveillance system in china, : the need to increase clinician participation in the detection and reporting of emerging respiratory infectious diseases date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: hptjqmrl background: we sought to assess reporting in china’s pneumonia of unknown etiology (pue) passive surveillance system for emerging respiratory infections and to identify ways to improve the pue surveillance system’s detection of respiratory infections of public health significance. methods: from february –may , , we actively identified and enrolled patients in two hospitals with acute respiratory infections (ari) that met all pue case criteria. we reviewed medical records for documented exposure history associated with respiratory infectious diseases, collected throat samples that were tested for seasonal and avian influenza, and interviewed clinicians regarding reasons for reporting or not reporting pue cases. we described and analyzed the proportion of pue cases reported and clinician awareness of and practices related to the pue system. results: of ari admissions in two hospitals, ( %) met the pue case definition; none were reported. of specimens tested, ( %) were seasonal influenza virus-positive; none were avian influenza-positive. < % pue case medical records documented whether or not there were exposures to animals or others with respiratory illness. most commonly cited reasons for not reporting cases were no awareness of the pue system ( %) and not understanding the case definition ( %). conclusions: most clinicians have limited awareness of and are not reporting to the pue system. exposures related to respiratory infections are rarely documented in medical records. increasing clinicians’ awareness of the pue system and including relevant exposure items in standard medical records may increase reporting. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. china established the pneumonia of unknown etiology (pue) surveillance system in for timely detection of emerging respiratory infectious diseases [ ] , and the system has played an important role in detecting human infections with novel avian influenza viruses including a(h n ), a(h n ), a(h n ), and a(h n ) [ ] [ ] [ ] . nevertheless, a evaluation identified persistent underutilization of the pue surveillance system [ ] . more recently, inconsistent reporting occurred during the initial outbreak of low pathogenic avian influenza (lpai) a(h n ) [henceforth a(h n )], prompting public health authorities to allow clinicians to report cases directly without expert consultation committee approval. this change resulted in the reporting of cases in weeks compared with cases in the previous years [ ] . laboratory and case investigation resources were quickly strained and reporting procedures reverted to those used prior to the outbreak [ ] . as a result, case reporting subsequently decreased. a assessment of clinician and health administrator knowledge, attitude and practices related to pue surveillance conducted within healthcare facilities revealed a willingness to report pue cases, but identified limited awareness of the pue system, lack of understanding of the reporting process, and failure to follow the case definition [ ] . to evaluate these gaps, we piloted a -month active surveillance program in two hospitals to ) quantify the number of cases meeting the pue case definition and the number reported and ) to identify ways to improve the pue surveillance system's detection of respiratory infections of public health significance. national guidelines [ ] require all inpatient and outpatient healthcare facilities to report cases meeting the pue case definition. clinicians should report cases to an expert consultation committee, which after review of clinical and laboratory data determines whether to report the case to the pue surveillance system [ ] . if a case is reported to the pue system, the local center for disease control and prevention (cdc) will conduct a field investigation, collect respiratory specimens and send them to a national influenza surveillance network laboratory for testing of avian influenza viruses and, if associated with clusters of respiratory disease or relevant travel history, testing of severe acute respiratory syndrome coronavirus (sars-cov) and middle east respiratory syndrome coronavirus (mers-cov). a pue case is defined as an illness of unknown etiology with ) axillary temperature > °c, ) radiographic pneumonia, ) low or normal leukocyte count or low lymphocyte count during the early stages of disease, and ) no improvement or worsening symptoms after - days of antimicrobial treatment per clinical guidelines [ ] . participating hospitals were selected based on four criteria: if the facility ) admitted at least patients per month with a discharge diagnosis of pneumonia during february through may - ; ) used an electronic hospital information system; ) demonstrated willingness and capacity to collaborate with both national and local cdcs and ) was located within one of china's of provinces with previously identified h n and/or h n human cases. we selected two tertiary hospitals in anhui province: the second people's hospital of fuyang city, a -bed facility, which from february through may - , admitted an average of pneumonia patients per month, and lu'an city people's hospital, a -bed facility, which over the same time period admitted an average of pneumonia patients per month. fuyang hospital, an infectious disease hospital, had experience treating human infections with avian influenza, while lu'an hospital, a general hospital, did not. after reviewing the hospital information systems, the evaluation team developed a list of admission diagnoses that captured the majority of acute respiratory infections (ari) (additional file ). every day (including weekends) from february through may , , a designated, trained surveillance officer in each hospital ) reviewed the hospital admission registry database to screen all admission diagnoses from the prior days for diagnoses from the screening list (additional file ); ) reviewed admission medical records with a matching diagnosis to identify and enroll patients with illnesses meeting the pue case definition; ) days later, conducted a second medical record review for patients not enrolled during the first review to identify and enroll patients with illnesses newly meeting the case definition (for example, patients with no improvement or worsening symptoms after - days of antimicrobial treatment per clinical guidelines); and ) days later, conducted a third review of records for patients not enrolled during the first two reviews to enroll any remaining patients meeting the case definition. [fig. ]. for patients with illnesses meeting the pue case definition, the surveillance officer used a standard questionnaire to collect information from the hospital information system related to demographics and, if available, epidemiological risk factors, including exposures to poultry, patients with similar symptoms, and travel history. surveillance officers followed the pue surveillance protocol [ ] to investigate enrolled pue case-patients. surveillance officers conducted face-to-face patient interviews using a standard questionnaire to collect the same information described in the medical records review section above to determine both accuracy and completeness of medical records. surveillance officers collected throat swabs from all identified pue case-patients per the surveillance protocol [ ] . specimens were transported to the local cdc laboratory per standard procedures and tested for influenza viruses using real time reverse transcription polymerase chain reaction (rrt-pcr). if identified pue case-patients were part of a cluster of epidemiologicallylinked respiratory illnesses, specimens would also be tested for sars-cov and mers-cov. if a case-patient reported travel history to the middle east, specimens would be tested for mers-cov. the pue surveillance protocol describes a three-step procedure for reporting cases to the pue system: ) clinicians report identified pue cases to their supervisor; if the supervisor concurs, the case is reported to the director; ) the director determines whether to report the case to an expert consultation committee which usually includes specialists from the respiratory medicine department, the radiology department, and infection control; and ) the expert committee determines whether to report the case to the pue system. [ fig. ]. in our evaluation, after patients with illnesses meeting the pue case definition were enrolled, surveillance officers interviewed all clinicians who had primary medical responsibility for these patients. if the case were reported to the clinician's supervisor, the surveillance officer also interviewed the supervisor and a representative member of the expert committee. [ fig. ] surveillance officers used a standard questionnaire to collect ) demographic and occupational information about the clinician being interviewed and, when applicable, ) demographic and occupational information about the senior clinicians who received the case report, and ) reasons for reporting or not reporting pue cases. first, we described the number, proportion, ward distribution, and testing results of pue cases identified by surveillance officers. wilson score was used to calculate % confidence intervals (ci) for proportions. second, we described and compared epidemiologically-linked exposures documented by clinicians in the medical records and used chi-square tests to compare differences in the frequency of exposure histories collected by surveillance officer interviews. third, we described the number of pue cases reported at each of the tiers of the three-step reporting process. finally, we described factors associated with clinicians' reporting or not reporting pue cases. during the evaluation period, surveillance officers reviewed , hospitalization registrations, and screened patients with ari admission diagnoses. of those were from lu'an hospital and were from fuyang hospital. among all ari patients, ( %) had illnesses that met the pue case definition. the proportions of ari cases meeting the pue case definition in each of the two hospitals were similar [ % ( / ) vs. % ( / ), p = . ]. among the pue case-patients identified, % were male, and % were aged - years; most were from the pediatrics ( %) and pulmonary ( %) departments. [ table ]. the proportion of ari admissions meeting the pue case definition was highest in the icu ( %), followed by the infectious disease ( %), tuberculosis ( %), pediatrics ( %) and pulmonary ( %) departments. oral throat swabs, collected within hrs of pue case enrollment, were tested from ( %) of the identified pue case-patients. of the specimens, ( %) were positive for seasonal influenza viruses and none were positive for avian influenza viruses. none met sars-cov or mers-cov testing criteria. of pue case-patients with laboratory results, % ( / ) had documented exposures; % had documented histories related to contact with water potentially infected with parasites which is recorded as standard practice within the "personal history" section of the medical record in china. other exposures which were intermittently documented in the medical records included ( ) ( ) internal medicine emergency room ( ) ( ) internal medicine ( ) ( ) other departments ( ) ( ) animal exposure ( %), contact with persons with respiratory illnesses ( %), exposure to healthcare facilities caring for patients with respiratory illnesses ( %), and any travel history ( %). in addition, ( %) records documented occupation, a required item in the demographic section of the medical history, and identified two pue case-patients as healthcare workers [ table ]. among the enrolled pue case-patients interviewed by surveillance officers, ( %) had at least one exposure relevant to respiratory infections of public health significance, including animal exposure, contact with similar cases of respiratory disease, travel to/living in areas of novel respiratory epidemics, and occupational exposures. in the days before illness onset, ( %) had occupational exposure to poultry/livestock, ( %) were medical staff, ( %) had animal exposure ("exposure to poultry, pigs, etc."), ( %) had close contact with persons with similar respiratory disease symptoms, and ( %) had exposure to a healthcare facility caring for patients with respiratory illnesses [ table ]. although the relevant respiratory infectious disease exposures identified in the medical record and the surveillance officer interview were the same in > % for all exposures analyzed, there were discrepancies. occupational exposures, exposures to persons with similar respiratory symptoms, and animal exposures were identified less frequently through medical records compared to surveillance officer interviews [ table ]. none of the clinicians interviewed from lu'an hospital reported knowledge of the pue surveillance system compared with ( %) of the clinicians from fuyang hospital. at fuyang hospital, knowledge was highest among clinicians with > years ( %) and - years of work experience ( %) and lowest among those with < years of work experience ( %). none of the patients meeting the pue case definition were reported to the surveillance system. during the interviews with clinicians, the most common reasons clinicians cited for not reporting included: being unaware of the pue surveillance system ( %), not understanding the pue case definition ( %), and not accepting the pue case definition ( %) [ table ]. although none were reported to the national system, a clinician reported one pue case with acute respiratory distress syndrome (ards) and possible viral pneumonia to his supervisor. due to disease severity, the lack of a diagnosed pathogen, and no improvement on treatment, the supervisor reported the case to the director, and experts within the ward concluded that the illness met the pue case definition. they reported the case to the hospital's expert committee, which reported to the hospital's department of disease control, which reported the case to the local cdc and sent specimens for laboratory from february through may , , we conducted active surveillance in two hospitals and found that % of all patients admitted with ari met the pue case definition. none of the respiratory specimens tested were positive for avian influenza. only one pue case was reported to the local cdc; however, it was not reported to the national system because the specimen tested negative for avian influenza virus. our findings raise questions about the feasibility of using the existing pue case definition to identify respiratory infections of public health significance. extrapolating our results, if clinicians reported all illnesses meeting the pue case definition from china's more than , hospitals, the number of pue cases identified would be in the hundreds of thousands per year. such numbers would overwhelm the public health system's capacity for laboratory testing and epidemiologic investigations. the impracticality of the existing pue case definition is supported by both a prior study which found that % ( / ) of community-acquired pneumonia diagnoses met the pue case definition [ ] and by the pue surveillance experience in when streamlined reporting procedures led to a surge in cases that quickly strained response efforts [ ] . modifying the system to decrease the number of cases that meet the pue case definition but are not emerging respiratory infections of public health significance would increase the system's feasibility, acceptability, and usefulness. the extent of under-reporting in this pue assessment far exceeds the estimated - % under-reporting of cases of notifiable diseases identified during - in evaluations of the china national notifiable disease reporting system for notifiable diseases, for which reporting is required by law [ ] [ ] [ ] . during our evaluation, none of the identified pue cases was reported to the national system. one clinician reported one pue case appropriately, but the expert committee did not appropriately follow final reporting procedures by reporting the case to the pue system. the two most common reasons clinicians cited for not reporting pue cases to the system were not having knowledge of the pue system and not understanding the pue case definition. knowledge varied by hospital, with > % of clinicians from fuyang hospital reporting knowledge of the pue system compared with none from lu'an hospital. this difference may be explained by fuyang hospital's specialization in infectious disease and its recent experience treating one avian influenza a(h n ) virus infection and one avian influenza a(h n ) virus infection in and respectively. lu'an hospital, a general hospital, had no recent experience treating infections with avian influenza virus. pue surveillance system knowledge also varied significantly by clinicians' years in practice. china cdc conducted intensive, national clinician training on the pue system when the system was established and on-going trainings during the h n outbreaks through . in , the ministry of health required training on the new protocol of surveillance, investigation and management for pue for clinicians at all medical wanted to treat the patient longer prior to reporting ( , ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( ) ( , - . ) ( , - ) the patient was transferred to another hospital or ward ( , - ) ( , - . ) ( , - ) when a case is reported, the laboratory results are returned too late such that the testing is not helpful for diagnosis and treatment *ci refers to confidence interval institutions [ ] , however, national pue-related training has not occurred since . therefore, clinicians with < years work experience have not received systematic, indepth training on pue surveillance. another recent study among hospital clinicians in beijing found that new clinicians knew little about key public health concerns, such as infection control within healthcare facilities, without having received formal training [ ] . these findings highlight the importance of training clinicians on public health surveillance systems, reporting requirements and other key public health topics both as they enter the workforce and as refresher courses to improve their capacity to identify and report emerging and re-emerging infectious diseases. this evaluation found that clinicians did not document respiratory infection-related exposures. during the interviews, % identified pue case-patients reported recent exposures to animals and % reported exposures to patients with similar illnesses, yet these exposures were rarely documented in the medical record. these findings suggest that clinicians may not routinely assess respiratory infection-related exposures. about half of the medical records from identified pue cases documented any contact with parasites-infected water, because asking about "any potential exposure to parasite infected water" is a routine practice when completing the "personal history" section of the medical record [ ] . this finding suggests that clinicians are more likely to ask about specific exposures when they are part of routine practice as opposed to exposures that may only be asked intermittently if not part of routine practice. the widespread use of electronic medical records in china provides an opportunity for prompting clinicians to ask about certain exposures relevant to infectious diseases that can be documented in a standardized way in patient's medical record. by developing a checklist within the electronic medical record with questions related to exposures relevant to emerging respiratory diseases such as live poultry and swine for priority use in inpatient wards in china, clinicians would be prompted to routinely ask about these exposures. this may in turn improve detection and reporting of emerging respiratory infections. first, increasing documentation of relevant exposures may facilitate the addition of more specific epidemiologic criteria to the pue case definition to reduce the number of cases meeting the case definition that are not infections of public health concern. second, clinicians who identify concerning exposures in patients with respiratory diseases may be more likely to report these cases to the pue system. finally, since the majority of hospitals in china now use electronic medical records system, if exposure data were collected systematically as part of these systems, it is possible that cases with relevant epidemiological data could be automatically flagged for reporting. further investigations would be needed to assess these modifications to see if they have a positive impact on case reporting without overwhelming the pue system. this evaluation is subject to several limitations. first, the assessment occurred when there were no local reports of human infection with avian influenza virus. pue case identification and reporting may increase during outbreak periods. however, in , during the th epidemic of influenza a(h n ) which had the largest number of human infections to date, fuyang hospital did not report any cases to the national system despite sending potential pue case specimens to local cdc for testing, of which two were positive for influenza a(h ). during this same period, lu'an hospital did not send any potential pue case specimens to local cdc for testing, nor did it report any cases to the national pue system. lu'an hospital did, however, report one confirmed h n case to china cdc directly. second, the reporting practices within these two hospitals may not represent reporting practices throughout china. finally, the screening admission diagnosis list may not have captured all pue cases [additional file ]. our findings suggest that most clinicians are not reporting cases to the pue surveillance system. if clinicians were to report all cases meeting the pue case definition, the large number reported would likely overwhelm the public health system's capacity for laboratory testing and case investigation. of those reported, the vast majority of cases would not be emerging infections of public health concern. our findings lead to several recommendations that may increase the specificity of the pue case definition, increase clinician participation in the pue system, and contribute to the early detection of emerging respiratory infections in china. ) modifying the existing pue case definition by adding relevant exposure history may improve the specificity, feasibility and utility of case reporting. ) including exposure items related to emerging respiratory infectious diseases in the standard infectious disease history documented in medical records may increase the likelihood that clinicians will assess exposure histories relevant for emerging respiratory infections. including these items in the respiratory diseases department, the pediatric department and the intensive care unit, where pue cases are more common, may be most useful. ) providing clinicians with frequent publichealth related training and communications will ensure that clinicians are aware of public health reporting requirements. a multi-pronged approach to incorporating public health practice into clinical settings may include: offering training to clinicians as they enter the workforce followed by annual refresher courses, posting publichealth related updates and notices in clinical areas, incorporating public health guidance into hospital policies, engaging clinicians in the development of clinically-appropriate and easy-to-apply case definitions, and regularly sharing local and national public health data of interest with clinicians to highlight the public health importance of their work. ministry of health protocol of surveillance, investigation and management for pneumonia of unknown etiology investigation and analysis on the first case of human infected with avian influenza (h n ) in yunnan province epidemiological on the first human case with avian influenza a (h n ) virus in hubei province analysis on reporting of unknown etiology pneumonia cases in china the evaluation of surveillance system of highly pathogenic avian influenza virus infection in china. beijing: chinese center for disease control and prevention use of national pneumonia surveillance to describe influenza a(h n ) virus epidemiology, china assessing clinicians' acceptance of the pneumonia of unknown etiology surveillance system current status and clinical study of suspected pneumonia cases of unknown origin in china an investigational analysis of missing reports of notifiable diseases in medical facilities all over china in evaluation on management and quality of communicable diseases network direct reporting in china reporting quality of notifiable communicable diseases in hospitals in china the notice for the intensive training of local clinicians in different level of medical institutions for detection and reporting of avian influenza an analysis on knowledge survey of hiv occupational exposure and the influencing factors in medical workers at two tertiary general hospitals in beijing the notice of the ministry of health on the issuance of basic rules of medical records publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank the colleagues from china cdc, anhui province cdc, lu'an prefecture cdc, fuyang prefecture cdc, lu'an city people's hospital and the second people's hospital of fuyang city who supported project implementation for this assessment, including case screening and investigation, clinician interviews, specimen collection, laboratory testing, and logistical administration. authors' contributions nx, ys, yw, jw, cm. g, zf and ql contributed to the design of this study; nx, yw, jw, zd, js, wy, gg, rw, pg, zr, lg, px and dl contributed to the implementation and data collection of this study; nx, ys, yw, aj. m, cm. g, sz, dn, and ql contributed to data analysis, manuscript writing and manuscript revision. all authors have read and approved the final version of the manuscript. dr. nijuan xiang is an epidemiologist and chief of the surveillance, alert and risk assessment (sara) branch, public health emergency center, chinese center for disease control and prevention. her research interests include surveillance for emerging infectious diseases, prevention and control strategies for emerging infectious diseases, and surveillance and risk assessment for public health emergencies. this work was funded by a us cdc cooperative agreement ( u gh - ) and supported by the china-us collaborative program on emerging and re-emerging infectious disease. the fund body did not participate in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. all data generated or analysed during this study are included in this published article [and its supplementary information files]. the datasets generated and/or analysed during the current study are not publicly available due to the datasets containing personally identifiable information used for public health surveillance purposes. requests for data can be directed to the corresponding author.ethics approval and consent to participate this evaluation was approved by the chinese center for disease control and prevention institutional review board (number: ) and was determined to be non-research public health practice by us cdc. all patients participating in this evaluation, or their parent or guardian for patients < years of age, provided oral informed consent prior to interview and written informed consent prior to throat swab collection. not applicable. the authors declare that they have no competing interests. key: cord- -n tmn ph authors: cui, binglin; zhang, dangui; pan, hui; zhang, fan; farrar, jeremy; law, frieda; van doorn, h rogier; wu, beiyan; ba-thein, william title: viral aetiology of acute respiratory infections among children and associated meteorological factors in southern china date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: n tmn ph background: acute respiratory infections (aris) are common in children and mostly caused by viruses, but the significance of the detection of multiple viruses in aris is unclear. this study investigated respiratory viruses in aris among children and associated meteorological factors in shantou, southern china. methods: paired nasal/throat-flocked swabs collected from , children with aris, who visited outpatient walk-in clinics in a tertiary hospital between december and november , were examined for fourteen respiratory viruses - influenza viruses (flua, flub), respiratory syncytial viruses (rsv a and b), human coronaviruses (hcov: e, oc , hku , nl ), human metapneumoviruses (hmpv a and b), parainfluenza viruses (piv - ), human rhinoviruses (hrv a, b, c), enteroviruses (ev), adenoviruses (adv), human bocavirus (hbov), and human parechoviruses (hpev) - by multiplex real-time pcr. results: we identified at least one virus in . % ( / , ) and multiple viruses in . % ( / , ) of patients. ev and hrv were the most frequently detected single viruses ( . %, / and . %, / respectively) and co-detected pair ( . %, / ). overlapping seasonal trends of viruses were recorded over the year, with dual peaks for ev and single peaks for the others. by logistic regression analysis, ev was positively associated with the average temperature and humidity, hcov, and piv , but negatively with hrv, piv , and hbov. hrv was inversely associated with ev and piv . conclusions: this study reports high viral detection and co-detection rates in pediatric ari cases mainly due to ev and hrv. many viruses circulated throughout the year with similar seasonal trends in association with temperature, humidity, and wind velocity. statistically significant associations were present among the viruses. understanding the polyviral etiology and viral interactions in the cases with multiple viruses warrants further studies. electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory infections (aris) are one of the illnesses of highest morbidity and mortality in children worldwide [ ] [ ] [ ] . the pathogens causing aris vary geographically and by season, but globally viruses play a major role. respiratory syncytial virus (rsv) is by far the most common pathogen associated with severe respiratory diseases as bronchiolitis, exacerbation of asthma, or pneumonia in early life, and is a leading cause of hospitalization in children under two [ ] . influenza viruses have the greatest potential to cause severe respiratory diseases in the very young, the elderly and those with underlying chronic conditions [ ] . enteroviruses including human rhinoviruses (hrv) and human enteroviruses (ev), previously identified in childhood upper respiratory tract infections, are commonly associated with milder aris and have been suspected as major etiological agents of lower respiratory tract infections leading to bronchiolitis and pneumonia in infants [ ] . it has also been reported that human metapneumovirus (hmpv) causes approximately - % of all aris in children and adults [ ] and adenoviruses (adv) account for - % of respiratory infections in children [ ] . respiratory illnesses can be attributable to other viruses such as parainfluenza viruses (piv) and human coronaviruses hcov- e, oc [ ] . with rapid progress in molecular diagnostics, newly discovered viruses including human bocavirus (hbov), human coronaviruses (hcov-nl , hcov-hku ), human parechoviruses (hpev), and polyomaviruses wu (wupyv) and ki (kipyv) have also been detected in children with respiratory infections, with varying levels of proof of causation [ ] . hospital-based studies in children published over the last decade worldwide have identified viruses in up to % of ari episodes, with a single virus found in - % and multiple viruses in - % of infected patients [ , , ] . coinfection is reportedly related to the time of year when circulations of multiple viruses occur [ ] . some studies have shown that the prevalence of co-infections is not related to the absolute prevalence of individual viruses [ ] . factors such as young age, male gender, and history of immunosuppression are associated with an increased chance of viral co-infections [ , , ] . there could be likely interactions between climatic, environmental, and behavioral factors, and complex interplay between circulating viruses and population-level immunity regarding viral coinfections. understanding these factors may help us prevent transmission of these infections. recent etiologic studies on pediatric respiratory infections mostly report the prevalence in hospitalized children and the seasonality of viruses without elaborating viral co-infection. therefore, the significance of the detection of multiple viral pathogens in aris is unclear. here, we investigated fourteen common respiratory viruses among pediatric outpatients in southern china during - and their associations with meteorological factors. this study was conducted at the pediatric outpatient walk-in clinics, the first affiliated hospital of shantou university medical college. the pediatric department provides both primary and tertiary care (common practice in china) for approximately , children per year in the chaoshan region of southern china. the chaoshan region is in the subtropical zone with an average annual temperature of . °c and excellent to lightly polluted air quality levels (air quality index, aqi: - , in - ) . based on modified who standard case definition of aris [ ] , eligible participants were defined as a child - years of age presenting within days of onset of illness with at least two of the following: fever, sore throat, cough, rhinorrhea, nasal congestion, and hoarseness of voice. patients with any condition preventing swab collection were excluded. we recruited eligible patients in the morning, during which approximately % of patient visits are made, on a daily basis except public holidays from december to november . participants' demographic details and clinical features are shown in table . paired nose and throat-flocked swabs (copan, brescia, italy, cat. no. cs and cs ) were collected from each participant, combined in one tube, and stored within h of collection at − °c until further processing. multiplex real-time pcr was performed using roche, lightcycler ii (roche diagnostics, penzberg, germany) to identify the following respiratory viruses: influenza a (flua), influenza b (flub), respiratory syncytial viruses a and b (rsv), human coronaviruses e, oc , hku and nl (hcov), human metapneumoviruses a and b (hmpv), human parainfluenza virus types , , , and (piv , piv , piv , and piv ), human rhinoviruses a, b, and c (hrv), human enteroviruses (ev), human adenoviruses (adv), human bocavirus (hbov), and human parechoviruses (hpev). nucleic acid extraction was performed using the qiaamp viral rna mini kit (qiagen gmbh, hilden, germany, cat. no. ). reverse transcription and realtime pcr assays were performed as described previously [ ] , except for the primers and/or probes for hrv, hpev, and internal control equine arteritis virus (eav, see the sequences of viruses in additional file ). due to known cross-reactivity between enteroviruses [ ] [ ] [ ] , hrv was detected using two sets of primers and probes: hrv-v (version ) for screening and hrv-v (version ) for confirmation. real-time pcr results were interpreted as described previously [ ] . the pcr was considered positive or negative when the cp value was less than cycles or exceeded cycles, respectively, and the positive control showed the expected cp value, negative control was negative, and internal control showed the expected cp value. a negative internal control signal was accepted in case of a positive target sequence with correct positive and negative control signals. meteorological data, including the average daily temperature (°c), the average daily humidity (%), and the average daily wind velocity (km/h), were collected from the official website of shantou meteorology, tutiempo. net (http://www.tutiempo.net/en/climate/shantou/ / .htm). we used chi-square test to compare differences in the distribution of categorical variables, anova and kruskall wallis tests to compare medians, and the pearson correlation analysis to evaluate the associations between the meteorological factors and viruses and among viruses. the variables with significant associations were further analyzed in multivariate logistic regression models, in which symptoms and positivity of viruses were treated as dependent and independent variables to assess virussymptom associations; and individual viruses were treated as dependent variables with meteorological factors or other viruses as independent variables to investigate meteorological factor-virus and virus-virus associations. a two-tailed p-value of < . was considered significant. all these analyses were performed with spss statistics version . . the study was approved by the ethics committee of the first affiliated hospital of shantou university medical college and the oxford university tropical research ethical committee (oxtrec). written informed consent was obtained from parents or legal guardians of children enrolled in the study. of , children ( . % male) recruited, . % ( / , ) were > - years old (table ). at least one virus was identified in . % ( / , ) of the patients, with single virus in . % ( / , ) and multiple viruses in . % ( / , ). hpev was not detected. compared with virusnegative patients, virus-positive patients were less likely to have fever (or: . , % ci: . - . , p = . ). patients with multiple viruses were more likely to have rhinorrhea than those with single virus (or: . , % ci: . - . , p < . , table ). hcov (or: . , % ci: . - . ) and piv (or: . , % ci: . - . ) were more prevalent in the > year age group than in the ≤ year group (all p ≤ . ), while hbov (or: . , % ci: . - . ) and rsv (or: . , % ci: . - . ) were less frequently found in the > year group (all p < . ). chi-square test and multivariate logistic regression analysis showed that cough was positively associated with hrv and rsv, and negatively with ev; rhinorrhea was positively associated with hrv, piv , and hbov, and negatively with ev; fever was positively associated with ev, and negatively with hrv and piv ; and nasal congestion was positively associated with rsv, and negatively with ev and hcov (all p < . , table ). viruses detected alone or co-detected with other viruses are shown in table . the most frequently detected virus was ev ( . %, / ), followed by hrv ( . %, / ), and hcov ( . %, / ). ev and hrv were most commonly co-detected with other viruses (table ) and also the most commonly co-detected pair of viruses ( . %, / , see the distribution pattern of viruses in additional file ). screening with hrv-v identified cases co-positive for hrv and ev, and subsequent confirmation with hrv-v primers/ probes [ ] resulted in only positive cases ( . %, / ). the temporal circulation and co-circulation patterns of viruses are shown in figures and . there were overlapping seasonal trends of many viruses throughout the year, with dual peaks for ev in july and september and single peaks for the other viruses. both ev and hrv circulated throughout the year. hcov and piv circulated predominantly between april and may but sporadically throughout the year. piv , rsv, flua, and adv peaked in january, while hbov peaked in march. flub circulated mostly from february to july with a peak in april. codetection of - viruses occurred all in may (see additional file ). the optimal average daily temperature, humidity, and wind velocity for these viruses are shown in table . table shows the multivariate logistic regression models for independent associations between the viruses and meteorological factors and between the viruses. ev was positively associated with the average temperature and humidity and the presence of hcov and piv , but negatively with hrv, piv , and hbov. hrv was negatively associated with the presence of ev and piv . hcov was positively associated with the average temperature and humidity and the presence of ev and piv . piv was positively associated with the average humidity and the presence of rsv and flua, but negatively with the average temperature and wind velocity, and the presence of ev, hrv, and hbov. piv was positively associated with the average temperature and the presence of hcov and rsv, however, negatively with the wind velocity. hbov was positively associated with rsv and flua, but negatively with the average temperature and humidity and the presence of ev and piv . rsv was positively associated with the presence of piv - , hbov, and flua, but negatively with the average temperature and wind velocity. flua was positively associated with the presence of piv - , hbov, and rsv, but negatively with the average temperature. this is the first prospective study reporting the associations between meteorological parameters and co-circulation patterns of common respiratory viruses. the viral detection rate among pediatric outpatients with aris in this study ( . %, / , ) was higher than those reported from nanjing, china ( viruses, . %, / ) [ ] and other countries, including honduras ( viruses, . %, / ) [ ] and greece ( viruses, . %, / ) [ ] in the same study period. enteroviruses (ev, . % and hrv, . %) were most frequently detected in our outpatient children. influenza viruses and rsv, the leading pathogens in pediatric outpatients in only statistically significant results (p < . by chi-square test for individual comparisons of proportions within each group) are shown as "√" with reference(s) shown as "-". the optimal temperature for hcov and hbov was - °c. the optimal relative humidity was - % for piv and - % for hcov. similar studies [ , [ ] [ ] [ ] , were detected in . % and . % of our cases, with hcovs ( e, oc , hku , and nl ) in . %, and relatively recently discovered viral pathogens hbov and hmpv in . % and . % of cases, respectively ( table ) . the viral co-detection rate ( . %, / , ) was also high among our study population. reported rates of codetection vary widely, from . % among pediatric patients with influenza-like illness [ ] to % among infants with acute bronchiolitis [ ] . detection of dual viruses is common, and co-detection of five [ ] or even six viruses [ ] is not anecdotal. all the cases with - viruses in this study were in may, the end of the cold season in the chaoshan region. this may be in part due to past viral infections, as some viruses can still be detectable by pcr several weeks after infection [ , ] . most studies have shown that rsv is the predominant respiratory pathogen co-detected in hospitalized children, followed by hrv, piv, hmpv, hbov, and flua [ , ] . in this study, ev, hrv, hcov, and piv - were involved in the majority of co-detections, with ev-hrv as the most frequently co-detected pair ( % of codetections). ev and hrv were included in the panels in many studies globally [ , , , , , [ ] [ ] [ ] [ ] [ ] [ ] [ ] , and the ev-hrv pair was the most commonly detected pair among outpatient children with aris in finland ( . % of codetections) [ ] and infants with acute bronchiolitis in brazil [ ] . the co-detection rate of ev-hrv in this study is similar to that in finland [ ] . varying detection rates of multiple viruses in different studies may reflect the differences in the study period and location, study population, environmental factors, the number of respiratory pathogens tested, and/or the diagnostic methods/techniques used. likely reasons behind high detection rates of single and multiple viruses in this study could be due to improved recovery of viruses by using flocked swabs [ ] and/or combined nasal and throat swabs [ ] . there are advantages and disadvantages of multiplex pcr technique in diagnosing respiratory viral infections. while its high sensitivity and specificity facilitate simultaneous detection of a large spectrum of viruses, including those difficult to be identified by traditional methods [ ] , its capacity to detect low amounts of viral nucleic acids in some cases during viral incubation period, asymptomatic infection, or post-infectious shedding makes it difficult to interpret the results [ , ] . the development and validation of standardized quantitative pcr with clinically relevant cutoff values [ ] or combining qpcr with serology could be helpful for etiologic understanding of simultaneous presence of multiple viruses. certain host-specific risk factors may predispose a child to respiratory co-infection. younger age [ , , ] , male gender, and history of immunosuppression are associated with increased risk of viral co-detections [ ] . nonetheless, similar associations were not found in this study. viral co-detection is not random; clear associations for certain viral co-occurrence have been described [ ] . the viruses circulating at the same time of a year are more likely to accompany each other [ , , ] . this may be driven by meteorological factors which actually work behind seasonal variations, or by interactions of certain coexisting viruses. temperature, humidity, and wind velocity are the most commonly studied factors significantly associated with the overall number of ari hospitalizations and the prevalence of various respiratory viruses [ ] [ ] [ ] . the average temperature is the key climatic parameter associated with the prevalence of many respiratory viruses. some viruses survive and/or replicate better at low temperatures, having peak prevalence in the colder months. in our study, the detection rates of piv , rsv, flua, and adv were negatively associated with temperature and were highest at temperatures between °c and °c (tables and ), supporting the notion that low temperature is suitable for the survival of lipidenveloped air-borne viruses [ ] . low temperatures have been found to favor rsv in southeast china [ ] , malaysia [ ] , nepal [ ] , brazil [ ] and germany [ ] , influenza in japan [ ] and germany, and adv in germany [ ] ; however, high temperatures favored piv in southeast china [ , ] and nepal [ ] , rsv in singapore [ ] , hong kong [ ] , and indonesia [ ] , and adv in southeast china [ ] . no association between temperature and flua activity was found in nepal [ ] and brazil [ ] . in our study, other viruses such as ev, hcov, and piv were more often detected during months with higher temperatures, having peaks at temperatures between °c and °c (tables and ). in contrast to our findings, hcov was negatively associated with temperature, and no association between ev and temperature was found among children with aris in germany [ ] . association of humidity and viral detection rates has been reported from germany [ ] , singapore, hong kong, brisbane, and vancouver [ ] . in this study, three viruses (ev, hcov, and piv ) were positively associated with the average humidity ( table ). the optimal average humidity ranges for ev and hcov were - % and - % respectively, supporting a previous finding that high average humidity ( %) had a protective effect on the survival of hcov [ ] . our findings on piv are inconsistent with animal and laboratory observations that lipid-enveloped viruses such as piv survived better in cooler, less humid environment [ ] . hbov was negatively associated with the average humidity, and its optimal humidity was - % (tables and ). no climatic data is available for comparison regarding this virus. previously reported association between flua and the average humidity [ ] was not found in our study. wind velocity piv and piv have been reported to be negatively associated with wind velocity [ ] . in low wind speed environment, viruses can easily colonize in the epithelium of upper respiratory tract [ ] . an increased wind velocity is correlated with rsv activity in germany [ ] . in our study, piv , piv , and rsv were inversely associated with the wind velocity. although we observed higher rates of ev and flub but lower rate of hrv in low wind velocity, we could not confirm these associations by logistic regression analysis. the underlying reasons for the observed associations between virus circulations and meteorological factors are unclear. climate could have a direct or indirect effect on viral survival, transmission efficiency, host immunity, and social behavior change [ , ] . cold and dry conditions might favor the transmission of viruses, and cold or rainy days could decrease outdoor activities of children and increase the probabilities of close contact and transmission of infections [ ] . holidays (supported by our data with less cases in february as chinese new year and july-august summer holiday, figure and additional file ), could also play a role in an annual epidemic cycle [ ] . it is likely that several factors interact in complex ways in the development of observed epidemics under optimal climatic conditions and that the contributions of individual factors vary for different viruses. further investigations such as time series model over many years are needed to account for their inherent autocorrelations [ ] , and thus the observed associations between meteorological parameters and viruses in this exploratory analysis should be interpreted with caution. viral co-detection patterns may be the reflection of interactions between viruses. co-detection of viruses has been frequently reported [ , , , , , ] . here, we have assured their associations by mathematical models (table ) . we identified many pairs of viruses with positive associations, including ev-hcov, hcov-piv , piv -rsv, piv -flua, piv -rsv, hbov-rsv, hbov-flua, and rsv-flua. negative associations for ev-hrv, ev-piv , ev-hbov, hrv-piv , and piv -hbov were also found in this study. both belonging to the enteroviruses genus, hrv and ev have similarities in the highly conserved sequence of the ' noncoding region, which is the preferred site for molecular assay development [ , , , ] . cross-reactivity between the primers of hrv with evs has been reported and is among others attributable to ev-d , an emerging pathogen frequently undetected and misdiagnosed as hrv [ , , , ] . confirmation of cross-reacting ev types in this geographic region should be done in future studies. there is no consensus in the literature on the clinical implications of the viral detection and co-detection. some studies linked multiple viral detections with fever [ ] , or increased hospitalization and intensive care admission [ ] , while others described a very similar prognosis as in single infection [ , , ] , or even milder presentations [ ] . in this study, the virus-negative patients had fever more often, which may be caused by other pathogens such as bacteria. we also found that rhinorrhea was more frequently present in patients with multiple viruses than in those with a single virus, and some viruses were more (or less) likely to exist in certain age groups or were accompanied with certain symptoms. since we did not follow the cases, the associated clinical course and outcome (such as hospitalization) remain unknown. a better understanding on the clinical courses of single and multiple viral etiologies requires further studies. the current study has several limitations. the majority of outpatients enrolled in this study were mild and moderate cases. therefore, we could have missed pathogens responsible for severe aris. as healthy or asymptomatic controls were not included, their viral carriage burdens and the actual role of virus infections could not be elucidated. following up the cases for clinical burdens and serologic testing would be required in future studies. air quality indicators such as ozone and pm . , which might influence the host's susceptibility or virus circulation, should be included to investigate meteorological factors. in summary, this study reports a high viral carriage in pediatric ari cases with high viral co-detection rates mainly due to ev and hrv. there were overlapping seasonal trends of many viruses throughout the year. meteorological factors, including temperature, humidity, and wind velocity, were associated with the viral detection rates. statistically significant associations were present among the viruses. further studies are needed to address polyviral etiology and viral interaction in multiple virus positive cases. additional file : primers and probes of viruses. additional file : the distribution pattern of viruses in pediatric outpatients with acute respiratory infections, aris (n= ). the authors declare that they have no competing interests. authors' contributions blc and hp designed and performed the experiments, analyzed the data, and wrote the paper. dgz designed and performed the experiments, and analyzed the data. fz analyzed the data. jf conceived and designed the experiments, and analyzed the data. fl analyzed the data, and facilitated the study. hrvd analyzed the data. byw facilitated the study. 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publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we would like to thank the pediatricians from the pediatric department, the first affiliated hospital of shantou university medical college for their generous support, the children and their guardians for participation in this study, richard molenkamp at the university of amsterdam, academic medical center for technique and knowledge transfer to set up the multiplex real-time pcr, jieling chen at the shantou-oxford clinical research unit for technical assistance, and staff in the international institute of infection and immunity, shantou university medical college for their assistance with real-time pcr. this study was supported by the li ka shing foundation, shantou university medical college, and the university of oxford (grant no. b rsrt - ). the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. key: cord- -wszoi p authors: zhou, weimin; wang, wen; wang, huijuan; lu, roujian; tan, wenjie title: first infection by all four non-severe acute respiratory syndrome human coronaviruses takes place during childhood date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: wszoi p background: non-severe acute respiratory syndrome (non-sars)-related human coronaviruses (hcovs), including hcov- e, -hku , -nl , and -oc , have been detected in respiratory tract samples from children and adults. however, the natural prevalence of antibodies against these viruses in serum among population is unknown. methods: to measure antibodies to the spike (s) protein of the four common non-sars hcovs, recombinant s proteins of the four hcovs were expressed and characterised in t cell. an s-protein-based indirect immunofluorescence assay (ifa) was then developed to detect anti-s igg and igm for the four individual hcovs and applied to serum samples from a general asymptomatic population ( children and adults) in beijing. results: of blood samples tested, only ( . %) were negative for anti-s igg. the seropositivity of the four anti-s igg antibodies was > % within the general population. the majority of seroconversions to four-hcov positivity first occurred in children. both s-igg and s-igm antibodies were detectable among children and increased with age, reaching a plateau at years of age. however, no anti-s igm was detected in healthy adults. conclusion: large proportions of children and adults in beijing have evidence of anti-s igg against four the hcovs, and first infections by all four non-sars hcovs takes place during childhood. coronaviruses are positive-sense, single-stranded rna viruses found in humans and a wide variety of animals [ ] . in humans, four respiratory coronaviruses, namely human coronavirus (hcov) - e [ ] , -oc [ ] , -nl [ ] , and -hku [ ] , are endemic worldwide. these four common strains cause diseases from mild, febrile upper respiratory tract illnesses to severe outcomes, such as croup, bronchiolitis, and pneumonia, and attribute to about % of all upper and lower respiratory tract infections in hospitalised children [ , ] . in , a previously unknown coronavirus caused an outbreak of severe acute respiratory syndrome (sars) in humans [ , ] . more recently, the novel betacoronavirus species hcov-emc was identified from a man with pneumonia in saudi arabia [ ] . coronaviruses are phenotypically and genotypically diverse [ , ] . all coronaviruses possess a common genome organisation in which the replicase gene encompassing the ' two-thirds of the genome is comprised of two overlapping open reading frames, orf a and orf b. the structural gene region, which covers the ' third of the genome, encodes the canonical set of structural protein genes in the order '-spike (s) → envelope (e) → membrane (m) → nucleocapsid (n)- ' [ , ] . the s proteins is the most immunodominant cov protein [ , , ] . the most common method for diagnosing hcov infection is reverse transcription polymerase chain reaction (rt-pcr) or real-time rt-pcr using rna extracted from respiratory tract samples, such as nasopharyngeal aspirates [ , , ] . on the other hand, serological assays for detection of antibody to hcov are more complex to establish [ , ] . however, determining the levels of immunoglobulin m (igm) and immunoglobulin g (igg) antibodies in appropriate serum or plasma samples allows the researcher to define the point in time of primary infection as well as exposure rates; seroepidemiological studies are also important tools for hcov infection diagnosis and research [ , [ ] [ ] [ ] [ ] [ ] . previous reports have indicated that immunofluorescence assays (ifas) for the detection of seroconversion with igg antibodies against the main structural (n and s) proteins of sars-cov are useful for the diagnosis of acute sars-cov infection [ , ] . little is currently known about the prevalence of anti-s antibodies specific for non-sars hcov infection among children and adults. we propose that an ifa for the detection of igg against structural (n and s) proteins of non-sars hcov may be suitable for seroepidemiological studies. to expand the epidemiological knowledge of four non-sars-related endemic hcovs in china, we expressed s proteins in a eukaryotic system and established an ifa for the detection of igg or igm antibodies against these four viruses. we used this system to determine the prevalence of anti-s igg and igm antibodies against hcovs among a general population in beijing. serum samples were obtained from a general asymptomatic population ( months to years of age) who visited hospitals for medical examinations or vaccinations in beijing from to . all aspects of this study were performed in accordance with the national ethics regulations and approved by the institutional review boards of the centre for disease control and prevention of china and the ethics committee of peking union medical college hospital. participants received written information regarding the purpose of the study and of their right to confidentiality. written informed consent was obtained from all participants or their guardians. s-protein-coding gene optimisation was conducted and oligonucleotides synthesised (qingke bio-tech engineering service co., ltd., beijing) according to the s-protein sequences of four non-sars-related endemic hcovs in genbank ( enc_ , oc nc_ , hku nc_ , and nl nc_ ). the leader sequence of the original gene was removed and replaced by a human tpa leader sequence ( - aa) at the n-terminal of the s fragment. artificially synthesised s fragments of hcov-oc , - e, -hku , and -nl were cloned into the eukaryotic expression plasmid pvrc (a gift from dr. gary nabel, nih, usa) and named pvrc- e-s, -oc -s, -hku -s, and -nl -s, respectively ( figure a ). the plasmid pvrc- (from dr. gary nabel, nih, usa) [ ] , in which the s-protein-coding gene of sars-cov was constructed and used as a dna vaccine, was used as the control. female balb/c (h- d) mice between and weeks of age (animal care centre, chinese academy of medical science, beijing, china) were randomly assigned to four groups. in accordance with the institutional animal care and use committee (iacuc)-approved protocol, all mice were immunised at weeks , , and and bled at week . the mice were anaesthetised and primed with the above s-expression plasmid using intradermal injection into the lower dorsal side ( μg/ μl). gene delivery using in vivo electroporation was performed as described previously [ ] . serum samples were collected weeks after the last vaccination, and the pooled anti-s serum against individual hcovs was stored at − °c. ft cells were transfected with individual s-expression plasmids using lipofectinamine reagent (invitrogen company). at h post-transfection, the cells were lysed in ice-cold ripa buffer ( mm tris-hcl [ph . ], mm nacl, % triton x- , . % sds, and . % sodium deoxycholate) supplemented with a protease inhibitor mixture (sigma, st. louis, mo). the lysates were kept on ice for min, centrifuged, and resolved by sds/page in a % polyacrylamide gel. the proteins were transferred to a nitrocellulose membrane, blocked with % skim milk in pbs for h, and incubated for h at °c with anti-s mouse polyclonal antibody diluted to : in blocking buffer. the membrane was washed in pbs containing tween ( . %) and incubated for h with horseradish peroxidase-conjugated anti-mouse secondary antibody (pierce, rockford, il) diluted to : . the membrane was washed and the proteins were visualised with supersignal chemiluminescence substrate (pierce). an ifa was used to detect hcov s glycoprotein expression in t cells. briefly, t cells seeded on glass slides were transfected with pvrc- e-s, -oc -s, -hku -s, and -nl -s plasmids, respectively. after a -h incubation at °c in % co , the cells were fixed in % paraformaldehyde and blocked in % normal goat blood serum in % triton-x- pbs. the infected cells were incubated with anti-s mouse serum ( : ) for h, and then incubated with fitc-labelled goat anti-mouse igg (h + l; zhongahan co., beijing, china) for min. positive foci were identified by fluorescence microscopy (nikon, tokyo, japan) after evans blue duplicate staining. for serum anti-s igg or igm detection using ifa, an individual hcov s glycoprotein expression plasmid was used to transfect the ft cells in the -cm flask. forty-eight hours later, the transfected cells were washed twice with pbs and then dripped onto the slide. the cells were fixed using % paraformaldehyde for min, then permeabilised using . % tritonx- and washed three times with pbs. the anti-s-specific antibodies in sera (diluted to : ) were quantified using : -diluted fitc-labelled sheep anti-human igg (h + l; zhongahan co., beijing, china) or : -diluted fitc-labelled antihuman igm (μ-chain-specific), and the slide was viewed under an inverted fluorescence microscope (olympus, tokyo, japan). serum samples that reacted with hcov s protein at a dilution of > : were considered positive for anti-s antibodies when duplicate or triple test was consistent. furthermore, we confirmed that non-transfected t cells or those transfected with the control plasmid (pvrc , which expresses the s protein of sars-cov) did not react with the human serum samples tested. statistical analysis was performed using the statistic package for social science(spss) statistic package with χ -test and fisher's exact test. differences between the mean values of each group were considered significant at p < . when assessed using tukey's test. the demographic characteristics of the study subjects are summarised in table . of patients subjects studied, ( . %) were infants or children < years of age; ( . %) were male. the average age of the adults and children was . ± . years. the majority of patients in the child population were to years of age (n = , . %), and the majority of adults were to years of age (n = , . %). the structures of the s-fragments of individual hcov expression constructs used in this study are summarised in figure a . expression of the hcov s proteins was confirmed by western blotting ( figure b) . the segments were detected by anti-s mouse serum derived from dna vaccination with individual hcov s-expression plasmids. all four non-sars-related s proteins showed band sizes that were consistent with the predicted molecular mass ( - kda), presumably reflecting glycosylated forms of s protein. in addition, the expression of recombinant hcov s proteins was further confirmed by immunofluorescence staining using specific anti-s mouse serum, as noted above ( figure c ). all s proteins were expressed mainly in the cytoplasm and membrane. no cross-staining was observed when other hcov antiserum was used as the primary antibody, specifically concerning virus-antibody pairs of subgroup e/nl (alphacoronaviruses) or oc /hku (batacoronaviruses). furthermore, no false positive/negative result was observed in our study (data not shown). a representative anti-s-positive ifa result is shown in figure . using ifa based on ft cells expressing individual s proteins of hcov, we investigated the seroprevalence of these viruses in serum from various age groups of a healthy population in beijing. all samples were tested three using ifa and the result showed well reproducibility. of serum samples tested for anti-s igg by ifa, only ( . %) were negative for anti-s igg of hcov (table ) . ( . %) serum samples were positive for hcov- e, ( . %) for hcov-nl , ( . %) for hcov-oc , and ( . %) for hcov-hku . there were no significant differences between genders. the distribution and variation trend of anti-s igg to the four hcovs among the age groups are shown in figure . the seropositivity rate was . % for hcov- e and . % for hcov-oc , which are similar to the levels of hcov-hku ( . %) and hcov-nl ( %) among the -to -year-old group (p > . ). the proportions of hcov igg-positive samples increased with age during infancy, reaching levels > % at age > years. interesting, a significant drop in seropositivity rate was observed for hcov-oc in the -to -year-old group and for hcov-hku in the -to -year-old group. in the child population (n = ), the total positivity rate of anti-s igg antibodies was . % for e, . % for oc , . % for hku , and . % for nl ( figure ); no significant differences were observed with regard to igg to two novel hcovs among the children (nl , p = . ; hku , p = . ). in the adult population (≥ years of age), the seroprevalence of anti-s igg of hcov- e, -oc , and -nl- did not differ significantly among the age groups ( e, p = . ; nl , p = . ; oc , p = . ). for hcov-hku , a significant increase in the seroprevalence rate with increasing age was observed (p = . ). in the adult population (n = ), the igg positivity rate was . % for e, . % for oc , . % for hku , and . % for nl . the proportion of subjects with detectable igg antibodies to hcov- e was significantly greater than that to -oc and -nl (p < . ). furthermore, the number of subjects with igg to the three above-mentioned hcovs was also significantly greater than that to hcov-hku (p < . ). however, there were no significant differences in the anti-s igg seropositivity rates of individual hcovs with respect to gender in either the child or adult population (data not shown). the prevalence and variation trend of anti-s igm antibodies to individual hcovs among the child population is shown in figure . the total positivity rate of anti-s igm was . % for e, . % for oc , . % for hku , and . % for nl . the anti-s igm positivity rate of hcov- e was significantly lower than that of the other three hcovs (p = . ), while there were no significant differences in the anti-s igm seropositivity rates of individual hcovs with respect to gender (data not shown). there was a significant difference in the anti-s igm positivity rate for individual hcovs among age groups. the anti-s igm positivity rate of hcov-hku was significantly higher than that of the other three hcovs among the < -year-old groups (figure ). the positivity rate increased with age and began to drop after peaking in the school-age group (≥ years), which is similar to that described above for anti-s igg detection. no anti-s igm antibodies for the four individual hcovs were detected in the serum of the healthy adult population > years of age. this is the first comprehensive study to evaluate anti-s igg and igm for four non-sars hcovs among the general population in china. we first time show that anti-s igm is only present in children, indicating that for all viruses first infection takes place during childhood (age < years). the sars epidemic that originated from southern china in sparked interest in all areas of coronavirus research [ ] [ ] [ ] [ ] [ ] [ ] . four endemic non-sars-related hcovs (hcov-oc ,- e,-nl and -hku ) are major contributors to respiratory tract infections and other clinical manifestations [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] ] . however, specific and feasible serological surveys of these hcovs, especially for hcov-nl and -hku , have to date been reported among the general population only rarely in china. spike is the major structural protein of hcovs [ , ] . it contains multiple conformational epitopes that are major inducers of antibody neutralisation, and it has the lowest sequence conservation among coronavirus proteins, rendering it a specific target for serodiagnosis [ , , [ ] [ ] [ ] [ ] . we chose the hcov s gene for recombinant expression on the basis of current knowledge of immunodominant sars-cov antigens, which belongs to the same virus family as hcov. previous studies of sars-cov serology have successfully used the s protein in enzyme immunoassays, immunoblots, and ifa [ ] [ ] [ ] [ ] [ ] . in addition, comparison of the s protein sequences of four hcovs revealed that these proteins share < % similarity [ , ] . we hypothesised that the difference in the amino acid sequence is sufficiently high to ensure the usefulness of s protein as a specific antigen for antibody detection. the native surface s protein expressed in t cells can be recognised in post-infection population serum by ifa. some reports indicated that this cell-based s protein expression system can differentiate false-positive elisa results using the more cross-reactive nucleoprotein antigen [ , ] . furthermore, woo et al. reported that the rsbased igm elisa is more sensitive than the rn-based igm elisa for sars-cov pneumonia [ ] . our results showed that the s-based ifa enabled specific detection of igg or igm to four individual hcovs. using ifa, we investigated the natural seroprevalence of four non-sars-related hcovs in blood samples from a general population that comprised a variety of age groups. anti-s igg antibodies to these four non-sars-related hcovs were detected at high rates (> %) among healthy adults. both anti-s igg and igm antibodies were found in the child group, and their prevalence increased with age up to years, at which point it almost plateaued. these data suggest that exposure to hcov is common in childhood and first infections by all four non-sars hcovs takes place during children. moreover, we found evidence of anti-s igg against double and multiple hcovs in various combinations, which indicates that large proportions of the general population in beijing may experience infections with more than one hcov. the seroprevalence of hcov antibodies varies widely among studies, which used different antigens and methodologies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . previous studies of non-sars hcovs demonstrated that seroprevalence varies greatly depending on the age of the population [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . our ifa study showed that the prevalence of the four anti-s igg antibodies among this general chinese population were slightly higher than those in another study from germany using an escherichia coli bl -expressed recombinant n-based line immunoassay [ ] , which reported that the seropositivities in healthy blood donors were % for hcov-hku , % for -oc , % for - e, and % for -nl . on the other hand, the seropositivity rate of three individual hcovs excluding hcov-hku in this study was lower than that in another study of a us metropolitan adult population using baculovirus-expressed recombinant n-based elisa [ ] , which showed seropositivity rates of . % for hcov-oc , . % for - e, and . % for -nl , while that for -hku was . %. it was also supposed that the significantly different seropositivity rates for the various hcovs might result in individuals with different demographic factors (e.g. ethnicity, smoking status, and socioeconomic status) having different susceptibilities to individual hcovs [ ] . a recent study of older adult veterans with underlying chronic obstructive pulmonary disease at seven us sites showed that serum igg to hcov- e, -nl , and -oc was detected in at least % of subjects, while antibodies to -hku were identified in subjects ( %) [ ] . thus eia assays that use whole virus or n protein as the antigen, using which apparent cross-reactivity of hcov antibodies has been demonstrated previously [ , ] , may detect group-rather than type-specific antibody. our results regarding anti-s igg to four individual hcovs in children correlate with those of previous seroprevalence studies [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , although the data on hcov-nl and -hku were limited. shao et al. found that antibodies directed to hcov-nl and - e in children year of age and older were frequently detected using part of the c-terminal region of the n protein as an antibody capture antigen in an elisa [ ] . a study of children in the netherlands aged . - . years indicated that % and % of serum samples were positive for antibodies to hcov-nl and - e, respectively [ ] . we observed that the prevalence of these four non-sars-related hcov-directed igg antibodies among children > year of age were almost identical. information on the prevalence of anti-s igm to non-sars-related hcov is to-date lacking. we first investigated the prevalence of anti-s igm to four individual hcovs among a general population in beijing using ifa. anti-s igm to individual hcovs was detected in a portion of the asymptomatic child population. the anti-s igm seropositivity appeared to increase with age up to years and decline sharply after years of age. however, no anti-s igm to individual hcovs was detected in the healthy adult population. these results suggest that primary seroconversion to these viruses occurs mainly during childhood and youth; in addition, hcov infection might result in seroconversion in children with asymptomatic or subclinical manifestations. these results are also in agreement with hcov molecular epidemiological surveys [ , ] , which have indicated that primary exposure occurs mainly in childhood and youth. we conclude that s-based ifa might be a useful specific serological platform for epidemiologic investigation of hcov infection. high proportions of children and adults in beijing show anti-s igg seropositivity against the four hcovs, and anti-s igm antibodies were detected in the sera of asymptomatic children. these four non-sarsrelated hcovs appear be circulating in the general population, and sustained hcov infection becomes more likely with increasing age. this study may serve as a basis for the prevention and control of non-sars-related hcov infection. however, further research is needed to determine the false-positivity and -negativity rates associated with this anti-s ifa by determining antibody titres during the acute and convalescent phases after primary hcov infection. comparison of serological methods and antigen preparations as well as sample exchange will facilitate validation of the assays for individual hcov antibody determination. fields virology a new virus isolated from the human respiratory tract cultivation of a novel type of common-cold virus in organ cultures identification of a new human coronavirus characterization and complete genome sequence of a novel coronavirus, coronavirus hku , from patients with pneumonia update on rhinovirus and coronavirus infections identification of a novel coronavirus in patients with severe acute respiratory syndrome a novel coronavirus associated with severe acute respiratory syndrome severe respiratory illness caused by a novel coronavirus coronavirus genome structure and replication human coronavirus infections in rural thailand: a comprehensive study using real-time reverse transcription polymerase chain reaction assays epidemiology and clinical presentations of the four human coronaviruses e, hku , nl , and oc detected over years using a novel multiplex real-time pcr method a line immunoassay utilizing recombinant nucleocapsid proteins for detection of antibodies to human coronaviruses development of a nucleocapsid-based human coronavirus immunoassay and estimates of individuals exposed to coronavirus in a u.s. metropolitan population seroepidemiology of group i human coronaviruses in children human coronavirus nl and e seroconversion in children prevalence of antibodies to four human coronaviruses is lower in nasal secretions than in serum false-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid-based western blot assay were rectified by the use of two subunits (s and s ) of spike for detection of antibody to sars-cov development of an enzyme-linked immunosorbent assay-based test with a cocktail of nucleocapsid and spike proteins for detection of severe acute respiratory syndrome-associated coronavirus-specific antibody a dna vaccine induces sars coronavirus neutralization and protective immunity in mice enhanced delivery of naked dna to the skin by non-invasive in vivo electroporation a prospective hospital-based study of the clinical impact of non-severe acute respiratory syndrome (non-sars)-related human coronavirus infection recombinant protein-based assays for detection of antibodies to severe acute respiratory syndrome coronavirus spike and nucleocapsid proteins false-positive results in a recombinant severe acute respiratory syndrome-associated coronavirus (sars-cov) nucleocapsid enzyme-linked immunosorbent assay due to hcov-oc and hcov- e rectified by western blotting with recombinant sars-cov spike polypeptide differential sensitivities of severe acute respiratory syndrome (sars) coronavirus spike polypeptide enzyme-linked immunosorbent assay (elisa) and sars coronavirus nucleocapsid protein elisa for serodiagnosis of sars coronavirus pneumonia examination of seroprevalence of coronavirus hku infection with s protein-based elisa and neutralization assay against viral spike pseudotyped virus spike protein, s, of human coronavirus hku : role in viral life cycle and application in antibody detection effects of a "new" human respiratory virus in volunteers coronative antibody tires in sera of healthy adults and experimentally infected volunteers the time course of the immune response to experimental coronavirus infection of man prevalence of human coronavirus antibody in the population of southern iraq enzymelinked immunosorbent assay for detection of antibody in volunteers experimentally infected with human coronavirus strain e human coronavirus nl employs the severe acute respiratory syndrome coronavirus receptor for cellular entry effects of coronavirus infections in children submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the ms linglin zhang for samples collection and transport, and the medical and technical staffs from the peking union medical college hospital for their assistance and support. we also thank all the participants involve in this study for providing samples. the authors declare that they have no competing interests.authors' contributions tw created the original idea of this research and designed the study. zw and ww performed experiments. wh and lr provided important data analysis. zw and tw drafted the manuscript. all authors read and approved the final version of the manuscript. key: cord- -jb t p authors: suess, thorsten; remschmidt, cornelius; schink, susanne b; schweiger, brunhilde; nitsche, andreas; schroeder, kati; doellinger, joerg; milde, jeanette; haas, walter; koehler, irina; krause, gérard; buchholz, udo title: the role of facemasks and hand hygiene in the prevention of influenza transmission in households: results from a cluster randomised trial; berlin, germany, - date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: jb t p background: previous controlled studies on the effect of non-pharmaceutical interventions (npi) - namely the use of facemasks and intensified hand hygiene - in preventing household transmission of influenza have not produced definitive results. we aimed to investigate efficacy, acceptability, and tolerability of npi in households with influenza index patients. methods: we conducted a cluster randomized controlled trial during the pandemic season / and the ensuing influenza season / . we included households with an influenza positive index case in the absence of further respiratory illness within the preceding days. study arms were wearing a facemask and practicing intensified hand hygiene (mh group), wearing facemasks only (m group) and none of the two (control group). main outcome measure was laboratory confirmed influenza infection in a household contact. we used daily questionnaires to examine adherence and tolerability of the interventions. results: we recruited households ( control, m and mh households) with , and household contacts, respectively. in / all index cases had a influenza a (h n ) pdm infection, in / had an a (h n ) pdm and had a b infection. the total secondary attack rate was % ( / ). in intention-to-treat analysis there was no statistically significant effect of the m and mh interventions on secondary infections. when analysing only households where intervention was implemented within h after symptom onset of the index case, secondary infection in the pooled m and mh groups was significantly lower compared to the control group (adjusted odds ratio . , % ci, . - . ). in a per-protocol analysis odds ratios were significantly reduced among participants of the m group (adjusted odds ratio, . , % ci, . - . ). with the exception of mh index cases in / adherence was good for adults and children, contacts and index cases. conclusions: results suggest that household transmission of influenza can be reduced by the use of npi, such as facemasks and intensified hand hygiene, when implemented early and used diligently. concerns about acceptability and tolerability of the interventions should not be a reason against their recommendation. trial registration: the study was registered with clinicaltrials.gov (identifier nct ). since , the world health organisation (who) and other organisations have highlighted the need for controlled trials to assist in formulating recommendations on the use of non-pharmaceutical interventions (npi)such as facemasks or hand hygiene measures -as options to prevent influenza transmission, particularly in households [ , ] . as these measures are easily applicable and accessible for the general population and do not rely on microbiological data they could be available even in very early stages of a pandemic. several controlled trials testing npi have lately been undertaken in different settings and with different designs [ ] [ ] [ ] [ ] [ ] but have not been able to provide conclusive results. four of these studies were conducted within households [ ] [ ] [ ] [ ] , one among adults living in university residence halls [ ] . all but one [ ] took place during seasonal influenza epidemics. regarding interventions, one study compared facemasks to respirators [ ] , another evaluated facemasks only [ ] , while three studies assessed facemasks, and/or hand hygiene measures in various combinations [ , , ] . in intention-to-treat analysis, none of the four household based trials was able to show significant reductions in secondary attack rates (sar) when comparing intervention to control groups. however, one subgroup-analysis (restricting intervention households to those where intervention started within h of symptom onset) [ ] and one per-protocol analysis [ ] showed significant effects of the interventions. similarly, cumulative incidence in the study among university students yielded no significant differences between study arms, but survival analysis identified significant reductions of influenza-like-illness (ili) in weeks - after recruitment [ ] . in all publications, it was hypothesized that the effect of interventions may be more pronounced in the case of an influenza pandemic, due to higher public anxiety with resulting higher rates of adherence. this was supported by observations made in a comparable crisis, namely the sars epidemic [ ] . during the pandemic of influenza a (h n )pdm there was a considerable amount of uncertainty among public health officials if or which npi should be recommended. germany, in accordance with many other countries, did not encourage the widespread use of facemasks; hand washing had been generally recommended against the transmission of respiratory viruses already before the pandemic. further evidence based data on npi from controlled studies are still needed, as they are necessary to inform decision makers on the potential benefit of npi in influenza pandemics. between and we conducted a cluster randomised trial on the efficacy, adherence and tolerability of facemasks and intensified hand hygiene to prevent influenza transmission in households. study results from the / pandemic season concerning data about adherence and tolerability only have already been published [ ] . we conducted the study during two consecutive influenza seasons (november -january and january-april ) in berlin, germany. index patients were recruited by general practitioners or pediatricians. we cooperated with study sites in / and study sites in / evenly distributed in the city of berlin. we included index patients if they presented at the study sites within days of symptom onset, had a positive rapid antigen test for influenza (later to be confirmed by quantitative reverse transcription polymerase chain reaction [qrt-pcr]), and were at least years old. index cases also had to be the only household member suffering from respiratory disease within days prior to symptom onset. exclusion criteria were pregnancy, severely reduced health status and hiv infection. one person households were also not eligible for inclusion. we obtained written informed consent from all study participants. if these were less than years of age we asked their parents or legal guardians to provide proxy written consent, with additional written consent from those participants aged to years of age. children were defined as persons aged less than years, adults were at least years old. we used a cluster randomisation with the households serving as clusters. we prepared lists of random numbers with microsoft excel (mircosoft™ cooperation, seattle, usa) which were divided between the three intervention groups. each participating physician received a list of random numbers with the interventions represented in a : : ratio. eligible index patients were randomly assigned a number, which was then communicated to the study center. the resulting intervention was only communicated to the households with the physicians (as well as laboratory personnel) blinded from the randomisation results. intervention material was given to the study sites in closed boxes marked only with the randomisation number. recruiting physicians were not aware of the allocation of the numbers to the interventions and the boxes for the three intervention arms looked identical. after randomisation, participants were given their box by the physician's assistants. the following three intervention groups were used: (i) mask/hygiene (mh) arm: households were provided with alcohol based hand-rub (sterilium™, bode chemie, germany) and surgical facemasks in two different sizes, one for children aged younger than years (child's face mask, kimberly-clark, usa) and one for adults (aérokyn masques, lch medical products, france). if masks intended for participants younger than years did not fit properly (as assessed by study personnel during the first household visit), we asked them to wear adult masks instead. household also received information on the proper use of the interventions; (ii) mask (m) arm: we provided the household with surgical facemasks and information on their correct use; (iii) control (c) arm: no masks or hand rub was provided. all participating households received general written information on infection prevention [ ] . households received all necessary material (including a digital tympanic thermometer) on the day of recruitment and were called by study personnel immediately after leaving the study sites for instructions on how to use it correctly (provisional implementation of the intervention). trained study personnel visited the household no later than days after symptom onset of the index case. using written information, study personnel demonstrated the correct use of the intervention material (full implementation of the intervention). we asked participants of the mh group to always use the provided hand rub after direct contact with the index patient (or other symptomatic household members), after having touched household items being used by the index patients and/or other symptomatic household contacts, as well as after coughing/sneezing, before meals, before preparing meals and when returning home. we asked all participants of the mh and m groups to wear masks at all times when the index patient and/or any other household member with respiratory symptoms were together in one room with healthy household members. facemasks were to be changed regularly during the day and not to be worn during the night or outside the household. the observation period for each household lasted days, starting on the day of symptom onset of the index patient (day ). we visited households on days , , , , and (five times) or on days , , and (four times) depending on the day of recruitment. during these visits we obtained nasal wash specimens (or -if these were not possible -nasal swabs) from all participating household members. antiviral medication was given to index patients and secondary cases by their individual physicians based on their clinical evaluation independent of study procedures. by definition, a "timely" antiviral therapy started within days of symptom onset. when household members developed fever (> . °c ), cough, or sore-throat they were asked to adopt the same preventive behaviour as the index patient (i.e. use facemasks or hand hygiene measures as required by index patients to protect other healthy household members) until the end of the observation period. all participants self-recorded symptoms (fever, shivering, measured temperature, cough, sore throat) and daily routines (incl. the time spent at home, and within close range (i.e. < m) of the index patient) in a daily monitoring questionnaire. the primary outcome measure for secondary cases was qrt-pcr confirmed influenza infection. we defined a symptomatic secondary influenza virus infection as a laboratory confirmed influenza infection in a household member who developed fever (> . °c), cough, or sorethroat during the observation period. we termed all other secondary cases as subclinical. a secondary outcome measure was the occurrence of ili as defined by who [ ] as fever plus cough or sore throat. participants of the m and mh groups also recorded daily adherence with facemasks, i.e. if they wore masks "always", "mostly", "sometimes", or "never" in the situation they had been asked to wear them. in the season / they also recorded the number of masks used per day. participants of the mh households additionally noted the number of hand disinfections per day. on day nine, study personnel conducted an exit questionnaire with all participants collecting information on (preventive) behaviour during the past days, general attitudes towards npi, the actual amount of used intervention materials and -if applicable -problems with wearing facemasks. we did not address potential problems with intensified hand hygiene. parents answered the questionnaires on behalf of their children. for the final analysis, two definitions of adherence were used, the first based on daily observations, the second on the behaviour of participants during the five consecutive days after implementation. used intervention material per household member was calculated by dividing the amount used per household by the number of household members. participants received a reimbursement of € for the large number of respiratory samples obtained. for the collection of nasal wash, we used ml of isotonic saline, which were instilled into one nostril with participants heads tilted backwards. participants were asked to remain in this position for - s while making hard 'k' sounds without swallowing. subsequently, the participants were told to tilt their heads forward and the fluid was collected in a sterile cup [ ] . nasal swabs were collected by using virus transport swabs (mastas-wab™; mast diagnostica, reinfeld, germany). samples were stored refrigerated (at a temperature of approximately °c) before analysis [ ] . specimens were analysed by qrt-pcr at the centre for biological security, division of highly-pathogenic viruses (season / ) and the national reference centre for influenza (season / ) both part of the robert koch institute in berlin, germany. rna was extracted using either the magna pure dna and viral nucleic acid small volume kit (roche applied science, mannheim, germany) on magna pure instrument (roche applied science) according to the manufacturer's instructions. details about the pcr protocol as well as primer and probe sequences have been published elsewhere [ ] . we assumed a sar of laboratory confirmed infection of % in household contacts of the control group, based on data from another study of this group (sar % [ ] ) as well as other published data on seasonal ( . % [ ] ) and pandemic influenza ( . % [ ] , % [ ] ). assuming an average of household contacts per household [ ] and an intracluster correlation coefficient (icc) of . [ ] [ ] [ ] , we estimated that household members would be needed in each intervention arm to detect a % difference in secondary attack rates, i.e. % in the control group and % in the intervention groups, with % power and at a significance level of %. analysis was done by intention-to-treat. for descriptive analysis we used student's t-test and kruskal-wallis one-way analysis of variance for numerical and chisquare tests for categorical variables. the intention-totreat analysis was conducted in the following order: . comparison of sar between intervention groups via adjusted chi-square tests [ ] (overall and stratified by virus subtype, season and time of implementation of intervention) to account for the cluster design of the study. we used a cluster bootstrapping technique for the calculation of % confidence intervals ( % ci) [ ] . . we used the generalized estimating equations (gee) approach to fit logistic regression models [ ] for evaluation and comparison of sar between intervention groups. first, we calculated odds ratios (or) for the outcome "laboratory confirmed influenza" with the following independent variables: (i) intervention group, (ii) intervention group (with pooled data of m and mh group), (iii) one separate model for each individual variable that may have influenced household transmission of influenza (i.e. age, sex, timely antiviral therapy of the index, vaccination of household contacts, etc.) adjusted for intervention group. this corresponds to a univariable analysis with the exception of the adjustment for intervention group. . calculation of ors for the clinical case definition (otherwise as in .). . calculation of ors for the outcome "laboratory confirmed influenza" to analyse the effect of the interventions while adjusting for variables with possible influence on influenza transmission. in a further model we used the variable intervention group with pooled data of m and mh group. . calculation of ors for the outcome "laboratory confirmed influenza" adjusting for variables with possible influence on influenza transmission in the following subgroups: (i) only data from season / , (ii) only data from season / , (iii) only data from households with full implementation of intervention < h after symptom onset of the index case, (iv) only data from influenza a(h n )pdm cases. we used a forced-entry method adjusting for variables potentially associated with risk of secondary infection. sample sizes for these subgroup analyses were small and sometimes did not allow the inclusion of the full list of variables. the per-protocol-analysis was conducted in the same way as the intention-to-treat analysis but only with data from participants who had followed the assigned interventions. for all analyses, we used two-sided tests and considered p-values of < . as significant. we performed analyses with stata software version (stata corporation, texas, usa). ethics committee approval was obtained from the institutional review board of charité universitätsmedizin berlin. the study was registered with clinicaltrials.gov (identifier nct ). initially, we recruited households which were randomised into one of the three intervention groups during the two study periods in / and / ( figure ). after application of the exclusion criteria , and households remained in the control-, m-and mhgroups for analysis. the total number of study participants was , of whom were index patients and household contacts. the study flow according to the consort guidelines is shown in figure . table shows the baseline characteristics of index patients and household contacts of all analyzed households stratified by season of study participation and intervention group. one difference between the two study seasons was that in / all viruses belonged to a (h n ) pdm , while in / both a (h n ) pdm as well as b viruses circulated (p = . for comparison within season). another difference is that -compared to / -the number of index patients receiving timely antiviral therapy was significantly higher in / (p < . ). furthermore, in / a significantly larger number of both index patients (p = . ) and household contacts (p = . ) was vaccinated compared to / . finally, in / randomisation occurred significantly earlier after symptom onset compared to / (p = . ) and a higher proportion of households was visited by study personnel within h (p = . ). all other variables did not differ significantly between the two study seasons (table ). overall, there were ( %) secondary cases of qrt-pcr confirmed influenza and ( %) secondary ili out of a total of household contacts belonging to households. secondary laboratory confirmed cases occurred in households, including ( %) households with one secondary case, six ( %) with two secondary cases, and two ( %) with three secondary cases. when stratified by season, overall sar was % ( / ) in / and % ( / ) in / (p = . ). for laboratory-confirmed cases sar were not significantly lower in the m ( % ( / )) and mh group ( % ( / )) compared to the control group ( % ( / ); table ). in all stratified analyses (by influenza type, season, and implementation within h after symptom onset) sar of the m group was approximately reduced by % compared to the control group. in / the mh group had markedly different sar in a (h n ) pdm households compared to b households. in a (h n ) pdm households, sar was highest in the control group and similarly low in the mh and m groups; but in b households it was highest in the mh group. when considering only households with early "full implementation" of the intervention (within h after symptom onset), sar of the mh group was substantially lower than in the control group. sar measured by the ili case definition yielded somewhat lower results compared to those using the laboratory case definition, because not all laboratoryconfirmed cases were also ili cases. overall, differences in sar were not significant, neither for laboratory confirmed secondary cases nor for ili (table ) , neither in primary analysis nor after stratification for season, influenza virus (sub)type or timing of the first household visit ( table ) . in addition to the analysis of all three interventions groups, we also calculated odds ratios (or) for secondary infection when intervention (i.e. m-and mh-group) groups were combined (table ) . although or suggested a protective effect, this was not statistically significant. among other individual variables with possible influence on secondary infection (such as sex, age, time spent at home, timely antiviral therapy of the index patient, and vaccination of the household contact) one variable stood out: household members who spent at least h of the day at home were significantly more likely to develop laboratory confirmed influenza infection (or ili) compared to those who spent less time at home (table ) . in a subgroup analysis, we examined the effect of the interventions only in households with early full implementation, and secondly only in households with (pandemic) a (h n ) pdm infection. the first analysis was carried out in a subset of households where the intervention had been implemented no later than h after symptom onset of the index patient. in this subset of household contacts, we found a borderline significant protective effect of the mh intervention against laboratory confirmed influenza infection compared with the control group after adjustment for potential households randomised results of subgroup analyses using ili as outcome were comparable to those using laboratory confirmed cases, but none were statistically significant. considering that not all study participants followed the intended intervention in the group that they were assigned to, we conducted a (per-protocol) analysis among all participants who fully adhered to the study protocol. because we implemented two interventions (facemask use and intensified hand hygiene) it was possible that one person adhered to one intervention, but not to the other. we therefore considered only adherence to facemask use for this analysis. we excluded participants of the control group when they wore a facemask during the study period, and we excluded participants from the m and mh group when they reported not wearing masks at all throughout the study. apart from these exclusions of non-adherent study participants, we conducted the same analyses as for the intention-to-treat analysis. all or of the m and mh group were below , mostly between . and . relating to a protective effect of %- % for the interventions (table ). significant results were reached in the m group when analysing the complete data set and in the m as well as the mh group when considering only a(h n )pdm households. we used two definitions to describe adherence to wearing masks. the first evaluated daily adherence and considered a participant as "adherent" if they wore a mask "always" or "mostly" on each day as required by the study protocol (adherence definition ). the second definition evaluated behaviour of participants during the first days after implementation of the intervention [ ] . a participant was termed adherent if they wore a facemask "always" or "mostly" on each of the first days after full implementation of the intervention (adherence definition ). figures and display the data for adherence (according to definition ) to facemask use in the m and mh group separately for index patients ( figure ) and household contacts (figure ). in general, daily adherence was good, reaching a plateau of over % in nearly all groups (m and mh groups; / and / ) from the third day on (by then the intervention had been implemented in all households). a gradual decline towards lower adherence began around the sixth day of the index patient's illness. a further observation was that in / mh index patients were less adherent than m index patients (figure , right panel; difference not statistically significant) while the two groups were fairly similar during the / pandemic. similar differences can be observed when only the first days following full study implementation were considered (definition ) ( table ). adherence behaviour of household contacts was similar in both seasons. in both season, the majority of participants ( / , %) did not report any problems with mask wearing. this proportion was significantly higher in the group of adults ( / , %) compared to the group of children ( / , %) (p = . ). the main problem stated by participants (adults as well as children) was "heat/ humidity" ( / , % of children; / , % of adults) (p = . ), followed by "pain" and "shortness of breath" when wearing a facemask. for the daily evaluation of adherence to hand hygiene measures we used the number of hand disinfections as indicated by mh participants (adherence definition ). a mh participant was termed adherent according to definition if they had disinfected their hands at least five times per day on each of the days after full implementation of the intervention. similar to the low facemask adherence of mh index patients in / , this group also displayed low hand hygiene adherence compared to the index patients of / . as in facemask adherence, household contacts kept a relatively stable level of adherence in both seasons (figure ). also the -day adherence (adherence definition ) of index patients of the mh group dropped from % in / to % in / (p = . ) while it did not drop in household contacts (table ) . we validated reported data on adherence by comparing the indicated number of daily mask use and number of disinfections with the remaining intervention material in each household at the end of the study period. for both interventions, cumulative subjective information by participants correlated well with the objective measurements of the remaining material (facemasks: r = . , p < . (only possible for season / as information on the number of facemasks used per day was only collected in the season / ); hand hygiene: r = . , p < . ). we observed "contamination" between intervention groups in two control households only in the season / , one reported wearing masks, the other reported wearing masks and using alcohol based hand sanitizer. examination of further potentially relevant behavioural variables, such as daily time spent at home during the study period, time spent at close range of the index patient, sleeping in the same room or taking meals with the index patient, did not result in significant differences between the study groups or between influenza seasons (data not shown). we present results of a cluster randomised trial on the effectiveness of facemasks and hand hygiene in preventing household transmission of influenza. the trial was conducted in berlin during the first two seasons after the onset of the influenza a (h n )pdm pandemic. in primary intention-to-treat analysis of all data, the interventions did not lead to statistically significant reductions of sar in household contacts. however, in a a per-protocol analysis showed comparable results of lowered influenza transmission in the intervention groups which were statistically significant in the m group when analysing the complete dataset, andamong a (h n ) pdm households -in the combined analysis of the m and mh groups. the main drawback of the study was that we did not reach the number of households that we had aimed and planned for, one of the reasons being the at best moderate influenza season / . our sample size calculation was based on a % reduction of risk due to the interventions. this may seem questionably high in comparison to other studies, however based on experience from our pilot study we felt that adherence would be better than reported in the hong kong [ ] and bangkok [ ] studies. we therefore expected a larger effect size in our main study. the reason for the high sar of % in mh households from the / season (with % the sar was even higher when only households with influenza-b positive index patients were considered) remains unclear. however, we hypothesize that the particularly low adherence of mh index patients to both interventions during the / season might have influenced this observation. the fact that we observed a significant effect of the combined m and mh intervention only after restriction of analysis to households with early (< h after symptom onset) implementation of the interventions is in agreement with cowling et al. who had investigated a hand hygiene intervention as well as hand hygiene plus facemask use [ ] . the importance of early implementation of any intervention is plausible given high levels of viral shedding during the initial period of influenza infection [ ] as well as the short incubation period [ ] . recently, donnelly et al. quantified the probability of a transmission event by an infectious person relative to the onset of symptoms [ ] and showed that peak transmission occurred on days , and of the infectious patient's illness. merely % of transmission events took place more than two days after symptom onset of the index patient. an australian cluster-randomized household study conducted in a pre-pandemic winter season investigated the effect of the use of facemasks (surgical, or n ) on na not applicable, hd hand disinfection, sd standard deviation. * p < . between seasons. ** adherence to hand hygiene measure was defined as disinfection of hands at least times per day during the day after full implementation of the intervention (based on data from daily questionnaire). *** based on data from exit questionnaire. #only adults. ## adherence to facemask intervention was defined as wearing a facemask "mostly" or "always" on each of the days after full implementation of the intervention (based on data from daily questionnaire) the risk of respiratory infections with all index cases being children and having an influenza-like illness of any, even unknown, etiology [ ] . intention-to-treat analysis did not yield significant results, however, good adherence to facemask use proved to be significantly protective in a per-protocol analysis. two further cluster-randomized household studies failed to see any significant effects of intervention measures (facemasks or hand hygiene) even in secondary analyses. a french study investigated the efficacy of facemasks in the pre-pandemic influenza season / [ ] . although a planned second season was not followed through due to the onset of the influenza a (h n )pdm pandemic, reported sar after the first season were quite similar in intervention and control groups. adherence was reported to be good, but only a clinical case definition (ili) was used for secondary cases, thus probably missing a-and oligosymptomatic secondary cases. only index patients were supposed to wear the masks, and mean age of index patients in the intervention arm was years. since young children and infants may play a more important role in the (household) transmission of influenza [ ] [ ] [ ] , it is possible that these factors may have led to a cumulative underestimation of the real effect of facemasks. the second study failing to see an effect of npi in the household setting comes from bangkok, thailand and was conducted between april and august [ ] . interventions tested were facemasks combined with handwashing, and handwashing alone. although study size was large corresponding to high statistical power, the fact that % of index patients slept in the same bedroom as their parents without wearing facemasks during the night may have overcome any protective effect conferred by the interventions during daytime. in addition, authors describe a considerable amount of contamination between intervention groups, which may have further concealed true effects of the interventions. in our study adherence to both interventions was good. after full implementation of the interventions approximately % of m and mh participants (index and contacts) wore facemasks "mostly" or "always" during the daytime in situations as required by the study protocol, and mh participants disinfected their hands approximately - times per day; only mh index cases in / had lower adherence values for both interventions. comparison with other studies is difficult for a number of reasons, particularly because interventions differed. cowling [ ] defined facemask adherence similar to the present study and reported similar facemask figure adherence to hand hygiene measures by index patients and household contacts. frequency of daily hand disinfection (mean, standard deviation) in participants assigned to the mh group, stratified by season. symbols represent the mean frequency of hand disinfection before (green, hollow circles) and after (black squares) the intervention was fully implemented in the households. data of index patients are depicted by a continuous line, data of household contacts by a dashed line. adherence in index cases ( %), but worse in household contacts ( %). adherence to wearing facemasks in the canini [ ] study can be compared to our study for / , because the number of facemasks used per day was measured only in this season. the results were comparable in both studies. hand hygiene was part of the trial design only in the simmermann [ ] and cowling studies [ ] . mh index patients in our study disinfected their hands between . ( / ) and . times ( / ) per day, the index patients in the simmerman study washed their hands . times per day. adherence to hand disinfection by index patients over the course of the study (adherence definition ) ranged between % ( / ) and % ( / ) in our trial, compared to cowling et al. with % and % in the two groups assigned hand hygiene interventions. among household contacts in the mh group of our study adherence was higher in every parameter measured compared to simmerman et al. and cowling et al.. considering that facemask and perhaps also disinfectant use in household settings may be much less accepted in european compared to asian countries [ ] , the high overall adherence of both interventions in our study is remarkable. however, adherence data in all studies were based on self reporting and differences in reporting behaviour may have influenced results. compared to / adherence may have been higher during the pandemic season / , but differences were not statistically significant. increased use of or willingness to use preventive measures, such as facemasks or hand hygiene, was also documented during the sars epidemic as well as during the pandemic influenza a (h n )pdm [ ] [ ] [ ] . as we reported previously [ ] , adherence was good in adults and children alike, and although difficulties with facemasks were more frequently reported by children compared to adults, the numbers were not high. one notable exception with considerably lower adherence in / compared to / was observed in index patients of the mh group. because physical interventions used by infected children may have the largest effect on the reduction of spread [ ] and most index cases were children, it is possible that their reduced adherence has negatively affected transmission rates in mh households resulting in higher sar in this intervention group in / . in general, we believe that our data for adherence and tolerability would support a recommendation to use non-pharmaceutical interventions in a pandemic. several limitations may have influenced the results of this study. as in all previous studies on this subject, our study design resulted in delays between symptom onset of the index patients and implementation of the intervention. this delay could be as long as days in some households during the / season. although we tried to address this problem by calling the households for preliminary instructions directly after enrolment at their physicians' office, this does not substitute for a personal visit with a demonstration of the intervention in the household. this may have led to an underestimation of the true effect of the interventions. a further limitation of our study is that we cannot determine whether a possible protective effect of wearing facemasks is more attributable to their use by index patients or by household contacts (or both), nor can we say if intensified hand hygiene provides any additional protection. regarding the first question, there are data from a dutch experimental study suggesting that the use of masks may be more effective for inward than for outward protection which would favour the importance of healthy persons wearing them [ ] . this is in line with the results of the french trial [ ] which stated in its protocol that facemasks were only to be worn by index patients and which could not show any significant protective effect in this setting. regarding the role of hand hygiene, existing data from clinical trials are inconclusive. the study from thailand found no effect, neither for facemasks nor for hand hygiene [ ] . in the analysis of households where the intervention was applied within h the hong kong study saw a (nonsignificant) effect of hand hygiene alone which became stronger and significant in the mh arm [ ] . the investigators of a study among university students observed comparable reductions in ili both in the facemask only as well as the facemasks plus hand hygiene groups suggesting that the addition of a hand sanitizer did not increase the effect of facemasks, or at least not substantially [ ] . nevertheless, a recent cochrane review on the subject came to the conclusion that hand hygiene is generally effective in reducing the spread of respiratory viruses [ ] . a further limitation is the fact that laboratory testing of household contacts was only conducted for the virus subtype the index patient was infected with. this could have led to an underestimation of secondary cases. finally, we cannot rule out the possibility that behaviour of participating households may have been influenced by monetary incentives and frequent household visits. however, they did not differ in all three study arms so we do not expect this to have biased our results. furthermore, the other clinical trials had a similar design so that it should not endanger comparability of results. the strengths of this study include laboratory confirmation of primary and secondary cases with qrt-pcr, the serial testing of all household members over the study period irrespective of respiratory symptoms, and the low degree of contamination between the intervention groups. in conclusion, results of our study contribute to the body of evidence that npi may be effective in preventing transmission of influenza in households. prerequisites include early implementation of the intervention and good adherence. we were also able to show that the use of facemasks in particular is tolerable and acceptable for adults and children alike, both as household contacts and index cases, highlighting the fact that these measures could play an important role in the interruption of influenza transmission within households. future research should focus on the differential importance of facemask use by index cases or household contacts as well as the independent role of hand hygiene in the prevention of influenza transmission. preventing transmission of pandemic influenza and other viral respiratory diseases: personal 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surveillance oral oseltamivir in human experimental influenza b infection shedding and transmission of novel influenza virus a/h n infection in households-germany diagnostic approach for the differentiation of the pandemic influenza a(h n )v virus from recent human influenza viruses by real-time pcr secondary attack rate of pandemic influenza a(h n ) in western australian households transmission of pandemic influenza a (h n ) within households design and analysis of cluster randomization trials in health research bootstrapping clustered data longitudinal data analysis for discrete and continuous outcomes time lines of infection and disease in human influenza: a review of volunteer challenge studies emergence of a novel swine-origin influenza a (h n ) virus in humans serial intervals and the temporal distribution of secondary infections within households of pandemic influenza a (h n ): implications for influenza control recommendations household transmission of pandemic (h n ) risk factors of influenza transmission in households household transmission of influenza (h n - ) in japan: age-specificity and reduction of household transmission risk by zanamivir treatment journey through an epidemic: some observations of contrasting public health responses to sars perceptions and behaviors related to hand hygiene for the prevention of h n influenza transmission among korean university students during the peak pandemic period factors influencing the wearing of facemasks to prevent the severe acute respiratory syndrome among adult chinese in hong kong monitoring the level of government trust, risk perception and intention of the general public to adopt protective measures during the influenza a (h n ) pandemic in the netherlands physical interventions to interrupt or reduce the spread of respiratory viruses. cochrane database of systematic reviews professional and home-made face masks reduce exposure to respiratory infections among the general population this work was financially supported by the german federal ministry of health. the funding source had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. we thank all participating households and study physicians for their dedicated collaboration. furthermore, we highly appreciate the support of matthias an der heiden (robert koch institute) in data analysis and monika luchtenberg (robert koch institute) in study organisation. authors' contributions ts designed the study, co-ordinated the execution, executed the study, performed the statistical analysis, interpreted the results, and wrote the manuscript. ub developed the study idea, designed the study, supervised its co-ordination, data analysis and interpretation, and wrote the manuscript. cr designed the study, co-ordinated the execution, executed the study, and wrote the manuscript. sbs executed the study, and wrote the manuscript. ik analysed the data and wrote the manuscript. wh designed the study, and wrote the manuscript. gk provided important intellectual contributions to design, coordination and analysis of the study, and wrote the manuscript. bs, an, ks, jd, and jm carried out the laboratory analysis, interpreted data, and wrote the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -xagh jc authors: gunson, rory n; carman, william f title: during the summer outbreak of "swine flu" in scotland what respiratory pathogens were diagnosed as h n / ? date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: xagh jc background: during the april-july outbreak of h n / in scotland the west of scotland specialist virology centre (wossvc) in glasgow tested > clinical samples for h n / . most were from patients clinically diagnosed with h n / . out of these, % were positive. this study sought to determine what respiratory pathogens were misdiagnosed as cases of h n / during this time. methods: we examined the results from samples which were sent to the laboratory during april-july . all were from patients clinically diagnosed as having h n / (based on accepted criteria) and all were given a full respiratory screen using real time reverse transcriptase polymerase chain reaction (rtrt-pcr) assays. results: in total, respiratory pathogens were detected in . % ( % confidence interval, . - . %) of the samples submitted. numerous pathogens were detected, the most common of which were rhinovirus ( . % ( % confidence interval, . - . %)), parainfluenza ( . % ( % confidence interval, . - . %)) and ( . % ( % confidence interval, . - . %)), and adenovirus (( . % ( % confidence interval, . - . %)). conclusions: this study highlights the problems of using a clinical algorithm to detect h n / . clinicians frequently misdiagnosed common respiratory pathogens as h n / during the spring/summer outbreak in scotland. many undesirable consequences would have resulted, relating to treatment, infection control, and public health surveillance. on april th and th , a novel swine-lineage influenza a (h n / ) infection was reported to the world health organisation (who) by the centers for disease control and prevention (cdc) in atlanta. the virus was detected in two children from adjacent counties in southern california presenting with febrile respiratory illness [ , ] . these cases were not epidemiologically linked and neither child had exposure to swine. subsequent phylogenetic characterisation of h n / from the u.s. index case (a/california/ / ) showed that the virus had a unique genome composition that had not been previously identified. six genes (pb , pb , pa, ha, np, and ns) were similar to viruses previously identified in triple-reassortant swine influenza viruses in north american pigs. the remaining two genes (na and m) were derived from eurasian swine influenza viruses. this particular gene constellation had never been previously identified in humans or other reservoirs. following the original identification of influenza a/ h n / in the united states, sustained human-tohuman transmission was seen in other countries, and on june , , the who declared that the virus was responsible for the first influenza pandemic of the st century [ ] . the first cases of h n / in scotland were detected at the end of april in a couple returning from their honeymoon in mexico [ , ] . the initial public health response to the outbreak was a containment exercise aimed at preventing the spread of infection, detecting cases and taking action to prevent these cases from infecting others [ ] . the exercise was initially based on clinical and epidemiological criteria (table ) . patients who met these criteria were immediately given treatment and isolated while a rapid real time reverse transcriptase polymerase chain reaction (rtrt-pcr) for h n / was initiated. contact tracing was undertaken in order to treat those who had been in contact with confirmed cases. soon after the first detections, person to person transmission was confirmed as having occurred in scotland. consequently, the epidemiological criteria were no longer useful and the containment exercise was then based on clinical criteria only. testing continued to be carried out during this period. during the outbreak period (april-july ) the west of scotland specialist virology centre (wossvc) tested clinical samples for h n / ( figure ). of these, only were positive ( % overall for the period of april-july; range - % per month). consequently, the clinical diagnosis was found to be wrong in the majority of cases. a large number of viral infections, drugs and other diseases can cause disease presentations similar to those presented in table . this is especially true for respiratory pathogens. the present study sought to determine what respiratory pathogens were diagnosed as cases of h n / during the containment phase. background to the respiratory service in place during april-july during april-july all respiratory samples submitted from patients with a clinical diagnosis of h n / (as stated on the specimen request form), were initially tested using a universal influenza a real time rtrt-pcr assay and a h n / specific real time rtrt-pcr assay [ ] . if the sample was negative on these tests and was from a hospitalised patient, a patient deemed at risk of severe respiratory infection (e.g. an immunocompromised or a pregnant patient), or a patient attending gp services taking part in local or national surveillance schemes, a full respiratory screen was carried out (see below for details regarding the full respiratory screen). note that this information was derived from the sample request form. with regard to (i) samples from gp services not taking part in the surveillance schemes mentioned above, (ii) follow-up samples from known h n / positive patients, and (iii) samples from patients with no clinical/setting details, only the initial screening test was conducted. to determine which respiratory pathogens had been misdiagnosed as h n / , we examined the results of samples which had been given a full respiratory screen during the period april-july . all had been clinically diagnosed as having h n / , and all were found to be h n / negative. the samples submitted included gargles, throat swabs, nasal swabs, nasopharyngeal aspirates, sputums, and endotracheal secretions. the number collected each month and the age of the patients are shown in table . binomial % confidence intervals are shown for each age group. total nucleic acid was extracted from respiratory specimens using qiaamp viral rna kit (qiagen, crawley, united kingdom) according to the manufacturer's instructions. real-time rt-pcr was carried out in order to detect influenza a (a generic assay and a h n / specific assay [ ] ), b and c, rsv, rhinovirus, parainfluenza - , human metapneumovirus, coronavirus ( e, nl , hku and oc ), adenovirus, and mycoplasma pneumoniae. the oligonucleotide primers and probe (tib-mol-biol, berlin, germany) are outlined in table . the primers and probes for the influenza a generic assay and the h n / specific assay are described elsewhere [ ] . these assays have been developed by the wossvc and used as the frontline test for respiratory samples since . all assays have been shown to be sensitive and specific by in-house development procedures and via participation in numerous external quality assessment schemes (eqa), including those provided by the who, the health protection agency (hpa), and quality controls for molecular diagnostics (qcmd). all assays used the primers at a final concentration of . μm and the probe at . μm in a μl reaction volume. one-step rtrt-pcr was performed on μl of rna extract with the platinum one-step qrt-pcr kit (invitrogen life technologies, paisley, uk) on an abi prism sds real-time platform (applied biosystems). the following thermal profile was used: a single cycle of reverse transcription for min at °c, min at °c for reverse transcriptase inactivation and dna polymerase activation followed by amplification cycles of sec at °c and secs at °c each (annealing-extension step). data acquisition occurred at the annealing step of each cycle, and the threshold cycle (ct) for each sample was calculated by determining the point at which the fluorescence exceeded the threshold limit. the percentage detection rate for each pathogen was analysed monthly, and for the overall study period. binomial % confidence intervals were also determined for each detection rate. a chi-squared test was used for any comparisons of two data sets. please note that ethical approval was not required for this paper as the samples were collected as part of routine diagnostic work. examination of the detection rate over the four-month period shows that respiratory pathogens were detected in . % of all samples submitted ( % confidence interval, . - . %) ( table ). numerous pathogens were detected in the samples. the most commonly detected pathogen was rhinovirus which was detected in . % of all samples tested ( % confidence interval, . - . %), and was similarly detected in each of the months examined. adenovirus was also commonly detected ( . % ( % confidence interval, . - . %). parainfluenza showed its typical activity. it was detected in . % of all samples collected in april ( % confidence interval, - %) with decreasing activity thereafter ( . % in july ( % confidence interval, . - . %))(p < . )). interestingly, parainfluenza (a pathogen normally associated with winter activity) was present in an unexpectedly large number of samples during this period ( . % of all samples overall ( % confidence interval, . - . %) and mirrored the increasing activity of h n / (from . % in april ( % confidence interval, - %) to % in july ( % confidence interval, . - . %) (p = . )). the detection rate for the remaining pathogens was low (< . %) and showed no particular pattern over the months examined. this study highlights the problems of using a clinical algorithm to detect h n / during a period of low incidence. we found that clinicians frequently misdiagnosed common respiratory pathogens as h n / during the spring/summer outbreak in scotland. this finding is similar to results found in a recent audit of patients hospitalised with clinically diagnosed h n / in infectious disease units in scotland [ ] . the pathogens that were misdiagnosed as h n / were, for the most part, those viruses expected to be encountered during the spring/summer months. although all the pathogens included in the respiratory screen were detected on at least one occasion, rhinovirus was the most commonly detected pathogen. this is not a surprising finding, since rhinovirus is detected all the year round; moreover, it is recognised as a very common cause of the common cold and is increasingly being implicated in more severe clinical syndromes [ ] . pf , pf , adenovirus and human metapneumovirus were also frequently detected. an unexpected finding was the frequent detection of parainfluenza , a pathogen which is traditionally recognised as a winter pathogen. however, our data shows that there were numerous cases of parainfluenza during april-july , and these mirrored the activity of h n / . the unexpected summer activity of parainfluenza mirrors that of parainfluenza in , which was also unexpectedly detected during the summer months (data not shown). this suggests that the epidemiology of established viruses, such as parainfluenza , should be re-examined in the light of new, more sensitive, molecular assays. it should be noted that the majority (~ %) of samples submitted to the laboratory were found to be negative by real time rtrt-pcr. by participation in various eqa schemes, the assays used by the laboratory have been shown to be highly sensitive. consequently, the large number of negative results are unlikely to have been caused by the pathogens already tested for in the screen. however, the respiratory screen does not include an internal control. consequently, false negatives due to inhibition may have occurred. nevertheless, the number of samples affected is likely to be small, since a recent in-house audit found that inhibition occurs in~ % of throat and nasal swabs submitted to our laboratory. poor sampling may also have led to false negative results. however, as with the case of inhibition, the contribution of this factor is likely to be minimal. we cannot rule out that the possibility that these samples may have contained other respiratory pathogens not currently included in the respiratory screen (e.g. boca, hku or bacterial respiratory pathogens). in future, the use of a larger testing panel either by further developing the existing service or by using an alternative method (e.g. microarray) would be useful to examine negative samples. another possibility is that other infective and non-infective agents could have been present. for example, the previously-mentioned audit of patients presenting at infectious disease units found an alternative nonrespiratory diagnosis in~ % of patients initially clinically diagnosed as having h n / [ ] . another explanation could be that a number of samples were actually from the worried well, or from asymptomatic contacts of known or suspected cases of h n / . an audit similar to the one outlined in the publication above [ ] would help to clarify this issue. it should be noted that the samples examined are representative of our coding protocol and may not be representative of the population at large. consequently, certain patient groups either with or at risk of severe infection may be over-represented in the final data. whether or not this is the case, the present study shows that numerous respiratory pathogens were being misdiagnosed as h n / . the misdiagnosis of h n / would have had many undesirable consequences. for example, potentially serious conditions may have been wrongly diagnosed as h n / [ ] . in addition a large number of individuals are likely to have been unnecessarily treated with oseltamivir [ ] . this involves unnecessary cost, and would also have exposed individuals to the side effects of oseltamivir [ ] . it should further be noted that there could well have been emotional costs in being wrongly labelled as having "swine flu", especially at the early stage of the pandemic, when the severity and outcome of the illness was still largely unknown. in hospitalised patients, unnecessary infection control procedures may have been implemented [ ] [ ] [ ] . disease surveillance may also have been inaccurate, affecting public health measures and leading to increased panic/concern in the general public. one way to overcome these issues would be to incorporate a near-patient testing component into the algorithm outlined above. this would no doubt reduce the amount of unnecessary treatment and isolation, and would ensure that surveillance data was more accurate. such a test would need to be very rapid, sensitive, and specific. although a number of methods have been described, these can be expensive and -in comparison to pcr-based methods -can be insensitive and non-specific [ ] . as a result, pcr methods may be required to investigate influenza negative samples and in low prevalence periods, such as the time period examined in this study, pcr may also be required to confirm positive results. cdc: update: infections with a swine-origin influenza a (h n ) virus-united states and other countries novel swine-origin influenza a (h n ) virus investigation team: emergence of a novel swine-origin influenza a (h n ) virus in humans swine flu. after delays, who agrees: the pandemic has begun health protection agency; health protection scotland; national public health service for wales; hpa northern ireland swine influenza investigation teams: epidemiology of new influenza a (h n ) virus infection a: confirmation of first person to person transmission of swine flu in uk expected soon how well are we managing the influenza a/h n pandemic in the uk? development of a real-time rt-pcr for the detection of swine-lineage influenza a (h n ) virus infections hospitalised adult patients with suspected h n infection at regional infectious diseases units in scotland -most had alternative final diagnoses newly identified human rhinoviruses: molecular methods heat up the cold viruses how well are we actually managing the influenza pandemic? perhaps not so well possible harms of oseltamivir-a call for urgent action clinical diagnostic criteria for isolating patients admitted to hospital with suspected pandemic influenza performance of influenza rapid point-of-care tests in the detection of swine lineage a (h n ) influenza viruses pre-publication history the pre-publication history for this paper can be accessed here cite this article as: gunson and carman: during the summer outbreak of "swine flu the authors would like to acknowledge the following individuals. dr tony speekenbrink for advice on statistics. rhona miller, walt adamson, alasdair maclean and susan bennett for advice and comments relating to the paper. finally the authors would like to thank the scottish poet and english teacher donald adamson for proofreading the various versions of this paper. the corresponding author has the right to grant on behalf of all authors and does grant on behalf of all authors, an exclusive licence (or non exclusive for government employees). authors' contributions wfc conceived of the study. rg examined the data and wrote the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -n qe bh authors: saiki-macedo, stephanie; valverde-ezeta, jorge; cornejo-tapia, angela; castillo, maria esther; petrozzi-helasvuo, verónica; aguilar-luis, miguel angel; del valle, luis j.; cieza-mora, erico; bada, carlos; del aguila, olguita; silva-caso, wilmer; martins-luna, johanna; vasquez-achaya, fernando; del valle-mendoza, juana title: identfication of viral and bacterial etiologic agents of the pertussis-like syndrome in children under years old hospitalized date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: n qe bh background: acute respiratory infections (aris) represent an important cause of morbidity and mortality in children, remaining a major public health concern, especially affecting children under years old from low-income countries. unfortunately, information regarding their epidemiology is still limited in peru. methods: a secondary data analysis was performed from a previous cross-sectional study conducted in children with a probable diagnosis of pertussis from january to july . all samples were analyzed via polymerase chain reaction (pcr) for the following etiologies: influenza-a, influenza-b, rsv-a, rsv-b, adenovirus, parainfluenza virus, parainfluenza virus, parainfluenza virus, mycoplasma pneumoniae and chlamydia pneumoniae. results: a total of patients were included. the most common pathogen isolated was adenovirus ( %), followed by bordetella pertussis ( %) from our previous investigation, the most prevelant microorganisms were mycoplasma pneumonia ( %) and influenza-b ( . %). coinfections were reported in % of samples and the most common association was found between b. pertussis and adenovirus ( . %). conclusions: there was a high prevalence of adenovirus, mycoplasma pneumoniae and other etiologies in patients with a probable diagnosis of pertussis. despite the presence of persistent cough lasting at least two weeks and other clinical characteristics highly suspicious of pertussis, secondary etiologies should be considered in children under years-old in order to give a proper treatment. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory infections (aris) are a leading cause of morbidity, hospitalization, and mortality among children [ ] [ ] [ ] . according to world health organization (who), acute respiratory infections are responsible for . million annual deaths in children, mainly affecting patients under years old, with a higher incidence in those from low-income countries [ , ] . aris are mainly caused by a wide range of viruses and bacteria [ , ] . viruses are isolated in up to % of cases, the most common pathogens are the respiratory syncytial virus (rsv) a and b, influenza (flu) a, b and c, parainfluenza (piv) types , , and , coronavirus and rhinovirus [ , ] . classically, s. pneumoniae and h. influenzae type b are the most commonly isolated bacteria in both throat and nasopharyngeal specimens from patients with aris [ , ] . however, in resource-limited countries, atypical bacteria such as mycoplasma pneumoniae, chlamydia pneumoniae, and bordetella pertussis can play an important role in aris and can be detected in more than % of patients [ , [ ] [ ] [ ] [ ] . although numerous pathogens are associated with aris, their clinical manifestations are very similar, regardless of the causative agent. thus, laboratory identification of the etiological agent is key in order to give a proper treatment and avoid the overuse of antibiotics [ ] . moreover, aris due to atypical bacterial infections have become a global concern especially after their reemergence in low-income countries [ , ] . simultaneous infections with virus and bacteria species have become an obstacle for clinicians, their prevalence has significantly increased, with studies discovering co-infections in more than % of cases [ , [ ] [ ] [ ] . additionally, these coinfections have been associated with longer hospitalization periods, worse clinical outcomes and increased mortality, again highlighting the importance of molecular etiological confirmation [ , , ] . bordetella pertussis represents a persistent cause of morbidity and mortality in children [ ] . accounting for an estimated million cases and , deaths worldwide [ ] . in a previous study we conducted on children under -year-old with a probable diagnosis of pertussis from peruvian hospitals, we reported a prevalence of . % pertussis cases [ ] . with more than % of cases without an identified pathogen, hence a more comprehensive etiological analysis was required. the main objective of this study was to detect the presence of respiratory viruses (influenza-a, influenza-b, rsv-a, rsv-b, adenovirus, parainfluenza- , parainfluenza- and parainfluenza- ) and atypical bacteria (mycoplasma pneumoniae, chlamydia pneumonia), via polymerase chain reaction in samples from peruvian children under years-old previously analyzed for b. pertussis. a secondary data analysis was performed from a previous cross-sectional study conducted from january to july in peruvian hospitals: instituto nacional de salud del niño, hospital edgardo rebagliati martin, hospital de emergencias pediátricas, hospital nacional cayetano heredia and hospital regional de cajamarca. the original study enrolled children under years old admitted to the pediatric wards as probable pertussis cases. however, in this original study, only patients under -year-old were included in the final analysis. a probable case was defined if patients presented paroxysms of coughing, or inspiratory "whoop" or post-tussive vomiting in the setting of an acute cough illness of any duration in the absence of another more likely diagnosis. in patients under year-old apnea (with or without cyanosis) was also considered. all patients with chronic pulmonary conditions, cardiac disease or immunodeficiency were excluded. patient who received antibiotics days prior to the enrollement were also excluded. (fig. ) . this study included a total of children under years old hospitalized with a probable diagnosis of pertussis, requiring further comprehensive etiological identification. all samples were analyzed via pcr for the following etiologies: influenza-a, influenza-b, rsv-a, rsv-b, adenovirus, parainfluenza virus, parainfluenza virus, parainfluenza virus, mycoplasma pneumoniae and chlamydia pneumoniae. this study has been approved by ethics committees of the universidad peruana de ciencias aplicadas. parents and caregivers signed a written consent in the previous study which included a section that granted the investigators permission for a possible future use of the samples that could be given as an extension of the original research. nasopharyngeal samples were obtained by inserting a swab into both nostrils parallel to the palate (mini-tip culture direct, becton-dickinson microbiology system, md , usa) and a second swab from the posterior pharyngeal and tonsillar areas (viral culturette, becton-dickinson microbiology systems, md, usa). both nasal and pharyngeal swabs were placed into the same tube containing viral transport medium (minimal essential medium with % fetal bovine serum, amphotericin b μg/ml, neomycin μg/ml). two aliquots of each fresh specimen were stored at − °c to be used for posterior analysis of respiratory viruses and detect atypical pathogens by pcr. [ ] . amplified products were recovered from the gel, purified (spinprep gel dna kit; san diego, ca) and sent for commercial sequencing (macrogen, korea). a database was generated in microsoft excel® (microsoft corporation, california, usa), all data was then exported to stata® v . (statacorp, college station, texas, usa). quantitative variables were described as frequencies and percentages for each group. the association was established by the pearson correlation analysis (r) and the results presented in a scatter matrix with the ellipse plotted at % confidence. a total of patients under years old with a probable diagnosis of b. ertussis were studied thouroughly for specific etiological identification. more than % of our study population were infants between to months old with a slightly higher number of males ( . %). the group of infants between days - months-old ( . %) and the group between and months-old ( . %) were the most predominant, closely followed by the group between and months-old ( . %) (additional file : table s ). from our previous study, cases of bordetella pertussis were confirmed via pcr, leaving potentially % of samples without etiological identification. thus, all were analyzed for the presence of influenza-a table s -a). the identification of these infectious agents has made it possible to establish remarkably that coinfections were present at % ( / ) of patients. thus, cases of infection due to a single infectious agent were . % ( / ), and where the presence of adv was . % ( / ) and for b. pertussis was . % ( / ), followed by m. pneumoniae with . % ( / ). furthermore, the prevalence of these infectious agents were accumulated in children under months of age (additional file : table s -b). as indicated above, in infected children ( cases) coinfections stand out considerably (additional file : table s ). the coinfections found involve to different infectious agents, being the most frequent the coinfections of agents with . % ( / ) and those involving agents with frequency of . % ( / ). the most frequent association was the bacterial-viral coinfection, and the combination between bordetella-adv and mycoplasma-adv were the most common involvement reported with . % ( / ) y . % ( / ), respectively. however, the bordetella-mycoplasma association was very reduced (additional file : table s ), in addition, it is interesting to note that the associations in coinfections increase the frequency of infectious agents such as chlamydia, flu-b and rsv-a as observed in additional file : table s (compare a with b). regarding vaccination status, . % ( / ) of the positive cases were unvaccinated. however, the majority of these children ( / ) were under two months of age. an unknown vaccinated status was observed in . % ( / ) of patients positive for b. pertussis. a marked decrease was observed in children who had received at least one dose of vaccination, with a prevalence of . % ( / ) (additional file : table s ). in our population, the most common clinical symptoms registered at admission were vomiting ( . %), whooping ( . %) and shortness of breath ( . %), followed by fever ( . %) and cyanosis ( . %). a wide spread distribution of symptoms distribution was observed when patients symptoms were individually assessed based on etiological group. only pathogens had symptoms that were present in more than % of each group. for example, vomiting was more commonly reported among children with flu-a, rsv-a, parainfluenza- and b. pertussis (additional file : table s -a). however, the difficulty in establishing clear clinical symptoms associated with infectious causal agents is due to the high frequency of coinfections. therefore, additional file : table s -b has recorded the clinical symptoms of cases of infection with a single agent, and the association of these clinical symptoms are shown in fig. . thus, a clear non-association can be observed between mycoplasma and flu-b, adv or bordetella; the same happens for flu-b and bordetella or adv. this non-association means that the only infectious agent could be identified taking into account the clinical symptoms of the children as shown in additional file : table s -b. the most common complications were acute bronchial obstructive syndrome (abos) and pneumonia in . and . % of our population respectively. abos was the most frequent complications among patients with positive samples for rsv-a, flu-a, adv, m. pneumoniae, c. pneumoniae and b. pertussis. (additional file : table s -a). however, when the complications of children affected by a single infectious agent are analyzed, it is clearly demonstrated that abos is also a complication of flu-b. in addition, it is noteworthy that abos occurs in % ( / ) of the negative cases (additional file : table s -b) . finally, a seasonal distribution was described for each specific microorganism. positive samples for adv and mycoplasma pneumoniae were observed across the whole study period. on the contrary, most of the b. pertussis cases were detected from may to march . rsv-a and chlamydia were mostly detected from march to may ; however, the same distribution was not observed in the following year (fig. ). aris as the most common cause of morbidity and mortality in children, remaining a major concern, especially affecting children under years old from low-income countries [ ] [ ] [ ] [ ] . unfortunately, information regarding their epidemiology is still limited in peru [ , ] . in recent years, there has been evidence of pertussis resurgence in latin america, despite the introduction of the vaccine [ , , ] . this bacterium is highly contagious and virulent, about half of the infected children under one year of whooping cough require hospitalization. in peru, the national immunization program administers the combined pentavalent vaccine (dpt, hvb, hib) at , , months with reinforcements at months and years [ , ] . thus, we conducted a previous study on peruvian children with a probable diagnosis of pertussis and reported a bordetella pertussis prevalence of . % [ ] . however, the classical presentation of pertussis has proven to be not enough to achieve a definitive diagnosis and laboratory tests are of the utmost importance for etiological confirmation to avoid overdiagnosis [ , , ] . in the light of possible coinfections and more than % of patients without an etiological identification in our previous study, we conducted a new comprehensive analysis to detect viral and atypical bacterial etiologies in all our patients. from a total of samples analyzed from children under years old with a probable diagnosis of pertussis, the most common pathogen isolated was adv in % of samples, followed by b. pertussis in % from our previous analysis. although this study was conducted in patients with ari with a highly suspicious pertussis diagnosis, other studies on children with ari have identified adv as one of the most prevalent etiologies [ ] although, the viral and bacterial prevalence may vary widely depending on the population characteristics [ , , ] . interestingly, our population were children with a highly suspicious clinical diagnosis of pertussis and despite that whooping was present in . % of patients, adv was the most common etiology isolated. furthermore, a great number of patients with adv infection presented with clinical symptoms very common among patients with pertussis such as whooping ( . %), shortness of breath ( . %) and vomiting ( . %). similarly, a recent study has reported that gastrointestinal symptoms and difficulty breathing are among the most common type of presentations in children [ ] . additionally, adv has been historically identified as a major cause of pertussis-like syndrome, which results in the likelihood of a pertussis misdiagnosis in the absence of laboratory [ ] [ ] [ ] . in this way, we have shown that patients with infection by adv and b.pertussis as a single infectious agent have similar symptoms (fig. ) . it is also important to highlight the presence of mycoplasma pneumoniae ( %) and chlamydia pneumoniae ( . %) among our patients. in a previous study, in children with aris, we reported a very similar prevalence of mycoplasma pneumoniae and chlamydia pneumoniae in . and . %, respectively [ ] . demonstrating the high prevalence of these atypical bacteria among peruvian children with aris. our results from this current study also make noteworthy that clinical manifestations by mycoplasma pneumoniae and flu-b, adv, or b. pertussis are distinguishable when the infection is due to infectious agent alone. however, in coinfections the symptoms were undestinguibles (see table s ). additionally, in our previous study coinfections between these bacteria and viruses were also frequent; present in . % of samples, coinfections between were the most common combination and the association between mycoplasma pneumoniae with vrs-a was the most frequent one observed in . % of patients [ ] . surprisingly, in this study, we have observed % of coinfections in our samples, again being the viral-bacterial association the most frequent and the most commonly detected coinfection involving bordetella pertussis-adv and mycoplasma pneumoniae-adv with frequencies of . and . %, respectively. another study in children, although in patients with community-acquired pneumonia, also have reported coinfections in m. pneumoniae as the most common bacteria detected in association with a virus [ ] . thus, to avoid under-diagnosis, pertussis should be considered in patients with cough, especially if chronic, even when m. pneumoniae have been documented [ ] . another common coinfection was b. pertussis and flu-b present in patients. although viral-bacterial coinfections are commonly associated with worse clinical courses and longer hospitalizations [ , , ] . recent investigations have reported similar clinical outcomes in infants hospitalized with b. pertussis and another respiratory virus coinfection [ ] . however, noteworthy attention should be given to the b. pertussis and adv coinfection in infants. a study compared infants with rsv and rsv-b. pertussis coinfection reporting similar disease severity; however, patients with this coinfection clearly needed more respiratory care and nutritional support [ ] . consequently, our only patient with rsv-a and pertussis presented with cyanosis and required advance respiratory support. the variations in the rate and pattern of coinfection in patients with aris may be related to seasonal and geographical factors [ ] . in our study, we intended to describe all detected pathogens and their seasonal distribution. even though we were not able to describe any clear pattern, it is worth mentioning the high prevalence of adv and m. pneumoniae across all of the study period, as well as the increasing prevalence of b. pertussis on . our study had some limitations. as mentioned in our previous study, due to our study design we were not able to establish causality between the pathogens isolated in our samples and our patient's clinical presentation. additionally, missing samples, we were not able to perform an etiological analysis in samples from our previous study. we were not able to perform an etiological analysis in samples from our previous study. aditionally, there is still controversy if pcr alone can be used as a confirmatory method for m. pneumoniae diagnosis [ , ] . commercial pcr tests have high specificity and are currently a method of choice for direct pathogen detection [ , ] . however, studies recommend that pcr alone should not replace serology and the combination of both could be good screening tests for reliable and accurate diagnosis of m. pneumoniae [ ] . moreover, other more recent studies suggest that a positive pcr or serology for m. pneumoniae may be unable to differentiate between asymptomatic carriage and symptomatic infection [ ] . to date, the available published information regarding the etiological prevalence of aris in peruvian children is still limited. despite the high incidence of pertussis, especially in vulnerable populations such as infants, to date to establish a etiological diagnosis in low income countries is still challenging [ , , ] . moreover, the relationship between the clinical severity and coinfections in respiratory pathogens remains inconclusive. our study is among the first ones to describe multiple viral and bacterial etiologies in patients with a high clinical suspicion of pertussis [ , ] . further investigations should be conducted in order to understand the role of these pathogens in peruvian children. additional file : sampling was founded by grant of sanofi aventis del peru. further sample processing and analysis was founded by grants th incentive for research of the universidad peruana de ciencias aplicadas (exp- - -upc), lima-peru. the financing was used exclusively for obtaining and processing samples. in no case was there any influence of the entities in the analysis and interpretation of the data obtained, nor did it influence the preparation of the manuscript. abstraction format used in the study and dataset are available and accessible from the corresponding author upon request in the link: https:// figshare.com/articles/dataset_ - / . ethics approval and consent to participate this study has been approved by ethics committees of the universidad peruana de ciencias aplicadas. parents and caregivers signed a written consent in the previous study which included a section that granted the investigators permission for a possible future use of the samples that could be given as an extension of the original research. not applicable. on behalf of all authors, the corresponding author states that there are no competing interest or funding related to this study. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. estimates of world-wide distribution of child deaths from acute respiratory infections viral and bacterial etiologies of acute respiratory infections among children under years in senegal acute respiratory infections among under- children in india: a situational analysis global burden of acute respiratory infections in children: implications for interventions clinical utility of pcr for common viruses in acute respiratory illness seasonality of respiratory viruses causing hospitalizations for acute respiratory infections in children in nha trang 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viruses in clinical specimens by two multiplex reverse transcription nested-pcr assays alerta epidemiológica: tos ferina (coqueluche) bordetella pertussis: an underreported pathogen in pediatric respiratory infections, a prospective cohort study childhood whooping cough vaccine protects most children for at least years molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to bordetella pertussis and other bordetella subspecies detection of bordetella pertussis in infants suspected to have whooping cough incidence of respiratory viruses in peruvian children with acute respiratory infections epidemiology and seasonality of acute respiratory infections in hospitalized children over four consecutive years adenovirus respiratory tract infections in infants: a retrospective chart-review study pertussis-like syndrome associated with adenovirus presenting with hyperleukocytosis: case report whooping-cough due to adenovirus the role of adenoviruses in the pertussis syndrome etiologic spectrum and occurrence of coinfections in children hospitalized with communityacquired pneumonia pertussis accompanying recent mycoplasma infection in a -year-old girl pcr versus serology for diagnosing mycoplasma pneumoniae infection: a systematic review & meta-analysis laboratory diagnosis of mycoplasma pneumoniae infection incidence and burden of pertussis among infants less than year of age carriage of mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study respiratory viral infections in infants with clinically suspected pertussis respiratory viruses and b pertussis co-infections: a frequent occurrence in children hospitalized with b pertussis key: cord- -mymgnf z authors: nelson, megan m.; waldron, christopher l.; bracht, john r. title: rapid molecular detection of macrolide resistance date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: mymgnf z background: emerging antimicrobial resistance is a significant threat to human health. however, methods for rapidly diagnosing antimicrobial resistance generally require multi-day culture-based assays. macrolide efflux gene a, mef(a), provides resistance against erythromycin and azithromycin and is known to be laterally transferred among a wide range of bacterial species. methods: we use recombinase polymerase assay (rpa) to detect the antimicrobial resistance gene mef(a) from raw lysates without nucleic acid purification. to validate these results we performed broth dilution assays to assess antimicrobial resistance to erythromycin and ampicillin (a negative control). results: we validate the detection of mef(a) in raw lysates of streptococcus pyogenes, s. pneumoniae, s. salivarius, and enterococcus faecium bacterial lysates within – min of assay time. we show that detection of mef(a) accurately predicts real antimicrobial resistance assessed by traditional culture methods, and that the assay is robust to high levels of spiked-in non-specific nucleic acid contaminant. the assay was unaffected by single-nucleotide polymorphisms within divergent mef(a) gene sequences, strengthening its utility as a robust diagnostic tool. conclusions: this finding opens the door to implementation of rapid genomic diagnostics in a clinical setting, while providing researchers a rapid, cost-effective tool to track antibiotic resistance in both pathogens and commensal strains. combating antimicrobial resistance (amr) is a national and international priority. the u.s. national institutes of health [ ] , center for disease control [ ] , world health organization [ ] , and united nations [ ] have prioritized the issue. on sept. , former president barack obama issued amr-focused executive order [ ] , which was followed by a national action plan for combating antibiotic resistant bacteria [ ] . however, surveillance of antimicrobial resistance is a significant challenge [ , , ] , causing difficulties in obtaining a realistic threat measurement [ , ] , and impairing the ability to form future projections [ ] . current methods of assessing antimicrobial resistance are extremely slow, requiring days to weeks of culture time, and are also costly in terms of laboratory materials and technician effort [ ] . correspondingly, they are deployed unevenly, biasing our estimates of amr worldwide and inhibiting our ability to accurately assess this threat to human health [ ] . responding to calls for new diagnostic methods to address this unmet need [ ] , here we report a simple, rapid, culture-free genomic method for detecting antimicrobial resistance within min of assay time. we also validate a simple raw-lysate preparation method that does not require nucleic acid purification. together these innovations address a critical need in surveillance of antimicrobial resistance. recombinase polymerase amplification (rpa), an isothermal alternative to polymerase chain reaction (pcr), uses recombinase-primer complexes to identify and denature the genomic segment of interest, along with single-stranded dna-binding proteins to stabilize the open dna [ ] . detection is similar to taq-man hydrolysis probes [ ] except that the probe contains an internal abasic site analog, tetrahydrofuran, that is cleaved by endonuclease iv (nfo) [ ] during the course of amplification [ ] . the polymerase used is strand-displacing bsu [ ] , which is more resistant to chemical inhibition than taq, giving rpa more robustness than pcr [ ] . because dna denaturation is performed by proteins rather than heat, rpa occurs isothermally, usually °c - °c, and multiple reports document improved speed for rpa relative to pcr, often with detection within - min [ ] [ ] [ ] . in addition, rpa demonstrates extreme sensitivity, often detecting tens of copies of a nucleic acid target [ , [ ] [ ] [ ] [ ] . while rpa has not been widely implemented in clinical settings, it has been proven capable of detecting bacterial, viral, and protozoan human pathogens. eukaryotic pathogens detected with rpa include the blood-fluke schistosoma japonicum [ ] and the diarrheal protozoan pathogens giardia, cryptosporidium, and entamoeba [ , ] . viral pathogens detected by rpa include hiv [ , ] , chikungunya virus (chikv) [ ] , rift valley fever virus [ , ] , middle east respiratory syndrome coronavirus [ ] , foot-and-mouth disease virus (fmdv) [ ] , bovine coronavirus [ ] , and crimean-congo haemorrhagic fever virus (cchfv) [ ] . bacterial pathogens detected by rpa include mycoplasma tuberculosis [ , ] , neisseria gonorrhoeae, salmonella enterica, and methicillin-resistant staphylococcus aureus (mrsa) [ ] , chlamydia trachomatis [ ] , francisella tularensis [ ] , group b streptococci [ ] , orientia tsutsugamushi (scrub typhus), and rickettsia typhi (murine typhus) [ ] . in diagnostic applications rpa has been shown to be highly specific and thus resistant to false positives (type i errors). in several cases % specificity was shown [ ] [ ] [ ] ] . because of the health risks of erroneous detection and treatment, high specificity is an important characteristic of diagnostic assays. type ii errors (false negatives) are always possible if the pathogenic target is present at a low level in a sample, but the exquisite sensitivity of rpa (see above) minimizes this risk. in this study, we developed and tested a novel rpa assay for the detection of the macrolide efflux a, or mef(a) gene, an efflux pump rendering host bacteria resistant to -and -membered macrolide antibiotics (including erythromycin a and azithromycin) [ , ] . this gene can be found within streptococcus pyogenes, the largest member of the lancefield group a streptococci, where it is encoded on a transposon that is integrated into a prophage [ , ] . while initially identified in s. pyogenes and s. pneumoniae [ ] it has since been identified in an extremely wide range of gram-positive and negative bacteria worldwide [ ] consistent with horizontal transfer of antimicrobial resistance genes. using purified dna, a panel of bacteria cultures, and broth dilution antimicrobial resistance testing, we demonstrate extreme sensitivity and specificity of the rpa assay, and we confirm that positive results correctly predict antimicrobial resistance. our rpa assay uncovered an unexpected occurrence of the mef(a) gene within commensal streptococcus salivarius strain, and subsequent laboratory testing confirmed that this strain has genuine antimicrobial resistance. while s. salivarius has been known to frequently harbor antimicrobial resistance genes [ ] , this is the first case, to our knowledge, of antimicrobial resistance first discovered by rpa and confirmed by more traditional methods. presence or absence of mef(a) and ermb genes were assessed by local blastn against published genomes downloaded from the following genbank accessions: s. pyogenes mgas , accession cp . ; s. pyogenes mgas , accession cp . ; s. pneumoniae strain ga , accession ails . ; s. pneumoniae ga , accession agpe . ; s. pneumoniae strain np accession agqf . ; s. agalactiae sgbs , accession auwe . ; and enterococcus faecium strain accession ambg . . s. pyogenes, s. agalactiae, and s. salivarius were tested for their antimicrobial susceptibility by broth microdilution. ampicillin (cat # - ) was obtained from vwr (amresco) and erythromycin (cat # tce - g) was obtained from vwr (tci). bacteria were maintained on blood agar plates at °c, and single colonies selected for inoculation into liquid overnight cultures in sterile brain-heart infusion (bhi, vwr cat # - ). for each culture, ml of bhi media was inoculated in a sealed ml falcon tube for overnight incubation at °c (no shaking). gentle inversion was used to mix the cultures prior to setting up the assay. for the experiment, μl of overnight culture was mixed with ml of bmi media ( x dilution) in a sterile tray and gently mixed. this dilute culture was added at μl per well of a -well plate pre-loaded with μl of antibiotic solutions ranging, for erythromycin, from . to μg/ml ( x) to produce the desired final concentrations of . - . μg/ml. for ampicillin, the stocks were . μg/ml- μg/ml resulting in final concentrations of . μg/ml- μg/ml. the -well plate was then transferred to a filtermax f microplate reader for a h incubation at a temperature of °c, with readings taken every min. a -s orbital shaking was performed prior to each reading. for specificity testing, human dna was derived from primary adipose-derived cell line asc a (commercially obtained from zen-bio, raleigh, nc) cultured in a humidified % co incubator at °c. the growth media consist of dulbecco's modified eagle medium (dmem, thermofisher # ) supplemented with % fetal bovine serum (thermofisher # ), x penicillin / streptomycin (thermofisher # ), and x glutamax (thermofisher # ), changed every days. total dna was purified using the nucleospin tissue kit (macherey-nagel, düren, germany) and quantified on a qubit fluorometer (thermofisher), which was also used to measure bacterial dna liberated in crude lysates. primers and probe for the mef(a) rpa assay (table ) were designed following the instructions provided by twistdx (cambridge, uk). all primers and probes were synthesized by integrated dna technologies (coralville, iowa). for all rpa assays the twistdx nfo kit (tanfo -kit, twistdx, cambridge, uk) was used in agreement with manufacturer's instructions. for each reaction, a hydration mix was prepared including . μl of rpa primer pair ( . μl of each μm primer), . μl of probe ( μm), . μl of rehydration buffer, and . μl of sample containing dna or lysate to be tested ( . μl total). then the hydration mix was added to a reaction tube containing twistamp lyophilized enzyme pellet. the resulting mixture was mixed via pipetting - times carefully to avoid introduction of bubbles, and transferred to a qpcr -well plate (agilent cat # ). final concentration of primers was nm and the probe was nm. to activate the reaction, . μl of magnesium acetate stock solution ( mm) was added to the caps of the -well plate, rapidly mixed via inversion, immediately placed in a qpcr machine (agilent stratagene mx p). the reaction was maintained at constant temperature of °c for min, with fam signal recorded every s ( total readings). primers f and r (table ) were combined at a final concentration of nm with control dna (mgas ) dilutions at indicated concentrations, in x powersybr (thermofisher cat # ) and run on an agilent stratagene mx p. we used a -step program with cycles of s at °c and min at °c. the total program time was h min. bacterial identification was carried out using primers f and r with μl raw lysates prepared by boiling and diluting the overnight cultures. amplification was performed in a simpliamp thermocycler (applied biosystems) with a program of cycles with °c for s, °c for s, and °c for s. detection of mef(a) was performed by pcr using f and r primers and μl raw lysates as above. the program used was cycles of °c for s, °c for s, and °c for s. we designed a taq-man style hydrolysis probe incorporating fluorophore (fam) and quencher (iowa black) which doubles as a ′ end blocker. successful amplification leads to probe cleavage by endonuclease iv (nfo) at the abasic site, separating fam from the quencher and yielding detectable signal. earlier work used a quencher and fam internally, proximal to the abasic site [ ] ; our design simplifies this by using the quencher as a ′ end blocker (fig. a) . to assess assay sensitivity we ran a serial dilution of dna derived from mef(a)-positive streptococcus pyogenes serotype m strain mgas [ ] and found that confident detection was around genome copies (fig. b) . two-thousand genome copies corresponds to . picograms (pg) of dna, at a concentration of femtomolar (fm). while the fam signal crosses the threshold for , , and genome copies, these signals are probably nonspecific as demonstrated by negative controls showing similar late-rising (around min or later) signal (figs. b, c, and ). we conclude that the confident sensitivity limit of our assay is approximately genome copies, and that detection must be recorded before min to be considered real. the non-specific - -min signal was always easily distinguishable from real detection in our assays, which always came up quickly, around - min (compare figs. b, c, and ). we suggest the late-rising signal is analogous to qpcr's tendency to ubiquitously amplify even no-template controls by cycles. we performed sybr green based qpcr on the same dna dilution series using the same primers, and observed even greater sensitivity-relatively confidently down to genome copies-but it was significantly slower -the run took over h (fig. c) . as discussed later, the genome copy threshold may help distinguish diagnostically meaningful mef(a) gene loads, rather than mere colonizers [ ] . we next performed specificity testing with raw bacterial lysates from eight bacterial strains. mef(a) is present within the genomes of group a strep strain s. pyogenes mgas [ ] and s. pneumoniae strains ga and ga . known mef(a) negative strains include s. pyogenes mgas [ ] responsible for necrotizing fasciitis and puerperal sepsis, enterococcus faecium strain , s. pneumoniae strain np , and s. agalactiae sgbs . streptococcus agalactiae is resistant to macrolides by a different mechanism than mef(a): it hosts a target-site ribosomal methylase, ermb. methylation of the target site in the s rrna by ermb inhibits the interaction of antibiotic with the ribosome [ ] . we therefore predicted-and confirmed-that this species would show an absence of mef(a) by rpa but nonetheless display robust resistance to erythromycin (fig. g ). finally, we tested a patient isolate of s. salivarius with an unknown mef(a) status. the identities of s. salivarius, s. agalactiae, and s. pyogenes strains were confirmed by sequencing the s rdna locus. we developed a simple raw lysis method. individual bacterial colonies were inoculated into bhi media for overnight incubation at °c, followed by lysis by boiling at °c for minutes and -fold dilution into sterile h o. rpa was performed directly on this raw lysate (fig. a) . we tested eight bacterial strains in total: s. pyogenes ( strains), s. agalactiae, s. salivarius, s. pneumoniae ( strains), and e. faecium. rpa confirmed the presence of mef(a) within all known positive strains and none of the known negatives (fig. b, c) . rpa indicated the presence of mef(a) within s. salivarius, an unexpected result (fig. b) . while we had not expected this commensal species to contain mef(a), we nevertheless performed pcr which confirmed the gene's presence in mgas and s. salivarius (fig. a) . by sanger sequencing this product we observed that the s. salivarius gene has three single-nucleotide polymorphisms (fig. b) , suggesting that it has acquired a more divergent copy of the gene and confirming that the detections constitute independent mef(a) genes, not cross contamination. to test whether the mef(a) gene is functional, we performed broth dilution of both strains of s. pyogenes, s. salivarius, and s. agalactiae with erythromycin and ampicillin (a negative control) (fig. ) . this confirmed that s. pyogenes mgas , s. agalactiae, and s. salivarius are all resistant to erythromycin (mic greater than or equal to . μg/ml, table ) and mgas is susceptible (fig. ) . as reported by others, ermb gives stronger erythromycin resistance than mef(a) [ , ] , with s. agalactiae giving a mic > . μg/ml ( table ). all tested strains were susceptible to ampicillin as expected (fig. , table ). to evaluate assay specificity we constructed mixtures of nucleic acids as follows: a, b, and c contain ng of dna from non-mef(a) lysates (s. agalactiae plus mgas ) either by themselves (c) or spiked with . ng (a) or . ng (b) of mgas (mef(a)-positive). mixes a and b represent . and . % mef(a) positive, respectively. mixes d and e tested the effect of human dna, which might be expected to contaminate clinical samples. we therefore tested either ng human dna alone (d) or with . ng ( %) of mef(a)-positive mgas lysate (e). none of the non-specific dna had any apparent effect on the reactions, with only e, a, and b giving specific signal and in proportion to the total mef(a) gene present in the samples ( . ng, . ng, and . ng, respectively) (fig. ) . the mef(a)-negative c and d samples yielded no specific signal, giving non-specific time-to-threshold of . and . min, respectively (fig. ) . not only do these results show that the rpa assay was % specific and quantitative in the presence of non-specific dna, but also functions with a wide range of total dna in the mixture (from a few picograms, fig. b, to ng, fig. ), and is robust to the conditions of raw lysate including denatured proteins, lipids, and cell wall debris. genomic diagnostics offer the flexibility to in principle detect genetic material in any pathogen-bypassing the challenges associated with antibody-based assays which are much more cumbersome to produce while also being less sensitive than nucleic-acid based methods. for example, two meta-analyses of the rapid antigen-based test for group-a streptococcal pharyngitis found an % sensitivity [ , ] , so % of true positives are missed by this method. here we demonstrate a simple rpa-based genomic procedure offering flexibility and rapid detection within a similar timeframe as the rapid tests ( - min) that is suitable to a point-of-care application. we show that we can detect down to the femtomolar (fm) / picogram (pg) range (fig. b) . we found that spiking in up to x more non-specific dna than mef(a) + dna did not inhibit the assay, which remained extremely quantitative and specific to true target levels (fig. ). detection of antimicrobial resistance genes has been more frequently performed with loop-mediated isothermal amplification (lamp) rather than rpa. examples include detection of the beta-lactamase responsible for carbapenem resistance in acinetobacter baumannii [ , ] , the class integron-integrase gene inti from environmental samples [ ] , msra from staphylococcus aureus [ ] and mcr- from enterobacteriaceae isolates [ ] . in all cases, detection occurred within - min and generally sensitivity was in the picogram range. in contrast, rpa offers a simplified system with fewer primers that generally gives results in less than min, which may be a critical time advantage in certain settings like clinical applications. in contrast to lamp, genomic detection of antimicrobial resistance by rpa is still in its infancy and more progress has been made toward identifying single nucleotide polymorphisms that convey drug resistance. in one study, an hiv drug resistance allele was detected by rpa combined with an oligonucleotide ligation assay [ ] . another study identified multidrug resistant tuberculosis sequence variants using a nested rpa approach [ ] . a recent study demonstrated a thin film transistor sensor for rpa that significantly accelerates readout time, using ph changes during dna amplification as an electrical signal [ ] . the molecular targets in that study are beta lactamases conferring resistance to cephalosporins and carbapenems, and detection was achieved within - min; however those data do not include tests for specificity of the assay nor measurement of antimicrobial resistance levels in the bacteria [ ] . nevertheless these results broadly support our finding that rpa is a superior approach to genomic antimicrobial resistance testing. innovative readout technologies hold promise to further improve temporal performance of these assays beyond the - min detection times we demonstrate, while also providing more portable systems for point-of-care or field uses. our work is timely, given recent focus on the reservoirs of antimicrobial resistance genes ('resistomes') within oral [ , ] and gut [ ] [ ] [ ] microbial communities. our rpa assay for mef(a) is highly sensitive (down to picogram levels), and this sensitivity may offer new diagnostic potential. however, the existence of antimicrobial resistance genes within commensal strains of the oral cavity even of healthy individuals [ , ] raises concerns that a highly sensitive antibiotic-resistance test like ours may detect the genes when no infection is present. however, understanding the dynamics and inter-individual variation even in a healthy resistome is an important part of personalized medicine, which (see figure on previous page.) fig. bacterial panel for rpa assay and validation of raw lysate method. a schematic of culture and bacterial lysate method. b mef(a) rpa results for s. pyogenes, s. agalactiae, and s. salivarius. c mef(a) rpa results for s. pneumoniae and e. faecium. for panels b and c, dna concentration in raw lysates was measured and total amount of dna loaded into each reaction is indicated, and lines are labeled with species name and whether they are known mef(a) positive (+) or negative (−) includes the microbiome [ ] [ ] [ ] [ ] and associated mediators of antimicrobial resistance [ ] . because the microbiome is a dynamic entity in which antimicrobial resistance genes are shared among members [ ] , it is clinically vital to monitor levels of antibiotic resistance genes in commensal bacteria of healthy individuals that may contribute to more severe disease. for example, infections caused by cystic fibrosis are increasingly antibiotic resistant due to the horizontal transfer of resistance genes from commensal bacteria [ ] . to date there is no cheap, easy, rapid assay to measure mef(a) in a patient's healthy microbiome, but we provide such a tool, validated to show the genetic signature correlates with actual erythromycin resistance. furthermore, having insight into the presence of resistance genes in the (healthy) microbiome of a patient would properly inform clinicians should that person become sick, reducing both morbidity and therapeutic failure and re-treatment. in other words, a patient with intrinsically high levels of mef(a) in her healthy microbiome would the question of whether our rpa assay would distinguish infection from colonization is related to a larger debate in the diagnostic field: when is a molecular assay too sensitive? molecular detection methods like qpcr or rpa are much more sensitive than culture methods, often identifying many more microbes than culture [ , ] , leading some to conclude that the diagnostic utility of these methods is limited due to false positives [ ] . however, there are several strategies for mitigating this risk: for example, testing only at-risk populations, as applied to testing for c. difficile or group-a streptococcus (s. pyogenes) [ ] . this strategy minimizes the chance of a false-positive detection by not employing the test in cases unlikely to represent true infection. thus, a clinician might deploy our new mef(a) assay when a patient exhibits symptoms consistent with bacterial infection, to guide choice of therapeutic agent. a second, and more powerful strategy is to focus on levels of the genetic sequence observed. if mef(a) is helping a pathogen cause disease, it will be enriched to a higher copy number than it would be as a sporadic colonizer diluted into a healthy microbial community [ , ] . by providing quantitative data on relative levels of mef(a), our rpa assay is ideally suited to this approach, making the determination of an infection a matter of comparing the detected gene level with a threshold (after normalizing to total bacterial load). critically, future work must focus on empirically setting the threshold by testing many clinical samples, from both healthy and sick patients [ ] . by providing a validated, easy-to-use rapid molecular assay, the present study represents a vital first step in this process. mef(a) has been found in a wide variety of bacterial hosts [ ] , from neisseria gonorrhoeae [ ] to enterococcus faecalis [ ] and streptococcus pneumoniae and pyogenes [ ] , and it has recently been found within commensal strains including streptoccous salivarius [ ] as we independently confirmed using rpa. we anticipate the mef(a) assay we validated in this work will become an important tool in the diagnostic toolbox, offering physicians and scientists alike a rapid, accurate measure of macrolide resistance, whether hosted in the upper (s. pyogenes [ ] or s. salivarius [ ] ) or lower respiratory tract (streptococcus pneumoniae [ ] or staphylococcus aureus [ ] or others), or in other regions of the human microbiome. national institutes of health: niaid's antibacterial resistance program: current status and future directions cdc: antibiotic / antimicrobial resistance world health organization global action plan on antimicrobial resistance antimicrobial resistance: is the world unprepared executive order --combating antibiotic-resistant bacteria national action plan for combating antibiotic resistant bacteria better tests, better care: improved diagnostics for infectious diseases will million people die a year due to antimicrobial resistance by current and emerging techniques for antibiotic susceptibility tests dna detection using recombination proteins detection of specific polymerase chain-reaction product by utilizing the ′--- ′ exonuclease activity of thermus-aquaticus dna-polymerase homogeneous escherichia-coli endonuclease-iv -characterization of an enzyme that recognizes oxidative damage in dna rapid identification of quarantine invasive solanum elaeagnifolium by real-time, isothermal recombinase polymerase amplification assay a field-deployable reverse transcription recombinase polymerase amplification assay for rapid detection of the chikungunya virus recombinase polymerase amplification combined with a lateral flow dipstick for rapid and visual detection of schistosoma japonicum development of recombinase polymerase amplification assays 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sensitive to clindamycin: a common resistance pattern mediated by an efflux system molecular cloning and functional analysis of a novel macrolideresistance determinant, mefa, from streptococcus pyogenes structure and distribution of an unusual chimeric genetic element encoding macrolide resistance in phylogenetically diverse clones of group a streptococcus prophage association of mef(a) elements encoding efflux-mediated erythromycin resistance in streptococcus pyogenes molecular detection of the macrolide efflux gene: to discriminate or not to discriminate between mef(a) and mef(e) resistance genes and genetic elements associated with antibiotic resistance in clinical and commensal isolates of streptococcus salivarius progress toward characterization of the group a streptococcus metagenome: complete genome sequence of a macrolide-resistant serotype m strain evaluation of real-time pcr for the detection and quantification of bacteria in chronic obstructive pulmonary disease genome sequence of a serotype m strain of group a streptococcus: potential new insights into puerperal sepsis and bacterial disease specificity erythromycin resistance by ribosome modification in vitro activity of the new ketolide telithromycin compared with those of macrolides against streptococcus pyogenes: influences of resistance mechanisms and methodological factors resistance to macrolides and related antibiotics in streptococcus pneumoniae rapid diagnostic tests for group a streptococcal pharyngitis: a meta-analysis rapid antigen group a streptococcus test to diagnose pharyngitis: a systematic review and meta-analysis loop-mediated isothermal amplification: rapid and sensitive detection of the antibiotic resistance gene isaba -bla(oxa- -like) in acinetobacter baumannii rapid detection of pseudomonas aeruginosa and acinetobacter baumannii harboring bla(vim- ), bla(imp- ) and bla(oxa- ) genes by using loop-mediated isothermal amplification methods isothermal assay targeting class integrase gene for environmental surveillance of antibiotic resistance markers a rapid loop-mediated isothermal amplification (lamp) method for detection of the macrolide-streptogramin type b resistance gene msra in staphylococcus aureus evaluation of a loop-mediated isothermal amplification-based assay for the rapid detection of plasmid-encoded colistin resistance gene mcr- in enterobacteriaceae isolates ultra-fast electronic detection of antimicrobial resistance genes using isothermal amplification and thin film transistor sensors metagenomic insights into transferable antibiotic resistance in oral bacteria antibiotic resistome in a large-scale healthy human gut microbiota deciphered by metagenomic and network analyses the human gut resistome functional characterization of the antibiotic resistance reservoir in the human microflora microbiome at the frontier of personalized medicine introducing the microbiome into precision medicine the microbiome in precision medicine: the way forward precision antimicrobial therapeutics: the path of least resistance? a multifaceted 'omics' approach for addressing the challenge of antimicrobial resistance antimicrobial resistance in the respiratory microbiota of people with cystic fibrosis real-time pcr in clinical microbiology: applications for routine laboratory testing strategies for optimizing the diagnostic predictive value of clostridium difficile molecular diagnostics more pathogenicity or just more pathogens?-on the interpretation problem of multiple pathogen detections with diagnostic multiplex assays staphylococcus aureus and the ecology of the nasal microbiome acquired macrolide resistance genes in pathogenic neisseria spp a variety of gram-positive bacteria carry mobile mef genes distribution of mef(a) in gram-positive bacteria from healthy portuguese children we acknowledge generous bacteria culture advice from dr. jeffrey kaplan who also shared the s. salivarius strain with us. we also acknowledge the insightful comments on the manuscript provided by kate smoot bracht. this work was supported by nsf grant and nih grant k ca to j.r.b. these agencies had no role in the design of the study or in the collection, analysis, and interpretation of data, or in the writing of the manuscript. all authors consent to publication of this work. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. availability of data and materials all strains described in this work are available upon request. the sequences in fig. b obtained in this study (and marked with asterisks) either are identical to the corresponding region (antisense at position , , to , , ) of the deposited reference genome, accession cp . , or in the case of s. salivarius, are identical with the exception of the three single-nucleotide polymorphisms as shown in the figure. the study was approved by american university irb on march , under protocol # irb- - , "rapid genomic detection of antimicrobial resistance." in addition to the ethics approval, written informed consent for research was obtained from the individual who donated the s. salivarius tested in this work. the bacterial sample was de-identified prior to being used in this study. key: cord- -w eitw authors: mobaraki, kazhal; ahmadzadeh, jamal title: current epidemiological status of middle east respiratory syndrome coronavirus in the world from . . to . . : a cross-sectional study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: w eitw background: middle east respiratory syndrome coronavirus (mers-cov) is considered to be responsible for a new viral epidemic and an emergent threat to global health security. this study describes the current epidemiological status of mers-cov in the world. methods: epidemiological analysis was performed on data derived from all mers-cov cases recorded in the disease outbreak news on who website between . . and . . . demographic and clinical information as well as potential contacts and probable risk factors for mortality were extracted based on laboratory-confirmed mers-cov cases. results: a total of mers-cov cases, including deaths ( . %), were recorded in the disease outbreak news on world health organization website over the study period. based on available details in this study, the case fatality rate in both genders was . % ( / ) [ . % ( / ) for males and . % ( / ) for females]. the disease occurrence was higher among men [ cases ( . %)] than women [ cases ( . %)]. variables such as comorbidities and exposure to mers-cov cases were significantly associated with mortality in people affected with mers-cov infections, and adjusted odds ratio estimates were . ( % ci: . , . ) and . ( % ci: . , . ), respectively. all age groups had an equal chance of mortality. conclusions: in today’s “global village”, there is probability of mers-cov epidemic at any time and in any place without prior notice. thus, health systems in all countries should implement better triage systems for potentially imported cases of mers-cov to prevent large epidemics. middle east respiratory syndrome coronavirus (mers-cov) infection is considered to cause a new viral epidemic [ ] , and was first reported in a patient who died from a severe respiratory illness in a hospital in jeddah, saudi arabia, in june [ , ] . from . . to . . , world health organization (who) has notified a total of laboratory-confirmed cases of mers-cov, including at least deaths related to this infection from countries around the world [ ] . the origin of mers-cov has been widely discussed. initially, a bat reservoir was posited based on phylogenetic similarity of certain bat coronaviruses with mers-cov. however, there has been no clear bat source of infection or a consistent history of contact with bats in known cases of mers-cov to date [ , ] . another source such as dromedary was later introduced as a possible reservoir in some studies [ ] [ ] [ ] [ ] . some studies have declared that all cases of mers-cov were directly or indirectly linked to residence or travel to countries: saudi arabia, uae, jordan, qatar, kuwait, oman, yemen, egypt, iran, and lebanon [ , ] . the mers-cov infection has high mortality rates, especially in patients with comorbidities such as diabetes and renal failure, evoking global concern and intensive discussion in the media along with respiratory droplet route of its transmission [ ] . laboratory-confirmed mers-cov cases have been reported during hospital-based cluster outbreaks between . . to . . , and cases are still detected throughout the year [ ] . the occurrence of a large number of mers-cov cases and their associated deaths in the world indicate that this disease must be considered as a severe threat to public health [ ] because millions of pilgrims from countries converge in saudi arabia each year to perform hajj and umrah ceremony. upon their return to home, pilgrims hold a ceremony attended by family members and friends. oriental etiquette to share hospitality with others increases the transmission of probable mers-cov cases to others [ , ] . worldwide awareness of mers-cov is low, the disease has high intensity and lethality with unknown mode of transmission and source of mers-cov infection (i.e. whether zoonotic or human disease) [ ] . therefore, it is necessary to design and implement a research to identify some unknown epidemiological aspects and also determine the current epidemiological situation of mers-cov and its mortality risk factors in order to prevent, control and anticipate effective interventions. permission was obtained from who to conduct this analytical-descriptive epidemiological study. using census method, data related to laboratory-confirmed mers-cov cases between . . to . . were extracted from disease outbreak news on mers-cov from who website as follows. demographic information such as age, gender, reporting country, city, health care worker; clinical data and exposure status of mers-cov cases including comorbidities, exposure to camels, camel milk consumption, exposure to mers-cov cases, day/month of symptom onset, day/month of first hospitalization, day/month of laboratory confirmation, final outcome (dead or survived) of mers-cov cases were recorded. all statistical analyses were conducted using spss, version (ibm inc., armonk, ny, usa). quantitative measurement was expressed by medians and qualitative variables were presented as absolute frequency and percentage. logistic regression was used to calculate the odds ratio (or) with a % confidence interval in order to assess the probable relationship between risk factors and final outcome (dead/survived) of laboratory-confirmed mers-cov cases. p values of less than . were regarded as statistically significant. a total of mers-cov cases, including deaths ( . %), were recorded in the disease outbreak news on who website from . the median age of subjects was . years (range: - years). to assess the effect of several potential risk factors on death in morbid cases related to mers-cov infection, we used or index in order to better understand the mechanism of this relationship, and we reported both crude and adjusted or. based on this indicator, variables such as comorbidities and exposure to mers-cov cases were significantly associated with mortality in affected people with mers-cov infections ( table ) . six countries were affected with mers during the period of this study. the majority of cases (approximately . %) with highest mortality ( . %) as well as % of female cases have been reported from saudi arabia ( table ). the epidemic curve of laboratory-confirmed cases of mers between . . and . . is shown in fig. . it can easily be seen that two peaks are evident in this period: the first at the beginning of april and the second at the beginning of july . our results indicate that the number of mers-cov cases remained constant from the beginning of september to the end of january . the findings have important implications for infection control practice. especially, we found evidence that was contrary to many studies declaring that the high mortality rates are related to mers infection with increasing age [ ] [ ] [ ] . our results on mers-cov cases in global level showed that all age groups are somewhat at risk of death from this infection. the chance of mortality in mers-cov cases in all age groups is fairly equal. therefore, in the care and treatment of mers-cov cases, our results suggest that this important point is better to be considered on behalf of health care staff. in this study, we observed a higher disease occurrence and death of (table ) . a possible explanation for a higher disease occurrence and mortality of mers-cov among men is that men are likely to spend more time outdoors and hence have a higher risk of exposure to a source of infection. the evidence linking mers-cov transmission between camels and humans cannot be ignored. several studies have shown that persons with direct and indirect contact with dromedary camels had a significantly higher risk of mers-cov infection. our finding was inconsistent with other studies that did not mention such evidence (table ) . random error may be one of the reasons for obtaining this result since there were not details of exposure to camels and camel milk consumption for laboratory-confirmed mers-cov cases. our research is consistent with many studies that provided evidence of human-to-human transmission for mers-cov infection [ , , ] . figure shows two peaks during june until september, which coincides with the largest mass gathering of muslims around the world in saudi arabia to perform hajj and umrah ceremony. this finding highlights the effect of congregation in the spread of mers-cov infection. our findings in table and fig. show that most cases are reported from saudi arabia after about years since the start of mers-cov pandemic (june to january , ). so, it seems necessary that epidemiologic investigations are conducted by ministry of health in saudi arabia and international partners to better understand the transmission patterns of mers-cov. this study had a number of limitations. assessment of the relationship between mortality related to mers-cov infection and some potential risk factor requires reliable sources of mortality data. we used the data recorded in the disease outbreak news on mers-cov from who website. the quality and accuracy of this data depend primarily on quality of the recorded data reported by national ihr focal point from different countries to who. in this study, the researcher was unable to verify the accuracy of the data, which potentially results in information bias. in addition, information for some of the variables was not available and the number of missing data was high, which might introduce a negligible selection bias in results. another limitation of this research was that possible misclassification of cases may occur due to the respondent's declarations such as exposure to camels, camel milk consumption, and exposure to mers-cov cases, which potentially occurs as a result of measurement bias. despite the above limitations, the current analytical-descriptive epidemiological study may have a number of implications for health care policy by using the global data. it also reminds us that effective national and international preparedness plans should be in place as well as measures to prevent, control and predict such viral outbreaks, improve patient management, and ensure global health security. the results of this analytical-descriptive epidemiological study revealed and confirmed some potential risk factors for mers-cov cases, which were reported as a possible risk factor in previous research studies. in fact, it reminds us that there is probability of mers-cov epidemic at any time and in any place without prior notice in today's "global village". the pattern of middle east respiratory syndrome coronavirus in saudi arabia: a descriptive epidemiological analysis of data from the saudi ministry of health comparative epidemiology of middle east respiratory syndrome coronavirus (mers-cov) in saudi arabia and south korea the clinical and virological features of the first imported case causing mers-cov outbreak in south korea middle east respiratory syndrome coronavirus isolation and characterization of viruses related to the sars coronavirus from animals in southern china middle east respiratory syndrome coronavirus: review of the current situation in the world middle east respiratory syndrome coronavirus (mers-cov) in dromedary camels isolation of mers coronavirus from a dromedary camel dromedary camels and the transmission of middle east respiratory syndrome coronavirus (mers-cov) zoonotic origin and transmission of middle east respiratory syndrome coronavirus in the uae world organization health middle east respiratory syndrome estimating the severity and subclinical burden of middle east respiratory syndrome coronavirus infection in the kingdom of saudi arabia hadj ritual and risk of a pandemic transmission scenarios for middle east respiratory syndrome coronavirus (mers-cov) and how to tell them apart clinical aspects and outcomes of patients with middle east respiratory syndrome coronavirus infection: a single-center experience in saudi arabia middle east respiratory syndrome novel corona (mers-cov) infection. epidemiology and outcome update epidemiological, demographic, and clinical characteristics of cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus (mers-cov): summary of current situation, literature update and risk assessment first cases of middle east respiratory syndrome coronavirus (mers-cov) infections in france, investigations and implications for the prevention of human-to-human transmission the authors take this opportunity to thank all the who personnel as well as reporting countries with confirmed mers cases for data collection and sending the data to who. we thank rana sidani senior communication officer in who regional office for the eastern mediterranean (cairo, egypt) for their guidance, help and permission to extract the data. this paper is dedicated to arsam ahmadzadeh and anil ahmadzadeh. as a epidemiological analysis on who data, this study did not need financial support. the dataset used and/or analysed during the current study are available from the corresponding author on reasonable request.authors' contributions km and ja designed the study and performed the search and data extraction. ja analyzed the data. km and ja wrote the manuscript. ja edited the draft. both authors read and approved the final manuscript.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -s oqkqef authors: xu, lili; gao, hengmiao; zeng, jiansheng; liu, jun; lu, cong; guan, xiaolei; qian, suyun; xie, zhengde title: a fatal case associated with respiratory syncytial virus infection in a young child date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: s oqkqef background: respiratory syncytial virus (rsv) is the most common viral cause of pediatric bronchiolitis and pneumonia worldwide. risk factors for high mortality and prolonged morbidity after rsv infection include premature birth, bronchopulmonary dysplasia, congenital heart disease, and down syndrome. however, some previously healthy, full-term children who are infected with rsv also require hospitalization and even experience severe sequelae or death. case presentation: in this report, we present the case of an rsv-associated death of a child who was born at full-term and developed normally up to the age of years old. cardiopulmonary arrest occurred within days after the onset of symptoms, which included cough and high fever. complete brain edema was prominent, and encephalopathy was developing. viral antigen detection and microbiome analyses of oral swab and nasopharyngeal aspirate specimens verified an rsv infection, while bacterial culture of blood specimens yielded negative results. the rsv strain detected in this patient was subtyped as rsvb , and no mutation was found in the six antigenic sites for targeted drugs or vaccines. conclusions: the patient had a severe infection associated with rsv, which was very likely the cause of her central nervous system infection and acute neurological complications. respiratory syncytial virus (rsv) is the major cause of lower respiratory tract illness in children. for most children, an initial rsv infection normally occurred within the first years of life. in infants less than year of age and with lower respiratory infection, up to % are due to rsv [ ] . in most cases, the virus is not fatal. the most severe infections and well-defined high-risk groups, including infants with a history of premature birth, and those with chronic lung disease, congenital heart disease, cystic fibrosis and immunodeficiency [ ] . for those high-risk infants, palivizumab, a humanized monoclonal antibody which has produced favorable results to date, is strongly recommended to be administered prophylactically [ ] . however, most children with rsv infection were previously healthy, and it is often difficult to predict deterioration of rsv infection [ , ] . rsv-related encephalitis with acute encephalopathic symptoms such as seizure, severe sequelae and even death following rsv infection in children without underlying disease has sporadically been reported [ ] . rsv-related encephalitis usually develops within to days after the onset of clinical symptoms, such as high fever, cough, and fatigue. however, the mechanism underlying the rapid progression of related encephalitis remains unclear. central nervous system (cns) infection, coinfection with bacteria, and dysfunction of the host immune system may be possible causes. next-generation sequencing (ngs) accompanied with metagenomic analysis can be used as a nonselective method for pathogen discovery and is increasingly applied as a diagnostic tool to investigate the causes underlying unexplained encephalitis in patients. two key features of this methodology are: ( ) it makes no assumptions about the type of pathogen and has the potential to detect nucleotides from all species, and ( ) the causative pathogen may not necessarily be the most abundant signal in the ngs results and may sometimes be present as only a low-level signal compared to all other signals associated with commensal pathogens. it can, however, provide unbiased and sensitive identification. in this report, we present the case of a -year-old girl who was not born prematurely and had no underlying disease whose sudden death may have been related to an rsv infection identified by conventional methods and metagenomic analyses. the patient in this fatal case was a -year-old girl who was born full-term and had developed normally. she had no medical history of asthma or pneumonia and no familial history of immunodeficiency. she had no brothers or sisters. the patient was admitted to the hospital due to days of fever and min of respiratory and cardiac arrest. the symptoms started on november ( days before admission), with a fever (up to . °c) but no chills, rash or convulsions. ibuprofen was given orally. the body temperature decreased for to h but then climbed to . °c. shortness of breath accompanied the fever, and the body temperature did not decrease obviously after the oral administration of ibuprofen. wheezing caused by the retention of phlegm in the throat and a single paroxysmal cough occurred. the patient occasionally coughed up a small amount of yellow phlegm and had a slightly runny nose. she had no asthma, breathing difficulty or hemoptysis; she had lethargy and a poor appetite but no vomiting or diarrhea. on november ( days before admission), the patient still had a high fever, and her body temperature fluctuated around approximately °c. the patient's body temperature did not obviously decrease after she was given oral ibuprofen and acetaminophen alternately. acute infection was considered from then on. the patient received an intravenous infusion of . g zithromax and mg rographolide as well as aerosol inhalation of mg budesonide, but her fever persisted, and her body temperature rose to a peak of °c. on november ( day before admission), the patient still had a persistent fever and wheezing due to the retention of phlegm in her throat. she had shortness of breath, a light cough, and a drooping spirit, accompanied by a rash on the torso and limbs. her appetite was slightly improved, and she had no vomiting or convulsions. on november, the patient had sudden respiratory and cardiac arrest h before admission. she was immediately and continuously treated with cardiopulmonary resuscitation by physicians and the intravenous injection of adrenaline ( times). she was treated with trachea cannula and mechanical ventilation, and her heart beat recovered approximately min later, but the patient remained in a deep comatose state with no spontaneous breathing. then, the patient was transferred to our hospital and immediately underwent electrocardiogram (ecg) monitoring. bloody fluid was visible in the indwelling gastrointestinal decompression tube, and the blood-gas analysis showed metabolic acidosis. the patient was treated with sodium bicarbonate to correct the acidosis. she was diagnosed with an acute cns infection and brain hernia. after cardiopulmonary resuscitation, she was admitted to the pediatric intensive care unit (picu). the head ct scan showed extensive brain swelling, decreased brain parenchymal density, narrowed cerebral ventricles and cisterns. these findings prompted a diagnosis of extensive brain edema and hernia. the chest posteroanterior radiograph showed fuzzy, coarse bilateral lung markings, visible small patchy shadows in the right inferior lung and a clear pulmonary hilus. these findings were diagnosed as pneumonia. routine blood examination results suggested the presence of a bacterial infection; thus, the patient was treated with vancomycin and meropenem to control an infection. after that, immunoglobulin ( g/kg) was administered for immune support. the patient was still in a deep coma state, and light reflexes of both pupils were absent. the patient's spontaneous breathing was weak and irregular, and she had no response to painful stimulation. compared with earlier, her rash was reduced, and the pulmonary lesions shown on the chest posteroanterior radiograph were slightly absorbed. immunoglobulin ( g/ kg) was continuously administered to neutralize pathogens. on november ( days after admission), transcranial doppler ultrasound assessment showed that the patient's anterior and posterior cerebral circulation corresponded to the diagnostic criteria for brain death. on november ( days after admission), various organ functions failed, and the patient could not tolerate a spontaneous breathing test. her guardian chose to quit treatment, and the patient died. viral antigen detection based on both an immunofluorescence assay and the luminex xtag respiratory viral panel assay was positive for rsv in the patient's nasopharyngeal aspirates (which were collected on nov, the th day of disease onset and the nd day of admission) and negative for adenovirus, influenza a and b viruses, parainfluenza virus - , human metapneumovirus, enteroviruses and rhinoviruses, human coronavirus hku , e, nl and oc , and human bocavirus. because the patient's guardian refused to consent to lumbar puncture, cerebrospinal fluid (csf) was not available for the detection of cns infection. the blood biochemistry results are summarized in table . the amounts of red blood cells (rbcs), hemoglobin, and platelets continuously decreased after the onset of symptoms. extremely high levels of the percentage of t lymphocytes was . %, of which helper t cells and suppressor t cells accounted for . and . %, respectively. the ratio of cd /cd was . . the proportions of b lymphocytes and nk cells were . and %, respectively. all these immunological indexes indicated dysfunction of the patient's immune system. oral swab, nasopharyngeal aspirate, and serum specimens collected on nov (the th day of disease onset and the nd day of admission) were subjected to multiplex metagenomic analyses using an ngs platform. the nucleic acid library was constructed as previously described [ ] . the amplified nucleic acid libraries were then analyzed using an illumina hiseq sequencer for a single read of bp. the raw sequence reads were filtered using previously described criteria [ ] to obtain valid sequences. when bacteriophages, plant-origin sequences resulting from food debris in the oral cavity, and contamination from the reagents used in the sample processing step (murine leukemia virus (mlv), for example) were excluded, only human rsv (based on the ncbi taxonomy database) was identified, with at least one specific sequence from the oral swab ( reads) and the nasopharyngeal aspirate ( reads). no virus-related sequences were detected in the serum specimen. meanwhile, large numbers of sequence reads related to bacteria, including streptococcus mitis, streptococcus parasanguinis, streptococcus pneumoniae, streptococcus salivarius, streptococcus infantis, streptococcus suis, neisseria meningitidis, neisseria gonorrhoeae, haemophilus sputorum, haemophilus parainfluenzae, enterococcus cecorum, and other conditioned pathogenic bacteria were also detected in the oral swab, nasopharyngeal aspirate, or/and serum specimens (fig. ) . however, although the metagenomic analysis showed sequence reads assigned to kingdom bacteria, the bacterial culture of the blood specimens yielded negative results. based on the random distribution of reads of the rsv virus genome, the complete length of the genome was obtained using ngs methods and gap amplification. this rsv strain was subtyped as rsvb; it was found to cluster in the ba genotype and had the signature -bp duplication in the g gene. the newly identified virus was named rsvb/bch-y/ , and the full genome sequence was deposited in genbank under accession number ky . the phylogenetic analysis was conducted with representative sequences from nearly all rsvb subgroups (ba - , gb - , sab - , uru - ) from genbank; rsvb/bch-y/ belonged to ba subgroup (fig. ) . the nucleotide homology comparisons revealed that the g gene of this strain was most closely related (share . % homology) to strain rsvb/gz/ - , which was isolated from a child in guangzhou, china, in . for the six most important antigenic sites (Ø, i, ii, iv, v, vi) in the fusion protein for drug or vaccine (such as palivizumab) targeting [ , ] , no mutation was found in rsvb/bch-y/ . rsv infection generally causes symptoms such as fever, cough and wheezing. while respiratory failure may occur in severe cases, the fatality rate is not high in clinical cases. the patient in this case study was infected with rsv, and the disease progressed rapidly, with extensive cerebral edema and hernia. there are prior reports of neurological complications of rsv infection, which mainly include central apnea, seizures, and encephalopathy [ ] . apnea is a common occurrence in rsv-infected patients younger than months of age and is a frequent indication for intubation [ , ] . the mechanism of rsv-related apnea is unknown, but a significantly reinforced laryngeal chemoreflex or an immature respiratory center in infancy has been proposed [ , ] . evidence of an encephalitic pathology caused by rsv infection is sparse. the presence of rsv in csf, detected using polymerase chain reaction, was reported in a single case of a -month-old boy with febrile seizure [ ] . the pathogenesis of rsvrelated encephalitis is not yet fully understood. however, it has been hypothesized that rsv may enter the cns through the hematogenous/blood-brain barrier route or through invasion, causing the release of several humoral neurotoxic cytokine mediators [ ] . the diseases that we considered in the course of treatment included the following: ( ) severe hand, foot and mouth disease (usually caused by enterovirus, type ): this disease has a sudden onset in most cases, and symptoms include fever, rashes and herpes appearing on the oral mucosa, hands, and feet. this disease progresses quickly in a small number of cases, especially in children younger than years old. at - days after onset, complications such as meningitis, encephalitis, encephalomyelitis, pulmonary edema and circulatory disorders occur. a very small number of infections worsen and lead to death. however, symptoms such as herpes and pulmonary edema were not observed in this patient, and both viral pathogen analyses showed negative results for enteroviruses, so this speculation was not supported. ( ) high pathogenic influenza virus infection: the typical symptoms include fever, cough, sore throat, headache, body pain and fatigue. some patients progress quickly to severe pneumonia, acute respiratory distress syndrome (ards), shock and acute necrotic brain injury. however, this child underwent repeated influenza a virus antigen detection, and all results were negative; thus, this speculation was not supported. ( ) fulminant myocarditis: the initial symptoms primarily include fever, cough, fatigue and vomiting. the disease develops rapidly and can lead to sudden congestive heart failure, cardiogenic shock, and adam-strokes syndrome. ecg sometimes demonstrates st-t changes, myocardial infarction, and arrhythmia. the cardiac ultrasound may show left ventricular enlargement. however, no abnormalities were observed in the ecg or heart ultrasound analyses of our patient; thus, this speculation was not supported. because the patient's guardian refused to consent to lumbar puncture, a csf specimen was not available for further validation. we could not be sure whether the neuroinvasion of rsv did occur in this case because rsv has only rarely been identified in cns specimens [ ] . this finding is somewhat similar to that of influenza encephalitis. britton et al. have described cases of influenza-associated neurological disease without testing for influenza viruses in csf specimens [ ] . whether the influenza virus invades the cns or not is still controversial. fujimoto et al. reported that influenza virus rna was detected in the csf of . % ( / ) of patients who developed influenza-associated acute encephalopathy/encephalitis [ ] . however, in other reports, only a small number of patients were positive for viral rna in the csf and brain, and there was a lack of inflammation in the brain tissue of fatal cases [ ] [ ] [ ] [ ] . in the reported cases, no influenza rna was detectable in the csf. however, this finding does not exclude influenza as a cause of the encephalitis because the viral rna may have been cleared by the time the csf was taken. the fact that the rna is no longer detectable does not fig. heatmap based on the read numbers of pathogens derived from the oral swab, nasopharyngeal aspirate, and serum specimens. the clinical specimens are listed in the bottom row, and the pathogen names are presented in the left column. the boxes colored from blue to red represent the metagenomic sequencing reads observed (the reads varied between and ) exclude an earlier cns insult by the virus causing the current symptoms. for the case reported here, although direct evidence of rsv infection in the cns was not available, the clinical symptoms together with the laboratory findings and metagenomic analysis results suggested that the patient may have had severe sepsis that potentially resulting from an rsv infection with a high probability of cns infection and acute neurological complications. combining ngs with metagenomic analysis provides an important clinical tool to identify unexpected or novel pathogens in patients. the advantage of this methodology is maximized when the causative pathogen presents a low-level signal compared to all other signals representing environmental and commensal pathogens. improving the optimization and implementation of protocols suitable for clinical samples will no doubt improve microbial diagnosis in clinical practice. our findings, in conjunction with previously reported cases emphasize the need for more awareness of the neurological complications of rsv infection. its clinical manifestations may include seizures, encephalopathy, and focal neurological findings. early recognition of neurological complications of rsv infection is important for initiating effective treatment to reduce mortality and long-term morbidity. the datasets supporting the conclusions of this article are included within the article. authors' contributions llx performed the lab analysis and drafted the manuscript. hmg, jsz, jl and cl collected clinical data and monitored the patient. xlg performed the pathogen detection. zdx and syq participated in the study design and coordinated the drafting of the manuscript. all the authors read and approved the final manuscript. fig. phylogenetic analysis based on the complete g gene sequence of the rsv detected in this patient and other representative sequences from nearly all rsvb subgroups (ba - , gb - , sab - , uru - ) from genbank. the nucleotide phylogenetic tree was constructed using the neighbor-joining method with nucleotide p distances and bootstrap replicates in the molecular evolutionary genetics analysis program (mega, version . , usa) rsv infections: state of the art the burden of respiratory syncytial virus infection in young children updated guidance for palivizumab prophylaxis among infants and young children at increased risk of hospitalization for respiratory syncytial virus infection predicting deterioration in previously healthy infants hospitalized with respiratory syncytial virus infection examination of neurological prognostic markers in patients with respiratory syncytial virus-associated encephalopathy metagenomic analysis of viral genetic diversity in respiratory samples from children with severe acute respiratory infection in china deciphering the bat virome catalog to better understand the ecological diversity of bat viruses and the bat origin of emerging infectious diseases structure of rsv fusion glycoprotein trimer bound to a prefusion-specific neutralizing antibody neutralizing epitopes on the respiratory syncytial virus fusion glycoprotein extrapulmonary manifestations of severe respiratory syncytial virus infection-a systematic review risk factors for respiratory syncytial virus associated apnoea neurological complications of respiratory syncytial virus infection: case series and review of literature reflex apnoea response and inflammatory mediators in infants with respiratory tract infection detection of subgroup b respiratory syncytial virus in the cerebrospinal fluid of a patient with respiratory syncytial virus pneumonia respiratory syncytial virus-related encephalitis: magnetic resonance imaging findings with diffusion-weighted study the spectrum and burden of influenza-associated neurological disease in children: combined encephalitis and influenza sentinel site surveillance from australia pcr on cerebrospinal fluid to show influenzaassociated acute encephalopathy or encephalitis influenza-associated acute encephalopathy in japanese children in - neurologic complications associated with novel influenza a (h n ) virus infection in children encephalitis and encephalopathy associated with an influenza epidemic in japan detection of influenza virus rna by reverse transcription-pcr and proinflammatory cytokines in influenza-virus-associated encephalopathy this study was approved by the ethical review committee of beijing children's hospital. written informed consent for the further analysis that followed treatment of the patient and the sequencing that followed the routine care was obtained on the patient's behalf from her parents. written informed consent provided by the patient's parents was obtained on the patient's behalf for the publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor-in-chief of this journal. the authors declare that they have no competing interests. key: cord- - qd e r authors: zhao, xin-ying; xu, xuan-xuan; yin, hai-sen; hu, qin-ming; xiong, tao; tang, yuan-yan; yang, ai-ying; yu, bao-ping; huang, zhi-ping title: clinical characteristics of patients with coronavirus disease in a non-wuhan area of hubei province, china: a retrospective study date: - - journal: bmc infect dis doi: . /s - - -w sha: doc_id: cord_uid: qd e r background: since december , the coronavirus disease (covid- ) has expanded to cause a worldwide outbreak that more than , people infected and tens of thousands died. to date, the clinical characteristics of covid- patients in the non-wuhan areas of hubei province in china have not been described. methods: we retrospectively analyzed the clinical characteristics and treatment progress of patients diagnosed with covid- in jingzhou central hospital. results: of the patients diagnosed with covid- , cases ( . %) were severe and two patients ( . %) died. the severe disease group tended to be older ( . vs. . years; p = . ) and have more chronic disease ( % vs. . %; p = . ) relative to mild disease group. only . % of the patients were quantitative polymerase chain reaction (qpcr)-positive on their first tests, while typical chest computed tomography images were obtained for each patient. the most common complaints were cough (n = ; . %), fever (n = ; . %), fatigue (n = ; . %), and diarrhea (n = ; . %). non-respiratory injury was identified by elevated levels of aspartate aminotransferase (n = ; . %), creatinine (n = ; . %), and creatine kinase (n = ; . %) in laboratory tests. twenty-eight cases ( . %) suffered non-respiratory injury, including % of the critically ill patients and . % of the mild patients. conclusions: overall, the mortality rate of patients in jingzhou was lower than that of wuhan. importantly, we found liver, kidney, digestive tract, and heart injuries in covid- cases besides respiratory problems. combining chest computed tomography images with the qpcr analysis of throat swab samples can improve the accuracy of covid- diagnosis. since december , several cases of severe acute respiratory syndrome coronavirus (sars-cov- ) infection were first reported the virus has caused an outbreak in a short time by human-to-human transmission throughout china, especially in hubei province. the severe contagiousness and rapid disease progression of the coronavirus disease (covid- ) have drawn significant global public health attention. as of march , , more than , confirmed cases were reported worldwide, of which hubei province is the most affected area with greater than , cases and thousands of deaths confirmed from covid- . like severe acute respiratory syndrome (sars) [ ] and middle east respiratory syndrome (mers) [ ] , covid- not only causes infections in the respiratory tract but also in the digestive tract, liver, and heart [ ] [ ] [ ] . a considerable proportion of covid- patients develop severe pneumonia, pulmonary edema, acute respiratory distress syndrome, and even multiple organ failure within a short time. the mortality rate of patients in wuhan was as high as . % at the time of writing this report [ ] , but may be slightly lower in other areas. the clinical characteristics of covid- patients in non-wuhan areas of hubei province have not previously been described. in this study, we conducted a comprehensive exploration of the epidemiology and clinical features of patients with confirmed covid- admitted to jingzhou central hospital in jingzhou, one of the most severely affected cities in hubei province. we retrospectively analyzed patients diagnosed with covid- hospitalized from january , to february , . patients suspected of having covid- were admitted and quarantined, and throat swab samples were collected and tested for severe acute respiratory syndrome coronavirus (sars-cov- ) by quantitative polymerase chain reaction assay (qpcr). patients diagnosed with covid- were enrolled in this study and asked to sign a written informed consent form during hospitalization. this study was approved by the ethics committee of jingzhou central hospital. the patients have not been reported in any other submission by anyone else. the final date of follow-up was february , . clinical data [age, previous chronic disease, epidemiological history, symptoms, vital signs, computed tomography (ct) images, virus load, laboratory tests, complications, and treatment process] of the patients involved in this study were collected. acute respiratory distress syndrome was defined according to the berlin definition [ ] . liver injury was judged by alanine aminotransferase (alt) and aspartate aminotransferase (ast) levels. acute kidney injury was identified according to elevated creatinine (cr) and uric acid levels. the presence of cardiac injury was confirmed if the serum levels of cardiac biomarkers [cardiac troponin i (ctni), creatine kinase (ck), creatine kinase isoenzyme (ck-mb)] were elevated. the diagnosis of covid- was made by the comprehensive evaluation of epidemiological exposure, symptoms, laboratory tests, chest ct scan, and qpcr analysis. qpcr assay for sars-cov- rna for further tests was extracted from the throat swab samples, which were collected in virus preservation solution. next, μl of rna was added in a pcr reaction tube with μl of nucleic acid amplification reaction solution, μl of enzyme mixture, and μl of orf ab/n reaction solution (biogerm, shanghai, china). the cycle parameters for pcr amplification assay were set as follows: reverse transcription at °c for min; predenaturation at °c for min; cycles of denaturation at °c for s; and annealing, extending, and collection of fluorescence at °c for s. the open reading frame ab (orf ab) and nucleocapsid protein (n) gene regions of sars-cov- were simultaneously tested. primers for orf ab were as follows: forward primer ccctgtgggttttacact-taa, reverse primer acgattgtgcatcagctga, and the probe ′-vic-ccgtctgcggtatgtggaaagg ttatgg-bhq - ′. primers for n were as follows: forward primer ggggaacttctcctgctagaat, reverse primer cagacattttgctctcaagctg, and the probe ′-fam-ttgctgctgcttgacagatt-tamr a- ′. a cycle threshold value (ct value) of less than suggested a positive result, while a ct value of higher than indicated a negative result. and a ct value between and required retesting. the mann-whitney u test was used to compare continuous variables, while the chi-square test was adopted to compare categorical variables. the statistics were prepared using excel (microsoft corp., redmond, wa, usa) and graphpad prism software (graphpad software, la jolla, ca, usa), and analyzed using spss (ibm corp., armonk, ny, usa). a p-value of less than . was considered to be statistically significant. we collected clinical information of patients diagnosed with covid- in jingzhou central hospital. of these patients, ( . %) were assessed as being severely ill ( table ). the median age of our study population was . years, with severe patients being generally older with a median age of . years relative to that of . years among the mild cases (p = . ). slightly more than half ( . %) of the patients were male and there were no significant differences in the sex ratio between the severe and mild cases (p = . ). twenty-one patients ( . %) had one or more coexisting medical conditions, including hypertension, diabetes, chronic obstructive pulmonary disease, kidney disease, and malignancies. coexisting medical conditions were more prominent in the severe disease group ( % had chronic disease vs. . % in mild disease group; p = . ). the median duration from onset of symptoms to hospital admission was days. all patients were confirmed as qpcr-positive eventually, while only . % of patients were qpcr-positive when they first received the test. the qpcr-positive rate at different times was not different in the severe and mild disease groups (p = . ). in addition, the vital signs (pulse rate, temperature, and mean arterial pressure) between the two groups showed no significant differences. compared to the severe disease group ( . %), the mild disease group ( . %) had a higher proportion of discharged cases. of the two deceased patients who were critically ill, one had lung cancer and the other had hypertension. both suffered respiratory and acute renal failure on the day of admission. the mortality rate was . %. taken together, our results supported that older patients with chronic disease were more likely to become critically ill and negative qpcr results could not confidently exclude infection with the virus at symptom onset. at data cut off, considering evaluable patients, the most common symptoms were fever (n = ; . %) and cough (n = ; . %), followed by fatigue (n = ; . %), chest distress (n = ; . %), chill (n = ; . %), pharyngalgia (n = ; . %), and myalgia (n = ; . %) (fig. ) . additionally, some patients reported gastrointestinal problems, including diarrhea (n = ; . %) and nausea (n = ; . %) (fig. ) . symptoms of ). anorexia, arthrodynia, dizziness, and abdominal pain were also found ( fig. ). to explore the characteristics of laboratory tests from our patients with covid- , the baseline hematological and biochemical indices of the patients were analyzed ( table ). on admission, . % of patients had lymphopenia, which was more prominent in mild cases ( table ) . elevated ck levels were observed in . % of patients, while elevated alt and ast levels were recorded in . and . % of patients. severe cases had more prominently elevated cr ( . % vs. %; p < . ) and ck ( . % vs. . %; p = . ) levels. the prothrombin time was prolonged in . % of patients. levels of interleukin- and c-reactive protein, two biomarkers of inflammation, were increased in . and . % of patients (table ). these laboratory findings indicated that covid- resulted in liver, kidney, and cardiovascular injury. interestingly, separate from damage to the respiratory system, covid- patients showed signs of multiple organ injury on admission, including cases ( . %) of liver injury; cases ( . %) of cardiovascular damage with abnormal increases in troponin, ck, or ck-mb levels; five cases ( . %) of acute renal injury; and cases ( . %) of poor coagulation function (table ) . together, patients ( . %) suffered non-respiratory system injury, with an especially higher rate ( % vs. . %; p = . ) in the severe disease group (table ). further analysis revealed that severe patients tended to suffer damage to the cardiovascular system ( . % vs. . %; p = . ) and renal function ( . % vs. %; p = . ) ( table ) . angiotensin-converting enzyme ii (ace ) was proved to be the cell receptor of covid- [ ] , the same as sars infection [ ] . we performed bioinformatics analysis on the expression of ace receptors in different normal tissues sourced from the cancer microarray database oncomine, as shown in additional file . the data indicated that the highest level of ace was present in the ileum, followed by in the testis, diaphragm, heart, kidneys, seminal vesicle, colon, and respiratory tract. hence, we speculated that the high ace expression levels in the ileum, heart, kidneys, and colon caused the virus to pursue direct infection in these specific organs, which might explain the high rate of multiple organ damage caused by covid- . all of the patients in our study presented exudative changes, with different degrees of patchy consolidation or ground-glass opacities, on ct images (fig. a-c) . the processing of ct images (fig. a) revealed that the chest presented only scattered dotted shadows on the first day of admission, focal exudation on the fourth day, and diffuse ground-glass shadows on the th day. a good example of a case gradually improved with effective therapy is presented in fig. b . in addition, there were patients whose condition progressed rapidly within week (fig. c) . on the first day of admission, the chest ct scan was basically normal. however, on the seventh day, the chest ct scan showed patchy high-density shadow and diffused ground glass density shadow in both lungs. soon after, this patient died of multiple organ failure (fig. c) . these results suggest that ct imaging is an important method in differential diagnosis and evaluating the severity of covid- . most patients received antiviral therapy (n = ; . %), glucocorticoid therapy (n = ; . %), and antibacterial (table ). oseltamivir, lopinavir/ ritonavir, and umifenovir were common antiviral drugs used in our treatment protocols. in our study, . % of patients were treated with oseltamivir antiviral therapy (table ). lopinavir/ritonavir was more likely to be used in the mild disease group ( . % vs. . %; p = . ), while umifenovir was more likely to be used in the severe disease group ( . % vs. . %; p = . ) ( table ) . supportive treatment measures, including oxygen therapy (n = ; . %), mechanical ventilation (n = ; . %), the infusion of immunoglobulin (n = ; . %), and continuous renal replacement therapy (n = ; . %), were more likely to be applied to patients in the severe disease group (p < . ) ( table ). according to the data reported, the mortality rate in wuhan ( . %) [ ] is indeed higher than in other areas. as jingzhou ranks among the top three cities that have the most immigrant population from wuhan but does not confront the same challenges in wuhan, we contend that the cases described in this paper are more representative of the course of covid- . there are two main reasons accounting for the higher mortality rate reported in wuhan. although all covid- patients are treated in public hospitals and all expenses are borne by the government, patients in wuhan could not obtain prompt and adequate treatment as a result of the area hospitals being overloaded with large numbers of patients in a short time. further, we found that patients in the jingzhou central hospital were often younger, with a median age of . years relative to that of . years in wuhan. also, there were fewer patients with coexisting chronic diseases in this study, which assisted in lowering the mortality rate [ ] . not all of our patients were qpcr-positive after throat swab sampling during their first test. it took three times to obtain a positive qpcr result for . % of the patients in our study. false negatives often exist during qpcr testing. all patients presented typical ct imaging changes during the study, thus we could establish a clinical diagnosis decision using ct before positive qpcr results were obtained. hence, ct imaging is a favorable means for diagnosing covid- as well as evaluating the severity of the disease. in sum, the confirmation of covid- should be based on the symptoms and laboratory examinations of our patients, we found that, in addition to the respiratory tract, the digestive tract, liver, renal function, and cardiovascular system were affected. the mechanism of multiple organ damage in the context of covid- infection is currently unclear. the virus enters into the host cells by the recognition of spike glycoproteins. accumulated evidence has shown that ace is the cell receptor of choice for sars-cov- , same as in the sars-cov infection, which means that the virus infects cells expressing ace [ ] [ ] [ ] [ ] . it was also reported that anti-ace therapy blocked coronavirus replication during in vitro experiments [ ] . it is even proposed that angiotensin receptor blockers might be a treatment option for sars [ , ] , but there remains a lack of practice basis in this regard at present. ace was initially thought to be expressed only in the heart, kidneys, and testis, but has now been found to be widely expressed in the lungs, brain, and digestive tract [ ] [ ] [ ] . these results, together with the bioinformatics analysis in our study, might explain why the covid- caused multiple organ damage. other possible reasons, including hypoxia caused by respiratory failure and the immune response caused by virus, might also account for the multiple organ damage. due to the lack of effective antiviral drugs, some patients got worse and developed respiratory failure in seven to days. almost all the patients in this study received antibacterial agents, . % received antiviral therapy and . % received glucocorticoid therapy. oseltamivir is used to treat the influenza virus by inhibiting neuraminidase. the use rate of oseltamivir varies across different studies from . % in the study of zhong et al. [ ] to . % in that by wang et al. [ ] . in our study, . % of patients were treated with oseltamivir. at the beginning of the disease course, it can be difficult to distinguish the symptoms of patients with covid- from those with influenza. further, some patients tested positive for influenza virus antibodies, so oseltamivir antiviral therapy was used. in most cases, this drug was used in combination with other antiviral (lopinavir/ritonavir and umifenovir) and antibacterial agents. so far, there are no specific antiviral agents available to treat sars-cov- , sars, or mers. more prospective studies on specific antiviral therapy might help overcome this challenge. although the use of glucocorticoids in virus pneumonia is still very controversial, these medications are widely used in clinical practice. the . % frequency rate for use in our study is higher than in other reports [ ] [ ] [ ] . it is yet to be confirmed that the lower mortality in our study is correlated with the higher glucocorticoid utility ratio. chen et al. retrospectively analyzed patients, including critically ill patients, showing that glucocorticoids were effective in controlling the inflammatory response caused by sars [ ] . however, the multivariate analysis of another retrospective analysis suggested that corticosteroid therapy was significantly associated with a . times higher intensive care unit occupancy rate among patients with sars relative to sars patients who did not receive corticosteroids [ ] . thus, further prospective investigations are required to explore the benefits and side effects of glucocorticoid treatments in patients with viral pneumonia. multiple organ infection and the pathogenesis of sars efficacy of an automated multiple emitter whole-room ultraviolet-c disinfection system against coronaviruses mhv and mers-cov epidemiologic and clinical characteristics of novel coronavirus infections involving patients outside wuhan, china epidemiological and clinical characteristics of cases of novel coronavirus 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china treatment of severe acute respiratory syndrome with glucosteroids: the guangzhou experience the use of corticosteroid as treatment in sars was associated with adverse outcomes: a retrospective cohort study publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the medical staff in the department of infectious disease for their support in providing information about patients. we also thank the patients for their willingness to participate. finally, we thank letpub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. our single-center study of cases of confirmed covid- sampled from a distinct but representative location (jingzhou) other than wuhan feeds into the ongoing efforts to understand covid- , which should benefit the diagnosis, therapy, and control of the spread of the disease. further prospective investigations of effective antiviral therapies and sars-cov- vaccines may help remove this challenge. supplementary information accompanies this paper at https://doi.org/ . /s - - -w.additional file figure s .. ace distribution in different normal tissues from oncomine (nucleotide acc no.:aa ). authors' contributions zph and bpy conceived and designed the research. xyz and hsy analyzed data and wrote the paper. xxx analyzed data and modified the paper. qmh, tx, yyt, and ayy collected patient samples. all authors read and approved the final manuscript. not applicable. the datasets generated and/or analyzed during the current study are not publicly available due individual privacy of patients could be compromised, but are available from the corresponding author on reasonable request. this study has been approved by the ethics committee of jingzhou central hospital. written informed consents were signed during hospitalization. the data used in this study was anonymised before its use. not applicable. the authors declare that they have no competing interests to disclose. key: cord- -don qjpz authors: turner, paul; turner, claudia; watthanaworawit, wanitda; carrara, verena; cicelia, naw; deglise, carole; phares, christina; ortega, luis; nosten, francois title: respiratory virus surveillance in hospitalised pneumonia patients on the thailand-myanmar border date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: don qjpz background: pneumonia is a significant cause of morbidity and mortality in the developing world. viruses contribute significantly to pneumonia burden, although data for low-income and tropical countries are scarce. the aim of this laboratory-enhanced, hospital-based surveillance was to characterise the epidemiology of respiratory virus infections among refugees living on the thailand-myanmar border. methods: maela camp provides shelter for ~ , refugees. inside the camp, a humanitarian organisation provides free hospital care in a -bed inpatient department (ipd). between st april and th september , all patients admitted to the ipd with a clinical diagnosis of pneumonia were invited to participate. clinical symptoms and signs were recorded and a nasopharyngeal aspirate (npa) collected. npas were tested for adenoviruses, human metapneumovirus (hmpv), influenza a & b, and rsv by pcr. results: seven hundred eight patient episodes ( patients) diagnosed as pneumonia during the enhanced surveillance period were included in this analysis. the median patient age was year (range: < - ), and . % were aged < years. at least one virus was detected in . % ( / ) of episodes. virus detection was more common in children aged < years old (< year: or . , % ci . - . , p = . ; - years: or . , % ci . - . , p = . ). rsv was detected in / ( . %); an adenovirus in / ( . %); an influenza virus in / ( . %); and hmpv in / ( . %). twenty-eight episodes of multiple viral infections were identified, most commonly adenovirus plus another virus. rsv was more likely to be detected in children < years (or . , % ci . - . , p = . ) and influenza viruses in patients ≥ years (or . , % ci . - . , p = . ). ipd treatment was documented in / cases; all but one patient received antimicrobials, most commonly a beta-lactam (amoxicillin/ampicillin +/−gentamicin in / , . %). conclusions: viral nucleic acid was identified in the nasopharynx in half the patients admitted with clinically diagnosed pneumonia. development of immunisations targeting common respiratory viruses is likely to reduce the incidence of pneumonia in children living refugee camps and similar settings. pneumonia remains a leading cause of mortality globally: . million pneumonia deaths were recorded in [ ] . the highest incidence of disease occurs in young children [ , ] . an estimated million pneumonia episodes occur annually in children less than five years old. over % of these occur in the developing world, where the incidence of clinical pneumonia is estimated to be . episodes per child-year. almost three-quarters of childhood pneumonia deaths occur in sub-saharan africa and southeast asia [ ] . bacterial pathogens, most notably streptococcus pneumoniae and haemophilus influenzae type b, are important vaccinepreventable causes of pneumonia [ ] . viruses, in particular influenza and respiratory syncytial virus (rsv), are also responsible for a large number of pneumonia cases each year [ ] . using global population data for , for children under the age of five years, it was estimated that rsv was responsible for over million episodes of lower respiratory tract infections (lrti), with~ million of these requiring hospital admission, and , - , deaths [ ] . by similar analyses of data from , influenza viruses were estimated to cause million lrti and million severe lrti, with , - , deaths, in children aged less than five years [ ] . in both of these reviews, % of deaths from either influenza-or rsvassociated lrti occurred in the developing world. these viruses may be responsible for up to % of lrti (rsv % [ ] and influenza % [ ] ) in children under the age of five years. other viral pathogens associated with childhood pneumonia include adenoviruses, human metapneumovirus (hmpv), and parainfluenza viruses [ , ] . approximately one-third of the worldwide refugee population of million live in camps [ ] . these camps are often crowded with poor sanitation, providing ideal conditions for transmission of respiratory pathogens [ , ] . there have been refugees from myanmar (burma) living in camps in thailand since . in , lower respiratory tract infections were estimated to be the cause of % of deaths, and were responsible for % of all reported morbidity, in the under- age group of the border refugee population. the overall under- year mortality rate was per , , giving an estimated lrtispecific mortality rate of . per , [ ] . in , the us centers for disease control and prevention (cdc) and the shoklo malaria research unit (smru) established a respiratory virus surveillance programme in the burmese refugee population living in maela camp, northwest thailand. the programme included patients admitted to hospital with pneumonia during april -september . the aim of in-patient surveillance was to determine the relative burden of virusassociated pneumonia. the results of months of in-patient surveillance are presented here. maela camp is located in rural tak province approximately km from bangkok. it is the largest of the nine camps on the thailand-myanmar border, housing approximately , people in a km area, and has been in continuous operation since . karen is the predominant ethnicity in the camp population. general healthcare is provided by the non-governmental organisation première urgence-aide médicale internationale (pu-ami). camp residents receive world health organisation (who) expanded programme on immunisation (epi) immunisations, but immunisations against respiratory pathogens (haemophilus influenzae type b, influenza viruses, and streptococcus pneumoniae) are not available. from april to september , laboratory-enhanced respiratory surveillance was undertaken at the in-patient department (ipd) of the maela pu-ami hospital. throughout this period, trained local health workers reviewed ipd admission logs on six days each week to identify patients with an admission diagnosis of pneumonia, including those who were admitted on the seventh day. health workers invited all pneumonia patients to participate in enhanced surveillance and enrolled all who agreed. for enrolled patients, health workers completed a brief symptoms questionnaire by patient interview and record review, and collected nasopharyngeal aspirates (npa) as previously described [ ] . patient episodes were subsequently excluded from analyses if they (a) failed to meet the surveillance case definition for pneumonia (table ) , (b) occurred within days of previous episode in the same patient, or (c) lacked adequate laboratory specimens. surveillance case definitions were based on those devised by who for children under five years of age [ , ] . for older individuals, in whom a satisfactory clinical case definition is lacking, the surveillance case definition was based on that described by the british thoracic society [ ] . npa specimens, in ml viral transport medium (vtm, prepared in-house), were transported daily to the smru microbiology laboratory, which is located in the town of mae sot, approximately km from maela. specimens were placed into an insulated cool box immediately after collection and were transported back to the mae sot laboratory within eight hours of collection, where they were stored at− °c until analysis. viral nucleic acid was extracted from thawed npa-vtm specimens using commercial kits, following the manufacturer's instructions (qiaamp viral rna minikit [qiagen, hilden, germany], april until september ; viral nucleic acid extraction protocol of the magcore hf automated extractor [rbc bioscience, taiwan], october until september ). extracts were analysed by real-time reverse-transcription pcr (rrt-pcr) for adenoviruses, hmpv, influenza viruses (a and b, with typing of influenza a viruses to detect seasonal h /h and pandemic h strains), and rsv as described elsewhere [ , , ]. an internal control human rnasep pcr was included to confirm the specimen adequacy and to identify pcr inhibition [ ] . specimens were considered positive if a virus pcr ct value was < with appropriate run control results. to ensure the reproducibility of results approaching the assay limits of detection (approximately copies per reaction for each target; smru internal qc data), specimens with low positive pcr results (ct values of [ ] [ ] [ ] [ ] [ ] were repeated and only if the ct was < in both runs was the virus pcr considered positive. clinical and laboratory data were recorded on paperbased case record forms and subsequently entered into an access database (microsoft, redmond, wa, usa) and systematically checked for errors by comparison with the original case record forms. data were analysed by stata/ic version . (statacorp, college station, tx, usa). proportions were analysed by chi-squared or fisher's exact tests as appropriate. logistic regression was used to calculate odds ratios (or) and their % confidence intervals (ci). multivariate models were constructed to determine relationships between age, viral detection, pneumonia severity (< years old only), and antimicrobial use prior to admission. two-tailed p-values of < . were considered significant. this surveillance program underwent ethical and regulatory review at cdc, and was determined not to meet the definition of research. local ethical review in maela was not possible at the commencement of surveillance. however, the surveillance activity was discussed with pu-ami staff, and all concerns were addressed, prior to the beginning of the project. verbal consent was obtained from each potential participant, or their parent/legal guardian in the case of children aged < years, prior to enrolment in the surveillance programme. among all ipd pneumonia admissions, patient episodes were enrolled in enhanced surveillance. after review of the symptom questionnaire, patient episodes were excluded because of failure to meet the case definition and one episode was excluded for a patient who presented twice within days. in three episodes, npa specimens were not collected and in another six, the specimens were technically inadequate (internal control pcr negative). the remaining patient episodes were included in the following analyses. a total of individuals were sampled ( patients with single episodes, eight patients with two episodes, and one with three episodes). the median age at presentation was one year (iqr < - ; range < to ). six hundred and forty patients ( . %) were aged < years (table ). there were significantly more males in the < year age group compared with the older patients ( . % vs. . %, p = . ). patients presented at a median of days after symptom onset (iqr - ). eighty five percent of pneumonia episodes in the patients aged < years were classified as severe or very severe; this did not vary by gender or the duration of illness prior to admission (data not shown). children aged - years were less likely to have severe or very severe pneumonia compared with those aged < year (or . , % ci . - . , p = . ). treatment was documented for / ( . %) episodes: all but one patient received an antimicrobial drug, most commonly amoxicillin or ampicillin +/−gentamicin ( / , . %). viral nucleic acid was detected in . % ( / ) npa specimens. the rank order of detection was rsv ( , . % of npa), adenovirus ( , . %), influenza a ( , . %), hmpv ( , . %), and influenza b ( , . %). detection of viruses varied considerably by age (table ). patients aged < years were more likely to have viral adenoviruses were also more likely to be detected in those aged one year or more ( - years old: or . , % ci . - . , p = . ; ≥ years old: or . , % ci . - . , p = . ) (figure ). diagnosis of severe or very severe pneumonia in the < year age group was associated with detection of rsv (or . , % ci . - . , p = . ). the trend remained the same, although the association became statistically non-significant, when controlling for age and detection of other viruses in a multivariate logistic regression model (aor . , % ci . - . , p = . ). in the same multivariate model, detection of adenovirus (aor . , % ci . - . , p = . ) or an influenza virus (aor . , % ci . - . , p = . ) were associated with a lower likelihood of severe or very severe pneumonia diagnosis. multiple viruses were detected in . % ( / ) specimens. two viruses were detected in specimens ( adenovirus + rsv; influenza + rsv; adenovirus plus influenza; adenovirus + hmpv) and three viruses were detected in two specimens ( adenovirus + influenza + hmpv; adenovirus + influenza + rsv). there were no associations between multiple virus detection with age or severity of pneumonia (data not shown). virus detection varied by season. rsv, influenza viruses, and hmpv were all detected in the wet (june-october) and cool (november-february) seasons, whereas adenovirus detection occurred year round and peaked in the late cold and hot (march-may) seasons ( figure ). interestingly, in an age-adjusted analysis, patients who had received an antimicrobial in the two weeks preceding admission were more likely to be rsv pcr positive (aor . , % ci . - . , p = . ). no such association was seen for the other viruses. (figure ). twenty five patients with influenza a associated pneumonia (admitted may-october ) have been described in detail in a previous manuscript [ ] . laboratory-enhanced surveillance has documented the contribution of respiratory viruses to hospitalised clinical pneumonia episodes occurring in a crowded refugee camp on the thailand-myanmar border during april to september . as expected, the vast majority of patients were aged less than five years [ ] . viruses were detected in the nasopharynx of half of the cases. virus detection was significantly more likely in infants compared to older children and adults, although the number of adult cases was small. during the same period, routine hospital surveillance detected , in-patient lrti episodes, including ( . %) deaths and , ( . %) episodes in children under five years [ ] . therefore, ipd pneumonia episodes under enhanced surveillance reported here accounted for . % of all ipd lrti episodes ( . % of ipd lrti episodes in children aged less than five years and . % of episodes in persons aged five years or older). the maela data add to the scant data on the aetiology of pneumonia in refugee populations. the results are broadly consistent with a similar surveillance programme conducted in two kenyan refugee camps [ ] , where . % patients with severe acute respiratory infection (sari) had at least one of adenovirus, hmpv, influenza a/b, parainfluenza virus - , or rsv detected. in the kenya surveillance, adenovirus was the commonest virus detected ( . % of specimens), followed by rsv ( . %). an influenza virus was detected in . % of specimens. the majority ( %) of hospitalised sari cases were children less than five years of age. results from pneumonia aetiology studies from various locations in the developing world have confirmed the high prevalence of virus detection in hospitalised pneumonia episodes in young children [ , ] . rsv has been frequently identified as the commonest virus and is associated with severe infections [ ] [ ] [ ] [ ] . a significant proportion of rsv infections occur in the first year of life, although both primary infection and re-infections are common in older children [ , , ] . rsv has previously been documented to be a significant pathogen in maela, with an incidence of rsv-associated pneumonia of . episodes per child-year at risk in a cohort of infants followed from birth until two years of age [ , ] . our results confirm that rsv is the leading virus associated with pneumonia in the general maela camp population. the majority of infections were in young children and only two ( / , . %) rsv infections occurred in patients aged five years or older. influenza viruses were detected in almost % of patient episodes of pneumonia. this figure is consistent with previously published data on influenza hospitalisations. simmerman and uyeki determined that influenza viruses were detected in - % of hospitalised pneumonia cases in a recent review of east and southeast asian data [ ] . the role of adenoviruses in the aetiology of pneumonia remains unclear. frequent re-infection and persistence in young children makes their detection in npa specimens at the time of pneumonia diagnosis difficult to interpret [ , ] . a recent case-control study from kenya, undertaken as part of a multi-centre paediatric pneumonia aetiology study (perch), did not find a higher odds of adenovirus detection in pneumonia cases compared with controls [ ] . in that study, only the detection of rsv in the upper respiratory tract had a significant association with hospitalisation for pneumonia. who definitions for clinical pneumonia in childhood were used in our enhanced surveillance in maela. these definitions were designed to have optimal sensitivity for the diagnosis of potentially life-threatening bacterial infection in resource-poor settings [ ] . it was accepted that some over-diagnosis and unnecessary treatment would occur [ ] . as was demonstrated in a south african study, where virus-associated pneumonias were reduced by a third in children given a nine-valent pneumococcal conjugate vaccine, a proportion of virus-positive individuals would have had a secondary bacterial infection [ ] , but many will have received an unnecessary course of antibiotics. recent work from pakistan has confirmed that placebo and amoxicillin had equivalent efficacy for who non-severe pneumonia and that oral amoxicillin at home is an acceptable alternative to hospital admission and parenteral antibiotics for severe pneumonia [ , ] . in maela, almost all patients were treated with at least one antimicrobial drug, yet over a third (excluding adenovirus) had a proven viral infection. collectively, these findings point to the likely over treatment of viral infections in children with who defined pneumonia, which may contribute to the rising prevalence of antimicrobial resistance in the developing world [ ] . the enhanced surveillance system had several limitations. not all hospitalised pneumonia episodes were captured and therefore it was not possible to calculate virus-specific incidence rates. estimating the representativeness of the patients enrolled by comparison of enhanced surveillance results with routine surveillance figures is problematic. however, as we note, if a direct comparison is made, enhanced surveillance included . % ( / , ) of all lrti episodes identified through routine surveillance in children under five years of age but only . % ( / ) of episodes in patients aged five years or older. differences in case definitions used in the routine surveillance compared with enhanced surveillance is the most likely explanation for this discrepancy [ ] . for the under- age group the definitions were almost identical (i.e. who-based) but for the older age group lrti was defined in routine surveillance by the presence of a fever plus cough or sore throat and shortness of breath/difficulty breathing. absence of a requirement for abnormal chest signs likely resulted in inclusion of many non-pneumonia cases within the lrti category. the low number of deaths recorded in the his system suggests that severity was not a significant reason for non-inclusion. also, since pu-ami hospital was the only general medical admissions unit within the camp, there were not significant numbers of patients missed as a result of admission to other hospitals. however, it remains possible that the older participants were not representative of the entire hospitalised pneumonia patient group. additional severity data (e.g. need for supplemental oxygen and length of stay) and outcome data were not collected, which further limit the conclusions that can be drawn from the surveillance and the comparisons that can be made with other studies. the panel of viruses tested for included the key pathogens for which evidence of an association with pneumonia is proven, but inclusion of pcr assays to detect additional respiratory viruses would have added value and may have identified a higher prevalence of multiple infections. studies using multiplexed virus pcr assays have detected both an increased proportion of children with single and multiple viral lower respiratory infections [ ] . both human bocavirus and rhinoviruses may be detected in a large proportion of pneumonia cases, although data regarding causality from case-control studies are still limited [ , ] . despite this, it was demonstrated that influenza virus and/or rsv were associated with a third of hospitalised pneumonia episodes in maela. the cost of in-patient pneumonia treatment is high. in two recent studies, the estimated average cost per district hospital pneumonia admission was us$ . in kenya and us$ . in thailand [ , ] . influenza infections are vaccine preventable. a vaccine to prevent rsv infection remains elusive, although progress continues to be made [ , ] . inclusion of influenza immunisation, and rsv immunisation should it become available, in the immunisation programme for refugees on the thailand-myanmar border would likely significantly reduce the burden of pneumonia requiring hospitalisation. viruses were commonly identified in burmese refugees admitted to hospital with clinically-diagnosed pneumonia. use of influenza immunisation and the development of vaccines targeting other common respiratory viruses would be likely to reduce the incidence of pneumonia in children living in refugee camps and similar settings. who: the global burden of disease: update. geneva: world health organisation unicef: pneumonia: the forgotten killer of children. geneva: unicef/who childhood pneumonia mortality-a permanent global emergency epidemiology and etiology of childhood pneumonia respiratory tract infections in children in developing countries viral pneumonia global burden of acute lower respiratory infections due to respiratory syncytial virus in young children: a systematic review and meta-analysis global burden of respiratory infections due to seasonal influenza 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pneumoniae in virus-associated pneumonia comparison of oral amoxicillin with placebo for the treatment of world health organization-defined nonsevere pneumonia in children aged - months: a multicenter, double-blind, randomized, placebo-controlled trial in pakistan effectiveness of community case management of severe pneumonia with oral amoxicillin in children aged - months in matiari district, rural pakistan: a cluster-randomised controlled trial antimicrobial resistance in developing countries part ii: strategies for containment spectrum of respiratory viruses in children with community-acquired pneumonia human bocavirus infections the economic burden of inpatient paediatric care in kenya: household and provider costs for treatment of pneumonia, malaria and meningitis the incidence of pneumonia in rural thailand new strategies for control of respiratory syncytial virus infection an evaluation of the emerging interventions against respiratory syncytial virus (rsv)-associated acute lower respiratory infections in children respiratory virus surveillance in hospitalised pneumonia patients on the thailand-myanmar border we are grateful for the hard work of say paw and mallika (smru clinic, maela) and for the support of the clinical staff at the pu-ami hospital in maela. this work was supported by a us-cdc cooperative agreement ( u ci ). smru is part of the mahidol oxford university tropical medicine research unit, supported by the wellcome trust of great britain. the findings and conclusions in this report are those of the authors and do not necessarily represent the official position of the centers for disease control and prevention. the authors declare that they have no competing interests.authors' contributions pt, vc, ct, cd, cp, lo, and fn conceived the surveillance project. nc and ct were responsible for specimen and data collection. ww performed the laboratory work. pt and vc did the data analysis. pt prepared the first draft of the manuscript. all authors reviewed and contributed to revisions of the manuscript. all authors read and approved the final manuscript. key: cord- -ysatxwph authors: wang, le; feng, zhishan; shuai, jinfeng; liu, jianhua; li, guixia title: risk factors of -day rehospitalization following discharge of pediatric patients hospitalized with mycoplasma pneumoniae pneumonia date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: ysatxwph background: among pediatric patients hospitalized for mycoplasma pneumoniae pneumonia (mpp), the risk factors for -day readmission after discharge is undefined. methods: we conducted a retrospective observational study of patients < years of age who were discharged with a diagnosis of mpp between january and february . we collected clinical, laboratory and radiographic variables at the time of initial admission. we assessed pneumonia-related readmission within -day after discharge. risk factors independently associated with rehospitalization were identified using multiple logistic regression models. results: of the mpp hospitalizations, ( . %) were readmitted within days and were mainly diagnosed with pneumonia. patients with younger age or coinfection with influenza a were more likely to be readmitted. in addition, compared with children without readmission, the readmission ones showed different clinical and laboratory characteristics at the index hospital admission. multiple logistic regression analysis identified age (or . , %ci . – . ) and body temperature (or . , %ci . – . ) were significantly associated with lower risk of -day readmission. coinfection with influenza was independently associated with a greater likelihood of -day readmission (or . , %ci . – . ). conclusions: readmission after mpp are common and is related to patients’ age, body temperature and influenza a coinfection during initial hospital stay, indicating potential targets could be noticed to reduce the rehospitalization after pediatric mpp. readmission of patients initially hospitalized for community acquired pneumonia (cap) is relatively common [ ] [ ] [ ] . both of preventable and non-preventable risk factors have been explored, but the main participants in these studies were the elderly and patients with multiple comorbidities, not children [ ] [ ] [ ] [ ] . the previous three studies described days [ ] , days [ ] readmission rates (range of . - %) for children with pneumonia or lower respiratory infections (lris) [ ] . in these studies, one of the consistent identified risk factors was chronic medical conditions. but in fact, a large number of children who are rehospitalized are caused by acute diseases [ ] . if patients with chronic conditions are excluded, the difficulty is to detect readmission risk factors associated with the current acute infection. in addition, children with lris, a relatively broad diagnosis, or pneumonia with an underlying chronic illness may bring compounding factors conferring susceptibility for readmission. a promising approach to resolve this problem is to narrow down study sample according to the pathogenic or clinical features, such as mycoplasma pneumoniae pneumonia (mpp), which accounts for up to % pediatric cap [ ] , and its diagnosis is based on etiology and clinical evidence, thereby elevating the power to detect readmission risk factors associated with the current acute infections. we hypothesized that mpp children may have different characteristic during the first hospital stay between patients with and without readmission. this study was therefore conducted to enroll pediatric mpp patients without other underlying chronic diseases. our aims were to ( ) describe the incidence and type of readmission after mpp discharge, ( ) investigate the differences between patients with and without readmission at the initial hospital stay, ( ) examine the risk factors for -day pneumonia-related rehospitalization. this retrospective, observational study was conducted at children's hospital of hebei province, a -bed teaching hospital in hebei province (northern china) that serves a population of , , inhabitants, including . % children. patients with a discharge diagnosis of mpp were evaluated. the project was approved by the ethics review board of the hospital. because data in this report were collected from inpatient electronic medical records, there was no need to collect new specimens or the corresponding written informed consent. children ≤ years of age who were admitted to children's hospital of hebei province with a diagnosis of mpp from january , to february , were consecutively enrolled into the study. the diagnosis of mpp needs to meet the following first points plus either third or fourth point [ ] : ) a new infiltrate on a chest radiograph; ) fever, cough and abnormal lung auscultation; ) positive serology laboratory results specific mp antibody titer≥ : detected by a micro-particle agglutination test [ ] ; ) positive pcr laboratory results, mp-dna positive detected in sputum, nasopharyngeal aspirate or bronchoalveolar lavage fluid (balf) by pcr [ ] . patients were excluded from the study if they were known to be chronically immunosuppressed, or with chronic cardiopulmonary conditions or had been hospitalized for the previous days. if a patient had more than one episode of pneumonia during the index hospitalization, only the first one was included in the analysis. the following patient characteristics were evaluated: age, sex, signs and symptom before admission (wheezing, cough and diarrhea). the laboratory data and radiological findings were also measured and retrospectively investigated from inpatient electronic medical records system. clinical symptoms included wheezing, cough and diarrhea. ill day and febrile day before admission, hospital days, febrile day and readmission rate were also recorded. the time frame of readmission was set as day from original discharge. body temperature was examined at the beginning of admission and every h thereafter. a febrile day was defined as the body temperature exceeded . °c at least once [ ] . patients were asked to cough, and the expectorated sputum was collected. if the child is too young to cough, a sterile negative pressure suction catheter is applied to obtain the oropharyngeal suction (ops). the storage, transportation and nucleic acid extraction procedure were reported elsewhere [ ] . the paired serum samples were taken at the presentation of pneumonia and at least days after the first collection of serum. the serum was obtained from ml whole blood by the separation gel tube. the gexp assay (genomelab gexp genetic analysis system) was performed on all specimens for the type/ subtypes of common respiratory pathogens including m. pneumoniae. the multiplex-pcr was performed as previously described elsewhere [ ] . the bacteria infection was examined by standard culture methods from sputum specimens [ ] . the determination of mp-specific antibody was performed using a commercially available micro-particle agglutination test serodia-mycoii kit (fujirebio, tokyo, japan) [ ] . diagnosis criteria were defined as ≥ -found rising for paired sera or single serum of titer ≥ : [ ] . the chi-squared test was used to compare categorical variable in subgroups. and for those continuous variables with normal and non-normal distributions, mean or median values were compared using the t test or mann-whitney u test. spss . statistics package (spss inc., chicago, usa) software was used for all statistical analysis. p < . was considered statistically significant. during the study period, inpatients met our study eligibility criteria for mpp: patients were excluded due to the missing sex, age or diagnostic data, and patients were not readmitted to hospital within days ( fig. ) . of the patients who were readmitted in days, were diagnosed with pneumonia-unrelated diseases including epilepsy, encephalitis, carditis and arthritis. therefore, ( . %) children were readmitted due to pneumonia-related diseases in days of initial mpp discharge. of the re-admitted cases, ( . %) were reinfected with mp, ( . %) were negative for mp, and ( . %) did not receive pathogen detection test. the most common cause of readmission was pneumonia ( . %), followed by bronchial pneumonia ( . %) bronchitis ( . %) and one ( . %) refractory mycoplasma pneumoniae pneumonia (rmpp) ( table ) . among the enrolled patients, there were cases were detected to be positive by pcr alone, were serology alone (with cases as seroconversion and cases as single high titer) and were positive by both pcr and serology assays. demographic characteristics included age (median age . , range in . - ) and gender ( boys and girls). as shown in table , patients who were readmitted in days were significantly younger than those without readmission (median age: v.s. . years, p < . ) and were more likely to show wheezing symptoms (p < . ), as well as a lower body temperature on admission (p < . ), shorter febrile days during hospital stay (p < . ). similarly, more patients with readmission showed normal (p = . ) or light diffuse shadowing (p = . ) radiological findings. crp, ldh, hbdh and neutrophil percentage levels were lower, but the percentage of lymphocyte was significantly greater (all p < . ) ( table ) . coinfection was observed in ( . %) cases, and children infected with influenza a were more likely to be admitted again (p < . , table ). coinfection rates of other pathogens (rhinovirus, parainfluenza, influenza b, respiratory syncytial virus, adenovirus, coronavirus, human metapneumovirus, s. pneumoniae and human bocavirus) did not vary significantly between patients with and without rehospitalization. multiple logistic regression analysis identified age (or . , %ci . - . ) and body temperature (or . , %ci . - . ) during initial hospital stay were significantly associated with lower risk of -day readmission. coinfection with influenza a at index admission was independently associated with a greater likelihood of day readmission (or . , %ci . - . ) ( table ). generally, readmissions after pneumonia are common. in terms of safety and cost, it is important to assess the relationship between initial hospital stay and readmission outcomes [ , ] . although the investigation of risk factors is challenging, significant progress has been made on the elderly, that the readmission was found to be largely depends on the comorbidities and factors external to the patient [ , , , ] . this has also been observed in children, and one of the identified risk factors is chronic medical conditions such as underlying pulmonary or cardiovascular disease [ ] [ ] [ ] . to date, research on potential risk factors for readmission has hardly focused on current acute infections or specific pathogens. because pneumonia is a complex heterogenous disease that can be caused by a variety of pathogens, ) studying pneumonia patients with the same infectious pathogen can reduce heterogeneity, ) exclusion of patients with underlying diseases can improve the ability to detect readmission risk factors associated with the current acute infections. therefore, we investigated the rate of -day pneumoniarelated readmission in hospitalized children with mpp who had no basal or chronic disease. after comparing the clinical information of the first hospitalization between patients who were readmitted and not readmitted to the hospital, we obtained the following findings: ) ( . %) children were readmitted within days of the first mpp there are mixed infections with more than one pathogen, the total cases is not equal to . there are cases of single pathogen coinfection, cases of two pathogens, cases of pathogens discharge; ) at the index hospital stay, readmission patients manifested different characteristics; ) co-infection with influenza a increased the risk of -day readmission. in this study, the readmission rate after mpp discharge was . %. pneumonia and bronchial pneumonia were the majority diagnoses on readmission. nakamura et al. identified that . % cases readmitted after lri hospitalization, and the most common readmission diagnose was lri ( . %) [ ] . similar to the pattern observed by neuman and colleagues, nearly half of the % of patients who were discharged from initial pneumonia hospitalization were also associated with pneumonia [ ] . previous work has shown that % pediatric readmissions are potentially preventable, especially the index admission and readmission are causally related [ ] . this is one of the reasons we focus on pneumonia-related readmission. meanwhile, we observed a trend in patients with relatively mild radiological symptoms and lower levels of acute inflammatory markers that are more likely to be rehospitalized. the cause of this phenomenon may be related to two points, including different host immunity and treatment strategies. first, clinical presentation depends on the host's immune response, rather than direct microbial destruction during the progression of m. pneumoniae infection [ ] . patient with a reduced immune system, such as younger ones who have had less time to develop immunity, may be characterized by mild clinical symptoms but with a prolonged recovery period. second, pediatricians will adopt different treatment strategies for patients with severe or mild symptoms. patients at risk of readmission may receive different medication times due to m. pneumoniae virulence or host immune response. in this present study, we found that influenza coinfection increased the risk of readmission, which is consistent with previous investigations of children with complicated pneumonia. william et al. found that although there was a trend to increase mortality, patients with flu coinfection were less likely to readmitted in weeks readmission, [ ] . brogan et al. observed that children who were infected with influenza during the initial hospital stay had a higher rate of readmission than children who were not infected with influenza [ ] . regarding the elderly, researches show that influenza vaccination is associated with a lower likelihood of readmission [ , , ] . in view of these findings, influenza vaccination should be promoted not only in pediatric hospitals at cap discharge, but also for all people, particularly in high risk groups including children under years old, and those with asthma. in addition, we observed that younger children are liable to readmit, which is consistent with previous findings, demonstrating a higher rate of readmission for children under year of age [ , ] . as explained by gay jc et al., pneumonia in young patients usually has protracted and waning course, leading to structural lung damage or immune paresis and further pneumonia episodes [ ] . second, younger patients may be more prone to new infections due to higher exposure during nursery attendance and the previous lack of immunity to respiratory pathogens, which will be resulting in rehospitalization. furthermore, studies of children with asthma have found that the rate of readmission of children under year of age in higher, further highlighting the need to improve inpatient decision-making for young patients. to our knowledge, this is the first study to explore the factors of readmission for pediatric mpp patients. further research in larger cohorts is needed to validate the data. meanwhile, some questions remain to be answered: first, it has been reported that pneumonia attributed to potentially antibiotic-resistant bacteria is associated with an increased risk of readmission [ ] , we strongly felt that macrolide resistance has a role on the risk of readmission, but what is the role? second, coinfection with influenza a will increase the risk of readmission, what is the underlying mechanism? limitations this study has several limitations. first, the sample size may be small because only patients were rehospitalized within days. second, if the child is readmitted to another institution, we may underestimate the rate of readmission. third, in the absence of the information after first discharge, the interference factor related to age may be introduced into this research, as the rehospitalization may be caused by a new infection during nursery attendance. fourth, other clinical information to document severity is not included in the study, such as oxygen requirement, antibiotic or corticoid duration. fifth, a potential limitation would be that the serological assays have a high false positive detection rate and it is difficult to obtain the second serum. in our report, only children provided paired sera, and the other patients ( / , %) had positive serological results with only a single high titer, but the pcr results were negative. last, although there were significant differences in crp, ldh levels or patient characteristics between rehospitalized and non-rehospitalized patients, these factors cannot be controlled and of low value in clinical practice. in conclusion, rehospitalization after mpp is relatively common and is related to patients' age and co-infected pathogens. careful attention to clinical variables may reduce the frequency of rehospitalization of pediatric patients after discharge on mpp. abbreviations cap: community acquired pneumonia; gexp assay: genomelab genetic analysis system; mp or m. pneumoniae: mycoplasma pneumoniae; rt-pcr: reverse transcription polymerase chain reaction predictors of rehospitalization after admission for pneumonia in the veterans affairs healthcare system on-behalf of the project fispiwg: factors associated with -day readmission after hospitalisation for communityacquired pneumonia in older patients: a cross-sectional study in seven spanish regions risk factors for communityacquired pneumonia in adults in europe: a literature review predictors of short-term rehospitalization following discharge of patients hospitalized with community-acquired pneumonia discharge disposition as an independent predictor of readmission among patients hospitalised for community-acquired pneumonia causes and risk factors for rehospitalization of patients hospitalized with communityacquired pneumonia a prospective cohort study of healthcare visits and rehospitalizations after discharge of patients with community-acquired pneumonia variability in processes of care and outcomes among children hospitalized with community-acquired pneumonia readmissions among children previously hospitalized with pneumonia pediatric readmissions after hospitalizations for lower respiratory infections pediatric readmission prevalence and variability across hospitals pediatric respiratory infections by mycoplasma pneumoniae a randomized controlled trial of filgrastim as an adjunct to antibiotics for treatment of hospitalized patients with community-acquired pneumonia. cap study group evaluation of serodia myco ii particle agglutination test for detecting mycoplasma pneumoniae antibody: comparison with mu-capture elisa and indirect immunofluorescence comparison and evaluation of real-time pcr, real-time nucleic acid sequence-based amplification, conventional pcr, and serology for diagnosis of mycoplasma pneumoniae clinical evaluation of macrolide-resistant mycoplasma pneumoniae a comparison study between gexp-based multiplex-pcr and serology assay for mycoplasma pneumoniae detection in children with community acquired pneumonia a gexp-based assay for simultaneous detection of multiple viruses in hospitalized children with community acquired pneumonia standardization of laboratory methods for the perch study hospital readmissions: measuring for improvement, accountability, and patients pneumonia pathogen characterization is an independent determinant of hospital readmission potentially preventable -day hospital readmissions at a children's hospital mycoplasma pneumoniae and its role as a human pathogen influenza coinfection and outcomes in children with complicated pneumonia hospital readmissions among children with h n influenza infection instability on hospital discharge and the risk of adverse outcomes in patients with pneumonia preventability and causes of readmissions in a national cohort of general medicine patients epidemiology of -day readmissions to a children's hospital publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we sincerely thank all the participants for their support. we also thank dr. sukun lu and lijie cao from the no. authors' contributions lw performed the statistical analysis and drafted the manuscript. zf participated in the validation. gl and jl participated in the design of the study. gl conceived of the study, and participated in its design and coordination, js helped to draft the manuscript. all authors read and approved the final manuscript. not applicable. the datasets generated and/or analyzed during the current study are available in the (figshare) repository (https://figshare.com/articles/mp_ readmission/ ). the data showed cases with readmission and index hospitalization after mpp. the protocols used in this retrospective study was reviewed and approved by the institutional review board of children's hospital of hebei province. because there was no need to collect new specimens and the clinical data was de-identified, so the consent was waived by irb of children's hospital of hebei province. after obtaining the irb' permission, we can review patient records and use these data, which were all de-identified. not applicable. all the authors declared that they have no competing interests. key: cord- - m ygzn authors: chen, yin-yin; chen, liang-yu; lin, seng-yi; chou, pesus; liao, shu-yuan; wang, fu-der title: surveillance on secular trends of incidence and mortality for device–associated infection in the intensive care unit setting at a tertiary medical center in taiwan, – : a retrospective observational study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: m ygzn background: device–associated infection (dai) plays an important part in nosocomial infection. active surveillance and infection control are needed to disclose the specific situation in each hospital and to cope with this problem effectively. we examined the rates of dai by antimicrobial-resistant pathogens, and –day and in–hospital mortality in the intensive care unit (icu). methods: prospective surveillance was conducted in a mixed medical and surgical icu at a major teaching hospital from through . trend analysis was performed and logistic regression was used to assess prognostic factors of mortality. results: the overall rate of dais was . episodes per device–days. the most common dai type was catheter–associated urinary tract infection ( . per urinary catheter–days). there was a decrease in dai rates in and rates of ventilator–associated pneumonia (vap, . per ventilator–days) have remained low since then (p < . ). the crude rates of –day ( . %) and in–hospital ( . %) mortality, as well as infection by antibiotic-resistant vap pathogens also decreased. the most common antimicrobial-resistant pathogens were methicillin–resistant staphylococcus aureus ( . %) and imipenem–resistant acinetobacter baumannii (p < . ), which also increased at the most rapid rate. the rate of antimicrobial resistance among enterobacteriaceae also increased significantly (p < . ). after controlling for potentially confounding factors, the dai was an independent prognostic factor for both –day mortality (or . , % confidence interval [ci] . – . , p = . ) and in–hospital mortality (or . , % ci . – . , p < . ). conclusions: the decrease in the rate of dai and infection by resistant bacteria on the impact of severe acute respiratory syndrome can be attributed to active infection control and improved adherence after . surveillance of nosocomial infections (nis) has become an integral part of infection control and quality assurance in many countries. gastmeier et al. reported that effective surveillance could reduce the ni rate on average about - % [ , ] . surveillance programs provide data on the microbes causing specific nis and their resistance to antibiotics. moreover, such programs can guide clinical practices and ni prevention efforts in different geographic regions and clinical settings. the surveillance of device-associated infections (dais) in intensive care units (icus) has become more important owing to the more frequent employment of invasive advanced life support devices, especially after the introduction in of surviving sepsis bundles [ , ] . nevertheless, according to the three largest surveillance systems, the pooled mean rates of dais were: ventilator-associated pneumonia (vap), . - . per ventilator-days; central line-associated bloodstream infection (clabsi), . - . per catheterdays; and catheter-associated urinary tract infection (cauti), . - . per catheter-days [ ] [ ] [ ] . in addition, dais have been associated with significant cost and mortality [ , , ] . the crude mortality rates of icu patients with dai were . - . % [ ] . moreover, as indicated by the message "bad bugs, no drugs" released by the infectious disease society of america in , the emergence of antibiotic resistance threatens to exacerbate the problem of nis in critically ill patients. decreased susceptibility of both gram-positive and gram-negative microbes to antibiotics has been well described in several surveillance studies over the past decade, and increases in the rate of bloodstream infection caused by multi-drug resistant (mdr) gramnegative bacteria have been reported to be -fold [ , [ ] [ ] [ ] [ ] . in addition, both the morbidity and mortality rates have increased [ ] [ ] [ ] . in this study, prospective surveillance was conducted to determine the dai rate and prevalence of antibiotic-resistant isolates at an adult medical-surgical icu (ms icu). our aim was to analyze the secular trend of incidence for different types of dais, determine the common pathogens involved, and determine the rates of antimicrobial resistance and overall -day and in-hospital mortality during the period - . this study was conducted in a -bed adult medicalsurgical icu with more than admissions (age years or older) per year located in a -bed major teaching hospital in the northern part of taiwan. the hospital-wide infection surveillance and control program was established in , with one infection control practitioner (icp) for every beds. all patients admitted to the icu in the period - who developed infections more than hours after admission (i.e., nosocomial infections) were eligible for the study. the protocol of this study was approved by the institutional review board of our teaching hospital. this icu-based surveillance was conducted according to the us centers for disease control and prevention (cdc) procedures. all patients in the unit were monitored for nis that affected particular body sites. infections at more than one site in the same patient were counted as separate infections. the antibiotic susceptibility of each pathogen involved was analyzed. the data were prospectively collected at least once a week in the icu by trained icps according to standardized protocols and definitions of the us cdc national healthcare safety network (nhsn; formerly the national nosocomial infection surveillance system [nnis]) [ ] . all dais of the outcome surveillance component were categorized using standard us cdc nhsn definitions that included laboratory and clinical criteria [ ] . the involved patient demographic information, the dates and sites of infection, device-utilization (du) ratio, pathogens, antimicrobial susceptibilities, invasive procedures, and overall -day mortality and in-hospital crude mortality were recorded. reports of cases of dai were also verified by an infectious disease specialist. data were also collected for each exposed patient in the icu from the prospective hospital database, including demographics and clinical characteristics. pneumonia was defined when a patient had a new or progressive infiltrate, consolidation, cavitation, or pleural effusion on chest radiograph and had the following signs or symptoms: new onset of purulent sputum or change in character of sputum. a vap was categorized as ventilator associated if the patient had been intubated and received ventilation for more than hours prior to the development of pneumonia. to detect vap microorganisms, tracheal aspirates obtained via endotracheal tube suction or tracheostomy tube suction methods were cultured. laboratory-confirmed bloodstream infection (bsi) was defined when a patient had a recognized pathogen cultured from one or more blood cultures and the microorganism cultured from blood was not related to an infection at another site. common skin contaminants (e.g., coagulase-negative staphylococcus [cons]) required culture from two or more blood cultures drawn on separate occasions or at least one blood culture for a patient with intravascular devices and microorganisms of the tip culture identical to those of the blood culture. a clabsi was considered central catheter-associated if a catheter had been in place for more than hours and a secondary site of infection was not present. to detect clabsi micro-organisms, a central catheter were removed aseptically and a -cm segment from the most distal end of the tip of the catheter along with paired peripheral blood samples were cultured. central catheter-tip colonization was defined as isolation of colony-forming units from a central catheter tip by using the roll-plate semiquantitative maki's culture technique. symptomatic uti was defined when a patient had one or more of the following signs or symptoms with no other recognized cause: fever (> °c), dysuria, urgency, frequency, or suprapubic tenderness and ( ) the patient had a positive urine culture, that is, ≥ microorganisms per cm , or urine with no more than two species of microorganisms, or ( ) pyuria (urine specimen with ≥ white blood cells /mm and a positive urine culture of ≥ and < cfu/ml with no more than species of microorganisms. a cauti was a symptomatic uti that occurred in a patient who had an indwelling urinary catheter in place within the hour period before the onset of the uti. to detect cauti organisms, a urine sample was aseptically aspirated from the sampling port of a urinary catheter and cultured quantitatively. pathogens were isolated from blood cultures using the bactec w nr- system (becton dickinson diagnostic instrument systems, spark, md, usa) between and and using the bact/alert d system (bio-mérieux, inc., marcy l'Étoile, france) between and . pathogens were isolated from other specimens using standard methods specified by the clinical laboratory standard institute (clsi) [ ] . antibiotic susceptibilities were determined using disk diffusion tests and interpreted according to the criteria specified by the clsi . the ni rate was defined as the number of nis per , patient-days. patient days were calculated as the number of icu days of the non-ni cohort or the number of icu days after the onset of ni. device-associated infection rates were calculated as the number of deviceassociated infections for a specified body site per , device days. the du ratio was calculated as the number of device-days per number of patient-days. secular trends of du ratio, antimicrobial resistant rates, -day mortality and in-hospital mortality rates were analyzed by chi-square test for linear trend. the overall and site-specific dai rates were analyzed by poisson regression analysis. logistic regression with a stepwise forward approach was used to assess prognostic factors of mortality, while controlling for potentially confounding variables (i.e., demographics, invasive devices, and laboratory data) [ ] . odds ratios (or) and % confidence intervals (ci) were calculated. a p-value < . was defined as statistically significant. statistical analysis was conducted using epi info tm version . . released by us cdc. graphs of secular trends, -day mortality and inhospital mortality rates were created using sigma-plot version . (systat inc., san jose, ca, usa). during the study period, , patient-days and , device-days were evaluated, and , nis and dais occurred in , patients admitted to ms icus with a mean apache ii score of . ± . . the crude mortality rate was . % during the study period. those patients who were admitted to ms icus had a mean age of . ± . years, and male gender accounted for . %. the length of icu stay was . ± . days in average. most patients were admitted due to major medical conditions ( %), such as neoplasms ( . %), digestive system problems ( . %), and respiratory system problems ( . %). patients with serum albumin < . g/dl were . % and blood creatinine > . mg/dl were . %. there were . % patients undergoing hemodialysis during their icu stay. the overall rates of nis and dais were . episodes per patient-days and . episodes per device-days, respectively. the most common dai type was cauti (mean figure ) . a total of , pathogens were isolated from clinical specimens ( table ) . acinetobacter baumannii ( %), pseudomonas aeruginosa ( . %), and staphylococcus aureus ( . %) were the three most common pathogens associated with vap, while s. aureus ( . %), a. baumannii ( . %), and candida albicans ( . %) accounted for the majority of clabsis. in contrast, non-albicans candida (nac) spp. ( %) rather than bacteria were the most common cauti pathogens, followed by enterococci ( . %) and escherichia coli ( . %). the rate of antibiotic resistance every year is presented in table (figures and ) . dai was an independent factor for -day mortality (or . , % ci . - . , p = . ) and in-hospital mortality (or . , % ci . - . , p < . ) by multiple regression analysis. other significant prognostic factors (p < . ) for mortality included apache ii score, service, length of stay after the onset of infection, serum albumin, blood creatinine, neoplasms and hemodialysis (table ). the mean rates of ni and dai in our adult ms icu during the study period were much lower than those reported by the inicc as well as for icus in developing countries [ ] , were similar to those reported by , hospitals in the us through the cdc nhsn [ , ] , and were slightly higher than those indicated by icus in the german surveillance system (icu-kiss) [ ] . reasons for these high dai rates in the inicc report and developing countries may include resource limitations, lack of legal enforcement of the infection control program, and poor adherence to infection control guidelines [ ] . the prospective hospital-wide surveillance and infection control program has been established for nearly years in our hospital, which made a great effort to control infection by implementing infection control bundles and educational programs. the increase of device-related infections is not obvious after , except for cauti. these strategies showed effectiveness in controlling dai rates and suggest the necessity of infection control bundles implementation. the common device-associated pathogens show geographic variation in distribution. a. baumannii, s. aureus, and p. aeruginosa were the three most common vap pathogens in our study, the us cdc nhsn study, and the sentry antimicrobial surveillance study, although their percentages differed between studies [ , ] . the percentage of isolates of mrsa (p = . ) and irpa (p = . ), but not isolates of irab (p < . ) remained relatively constant. however, any variation in these percentages would not be statistically significant and might rather be due to chance than to an actual variation. in contrast, higher rates of a. baumannii and c. albicans isolation compensated for the relatively low rates of cons and enterococcus spp. isolation in cases of clabsi, while c. albicans was replaced by nac spp in cases of cauti. differences in clinical setting, institution, study period, target population, and specific infection type might account in part for differences between studies. cons was less frequently identified in clabsi, because our criteria were slightly different from the us cdc definition for laboratory-confirmed bsi. the cdc defined skin contaminant bsi in and as 'the common skin contaminant (e.g., cons) is cultured from at least one blood culture from a patient with an intravascular line, and the physician institutes appropriate antimicrobial therapy'. however, cons bsis in our study were enrolled if the patient had only one blood culture of cons that was positive but then microorganisms cultured from the tip of the intravascular device that were also cons. the percentage of cons isolates was expected to be at least that of s. aureus isolates reported in previous studies [ , , , ] . however, the frequency of a. baumannii and candida spp. in specimens from patients with clabsi was also reported to be increasing in other hospitals in taiwan as well as several asian countries such as turkey and thailand [ ] [ ] [ ] [ ] [ ] , although the frequency of nac spp. represented by only one candida spp. has also been rising in specimens from patients with cauti. early and empirical usage of broad spectrum antibacterial agents in critically ill patients and preemptive administration of fluconazole are common factors contributing to the increase in frequency of isolation of these relatively resistant pathogens [ , , ] . use of indwelling catheters increases susceptibility to those multi-drug resistant pathogens and is associated with biofilm formation [ , ] . the high prevalence of mrsa is a common problem worldwide, and this situation was much more severe in our institute. our data showed a lower incidence density of s. aureus but a higher proportion of mrsa. the percentage of mrsa infections was . - . % in the inicc report [ ] , . - . % in the us cdc nhsn report [ ] , and, in the asia-pacific region, it was . % in the sentry antimicrobial surveillance program report ( ) ( ) [ ] and - % in the tigecycline evaluation and surveillance trial (test) report [ ] . mrsa rates were decreasing in many european countries but not in usa [ , ] . the more severe illnesses of patients and more frequent use of broad-spectrum antibiotics might account in part for the high rate of mrsa isolation from patients in icu at major teaching hospitals [ , ] . another possible explanation is the clonal spread of resistance genes or resistant strains [ , ] , but molecular analysis will be needed to prove this hypothesis. according to the infection control policies in our icu, when a patient was admitted to the icu, a multi-drug resistant (mdr) checklist was used to inquire about mdr pathogens including mrsa infection or colonization. if mrsa had been isolated, then contact precautions were implemented. furthermore, we have promoted hospital-wide hand-washing activity from to the present. the infection control team also carries on the non-warning investigation of hand-washing and of isolation precautions in each season, and gives feedback of the results to the unit. infectious disease doctors assist in carrying on the infectious disease treatment and the antibiotic use in the icu. mrsa infection rates have been reduced by year from . interestingly, the rates of antibiotic resistance for pathogens other than mrsa was lower at our hospital than those in previously published reports and lower than those for all nis reported by the test and sentry antimicrobial surveillance programs [ , , , ] . however, despite the carbapenems being the most active antimicrobials against acinetobacter species, the increasing development of significant carbapenem resistance among acinetobacter species has been reported [ , , ] . in the present study, the average percentage of a. baumannii isolates resistant to imipenem was . %. the rate of icu patients with irab dai has been rapidly rising (from . % to . %). among enterobacteriaceae, ciprofloxacin-r e. coli and ceftazidime-r k. pneumoniae from , and ceftazidime-r e. coli from , had significant increases. this finding revealed that the resistance of gram-negative bacteria has increased, the development of which should require closely monitored. aside from the fact that dai is an important prognostic factor of mortality., several previous studies have shown that the mortality rate attributed to dai is . - . times higher than that attributed to no infection [ , , ] . our study supports the findings of these published reports. in the present study, the multiple regression analysis indicated that patients with dais (compared to patients with no hai) had significantly increased likelihood of mortality (p < . ). moreover, the annual -day mortality rates of cautis and clabsis had significant changes over the period through . these results may be caused by chance, because this study period did not change substantially in terms of medical care, novelty medical technology, and patient disease severity. in addition to the above-mentioned findings, we used a multiple regression analysis approach to adjust covariables, in addition to demographics, invasive devices, and laboratory investigations. we also identified severity of illness using apache ii scores as a predictor of mortality, with the results indicating that the hazard of mortality is associated with increasing scores. patients who died with dai infection were usually already severely ill and their existing illness, rather than the dai, was often the main cause of death. thus, an important prognostic factor was the severity of their illness, which resulted in an increased likelihood of mortality [ , ] . we also found that patients with blood creatinine over . mg/dl were the highest risk group for dying. excluding an endogenous effect, the reason may be that many patients in this group were receiving hemodialysis with cvcs inserted. the rates of dais of all types decreased during the period - , but this decrease was maintained [ ] [ ] [ ] . some limitations of the present study should be noted. the study was performed at a single medical center. however, the results could be provided to the hospital as a part of the teaching or research mission. this study was a retrospective nine-year survey which might have some potential biases. in the analysis of long-term changes in infection rates or mortality rates, we must consider whether changes in the population, advances in laboratory diagnostic techniques, changes in exposure to risk factors, microbial culture and other factors lead to increased or decreased rates. however, there did not occur any outbreaks of dais during the study period, except for the sars outbreak. we have presented here the secular trend of dais at our institution in northern taiwan, and the great achievement of our infection control and surveillance program, which was the maintenance of a low dai incidence despite high device-utilization ratios. the incidence of dais decreased in . the incidence of vap remained low, and the rate of antimicrobial resistance of the three most common pathogens causing vap decreased. 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respiratory syndrome among healthcare workers submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank the icps in the department of infection control, taipei veterans general hospital, for data collection during the study period. this study was supported by a part of research grant from taipei veterans general hospital (v b - ), taipei, taiwan. the funding institutes did not have any role in study design, data collection/analysis, the writing of the manuscript and the decision to submit the manuscript for publication. the authors declare that they have no competing interests.authors' contributions yyc participated in the design, data collection and analysis, and drafted the manuscript. lyc participated in the analysis and drafted the manuscript. syl and pc commented on drafts of the manuscript. syl participated in the data collection. fdw conceived of the project, participated in the design and helped to draft the manuscript. all authors approved the final manuscript. key: cord- -gwd qin authors: chiu, rossa wk; jin, yongjie; chung, grace ty; lui, wing-bong; chan, anthony tc; lim, wilina; dennis lo, ym title: automated extraction protocol for quantification of sars-coronavirus rna in serum: an evaluation study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: gwd qin background: we have previously developed a test for the diagnosis and prognostic assessment of the severe acute respiratory syndrome (sars) based on the detection of the sars-coronavirus rna in serum by real-time quantitative reverse transcriptase polymerase chain reaction (rt-pcr). in this study, we evaluated the feasibility of automating the serum rna extraction procedure in order to increase the throughput of the assay. methods: an automated nucleic acid extraction platform using the magna pure lc instrument (roche diagnostics) was evaluated. we developed a modified protocol in compliance with the recommended biosafety guidelines from the world health organization based on the use of the magna pure total nucleic acid large volume isolation kit for the extraction of sars-coronavirus rna. the modified protocol was compared with a column-based extraction kit (qiaamp viral rna mini kit, qiagen) for quantitative performance, analytical sensitivity and precision. results: the newly developed automated protocol was shown to be free from carry-over contamination and have comparable performance with other standard protocols and kits designed for the magna pure lc instrument. however, the automated method was found to be less sensitive, less precise and led to consistently lower serum sars-coronavirus concentrations when compared with the column-based extraction method. conclusion: as the diagnostic efficiency and prognostic value of the serum sars-cov rna rt-pcr test is critically associated with the analytical sensitivity and quantitative performance contributed both by the rna extraction and rt-pcr components of the test, we recommend the use of the column-based manual rna extraction method. the severe acute respiratory syndrome (sars), etiologically related to a newly emerged coronavirus (sars-cov) [ ] , caused an epidemic in with reported cases in countries around the world [ ] . a factor that is important in the effective control of an epidemic involves the early identification and isolation of infected individuals [ ] . we have previously reported the development of a diagnostic test based on the detection of the sars-cov rna in serum/plasma by real-time quantitative reverse transcriptase polymerase chain reaction (rt-pcr) [ , ] . eighty percent of infected individuals were shown to be positive by the test on the first day of hospital admission with no false-positive results [ , ] . the serum sars-cov rna concentration detected upon admission has also been shown to be predictive of the requirement for subsequent intensive care [ ] . the approach has been demonstrated to be useful for serial monitoring of treatment efficacy [ ] . the analytical protocol that had been developed involved the use of a manual rna extraction method. as the demand for diagnostic testing at times of infectious disease outbreaks would be high, strategies that may enhance the throughput of analytical procedures would be advantageous. in this study, we assessed the feasibility of automating the rna extraction procedure of the serum sars-cov rna test by evaluating the performance of an automated extraction system. the aim of the study was to compare the efficacy of sars-cov rna extraction based on the use of the qiaamp viral rna mini kit (qiagen, hilden, germany) with protocols adapted for the magna pure lc instrument (roche diagnostics, basel, switzerland). the former protocol is a manual column-based method based on silica-adsorption. on the other hand, the magna pure lc instrument extracts nucleic acids from biological specimens based on magnetic bead separation. the principle and general setup of the instrument had been previously described [ ] . the main objective of this study was to compare the resultant analytical sensitivity and quantitative performance of the serum sars-cov rna test when either the manual or automated extraction protocol was used. according to the manufacturer's information, kits, namely the magna pure lc total nucleic acid isolation kit (roche diagnostics) and the magna pure total nucleic acid large volume isolation kit (roche diagnostics), are recommended for use with the magna pure lc instrument for the extraction of viral dna or rna from serum or plasma. the main differences between the two kits lie in the starting sample volume and whether an external lysis protocol is available. the former kit processes µl of serum and is compatible with the use of an external lysis protocol preinstalled in the accompanying software (magna pure lc software v. . , roche applied science) of the magna pure lc instrument. the latter kit, however, processes µl of serum and no external lysis protocol had been predefined. external lysis is a processing step whereby lysis buffer can be added to clinical specimens manually prior to the transfer of the sample and buffer mixture to the automated instrument for further downstream processing. external lysis is a desirable step for the processing of potentially infectious specimens whereby the specimens could be processed according to the recommended biosafety precautions until the pathogens are lysed and the specimen rendered safe for further processing by the magna pure lc instrument. however, the sensitive detection of sars-cov rna from serum may be dependent on a large starting volume of serum. therefore, we evaluated an in-house modification of the manufacturer's protocol for the "large volume" kit with the addition of an external lysis step. to minimise the infectious risk to the laboratory personnel, all analyses were performed using aliquots of a sars-cov culture isolate that had been inactivated by procedures previously described [ ] . inactivated sars-cov was spiked into transport medium so that the resultant mixture contained copies/ml of the virus. viral rna was extracted from aliquots of this mixture in triplicate both according to the standard as well as modified protocols for the large volume kit. for the standard protocol, laboratory personnel were only involved with the initial transfer of µl of each specimen to individual sample cartridges placed inside the instrument after which proteinase k and buffers, including µl of lysis buffer, were added by the instrument in a sequential and automated fashion. for the modified protocol, µl of lysis buffer was first added to µl of specimen in a biosafety cabinet to make up a final volume of µl. after vortexing, the mixture was transferred in ice to the sample cartridges on the magna pure lc instrument. the instrument was then activated to run as per the standard protocol. the externally-added lysis buffer ( µl) when mixed with the volume of lysis buffer pre-loaded on the magna pure lc instrument ( µl) amounts to a total of µl of lysis buffer for µl of specimen and thus contributes to a lysis buffer:sample ratio which largely resembles that adopted in the external lysis protocol of the total nucleic acid isolation kit. all viral rna preparations extracted in this study were quantified using a real time quantitative one-step rt-pcr assay targeting the nucleocapsid-gene of the sars-cov as previously described [ ] . briefly, the assay involves the use of the ez rtth rna pcr reagent set (applied biosystems, foster city, california) on an applied biosystems sequence detector. µl of extracted viral rna was used for amplification in a reaction volume of µl. the modified large volume protocol with the external lysis step was further compared with the external lysis protocol of the total nucleic acid isolation kit using a transport medium mixture containing copies/ml of inactivated sars-cov. quadruplicate extractions were performed. we also assessed the analytical sensitivities of both protocols by comparing the detection rates for aliquots of transport medium diluted to contain , and copies/ml of inactivated sars-cov, respectively. five extractions were performed for each concentration. we then addressed the possibility of carry-over contamination within the magna pure lc instrument by introducing nine aliquots of transport medium containing inactivated sars-cov ranging from to copies/ml alternating with aliquots of plain transport medium on the instrument and using the modified large volume protocol for extraction. the modified large volume protocol was selected for further comparison with the performance of the columnbased manual method. the manual extraction method was performed according to the manufacturer's instructions. transport medium and pooled sera were mixed with serially diluted aliquots of inactivated sars-cov to produce samples containing sars-cov with concentrations ranging from to copies/ml. the pooled sera were first confirmed to be negative for sars-cov by the quantitative rt-pcr assay. viral rna was extracted from the serial samples by both the automated and manual methods and sars-cov rna concentrations were determined by the quantitative rt-pcr test [ ] . viral rna concentrations were compared using passing-bablok regression [ ] . we next compared the effects of the rna extraction methods on the overall assay sensitivity. pooled sera spiked with , and copies/ml of inactivated sars-cov were extracted by both methods. ten replicate extractions were performed for each concentration. the detection rate at each concentration was compared amongst the two extraction methods. lastly, we assessed the reproducibility of both protocols by performing replicate analyses (n = ) of two pooled sera containing and copies/ ml of inactivated sars-cov, respectively. the mean and coefficient of variation were determined and compared. statistical analysis was performed using the medcalc software version . . triplicate viral rna extractions of the transport medium mixture containing copies/ml inactivated sars-cov by the standard protocol of the large volume kit yielded sars-cov rna concentrations of . × , . × and . × copies/ml. by incorporating an external lysis procedural step, the modified protocol yielded . × , . × and . × copies/ml. as the modified large volume protocol yielded results comparable to that of the standard large volume protocol, the former was used for further comparison with the external lysis protocol of the "total nucleic acid" kit which uses a starting sample volume of µl. quadruplicate analysis of the transport medium mixture containing copies/ml sars-cov yielded . × , . × , . × and . × copies/ml when the large volume kit was used. when the total nucleic acid kit was used, the results were . × , . × , . × and . × copies/ ml. the analytical sensitivities contributed by the two kits were next compared. viral rna extracted by both kits was detectable in all five replicates when the sample contained copies/ml sars-cov. however, when the sample contained copies/ml sars-cov, the large volume kit yielded positive detection in all five replicates, while sars-cov was only detected from three replicates when extracted by the total nucleic acid kit. furthermore, when the sample contained copies/ml sars-cov, the large volume kit yielded positive detection in four replicates, while viral rna extractions from the total nucleic acid kit was only positive in one replicate. these data suggest that the modified protocol of the large volume kit has comparable performance with the total nucleic acid kit for the extraction of samples containing high sars-cov concentration, but enabled more sensitive detection when samples containing low levels of sars-cov were extracted. by aligning samples positive and negative for sars-cov in an alternating manner for extraction by the modified protocol of the large volume kit, there was no evidence of carry-over contamination. all the negative samples were indeed tested negative regardless of the magnitude of the sars-cov concentrations (ranging from to copies/ml) in the adjacent wells. thus, the modified protocol of the large volume kit was selected for further evaluation for sars-cov rna extraction from serum. serially diluted inactivated sars-cov isolate in transport medium was extracted by both the column-based manual method and the magna pure lc instrument using the modified large volume protocol with external lysis. the sars-cov concentrations from both series of viral rna extracts were compared using the passing-bablok regression method [ ] . the passing-bablok procedure is a linear regression method developed for method comparison evaluations without dependence on the assignment of either one of the two compared methods as the reference. furthermore, it makes no assumption on the distribution of sample data and measurement errors of the compared methods. the passing-bablok comparison of the sars-cov concentrations in transport medium as extracted by the two methods is presented in figure a . the quantitative relationship can be described by y = - . + . x ( % confidence intervals for the slope, . to . ; and y-intercept, - . to . ). the slope of the regression line being less than suggests there is a proportional negative bias in sars-cov concentration extracted by the automated method when compared with the manual method. a similar comparison was performed for serially diluted sars-cov mixture in pooled sera. figure b illustrates the passing-bablok comparison. the quantitative relationship can be described by y = - . + . x ( % confidence intervals for the slope, . to . ; and y-intercept, - . to . ). the slope of the regression line being less than also suggests the presence of a proportional negative bias in serum sars-cov concentration extracted by the automated method when compared with the manual method. the effect of the automated and manual methods on the overall assay sensitivity was next compared. results for this part of the study are summarized in table . sars-cov rna was detectable from all ten replicates when the serum aliquots containing copies/ml sars-cov were extracted by either methods. for serum containing copies/ml sars-cov, nine and seven of the replicates were tested positive when viral rna was extracted by the manual and automated methods, respectively. for serum containing copies/ml sars-cov, four of the replicates were positive when extracted by the manual method and only one was positive when the replicates were extracted by the automated method. to assess the effects of the two extraction protocols on the precision or reproducibility of the quantitative sars-cov rt-pcr assay, rna extractions by each protocol were repeated times for serum aliquots containing sars-cov concentration well above the detection limit of the assay, namely and copies/ml. results for this part of the study are summarized in table . for serum sars-cov concentration at copies/ml, the overall assay coefficient of variation (standard deviation/mean × %) was . % and . % when rna extractions from the manual and automated methods were quantified, respectively. for serum sars-cov concentration at copies/ml, the assay coefficient of variation was . % for the manual rna extraction and . % when automated rna extraction was used. in an attempt to increase the throughput of a previously developed quantitative serum sars-cov rna rt-pcr assay [ , ] , we evaluated the feasibility of automating the rna extraction procedure through the use of the magna pure lc instrument (roche diagnostics). reagent kits suitable for the extraction of viral rna from serum and plasma as recommended by the instrument manufacturer were evaluated. as the extraction procedure should conform to the biosafety practices recommended by the world health organization [ ] , a modified protocol which incorporates an external lysis processing step for the magna pure lc total nucleic acid large volume kit (roche diagnostics) was developed. the world health organization recommends that nucleic acid extraction procedures for sars-cov involving untreated specimens should first be performed under biosafety level facilities with additional level work practices [ ] . after the viral particles had been lysed or inactivated, the specimens could be handled according to standard level practices. we showed that the use of the large volume kit resulted in better analytical sensitivity when compared with the total nucleic acid kit as evident by the higher rates of positive detection among samples containing low concentrations of sars-cov. furthermore, the magna pure lc system was shown to be free from problems of carry-over contamination. the automated extraction method involving the use of the large volume kit with the external lysis procedure was further compared with the quantitative performance of a previously described manual viral rna extraction method based on the use of the qiaamp viral rna mini kit (qiagen). viral rna extracted from the automated method led to sars-cov concentrations that were consistently lower than that extracted by the manual method across a wide range of sars-cov concentrations in both transport medium and serum. furthermore, better detection rates were observed for serum containing low concentrations of sars-cov when extracted by the manual method in comparison with the automated method. the manual method also contributed to better overall analytical precision as evident by the lower coefficients of variation. we have developed a modified protocol based on the use of the magna pure lc large volume kit (roche diagnostics) which is more sensitive than the predefined external lysis protocol of the magna pure lc total nucleic acid kit (roche diagnostics). albeit the convenience and poten-tial improvement in throughput offered by an automated protocol, our evaluation revealed that the automated viral rna extraction protocol is less sensitive, less precise and produced quantitative results that were consistently lower than those of the column-based manual extraction method. though the reasons for the observed differences in kit performance is not known at present, we recommend the use of the column-based manual rna extraction method as the diagnostic performance of the serum sars-cov rna quantitative rt-pcr test [ , ] is critically associated with the analytical sensitivity contributed both by the rna extraction and rt-pcr components of the test,. furthermore, as it has been previously shown that the serum sars-cov concentration has prognostic implications [ ] and serial assessment is useful for the monitoring of patient progress [ , ] , the superior quantitative performance and precision of the column-based extraction method are additional features that favour its use over the automated protocol. who: summary of probable sars cases with onset of illness from a major outbreak of severe acute respiratory syndrome in hong kong quantitative analysis and prognostic implication of sars coronavirus rna in the plasma and serum of patients with severe acute respiratory syndrome serial analysis of the plasma concentration of sars coronavirus rna in pediatric patients with severe acute respiratory syndrome effects of early corticosteroid treatment on plasma sarsassociated coronavirus rna concentrations in adult patients fully automated nucleic acid extraction: magna pure lc sars molecular detection external quality assurance a new biometrical procedure for testing the equality of measurements from two different analytical methods. application of linear regression procedures for method comparison studies in clinical chemistry, part i who: who post-outbreak biosafety guidelines for handling of sars-cov specimens and cultures the project team is supported by the research fund for the control of infectious diseases (rfcid) from the health, welfare and food bureau of the hong kong sar government. sars, severe acute respiratory syndrome; sars-cov, sars-coronavirus, rt-pcr, reverse transcriptase polymerase chain reaction. patent applications covering aspects of the serum sars-cov rna quantitative rt-pcr test have been filed by the chinese university of hong kong. rwkc and ymdl designed the study. rwkc interpreted the data and drafted the manuscript. yj, gtyc and wbl performed the molecular and data analyses. wl provided the inactivated viral material. atcc provided the expertise for the use of the magna pure lc instrument. all authors read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -h w o authors: cabrera alvargonzalez, jorge julio; rey cao, sonia; pérez castro, sonia; martinez lamas, lucía; cores calvo, olaia; torres piñon, julio; porteiro fresco, jacobo; garcia comesaña, julio; regueiro garcia, benito title: pooling for sars-cov- control in care institutions date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: h w o background: workers and residents in care homes are considered at special risk for the acquisition of sars-cov- infection, due to the infectivity and high mortality rate in the case of residents, compared to other containment areas. the role of presymptomatic people in transmission has been shown to be important and the early detection of these people is critical for the control of new outbreaks. pooling strategies have proven to preserve sars-cov- testing resources. the aims of the present study, based in our local experience, were (a) to describe sars-cov- prevalence in institutionalized people in galicia (spain) during the coronavirus pandemic and (b) to evaluate the expected performance of a pooling strategy using rt-pcr for the next rounds of screening of institutionalized people. methods: a total of , nasopharyngeal swab samples from the total of the residents and workers at care homes in galicia (march to may ) were individually tested using rt-pcr. prevalence and quantification cycle (cq) value distribution of positives was calculated. besides, pools of samples and pools of samples were tested using rt-pcr as well ( positive/pool). pooling proof of concept was performed in two populations with . and % prevalence. results: distribution of sars-cov- infection at care homes was uneven ( – %). as the virus circulation global rate was low in our area ( . %), the number of people at risk of acquiring the infection continues to be very high. in this work, we have successfully demonstrated that pooling of different groups of samples at low prevalence clusters, can be done with a small average delay on cq values ( and . cycles for pools of and samples, respectively). conclusions: a new screening system with guaranteed protection is required for small clusters, previously covered with individual testing. our proposal for care homes, once prevalence zero is achieved, would include successive rounds of testing using a pooling solution for transmission control preserving testing resources. scale-up of this method may be of utility to confront larger clusters to avoid the viral circulation and keeping them operative. severe acute respiratory syndrome coronavirus (sars-cov- ) has caused more than , deaths since late [ ] . screening of care homes has been critical to limit the mortality rate in galicia (spain). direct viral detection by real time rt-pcr was useful to identify people with potential sars-cov- transmission risk. limited stocks and restrictions in test capacity prevented a higher number of rt-pcr tests per day. pooling strategies have proven to preserve sars-cov- testing resources and time with an increase in testing capability of the % for an incidence rate of sars-cov- infection of % or less [ ] [ ] [ ] [ ] [ ] [ ] [ ] , but it could be associated with a decrease in detection [ , ] . main limitations could be the preanalytical step, the sample viral load or the increase of the limit of detection of the individual sample [ ] . the rationale in this study is to develop a new strategy based on initial individual identification of positive coronavirus cases in order to organize low prevalence clusters, followed by a serial pooling strategy testing of these clusters, in order to control areas free of virus circulation, allowing them to be fully operative. nasopharyngeal swab samples were obtained from residents and workers at care homes in galicia (march to may ) and conserved in viral transport medium. the study protocol ( / ) was approved by the galician network of committees of research ethics. samples were mixed : with cobas® omni lysis reagent ( % guanidine thiocyanate) for viral inactivation before individual testing. the open reading frame (orf) /b non-structural region of sars-cov- and the envelope e-gene of sarbecovirus were detected with the cobas® sars-cov- test (roche diagnostics, nj, usa) on the cobas® system (roche diagnostics). for all rt-pcrs in this study, a sample was considered positive if at least one target was detected (quantifying cycle -cq-below ). pooling of samples was performed by the qiagility instrument (qiagen) using - μl of each sample. for positivity assessment, selected positive samples were processed individually and by pooling ( positive/pool) using the magcore® hf plus system (rbc bioscience) and the allplex™ -ncov assay (seegene in, seoul, south korea) on the cfx- system (bio-rad laboratories, hercules, ca, usa). positive samples detected during care homes screening with cq value below the third quartile were selected. for a proof of concept, screening of selected care homes was performed using a pooling strategy by the starlet instrument (microlab) with starmag × universal cartridge kit for automated extraction and pcr set-up. the rna-dependent rna polymerase (rdrp) and nucleocapsid (n) genes of sars-cov- and the e gene were detected. selection was performed by prevalence observed during the screening step. global sars-cov- prevalence and % confidence interval were calculated. distribution of care institutions by sars-cov- prevalence and a summary of cq values of positive samples were calculated. differences in cq values (mean and range) obtained by individual and pooling testing strategies were calculated for each target. the cq values were considered as in case of undetectable result. global sensitivity and reduced number of tests were calculated for screening with pooling. r version . . http://www.r-project.org/ during the coronavirus pandemic, sars-cov- prevalence was obtained by individually testing of , people from galician care homes: , residents, workers and not specified. the mean age of workers and residents was . years (min , max ) and . years (min , max ), respectively. sars-cov- was detected in people ( . , % ci: . - . %). the distribution of institutions by sars-cov- prevalence is shown in fig. . a total of institutions ( , people) had sars-cov- prevalence < %, including institutions ( , people) with prevalence zero. prevalence from to % was observed in institutions ( people), from to % in institutions ( people) and from to % in institutions ( people). cq value distribution for positive samples was as follows: minimum the selection of the optimal pool size should be made before the implementation of pooling testing. with nonoverlapping pools, only positive pools will be retested. the reduction of the expected number of tests depends on the prevalence, the initial pool size and the number of stages for the pooling algorithm. in fact, it is generally accepted that % could be the prevalence threshold to achieve a % reduction in the expected number of tests per individual. on the other hand, the sensitivity and specificity of the global process depends on the analytical characteristics of the test and on the number of times one sample is retested. differences in the expected number of tests per individual, based on mathematical simulations, could help to choose the best set of pool sizes. according to other authors [ ] , for prevalence between and %, sensitivity % and specificity %, the optimal pool size would be between and samples and the optimal sub pool size would be between and samples. in order to minimize the false negative factor for pooled testing recently defined [ ] and to standardize the pooling method, pools of twenty samples (p ) and sub pools of five samples (sp ) were selected. test performance of twenty-six p and fourteen sp was studied. each pool included one positive sample. a total of twenty-six positive samples were tested. mean cq values were . and . for orf /b and e gene, respectively. a boxplot of paired cq values is shown in fig. . all positive samples yielded a global positive result when tested in p or sp . sensitivity of e, rdrp and n gene was, respectively, . % ( / ), . % ( / ) and . % ( / ) for p . sensitivity was . % ( / ) for the three targets for sp . mean delay in the cq values (cq pool-cq positive sample) was . cycles for the p and . cycles for the sp (table ). an example of the amplification curves obtained for one particular sample is shown in additional files , and . the n gene was not detected by allplex™ -ncov assay in one specific sample independently of pooling or individual testing. samples from care homes selected by prevalence were retrospectively tested in pools using the following algorithm: p , sp when positive, individual analysis when positive. a first simulation was performed with samples from % ( % ci: . - . %, / ) prevalence care homes. five p were tested. as positive pools were obtained, sp were processed. two sp were positive, so samples were tested individually. two samples were positive. number of tests was reduced % ( . tests per individual). a second simulation included samples from . % ( % ci: . - . %, / ) prevalence institutions. three p , sp and individual samples were tested. one sample was positive. number of tests was reduced by % ( . tests per individual). a global sars-cov- seroprevalence of % in spain [ ] and a global viral prevalence around % at care homes reported in the present study, suggest that the number of people at risk of acquiring the infection continue to be very high. the role of transmission before symptoms has been shown to be important, presymptomatic / asymptomatic individuals may contribute to it [ , ] . for these reasons their early detection seems critical to prevent further outbreaks. to control the spread of the virus, it is essential to detect as many infected individuals as possible, as quickly as possible to trace down and test possible contacts [ ] . we performed the screening of care homes ( , determinations) in workers and residents using individual testing by rt-pcr. with a prevalence < % for [ , , [ ] [ ] [ ] ] . two tests authorized by the food and drug administration were available at our laboratory. both have shown suitable specificity and sensitivity for clinical diagnosis, but specific studies will be required for assessing their performance in pooling conditions. the choice of the allplex™ -ncov assay was due to the flexibility and adaptability in the automation process useful for future interventions. additionally, although it has been established a moderate mutation rate of sars-cov- [ ] [ ] [ ] , the possibility of detecting three targets could increase the possibilities of detection [ , ] . as previous studies [ , , ] , our results using pools showed an increase of - cycles in the cq value between pooled tests and individual positive samples. the pooling strategy was associated with a decreased sensitivity for individual targets ( - %). nevertheless, it has not carried out loss of global sensitivity in pools for samples included in this study. samples of this study have been selected in order to represent those with cq value within the first three quartiles observed in our population. a % global sensitivity was also achieved when testing care homes with prevalence around %, reducing until % the number of tests. here there is our proposal for introducing the pooling strategy for screening purposes in care institutions: when an institution with prevalence zero is characterized, successive rounds of pooling testing would be the option for transmission control. the maximum interval between rounds would be adjusted to avoid the loss of detection of infected people who could be in a phase of low viral load. the incubation period has been reported to be highly variable with an estimated average of - days [ , [ ] [ ] [ ] [ ] limitations of this study were the limited number of samples included. testing more negative samples would allow us to assess specificity and the risk of contamination along the processing. there is a likelihood of obtaining false negative results when a pooling strategy is introduced. mainly, low quality samples cannot be discarded from the pools and the dilution could reduce the arn concentration below the limit of detection. in this study we have focused on demonstrating that any pool containing until individual samples from highly infectious people would be detected. this work has shown the prevalence of sars-cov- in spanish care homes during the coronavirus pandemic. prevalence differences shown between institutions should address the interventions for viral transmission control. few studies have assessed the performance of pooling for sars-cov- detection by rrt-pcr in real conditions, especially when aiming to keep areas free of virus circulation to be operative and functional. sample pooling could be a new testing strategy relevant for maintaining low level or no transmission among 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biosensors for covid- mass testing large-scale implementation of pooled rna extraction and rt-pcr for sars-cov- detection evaluating the efficiency of specimen pooling for pcr-based detection of covid- pooling of nasopharyngeal swab specimens for sars-cov- detection by rt-pcr coronavirus: los primeros datos de seroprevalencia estiman que un % de la población ha estado contagiada, con variabilidad según provincias estimating the generation interval for coronavirus disease (covid- ) based on symptom onset data temporal dynamics in viral shedding and transmissibility of covid- pattern of early human to human trensmission of wuhan novel coronavirus pooling of samples for testing for sars-cov- in asymptomatic people epidemiological and genomic analysis of sars-cov- in ten patients from a mid-sized city outside of hubei, china. infect dis (except hiv/aids). ; abr the establishment of reference sequence for sars-cov- and variation analysis evolutionary history, potential intermediate animal host, and cross-species analyses of sars-cov- failure of the cobas® sars-cov- (roche) e-gene assay is associated with a c-to-t transition at position of the sars-cov- genome incubation period of novel coronavirus ( -ncov) infections among travellers from wuhan, china incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data the incubation period of coronavirus disease (covid- ) from publicly reported confirmed cases: estimation and application sars-cov- viral load in upper respiratory specimens of infected patients publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations to the servizo galego de saúde, consellería de sanidade and xunta de galicia for supporting this study. to dr. vázquez almuiña for his suggestions. to all the professionals who collaborated in information management, sampling, supplies, contact tracing and organization at all levels. especially to the technician staff of the microbiology department of the chuvi. to laura regueiro for her assistance with the manuscript. received: july accepted: september authors' contributions jg-c and bjr have coordinated with consellería de sanidade the screening in care institutions. bjr, jg-c, jp, jt, jjc, sr, sp participated in the study design. bjr, jjc and sp wrote the first draft. jjc, sr, sp, lm-l, oc-c have processed samples, collected data, participated in the analyses and reviewed the draft. all authors agreed on the final version of the manuscript. no applicable, no external funding. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. all data and materials were obtained working under servizo galego de saúde, consellería de sanidade of xunta de galicia (institutions belonging to our national health public system) and are under their regulations.ethics approval and consent to participate informed consent was not obtained since the samples were remnants of the clinical diagnosis (no additional samples were obtained for this study) and personal information was pseudonymized by people unrelated to the study investigators. this study was approved by galician network of committees of research ethics (protocol number - ). no applicable. the authors: no reported conflicts of interest.author details key: cord- - mtmw z authors: sadeghi, christine d; aebi, christoph; gorgievski-hrisoho, meri; mühlemann, kathrin; barbani, maria teresa title: twelve years' detection of respiratory viruses by immunofluorescence in hospitalised children: impact of the introduction of a new respiratory picornavirus assay date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: mtmw z background: direct immunofluorescence assays (dfa) are a rapid and inexpensive method for the detection of respiratory viruses and may therefore be used for surveillance. few epidemiological studies have been published based solely on dfa and none included respiratory picornaviruses and human metapneumovirus (hmpv). we wished to evaluate the use of dfa for epidemiological studies with a long-term observation of respiratory viruses that includes both respiratory picornaviruses and hmpv. methods: since all children hospitalized with respiratory illness at the university hospital bern have been screened with dfa for common respiratory viruses including adenovirus, respiratory syncytial virus (rsv), influenza a and b, and parainfluenza virus - . in assays for respiratory picornaviruses and hmpv were added. here we describe the epidemiological pattern for these respiratory viruses detected by dfa in ' nasopharyngeal aspirates collected from ' patients during a -year period ( - ). results: addition of assays for respiratory picornaviruses and hmpv raised the proportion of positive dfa results from % to % (p < . ). respiratory picornaviruses were the most common viruses detected among patients ≥ year old. the seasonal patterns and age distribution for the studied viruses agreed well with those reported in the literature. in , an hmpv epidemic of unexpected size was observed. conclusions: dfa is a valid, rapid, flexible and inexpensive method. the addition of assays for respiratory picornaviruses and hmpv broadens its range of viral detection. dfa is, even in the "pcr era", a particularly adapted method for the long term surveillance of respiratory viruses in a pediatric population. respiratory infections are a major cause of morbidity and hospitalizations in children [ ] , of which a significant proportion are caused by viruses [ , ] . surveillance of respiratory viruses is important to predict seasonal epidemics, to define patient risk groups and to allocate hospital resources, as well as to describe the burden and characteristics of emerging viruses [ ] . in the field of virology, the most commonly used diagnostic methods for virus detection are culture, rapid culture (such as shell vial assay), direct immunofluorescence staining of clinical specimens (dfa), and pcr. pcr is attractive due to its high sensitivity and broad range of virus detection. pcr-based studies have suggested the important role of respiratory picornaviruses (rhinovirus and enterovirus) as a leading cause of lower respiratory tract infections in children [ ] , in particular wheezing illnesses such as bronchiolitis [ , ] , wheezy bronchitis [ ] and asthma exacerbations [ ] , but also pneumonia [ ] . in addition, pcr has allowed for the detection of new respiratory viruses, such as hmpv [ ] , which has been implicated in upper and lower respiratory tract infections in children [ ] [ ] [ ] . it is widely believed that nowadays, epidemiological studies on respiratory viruses can only be done with pcr. however, the high sensitivity of pcr is also a limitation of the technique. a significant proportion of asymptomatic children test positive by pcr to respiratory viruses [ ] [ ] [ ] , and picornavirus rna can be detected by pcr up to weeks after an acute infection [ ] . therefore, epidemiological studies based on pcr may overestimate the burden of certain viruses, in particular the common respiratory picornaviruses. dfa has lower sensitivity than pcr, but this may be an advantage for the detection of clinically relevant infections [ , ] . moreover, dfa is more rapid and less expensive than pcr and can therefore be used for real-time, routine surveillance of respiratory viruses, which would be difficult by pcr because of the high costs [ , ] . nevertheless only few epidemiological studies have been published based solely on dfa [ , ] , and none of them included testing for respiratory picornaviruses or hmpv, because antibodies for the detection of hmpv have only recently become available and there are no commercial antibodies for the detection of respiratory picornaviruses. recently, our group reported the validity of immunofluorescence for the detection of picornaviruses directly in respiratory samples using monoclonal antibodies originally designed for the identification of enterovirus in culture [ ] . we aimed to evaluate the use of dfa for epidemiological studies of respiratory viruses, now that assays for respiratory picornaviruses and hmpv are available. we performed a retrospective analysis among pediatric patients hospitalized with respiratory tract infections between and at the university hospital bern. prospective dfa testing in nasopharyngeal aspirates has been used routinely in this institution for adenovirus (adv), respiratory syncytial virus (rsv), influenza a and b (ifa and ifb), and parainfluenza - (piv - ) since , and additionally for hmpv and respiratory picornaviruses since . the study was approved by the ethics committee of the university hospital of bern in accordance with cantonal ethical regulations (nr. e - - ). the study included consecutive respiratory tract samples from children under the age of years, who were hospitalized at the department of pediatrics, university hospital bern, between may st and april th . during the entire study period the pediatrics department had the policy of screening children for respiratory viruses if they were hospitalized with a respiratory illness or if they developed respiratory symptoms during their hospital stay. a total of ' respiratory samples were collected. after exclusion criteria, ' samples remained for the retrospective analysis of dfa results. the exclusion criteria were as follows: samples other than nasopharyngeal aspirates, samples containing less than epithelial cells and samples not tested against the whole viral panel; results of samples from the same patient taken within a time period of days (considered part of the same respiratory episode); results for the month of august , since during this time period practically all respiratory samples were tested by pcr rather than dfa due to the influenza a h n pandemic. all samples were analysed at the institute for infectious diseases, university of bern. the methods used have previously been described [ ] . between blend" (chemicon international/millipore) was introduced for the detection of respiratory picornaviruses. this assay is formally an indirect immunofluorescence assay, as described elsewhere [ ] , and does not allow the differentiation between rhinoviruses and enteroviruses. in november , the dfa metapneumovirus identification kit (diagnostic hybrids) was added to the screening. an epidemiological year was defined as may st to april th of the following year. summer was defined as the months july to september, and winter as january to march. epidemiological years were designated "odd" if the month of january was in an odd year, and they were labelled "even" if the month of january was in an even year. all statistical analyses were performed with the graphpad prism ® software tool (graphpad software, inc.). proportions were compared using the chi-square test. medians were compared with the kruskal-wallis test and dunn's multiple comparison test. a cut-off of p ≤ . , two tailed, was used for all statistical analyses. a total of ' samples from ' patients were analysed. the median age of the study population was months (range - years) and . % were boys. before the addition of dfa for picornavirus and hmpv, the rate of viral detection was dominated by the rsv season, with a yearly average rate of %, peaks of up to % (average %) in winter seasons, and troughs as low as % (average %) during summer time. the addition of dfa for picornaviruses increased the positivity rate, and dampened the seasonal variations. the positivity rate after was on average % in winter and % in summer, with a yearly average of % (versus %; p < . ) ( figure , table ). for comparison, analysis performed on specimens in parallel to dfa screening with the xtag respiratory viral panel (luminex molecular diagnostics) between november and september yielded a positivity rate of %. the higher detection rate by pcr could mostly be attributed to increased detection of respiratory picornaviruses; from the additional positive results were respiratory picornaviruses (unpublished data). respiratory picornaviruses were the most common pathogens detected overall in our study population after the introduction of the assay ( % versus % for rsv; p < . ) ( table ) . they were present year round, with peaks in the spring and the fall. during the summer time, respiratory picornaviruses also accounted for the majority of viral respiratory infections (figure ). low prevalence of respiratory picornaviruses during winter time coincided with the winter peaks caused by rsv, influenza, or hmpv, and this was the only time during the year when respiratory picornaviruses were not the most commonly detected respiratory virus ( figure ) . rsv was the second most commonly detected pathogen after picornaviruses (overall prevalence of %), but the most prevalent virus during the winter months (table , figure ). it manifested a biennial pattern, with large winter seasons in odd years alternating with smaller ones in even years. influenza figure ) . hmpv was detected in . % of all samples collected after november (table ) . yearly hmpv activity varied from being almost absent during the winter season to , to causing yearly winter outbreaks during the following years ( figure , figure ). in early , viral activity surpassed previous years, with more cases observed within months ( cases between december and april ) than during the entire previous period since introduction of the dfa test ( cases between november and november ). we compared the proportion of samples positive for a given virus by age (figure ). respiratory picornaviruses were the most common pathogens in children ≥ years ( - years: % versus % rsv, p < . ; - years: % versus < % other viruses, p < . ; - years: % versus % influenza a, p = . ), and rsv was the most common detected in children < year ( % versus % picornavirus, p < . ). influenza a showed growing importance with age, and was the second most common virus detected in children > years old ( % influenza a versus < % other viruses, p < . ). out of the total of ' samples analysed, were positive for two viruses ( table ) . no sample was positive for more than two viruses. this corresponds to a codetection rate of . % with dfa. with the xtag respiratory viral panel (luminex) . % of samples were positive for two viruses (in % of these a respiratory picornavirus was present). in . % of samples we detected three respiratory viruses. in order to determine the value of dfa in conducting epidemiological studies on respiratory viruses now that assays for respiratory picornaviruses and hmpv are available, we retrospectively analysed the results of years of dfa screening of viral pathogens in hospitalized children with respiratory disease. respiratory picornaviruses were the most common viral pathogens detected overall in our study, with the exception of patients < year in whom rsv was detected more often, confirming the results of recent studies based on molecular methods [ , ] . pcr detection of respiratory picornaviruses suggests a previously unexpected role [ , ] in severe respiratory disease [ , , ] , but this issue is still debated, given that viral genome can also be detected by pcr many weeks after an acute viral infection [ ] , or even in entirely asymptomatic children [ , ] . dfa assays need a high viral load to score positive, so a positive result may be more indicative of an acute infection caused by the virus [ , ] . our high detection rate of respiratory picornaviruses by dfa in hospitalized patients therefore supports their high burden of disease. the introduction of the hmpv and respiratory picornavirus assays in increased the positivity rate of our dfa screening from % to %. for comparison, pcr methods in our laboratory and in the literature usually reach positivity rates of well over %, in large part due to a higher detection of respiratory picornaviruses [ , , , ] . dfa's lower sensitivity, in particular for respiratory picornaviruses, can however be seen as an advantage considering the difficulty in interpreting the clinical significance of pcr-positive results, as described above. another common issue in pcr-based studies is the high codetection rate, with on average about % of samples being positive for two or more viruses [ , ] . with the xtag respiratory viral panel we detected more than one virus in % of samples. in % of these a respiratory picornavirus was present. the clinical significance of these "coinfections" remains unclear because of the high sensitivity of pcr [ , , ] , especially for respiratory picornaviruses. it is difficult to determine whether both or only one and which of the codetected pathogens is causing the acute illness [ ] . our lower rate of codetection by dfa suggests that most codetections detected by pcr may indicate consecutive infections. with the exception of hmpv, our study confirms known patterns of seasonality and age distribution for the studied viruses [ , , , ] . it has been postulated that hmpv has a biennial "large-early" and "small-late" season cycle [ , [ ] [ ] [ ] . we observed an unexpectedly large epidemic in early , which was observed simultaneously in many cities throughout germany (personal communication, prof. o. adams, university of düsseldorf). given the recent discovery of hmpv [ ] , epidemiological studies so far have covered a short time interval and continued monitoring is necessary. currently, pcr is considered the most adapted technique to conduct epidemiological studies on respiratory viruses. in contrast to molecular methods, dfa is low in cost and has a rapid turnaround time [ ] . assays can be performed many times a day, and one does not have to wait for a certain number of samples to be collected to start a run. the samples can be screened for many different viruses simultaneously ("multiplex"). results are usually available within - hours [ ] . these aspects make dfa a method widely and often used in clinical routine, and this concurrently provides the data for ongoing, real-time surveillance of circulating viral pathogens on a large scale. our systematic monitoring led for example to the early detection of the unexpectedly large hmpv epidemic mentioned above. in conclusion, dfa's clinical relevance, flexibility and capacity to conduct "multiplex" assays at very low cost make it a valuable diagnostic tool, and now that its range of viral detection has been broadened to include hmpv and especially respiratory picornaviruses, allows for long-term, systematic, real-time monitoring of local epidemiology in pediatric populations. abbreviations dfa: direct immunofluorescence assay; adv: adenovirus; rsv: respiratory syncytial virus; ifa: influenza a; ifb: influenza b; piv - : parainfluenza viruses - ; hmpv: human metapneumovirus; picorna: respiratory picornaviruses; gender disparity in paediatric hospital admissions etiology of community-acquired pneumonia in hospitalized children respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology why diagnose respiratory viral infection? rhinoviruses infect the lower airways association of respiratory picornaviruses with acute bronchiolitis in french infants association of rhinovirus infection with increased disease severity in acute bronchiolitis rhinovirus-associated wheezing in infancy: comparison with respiratory syncytial virus bronchiolitis community study of role of viral infections in exacerbations of asthma in - year old children osterhaus ad: a newly discovered human pneumovirus isolated from young children with respiratory tract disease epidemiological and clinical features of hmpv, rsv and rvs infections in young children human metapneumovirus and lower respiratory tract disease in otherwise healthy infants and children respiratory viral infection in lower airways of asymptomatic children respiratory pathogens in children with and without respiratory symptoms a case-control study of acute respiratory tract infection in general practice patients in the netherlands persistence of rhinovirus and enterovirus rna after acute respiratory illness in children methods in virus diagnosis: immunofluorescence revisited immunofluorescence versus xtag multiplex pcr for the detection of respiratory picornavirus infections in children epidemiological and clinical study of viral respiratory tract infections in children from italy comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness use of monoclonal antibodies for rapid diagnosis of respiratory viruses in a community hospital viral pathogens of acute lower respiratory tract infection in china rapid detection of respiratory picornaviruses in nasopharyngeal aspirates by immunofluorescence assay respiratory picornaviruses and respiratory syncytial virus as causative agents of acute expiratory wheezing in children ten years' experience with year-round active surveillance of up to respiratory pathogens in children epidemiology of viral infections and evaluation of the potential benefit of om- bv on the virologic status of children attending day-care centers human rhinoviruses: the cold wars resume rhinovirus associated with severe lower respiratory tract infections in children human picornavirus and coronavirus rna in nasopharynx of children without concurrent respiratory symptoms frequency of detection of picornaviruses and seven other respiratory pathogens in infants single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants two-year periodicity of respiratory syncytial virus epidemics in switzerland monto as: epidemiology of influenza biennial spring activity of human metapneumovirus in austria human metapneumovirus infections-biannual epidemics and clinical findings in children in the region of basel, switzerland epidemiologic characteristics and seasonal distribution of human metapneumovirus infections in five epidemic seasons in prospective study of human metapneumovirus detection in clinical samples by use of light diagnostics direct immunofluorescence reagent and real-time pcr we gratefully acknowledge the technical support of the staff of our virology laboratory. authors' contributions cds participated in statistical analysis, data interpretation, drafting of the manuscript, and critical revision of the manuscript. ca participated in study design and sample acquisition. mgh participated in study design, data interpretation, and critical revision of the manuscript. km participated in study design and critical revision of the manuscript. mtb participated in study design, data interpretation, drafting of the manuscript and critical revision of the manuscript. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -z ygy e authors: mccaw, james m; howard, peter f; richmond, peter c; nissen, michael; sloots, theo; lambert, stephen b; lai, michael; greenberg, michael; nolan, terry; mcvernon, jodie title: household transmission of respiratory viruses – assessment of viral, individual and household characteristics in a population study of healthy australian adults date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: z ygy e background: household transmission of influenza-like illness (ili) may vary with viral and demographic characteristics. we examined the effect of these factors in a population-based sample of adults with ili. methods: we conducted a prospective cohort study in community-dwelling australian adults nested within an influenza vaccine effectiveness trial. on presentation with ili, participants were swabbed for a range of respiratory viruses and asked to return a questionnaire collecting details of household members with or without similar symptoms. we used logistic and poisson regression to assess the key characteristics of household transmission. results: participants from multi-occupancy households experienced ili episodes and returned a questionnaire. of these, were the primary case in the household allowing assessment of factors associated with transmission. transmission was significantly associated in univariate analyses with female sex ( % vs. %, risk ratio (rr) = . ( . , . )) and the presence of a child in the house ( % vs. %, rr = . ( . , . )). the secondary household attack proportion (shap) was . , higher if influenza was isolated (rr = . ( . , . )). vaccinated participants who nonetheless became infected with influenza had a higher shap (incidence rr = . ( . , . )). conclusions: the increased shap in households of vaccinated participants who nonetheless had confirmed influenza infection supports the hypothesis that in years of vaccine mismatch, not only is influenza vaccine less protective for the vaccine recipient, but that the population’s immunity is also lower. improved characterisation of the determinants of household transmission of influenza-like illness (ili) remains an important public health priority, particularly in light of the past decade's events in which we have witnessed the emergence of severe-acute-respiratory-syndrome (sars) and the h n influenza pandemic. the evidence base for pandemic influenza public health interventions such as home-quarantine, provision of antiviral agents for post-exposure prophylaxis, school-closure and vaccination builds upon an appropriate understanding of the patterns and timing of infection within the household unit [ ] [ ] [ ] [ ] [ ] [ ] . while influenza viruses, rhinoviruses (hrvs), adenoviruses, respiratory syncytial virus (rsv) and parainfluenza viruses (pivs) are the most common aetiological agents in acute-respiratory-infection (ari) episodes [ , ] , in - % of all ari episodes no known respiratory virus can be identified [ , ] . this is despite discovery of a number of previously undescribed viruses since from clinical specimens from the human respiratory tract (human metapneumovirus [ ] , sars coronavirus [ ] , coronavirus nl [ ] , coronavirus hku [ ] , novel rhinoviruses [ ] , human bocaviruses [ ] and k and wu polyomaviruses [ , ] ). reflecting the need to improve our understanding of household transmission of ari, the literature examining factors associated with household transmission of influenza [ , [ ] [ ] [ ] [ ] has expanded significantly since the h n influenza pandemic [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , including a systematic review and meta-analysis [ ] . donnelly et al. estimated the serial interval for all ili (without laboratory confirmation) from case reports during the pandemic [ ] . only two studies of which we are aware explicitly consider the impact of virus type on infectiousness. principi et al. found less onwards transmission to household members from influenza-negative than influenza-positive children presenting to a hospital emergency department [ ] . similarly, in a cohort study of ari in young children, lambert et. al. observed significant heterogeneity in the proportion of participants' households in which one or more illness events were observed (ranging from % for isolation of hmpv from the child to % for isolation of influenza) [ ] . here we report on the household transmission of a range of viruses in a cohort study of healthy, community-dwelling adults reporting symptoms of influenza-like illness (ili). the study population was sourced from a large, industry sponsored placebocontrolled phase iv efficacy trial of a licensed seasonal trivalent influenza vaccine (fluvax w , csl ltd), conducted prior to the pandemic between march and november . in a previous article [ ] we have described the viral aetiology of ili in the cohort, and examined the influence of virus type, host and spatiotemporal factors on disease symptomatology. full details of subject recruitment and selection for the primary phase iv vaccine efficacy trial have been described previously [ ] . briefly, across study sites in australia and new zealand, healthy adults aged ≥ to < years were recruited for a placebocontrolled trial of a licensed trivalent influenza vaccine (fluvax w , csl ltd) in (clinicaltrials.gov #nct ). study participants were randomized to receive either placebo or vaccine in a : ratio prior to the southern hemisphere influenza season. from an available pool of participants from the primary study at study sites, we consider the persons (adults) who experienced at least one ili episodemeeting the case definition of at least one respiratory symptom (cough, sore throat, runny nose or nasal congestion) and at least one systemic symptom (fever (oral temperature ≥ . °c), feverishness, chills or myalgia) [ ] , and who provided written informed consent for participation in the nested cohort-study which required contribution of a valid biological sample (copan tm dry flocked swab). samples were tested for a range of respiratory viruses using a combination of multiplexed and uniplexed conventional and real-time polymerase chain reaction (pcr) assays [ ] . of those, were members of multi-occupancy households, allowing investigation of transmission within the household. using a non-specific and sensitive ili definition, they reported an episode of ili on occasions. for each episode they returned a study questionnaire (additional file ) detailing respiratory symptoms (see [ ] for details), health seeking behaviour (health care provider consultations, hospital admission, time off work), household characteristics (number of adults (≥ years) and children (< years)) and temporally associated symptoms of ili (if any) in other household members. from herein, we consider the illness episode as the primary unit of analysis. the virology results were classified into virus groups [ ] : influenza (influenza a, influenza b), coronaviruses (oc , e, nl , hku ), picornaviruses, other viruses (parainfluenza viruses ( , , ), adenoviruses, human metapneumovirus (hmpv), bocaviruses, rsv and ki and wu polyomaviruses) or none, where none indicates that no 'tested-for' virus was detected in the participant's sample, as opposed to a missing sample or inconclusive result. study participants' vaccination status (as determined by the primary phase iv trial intervention), physical location (i.e. study site), and socio-demographic characteristics were also recorded. the relevant outcome measure for this sub-analysis was evidence of transmission within households based on experience of symptomatic illness in at least one other member of the study participant's household. study participants were asked to complete the diary on the day following cessation of their own symptoms. they recorded the date of onset of symptomatic ilis in household members from between days prior to the study participant's illness through to the day of diary completion. note that the primary case in the household may or may not be the study participant. for household ili events in which the participant was the primary case ( of ), transmission may or may not have occurred in the household and so it is statistically valid to develop univariate and multivariate explanatory models. for participants who reported recurrent ili episodes during the study in which the same virus was isolated, we exclude all but the first episode. events with co-introduction, defined as onset of symptoms in the participant and one or more household members on the same day, were also excluded. following these exclusions, episodes remained for analysis. for household ili events in which the participant was not the primary case ( of ), while we do know who the introducer was for these events (via the questionnaire data), other household ili events initiated by that introducer that did not involve the participant are unobserved. that is, any events in which a child (or for that matter, any other adult member of the household) introduced an infection that did not infect the study participant are not captured by the study protocol. this observation necessitates the exclusion of all household ili events in which the participant was not the primary case from the analyses. in the one remaining household ili event, the status of the participant (primary or not) was unknown, so the episode was excluded from the analysis. for the episodes in which the participant was the primary case, logistic regression models were used to explore associations between host, demographic or virus variables with any observation of within-household transmission (outcome variable = transmission in household for each recorded ili episode in a study participant). the secondary household attack proportion (shap) was calculated as the proportion of potentially exposed household members (assumed susceptible) experiencing illness, averaged over all recorded episodes. we present descriptive statistics for the shap and its variation by virus, participant and demographic variables. poisson regression models were used to assess the influence of virus, participant and demographic variables on the number of secondary cases within a given household, offset against the number of potentially exposed household members (outcome variable = number of secondary cases in household for each recorded ili episode in a study participant). vaccination status of participants was not included in the primary logistic and poisson statistical analyses due to its known mitigating effect on the likelihood of influenza acquisition [ ] . investigation of the influence of prior immunisation on influenza transmission in 'breakthrough cases' was explored in a secondary analysis by inclusion of a statistical interaction term between vaccination and influenza-identification status. we make an empirical calculation of the mean time between the onset of symptoms in the primary case and the onset of symptoms in the household contacts (the serial interval), for all household ili events, events in which the participant was the primary case, and events in which influenza was isolated from the participant's virological sample. all statistical analyses were conducted in stata/ic . . figure reports characteristics of the multioccupancy households in which transmission did and did not occur. study participants reported two or more ili episodes during the course of the study. for two participants, who both experienced two episodes, picornavirus was isolated on both occasions. we only retain the first episode for each participant. the distribution of household size is dramatically different based on the absence or presence of children within the household ( figure ). in households without children, the distribution is left-skewed (mean household size = . , standard deviation (sd) = . , skewness = . ), while in households with children there is minimal skew (mean household size = . , sd = . , skewness = . ). table summarizes the descriptive statistics (and logistic model results) associated with presence or absence of transmission in the household for the household ili events in which the participant was the primary case. there is marked co-linearity between the variables 'presence of child in household' , 'age-category' and 'household size'. for example, respondents aged - years had significantly greater odds of having a child in the household than those aged - years (or . ( . , )), while no participant aged more than years lived with a child. the relationship between the household size distribution and presence or absence of children is depicted in figure . we retained 'presence of child in household' in the final multivariate model for transmission due to its strong predictive role, intuitive appeal, presumed causal role in our observed (univariate) association with age-category, and previous research indicating an association between transmission and children [ , , , , ] . in the multivariate model, the observed increased risk of transmission with female sex remains (or = . ( . , . ), p = . ). presence of children in the household is both the strongest and most statistically significant factor associated with transmission (or = . ( . , . ), p = . ). within multi-occupancy households, primaryparticipant introductions gave rise to secondary cases among potentially exposed individuals, a secondary household attack rate (shap) of . . of exposed children, in households in which the participant was female, secondary infections were reported among exposed household members (shap = . ), compared with secondary cases among contacts in households in which the participant was male (shap = . ), a riskratio of . ( . , . ), p = . ( -sided fisher's exact). in households with children, secondary infections were reported among exposed household members (shap = . ), compared with secondary cases among contacts in households without children (shap = . ), a risk-ratio of . ( . , . ), p = . . a multivariate poisson regression model ( table ) was used to consider the influence of virus group and demographic characteristics on the number of reported secondary cases within a given household, offset against the number of potentially exposed household members. in correspondence with the logistic regression model for transmission, we include presence of children in the in a secondary analysis, we considered the influence of prior vaccination on the reported number of secondary household cases among participants testing positive for influenza compared with all other participants. in a poisson model for secondary attacks including a statistical interaction between influenza detection (true/false) and vaccination status (placebo/vaccine), the irr for influenza positive cases in those receiving placebo was . ( . , . ), p = . . the factor increase (interaction term) for the irr for vaccinated participants was . ( . , . ), p = . , yielding a net irr for vaccinated influenza-positive participants relative to vaccinated influenza-negative participants of . ( . , . ), p < . ). under the simplifying assumption that the introducer of infection into the household is responsible for all subsequent infections, we may calculate an empiric serial interval, the time from symptom onset in one individual until symptom onset in another. we first consider infections to be related if symptoms are reported within days following onset in the primary case. across all virus-type isolations, we calculate a mean serial interval of . days (sd = . ) for all household ili events (where the study participant was the primary case or otherwise), and . days (sd = . ) for the events in which the participant was the primary case. for the five events in which the primary participant had virologically confirmed influenza and transmission occurred, the mean serial interval was . days (sd = . ). if we limited the maximum serial interval to seven days, the mean was reduced to . days (sd = . ) for all household ili events and . days (sd = . ) for events in which the in our main analysis, we made two assumptions that we now subject to a sensitivity analysis. of the events in which our participant was the primary case, were classified as co-introductions as (at least) one other household member recorded symptoms beginning on the same day. as the latent period for respiratory infections may vary from individual to individual, here we exclude a further episodes in which there was a day interval from onset of symptoms in the study participant to onset of symptoms in another household member. the resulting multivariate models (equivalent to tables and ) are materially unchanged, with the expected slight reduction in statistical power (data not shown). a second assumption made was that, for participants who reported multiple ili episodes during the study period, we only excluded the latter ili episode where the same respiratory pathogen was isolated on both occasions. however, if we conservatively exclude all ili episodes except for the first ( episodes excluded (by virus type: "none", "picornavirus", "influenza", "coronavirus")), again we find no material change in either the logistic or poisson analyses (data not shown). this study, notable in its consideration of a broad range of respiratory pathogens in addition to influenza, demonstrates that household transmission of ili is most strongly associated with host and demographic factors: female sex and the presence of children within the household (tables and ) . the observation that female sex may be associated with increased transmission in the absence of children (rr = . ( . , . ), p = . ) is novel, perhaps suggesting that females are fundamentally more infectious, and not simply more connected to children (in terms of both their susceptibility compared with males if a child introduces infection, and their infectiousness to children if they are the primary household case). behavioural differences whilst ill may drive such an observation. alternatively, mechanisms by which influenza pathogenesis is sex dependent have been investigated [ ] ; whether or not differences extend to infectiousness and susceptibility is not clear. barbara et al. have recently identified that the reporting of respiratory symptoms may be linked with risk perception [ ] and hence gender [ ] . clearly, we cannot exclude the possibility of gender difference in the reporting of within household transmission. the association between transmission and the presence of children within the household is consistent with many other studies [ , , , , ] . the logistic and poisson model findings (tables and ) are consistent with an increased susceptibility for children. this is further supported by the observed increased shap in children compared to adults ( . compared to . , a risk-ratio of . ( . , . ). the shap in adults did not differ by whether or not their household contained children, suggesting that other 'indirect' effects of children are less likely. as our study design limited the analysis to household events with an adult introducer, we were unable to assess the hypothesis that children may be more infectious than adults. our poisson regression analysis on the number of secondary cases given that the participant was the primary case ( table ) indicates that isolation of influenza in the introducer of infection to the household is associated with an increase in the number of secondary cases. we explored this finding more deeply using a statistical interaction model. while somewhat limited by sample size, we found that in placebo recipients identification of influenza was not significantly associated with an increase in the number of secondary cases (irr = . ( . , . ), p = . ), while in vaccine recipients the irr (relative to identification of any other virus, including 'none') was . ( . , . ) , p < . . note that our previous analysis confirms that vaccination is associated with a reduced probability of influenza virus identification [ ] . additionally, 'breakthrough' influenza cases have similar symptoms compared to unvaccinated individuals [ ] . we therefore suggest that our finding of increased transmission may be explained by infection with an influenza virus mismatched to the vaccine-strain (known to be in circulation during the year of study [ ] ), which furthermore may be relatively antigenically novel and to which household members may be expected to have heightened susceptibility. with no virological samples available from other household members and the small number of vaccinated participants who were infected with influenza we are unable to explore this hypothesis further. across all virus types isolated and all household ili events, and assuming that all secondary cases within the household are directly infected by the introducer, we calculate a serial interval of . days. restricting to events in which the participant was the primary case and in which influenza was isolated, we calculate a serial interval of . days. this simple approach, as taken by others [ , ] , cannot account for two important factors: community importation and infection of household members by other non-introducing members (i.e. tertiary cases). while others have partially accounted for these effects [ , , ] , a mechanistically-motivated statistical model is required to fully account for such possibilities, for example as introduced by cauchemez et al. [ ] who determined a serial interval for influenza of . days (sd = . ) compared to . days if calculated directly from empirical observations. with just events, application of these more advanced model-based techniques is not justified for our data. of primary interest for this sub-analysis focussed on transmission is the complication introduced by the monitoring and assessment of ili in an individual rather than a household. ideally, a protocol such as that suggested by klick et al. would have been employed [ ] . the lack of virological assessment of household secondary cases and the broad nature of the question used to establish the secondary case count in each house also contributes to uncertainty with regards to our assignment of temporally associated ili to within-household transmission. both of these limitations were an unavoidable consequence of the nesting of the data-collection protocol within a randomized placebo-controlled trial. furthermore, due to the requested timing for completion of the questionnaire, we cannot exclude the possibility that late onset of secondary (or tertiary etc.) cases may have been missed, particularly if a participant's experience of symptoms was of short duration. similarly, because the study protocol and analyses effectively assume that individuals are infectious until the end of their symptoms, any systematic differences (by virus type) in this relationship may influence the results. however the prompt to return the diary upon symptom cessation was in an effort to ensure timely reporting of questionnaire information to minimise recall bias. conversely, our poisson model implicitly assumes independence among household members, attributing all household infections to the primary case. more advanced model based methods that account for tertiary (and subsequent) cases and community introduction would be warranted with more complete data sources. as with all protocols based purely on symptomatic presentation (as opposed to active surveillance for nonclinical signs of infection such as virological or immunological measures [ , ] ), we are unable to account for potential sub-clinical infection routes, with potential impact for our assessment of whether or not transmission did occur, the primary case status of our participants and determination of the size of the susceptible pool within a given household. conversely, taking a nonsimulation approach to analysis, we are unable to discount our estimate for the shap due to the effects of community introduction into the household, or account for community introduction and tertiary cases in our estimate for the serial interval [ ] . our study sample had an over-representation of females ( of ( . %) individuals for the captured episodes; of ( . %) individuals for the primary-participant introductions). furthermore, it should be noted that the study population were originally volunteers in a randomized controlled trial and as such more likely to represent a group who were more concerned with their health than the general population. eligibility was restricted to healthy adults without recognized risk factors for severe influenza infection. in the context of a literature focussed on the transmission characteristics of laboratory confirmed influenza, our study is the only one that we know of to systematically explore the relationship between transmission and virus aetiology. the analyses suggest that influenza is more transmissible than other causative agents of ili, at least when introduced to the household by an adult. host and demographic factors are also of importance. further studies combining active surveillance of all household members with specimen collection and testing for a range of respiratory pathogens are warranted to elucidate these relationships. additional file : illness visit questionnaire. competing interests pcr has previously served on a scientific advisory board regarding influenza vaccines for csl ltd and has received a grant for an investigator initiated epidemiological study of otitis media from glaxosmithkline australia. he has also received travel support for himself and staff employed by the vaccine trial group to attend and present data at scientific meetings from baxter, glaxosmithkline, sanofi and pfizer. mdn has received travel grants from wyeth australia to present independent research at international meetings, and currently and previously has been the principal investigator for clinical trials sponsored by abbott, baxter, csl, gsk, medimmune, merck, novartis, sanofi-pasteur, wyeth, and pfizer. ml is an employee of csl limited and has an equity interest in the company. authors' contributions jmc, ph and jmv conducted the statistical analyses, provided the primary interpretation of the results and wrote the manuscript. pr was principal investigator on the vaccine efficacy trial within which the sub-study was conducted. tn, jmv, ts, mn, sl and pr conceived the sub-study and secured funding for its conduct, in partnership with csl limited represented by ml and mg. jmv coordinated conduct of the study at multiple sites and oversaw collation of the questionnaire data. ts, mn and sl oversaw conduct of and reporting of the virological testing at the queensland paediatric infectious diseases laboratory. ml was medical monitor for the main vaccine study and a partner investigator on the sub-study, as was mg. all authors contributed to critical revision of the manuscript and have seen and approved the final version of the manuscript. reducing the impact of the next influenza pandemic using household-based public health interventions the transmissibility and control of pandemic influenza a (h n ) virus effective, robust design of community mitigation for pandemic influenza: a systematic examination of proposed us guidance estimating antiviral effectiveness against pandemic influenza using household data modeling targeted layered containment of an influenza pandemic in the united states 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and medical records on signs and symptoms of respiratory illness gender differences in risk perception: theoretical and methodological perspectives annual report of the national influenza surveillance scheme estimation of the serial interval of influenza optimal design of studies of influenza transmission in households. i: case-ascertained studies submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution we thank study staff at the sites for the recruitment of participants and collection of and processing of samples for this sub-study. we thank dale key: cord- -nwnt tde authors: wang, yi; yao, lin; zhang, jian-ping; tang, pei-jun; ye, zhi-jian; shen, xing-hua; xu, jun-chi; wu, mei-ying; yu, xin title: clinical characteristics and laboratory indicator analysis of covid- pneumonia patients in suzhou, china date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: nwnt tde background: sudden exacerbations and respiratory failure are major causes of death in patients with severe coronavirus disease (covid- ) pneumonia, but indicators for the prediction and treatment of severe patients are still lacking. methods: a retrospective analysis of collected cases was conducted and included approximately patients with covid- pneumonia who were admitted to the suzhou fifth people’s hospital from january , to february , . the epidemiological, clinical and imaging characteristics as well as laboratory data of the patients were analyzed. results: the study found that fibrinogen (fib) was increased in ( . %) patients, and when fib reached a critical value of . g/l, the sensitivity and specificity、da, helping to distinguish general and severe cases, were and %、 . %, respectively, which were significantly better than those for lymphocyte count and myoglobin. chest ct images indicated that the cumulative number of lung lobes with lesions in severe patients was significantly higher than that in general patients (p < . ), and the cumulative number of lung lobes with lesions was negatively correlated with lymphocyte count and positively correlated with myoglobin and fib. our study also found that there was no obvious effect of hormone therapy in patients with severe covid- . conclusions: based on the retrospective analysis, fib was found to be increased in severe patients and was better than lymphocyte count and myoglobin in distinguishing general and severe patients. the study also suggested that hormone treatment has no significant effect on covid- . novel coronavirus pneumonia is an acute infectious disease caused by severe acute respiratory syndrome coronavirus (sars-cov- ) infection and is mainly transmitted by respiratory particles [ ] . since the first novel coronavirus pneumonia case was reported in wuhan, china, the new coronavirus spread rapidly across the country, and it is also endemic in many countries around the world, including japan, singapore, thailand and the united states [ ] . thus far, thousands of cases have been confirmed. on february , , the world health organization (who) officially announced that the cause of the new coronavirus pneumonia is a new variant of coronaviruses and named the disease that it caused as coronavirus disease (covid- ). on february , , the national health commission of china temporarily named the pneumonia caused by the new coronavirus new coronavirus pneumonia (ncp). sars-cov- is a coronavirus that belongs to the genus betacoronavirus, with an envelope and particles that are round or oval and often polymorphic, with a diameter of - nm. its genetic characteristics are different from those of severe acute respiratory syndrome coronavirus (sarsr-cov) and respiratory syndrome coronavirus in the middle east (mersr-cov) [ ] . current research shows that it has more than % homology with bat sars-like coronavirus (bat-sl-covzc ) [ ] . when isolated and cultured in vitro, sars-cov- can be found in human respiratory epithelial cells in approximately h, while it takes approximately days to isolate and culture in veroe and huh- cell lines. thus far, we are not fully aware of the pathogenesis of covid- pneumonia, its development process in the body, or its route of transmission. the gold standard for the diagnosis is real-time fluorescence rt-pcr to test whether samples are positive for the nucleic acid of sars-cov- , but this method is time-consuming and has the possibility of false negatives. understanding the early epidemiological and clinical characteristics of covid- pneumonia patients is extremely important for diagnosis; therefore, we conducted a retrospective analysis of cases of covid- pneumonia. a total of cases were collected, including healthy donors, tuberculosis patients and patients with covid- pneumonia. the healthy donors were from the physical examination center and exclude tuberculosis, hepatitis b, hepatitis c, hiv infection and other pulmonary disease such as copd, bronchitis etc., the ages are to . tuberculosis patients are randomly selected inpatients with sputum culture positive about mycobacterium tuberculosis of our hospital and exclude hepatitis b, hepatitis c, hiv infection. covid- pneumonia patients were admitted to the pulmonary department building a of suzhou fifth people's hospital from january , to february , (the diagnosis conformed to the diagnostic criteria (nhc diagnostic criteria (v )) set out in the diagnosis and treatment of pneumonia infected by novel coronavirus ( th trial edition) issued by the general office of the national health commission on february , ). according to the nhc diagnostic criteria (v ), cases were classified as mild, cases as general, cases as severe, and cases as fatal. due to the limited sample size and to reduce sampling error, we combined the mild and general cases into group a, and we combined the severe and fatal cases into group b. we obtained informed consent from the subjects, and the study was approved by ethics committee of suzhou fifth people's hospital (clearance number: - ). covid- pneumonia patients present with chest ct imaging abnormalities, even asymptomatic patients, with rapid evolution from focal unilateral to diffuse bilateral ground-glass opacities that progress to or coexist with consolidations within - weeks. the assessment of both imaging features and clinical and laboratory findings could facilitate the early diagnosis of covid- pneumonia. the classification of the severity of covid- conforms to the nhc diagnostic criteria (v ) and is set out as follows: mild type: clinical symptoms are mild and no pneumonia is present in chest ct images; general type: fever, respiratory tract and other symptoms, chest ct images show pneumonia; severe type (meets any of the following): ① respiratory distress and rr ≥ breaths/min; ② oxygen saturation at rest≤ %; or ③ pao /fio ≤ mmhg; and fatal type (meets any of the following): ① respiratory failure and the need for mechanical ventilation; ②shock; or ③ the combined failure of other organs requires icu monitoring and treatment. blood samples of group a and group b were taken within - h of admission. blood tests were performed, and c-reactive protein, routine biochemical and coagulation parameters, and myoglobin levels were tested. in the control group, fasting venous blood samples were collected for examination on the day of the physical exam. the instrument used to analyze routine blood samples was a sysmex-xn blood analyzer from japan sysmex corporation, and the reagent used was a supporting product of the company. the instrument used to detect crp was a jet-istar immunoassay analyzer from zhonghan shengtai biotechnology co., ltd., and the reagent was a supporting product of the company. the instrument used to detect the coagulation index was a ca hemagglutination apparatus from japan sysmex corporation, and the reagent was a supporting product of the company. the instrument used to detect myoglobin was a roche , and the reagent was a supporting product of the company. statistical processing spss . statistical software was used for data processing. measurement data for normal distributions is expressed as ±s; comparisons between groups were performed by t test, the test level was α = . (both sides); the difference was statistically significant with p < . ; the measurement data of skewed distribution is expressed as "median (quartile) [m (q , q )]", and the differences between groups were compared by using the rank sum test. spearman's correlation was used for correlation analysis. the receiver operating characteristic (roc) curve were drawn, the area under the roc curve (auc) were calculated, and the absolute value of lymphocytes, fibrinogen, myoglobin and other indicators selected to alert the best cutoff value of the ncp and the corresponding sensitivity and specificity. comparison of the general patient information of group a and group b sixty-six patients with covid- pneumonia manifested chest ct imaging abnormalities, even asymptomatic patients, with rapid evolution from focal unilateral to diffuse bilateral ground-glass opacities that progressed to or coexisted with consolidations within - weeks. combining the assessment of imaging features with clinical and laboratory findings could facilitate the early diagnosis of covid- pneumonia. sixty-seven patients were collected in the study, the average age was years. there were patients in group a, with males, and patients in group b, with males. no significant difference in sex was observed between the two groups (p > . ). ninety percent of the patients in this study had a history of exposure to the hubei epidemic area, with an incubation period of - days and a median incubation period of . days ( . - . ). twenty-two percent of patients had chronic underlying diseases, of which hypertension and diabetes accounted for the highest proportion of chronic diseases, and there was no significant difference between the two groups (p > . ) ( table ) . of the patients, % had fever symptoms. there were more fever patients (> . °c) in group b; the duration of fever was longer in group b than in group a, and the difference was statistically significant (p < . ). in addition, cough ( . %), fatigue ( . %) and shortness of breath ( . %)were the most common symptoms, and a few patients had diarrhea ( %), which was more common in group a than in group b. according to the imaging findings, . % of the patients had lung lesions, among whom the cumulative number of lung lobes with lesions in group b was statistically significantly higher (p < . ) than that in group a (fig. ). the median time for the progression of lesions in covid- pneumonia patients was . days ( . , . ), and there was no significant difference between the two groups (p > . ) ( table ) . among the covid- pneumonia patients, patients had leukocyte abnormalities, of whom ( . %) had decreased white blood cells and ( . %) had increased white blood cells. a total of ( . %) patients had an increased neutrophil ratio, and the absolute value of lymphocyte counts was decreased in ( . %) patients ( ( . %) in group a and ( . %) in group b). the differences in the above indicators were statistically significant (p < . ). c-reactive protein increased in of patients, especially in group b, and the difference was statistically significant (p < . ). abnormal liver function was found in ( . %) patients (alanine aminotransferase (alt) and aspartate aminotransferase (ast) increased in and patients, respectively, with the maximum value of alt being u/l and that of ast being u/l). liver function indexes in group b were higher than those in group a, with statistically only patients had increased renal function. in addition, fibrinogen (fib), d-dimer, and myoglobin were significantly increased in patients in group b, which was significantly different from group a (p < . ) ( table ) . in a comparison of patients in group a with patients in group b, the lymphocyte count absolute value (l), myoglobin (mb) and fibrinogen of covid- pneumonia patients in group a were significantly higher than those in group b, with statistically significant differences (p ≤ . ) ( table ). in a comparison of group a with the healthy control group, the l was significantly higher in group a than in the healthy control group (p < . ). compared with group a, there was no significant difference in the absolute value of lymphocytes and fibrinogen in the tuberculosis group (p > . ) ( table ) . with the data of both group a and group b plotted in a receiver operating characteristic (roc) curve, the data of group a and group b were compared. the roc curve was used to evaluate the l, mb, fib and other indicators for the prediction of severe disease in patients with covid- pneumonia (fig. ) . the optimal truncation values (with maximum youden index) were selected, and the optimal truncation values of l, mb and fib were calculated as . * ^ /l, . ng/ml and . g/l, respectively. the area under the roc curve (auc) for l was . , for mb was . and for fib was . . when the optimal truncation value of the fib index was selected, the sensitivity, specificity and da for the prediction of severe disease in covid- pneumonia patients were %, . and . %, respectively, and the sensitivity, specificity and da of fib were significantly higher than those of l and mb (table ) . an analysis of the correlation of the cumulative number of lung lobes with lesions with l, mb, and fib in covid- pneumonia patients indicated that the cumulative number of lung lobes with lesions was positively correlated with mb and fib and negatively correlated with l ( table ) . sixty-one ( . %) patients were treated with oxygen therapy, among which nasal catheter oxygen was the main treatment in group a, while noninvasive ventilator-assisted ventilation or high-flow oxygen was needed for some patients in group b who were in respiratory failure, and the difference was statistically significant (p < . ). only ( . %) patients were treated with hormone therapy, of whom the proportion in group b was larger; the time of hormone use was longer in group b than in group a, and the difference was statistically significant (p < . ). the vast majority ( . %) of patients received antiviral treatment immediately after admission, with no statistically significant difference between the two groups (p > . ) ( table ) . at the same time, we also analyzed the effects of hormone therapy and nonhormonal therapy. compared with nonhormonal therapy, hormone therapy did not promote key indicators (fig. ). current research indicates that sars-cov- is more than % homologous to bat-sl-covzc [ ] . therefore, it is considered that sars-cov- was transmitted by bats [ ] , which needs further research confirmation. for describing the clinical and laboratory characteristics and analysis the oxygen therapy and hormone therapy, we conducted a retrospective analysis of cases of covid- pneumonia in suzhou, china. this study included patients with covid- diagnosed in the suzhou fifth people's hospital, which shows that, similar to chen's study [ ] , patients with sars-cov- infection were mainly middle-aged and elderly individuals, with a median age of years ( . . ), and ( . %) were males. ninety percent of the patients had a history of exposure to the hubei epidemic area, with an incubation period of - days and a median incubation period of . days ( . - . ), which is also similar to wang's study [ , ] . a total of . % of patients had underlying chronic diseases, of which hypertension and diabetes accounted for a higher proportion of chronic diseases, and there was no significant difference between the two groups (p > . ). fever is the most common clinical symptom of covid- pneumonia, with ( . %) cases in patients observed. fever occurred in the early stage of the disease, and ( . %) patients had a body temperature ≤ . °c, which was more common in group a. ten patients ( . %) had a body temperature ≥ . °c in group b, which was statistically significantly higher (p < . ) than that in group a. in addition, the duration of fever in group b was significantly longer than that in group a; the median fever duration in group b was . days ( . - . ), and the difference was statistically significant (p < . ). cough ( . %), fatigue ( . %) and shortness of breath ( . %) were also common, among which shortness of breath was more common in group b, while fatigue was more common in group a, and the differences were statistically significant (p < . ). these symptoms are considered to be related to lung lobe invasion in severe patients. diarrhea ( . %) and muscle soreness ( . %) were less common in patients; however, we still need to be alert to the patients who are diagnosed with mainly gastrointestinal symptoms, pay attention to strengthening protection, and conduct timely sars-cov- nucleic acid testing for patients with a history of epidemiology. the retrospective analysis of patients with covid- pneumonia indicated that ( . %) patients showed lung lesions on ct chest images, with multiple sites of distribution, and lesions were seen in both lungs and subpleural areas [ ] , mostly showing ground-glass note: comparing with group b, a p< . for indicators in group a, b p< . for indicator of l in healthy control group, comparing with healthy control group, p< . for indicator of l in comparing with tuberculosis group, comparing with group a, c p> . for indicators in tuberculosis group opacities, consolidation, interstitial changes, and interlobular septal thickening [ , ] (fig. ) . the median time from illness onset to lesion progression in covid- pneumonia patients was . days ( . , . ). the median number of cumulative lung lobes with lesions was . ( . , . ), and the cumulative number of lung lobes with lesions in group b was significantly higher than that in group a, with a statistically significant difference (p < . ). laboratory data showed that the wbc, n%, and crp in group b covid- pneumonia patients were significantly higher than those of group a patients, and the differences were statistically significant (p < . ). the increase in these levels is considered to be caused by systemic inflammation, which was relatively obvious in severe patients, but the possibility of bacterial infection or secondary fungal infection in some severe patients could not be ruled out. the absolute value of lymphocyte counts decreased significantly in . % of covid- pneumonia patients, especially in group b, compared with group a, with a statistically significant difference, suggesting that cellular immune function decreased in the early stage in covid- pneumonia patients, especially in severe patients. in addition, the l in the group a and tuberculosis group has no significantly different, but tuberculosis mainly causes the reduction of cd + t cells, while the covid- is cd + t cells. although the performance of them is similar, the types of lymphocytes which decrease are different. a total of ( . %) patients had abnormal liver function (alt maximum value of u/l, ast maximum value of u/ l). the liver function index of group b was statistically significantly higher (p < . ) than that of group a, suggesting that severe patients are more likely to have liver dysfunction. most patients had normal renal function, and only ( . %) had abnormal renal function indicators, both of whom were severe patients, suggesting that sars-cov- may not cause significant kidney damage. thirty-six ( . %) patients had hypoproteinemia, and serum albumin (alb) in group b was significantly lower than that in group a, suggesting that the function of synthetic alb was decreased by liver function damage in severe patients. in addition, the basal metabolic rate and resting energy consumption of severe patients were high, and alb catabolic metabolism was accelerated. therefore, attention should be paid to the treatment of decreased albumin levels in severe patients. fib is a coagulation factor mainly secreted into the blood by liver cells. it is involved in the blood coagulation process and is a key factor in thrombosis. in addition, fib is also a stress response protein fib [ ] . a total of ( . %) patients had elevated blood fib content, suggesting that sars-cov- infection could lead to a stress response in the body, promote the synthesis and release of fib by liver cells and macrophages, and thereby increase serum. in addition, fib in group b was significantly higher than that in group a (p < . ). the increase in fib in covid- patients is considered to be caused by systemic inflammation and is relatively obvious in severe patients. d-dimer is a product of fibrinolytic crosslinked fibrin clot formation. elevated d-dimer levels indicate high blood clotting and are a sensitive marker of acute thrombosis. this study shows that the value of ddimer in group b was significantly higher than that in group a (p < . ). it is considered that harmful substances such as viruses and endotoxins can activate coagulation factor xii after entering the blood, activate the endogenous coagulation system, and activate the fibrinolytic system, which leads to an increase in d-dimer. severe patients often have systemic inflammation, which could cause endothelial function to be impaired, resulting in platelet aggregation and the release of coagulation factors, thereby leading to the hyperfunction of the fibrinolytic system. the increase in fib and d-dimer indicates that preventive anticoagulation therapy should be given to covid- pneumonia patients, especially severe patients. treatment results indicated that ( . %) patients were treated with oxygen therapy, among which nasal catheter oxygen was the main treatment ( ( . %) patients in group a, ( . %) patients in group b). some patients in group b were associated with respiratory failure; thus, noninvasive ventilator-assisted ventilation or high-flow oxygen was needed, and the difference was statistically significant (p < . ). all patients received antiviral treatment immediately after admission, while only ( . %) patients were treated with hormone therapy, most of whom were from group b. additionally, the time of hormone use was statistically significantly longer (p < . ) in group b than in group a. in this study, l, mb and fib were selected as the meaningful laboratory indicators to help distinguish between general and severe covid- . the results showed that the values of l, mb, and fib in group b were statistically significantly different (p < . ) from those in group a. when fib reached a critical value of . g/l, the sensitivity and specificity were and . %, respectively, which were significantly better than those of l and mb. it can be seen from the roc curve that fib had the largest area under the roc curve ( . ), indicating that fib could be used as an effective laboratory indicator to help distinguish general and severe covid- , but the specificity of fib was low. therefore, a comprehensive diagnosis should be made based on clinical manifestations and meaningful data. for patients with chest tightness, l < . * ^ /l, and fib significantly higher than . g/l, we should be alert to the possibility that they may subsequently progress into severe covid- or have severe tendencies, which will help in the timely clinical assessment of the condition and the adjustment of treatment. our study has some limitations. this study did not cover all the covid- patients in our hospital, some patients were excluded but were not diagnosed, the number of selected patients was relatively small, and there might be biasing factors in the case selection. therefore, the findings of statistical tests and p values should be interpreted with caution, and it is important to note that nonsignificant p values do not necessarily rule out the difference between group a and group b patients. in addition, the patients' symptoms of discomfort are highly subjective; therefore, there might be errors in the reporting of clinical symptoms. some patients did not seek medical treatment in time; thus, the imaging performance may be lagging. therefore, further research is needed to obtain a full picture of covid- pneumonia. our study demonstrates the clinical features of patients with severe and mild disease. patients with severe disease higher peaks of fever, longer periods of fever and more lung lesions. we also found that fib would be a better marker for indicating the progression of this disease, and with better sensitivity and specificity than lymphocyte counts and myoglobin tests. this study further characterizes the clinical features of covid- pneumonia patients and shows that fib would be a potential clinical predictor for covid- patients. novel coronavirus outbreak in wuhan, china, : intense surveillance is vital for preventing sustained transmission in new locations the progress of novel coronavirus event in china sars-cov- , sars-cov, and mers-cov: a comparative overview potent binding of novel coronavirus spike protein by a sars coronavirus-specific human monoclonal antibody overlapping and discrete aspects of the pathology and pathogenesis of the emerging human pathogenic coronaviruses sars-cov, mers-cov, and sars-cov- _ncov: rapid classification of betacoronaviruses and identification of traditional chinese medicine as potential origin of zoonotic coronaviruses epidemiological and clinical characteristics of cases of novel coronavirus pneumonia in wuhan, china: a descriptive study incubation period and other epidemiological characteristics of novel coronavirus infections with right truncation: a statistical analysis of publicly available case data clinical characteristics of hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china imaging and clinical features of patients with radiological findings from patients with covid- pneumonia in wuhan, china: a descriptive study analysis of ct features of children with novel coronavirus infection aberrant coagulation causes a hyper-inflammatory response in severe influenza pneumonia publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank all patients, clinicians, and support staff who participated in this study.authors' contributions yw, xy, jcx and myw conceived and designed the study. yw, ly and jcx contributed to the literature search. yw, ly and xy contributed to data collection. yw jcx and xy contributed to data analysis. xy and myw contributed to data interpretation. yw and jcx contributed to the figures. jpz, xhs, pjt, zjy, and myw contributed to writing of the report. all authors read and approved the final manuscript. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. all procedures performed in studies involving human participants were in accordance with the ethical standards of the institutional and/or national research committee and with the helsinki declaration and its later amendments or comparable ethical standards. all participants written informed consent for participation in the study was obtained, if the participants are children (under years old) informed consent will from their parent or guardian. the study was approved by ethics committee of suzhou fifth people's hospital and the clearance number was - . not applicable. the affiliated infectious hospital of soochow university, , guangqian road, suzhou, jiangsu, p. r. china . the fifth people's hospital of suzhou, suzhou, china.received: may accepted: october the authors declare that they have no competing interests. key: cord- -fawcn em authors: liu, chunyan; xiao, yan; zhang, jing; ren, lili; li, jianguo; xie, zhengde; xu, baoping; yang, yan; qian, suyun; wang, jianwei; shen, kunling title: adenovirus infection in children with acute lower respiratory tract infections in beijing, china, to date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: fawcn em background: human adenoviruses (hadv) play a significant role in pediatric respiratory tract infections. to date, over types of hadv have been identified. here, hadv types are characterized in children in the beijing area with acute lower respiratory tract infections (alrtis) and the clinical features and laboratory findings of hospitalized hadv-infected cases are described. methods: respiratory specimens were collected from pediatric patients with alrtis in the emergency department or from those admitted to beijing children’s hospital between march and december . infections with common respiratory viruses were determined by pcr or rt-pcr. hadv positive samples were further typed by pcr and sequencing. results: among patients with alrtis, ( . %) were found to have hadv infection. hadv infection was primarily confined to children ( . %) less than years of age. a total of different types of hadv were detected throughout the study period, with hadv-b ( . %) and hadv-b ( . %) as the most prevalent types, followed by hadv-c ( . %) and hadvc ( . %). newly emerging and re-emergent types or variants, hadv-b (n = ), hadv-c (n = ), and hadv-b p (n = ), were identified. results also included the reported first case of co-infection with hadv-c and hadv-c . clinical entities of patients with single hadv infection (n = ) were similar to those with mixed hadv/respiratory syncytial virus (rsv) infections (n = ). patients with hadv-b infection had longer duration of fever and higher serum levels of muscle enzymes than hadv-b -infected patients. conclusions: during the study period, hadv-b and hadv-b were the predominant types identified in pediatric alrtis. hadv-b infection tends to have more severe clinical consequences. the presence of newly emerging types or variants and co-infection with different types of hadv highlights the need for constant and close surveillance of hadv infection. acute lower respiratory tract infections (alrtis) are the leading cause of pediatric morbidity and mortality worldwide, particularly in developing countries. in infants and young children, alrtis are most frequently caused by respiratory viruses. one such virus, human adenovirus (hadv), plays a significant role in pediatric respiratory tract infections, accounting for - % of the overall respiratory illnesses and - % of the pneumonias [ , ] . although most cases are mild and indistinguishable from other viral causes, alrtis caused by hadv can be severe, or even fatal, and are associated with the highest risk of long term respiratory sequelae [ ] . thus, hadvassociated alrtis are of particular interest to both clinicians and researchers. hadv are responsible for a wide spectrum of clinical diseases, including respiratory illness (both upper and lower respiratory tract), pharyngoconjunctival fever, conjunctivitis, cystitis, gastroenteritis, and neurologic and venereal disease [ ] . hadv were first isolated in as respiratory pathogens [ , ] . to date, over types of hadv have been identified and classified into seven species (a to g) [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . cases of severe infection, outbreaks in closed populations, and even epidemic outbreaks have been associated with the newly emerging or re-emergent types or variants [ ] [ ] [ ] . interestingly, different types of hadv display various tissue tropisms that correlate with different clinical manifestations of infection. hadv infections of the respiratory tract are predominantly caused by hadv-b (including subspecies b and b ), hadv-c, or hadv-e. the predominant types vary among different countries and regions and they change over time because transmission of novel strains between countries or across continents may occur [ ] . type identification is critical to epidemiological surveillance, detection of new strains, and understanding of hadv pathogenesis. however, because most clinical laboratories do not type the isolates, there is little published information about epidemiologic and clinical features of hadv infections by type in children with alrtis. to identify hadv types and species in children with alrtis in beijing area and to characterize clinical features and laboratory findings of hospitalized hadvinfected cases, respiratory specimens were collected from hospital-admitted pediatric patients with alrtis and typed hadv positive samples using pcr and sequencing. the study protocol was approved by the ethical review committee of beijing children's hospital. individual written informed consent was obtained from the parents or guardians of all participants. from march to december , pediatric patients with alrtis who presented in emergency department or were admitted to respiratory department or intensive care unit, beijing children's hospital, were recruited for the study. the study site hospital is a tertiary comprehensive pediatric hospital with over beds and more than twenty clinical departments. alrtis were defined as the presence of signs and symptoms of respiratory tract infection (i.e., fever, coughing, rhinorrhea, oropharyngeal hyperemia, swelling of tonsils), and lower respiratory signs (tachypnea, dyspnea, retractions, or wheezing/rales upon auscultation). the patients were diagnosed with bronchitis, bronchiolitis or pneumonia. chest x-rays were taken for all patients and the criteria for diagnosing pneumonia are the presence of lung infiltrates indicated by chest radiography. nasopharyngeal aspirate or throat swab specimens were collected in virus transport media from each patient. no repeated samples were collected from any patient. all samples were stored at − °c prior to use. total nucleic acids (dna and rna) were extracted from μl nasopharyngeal aspirate or throat swab specimens using the nuclisens easymag™ automated extraction system (biomérieux, marcy l'etoile, france) according to the manufacturer's instructions and eluted in μl elution buffer. the presence of common respiratory viral agents, including parainfluenza virus (piv) type - , influenza virus (ifv), respiratory syncytial virus (rsv), human rhinovirus (hrv), enterovirus (ev), human coronavirus (hcov e, nl , hku , and oc ), human metapneumovirus (hmpv), human bocavirus (hbov), and hadv was determined by multiplex rt-pcr, single rt-pcr, or pcr assays as previously described [ , ] . blank virus transport media here served as a negative control for nucleic acid extraction and pcr. hadv positive samples were further amplified using a nested pcr procedure that targeted hyper variable regions - of the hexon gene as described by lu and erdman [ ] . expected amplicons ranged from bp to bp (secondary amplification) in length. sequencing was performed in both directions using the amplification primers. sequences were proof read and assembled using seqman software v . . (dnastar inc., wi, u.s.). for assignment of molecular identity and identification of the closest match, sequence alignment was performed using the basic local alignment search tool (blast) against ncbi genbank database (http://www.ncbi.nlm.nih.gov). clinical data were retrospectively recorded by careful analysis of patient medical files in beijing children's hospital, using a predefined microsoft excel spreadsheet. patients' demographic, clinical, and radiologic findings were collected. continuous variables were summarized as means ± standard deviations (sd) or medians. for categorical variables, percentages of patients in each category were calculated. differences between groups were assessed using pearson's chi square test or fisher's exact test for categorical variables and the one way anova, independent-samples t test, mann-whitney u test, and kruskal-wallis test for continuous variables. all analyses were performed using spss software, version . (ibm corporation, ny, u.s.). all tests were calculated in a two-tailed manner and a p value of < . was considered statistically significant. from march through december , a total of patients with alrtis ( with pneumonia, with bronchitis and with bronchiolitis) were enrolled in this study. the mean age of study participants was . ± . years (median year; age range, . month to years and months). there were male participants with a male-to-female ratio of . : . at least one respiratory virus was detected in nasopharyngeal aspirate or throat swab specimens of ( . %) enrolled participants. rsv ( . %) was the most commonly detected viral pathogen, followed by hrv ( . %) and piv ( . %). one hundred and ninety-four patients ( . %, / ) were found to have hadv infection, representing . % ( / ) of patients with positive respiratory samples. male paticipants were more likely to be infected with hadv ( boys and girls, male to female ratio = . : ). the mean age of infection was . ± . years (median, year; age range, month to years). most of hadv-infected cases ( . %) were under years of age and the highest percentage of hadv infections ( . %) occurred in infants (age group -< year), followed by the age group -< years ( . %). additionally, one or more other respiratory viruses were detected in . % (n = ) of hadv-infected participants. dual viral infection was identified in cases, triple infection in cases, quadruple in and quintuple in . rsv (n = ) was the most frequently co-detected virus, followed by hrv (n = ) and piv (n = ). hbov (n = ), hcov (n = ), ifv (n = ), ev (n = ), and hmpv (n = ) were also found to be co-infected with hadv. one hundred and ninety-four hadv-positive specimens were all successfully typed by hexon gene amplifying and sequencing. throughout the study period, four species (a, b, c, e) of hadv, including different types were identified. additionally, hadv-b (n = ; . %) and hadv-b (n = ; . %), which belong to species b, were the most prevalent hadv types, accounting for . % of all hadv-associated infections. hadv-c ( . %), hadv-c ( . %), hadv-c ( . %), hadv-b ( . %), hadv-c ( . %), hadv-c ( . %), hadv-a ( . %), hadv-b ( . %), and hadv-e ( . %) were also detected. interestingly, sequencing results from one specimen showed superimposed peaks in the chromatograms. to confirm the possibility of multiple hadv strains in that sample, pcr products were cloned and sequenced further. distinct hexon genes of different types (hadv-c and hadv-c ) were verified. hadv detection rate varied through the years, ranging from . % in to . % in (fig. ) . additionally, although hadv was detected throughout the year, cases commonly peaked in winter and spring season (fig. ) . furthermore, different types of hadv did not remain constant across the whole study period (fig. ) . specifically, hadv-c , −c , −b , and -b were detected throughout the study; hadv-c in all years except ; hadv-c and hadv-c in years , , among the hadv-positive cases, hospitalized cases were included in the clinical analysis, and cases from the emergency department for which the details of the medical records were not available were excluded. pneumonia (n = , . %) was the most common clinical diagnosis, followed by bronchitis (n = ) and bronchiolitis (n = ). additionally, almost all hospitalized hadv-infected patients presented with fever ( / , . %) and coughing ( / , . %) ( table ). the mean peak body temperature was . ± . °c (n = , range . − . °c) and febrile seizures were noted in two febrile patients. in addition to respiratory symptoms, diarrhea, vomiting, skin rash, and conjunctivitis were noted in . %, . %, . % and . % of the patients respectively. twenty-two patients ( . %) had underlying diseases, which included congenital heart disease ( patients), airway anomaly (malacia, stenosis, patients), bronchopulmonary dysplasia ( patient), asthma ( patients), or primary immunodeficiency ( patient). seventeen patients ( . %) required admission to the intensive care unit and patients ( . %) received mechanical ventilation including both noninvasive (n = ) and invasive (n = ) modes. analysis revealed that the mean value of white blood cell (wbc) count was . ± . × /l ( table ) . leukocytosis (wbc > . × /l) was observed in ( . %) patients. eighty-one patients ( . %) had elevated serum c-reaction protein (crp). given rsv was the virus most frequently co-detected with hadv, differences among patients with single hadv infection (n = ) and those with hadv/rsv coinfections, including both dual infections (n = ) and multiple infections (hadv/rsv with one or more other respiratory viruses, n = ), were assessed ( table ). the mean age of patients with multiple infections ( . ± . ) and dual infections ( . ± . ) was significantly younger than those with single hadv infection ( . ± . ) (p = . ). however clinical characteristics and laboratory findings showed no significant differences among different groups. because hadv-b and hadv-b were the most predominant type among patients with hadv infection, the clinical entities of patients with single hadv-b infection (n = ) and those with single hadv-b infection (n = ) were also compared to exclude the possible effect of other respiratory virus infection (table ) . patients with single hadv-b infection showed longer duration of fever ( . ± . vs . ± . , p = . ) than patients with hadv-b alone. immunoglobulin was more frequently used in single hadv-b infected patients than in single hadv-b group (p = . ). patients with hadv-b alone also tend to require longer hospital stay ( . ± . vs . ± . , p = . ) than those with single hadv-b infection, although no significant difference was found. biochemical tests demonstrated aspartate aminotransferase (ast), alanine aminotransferase (alt), lactate dehydrogenase (ldh) and hydroxybutyrate dehydrogenase (hbdh) levels were significantly higher in the single hadv-b infected group. two patients died in-hospital. both of them required icu admission and died of multiple organ failure. one was a month-old boy with multiple underlying conditions of complex congenital heart disease and tracheobronchial malformation. the other was a previously healthy month-old boy. analysis indicated that both fatal patients were infected with hadv-b but no other respiratory viruses. hadv is a significant causative agent of respiratory tract illnesses in both children and adults. here, the molecular showed that the hadv infection rate in the current study population was . %, which was consistent with previous reports from china and other countries [ ] [ ] [ ] . results showed that most patients with hadv infection were younger than years ( . %), which is similar to numbers reported in previous studies [ , , , [ ] [ ] [ ] . this may because the immune systems of young children are not well developed, which leaves them prone to more severe hadv disease. this may also suggest that schoolage children are exposed to the most common endemic types of hadv early in life, thereby establishing a protective immunity resulting only in mild clinical symptoms, such that upper respiratory tract infection does not require care in an emergency department or hospital in this age group. over a period of years, different types of hadv belonging to species (hadv-a, b, c, e) were identified in respiratory specimens from children with alrtis. hadv- and hadv- of species b comprised the most prevalent types and presented throughout the duration of the study. although these results were consistent with previous reports from korea and argentina [ , , ] , investigations from croatia, peru, canada, france showed that species c predominated [ , , , ] . this difference in type prevalence may be attributed to difference in regions, year of study, and population recruited. notably, some newly emerging or re-emergent types or variants were here identified, although only in rare cases. five patients were found to have hadv-b (formerly named hadv- a), which is an uncommon re-emergent type that once caused an outbreak of respiratory tract infection in a senior high school in shanxi province, china in , including one fatal case [ ] . subsequently, hadv-b has been associated with several outbreaks of respiratory disease in other provinces in china [ ] . an emerging variant, hadv-b p (formerly known as a), was also found. recently, hadv-b p has been associated with several large outbreaks of acute respiratory infection, which included severe and even fatal cases in the united states and europe [ , ] . additionally, in , an outbreak of febrile respiratory illness that affected students in gansu province, china was reported to be caused by hadv-b p [ ] . one hadv-b infected patient who presented with bronchopneumonia and required hospitalization in april was identified. by further sequencing the fiber gene (data not shown), this strain was confirmed to be hadv-b p because it contained a unique characteristic -nuleotid deletion in fiber knob region as reported by kajon et al. [ ] . last, this is the first report of detection of hadv-c in respiratory samples collected from pediatric patients with alrtis and the first of co-detection of hadv-c with hadvc- . hadv-c (formerly designated strain ) was first isolated from the feces of a healthy child as part of an acute flaccid paralysis surveillance program. computational genomic and bioinformatic analysis showed hadv-c to be a recombinant virus with fiber gene nearly identical to hadv-c and a unique hexon distinct from all viruses in species hadv-c [ , ] . out of the three hadv-c -infected cases identified here, one was a previously healthy month-old male who presented with bronchopneumonia and conjunctivitis requiring hospitalization. because only a small number of hadv-c positive cases were found here and all were co-infected with other respiratory viruses, the pathogenic role of hadv-c in respiratory infections will require further investigation. hadv type is traditionally determined by virus isolation and subsequently serum neutralization tests, in which antibodies raised against specific type are used to suppress cytopathic effects in tissue culture assays. by nature of its design, this test can only reveal the dominant type. by applying pcr-based identification targeting hexon or fiber genes, co-infections with multiple hadv types (types from same or different species) have been reported in both immunocompromised and immunocompetent patients [ , , [ ] [ ] [ ] . in current study, results showed that one specimen contained both hadv-c and hadv-c by cloned sequencing the pcr products. these were amplified directly from respiratory samples using universal primers of hexon gene. this co-infected phenomenon was confirmed using the fiber gene sequencing results with type-specific primers (data not shown). the specimen was collected from a previously healthy . -year old boy, presenting with fever, coughing and seizure at emergency department on december , . co-infection of different hadv types has never been reported in any previous studies of mainland china. the clinical implications of such co-infection remain unclear, and its role in hadv pathogenesis and evolution will require further study. consistent with the report from guangzhou, southern china [ ] , results here showed that . % of hadvinfected participants were co-infected with one or more other respiratory tract viruses and that rsv was the most frequently co-detected virus. however, no significant differences in clinical characteristics and laboratory findings were found between patients with single hadv infection and those co-infected with rsv except that coinfections were more frequently observed in younger children. similarly, a study from peru also did found no higher prevalence of any clinical manifestations in coinfected patients than in those infected with hadv alone [ ] . the results of another report from chile showed the clinical severity to be the same in patients with single hadv infection and those with mixed rsv-hadv infections [ ] . these data demonstrate that, as more sensitive molecular methods become more frequently used to identify pathogens, co-detection of different viruses in the same specimen may also become more common. however, the clinical role of such coinfections will still require independent investigations. both hadv-b and hadv-b may cause severe or even fatal pneumonia in even immunocompetent children. several previous studies showed that patients infected with hadv-b tend to have higher case-fatality rates than those with hadv-b [ , ] . two fatal cases were recorded during the study period, and both of these patients were infected with hadv-b alone. analysis revealed that patients with hadv-b infection had longer duration of fever and higher serum levels of muscle enzymes than hadv-b -infected patients. patients with hadv-b infection also tended to require longer hospital stays although no significant difference was found. these differences have excluded the possible interference by any other co-infected respiratory viruses since this work only evaluated the patients with hadv infection alone. such results may suggest that hadv-b infection tended to cause more extrapulmonary tissue damage (such as liver and heart) and may have more severe clinical consequence. this is a cross-sectional study. only one respiratory sample was collected from each patient and no viral load analysis was performed. although hadv is a pathogen that for long has been known to cause respiratory tract infection, asymptomatic carriage of the virus may persist for weeks [ ] . the detection of hadv in nasopharyngeal aspirate or throat swab with the use of a pcr assay could represent convalescent-phase shedding, so detection may not suggest the current infection. in summary, a total of different types of hadv were identified in children with alrtis and hadv-b and hadv-b were the most predominant types. clinical entities of patients with single hadv infection were 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outbreaks of lower respiratory tract infections in children adenovirus serotype and infection with acute respiratory failure in children in taiwan we would like to thank all participating physicians and nurses of beijing children's hospital for their assistance and collaboration in the sample and clinical data collection. this study was supported by grants from the national major s & t research projects for the control and prevention of major infectious diseases in china ( zx - ) and national science and technology supported projects ( bai b ). the authors report no conflicts of interests.authors' contributions cl analyzed data, performed statistical analysis, drafted and reviewed manuscript. yx and jz carried out the molecular studies. lr participated in study design and coordination and helped to review the manuscript. jl designed the primers of sequencing typing. zx analyzed data and reviewed manuscript. bx, yy and sq collected samples and data. jw and ks conceived of the study. all authors read and approved of the final manuscript. availability of data and materials not applicable submit your next manuscript to biomed central and take full advantage of: key: cord- -jozrgcq authors: tan, xin quan; zhao, xiahong; lee, vernon j; loh, jin phang; tan, boon huan; koh, wee hong victor; ng, sock hoon; chen, mark i-cheng; cook, alex richard title: respiratory viral pathogens among singapore military servicemen – : epidemiology and clinical characteristics date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: jozrgcq background: few studies have comprehensively described tropical respiratory disease surveillance in military populations. there is also a lack of studies comparing clinical characteristics of the non-influenza pathogens with influenza and amongst themselves. methods: from may through october , consenting cases of febrile respiratory illness (fri) (temperature [greater than or equal to] . degreesc with cough or sorethroat) and controls in the singapore military had clinical data and nasal washes collected prospectively. nasal washes underwent multiplex pcr, and the analysis was limited to viral mono-infections. results: % of cases tested positive for at least one virus, of whom % had multiple infections. % of the fri cases fulfilled the definition of influenza-like illness (ili), of whom % were positive for at least one virus. the most frequent etiologies for mono-infections among fri cases were influenza a(h n )pdm ( %), influenza b ( %) and coxsackevirus ( %). the sensitivity, specificity, positive predictive value and negative predictive value of ili for influenza among fri cases were %, %, % and % respectively. on logistic regression, there were marked differences in the prevalence of different symptoms and signs between viruses with fever more prevalent amongst influenza and adenovirus infections than other viruses. conclusion: there are multiple viral etiologies for fri and ili with differing clinical symptoms in the singapore military. influenza and coxsackevirus were the most common etiology for fri, while influenza and adenoviruses displayed the most febrile symptoms. further studies should explore these differences and possible interventions. influenza-like illness (ili) is often used for influenza surveillance [ ] , as influenza is a disease of global interest with % of adults developing symptomatic disease annually and with case fatalities of . % in susceptible populations [ ] . while influenza surveillance remains a priority, ili can also be caused by a wide range of viral pathogens that present with a spectrum of respiratory symptoms [ ] [ ] [ ] [ ] . in the tropics, viral respiratory pathogens have been reported to exhibit different seasonality and transmission characteristics compared to temperate climates [ , [ ] [ ] [ ] . this necessitates a better understanding of their epidemiology to assess the utility and importance of surveillance in these settings. the year-round circulation of respiratory viruses in the tropics may also predispose patients to coinfection with multiple pathogens, with implications for severity of disease [ , ] and secondary bacteria infection [ , ] . while there have been studies comparing differences in clinical presentation between influenza and non-influenza cases [ ] , few describe the epidemiology and differences in clinical presentation among various non-influenza respiratory viruses. as influenza viruses have accounted for only between . % to . % of all ili cases [ ] [ ] [ ] , it is important to understand the contribution of other respiratory pathogens to overall morbidity and to determine their epidemiological distribution and clinical presentation. to address these issues, this study explores data obtained from a respiratory disease sentinel surveillance system in the singapore military to examine the etiologic viral agents of respiratory illnesses in a tropical environment, to determine the viruses that circulate post-influenza vaccination, and to compare the differences in clinical presentation. singapore is a city-state in tropical south-east asia with a population of . million people (mid-year ). the singapore military is based on national service in which all male citizens and liable permanent residents serve for two years after high school. servicemen typically live in barracks-style accommodation on weekdays and return home on weekends. the singapore military started a sentinel respiratory disease surveillance program in major camps (including a recruit training camp) on may , tracking febrile respiratory illness (fri) cases (temperature ≥ . °c with cough or sore throat). the definition of fri contrasts with influenza-like illness (ili, defined as fever ≥ . °c with cough or sore throat) to broaden the capture of other febrile cases that also result in absenteeism while limiting cases to those with fever as an indicator of severity. this allows for detection of a larger number of respiratory pathogens. patients who visited the primary healthcare clinics in the camps between may and october during regular consultation hours who met the fri criteria were recruited. healthcare workers obtained written informed consent, administered a questionnaire, obtained clinical specimens and performed a clinical examination on partcipants. repeat consultations were excluded if the healthcare worker determined that the patient had not recovered from the first illness episode. we also obtained samples from controls (those without respiratory symptoms or acute infections), who were recruited across the year at between to persons per week. informed consent, the baseline questionnaire, and clinical specimens were obtained. from december , all recruits were administered with the influenza a(h n )pdm (flu-a(h n )pdm ) vaccine. the trivalent seasonal influenza vaccination was first introduced to recruits in december , followed henceforth by all other personnel in november . nasal washes from each side of the nose were taken from consenting participants by trained medical staff, placed in viral transport media and refrigerated. the samples were transported to the laboratory on ice for etiological testing within hours. laboratory analysis was performed in an iso accreditated laboratory for molecular diagnostics which regularly takes part in external proficiency programs such as qcmd eqa programs. detailed laboratory methods were previously described [ ] . we used the multiplex pcr strategy based on the resplex assays described below, and performed additional singleplex pcr assays to determine the influenza subtype. total nucleic acids were extracted from each specimen using the dna minikit (qiagen, inc, valencia, ca, usa) according to manufacturer's instructions. a total of μl of extract were tested with resplex i and ii (version . , qiagen, inc., valencia, ca, usa) [ ] for respiratory micro-organisms on the liquichip workstation, according to manufacturer's instructions. the resplex i and ii (version . ) assays are multiplex pcr assays coupled with bead array detection technology and can simultaneously detect and subtype different pathogens including influenza a (flu-a) and influenza b (flu-b). specimens that were resplex ii positive for flu-a were further subtyped with real-time pcr for h or h (singapore ministry of health), or for flu-a(h n )pdm . briefly, μl of total genetic extracts were tested using an in-house developed assay based on the one-step superscriptiii/platinum taq kit (invitrogen, carlsbad, ca, usa) following manufacturer's instructions on the lightcycler machine from roche or the applied biosystems real-time pcr machine ( ). the analysis was limited to viral mono-infections amongst cases to discern clinical presentations and symptom complexes associated with each pathogen. we excluded viruses with fewer than cases ( . % of the total), as the number was too small to have a reasonable sample sizethese were coronavirus hku (cov-hku ), parainfluenza (hpiv- ), hpiv- , hpiv- , influenza a(h n ) (the prepandemic strain), respiratory syncytial virus a (rsv a), rsv b, cov and bocavirus (bv). this left viruses for the subsequent analyses. the main aim was to compare the differences in clinical expressions, including individual symptoms (or signs), pairs of symptoms, and overall symptom load between patients with different viral infections. we counted the clinical symptoms/signs and calculated the corresponding empirical proportions with % confidence intervals (cis) to evaluate the overall symptom load. logistic regression analysis was used to investigate the differences in symptom expressions for each pair. differences were identified at a significance level of . . to assess the presence of paired symptoms/signs for all viruses, we conducted binomial tests to compare the joint proportions of symptom pairs occurring together to the expected proportions assuming independence of symptoms. the ratio of the observed proportion of symptom pairs relative to the product of the marginal proportion of each symptom is defined as the excess probability ratio which measures effect size. multivariate logistic regression analysis was performed to compare the risk of having an individual symptom/sign among viral mono-infections by assigning a categorical variable for all viruses as the primary predictor. potential confounding was addressed by adjusting the model for age, smoking status, asthma and heart disease. non-significant variables were dropped at a significance level of . to obtain the final model. statistical analyses were performed using the r statistical software (version . . ) [ ] . ethics approval was given by the singapore military's joint medical committee for research, and the national university of singapore's ethics review committee. the basic demographic data are described in table . participants were mostly young male adults, with other characteristics largely similar. however, there were significantly less recruits amongst controls than amongst other groups. the temporal distribution of cases is described in figure . no obvious overall seasonal pattern can be observed. the peak in june and july corresponds to the flu-a(h n )pdm pandemic [ ] . as this peak tailed off, we observed an increase in flu-b cases (starting feb-mar ). subsequently, as the flu-b cases fell, adenovirus e (adv-e) cases started to increase. coxsackie/echovirus (cv) and rhinovirus (rv) infections were consistently present in the earlier periods but appeared to tail off by , corresponding to the rise in fri cases due to other viruses. the etiologies of selected infections are illustrated in table . at least one virus was detected in of the fri cases ( . %). in ( . %) of the cases, more than one virus was detected and these were excluded. ( . %) of the fri cases fulfilled the definition of ili; ( . %) of these ili cases were positive for at least virus. of the fri mono-infection cases, ( . %) were viral. ( . %) of these cases met the definition of ili. we examined the proportion of ili cases among those with viral mono-infections (table ) . influenza viruses accounted for only % of ili. among fri cases, more than % of patients with influenza and adenovirus infections presented with ili. however, several other viral infections led to high rates of ili, including cv, human metapneumovirus (hmpv) and covs. the sensivity, specifity, positive predictive value (ppv) and negative predictive value (npv) of ili for influenza was . %, . %, . % and . % respectively. on from the multivariate analysis ( figure ), compared to most other viruses, a flu-a(h n )pdm and flu-a (h n ) less commonly resulted in sorethroat. running nose was more common in enterovirus (ev) and rv cases and less common in adv. flu-b was more likely than a majority of the other viruses to cause dry cough while ev and adv-e were less likely to cause dry cough. cov-oc and hmpv were more likely to cause cough with phlegm than the most other viruses. influenza viruses and adenoviruses were more likely to cause fever ≥ . °c. in figure , we explored the associations (and dissociations) between different clinical symptoms and signs across all viruses. some are expected, such as association of fever ≥ . °c and fever ≥ . °c and dissociation of dry cough and cough with phlegm. fever ≥ . °c was also associated with systematic complaints, such as chills, bodyache, headache and eye pain. sorethroat was associated with an injected pharynx. our study shows the different viral etiologies of ili and compares the clinical characteristics of different viral etiologies in a tropical setting. this data series only spanned three years, and initial observations showed no clear seasonal variation compared to temperate regions, similar to previous reports of overall tropical respiratory disease patterns [ , , ] . the initial peak corresponded to the flu-a(h n )pdm pandemic [ ] , with the subsequent lower incidence in the recruit population likely due to vaccination with the pandemic vaccine a year before annual seasonal vaccination was started across all personnel [ ] . the number of fri cases remained fairly consistent throughout the study period (except the pandemic). however, prevalence of pathogens varied throughout, with some negative correlation observed between the virusese.g. a drop in flu-a followed by a rise in flu-b activity, and a drop in flu-b cases followed by a rise in adenovirus activity. correlation of viral activity have previously been reported -wang et al [ ] reported negative association between rv and adv rates, while bellei et al [ ] and razanajatovo et al [ ] described concomitant rise of influenza and rv, and influenza and adv activity respectively. in addition, kasper et al reported that ili rates remained constant despite varying prevalences of influenza [ ] . this supports our findings that multiple agents are capable of causing ili, and a decrease in the prevalence of one virus was replaced by an increase in prevalence of another. further studies across a longer time period are necessary, especially for vaccine effectiveness evaluation. . % of fri and . % of ili cases were positive for a virus, similar to the . % and . % reported by studies targeting similar panel of organisms [ , , , , , ] . the remaining fri cases may be due to non-viral agents, agents beyond the ability of the test, non-infectious causes, and possible sampling errors. viruses most commonly detected in ili cases were flu-a, flu-b, and cv in that order. influenza was also the top etiologic agent for ili in some studies [ , , , , ] although other pathogens have been identified to be most prevalent in different settings, such as rsv and hmpv in france [ ] , influenza and rsv in the usa [ ] , influenza and rv in central america [ ] and china [ ] and in italy, influenza and adv [ ] . the range of pathogens indicates a need to perform local continual surveillance since prevailing pathogens differ across different populations, geographic regions and climates. the high incidence of cv warrants further studyhand, foot and mouth disease is endemic to singapore and cv is frequently identified in pediatric samples [ ] , and it is possible that cv circulates at high levels in adults also. previous studies have identified a co-infection rate of . to . % [ , , , , ] for viruses, higher than the . % found in this study despite some studies using a less extensive diagnostic panel in a similar age group. possible reasons include our highly influenza-vaccinated population or a warmer climate with higher relative humidity resulting in lower virus circulation [ ] and a study population that did not include children (studies reported higher co-infection rates amongst pediatric patients [ , ] ). in both univariate and multivariate analysis, adv and influenza viruses were more likely to cause fever (≥ . °c). this finding has been demonstrated in other studies [ , , [ ] [ ] [ ] [ ] . fever also tends to be associated with other systemic complaints such as eye pain, bodyache and headache; this may be due to cytokine mediated systematic inflammatory response [ ] and could indicate more severe disease. bellei et al's [ ] study in brazil also found that ev, rv and cov were least likely to cause fever, and that rv and ev cases were the most likely to present with rhinorrhea. cough with sputum in flu-a(h n ) and flu-a were less prevalent than in other viruses, in contrast to reports in other settings [ , [ ] [ ] [ ] . we found that flu-b cases were more likely to report dry cough, similar to other studies [ , [ ] [ ] [ ] . the heterogeneity of results across different studies highlight the difficulty of using clinical symptoms in determining the etiology of ili. we also detected a small proportion of asymptomatic individuals who tested positive for the various viruses ( table ). these could represent carriage without infection or subclinical/asymptomatic infections during periods of virus circulation. although ili is widely used to identify influenza, the traditional definition would have picked up only % of influenza infections (except untyped flu-a) that were identified as fri. adv infections also frequently fulfilled the ili definition ( . % of adv-b and . % of adv-e), as did substantial fractions of cv, hmpv, and cov. only . % of viral mono-infections that met the ili definition were due to influenza. we found a fairly high sensitivity ( . %) of ili for influenza, but a low specificity ( . %) in keeping with sensitivities of . - . % and specifities of . - % reported elsewhere [ , , , ] . color cells represent variables that are significant at the % level, and the thickness of the cell wall represents the p-value (thin means . < p < . ; medium, . < p < . ; and thick, p < . ). the odds ratios are encoded by colors where a red cell indicates an odds ratio > ; and blue otherwise. for example, for a sore throat, flu-a(unknown), flu-b, cv, rv, adv-e, cov-oc and cov-nl have more of the sore throat than iflu-a(h n )pdm indicated by the red cells in the row for flu-a (h n )pdm and corresponding columns. ppv of ili was low ( . %) compared to other studies ( . - . %) likely because these studies were conducted during influenza seasons. this is supported by the higher npv ( . %) compared to other studies ( . - . %) [ , , , ] . two tropical studies [ , ] also report low ppv of . - % and high npv of - . %. adv are as likely as influenza viruses to present with fever and tend to be captured by the ili definition. however, they are more likely to present with cough and sorethroat than influenza. it may be possible to differentiate adv and influenza infection based on rhinorrhea and other symptoms. this could be useful in surveillance and clinical management, especially when deliberating whether to start antivirals. early etiologic diagnosis of influenza has been shown to be cost effective [ ] with reduced antibiotic use and may reduce complications with early antivirals. it may be possible to combine a clinical diagnostic model with rapid testing to achieve these goals. the analysis was limited to viral mono-infections and future studies should explore co-infections and bacterial infections. this study involved predominantly young adult males, and results may not be generalizable to the overall population, necessitating further studies among various age groups and gender. there were also less recruits amongst controls than amongst other groups, and this would be an important consideration when comparing the two groups in the future. finally, the actual clinical impact of differentiating between various viral etiological agents may be limited, and we could not determine the relative severity of symptoms other than fever. our study highlights the varied etiology for fri and ili in the tropical settinginfluenza and adv and cv were all common. influenza and advs tend to present with higher fever, and vaccination should be considered. the utility of ili for tropical surveillance of influenza needs to be reviewed given the low ppv and high npv compared to temperate regions. the surveillance system has enabled the singapore military to understand the etiologic agents affecting servicemen, hence implementing and evaluating controls measures such as vaccination. figure correlation of symptoms and signs across all viruses. clinical signs or symptoms are listed by average frequency from the most to the least. binomial test is used to assess the discrepancy between the observed proportion of symptom pairs and the expected proportion of symptom pairs which is the product of the two marginal distributions by assuming symptoms develop independently. color cells represent differences that are significant at the % level, and the thickness of the cell wall represents the p-value (thin means . < p < . ; medium, . < p < . ; and thick, p < . ). the excess probability encoded by colors measures the effect size. if the observed 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key: cord- -flsaa wx authors: aldohyan, meshal; al-rawashdeh, nedal; sakr, farouk m.; rahman, saeed; alfarhan, ali i.; salam, mahmoud title: the perceived effectiveness of mers-cov educational programs and knowledge transfer among primary healthcare workers: a cross-sectional survey date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: flsaa wx background: knowledge transfer of middle east respiratory syndrome coronavirus (mers-cov) involves the dissemination of created/acquired information on mers-cov in hospitals, making this information accessible to all healthcare workers (hcws). this study evaluated the perceived effectiveness of mers-cov educational programs and knowledge transfer among primary care hcws at a hospital in saudi arabia that witnessed the largest outbreak of confirmed mers-cov cases in this country. methods: a survey was distributed among primary care hcws at five clinics in saudi arabia in . those with non-direct patient care responsibilities were excluded. their knowledge was evaluated against facts published by mayo clinic foundation, and its percentage mean score (pms) ± standard deviation was calculated. hcws’ perceived effectiveness of educational programs and knowledge transfer was classified as negative or positive. results: sample comprised of hcws, of which % were females and % were males. almost % were ≤ years old, and % had > years of work experience. almost . % were nurses, . % physicians, . % were pharmacists, and . % were technical staff. pms for knowledge was . ± . . the prevalence of negative perceptions towards educational programs was . % and of knowledge transfer was . %. older(> years of age) and more experienced(> years) hcws had the highest pms for knowledge( . ± . ,p = . and . ± . ,p < . respectively). negative perceptions of educational programs ( . ± . ; p < . ) and knowledge transfer ( . ± . ; p = . ) were associated with a lower knowledge pms. males were . [ % confidence interval . – . ] times and . [ . – . ] times more likely to have negative perceptions of educational programs and knowledge transfer (adjusted (adj.)p = . and adj. p = . , respectively). physicians/pharmacists were . [ . – . ] and . [ . – . ] times more likely to have negative perceptions of both outcomes (adj. p = . and adj. p = . , respectively). less experienced hcws were . [ . – . ] times and . [ . – . ] times more likely to exhibit negative perceptions of the two outcomes (adj. p < . each). conclusions: a negative perception of the effectiveness of mers-cov knowledge transfer was associated with poorer knowledge and was more prevalent among male hcws, physicians/pharmacists and less experienced hcws. hospitals should always refer to efficient knowledge sharing and educational strategies that render beneficial outcomes to patients, hcws, and the public community. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. middle east respiratory syndrome coronavirus (mers-cov) has created an epidemiological and clinical crisis within countries in north africa, europe, asia and the usa but mainly in the middle east (kingdom of saudi arabia) [ ] . it is a viral respiratory illness, initially discovered in and speculated to have originated from camels or bats in saudi arabia, with subsequent spread to humans and across borders [ ] . since , a minimum of laboratory-confirmed cases have been reported in saudi arabia, of which patients have died, have recovered and two were under treatment [ ] . high-risk groups were those in close contact with camels, geriatric persons, pregnant women, healthcare workers (hcws) and those with pre-existing comorbidities [ ] . mers-cov infection ranged from asymptomatic or mild respiratory symptoms to severe acute respiratory disease and even death, which was reported in three to four out of every reported mers-cov cases [ ] . biologic samples of subjects with a suspected mers-cov infection (based on clinical symptoms) and of those exposed to reported mers-cov cases are tested using real-time reverse transcription polymerase chain reaction (rrt-pcr) assays. serology, such as an enzyme-linked immunosorbent assay and immunofluorescence assay, is also used to confirm mers-cov by the presence of antibodies [ ] . in saudi arabia, a series of modifications was applied to the patient pathways while visiting the emergency departments or admitted as in-patients. this included segregating patients during triage based on prioritizing the inflow of patients by their chief complaints, bed availability and screening of flu symptoms/history of exposure. the infrastructure of the medical facility, particularly the ventilation system and isolation capacity of rooms, was subject to changes. some hospital wards and staff (especially nurses) were dedicated specifically to confirmed mers-cov cases to limit the chance of cross-contamination across wards and hcws. the infection prevention and control department (ipcd) at smng-ha, in particular, was on high alert for such mers-cov outbreaks, especially with the evident transmission of viral infections between patients and hcws at smng-ha. crisis management required a rapid implementation of adequate infection prevention, control procedures and case isolation, in addition to collaboration and coordination with local and international consultations. exceptional efforts have been made by the ipcd to apply the latest and most effective means of universal standard precautions throughout the mers-cov crisis. rules and regulations pertinent to infection control and prevention have been revisited and environmental surveillance has been carried out regularly to ensure that all wards are equipped with suitable protection and precautionary gear. numerous seminars, workshops and awareness campaigns have been launched for hcws of all disciplines to boost their knowledge on mers-cov, as well as their morale, to maintain a high-quality, safe and dedicated service for the patients. the latest updates issued on mers-cov from the world health organization (who), the centers for disease control and prevention (cdc), collaborative task forces (local and regional) and researchers have been circulated regularly among all hcws and across all managerial levels. numerous research studies have been conducted and published on the perception, knowledge and attitude of hcws towards mers-cov. it is rare to find a hcw who has not attended an educational program on mers-cov in saudi arabia. dissemination of mers-cov information/updates or knowledge transfer within a healthcare organization is a process in which this information is created, generated or acquired, and then organized and distributed within the system to ensure it is accessible to all hcws. one of the mechanisms of knowledge transfer is personalization whereby knowledge is transferred from one individual to another, while the other is codification where knowledge is converted into products such as documents, images and videos [ ] . the need to transfer efficiently the precautionary regulations and updates about mers-cov to large numbers of hcws necessitates the mechanism of codification [ ] . in addition, knowledge transfer or information sharing was found to be positively associated with job satisfaction [ ] . authors hypothesized that although the dissemination of knowledge and updates on mers-cov among hcws has been given full consideration, these hcws might have reservations on the effectiveness and quality of mers-cov related educational offerings. therefore, there was an emerging need to evaluate the perceived effectiveness of mers-cov educational programs and knowledge transfer from the hcw's perspective, in a setting that witnessed the largest outbreak of confirmed mers-cov cases in saudi arabia. this was a cross-sectional study, based on an anonymous survey in english distributed among hcws at the primary healthcare centers in smng-ha medical centers, in riyadh, saudi arabia, between october and december . the smng-ha is the second-largest healthcare sector in the country, second only to the saudi ministry of health, and provides healthcare services to the community of national guards, their dependents and employees [ ] . the targeted primary healthcare centers were five randomly selected clinics out of clinics that employ physicians, pharmacists, technicians and nurses. these clinics serve a population of , registrants, with a rate of four visits per registrant annually. eligible primary care hcws were targeted as being in frontline contact with potential confirmed cases of mers-cov. those occupying positions of management, education or non-direct patient care were excluded. medical and nursing students were also excluded. this study was ethically approved by the institutional review board at king abdullah international medical research center, king saud bin abdul-aziz university for health sciences, sp- / . the provisioned sample size in this study was calculated based on a reported level of knowledge between . and . % by alkot et al. among hcws in the western region of saudi arabia. assuming an expected level of knowledge of %, with a % confidence limit (z = . ), and a margin of error %, the estimated sample size for this study was . for convenience, all eligible hcws at the targeted setting were invited to participate in this survey, to overcome a % nonresponse rate. the survey was provided in a sealed envelope with a cover letter that described the objectives of the study. the survey was in english language, as the targeted study participants were english literate and the educational offerings provided at the targeted setting were also in english. participants who agreed to enroll in this study hand-signed an agreement statement at the end of an informed consent, with no need for any personal identifier. the data collection tool comprised the characteristics of the hcw, principally gender, age category (years), job position and experience (years) [ ] . the knowledge of hcws was measured using statements based on undisputed facts published in the literature and issued by the mayo foundation for medical education and research in [ ] . correct answers were scored " ", while wrong/don't know answers were scored " ". the percentage mean score (pms) of knowledge was calculated by adding the correct responses of the statements, dividing the score by and multiplying it by (range of score to ). the perceived effectiveness of the mers-cov educational programs was measured using one statement: "prevalence of mers can be reduced by active participation of healthcare workers in the hospital infection control program", while the perception of knowledge transfer was measured by one statement: "any related information about mers should be disseminated among healthcare workers". both statements were rated on a four-point likert scale (strongly disagree, disagree, agree and strongly agree). those who responded by disagree or strongly disagree were classified as having a negative perception, while those who responded agree or strongly agree were classified as having a positive perception. the negative perception rate was calculated by dividing the number of participants who had negative responses over the total number of respondents. in addition, participants were asked about the source of mers-cov information. the survey was piloted on a group of five hcws, and their subjective comments were considered. the internal consistency or reliability (cronbach's alpha) of the knowledge domain measured . (additional file ). data were analyzed using the statistical package for social studies (spss ; ibm corp., new york, ny, usa). hcw characteristics, perceptions (negative vs positive) and incorrect knowledge response statements were presented in terms of frequencies and percentages, while the pms of knowledge was presented as the mean ± standard deviation. missing data were replaced by the average of the total, and outliers were dropped out. pearson's chi-square test was used to test categorical outcomes across hcw characteristics, while a mann-whitney test and a kruskal-wallis test were used to test the non-normally distributed pms of knowledge scores. two binary logistic regression models were constructed to determine the factors significantly associated with negatively perceived effectiveness of mers-cov educational programs and knowledge transfer. due to the small subgroup size of job positions, nurses were grouped with technicians, while pharmacists were grouped with physicians. these two subgroups had job positions comparable in terms of the educational levels, scope of practice and nature of patient care. the adjusted odds ratios [ % confidence interval] were calculated, and statistical significance was set at a value of p < . . initially, surveys were distributed among hcws; participants agreed to enroll and completed the survey (response rate . %). those who did not participate were mainly either off duty or busy with their workload. females constituted . % of the sample, while males comprised . %. almost % were ≤ years old, % were - years old and % were > years old. the majority ( . %) were nurses, followed by physicians ( . %), pharmacists ( . %) and technical staff ( . %). most hcws ( . %) had accumulated > years of work experience, with . % having < years of experience and . % having - years (table ) . overall, . % of respondents claimed that their main source of information was the internet, while . % reported more than one source, including research studies, books, media and others. the pms of knowledge score was . ± . . the most common incorrect response to the statements ( . %) was that for "incubation time for virus", followed by . % with an incorrect response to the statement that "antibiotics are the first-line treatment for the management of mers-cov". other incorrect responses to statements are listed in chronological order in table . overall, . % of participants reported a negative perceived effectiveness of mers-cov educational programs, while . % had a negative perception of knowledge transfer. with regard to the perceived effectiveness of mers-cov educational programs, male hcws had significantly a more negative perception than female hcws (n = , . %, vs n = , . %, respectively; p < . ). pharmacists (n = , . %) and physicians (n = , . %) reported more negative perceptions than technical staff (n = , . %) and nurses (n = , . %) (p = . ). hcws with work experience of < years had the most negative perceptions in comparison with the other groups (p = . ). a number of factors were associated with a negative perception of knowledge transfer of mers-cov information. male hcws had a greater negative perception than females (n = , . %, vs n = , . %, respectively; p < . ). physicians (n = , . %) and pharmacists (n = , . %) had more negative perceptions of knowledge transfer in comparison with technical staff (n = , . %) and nurses (n = , . %) (p < . ). junior hcws with work experience of < years ( . %) had the highest rate of negative perception of knowledge transfer (p < . ) ( table ) . hcws > years old (pms . ± . ) had the highest knowledge scores in comparison with the other age groups (p = . ). more experienced hcws (> years) also had the highest knowledge scores (pms . ± . ; p < . ). those who had a negative perception of the effectiveness of mers-cov educational programs (pms . ± . ) and of knowledge transfer of mers-cov updates (pms . ± . ) both had lower knowledge scores in comparison with the positive-perception group (p < . and p = . , respectively), table . logistic regression analyses showed that male hcws table . mers-cov educational programs at healthcare institutions are a formal and reliable channel to deliver essential knowledge to hcws. for the sake of personal safety, job satisfaction and work morale, hcws should not pass up any opportunity to increase their theoretical knowledge and practical skills. hospital administrators do not necessarily face the challenge of producing new information, as an immense amount of valuable information already exists in the literature. the problem arises from the fact that current knowledge is either poorly structured or inaccessible to hcws [ ] . for example, advanced practice nurses are observed to be "knowledge brokers" in a sense that they act as disseminators of knowledge among the nursing body. furthermore, health educators retrieve different types of evidence, synthesize it in different forms, translate it by evaluation, interpret it and then distribute it among nurses [ ] . health education can improve levels of awareness and perception among hcws towards mers-cov infections [ ] , and these higher levels of knowledge can aid in the control n frequency, % percentage of disease outbreaks [ ] . however, published evidence in saudi arabia has shown that there is limited knowledge on mers-cov (both microbiological and virological aspects) among hcws in southern saudi arabia [ ] . another study also claimed that knowledge about emerging infectious diseases was poor, and that infection control practices were suboptimal and also seemed to be overestimated [ ] . the association between younger age and less experience on one hand and lower knowledge scores on the other was a reasonable finding. similar to literature findings, the knowledge of hcws in this setting was suboptimal and gaps remain that should direct the focus towards the mechanisms and quality of knowledge transfer. dissemination of mers-cov updates using e-mail, the internet, institutional announcements, employee meetings, the media and even personal communications are all methods of knowledge transfer. hcws can experience knowledge transfer both passively, absorbing information unconsciously, and actively. investigators in this study were curious to know how hcws perceived the transfer of knowledge about mers-cov and why this would be of concern to hospital administrators. for instance, knowledge transfer has been adopted with regard to smoking as a health hazard, hiv transmission as a sexual risk and seat belts in motor vehicles as a safety measure. people are exposed almost daily to precautionary advice by a variety of methods but unfortunately still undertake high-risk activities and are exposed to these hazards. this occurs regardless of the duration, frequency and quality of awareness campaigns. therefore, it is an aggravating concern that the repetitive exposure of hcws to mers-cov campaigns might have created some sort of "tolerance". hcws might disremember or take lightly the acquisition of current or new updates about mers-cov precautions due to routine attendance of educational programs or repetitive circulation of e-mails. knowledge and skills must be passed on in a systematic way from expert to novice employees in a way that makes sense [ ] . managers who support work-empowering environments are actually boosting the engagement of participations in terms of knowledge transfer [ ] . in fact, one of the key elements in seeking accreditation or managing crises such as mers-cov is knowledge communication, in the sense that effective communication ensures a purposeful exchange of information, thus allowing a more thorough understanding of the outbreak [ , ] . interactive workshops remain highly recommended for the sharing and transferring of knowledge among hcws. however, one study noted that, although those who attended such workshops valued the expert input and discussions, after few months their sustainability of attendance was lost [ ] . some barriers to mers-cov knowledge transfer could be the inability of hcws to recognize and articulate the instructions, personal opposition or resistance to change, the quality of the communication technologies, the absence of visual representations, language and cultural differences, deficiency in expertise, the work environment, a lack of job incentive/motivation, the organizational culture and others [ , ] . current efforts to manage the mers-cov crisis are directed towards developing educational programs that target both the community and hcws [ ] . a negative perception of mers-cov educational programs in this setting might result in outdated knowledge among hcws, which jeopardizes their compliance with disease precautionary and control measures. a mers-cov task force committee pointed out that the saudi arabian ministry of health has posted updates on mers-cov through videos, posters, handouts, posters and an official website. resilience against mers-cov increases with enriched education and awareness [ ] . a saudi arabian study reported that hcws were unaware of the availability of mers-cov information at their work areas; they did not feel they had sufficient training and were not confident about infection control guidelines. these factors may also contribute to having a negative perception of mers-cov-related educational programs [ ] . one study reported that the interest in following disease updates among hcws improved significantly after the implementation of a mers-cov educational program [ ] . these programs improved the attitude of the hcws towards governmental measures taken regarding the crisis [ ] . hcws often grasp their mers-cov educational information primarily from watching tv reports, or from the internet. a negative perception of knowledge transfer might be due to a pre-existing lack of trust in the media or in websites that might, to some degree, lack scientific credibility in comparison with educational programs provided in healthcare centers [ ] . knowledge itself is complex, and its transfer process within healthcare institutions carries many challenges [ ] . one way to overcome these challenges is to determine the characteristics of hcws who might be more likely to exhibit negative perceptions of knowledge transfer for significant mers-cov updates. in the literature, knowledge transfer has been investigated more frequently in manufacturing industries and firms, or among the public community. it has been seldom evaluated among hcws [ ] , and never in a middle eastern setting or related to a mers-cov outbreak. a crossnational study suggested that organizational culture was a significant influence on the capacity of hcws to engage in knowledge transfer [ ] . a systematic review paper study stated that knowledge transfer could streamline productivity and coordinate the use of resources more efficiently [ ] . this review paper claimed to be the first to review published research focused on the perceptions of hcws about knowledge management [ ] . knowledge management was defined as having an efficient idea or new practice accepted and adopted by an individual or a group through communication channels (successful diffusion of ideas) [ ] . this definition also applies to the dissemination of updated regulations on the outbreak of mers-cov. this information, once absorbed by people, should be sustained for as long as it is useful, and not decay over time [ ] . accordingly, a negative perception of the importance of knowledge transfer could be a warning sign of an interruption in this sustainability of retained information about mers-cov. signs of information decay were evident among hcws in this study, as those who had negative perceptions had lower knowledge scores about mers-cov in comparison with those who had positive perceptions. one of the key goals of knowledge transfer is to educate and train the less experienced and/or the newly hired hcws [ ] . this phase of staff development is crucial yet stressful for novice hcws, who are expected to acquire skills and competencies rapidly to ensure that a safe and quality service flow is maintained at the institution. this explains why hcws with less work experience (< years) had significantly more negative perceptions of knowledge transfer and the perceived effectiveness of mers-cov educational programs. as they gain more work experience, this perception improves as they realize the importance of education not only for their patients but also for their career advancement. the level of knowledge on mers-cov among hcws in primary healthcare clinics in this setting was found to be less than optimal. as the frontline in the battle of disease prevention and control, hcws are expected to be equipped with the relevant theoretical updates about mers-cov. special consideration should be paid to younger and less experienced hcws whose knowledge on mers-cov was moderately low. a negative perception of the knowledge transfer of mers-cov information and educational programs was associated with poorer knowledge. this negative perception was more prevalent among male hcws, physicians/pharmacists and less experienced hcws. this study has been conducted at one setting, yet the struggle against mers-cov has not ended and will continue against future emerging strains of viruses and bacteria causing communicable diseases in other settings too. knowledge is a valuable asset, and its holders within any healthcare institution should be retained and motivated so that they continue to spread their benefit among other hcws. all healthcare institutions should always identify and refer to reliable sources of knowledge. for instance, the center for disease control and prevention is a leading national public health institute and accountable for disseminating up-to-dates on various infectious topics. in saudi arabia, the ministry for public health has designated communication channels to release updates on mers-cov on their websites, through scientific arenas, memorandums and helpdesks. knowledge sharing and management strategies in the healthcare sector can render beneficial outcomes to patients, hcws, the organization and the public community [ ] . in addition to the attendance of seminars or workshops, other methods of knowledge dissemination might involve launching of journal clubs among hcws to discuss updates on mers cov. audiovisuals at hospitals, such as educational videos on tv screens in lobbies or corridors, constantly enlighten hcws. deeper understanding of the negativity in the perception towards the quality or method of knowledge transfer necessitates a qualitative methodological approach, as face to face interviews with hcws aid in determining the underlying reasons and at a more personal level. furthermore, the execution of these strategies needs to be routinely monitored and evaluated so that the transfer of knowledge is time efficient and effective. optimal theoretical knowledge and practical competence are two main indicators of successful knowledge transfer among hcws. last but not least, a number of key points can be noted: as well as their morale, to maintain a high-quality, safe and dedicated service for patients. -the perceived effectiveness of mers-cov educational programs and knowledge transfer among health workers in this high risk setting was evaluated. -primary health workers were expected to be aware of the most recent updates on mers-cov, yet younger and less experienced hcws had moderate knowledge. -a negative perception of the effectiveness of mers-cov knowledge transfer was associated with poorer knowledge, and was more prevalent among male hcws, physicians/pharmacists and less experienced hcws. global summary and risk assessment middle east respiratory syndrome effects of educational program on mers-coronavirus among nurses students at jazan university - an observational, laboratory-based study of outbreaks of middle east respiratory syndrome coronavirus in jeddah and riyadh, kingdom of saudi arabia laboratory testing for middle east respiratory syndrome coronavirus (mers-cov). cdc what's your strategy for managing knowledge? knowledge management research & practice. journal article does perception of knowledge sharing ,transfer and recognition have an impact on job satisfaction? an empirical study in saudi arabia smoking cessation advice: the selfreported attitudes and practice of primary health care physicians in a military community, central saudi arabia predictors of attitude and intention to use knowledge management system among korean nurses what is mers-cov, and what should i do? : mayo clinic implementing knowledge management practices in hospital-in-the-home units the role of advanced practice nurses in knowledge brokering as a means of promoting evidence-based practice among clinical nurses knowledge, attitude, and practice toward mers-cov among primary health-care workers in makkah al-mukarramah: an intervention study knowledge and perception of health practitioners towards mers-cov in hail region, kingdom of saudi arabia knowledge and attitude towards the middle east respiratory syndrome coronavirus among healthcare personnel in the southern region of saudi arabia knowledge, attitudes and behaviours of healthcare workers in the kingdom of saudi arabia to mers coronavirus and other emerging infectious diseases sense making and knowledge transfer: capturing the knowledge and wisdom of nursing leaders nurses' participation in personal knowledge transfer: the role of leader-member exchange (lmx) and structural empowerment knowledge communication: a key to successful crisis management l'accréditation, source de connaissance et d'enrichissement using interactive workshops to prompt knowledge exchange: a realist evaluation of a knowledge to action initiative culture as an issue in knowledge sharing: a means of competitive advantage academic conferences limited understanding change and change management processes: a case study an educational programme for nursing college staff and students during a mers-coronavirus outbreak in saudi arabia questionnaire-based analysis of infection prevention and control in healthcare facilities in saudi arabia in regards to middle east respiratory syndrome middle east respiratory syndrome-related knowledge, preventive behaviours and risk perception among nursing students during outbreak intra-firm knowledge transfer-a qualitative case study of knowledge transfer and its implications in a soft service firm knowledge management practices in healthcare settings: a systematic review the importance of knowledge transfer between specialist and generic services in improving health care: a cross-national study of dementia care in england and the netherlands diffusion of innovations impaired memory retrieval correlates with individual differences in cortisol response but not autonomic response expatriate knowledge transfer, subsidiary absorptive capacity, and subsidiary performance this study was approved and monitored by king abdullah international medical research center, king saud bin abdulaziz university for health sciences, riyadh, saudi arabia. the authors would like to thank the research office and the institutional review board for their tremendous support. none to declare. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request. all authors conceptualized and designed the study. md, fs, smr and aaf supervised the conduct of the study and data collection. md, smr and aaf undertook the recruitment of subjects and managed the data. fs, smr and aaf were accounted for the quality control. nar and ms provided statistical advice on study design, data analysis and responded to reviewers' comments. all authors drafted the manuscript, and contributed substantially to its revision as submitted and agree to be accountable for all aspects of the work. ethics approval and consent to participate a self-explanatory letter of invitation to participate was presented to each of the participants. all participants had given written informed consents for their participation in the research presented in this manuscript with full knowledge of the possible risks and benefits of participation. participants consented by ticking "agree", indicating their agreement to provide their feedback for this research study. study was approved by the institutional review board of the saudi ministry of national guard health affairs, riyadh, saudi arabia (protocol # sp / ). this study followed the recommendations of the international conference on harmonization for good clinical practice (ich-gcp). not applicable. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.author details key: cord- - fh pe i authors: reyes, leticia; reinhard, mary; brown, mary b title: different inflammatory responses are associated with ureaplasma parvum-induced uti and urolith formation date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: fh pe i background: epidemiologic studies show a strong association between ureaplasmas and urogenital tract disease in humans. since healthy humans can be colonized with ureaplasmas, its role as a pathogen remains controversial. in order to begin to define the role of the host in disease, we developed a rodent model of urinary tract infection (uti) using fischer (f ) rats. animals were inoculated with sterile broth, ( ), ( ), ( ), ( ), or ( )log cfu of a rat-adapted strain of ureaplasma parvum. results: infected animals exhibited two distinct profiles, asymptomatic uti and uti complicated with struvite urolithiasis. inoculum dose of u. parvum affected the incidence of uti, and % to % of animals inoculated with ≥ ( )cfu of u. parvum remained infected (p < . ). however, inoculum dose did not influence immune response to u. parvum. asymptomatic uti was characterized by a minimal immune response that was predominantly monocytic and lymphocytic, with limited lesions, and elevated urinary levels of ifn-γ, il- and mcp- (p ≤ . ). uti complicated with struvite formation was characterized by an exaggerated immune response that was mostly neutrophilic (p ≤ . ), with lesions that showed extensive uroepithelial hyperplasia (p ≤ . ), and a predominance of il- α, il- β, and gro/kc in the urine (p ≤ . ). animals with asymptomatic uti also had a significantly high rate of kidney infection (p ≤ . ). conclusion: complications associated with u. parvum infection are primarily dependent upon host-specific factors rather than ureaplasma microbial load. the immune response in f rats is similar to that which occurs in humans with ureaplasmal associated disease. therefore, this model of infection is a useful tool for elucidating u. parvum-host interactions that confer uti and disease. vated pro-inflammatory cytokines, most notably il- α, il- β, il- , il- , mcp- and tnf-α, accompanied by infiltration of neutrophils and macrophages at sites of infection [ , [ ] [ ] [ ] . however, little work has been done to characterize the immune response during uncomplicated infections. therefore, the complex interactions between ureaplasma and the host that lead to simple colonization versus inflammation and disease are largely unknown. in a recent study, we showed that the inbred rat strain fischer (f ) is susceptible to uti induced by a rat adapted strain of ureaplasma parvum isolated from the urine of a patient with recurrent uti [ ] . as part of that study, we found that % of infected f rats developed struvite uroliths, which were associated with an exaggerated inflammatory response that is similar to what has been reported in other disease states caused by ureaplasma infection [ , , , , ] . interestingly, the other % of f rats developed uncomplicated uti that was characterized by low concentrations of pro-inflammatory cytokines in urine as well as mild to moderate lesions in the lower urinary tract. since f rats are an inbred strain, this particular infection model would be useful for identifying the host/ureaplasma interactions that confer disease or asymptomatic infection without confounding variables that would be introduced by genetic variability. in the study reported here, we examined the innate immune response to uti induced with varying microbial concentrations of u. parvum in the f rat. by applying an integrated approach that combines histopathology with cytokine profiling, we were able to identify innate immune response profiles that were significantly different between an uncomplicated uti and a uti accompanied by struvite formation. our findings provide insights into innate immune responses that are likely involved in the development of complicated disease with ureaplasma. ureaplasma preparation and culture a host-adapted strain of u. parvum, designated strain - was used for the entire study [ ] . fifty mls of u. parvum in logarithmic growth phase was aliquoted into ml volumes and stored at - °c. this stock was used for all experiments. for infection studies, one ml of the working stock was grown in ml of b broth for to hours at °c. the ureaplasma culture was pelleted by centrifugation at , × g, at °c, for minutes. due to the delicate nature of ureaplasma, the pellet was resuspended in to ml of fresh b broth instead of saline, to give a final concentration of cfu per ml then serially diluted to produce various inocula that contained , , , and cfu per ml. the cfu of all inocula (including all serial dilutions) were confirmed by culture on a agar. for each infection experiment, at least two animals were included in each u. parvum dose group and experiments were replicated a minimum of times. inocula and animal tissues were serially diluted -fold in b broth to - and - , respectively. for cfu determination, μl from each sample and its corresponding dilutions were plated on a agar. agar plates were incubated at °c in % co ; broth cultures were incubated at °c in ambient air. agar cultures were incubated for at least days before colonies were counted to determine cfu. specific pathogen free f virgin female rats were purchased from a commercial vendor (charles river, indianapolis, in). all animals ranged in weight from - grams. animal colonies were monitored and found free of the following pathogens: sendai virus, h- virus, rat corona virus, sialodacroadenitis virus, reovirus type , kilham rat virus, hantaan virus, m. pulmonis, respiratory and enteric bacterial pathogens, endoparasites and ectoparasites. all animals were handled in accordance with procedures approved by the university of florida institutional animal care and use committee. all animals were handled within a biosafety laminar flow hood. rats were housed in microisolator ® (lab products, inc., maywood, nj) cages in the same room under the same temperature and light conditions. control animals were always handled before infected and housed in separate microisolator cages in order to prevent contamination with ureaplasma. all food, water, bedding, and caging were autoclaved before use. rats were anesthetized and inoculated with sterile broth or u. parvum inoculum into the bladder as previously described [ ] . for each infection experiment, a minimum of two rats per inoculum dose were infected, so that each dose was represented in each experiment. rats were necropsied at two weeks post-infection as previously described [ ] . prior to euthanasia, free catch urine was collected for cytokine analysis. the bladder was processed for histopathologic evaluation. each kidney was transected sagittally so that a portion of the renal pelvis was present in each section. one half of each kidney was processed for histopathologic evaluation. the remaining halves of the right and left kidneys for an individual animal were combined, minced in sterile b broth, and the medium was aseptically removed and cultured for u. parvum. bladder calculi were submitted to a commercial laboratory (louis c. herring and co., orlando, fl) and analyzed by integrated crystallography. bladder and kidney tissues were fixed in a paraformaldehyde-lysine-periodate [ ] solution for hours, then washed times in sterile saline and transferred to % ethanol prior to processing. tissues were processed routinely and stained with hematoxylin and eosin (h&e). bladder lesions were scored by a system developed in a previous study [ ] . epithelial changes in bladder tissues were scored as: for none, for minimal hyperplasia, ulceration or effacement of epithelium by inflammation; for mild hyperplasia and rare dense inflammatory infiltrates, and for the same changes noted in a score of but accompanied with marked erosion and/or ulceration of the epithelial surface. scoring for cell types that comprised the inflammatory infiltrate was: for primarily mononuclear cells (lymphocytes, plasma cells and macrophages), for mononuclear cells and neutrophils, and for mononuclear cells, neutrophils and fibrous infiltrates. kidney tissues were scored on the basis of total area affected, which was: for less than %, for to %, and for greater than %. urine from control and infected rats was analyzed for the presence of cytokines with a multiplex antibody-immobilized bead immunoassay (lincoplex kit, linco research, inc., st. charles, mo). the manufacturer's protocol was followed for the simultaneous detection of the following cytokines and chemokines: gm-csf, il- α, il- β, il- , il- , il- p , ifn-γ, il- , gro/kc (growth related oncogene/keratinocyte chemoattractant-the rat analog for human il- ), and tnf-α within the same aliquot of urine. briefly, a standard cocktail was serially diluted in order to develop a standard curve for each analyte that ranged from . to pg/ml. urine samples were diluted in assay buffer to obtain a total volume of μl per well, and run in duplicate as previously described [ ] . data from multiple experiments were grouped together in order to make statistical analysis possible. wherever possible, data were analyzed by one-way anova when more than two groups were included in the analysis. fisher's plsd test was used when the anova indicated a significant difference among group means. an unpaired student's t test was used for comparisons between two groups. contingency table analysis was used for comparisons of groups involving nominal data (positive vs. negative). cytokine pattern recognition analysis was performed using jmp genomics . (sas institute, cary, nc). datasets were initially evaluated by distribution analysis and normalized prior to analysis by one-way anova using row by row modeling and fischer c correction for multiple comparisons. for all analyses a probability of p < . was considered significant. ureaplasmas were not isolated from any site from any control rat (data not shown). neither uroliths nor crystals were detected at any site in animals inoculated with sterile b broth. there was no statistical difference in the log cfu of u. parvum isolated from culture positive animals among the various inoculum groups. the log cfu isolated from the bladder tissue of culture positive animals was . ± . (mean ± sd), and . ± . from kidney tissue. however, there was a statistical difference in the frequency of animals that remained infected weeks postinoculation (table ) . animals inoculated with or higher cfu had the greatest frequency of bladder infections at time of necropsy (p ≤ . ). animals inoculated with u. parvum were divided into three groups (negative, uti, or struvite) stratified on the basis of culture status and urolith status ( table ). the negative group consisted of animals found to be culture negative and urolith negative at time of necropsy. as expected, the frequency of animals that were culture negative after inoculation with u. parvum decreased as the log cfu of the inoculum increased. the uti group represents ( ) / ( ) / ( ) / ( ) / ( ) ns a) data was collected at weeks post-inoculation, and is a combination of separate experiments. b) probability values were derived by g statistic, p values greater than . were considered not significant (ns). c) both sites refer to bladder and kidney. animals that were culture positive in the bladder and/or kidney at time of necropsy but were found to be negative for uroliths. eleven of ( . %) animals within the uti group had kidney infections at time of necropsy, which was significantly greater (p ≤ . ) than the number of renal infections within the urolith group [ of ( %) animals]. all animals within the struvite group had bladder stones, which were composed of to % magnesium ammonium phosphate (struvite), to % calcium phosphate (carbonate apatite) and to % protein and blood. no animal had macroscopic evidence of kidney stones at time of necropsy. although not statistically significant (p ≤ . ), animals inoculated with or greater cfu tended to have the highest frequency of struvite uroliths. the inoculum dose of u. parvum did not impact the distribution of animals that developed either uti or struvite uroliths (table ) . further, there was no statistical difference between the log cfu cultured from bladder tissue among these two clinical groups. the log cfu (mean ± sd) of u. parvum isolated from the bladder of animals within the uti group was . ± and . ± from the bladder of animals within the struvite group. the extent and severity of bladder lesions as well as the types of inflammatory cells present differed among groups. there were no detectable lesions in bladder tissue from control rats (see figure , panel a). in animals inoculated with u. parvum, bladder lesions associated with inflammation were highly variable. when present, lesions consisted of infiltrates of lympho-plasma cells, macrophages, and neutrophils that were primarily located within the epithelial layer ( figure , panel b and d). mast cells could be found within the submucosa regardless of clinical profile (data not shown). uroepithelial changes ranged from spongiosis of epithelial cells with some necrosis, exfoliation of uroepithelium, or hyperplasia (see figure , panels b, c, and d). animals with struvite uroliths had the most extensive lesions and the highest lesion scores ( figure ). in these animals, inflammation extended into the submucosa and muscularis layers, and was occasionally accompanied by venous congestion and edema with a fibrinous reaction ( figure , panel b). neutrophilic infiltrates were present in all of the animals within the struvite group. in contrast, infiltrates in animals within the negative and uti groups were predominantly lymphocytes, plasma cells and macrophages ( figure , panel b) . animals with uroliths also had the most extensive uroepithelial hyperplasia as shown in figure , panel c. animals within the negative and uti groups had the mildest inflammatory changes ( figure ). however, somewhat surprisingly, more animals within the negative group had a higher degree of both inflammation and epithelial change than did animals within the uti group. most of the animals in the uti group exhibited exfoliation of uroepithelium with some spongiosis. both the inflammatory cell type score and inoculating dose of u. parvum influenced the lesion scores within the negative group. there was no difference in the lesions scores pertaining to degree of inflammation and degree of epithelial change among the inoculating dose groups (data not shown). however, there was a significant difference (p ≤ . ) in the inflammatory cell type score and inoculating dose of u. parvum (figure , panel d) . a significant number of animals that received log cfu had a mixed inflammatory cell infiltrate that included neutrophils as well as mononuclear cells (lymphocytes, plasma cells, and macrophages). kidney tissue from f rats were also evaluated for the presence of inflammation. there were no significant lesions present in the collecting ducts and renal pelvis of control rats (fig , panel a) . histopathologic findings in kidney tissue from rats inoculated with u. parvum ranged from minimal changes to varying degrees of inflammatory infiltration that consisted of lymphocytes, plasma cells, macrophages and neutrophils. in some animals, lesions were characterized by scant multifocal areas of predominantly mononuclear cells that were present in the subepithelial region of the renal pelvis ( figure , panel b). in ( ) / ( ) / ( ) these animals, the uroepithelial lining the pelvic space was hyperplastic. animals with the most severe kidney lesions had extensive erosion of the uroepithelium, as shown in figure , panel d. in these animals, erosion of the uroepithelial barrier was accompanied by hemorrhage and infiltration with inflammatory cells (predominantly neutrophilic) that spanned the pelvic luminal space, through the epithelial layer and into the sub-epithelial region of the pelvis (figure , panel d). other animals, had inflammatory infiltration of the renal interstitium as shown in figure , panel c scoring of kidney tissues on the basis of total area affected did not reveal any patterns that could be correlated to local/active infection or clinical profile. specifically, there was no correlation between the total area affected score and u. parvum culture status (data not shown). there also was no correlation between the total area affected score summary of the bladder lesions found in f rats experimentally infected with u. parvum figure summary of the bladder lesions found in f rats experimentally infected with u. parvum. panel a is a × magnification of bladder tissue from a control rat. this sample represents a lesion score of for degree of inflammation, degree of epithelial change and inflammatory cell type. panels b, c, and d are tissue sections from animals inoculated with u. parvum that had a lesion score of for degree of inflammation, degree of epithelial change and inflammatory cell type. panel b is a × magnification of bladder tissue from an animal within the struvite group. the asterisk demarcates the extensive edema and fibrinous exudate infiltrating the submucosa. the black arrow points to uroepithelial effacement. panel c is a × magnification of extensive uroepithelial hyperplasia. panel d is a magnified inset of panel b that highlights the array of white blood cells that comprised in the inflammatory cellular infiltrate in the tissues of infected animals. the blue arrow is pointing to a neutrophil. the green arrow is pointing to a tissue macrophage. the yellow arrow is pointing to a plasma cell. the red arrow is pointing to a lymphocyte. and clinical profile (negative, uti, or struvite), additional file . the relationships between specific cytokines and bladder lesion scores among animals inoculated with sterile b broth or u. parvum were examined by spearman correlation analysis. there were no correlations between urine cytokine levels and degree of inflammation score among animals inoculated with sterile b broth (data not shown). however, there was a significant correlation (summarized in table ) between the degree of inflammation score and urine concentrations of gro/kc, il- α, il- β, il- and tnf-α. there was also a significant correlation between degree of epithelial change and urine concentrations of gro/kc and il- β (table ). mcp- levels negatively correlated with degree of epithelial change ( table ). the cytokine profile in urine differed among groups. absolute concentrations of each individual cytokine in urine were compared among clinical profiles (control, negative, uti and struvite). control animals had significantly higher levels of mcp- than animals in either the negative or uti groups (figure ) . animals in the struvite group had the highest levels of gro/kc (figure ) , il- α, il- β, il- , il- and tnf-α ( figure ) than did animals in all other groups. there was no statistical difference in the absolute amounts of urine gm-csf, ifn-γ, il- , il- , il- , and il- among groups (data not shown). in order to identify distinctive chemokine/cytokine patterns between clinical profiles associated with active infection, samples from animals within the negative group were excluded from this analysis. each urine cytokine multiplex from each animal was normalized prior to analysis by one-way anova using row by row modeling and fischer c correction for multiple comparisons. figure is a clustered heat map illustrating two cytokine profile clusters that significantly differed between control, uti and struvite groups (p ≤ . ). both control and uti groups showed a significant emphasis in il- and mcp- , whereas the struvite group showed a significant emphasis in il- α, il- β, and gro/kc. only the uti group showed a significant emphasis in ifn-γ. urine cytokine data from control and culture negative animals was normalized prior to statistical analysis and analyzed as described above. there was a significant difference in the overall pattern of il- , il- , il- , tnfα, ifn-γ, and gro/kc in the urine of culture negative animals that received different inoculating doses of u. parvum (p ≤ . ). figure is a clustered heat map illustrating lesion scores of bladder tissue from f rats inoculated with u. parvum two distinct cytokine profile clusters (green and red cluster trees) among these animals. with the exception of log and log inoculating groups, there was an inverse relationship in the pattern of expression between the two cytokine clusters. ureaplasmas are an underappreciated pathogen of the urogenital tract. despite strong epidemiological evidence and even experimental infections in humans that fulfilled koch's postulates [ ] , the etiologic role of ureaplasmas is confounded by the isolation of the microbe from the lower urogenital tract of normal, asymptomatic individuals. in addition, the severity of disease for most mycoplasmal infections depends on the host immune response. therefore, experimental infections in genetically defined animal models will be critical to unraveling the key interactions in the host/parasite relationship that contribute to disease severity. by using a combined approach involving histopathology and cytokine profiling, we were able to further characterize the immune response associated with asymptomatic uti and uti complicated with struvite formation. summary of the kidney lesions found in f rats experimentally infected with u. parvum figure summary of the kidney lesions found in f rats experimentally infected with u. parvum. panel a is a × magnification of kidney tissue from a control rat and demonstrates the lack of inflammatory lesions that are characteristic in animals inoculated with u. parvum. panels b, c, and d are tissue sections from animals inoculated with u. parvum that had a lesion score of for total area affected. panel b is a × magnification demonstrating the inflammatory infiltrate extending from the renal pelvic space into the interstitium with uroepithelium largely intact. the black arrow is pointing to uroepithelial hyperplasia. panel c is a × magnification of renal tubules. the black arrow is pointing to the extensive inflammatory infiltrate throughout the renal tubular interstitium. the yellow arrow is pointing to a glomerulus. panel d is a × magnification of renal uroepithelium at the edge of the pelvic space. the black arrow is pointing to extensive hemorrhage and disruption of the uroepithelial barrier by a fibrinous inflammatory infiltrate. the f rat strain is highly susceptible to development of complicated uti following experimental inoculation with u. parvum [ ] . in this study, we showed that varying the inoculum dose of u. parvum significantly affected the frequency of animals that remained colonized two weeks after inoculation. therefore, the initial microbial load is important in establishing infection. however, once infected, the proportion of animals that developed complicated uti in response to varying inocula of u. parvum did not show a definitive dose response effect. more importantly, the immune response to infection in this group of animals with complicated uti was consistent, regardless of initial inoculating dose. for example, the cytokine profile and urinary tract pathology of a struvite positive animal that was inoculated with cfu was indistinguishable from a struvite positive animal that was inoculated with cfu. this suggests that the initial microbial load of u. parvum is not a critical factor in the development of complicated uti in this rat strain. further, it supports the concept that, once infection is established, the host inflammatory response is a key determinant of lesion severity in the urinary tract. as previously reported [ ] , animals with asymptomatic uti had significantly less pro-inflammatory urine cytokines and tissue damage when compared to rats with struvites. by profiling the entire cytokine milieu, we were able to identify a significant predominance of cytokines such as ifn-γ, il- , and mcp- in the uti group that work synergistically to regulate monocyte/macrophage activation [ ] [ ] [ ] . an intriguing finding was the significant emphasis of ifn-γ in the urine of animals with asymptomatic uti, since this cytokine is a potent priming agent for macrophages [ ] . this cytokine profile also coincides with the cellular immune response in these animals that consisted of macrophages, lymphocytes, and plasma cells, which resembles a profile that may be seen during the healing or resolution phase of infection. we cannot rule out that these animals could be displaying a pre-resolution phase to infection, but there are indicators suggesting that these animals have compromised immune defense. for example, the immune profile of these animals was obtained while they were actively colonized with u. parvum, and % exhibited an ascending infection into the kidneys. further, the microbial load of u. parvum in animals with asymptomatic uti was equivalent to animals in the struvite group. another intriguing feature in animals with asymptomatic uti was the overall lack of uroepithelial proliferation that was present in varying degrees in the negative group as well as the struvite group. a primary defense mechanism of uroepithelium exposed to bacteria involves desquamation, necrosis or apoptosis followed by proliferation [ ] . therefore, the overall lack of this response in animals with asymptomatic uti also implies that uroepithelial defense mechanisms may be perturbed by u. parvum. f rats with struvite uroliths had a similar clinical profile to what we have previously described [ ] . specifically, these animals had the greatest concentration of proinflammatory cytokines in their urine (gro/kc -the rat analog of human il- , il- α, il- β, il- and tnf-α) and the most extensive inflammatory changes in bladder tissue. since il- β is a known inducer of il- and gro chemokines in human and murine epithelial cells [ ] [ ] [ ] , it is not an unexpected finding that these cytokines are closely linked in their expression. cytokine pattern analysis showed this cytokine cluster is unique to animals with struvites. moreover, there is a significant positive correlation between il- β, gro/kc and the degree of histopathologic change, which suggests that il- β and gro/ kc are critical elements in a pro-inflammatory loop that leads to chronic active inflammation, epithelial hyperplasia and struvite formation as seen in struvite positive f rats. most of the animals within the struvite group had uroepithelial hyperplasia or erosion with hemorrhage and inflammation within the kidneys, yet none of these animals had uroliths in the renal pelvis that could account for these lesions. therefore, although mechanical irritation by the urolith itself may partially contribute to epithelial erosion or hyperplasia in the bladder, it cannot entirely account for the lesions that were present in the urinary tract of these animals. the immune response of animals within the negative group was highly variable and most likely comprises a mixed population of rats, including animals that never became colonized as well as animals that cleared the infection at various time points post-inoculation. therefore, interpretation of data from this group of animals is difficult and is done with caution. in spite of this limitation, profiling urine cytokine data and bladder lesion scores by inoculum dose was informative. the threshold dose for successful colonization appears to be between log and log cfu, since % and % of rats respectively were culture negative weeks post-inoculation. the animals within the log and log cfu inoculation groups also had the greatest flux in both pro-inflammatory (tnfα, ifn-γ and gro/kc) and anti-inflammatory (il- , and il- ) cytokines. interestingly, pattern analysis of urine cytokine data showed two distinct clusters. the first cluster identified in the negative group included il- , il- , il- , and tnf-α; these cytokines were notable as they were not part of the cytokine cluster groupings of u. parvum infected animals. therefore, these cytokines may be critical in directing a more efficient immune response that leads to bacterial clearance with minimal tissue damage. the second cytokine cluster in the negative group included ifn-γ and gro/kc, which are significant cytokines in the uti and struvite groups, respectively. except for animals that were inoculated with log cfu of u. parvum, the expression pattern of ifn-γ and gro/kc was not inversely related as they were in culture positive animals. this may be reflecting a more balanced immune response than what is seen in u. parvum positive animals, and we suggest that this balanced response may be critical to resolution of infection and prevention of severe disease. the variable clinical outcome to experimental inoculation with u. parvum in the f rat is an interesting phenomenon since this is an inbred strain. in this study, both genetic and environmental influences on disease were minimized to the extent possible. all of the animals in this study originated from the same colony. further, rats were housed under the same barrier maintained conditions in order to minimize environmental variability. despite our efforts, it was common to find that a rat that developed asymptomatic uti had co-habited the same cage with a rat that developed struvites or was culture negative at time of necropsy. therefore, external environment could not account for the varying clinical outcome in our study. however, our experimental inoculation procedure may be a critical source of variability. although attempts were made to reduce mechanical trauma caused by catherization, it is possible that the trauma may have been sufficient to shift the immune response towards a proinflammatory profile in a subset of animals. once this occurred, the pro-inflammatory cycle progressed until infection was resolved (negative group) or the study was terminated (struvite group mcp- pg/ml complex interactions between most mucosal pathogens and the host that lead to uncomplicated colonization versus inflammation and disease are largely unknown. therefore, this model may be particularly useful for identifying the molecular events that confer asymptomatic infection, complicated infection as well as resolution of infection with an opportunistic pathogen of the urogenital tract. the complex interactions between ureaplasma and the host that lead to uncomplicated colonization versus inflammation and disease are largely unknown. we characterized the f rat immune response in the urinary tract to varying inoculum concentrations of u. parvum. establishment of uti was influenced by microbial load, but the host immune response was independent of microbial load. two distinct innate immune profiles were identified with two different clinical outcomes: asymptomatic uti and complicated uti with struvite formation. asymptomatic uti was characterized by a minimal immune response that was predominantly monocytic and lymphocytic and was accompanied by a significantly high rate of kidney infection. uti complicated with struvite formation was characterized by an exaggerated immune response that was predominantly neutrophilic and was accompanied by uroepithelial hyperplasia and extensive tissue damage. the authors declare that they have no competing interests. urine cytokines detected in f rats inoculated with sterile b or u. parvum figure urine cytokines detected in f rats inoculated with sterile b or u. parvum. data represent the mean ± sd of a combination of separate experiments. urine cytokine concentrations were grouped according to clinical profile, control (n = ), negative (n = ), uti (n = ), and struvite (n = ). p values within each graph were obtained by one-way anova. fisher's plsd test revealed that il- α, il- β, il- , and tnf-α concentrations in the struvite group were significantly greater than control, negative, and uti groups. fisher's plsd test revealed il- concentrations in the struvite group were significantly greater than negative and uti groups. global profiling of urine cytokines detected in control and u. parvum infected f rats figure global profiling of urine cytokines detected in control and u. parvum infected f rats. the clustered heat map represents the standardized ls means for each cytokine that had a significantly different pattern of expression among clinical groups (p ≤ . ). values were obtained by one-way anova using a row by row modeling with fischer c correction for multiple comparisons. two main cytokine cluster patterns were identified in the analysis and are demarcated by the green and red cluster tree. the number of biological replicates were n = for control, n = for uti group, n = for the struvite group. ureaplasma infection and neonatal lung disease ureaplasma urealyticumharmless commensal or underestimated enemy of human reproduction? a review comparative randomized pilot study of azithromycin and doxycycline efficacy and tolerability in the treatment of prostate infection caused by ureaplasma urealyticum uropathogens and urinary tract concretion formation and catheter encrustations antenatal ureaplasma urealyticum respiratory tract infection stimulates proinflammatory, profibrotic responses in the preterm baboon lung characterization of a murine model of ureaplasma urealyticum pneumonia biochemical and histologic findings in experimental pyelonephritis due to ureaplasma urealyticum concrement formation in the urinary bladder in rats inoculated with ureaplasma urealyticum morphological lesions of the rat urinary tract induced by inoculation of mycoplasmas and other urinary tract pathogens epidemiology and causes of preterm birth intrauterine infection and prematurity. mental retardation and developmental disabilities research reviews disordered pulmonary myofibroblast distribution and elastin expression in preterm infants with ureaplasma urealyticum pneumonitis rat strains differ in susceptibility to ureaplasma parvum-induced urinary tract infection and struvite stone formation immunohistochemical detection of cytokines in paraffinembedded mouse tissues human intra-urethral inoculation of ureaplasmas interleukin- and host defense against infection. the journal of infectious diseases the role of gamma interferon in antimicrobial immunity il- induces monocyte chemotactic protein- production in macrophages through the phosphatidylinositol -kinase/akt and mek/erk / pathways regulation of macrophage activation bad bugs and beleaguered bladders: interplay between uropathogenic escherichia coli . and innate host defenses the effect of interleukin- on cytokine gene expression by human corneal epithelial cells keratinocyte chemoattractant (kc)/human growth-regulated oncogene (gro) chemokines and pro-inflammatory chemokine networks in mouse and human ovarian epithelial cancer cells. cancer biology & therapy interferon gamma induces differential upregulation of alpha and beta chemokine secretion in colonic epithelial cell lines this work was supported by the national institutes of health grants r ai and k dk . we also wish to acknowledge ms. janet stevens and lj mcdonnell for their assistance with microbial cultures and cytokine immunoassays. lr designed and executed animal infection studies, data analysis and manuscript preparation. mr performed histopathologic evaluation of tissues and developed the lesion scoring system implemented in this study. mbb participated in the design and coordination of the study, and helped draft the manuscript. all authors read and approved the final manuscript. profiling the inflammatory response to different doses of u. parvum in culture negative f rats figure profiling the inflammatory response to different doses of u. parvum in culture negative f rats. panel a is a clustered heat map representing the standardized ls means for each cytokine with a significantly different pattern of expression among infection dose groups (p . ). values were obtained by one-way anova using a row by row modeling with fischer c correction for multiple comparisons. two main cytokine cluster patterns were identified in the analysis and are demarcated by the green and red cluster tree. the number of biological replicates were n = for control, n = for log cfu, n = for log cfu, n = for log cfu, n = for log cfu, and n = for log cfu. the red arrow is highlighting the pattern of cytokines present in the urine of culture negative rats that were inoculated with log cfu. animals within this dose group were the only animals to exhibit an obvious inflammatory cell infiltrate comprising a mixture of mononuclear cells with neutrophils (p . , figure the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -n r pzan authors: lau, joseph tf; kim, jean h; tsui, hi yi; griffiths, sian title: anticipated and current preventive behaviors in response to an anticipated human-to-human h n epidemic in the hong kong chinese general population date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: n r pzan background: the prevalence of self-reported preventive behaviors in response to an anticipated local human-to-human h n transmission outbreak and factors associated with such behaviors have not been examined. methods: a random, anonymous, cross-sectional telephone survey of hong kong chinese adults. results: the public in hong kong is likely to adopt self-protective behaviors (e.g., wearing face mask in public venues ( . %), increasing the frequency of handwashing ( . %)) and behaviors that protect others (e.g., wearing face masks when experiencing influenza-like illness (ili, . %), immediately seeking medical consultation ( . %), making declarations when crossing the border with ili ( . %), complying to quarantine policies ( . %)). multivariate analyses indicated that factors related to age, full-time employment, perceived susceptibility, perceived efficacy of preventive measures, perceived higher fatality as compared to sars, perceived chance of a major local outbreak, and being worried about self/family members contracting the virus were significantly associated with the inclination to adopt self-protective measures. similar analyses showed that education level, variables related to perceived efficacy, perceived major local outbreak and such were significantly associated with various behaviors directed towards protecting others. conclusion: in the event of a human-to-human h n outbreak, the public in hong kong is likely to adopt preventive measures that may help contain the spread of the virus in the community. it has been estimated that . million deaths may result worldwide [ ] . during the sars epidemic, preventive measures were commonly practiced [ ] and a number of these behaviors [ ] [ ] [ ] have been sustained by a large proportion of the public even after the sars epidemic subsided in hong kong [ ] . preventive behaviors such as face mask use and handwashing have been suggested to be effective in the control of the sars epidemic [ ] . understanding the correlates of these behavioral changes would facilitate formulation of policies and campaigns to promote appropriate behavioral responses in the event of a human-to-human h n outbreak. this study investigated the prevalence of self-reported preventive behaviors in response to a hypothetical local outbreak of human-to-human h n transmission. these behaviors included both self-protective measures as well and those protecting others from contracting the virus. factors associated with these behaviors were investigated. the study population was comprised of male and female hong kong chinese adults aged - years. an anonymous cross-sectional telephone survey using a structured questionnaire was conducted in november (n = ). table summarizes the background characteristics of the respondents. random telephone numbers were selected from up-to-date telephone directories. telephone surveys have been used in a number of sars and avian influenza studies [ ] [ ] [ ] [ ] . telephone calls were made by trained interviewers from : pm to : pm each night to avoid over-representation of unemployed persons. for unanswered calls, at least other independent calls were made. the household member whose birthday was closest to the date of the interview was invited to join the study. verbal informed consent was obtained from the respondents and ethics approval was obtained from the ethics committee of the chinese university of hong kong. the response rate, defined as the number of completed interviews divided by the number of eligible households, was approximately %. respondents were asked how likely they would be to adopt the following preventive behaviors if a local human-to-human h n outbreak (defined as "if - new human-to-human transmission of h n cases were to be reported in hong kong") were to occur: face mask use in public venues, increased frequency of handwashing, avoidance of eating poultry, declaration of influenzalike illness (ili) symptoms at border health checkpoints, the seeking of medical consultation immediately with the onset of a fever, face mask use in public venues when having ili symptoms and compliance with any quarantine policies. responses were recoded into categories (yes or no) from a -point likert scale. respondents were asked about perceptions related to human-to-human h n transmission, including perceived modes of transmission (whether human-to-human transmission of the h n virus could occur via respiratory droplets, bodily contact, contaminated objects, eating well-cooked poultry), perceived susceptibility to h n in different groups of people (self, family members, children, adults, older people, health care workers, food handlers, food vendors and the general public), perceived chance of having a major outbreak in hong kong in the next months and perceived efficacy of various prevention measures (quarantine of infected people, face mask use in public venues, frequent handwashing, home disinfection, mass extermination of poultry). respondents were also asked their perceptions of the current availability of effective treatments, whether they believed health outcomes would be worse than those of sars (higher fatality and lower treatment efficacy) and the degree of concern they had about oneself or one's family contracting the virus if - new human-to-human h n were to be reported in hong kong. univariate odds ratios of the associations between the studied perception variables and individual studied preventive behaviors were derived. variables that were significant in the univariate analyses were further analyzed using multivariate logistic regression analyses. statistical significance was set at p = . and spss software version . . (spss inc, chicago, il, ) was used for data analyses. the majority would adopt self-protecting behaviors such as wearing face mask in public venues ( . %), increasing frequency of handwashing ( . %), avoiding eating poultry ( . %), wearing face mask in public venues when having ili symptoms ( . %), seeing a doctor immediately when having a fever ( . %), making declarations at border health checkpoints when traveling with ili ( . %), and full compliance with any quarantine policies ( . %). gender and educational differences were, in general, non-significant, whereas some significant age differences were noted. overall, . % of the respondents would practice all types of protective behaviors ( table ) . the majority of the respondents believed that various groups of people would be highly susceptible to the virus. of the respondents, . % believed themselves and . % believed their family members or the general public to be highly susceptible (table ). over % of the respondents perceived "high"or "very high" efficacy in various preventive practices, and . % perceived mass extermination of poultry as an efficacious measure. of the respondents, . %, . %, . % and . %, respectively, believed that respiratory droplets, contaminated objects, body contacts and eating well-cooked poultry to be transmission modes of the virus (table ) . with regard to the impact of the disease, % believed that there would be a major human-to-human h n outbreak in hong kong in the coming year; . % believed that it would have higher fatality as compared to sars and . % would be very worried about oneself or one's family members contracting the virus if an outbreak were to occur. further, half of the respondents ( . %) believed that effective drugs are now unavailable, fewer ( . %) thought that the efficacy of treatment for this disease would be worse that that of sars (table ) . certain factors were significantly associated with self-protective behaviors at the multivariate level. these include older age, full time employment, higher degree of perceived susceptibility, perceived efficacy of using face masks in preventing the disease, higher degree of perceived efficacy of the preventive measures, perceived higher fatality of h n than sars, anticipation of risk of a major outbreak in hong kong in the coming year, and concern about oneself/one's family in contracting the virus (or = . to . , p < . , table ). perceived efficacy related to mask use, handwashing and mass extermination of poultry, perceived higher fatality rate as compared to sars and worry about oneself/one's family in contracting the virus were significant in the multivariate analysis in predicting perceived higher frequency of handwashing (or = . to . , p < . , table ). having ever been married, perceived susceptibility for food handlers, perceived efficacy of face mask use, perception that well-cooked poultry is a mode of transmission and perceived major outbreak in hong kong were multivariately predictive of avoidance of eating poultry (or = . to . , p < . , table ). multivariate results in table indicate education level, perceived efficacy related to face mask and perceived major outbreak in hong kong in the next year were predictive of anticipated use of face mask in public venues when having ili symptoms (or = . to . , table ); perceived likelihood of a major outbreak in hong kong in the next year and perceived efficacy of disinfection of living quarters were multivariately associated with declaring ili symptoms at cross-border checkpoints (or = . and . , respectively); perceived efficacy of mass extermination of poultry was the only factor predicting immediate doctor consultation when having fever (or = . ). evermarried status, perceived susceptibility related to children, perceived efficacy of face mask use were multivariately associated with intended full compliance with any quarantine policies (or = . to . ). these data indicate that the majority of the hong kong general public would adopt preventive measures, even in the event of - reported human-to-human h n transmissions in hong kong. during the sars epidemic, the prevalence of similar preventive behaviors increased sharply in the initial weeks of the outbreak [ ] . currently, we estimate that reasonably half of the general population is washing their hands over times a day (unpublished data). an even higher frequency of handwashing is expected if the anticipated outbreak occurs. this is consistent with the local government campaigns for promoting handwashing [ ] . handwashing has been efficacious in preventing influenza [ , ] and sars [ ] and the vast majority of the respondents ( %) believed that it would be efficacious in preventing human-tohuman avian influenza. such a belief was, in turn, associated with anticipated higher frequency of handwashing. handwashing may have become a commonly accepted means of preventing infectious respiratory diseases. approximately % of the general population reported face mask use in public venues during the peak of the sars epidemic [ , ] . the majority of the respondents would do so if there was a human-to-human h n outbreak in hong kong. many believed that face mask use in public venues was an efficacious method of human-tohuman h n prevention. however, while . % of the respondents reported that they would report ili symptoms at health checkpoints as required at times of a human-to-human h n outbreak, . % of the respondents did not do so during the period of april to january (during and shortly after the sars epidemic) (unpublished data) [ ] . studies have demonstrated the limited effectiveness of such measures implemented during the sars epidemic [ , ] . it is shown that about % of the general population with ili symptoms during the sars epidemic were wearing face masks [ ] , which is comparable to the results of this study showing that about % would wear face masks. the similarities between various public health responses related to sars and avian influenza are reported and it is speculated that the public is modeling their responses to avian influenza outbreak to those of the sars epidemic. during the sars epidemic, the reported prevalence of face mask use when having ili while traveling abroad was not very high, indicating that a substantial proportion of the general population was not practicing behaviors directed towards protecting others [ ] . this is in contrast to the results of this study, which noted very high proportion of respondents stating their intent to adopt of behaviors directed towards protecting others. the perceived severity of h n may elicit more behaviors directed at protecting others. quarantine was an effective means contributing to the control of sars [ , ] but a comparatively low percentage of the respondents ( . %) believed quarantine to be an effective public health measure for control of avian influenza, and . % of the respondents would not fully comply with government quarantine policies indicates there is still a need for education. as perceived efficacy was or m = odds ratios obtained from multivariate stepwise logistic regression using univariately significant (including marginally significant) variables as candidate variables. . < p < . ; *p < . ; **p < . ; ***p < . . ns = not significant. ---univariately non-significant and was not considered in the multivariate stepwise logistic regression analysis. would worry that it is likely/very likely for oneself or one's family members to contract h n (if - new cases were to be reported in hong kong. gender, age group, and perception that the impacts of human-to-human h n would be worse than that of sars in terms of fatality rate and efficacy of treatment were not associated with any of the dependent variables and were hence not tabulated. or u = univariate odds ratio. or m = odds ratios obtained from multivariate stepwise logistic regression using univariately significant (including marginally significant) variables as candidate variables. . < p < . ; *p < . ; **p < . ; ***p < . . ns = not significant. ---univariately non-significant and was not considered in the multivariate stepwise logistic regression analysis. table : factors associated with anticipated preventive behaviors to protect others if to new human-to-human h n cases were to be reported in hong kong (continued) univariately associated with anticipated compliance with any quarantine policies, dissemination of information about its efficacy may be useful. single respondents were less likely to comply fully with quarantine policies are also generally more mobile. these people may have less support if quarantined, and this should be considered by policy makers. from until the present, the reported fatality rate of avian influenza in human has been over % [ ] , which is markedly higher than the . % case fatality of rate sars [ ] . however, only about % of the study's respondents considered human-to-human h n fatality rate to be higher than that of sars. hence, the actual magnitude of behavioral responses might be even stronger, since we found perceived fatality to be significantly associated with self-protective behaviors. it is particularly interesting to note that while associations with self-protective behaviors such as mask use and handwashing were highly significant, perceived fatality rate in comparison to sars was not significantly associated with any of the behaviors directed to protecting others. therefore, different considerations may be involved in making decisions of whether to practice preventive behaviors, depending on whether such behaviors are self-directed or directed towards others. with full population compliance with quarantine policies, the critical battle front of the avian influenza epidemic would shift to effective hospital infection control. it is unlikely that health care workers will be able to comply to all the stringent occupational guidelines in the event of nosocomial human h n outbreaks. high fatality rates may occur in these health settings if panic or widespread non-adherence to safety measures occur. research, counseling and emergency plans are essential to ensure that front-line health care workers are psychologically prepared and that the operations of health systems will not be disrupted. the public regarded older people, children, health care workers and food vendors as particularly at risk of contracting the virus. during the sars epidemic, it was reported that discriminatory attitudes have been expressed toward some at-risk groups, such as health workers [ , ] . it is worth noting that certain social groups, such as health care workers or food vendors, may be stigmatized similarly during an h n influenza. preventing or minimizing this should thus be focus of future study. approximately one-quarter of the respondents believed that they or their family members would likely/very likely be affected by an h n outbreak whereas approximately one-third of the respondents believed this to be true for the general population. it was reported that during the sars epidemic, the general public worried about themselves or their family members' contracting the virus [ ] . this is expected to be repeated if a human-to-human avian flu outbreak occurs. these perceptions were also associated with anticipated preventive behaviors. many studies have documented severe distress in the community during and after the sars epidemic [ , ] and attention should be given in reduce panic at times of a human-to-human h n outbreak. with the potentially high fatality and infectivity, high level of distress in the public is expected. although prevalence of health-seeking behaviors are usually higher among females than males, gender was not associated with any of the studied behaviors in this study. the health threat in this case may have overridden the aforementioned gender differentials. education level was not associated with self-directed preventive behaviors but was associated with one of the others-directed preventive behaviors. it is possible that altruism is associated with education level. the reverse was true with age. another study indicated that higher age was associated with the more use of preventive measures [ ] . in general, factors related to perceived susceptibility, perceived clinical severity of outcomes (e.g. fatality rate, perceived availability and efficacy of treatments) were associated with anticipated preventive behaviors. such variables are the key factors prescribed by the health belief model (hbm), which stated that adoption of health behaviors is a function of an individual's attitudes and beliefs about the health issue/behavior of concern [ ] . these variables were significant in predicting preventive behaviors related to sars and influenza [ , , ] . the hbm is therefore applicable to understanding behaviors for preventing the spread of emerging infectious diseases. the study has a number of limitations. first, the study was conducted using telephone surveys and some households may not have been included. in hong kong, however, almost all households have telephones [ ] and a large number of local published studies on sars [ ] and avian influenza [ , , ] have utilized this method. second, the response rate of the study was not very high. nevertheless, the response rate was similar to many of other published local studies [ , , ] . with the distributions of - and - year age groups being very comparable to those obtained from the census data ( . % and . %, respectively). the study's gender distribution was also comparable to the census distributions ( . % male and . % female) [ ] . adoption of behavioral responses was self-reported and had not been validated. however, during this pre-outbreak stage, it is unlikely that social desirability strongly biased the reporting of these behaviors. while the results of this study may have meaningful regional policy implications, caution should be given when generalizing the results of this study to other countries. it is less clear whether populations which were relatively unaffected by sars and populations that demonstrated less frequent face mask use and other relevant public health measures would exhibit the same magnitude of intended behavioral responses as the population of hong kong. cultural and perceptions factors (such as perceived efficacy of prevention means) would also result in different prevalence of behavioral responses. international comparisons are therefore also greatly warranted. in the event of a human-to-human h n outbreak, the hong kong public is very likely to adopt strong preventive measures in order to protect themselves and others. the magnitude of these behavioral responses may be even greater than those witnessed during the sars epidemic and would be likely to increase if a high fatality rate or high infectivity rate were reported. these preventive behaviors may be an effective firewall to the continued spread of the virus in the community. surveillance of public responses is an integral part of the government's h n preparedness plan. it should address issues related to the potentially underestimated fatality associated with human h n cases. surveillance should also closely monitor prevalence of important public health behaviors such as quarantine compliance and cross-border preventive measures. up-to-date surveillance information is necessary for the government to implement and make rapid adjustments these public health measures. publish with bio med central and every scientist can read your work free of charge world organization for animal health: update on avian influenza in animals (type h world health organization: cumulative number of confirmed human cases of avian influenza a/(h n ) reported to who probable person-to-person transmission of avian influenza a (h n ) who confirms human-to-human avian flu transmission avian influenza viruses and their implication for human health pattern 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are extended to ms. m.w. chan for her assistance in data collection. the study was supported by the li ka shing institute of health science. the author(s) declare that they have no competing interests. jl conceptualized, oversaw the project, drafted and made final revisions of the manuscript. jhk helped to draft the manuscript and assisted in the statistical analysis. hyt participated in the design and coordination of the study and performed the statistical analysis. sg was involved in the conceptualization of the study andediting of the manuscript. all authors read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -r bmtloy authors: jendrny, paula; schulz, claudia; twele, friederike; meller, sebastian; von köckritz-blickwede, maren; osterhaus, albertus dominicus marcellinus erasmus; ebbers, janek; pilchová, veronika; pink, isabell; welte, tobias; manns, michael peter; fathi, anahita; ernst, christiane; addo, marylyn martina; schalke, esther; volk, holger andreas title: scent dog identification of samples from covid- patients – a pilot study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: r bmtloy background: as the covid- pandemic continues to spread, early, ideally real-time, identification of sars-cov- infected individuals is pivotal in interrupting infection chains. volatile organic compounds produced during respiratory infections can cause specific scent imprints, which can be detected by trained dogs with a high rate of precision. methods: eight detection dogs were trained for week to detect saliva or tracheobronchial secretions of sars-cov- infected patients in a randomised, double-blinded and controlled study. results: the dogs were able to discriminate between samples of infected (positive) and non-infected (negative) individuals with average diagnostic sensitivity of . % ( % confidence interval [ci]: . – . %) and specificity of . % ( % ci: . – . %). during the presentation of randomised samples, the dogs achieved an overall average detection rate of % (± . %) with correct indications of positive, correct rejections of negative, incorrect indications of negative or incorrect rejections of positive sample presentations. conclusions: these preliminary findings indicate that trained detection dogs can identify respiratory secretion samples from hospitalised and clinically diseased sars-cov- infected individuals by discriminating between samples from sars-cov- infected patients and negative controls. this data may form the basis for the reliable screening method of sars-cov- infected people. the ongoing covid- pandemic highlights the importance of fast and reliable testing for accurate identification of symptomatic and asymptomatic carriers to reduce spread of infection effectively [ ] . current testing regimens usually require nasopharyngeal swabs applied by a trained person and a reverse transcription polymerase chain reaction test (rt-pcr) for pathogen identification. obtaining rt-pcr results is time consuming and can be cost-prohibitive, especially for developing countries, and is therefore currently often used in a targeted fashion, testing predominantly patients with covid- specific symptoms [ ] . there is therefore a need for an additional faster, reliable, noninvasive, and versatile screening tool, especially to identify asymptomatic and pre-symptomatic individuals. several studies have proven the canines' extraordinary olfactory acuity to detect persons with infectious and non-infectious diseases like different types of cancer [ ] , malaria [ ] , bacterial, and viral infections [ ] [ ] [ ] , with usually high rates of sensitivity and specificity [ ] . a pathogen-specific odour that can be detected by dogs may be composed of specific patterns of volatile organic compounds (vocs). compared to bacteria, viruses have no own metabolism, and therefore vocs are released by infected body cells as a result of metabolic host processes [ ] . different technical approaches have used the detection of vocs to discriminate infectious diseases successfully, but none is being used routinely in clinical practice [ ] . as dogs can be trained quickly, the aim of the present study was to test the concept of using dogs reliably and in real-time to discriminate between samples of sars-cov- infected patients and non-infected controls. this method could be employed in public areas such as airports, sport events, borders or other mass gatherings as an alternative or addition to laboratory testing, thus helping to prevent further spreading of the virus or further outbreaks. saliva samples and tracheobronchial secretion samples were collected from hospitalised covid- patients that showed clinical symptoms and were diagnosed as sars-cov- positive using nasopharyngeal swab samples. negative control samples were obtained from sars-cov- rt-pcr negative people with no previous history of covid- , nor had the individuals any history of a recent cold or infection. none of the samples were screened for different human coronaviruses like beta coronavirus hcov-oc or alpha coronavirus hcov- e. after the sample acquisition, the anonymised samples were transported to the university of veterinary medicine hannover. all collected samples were confirmed as positive or negative using the rt-pcr sars-cov- -ip assay from institut pasteur (recommended by the world health organization [ , ] , including an internal control system and protocol as described [ , ] . samples from covid- patients (irrespective of the final rt-pcr result) were further subjected to virus quantification (end point dilution assay) and virus isolation analysis using vero e cells under biosafety level conditions. the cell layers were assessed for cytopathic effects and final results were obtained days after cell infection. since dogs are susceptible to sars-cov- [ ] all samples from covid- patients were inactivated using beta propiolactone (bpl) in order to protect the dogs and their handlers from infection during training. briefly, samples and reagents were kept at °c, μl/ml nahco ( . %) was added, and samples were incubated for min at °c. after addition of μl/ml of % bpl, samples were incubated at °c for to h. hydrolysis of bpl was conducted at °c for to h. samples that showed a cytopathic effect before bpl inactivation using virus isolation or end point dilution assay were tested again after bpl inactivation and were confirmed to be inactivated. only bpl inactivated samples from covid- patients were used for the dog training. furthermore, detection dogs were provided both negative control samples with and without previous bpl treatment to exclude hydrolysed bpl as a potential distracting reagent. for the dog training, a volume of μl per sample was pipetted onto a cotton pad, which was placed into a ml glass tube. the presentation of the samples to the dogs was conducted via a device called detection dog training system (ddts; kynoscience ug, germany), which can present samples in a randomised automated manner without trainer interference. for a short video sequence, see additional file . ddts was utilised for training and testing. the device is composed of seven scent holes. behind each hole two tubes are leading to two metal containers. in the study, the first container enclosed the target sample and the second one carried the control sample. only one container is presented in each sniffing hole at any given time as the pairs of containers are situated on movable slides inside the device. the metal containers were covered with grids, which allowed the odour to escape and reach the sniffing hole. each tube extension was identical and lshaped, which prevented dogs from physical contact with the samples and excluded any visual cues that may have enabled further detection capabilities. for each trial run, only one hole presented a sars-cov- positive sample at a time while the other six holes presented negative samples. after the indication of the hole with the positive sample, the dog was automatically rewarded by the device with food or ball. the indication time was changed during successful training from s to s. while the reward was eaten, the device's software randomly and automatically assigned new positions to the slides for the following session with again only one hole presenting the positive odour sample. the dog, its handler and a person observing the study were blinded during the double-blinded study. all personnel stood behind the dog during the test runs to avoid distraction. the device recorded automatically the number and time length of each nose dip into the scent holes and the location of the positive and negative samples. this was verified by manual time-stamped video analysis. after a weeks habituation process to the ddts, the eight dogs needed days of training in total until the detection rate was above chance. an additional spreadsheet provides background information of the dogs used in the study (see additional file ). the controlled doubleblinded detection study was then conducted after days of training and in total , sample presentations ( table ) . on each training day, unknown and known positive samples and negative control samples were presented to the canines. the response to the new sample was used in order to evaluate if the generalisation process has been achieved. while the dogs had only achieved an average detection rate of % on the second day of training, the values increased to % on day five and even % on day seven indicating a successful generalisation process. after completion of the training process, the detection accuracy of the eight trained dogs was evaluated in a randomised, double-blinded, and controlled study ( table ) . samples from seven infected and seven healthy individuals were used in this study. two of the positive samples were tracheobronchial secretion, the other samples consisted of saliva. within randomised and automated sample presentations, dogs achieved an overall average detection rate of % (± . %) with correct indications of positive, correct rejections of negative, false positive and false negative indications. the canines discriminated between infected and non-infected individuals with an overall diagnostic sensitivity of . % ( % confidence interval [ci]: . - . %) and specificity of . % ( % ci: . - . %). sensitivity ranged from . to . % and specificity from . to . % (fig. ) . there was no notable difference in detection ability between saliva and tracheal secretion (average hit rates . and . %, respectively). timely and accurate detection of sars-cov- infected individuals is of uttermost importance for a society to control the pandemic. our data indicate that detection dogs can be trained in just about a week to discriminate between samples of people infected and non-infected by sars-cov- . the average detection rate was %. analysis for accuracy and precision revealed a diagnostic sensitivity of . % ( % ci: . - . %) and a high diagnostic specificity of . % ( % ci: . - . %) for all dogs. all dogs had a high diagnostic specificity with a small range in variation, which could be important for population screening to avoid false positive results. however, there was quite a range in variation of sensitivity for the individual dog and inbetween dogs. this can in part be explained with the dogs' variable training background (see additional file ), signalment, personality traits and short training period of days. to avoid a bias concerning hospital specific smells, positive samples were obtained from two different hospitals to include a variation in a covariate factor and this appears to have not influenced the current results. understanding better why there is this range in sensitivity and how to potentially improve it would be important prior to considering the use of detection dogs in the field. in comparison, the current gold standard diagnostic rt-pcr test of a nasopharyngeal swab can, in trained hands, have a false detection rate of % and a false positive rate of . - . % [ ] . a new, not yet published study indicated a clear, nearly % voc specific pattern of sars-cov- infected individuals compared to negative controls and individuals infected by the influenza virus using multicapillary column coupled ion mobility spectrometry of breath [ ] . this provides further indications that unique voc imprints exist and can be used for the development of diagnostic procedures. the current study results are promising, although they should be regarded as preliminary and suitability for this detection method in the field can only be acquired after further research has been conducted. our work provides the very first steps of the development of a new sars-cov- screening method. our inclusion criteria for the samples collected were rather non-specific and not stratified by severity of symptoms, disease status or virus load. future studies are needed to address this including a higher number of different samples to evaluate the analytical sensitivity (e.g. dilution of samples/detection level, different disease phenotypes and stages) and analytical specificity (differentiation to other lung diseases or pathogens such as cancer or infection with other seasonal respiratory virus infections, e.g. influenza, respiratory syncitial virus, adenovirus, other than sars-cov- coronaviruses, rhinovirus). in the current study negative control samples were acquired from healthy individuals without clinical signs of respiratory disease. the individuals were only tested for sars-cov- virus and therefore one cannot exclude that a former infection, especially with another human coronavirus like hcov-oc resulted in false positive indications of the dogs and that cross detection occurred. on the other hand, samples included in the current study were from severely affected, hospitalised covid- patients, but one of the main challenges in controlling the current pandemic is to identify pre-symptomatic covid- patients and asymptomatic carriers, which may constitute most covid- cases [ ] . the sensitivity of detection by dogs may also vary across the course of the disease. future research should therefore focus on the ability of dogs to identify the different covid- disease phenotypes and phases of disease expression, such as asymptomatic, pre-symptomatic, mild and severe clinical cases as well as to test samples of the same individuals at different timepoints across the course of the disease. one of the most important requirements regarding handling of infectious samples is infection prevention and control. initially, it was assumed that dogs cannot get infected by sars-cov- , but recent single cases have been reported showing that dogs can get infected by sars-cov- and could potentially play a role in viral spread [ , ] . there is evidence of human-to-animal transmission with a subsequent infection of dogs. it is still unclear whether dogs can function as spreaders of the virus by infecting other animals or humans [ , ] . nevertheless, this needs to be considered when using dogs for detection of infected material or people. it is also unclear how an infection in the dog will alter its sense of smell. in the current study we chose to use an inactivation procedure which should not affect vocs. however, this is not practical for testing in the field and we are currently developing new strategies for a secure presentation of non-inactivated samples. this would eliminate potential risks of virus transmission by detection dogs when used in a non-laboratory setting. detection dogs were able to discriminate respiratory secretions of infected sars-cov- individuals from those of healthy controls with high rates of sensitivity and specificity. the current pilot study had major limitations which needs to be elucidated in future studies. sars-cov- detection dogs may then provide an effective and reliable infection detection technology in various settings like public facilities and function as an alternative or addition to regular rt-pcr screening. in countries with limited access to diagnostic tests, detection dogs could then have the potential to be used for mass detection of infected people. further work is necessary to better understand the potential and limitation of using scent dogs for the detection of viral respiratory diseases. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file : additional video. detection dog working with ddts. the video (additional file ) shows the labrador retriever "seven" can be seen at the bottom of the video. the scent hole with a sample of an sars-cov- infected individual is marked in green on the video (please note the green mark was not seen by the dog and was only used in the video as a visualisation tool for the viewer to demonstrate the dog's search and detection behaviour). at each detection trial run only one hole is presenting the target scent with the other six holes presenting saliva samples from sars-cov- negative tested individuals. when the dog detects the target scent, the nose will be left within the hole for ≥ s to indicate the detection. this will be recorded by the device. a beeping sound announces the food or ball reward, which is automatically ejected by the device, distracting the dog for a short time period. in the meantime, the device rearranges the sample presentation in an automatic and random fashion, presenting one other scent hole with a sample of a sars-cov- positive tested individual and six control scent holes with negative control samples. pathophysiology, transmission, diagnosis, and treatment of coronavirus disease (covid- ): a review diagnostic accuracy of canine scent detection in early-and late-stage lung and breast cancers trained dogs identify people with malaria parasites by their odour using dog scent detection as a point-of-care tool to identify toxigenic clostridium difficile in stool canine detection of the volatilome: a review of implications for pathogen and disease detection realtime detection of a virus using detection dogs biomedical scent detection dogs: would they pass as a health technology? the human volatilome: volatile organic compounds (vocs) in exhaled breath, skin emanations, urine, feces and saliva the scent of disease: volatile organic compounds of the human body related to disease and disorder protocol: real-time rt-pcr assays for the detection of sars-cov- sensitivity assessment of sars-cov- pcr assays developed by who referral laboratories a universal heterologous internal control system for duplex real-time rt-pcr assays used in a detection system for pestiviruses infection of dogs with ars-cov- sensitivity, specificity, and predictive values: foundations, pliabilities, and pitfalls in research and practice. front pub health false positives in reverse transcription pcr testing for sars-cov- . medrxiv rapid detection of sars-cov- infection by multicapillary column coupled ion mobility spectrometry (mcc-ims) of breath. a proof of concept study medrxiv community prevalence of sars-cov- virus in england during may : react study the risk of sars-cov- transmission to pets and other wild and domestic animals strongly mandates a onehealth strategy to control the covid- pandemic. one health susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the members of the id-uke-covid- study group marylyn m. addo, etienne bartels, thomas t. brehm, christine dahlke, anahita fathi, monika friedrich, svenja hardtke, till koch, ansgar w. lohse, my l. ly, stefan schmiedel, l. marie weskamm, julian schulze zur wiesch at the university medical-center hamburg-eppendorf by helping us with recruitment of patients and sample collection. we further would like to thank stefan hampel, sina knisel and miguel acosta for support at the bundeswehr school of dog, german armed forces, ulmen during dog training and leander buchner from the central institute of medical services, german armed forces in koblenz for the support in getting samples. a special thanks goes to hans ebbers, kynosciences, for providing the ddts free of charge and for the support in dog training. we would like to thank our doctoral student saskia irene peek for her support during sample collection. special thanks go to our "doggy noses" coyote, elli, lotta, donnie, hoss, luigi, jo and seven. heartfelt thanks go to all the people providing us with samples, especially to the sars-cov- infected persons and their relatives with the sincere intention to contribute to the containment of covid- and to scientific progress. we wish you lots of strength and full recovery during the current pandemic. authors' contributions pj participated in the planning of the study, carried out the main practical work, data analyses and drafted the manuscript. cs participated in the planning of the study and carried out the laboratory work including rt-pcr and virus inactivation, as did vp. ft, sm and hav designed and coordinated the study, drafted the manuscript, conducted and coordinated (ft) the sample acquisition and were responsible for data analyses. mvkb and admeo participated in the planning of the laboratory part of the study and were in charge for the legal permission for sample processing. je programmed the ddts software. ip, tw, mpm, af and mma were in charge for the ethical approval, patient recruitment and sample collection (ip, af) at hannover medical school (ip, tw, mpm) and university medical-center hamburg-eppendorf (af, mma). es participated in the planning of the study, was responsible for the dog training and helped with data analyses. all authors have read and approved the final manuscript. not applicable. the datasets used and/or analysed during the current study are available at jendrny, paula, twele, friederike, schulz, claudia, meller, sebastian, von köckritz-blickwede, maren, volk, holger andreas. ( ). sars-cov- detection dogs -a pilot study [data set]. zenodo. https://doi.org/ . /zenodo. ethics approval and consent to participate the study was conducted according to the ethical requirements established by the declaration of helsinki. the local ethics committee of hannover medical school (mhh) and hamburg medical association at the university medical-center hamburg-eppendorf (uke) approved the study (ethic consent number _bo_k_ and pv , respectively). written consent from all people were collected before sample collection. not applicable. the authors declare that they have no competing interests. key: cord- -qys r jo authors: liu, nan; xie, jing; qiu, xiaoli; jia, leili; wu, zhihao; ma, yuhua; wang, zhongqiang; li, peng; ren, xingbin; hao, rongzhang; wang, ligui; wang, yong; qiu, shaofu; song, hongbin title: an atypical winter outbreak of hand, foot, and mouth disease associated with human enterovirus , date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: qys r jo background: to analyze the epidemiological characteristics and pathogenic molecular characteristics of an hand, foot, and mouth disease (hfmd) outbreak caused by enterovirus in linyi city, shandong province, china during november to december , . methods: one hundred and seventy three stool specimens and throat samples were collected from hospitalized cases. epidemiologic and clinical investigations, laboratory testing, and genetic analyses were performed to identify the causal pathogen of the outbreak. results: among the cases reported in december , the male–female ratio was . : ; cases ( . %) were severe. the majority of patients were children aged < years ( . %). some patients developed respiratory symptoms including runny nose ( . %), cough ( . %), and sore throat ( . %). one hundred and thirty eight ev positive cases were identified based on real time reverse-transcription pcr detection and isolates were sequenced with the vp region. phylogenetic analysis of full-length vp sequences of linyi ev isolates showed that they belonged to the c a cluster of the c subgenotype and were divided into lineages (lineage i, ii and iii). the two amino acid substitutions (gly and gln for glu) at position within the vp region are more likely to appear in ev isolates from severe cases ( . %) than those recovered from mild cases ( . %). conclusion: this outbreak of hmfd was caused by ev in an atypical winter. ev strains associated with this outbreak represented three separate chains of transmission. substitution at amino acid position of the vp region of ev might be an important virulence marker for severe cases. these findings suggest that continued surveillance for ev variants has the potential to greatly impact hfmd prevention and control. hand, foot, and mouth disease (hfmd), primarily a disease of young children, is caused by a virus belonging to the group enteroviruses. ev is known to be a causative agent of hfmd, herpangina, aseptic meningitis, paralysis, and meningoencephalitis [ ] . hfmd usually presents with symptoms including fever and a characteristic rash associated with the limbs, mouth and skin. in severe disease such as caused by ev , however, the patients may develop nervous system diseases such as aseptic meningitis, encephalitis, brainstem encephalitis, encephalomyelitis, and poliomyelitis-like syndrome as well as neurogenic pulmonary edema and myocarditis, resulting in high morbidity and mortality [ ] . ev was first isolated in the united states (california) in and by the mid- s, ev outbreaks, characterized by central nervous system complications occurred in succession in bulgaria and hungary [ ] . in the late s, ev emerged in east asia. in , cases in malaysia were reported, with deaths [ ] . one year later, an estimated . million cases occurred in taiwan; cases developed severe neuropathic complications with deaths [ ] . in , an ev pandemic occurred in perth, australia; cases were reported in months; cases developed severe disease [ ] . subsequently, numerous outbreaks have been documented in eastern and southeastern asia, including singapore [ ] , south korea [ ] , malaysia [ ] , japan [ ] , vietnam [ ] and mainland china. in , a large scale outbreak of hfmd occurred in anhui province, china; cases were reported with deaths [ ] . hfmd outbreaks associated with ev exhibit a significant seasonal pattern with a peak in summer and low incidence in winter [ , ] , however the number of hfmd cases increase significantly with increasing temperature and relative humidity [ , ] . in this study, we describe an atypical winter outbreak of hfmd from november to december , , in linyi city, shandong province, china, during which cases were admitted to the linyi people's hospital. in this study, we analyzed the epidemiological characteristics of this outbreak and the molecular epidemiology of ev , with an attempt to provide scientific evidence for the prevention and control of hfmd. one hundred and seventy three patients admitted to linyi city people's hospital were enrolled in the study. case information was obtained from the hospital information system including age, gender, onset date, place of onset and clinical information. all cases were diagnosed according to a standardized clinical case definition of hmfd [ ] . hfmd was defined as fever, accompanied by herpangina and rashes on the hands and feet, with or without buttock involvement. severe hfmd patients presented with obvious symptoms of nervous system involvement and severe complication, such as myoclonus, encephalitis, acute flaccid paralysis, pulmonary edema, or heart failure. to confirm the diagnosis of hmfd, specimens ( stool and throat swabs) were collected for enterovirus detection and molecular typing. throat swabs were immediately immersed into sterile tubes containing viral transport medium (vtm). the study protocol was approved by the medical ethics committee of academy of military medical sciences. approximately . g of a fecal sample was put into a . ml centrifuge tube with a glass bead. after addition of ml of phosphate buffer, the solution was vortexed for min and centrifuged at rpm for min. fecal supernatants and the vtm from tubes containing throat samples were filtered through a . or . μm syringe filter (pall, ann arbor, mi, usa). filtrates were cultured with human rhabdomyosarcoma cells (rd cells) at °c and % co for days. cells were observed daily for cytopathic effects (cpe). each specimen was passaged blindly at least times. cells demonstrating an observed cpe were repeatedly frozen and thawed times to allow for rna extraction and identification using molecular biology approaches, and then cell cultures and rnas were stored at − °c. if no specific cpe was observed cell culture was interpreted as negative. nucleic acid extraction and molecular typing viral rna was extracted from clinical specimens and viral cultures using a qiaamp viral rna mini kit (qiagen, germany) according to the manufacturer's instructions. all rna samples were examined by real time reverse-transcription pcr (rt rt-pcr) using a set of pan-ev (ev universal primer) probe and primers; positive samples were tested by rt rt-pcr for ev and ca using specific primers and probes [ ] . rt rt-pcr was performed using agpath-id™ one-step rt-pcr reagents (applied biosystems, foster, ca, usa). total dna was extracted from throat specimens using qiaamp dna blood mini kit (qiagen, germany). commercial available multiplex pcr assays (seeplex® rv ace, korea) was used for testing respiratory viruses of throat specimens, including human adenovirus, coronaviruses, parainfluenza viruses, influenza a virus, influenza b virus, respiratory syncytial virus a and b, and bocavirus. amplification was performed using an iq™ quantitative real-time pcr system (biorad, hercules, ca, usa). full-length vp sequences from ev isolates were amplified with the primers ev -vp -f, ggkgcrcc caayacwgcyt and ev -vp -r, ccvccrcaat chccwggyt, resulting in a -bp amplicon. pcr was performed using a geneamp thermal cycler (applied biosystems). pcr products were detected by agarose gel electrophoresis, and products were purified and sequenced with an abi prism genetic analyzer (applied biosystems). an online blast search tool (http://blast.ncbi.nlm.nih.gov/blast.cgi) was used to compare the sequences and determine the types. the constituent ratio of amino acid variation between severe cases and mild cases strains was compared with chisquare test by sas software (version . ; sas institute). multiple sequence alignments were performed by using the mega software version . and the clustal w program to determine nucleotide and amino acid sequence similarities. phylogenetic trees were constructed in mega using the neighbor-joining (nj) cluster algorithm with evolutionary distances estimated using the kimura -parameter model; bootstrapping was performed using , pseudo-replicates [ ] . the full sequences of vp from this study are deposited in genbank with the following accession numbers: kf -kf , kj -kj . a total of hfmd cases (including with severe presentation) were diagnosed and treated in linyi people's hospital from november to december , ( figure ). the majority of patients were children aged < years ( . %) with the male-female ratio of . : . cases presented from counties or districts; although the incidence was greatest in lanshan district ( . %) and fei county ( . %). all cases had fever and the characteristic rashes associated with the limbs, mouth and skin. severe cases were associated with meningitis (n = ) or more severe neurological complications (meningoencephalitis, n = ; neurogenic pulmonary edema, n = ); no deaths were observed. although some patients developed respiratory symptoms including runny nose ( . %), cough ( . %), and sore throat ( . %), testing with multiplex pcr assays of a subset of throat samples (n = ) for respiratory viruses including human adenovirus, coronaviruses, parainfluenza viruses, influenza a virus, influenza b virus, respiratory syncytial virus a and b, and bocavirus were all negative. a small number of patients also reported nausea, or experienced vomiting, diarrhea, and other gastrointestinal symptoms. most cases were accompanied by characteristic neurological symptoms including drowsiness and convulsions (table ) . all rna samples were examined by rt rt-pcr for the presence of any detectable enterovirus (pan ev screen) and then specifically for ev and ca . among the specimens, an amplicon was evident in of the samples, taken from cases, using the pan-ev primers and probes. using primers and probes specific for ev , the pan ev-positive specimens were also positive; stool specimens were positive from cases, and cases who had a throat swab and fecal sample both tested positive. primers and probes specific for ca were negative in all the samples. viral samples from the ten patients with two positive samples were indistinguishable. in total, / ( . %) samples from individual cases were culture-positive for ev ; identification was confirmed by rt rt-pcr amplification and sequencing of vp regions; isolates ( . %) were recovered from severe cases and isolates ( . %) from mild cases. in order to analyze molecular epidemiological characteristics of the ev isolates in this study, ev isolates were selected from this outbreak, and vp sequences were compared to those from ev isolates circulating in china and additional ev strains (a, b -b , and c -c ) isolated from children living in other countries (figure ). sequence alignments showed the linyi ev vp sequences shared . - % nucleotide identity. sequences of the vp region of the linyi ev isolates were closely related to the predominant ev isolates from china, and all isolates belonged to subgenotype c . subgenotype c can be divided into two groups (c a and c b) [ ] . according to the phylogenetic analysis (figures and ) , all of the linyi ev isolates belong to c a; isolates were further divided into three lineages (lineage i, ii, and iii) with mean intralineage p-distances of . , . and . , respectively. the majority of the outbreak isolates ( / ) belonged to lineage i which contained all of the isolates from the severe cases; these isolates were mainly found in three districts or counties of linyi city, shandong province (lanshan district, fei county and hedong district). lineage ii and lineage iii had three and seven outbreak isolates, respectively. vp nucleic acid dissimilarity among the three subgroups ranged from . %- . %; these results suggest that the outbreak likely consisted of three main transmission chains, with lineage i being the predominant contributor. comparison of the translated vp amino acid sequences ( aa) of the ev isolates used in this study indicated that the isolates were . % identical to one another; variable amino acid residues were at positions , , , , , and . substitution at amino acid position of vp appeared to be significantly different between severe cases and mild cases strains. two amino acids, gly and gln, were significantly more likely to appear in the vp of ev recovered from severe cases figure dendrogram showing the phylogenetic relationships of ev isolates in this outbreak and other genotypes of ev isolates from genbank based on vp sequence alignment, which was constructed using mega software (version . ). ( / , . %) than in mild cases ( / , . %; p < . ) (figure ). hfmd epidemics in china mainly occur in the spring and summer [ ] [ ] [ ] , when hot and humid weather is conducive to the propagation and spread of the virus [ ] . however, the linyi hfmd outbreak occurred in december, which is relatively uncommon. according to meteorological parameters provided by the meteorological bureau of shandong province, the mean temperature of linyi city during the winter of was . °c higher than that in . flett et al. suggested that atypical seasonality of hfmd outbreaks during winter might be related to unusually mild temperatures [ ] . research in hong kong has also indicated that altered disease etiology of hfmd might be explained by increased winter temperatures [ ] . it is reasonable to speculate that the short-term changes in weather variables may affect the seasonality of hfmd. however, cold weather increases the probability that populations will gather in confined spaces such as houses and indoor play areas; increasing the likelihood of unwanted exposures. in addition, more upper respiratory tract infections occur during the winter, resulting in a decrease in immune functions among pediatric patients, which might contribute to increasing incidence of hfmd. the epidemiology of ev in china since this virus was first detected in has shown that only subgenotype c is in endemic circulation [ ] . phylogenetic analysis suggests that three ev variants contributed to the linyi outbreak and share strong similarities to some ev viruses circulating previously in shandong province. however, no obvious evidence revealed the transmission relationship between strains from this outbreak and other provinces in china. therefore, we speculate that this uncommon winter outbreak did not occur in isolation, but was related with local epidemic occurring in the region during the spring and summer of . it is probable that the change of climatic parameters contributed to the occurrence of this atypical seasonal outbreak but the extent of this contribution needs to be further examined. subgenotype c a is geographically broadly distributed and genetically variable; it is estimated to undergo . × − substitutions per nucleotide per year, a rapid rate of change [ ] . phylogenetic analysis shows that subgenotype c should be re-designated as a novel figure phylogenetic tree of ev isolates in this outbreak and related c a subgenotype strains. accession numbers are given in parentheses. ▲, isolates from severe cases. ly, isolates from this study; sd, shandong province; zj, zhejiang province; bj, beijing city; js, jiangsu province; cq, chongqing city. genotype, d [ , ] . thus, the three lineages may represent different ev transmission modes in this region; this hypothesis should be clarified by further molecular epidemiological studies. amino sequence analysis showed one primary variation between viruses isolated from severe and mild cases of hfmd, substitution of the amino acid residue at position . several studies have revealed that this change in the vp region of ev may play an important role in virulence. residue is reported to be located in the de loop which contains neutralizing antigenic sites; this loop is located in the canyon rim, a region involved in the receptor binding for enterovirus and rhinovirus [ ] [ ] [ ] [ ] . selection pressure analysis of many vp sequences of ev showed that amino acid was a positive selection site [ , ] . mutation of glutamine to glutamic acid on vp region cooperatively promote viral binding and rna accumulation of ev , contributing to viral infectivity in vitro and mouse lethality in vivo [ ] . substitution of glycine to glutamic acid at the same position might increase the efficiency of uncoating upon specific binding of the virion to the receptor molecule on the target cells in nod/scid mice and subsequently facilitate the infection of a mouse adapted ev strain [ ] . although it has been demonstrated that the amino acid substitution of gly to glu at position of vp could increase ev virulence in mice, glu to gly/gln/arg substitutions may enhance virulence in humans [ ] . consistent with this result, isolates from severe cases in our investigation were significantly more likely to carry the gly/gln at amino acid than those from mild cases. this substitution may influence the virus binding to antibody or receptor, thereby affecting the virulence of the virus [ ] . in conclusion, hfmd caused by ev has become a global concern in recent years. in february , hfmd figure amino acid sequence alignment of the full length vp region of the ev virus isolates in linyi city, shandong province, . amino acid sequences were numbered according to the sequence of ev _brcr (genbank accession number: etu ). identical residues are indicated as dots. amino acids that differ from the consensus sequence are shaded. ▲, isolates of severe cases. was officially categorized as a class c infectious disease in china. further studies on the transmission patterns of ev using approaches in molecular biology are therefore warranted, and recognizing ev isolates with increased virulence should be a priority for hfmd prevention and control efforts. moreover, monitoring the genetic variation of ev may be useful in facilitating the development of effective ev vaccine candidates on chinese market and evaluating their effects of prevention and control of the ev infections. frequent importation of enterovirus from surrounding countries into the local community of yamagata neural pathogenesis of enterovirus infection an apparently new enterovirus isolated from patients with disease of the central nervous system molecular detection of enteroviruses from an outbreak of hand, foot and mouth disease in malaysia in an outbreak of enterovirus infection in taiwan, : epidemiologic and clinical manifestations neurological manifestations of enterovirus infection in children during an outbreak of hand, foot, and mouth disease in western australia complete sequence analyses of enterovirus strains from fatal and non-fatal cases of the hand, foot and mouth disease outbreak in enterovirus infection with central nervous system involvement identification of enterovirus isolates from an outbreak of hand, foot and mouth disease (hfmd) with fatal cases of encephalomyelitis in malaysia outbreak of central nervous system disease associated with hand, foot, and mouth disease in japan during the summer of : detection and molecular epidemiology of enterovirus epidemiologic and virologic investigation of hand, foot, and mouth disease, southern vietnam epidemiology of hand, foot, and mouth disease and genotype characterization of enterovirus in jiangsu china the association between enterovirus infections and meteorological parameters in taiwan hand, foot, and mouth disease in china: patterns of spread and transmissibility the influence of temperature and humidity on the incidence of hand, foot, and mouth disease in japan ministry of health of the people's republic of china: guideline for the diagnosis and treatment of hand, foot, and mouth disease epidemiological and etiological characteristics of hand, foot, and mouth disease in molecular epidemiology and dual serotype specificity detection of echovirus strains in finland an outbreak of hand, foot, and mouth disease associated with subgenotype c of human enterovirus in shandong china epidemiological characteristics analysis and prevention strategies of hand-foot-mouth disease -in zaozhuang of shandong province in meteorological factors and hfmd response analysis a exploration and study of the relationships of hand-foot-mouth disease (hfmd) and the climate short term effects of weather on hand, foot and mouth disease hand, foot, and mouth disease caused by coxsackievirus a changing epidemiology of hand, foot, and mouth disease in hong kong emergence and transmission pathways of rapidly evolving evolutionary branch c a strains of human enterovirus in the central plain of china phylogenetic designation of enterovirus genotypes and subgenotypes using complete genome sequences emergence of enterovirus "double-recombinant" strains belonging to a novel genotype d originating from southern china: first evidence for combination of intratypic and intertypic recombination events in ev characterization of the n-terminal part of the neutralizing antigenic site i of coxsackievirus b by mutation analysis of antigen chimeras induction of neutralizing antibodies by synthetic peptides representing the c terminus of coxsackievirus a capsid protein vp molecular evolution of the human enteroviruses: correlation of serotype with vp sequence and application to picornavirus classification structure of a human rhinovirus complexed with its receptor molecule reemergence of enterovirus in in taiwan: dynamics of genetic and antigenic evolution from to positive selection analysis of vp genes of worldwide human enterovirus viruses mutations in vp and vp capsid proteins increase infectivity and mouse lethality of enterovirus by virus binding and rna accumulation enhancement cooperative effect of the attenuation determinants derived from poliovirus sabin strain is essential for attenuation of enterovirus in the nod/scid mouse infection model molecular analysis of virulent determinants of enterovirus an atypical winter outbreak of hand, foot, and mouth disease associated with human enterovirus we are grateful to dr john d. the authors declare that they have no competing interests. key: cord- -mm d jkh authors: ying, mingliang; lu, bin; pan, jiangfeng; lu, guanghong; zhou, shaobin; wang, dingjun; li, lu; shen, junkang; shu, jiner title: covid- with acute cholecystitis: a case report date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: mm d jkh background: the novel coronavirus (covid- ) presents a major threat to public health and has rapidly spread worldwide since the outbreak in wuhan, hubei province, china in . to date, there have been few reports of the varying degrees of illness caused by the covid- . case presentation: a case of -year-old female with covid- pneumonia who had constant pain in the right upper quadrant of her abdomen during her hospitalization that was finally diagnosed as acute cholecystitis. ultrasound-guided percutaneous transhepatic gallbladder drainage (ptgd) was performed, and the real-time fluorescence polymerase chain reaction (rt-pcr) covid- nucleic acid assay of the bile was found to be negative. ptgd, antibacterial and anti-virus combined with interferon inhalation treatment were successful. conclusion: the time course of chest ct findings is typical for covid- pneumonia. ptgd is useful for acute cholecystitis in covid- patients. acute cholecystitis is likely to be caused by covid- . in december , an outbreak of the novel coronavirus (covid- ) occurred in wuhan, hubei province, china, and rapidly spread throughout the world [ , ] . patients had clinical manifestations of fever, cough, and chest stuffiness in addition to other non-specific symptoms, including diarrhea, vomiting, abdominal pain and so on. abdominal pain was uncommon [ ] . here, we report a confirmed case of a female with covid- pneumonia who had constant pain in the right upper quadrant of her abdomen during her hospitalization that finally diagnosed as acute cholecystitis. written informed consent was waived by the jinhua hospital of zhejiang university institutional review board. on january , , a -year-old woman presented to the hospital with a -day history of fever, chest stuffiness and diarrhea without chills, cough, or nasal discharge. she had a history of good physical health and no underlying diseases. she had stayed with her son who had been diagnosed with novel coronavirus (covid- ) pneumonia after business travel to wuhan, china. her body temperature was elevated to . °c ( . °f) for days before her hospitalization. the realtime fluorescence polymerase chain reaction (rt-pcr) assay of pharyngeal swabs and feces was positive for covid- nucleic acid upon hospitalization. the patient's temperature on admission was normal at . °c ( . °f), and there were coarse breath sounds from both lungs on auscultation. laboratory studies showed a normal white blood cell count of . × /l (normal range, . - . × /l), differential neutrophil count of . % (normal range, . - . %), and lymphocyte count of . % (normal range, . - . %). the alanine in the present case, the chest ct findings were typical for covid- pneumonia ( fig. a-f ). at first, the patient did not show any abdominal symptoms except diarrhea; however, she developed constant pain in the right upper quadrant of her abdomen and murphy's sign after days of hospitalization, and her body temperature was elevated to . °c ( . °f). the laboratory examinations indicated elevated c-reactive protein ( . mg/l; normal range, - mg/l). we considered acute cholecystitis or cholangitis and performed an abdominal plain ct scan, which revealed a distended gallbladder, hyperplasia of the gallbladder wall and biliary sludge (fig. g ) and did not show gallstones in the gallbladder. ultrasound-guided percutaneous transhepatic gallbladder drainage (ptgd) was performed on day , and approximately ml of green gallbladder bile was drained on day . the rt-pcr covid- nucleic acid assay of the bile was found to be negative. ptgd, antibacterial and anti-virus (lopinavir/ritonavir) combined with human interferon alfa- b inhalation treatment were successful. the patient was subsequently discharged from the hospital on day and referred to the clinic for follow-up. since december , a succession of patients in china have been suffering from pneumonia from unknown causes, later officially named covid- by the world health organization, based on the whole genome sequence analysis of the viruses in respiratory samples or feces from the patients [ , ] . most early patients had a history of exposure to the south china seafood market in wuhan city, and some patients demonstrated a family clustering feature. the clinical manifestations are mainly fever, fatigue, cough, and gradual dyspnea in some cases and acute respiratory distress syndrome in severe cases [ ] . in the present case, the patient had a clear history of contact with the patient diagnosed with covid- pneumonia, her lymphocytes were decreased, and the rt-pcr covid- nucleic acid assay of pharyngeal swabs and feces was positive; thus, she was definitively diagnosed with covid- . in the present case, the initial ct showed multifocal peripheral ground-glass opacities (ggos) in the right lower lobe, which may represent hyaline membrane formation. follow-up ct in this case demonstrated mild disease progression, as manifested by the increasing extent and multiple patchy consolidations, especially in the peripheral zones of the lungs, which may represent alveolar injury and inflammatory exudation. after symptomatic treatment, the consolidations and ggos were almost absorbed, leaving cord-like shadows, which represent an improvement of the disease. the time course of lung changes on chest ct images of this patient were typical, as described in previous studies [ ] [ ] [ ] . in our unique case, the patient developed constant pain in the right upper quadrant of the abdomen and murphy's sign after days of hospitalization, and her body temperature was elevated to . °c. because of the typical clinical presentation, this patient was likely to be diagnosed as acute cholecystitis or cholangitis in the clinic. acute cholecystitis was subsequently confirmed by plain abdominal ct scan. it was difficult to identify the cause of acute cholecystitis in covid- patients by abdominal ct imaging. and it could potentially cause severe gallbladder perforation, subsequently it was treated with ptgd. there has not been a study to report the rt-pcr nucleic acids for bile, while sputum or feces were with the highest positive rate of rt-pcr results. the precise mechanism of acute cholecystitis in covid- patients was unknown. the rt-pcr results failed to find virus of covid- in the bile. so our speculation about the potential association in the covid- patient developed acute cholecystitis was a possible complication of covid- . however it remains to envisage acute cholecystitis as a possible complication of covid- , pending further studies that could prove or disprove this hypothesis. it has not been reported whether the gallbladder might be vulnerable to covid- . we highlight a further complication possible with covid- . but the notable limitations of this study should be acknowledged. only one unique covid- patient presented an acute (see figure on previous page.) fig. chest and abdominal plain ct scans in a -year-old woman with covid- pneumonia and acute csholecystitis. a, b. transverse chest ct scan obtained on the first day after admission shows multifocal peripheral ggos in the dorsal (a) and posterior basal (b) segments of the right lower lobe. c, d. follow-up chest ct scan obtained on day after admission shows progression of the ggos. e, f. follow-up chest ct scan obtained on day after admission shows that the ggos were partly resolved in the dorsal segment of the right lower lobe (e) and completely resolved in the posterior basal segment of the right lower lobe (f). g. coronal mpr of the plain abdominal ct obtained on day shows a distended gallbladder, hyperplasia of the gallbladder wall, biliary sludge (arrow), and no gallstones in the gallbladder. h. coronal mpr of the plain abdominal ct obtained on day shows shrinkage of the gallbladder or its change in shape, a high-density drainage tube and a little bleeding in the gallbladder. ct = computer tomography, ggos = ground-glass opacities, mpr = multiplanar reconstruction cholecystitis, the pcr test of the bile show no evidence of covid- virus invasion of the gallbladder, and lack of pathological diagnosis for gallbladder tissue, which are not in favor of the possible association between covid- and acute cholecystitis, it remains however possible that the infection by covid- virus triggers the cholecystitis via a yet unknown mechanism. in conclusion, we report the clinical course of a female patient with covid- . the time course of chest ct findings is typical for covid- pneumonia. ptgd is useful for acute cholecystitis in covid- patients. acute cholecystitis is likely to be caused by covid- . emerging understandings of -ncov medical journals and the -ncov outbreak clinical characteristics of coronavirus disease in china emerging coronaviruses: genome structure, replication, and pathogenesis a novel coronavirus from patients with pneumonia in china chest ct findings in coronavirus disease- (covid- ): relationship to duration of infection pneumonia associated with novel coronavirus: can computed tomographic findings help predict the prognosis of the disease? time course of lung changes on chest ct during recovery from novel coronavirus (covid- ) pneumonia publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions my and bl were the main contributors to drafting the manuscript. gl, jp and sz contributed in diagnosing the disease, data collection and data analysis. ll and dw contributed to literature search, figure preparation. js and js contributed to study design and performed the final manuscript review. all authors have read and approved the manuscript. not applicable. this work was funded by the jinhua science and technology bureau, zhejiang province, grant number xg- . the funder (jinhua science and technology bureau) had no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript. the datasets used and/or analysed during the current study are available from the corresponding author on reasonable request. this study was approved by the ethics committee of jinhua municipal central hospital (zhejiang, china). written informed consent was obtained from the participant. written consent was obtained from the patient for publication of this case report and the accompanying images. the authors declare that they have no competing interests. key: cord- -mbwgi x authors: pang, junxiong; jin, jing; loh, jin phang; tan, boon huan; koh, wee hong victor; ng, sock hoon; ho, zheng jie marc; gao, qiuhan; cook, alex r; hsu, li yang; lee, vernon j; chen, mark i cheng title: risk factors for febrile respiratory illness and mono-viral infections in a semi-closed military environment: a case-control study date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: mbwgi x background: febrile respiratory illness (fri) results in substantial burden in semi-closed environments. tackling risk factors may reduce transmission and infection. however, risk factors involved in one setting may not be generalizable in all settings due to differences in climate, residential environment, population genetic and cultural backgrounds. this study aims to identify risk factors of fri and mono-viral infections in a tropical military environment. methods: from year to , military personnel with temperature ≥ . °c, cough and/or sore throat, and personnel with no fever or no respiratory symptoms were recruited as cases and controls, respectively. subjects provided nasal wash specimens and answered a standardized questionnaire. resplex assays were used to determine the viral etiologies. descriptive, univariate and multivariate analyses of the variables were performed using appropriate descriptive tests and logistic regression modelling, respectively, with r program. results: a total of , fri cases and , non-fri study controls were recruited. increasing age [adjusted odds ratio (aor) = . ; % confidence interval (ci) = . - . ], recruit camp (aor = . ; % ci = . - . ) and smoker (aor = . ; % ci = . - . ) were independent risk factors of fri. malay ethnicity was positively associated with influenza a(h n )pdm (aor = . ; % ci = . - . ) and coxsackie/echovirus (aor = . ; % ci = . - . ) mono-infection. significant contact risk factors were stay-out personnel with ill household member (aor = . ; % ci = . - . ), and stay-in personnel with ill bunkmate and household member (aor = . ; % ci = . - . ). staying in camp with none ill in bunk and at home was a protective factor against fri (aor = . ; % ci = . - . ). these contact risk factors were similarly observed for the five most common viruses detected, namely adenovirus, rhinoviruses, influenza a and b, and coxsackie/echovirus. conclusion: increasing age, smoker, recruit-camp, stay-out personnel with ill household members and stay-in personnel with ill bunkmates were independent risk factors of fri in a semi-closed military environment. early identification and isolation of ill personnel from their bunk may be effective to prevent and reduce transmission and disease burden. febrile respiratory illness (fri) results in substantial disease burden in semi-closed environments such as in the households [ ] and militaries [ ] [ ] [ ] . fri is most commonly caused by viral infections, as observed in military respiratory surveillance programmes in finland [ ] , united kingdom [ , [ ] [ ] [ ] [ ] , netherlands [ ] , france [ , ] , south korea [ ] [ ] [ ] , west africa [ ] , taiwan [ ] , china [ ] , singapore [ ] [ ] [ ] [ ] [ ] [ ] , and the united states [ , [ ] [ ] [ ] [ ] [ ] . identifying risk factors of infection may provide guidance on policies and strategies for the prevention and control of fri. previous documented risk factors of fri in other countries included body mass index equal or greater than kg/m , previous respiratory tract infections [ ] , overcrowding and closed units [ , [ ] [ ] [ ] , presence of sand and dust storms, extreme temperature changes [ , ] , smoking [ ] , female, navy service, poor latrine facilities, increasing age and higher rank [ ] . however, these risk factors may not be generalizable to different environments, and may differ between specific predominant aetiological agents. the predominant viruses reported in the singapore armed forces (saf) comprised adenovirus, rhinoviruses, influenza a and b, and coxsackie/echovirus between and [ ] . adenovirus-associated respiratory disease, outbreaks and death have been reported in several countries amongst military recruits [ , , , , , , , [ ] [ ] [ ] [ ] [ ] [ ] . males and close contact with a person with respiratory symptoms within days before their own onset of illness were associated with adenovirus infection, but sleeping adjacent to someone ill with respiratory symptoms did not present higher risk to infection [ ] . influenza a and b viruses have also resulted in much morbidity in outbreaks, particularly influenza a(h n )pdm virus infection [ , , , , , , ] . some of the risk factors proposed were crowded living quarters defined as more than three personnel and age group less than year old [ ] , asthma and obesity [ ] , age group less than years old and the high proportion of military who had being seroconverted [ ] . human rhinoviruses are known to cause common cold as well as more complicated respiratory infections [ , [ ] [ ] [ ] [ ] [ ] . all known human rhinoviruses have been reported to be present in military recruits during respiratory infection [ ] . association of rhinovirus with lower respiratory tract infections is well documented [ ] . viral interference has also been proposed between rhinovirus and adenovirus infection [ ] . stress factor due to adaptation to new and different surroundings for military recruits was also proposed as risk factor for rhinovirus infection [ ] . in this study, we investigate the risk factors associated with fri and the predominant viral aetiologies of fri in a semi-closed military environment of the saf. the saf started a sentinel respiratory disease surveillance program in four major camps (including a recruit training camp) in may [ , ] to track febrile respiratory illness (fri) cases defined as patients with temperature ≥ . °c with cough or sore throat. patients visiting primary healthcare clinics in the camps between may and october during regular consultation hours who met the fri criteria were recruited. the sentinel respiratory disease surveillance program includes the written informed consent obtained by healthcare workers, a questionnaire, clinical specimens collection and a clinical examination of the participants. repeat consultations were excluded if the healthcare worker determined that the patient had not recovered from the first episode of illness. we also obtained samples from controls (those without respiratory symptoms or acute infections), who were recruited from the same medical center during the same week as the recruitment of cases with about to controls per week. this is to ensure that both cases and controls had similar health-seeking behaviour, and similar chance of exposure to a particular respiratory pathogen circulating in the same environment around the same period of the year to minimize potential misclassification bias. moreover, the controls were not matched or restricted by barrack, sex, age or symptom-onset. this is because of the fact that the aim of the study is to evaluate most of these variables as potential risk factors of fri. informed consent, the baseline questionnaire, and clinical specimens were also obtained from these controls. the questionnaire covers demographics, co-morbidities, vaccination status, stay-in camp status and contact details of ill member in bunk (for stay-in personnel) and at home (for both stay-in and stay-out personnel). stay-in personnel stay in camp on weekdays and stay outside camp only on weekends, and hence, have household members and bunkmates as their key contacts. stay-out personnel do not stay inside camp on weekdays and have to travel in and out of camp on weekdays to work. these stay-out personnel hence only have household members but no bunkmates as key contacts. before the influenza a(h n )pdm pandemic in , trivalent inactivated seasonal influenza vaccine (pre-pdm tiv) was in use. then, the pandemic monovalent influenza a(h n )pdm vaccine [pdm-a(h n )v] was first introduced to saf and administered to all recruits only from december . this was superseded by the new trivalent influenza vaccine (post-pdm tiv) which included the influenza a(h n )pdm strain, first introduced to saf in october , and routinely administered to all recruits in december , and then all other military personnel in november (fig. ). nasal washes from both side of the nose were taken by certified medical staff and sent to the laboratory for etiological testing within h. detailed laboratory methods have been described in a previous study [ , ] . briefly, we used a multiplex pcr panel which included different respiratory viruses. they are as following-adenovirus e, influenza a(h n ), rhinovirus, coxsackie/echovirus, influenza b, influenza a(h n )pdm , enterovirus (ev), human metapneumovirus (hmpv), parainfluenza (hpiv- ), hpiv- , hpiv- and hpiv- , coronavirus oc (cov-oc ), cov-nl , cov- e, cov-hku , respiratory syncytial virus a (rsv-a) and rsv-b and bocavirus (bv). additional singleplex pcr assays were then performed to determine the influenza subtype. total nucleic acids were extracted from each clinical specimen using the dna minikit (qiagen, inc, valencia, ca, usa) according to the manufacturer's instructions. a total of μl of dna extract were tested with resplex i and ii (version . , qiagen, inc., valencia, ca, usa) for the presence of respiratory micro-organisms on the liquichip workstation, according to the manufacturer's instructions. specimens that were resplex ii positive for flu-a were further subtyped with real-time pcr for h or h , or for ph n . briefly, μl of total genetic extracts were tested using an in-house developed assay based on the one-step superscriptiii/platinum taq kit (invitrogen, carlsbad, ca, usa) following the manufacturer's instructions on either the lightcycler machine from roche or the applied biosystems real-time pcr machine ( ). we compared variables of all fri subjects, and subsets of subjects with mono-viral infection (mvi) for the five most common viral pathogens (case groups) against the non-fri patients without viral infection detected from the panel (control group). specifically, the five most common viruses were influenza b, influenza a (h n ) pdm , coxsackie/echovirus, adenovirus e and rhinovirus. univariate logistic regression was conducted to identify statistically significant variables of interest. selected variables with high co-linearity were dropped, but all other variables significant at p < . were then included in a multivariable logistic regression model to determine the independent factors. the best model was determined using backward stepwise regression method. power calculation showed at least % power to detect a true positive association with effect size of . with % of the controls having the exposure of interest as cases. all tests were conducted at the % level of significance. we report odds ratio (or) and corresponding % confidence intervals (ci) where applicable. all statistical analyses were performed using an open source statistical software r . . (r core development team). written informed consent was obtained from the study participants. this study was reviewed and approved by the singapore military's joint medical committee for research, and the national university of singapore's ethics review committee. a total of , fri cases were recruited. of these, there were , fri cases ( . %) with mono-viral infection (mvi). of the , mvi cases, the five most common mvi were due to influenza b with cases ( . %), influenza a (h n )pdm with cases ( . %), coxsackie/echovirus with cases ( . %), adenovirus e with cases ( . %), and rhinovirus with cases ( %); the number of cases observed in each month from may to october is shown in fig. . of the , non-fri subjects recruited, , subjects ( . %) were confirmed to be negative for the whole panel of respiratory pathogens tested, and these served as the study controls in all subsequent analysis. the mean age of fri cases was . (± . ) as compared to . (± . ) years old for controls (p < . ; table ). a significantly higher proportion of cases ( . %) came from the recruit camp as compared to the controls ( . %; p < . ). the proportion of fri cases who had pre-pdm tiv and post-pdm tiv were significantly lower and higher than the study controls, respectively ( . % vs . %, p = . , and . % vs . %, p < . , respectively; table ). in addition, there were significant differences in the smoking status among the cases compared to the controls (p = . ), and there were significantly higher proportion of fri cases than the study controls ( . % vs . %, p = . ). in terms of movement history, there was a significantly lower proportion of cases who had travelled to other camp in the last days before clinical presentation than that of study controls ( . % vs . %; p < . ), and there were significantly higher proportion of fri cases who stayed in camp compared to the controls ( . % vs . %, p < . ; table ). increasing age was observed to be an independent risk factor for fri [adjusted odds ratio (aor) = . ; % confidence interval (ci) = . - . ; fig. (table ) . similarly, asthma (cor = . ; % = . - . ) was a potential risk factor of fri, but it was not independently associated with fri after adjusting for potential confounding factors (table ) . of the five most common mvi, increasing age was positively associated with coxsackie/echovirus(aor = . ; % ci = . - . ; fig. personnel who travelled to the community in the last days before clinical presentation had a significantly lower risk of adenovirus mono-infection (aor = . ; % ci = . - . ; fig. ) compared to personnel who did not. however, personnel travelling overseas in the last days before clinical presentation had . times higher risk of adenovirus mono-infection (aor = . ; % ci = . - . ) compared with personnel who did not travel overseas. compared to stay-out personnel with no ill household members in the last days before clinical presentation, stay-out personnel with ill household members had . times higher risk of fri (aor = . ; % ci = . - . ). moreover, compared to stay-out personnel with no ill household members, stay-in personnel who had neither ill bunkmates nor household members had . times lower risk of fri (aor = . ; % ci = . - . ). however, there was a higher risk of fri for stay-in personnel with an ill member in bunk regardless of whether they had any ill household members (aor = . ; % ci = . - . ) or not (aor = . ; % ci = . - . ) in the last days before clinical presentation. results for the analysis on each of the five most common mvi were very similar to those for all fri analysis (figs. and ). there was significantly higher risk of infection for all of the five most common mvi in stay-out personnel with ill household members compared with those who did not (fig. ) . regardless of whether they had ill household members or not, stay-in personnel with no ill bunkmates were not at significantly increased risk for any of the five most common mvi compared to stay-out personnel (with no ill household members). stayin personnel with ill bunkmates but without ill household members had significantly increased risk of all the mvi except adenovirus e (aor = . ; % ci = . - . ), where there was a non-significant increase in risk; having ill household members further increased the risk for all [ ] had simultaneously document the risk of fri due to a range of specific pathogens. in this study, we had shown that the five most common viral pathogens within our military environment was strongly associated with contact history, and had fairly similar trend of the fri risk factors identified. increasing age, recruit camp, and smokers were demographic risk factors for fri. increasing age was also reported as a risk factor for ari in us military personnel in overseas deployments [ ] . additional analyses showed that the risk was higher with increasing age for all the five mvi in this study, but only significantly so for coxsackie/ echovirus. in contrast, increasing age was previously reported to be a protective factor for seroconversion against influenza a(h n ) pdm in the local military during the initial wave of infections from june to october [ ] . these discrepant findings may be due to the changing age distribution of susceptible population towards influenza a(h n )pdm infections [ , ] , which might have shifted to involve more older individuals over the study period presented here (up to october ). in addition, this may be due to the increased in herd immunity effects among the new young cohorts of conscripts, where vaccine (initially as a monovalent formulation, and then later as part of the post-pandemic trivalent inactivated vaccine) was administered to all military recruits since november [ , ] . moreover, the lack or waning immunity against influenza a(h n )pdm in the older cohorts may have attributed to this trend, even though the individual level effects of vaccination against influenza a(h n )pdm (which was found to be significantly protective) was accounted for in the multivariate model. personnel in the recruit camp were at higher risk of fri as well as all the five most common mvi, particularly adenovirus e infection. this is likely due to the higher contact exposure rate in semi-closed environments, and increased stressors [ , , , , , , , , ] . alternatively, it could be due to the fact that personnel in non-recruit camps are already protected due to the adaptive immune response developed from the previous infections in recruit camp, where recruits usually only stay on a short term basis, before posted to non-recruit camp. smoking has been shown to increase risk of upper respiratory infection among recruits [ ] , hajj medical mission personnel [ ] , infants and children exposed to parental smoking [ ] . hence, it is not surprising to observe smoking as a risk factor of fri in our study. there are some studies that had shown that cigarette smoking impairs oral and respiratory tract immunity [ ] [ ] [ ] . this may have predispose smokers to a higher chance of viral infection resulting in fri. however, further study is warranted to investigate mechanism behind this observation. malay ethnicity was positively associated with both influenza-a(h n )pdm and coxsackie/ echovirus monoinfections. we had previously also found malays in the community to be at higher risk of influenza a(h n )pdm infection [ ] . however, a previous study in the saf found that malays conscripts actually had significantly higher score in hygiene practices and knowledge towards pandemic influenza as compared to chinese and indians [ ] . hence, there may be a potential genetic basis for the higher risk of infection in malays as compared to chinese and indians, given differences in genetic backgrounds of the hla class region which have been shown to result in weaker immune response against pathogen antigens [ ] . nevertheless, other unmeasured sociocultural and behavioural factors might explain these observations, and further studies are needed to confirm these observations and to understand the basis for the association. the protective effects of the influenza vaccine was largely in line with expectations, with the pre-pdm tiv protecting against influenza b but not against influenza a(h n )pdm , the pdm-a(h n )v protecting against influenza a(h n )pdm but not influenza b, and the post-pdm tiv protecting against both pdm-a(h n )v subtypes, as observed in our previous study [ ] . however, there were also some unexpected findings. these includes a potential protective effect (aor = . ; % ci = . - . ) of the pdm-a(h n )v against rhinovirus, and an increased risk (aor = . ; % ci = . - . ) of adenovirus e infection with the post-pdm tiv. these findings may have been due to non-specific interactions and interference between respiratory viruses which have been suggested by others [ ] , but could also have been due to the periodic nature of respiratory virus outbreaks. in particular, the post-pdm tiv period included a period of heightened adenovirus e activity (see fig. ) which might have been unrelated to changes in the vaccination policy, but which we could not adjust for due to co-linearity between the timing of these adenovirus fig. contact risk factors for fri and the five most common mono-viral infections e outbreaks and the phased roll-out of the influenza vaccine formulations. these unexpected findings would still require more scientific and epidemiological evidence for further conclusion. travelling overseas in the last days before clinical presentation was associated with a significantly increased risk for adenovirus e infection. we were not able to distinguish these as either military or personal overseas trips, but a previous outbreak of b human adenovirus e a strain in a military camp in singapore was also reported to be highly similar to other asian strains involved in outbreaks, suggesting a potential import of this strain from the neighbouring regions [ ] . as such, implementation of adenovirus vaccination may be useful to prevent sudden surge of cases with adenovirus e outbreak, given the high incidence of adenovirus infection in south-east asia [ , ] . one key finding was the relatively lower risk of fri and the five most common mvi for stay-in personnel as compared with stay-out personnel. at least for influenza b and a(h n )pdm , this could be due to the lower proportion of members in the households and the community who had the seasonal influenza vaccination [ ] , as compared to the camps where vaccination programme was implemented for all military personnel since the end of [ ] . as such, this may have resulted in a smaller pool of susceptible individuals and a larger herd immunity effects in camps as compared to within the community. the other explanation maybe that stay-in personnel have less exposure to younger household members, which was previously found to have a significant risk for seroconversion to influenza a(h n )pdm , and the risk was accentuated if the household member had fri [ ] . this also concurs with our findings on the effect of exposure to ill household members and bunkmates, and the effects are influenced by the domiciliary status of the soldier. for the five most common mvi, an ill household member was a major risk factor for stay-out personnel. moreover, the increase in risk for stay-in personnel from having ill household members was not as marked and mostly not significant. however, stay-in personnel with an ill bunkmate had a substantial increase in risk of infection. while our current study design does not allow us to attribute the cause of infection to contact with these ill household members or bunkmates, our findings do suggest that some of the transmission of these pathogens is mediated through close contacts, and support the use of preventive measures for fri aimed at reducing transmission from ill household members and bunkmates. this could be in the form of issuing advisories to emphasize hygiene during outbreaks, and identifying and isolating ill personnel early to break the transmission of fri. moreover, this finding also has potential applications in surveillance. we had previously reported on how it would be difficult for syndromic surveillance systems to detect outbreaks in larger military units given the high baseline rates of respiratory illness [ ] . given that the relevance of ill bunkmates is consistent for the predominant viral agents of fri, outbreak detection methods could instead focus on clusters of illness in those who share the same quarters, or are from the same military subunit as a reasonable proxy. we believe such an approach to syndromic surveillance deserves a prospective validation study where such clusters of illness are systematically sampled. there are several limitations to this study. first, there was the influenza a (h n )pdm pandemic in june to september during the early part of the study period, where the force of infection for influenza a (h n )pdm is likely to be higher than usual. however, the pandemic spread was well-contained with prompt protective and preventive measures such as vaccination (fig. ) , enhanced respiratory hygiene measures, isolation, quarantine, "ring prophylaxis" with oseltamivir during this period. as such, these measure are also likely to limit the risk of transmission of other circulating respiratory viruses during this specific period compared to other periods in the study. since different vaccines were used promptly and appropriately during the different study periods (fig. ) , vaccine type was used as a surrogate to account for the potential bias due to the enhanced protective and preventive measures applied during the influenza a (h n )pdm pandemic. nevertheless, this bias should be minimal because the controls were also recruited in the same period and camp as the cases. second, hand washing behaviour, allergy and military rank were not evaluated as potential risk factors of fri. this is because it was very challenging to accurately assess how frequent hand washing was performed by the soldiers. moreover, the soldiers may also tend to report the expected favourable hand washing behaviour. hence, the likelihood of recall bias and information bias are likely to be high and would make any form of interpretation challenging. allergy was not evaluated because the symptoms are very broad to specifically define as an allergy, and there would be significant potential information bias as it is less likely to clinically diagnosed allergy as compared with asthma, diabetes, hypertension and heart disease. furthermore, the aim of this study is not to study clinical signs and symptoms that are associated with fri. we did not consider military rank due to fact that there is a significant number of cases that were recruited from the recruit camp, where the population is mainly made up of recruits as compared to non-recruit camp, where the population is mainly made up of higher ranks (table ; p < . ). as such, it would be biased to include military rank as one of the variables. third, our data is limited to febrile presentations of viral respiratory infections and may not be applicable to milder acute respiratory infections. fourth, there is a lack of clinical and laboratory confirmation of the ill household members and bunkmates, and such data are hence subjected to recall bias. fifth, the prevalence for fri and mvi is about % and % respectively. as such, or values as proxies to rr would be similar for mvi, whereas the or of the risk factors for fri is likely an overestimation, to some extent, relative to rr. sixth, the resplex i assay (qiagen) was designed to also detect six bacterial respiratory pathogens. they were mycoplasma pneumoniae, chlamydophila pneumoniae, legionella pneumomophila, streptococcus pneumoniae, neisseria meningitides and haemophilus influenza , , . however, fri subjects with bacterial causes were not excluded because one of the aims of the study is to determine the potential risk factors for fri, regardless of any detected or undetected respiratory virus and/or bacteria. lastly, this study involved predominantly young adult males in a military context, and hence, the results may not be generalizable to the overall population in the community, particularly for the contact risk factors. however, during the pandemic of influenza a(h n )pdm , clustering of febrile respiratory illness by classroom contact among school children [ ] and ill workplace contacts among healthcare workers were also observed [ ] . further studies in other settings such as nursing homes which collect contact history in a similar way should be attempted. increasing age, smokers, recruit camp, stay-out personnel with ill household members and stay-in personnel with ill bunkmates were independent risk factors of fri in a semi-closed military setting. early identification and isolation of ill bunkmates may be effective to prevent and to reduce further transmission in camp. public health campaigns and policy should take these risk factors into consideration to increase the effectiveness of interventions to reduce fri in the military environment. abbreviations fri: febrile respiratory illness; aor: adjusted odds ratio; ci: confidence interval; saf: singapore armed forces; mvi: mono-viral infection. frequency of acute respiratory illnesses and circulation of respiratory viruses in households with children over surveillance seasons epidemiology of community-acquired respiratory tract infections in adults. incidence, etiology, and impact recent trends of pneumonia morbidity in us naval personnel keep your hands clean influenza c virus infection in military recruits-symptoms and clinical manifestation respiratory infections in the military symptomatic respiratory syncytial virus infection in previously healthy young adults living in a crowded military environment respiratory syncytial virus: an important cause of acute respiratory illness among young adults undergoing military training viruses associated with acute 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epidemic of pandemic h n in singapore factors influencing infection by pandemic influenza a(h n )pdm over three epidemic waves in singapore acute respiratory tract infections among hajj medical mission personnel, saudi arabia the study protocol for a randomized controlled trial of a family-centred tobacco control program about environmental tobacco smoke (ets) to reduce respiratory illness in indigenous infants cigarette smoking impairs human pulmonary immunity to mycobacterium tuberculosis effect of smoking on immunity in human chronic periodontitis cigarette smoke effects on innate immune mechanisms in the nasal mucosa. potential effects on the microbiome risk factors for pandemic (h n ) seroconversion among adults knowledge, attitudes and practices towards pandemic influenza among cases, close contacts, and healthcare workers in tropical singapore: a cross-sectional survey ramifications of hla class i polymorphism and population genetics for vaccine development dramatic decline of respiratory illness among us military recruits after the renewed use of adenovirus vaccines an assessment of electronically captured data in the patient care enhancement system (paces) for syndromic surveillance teacher led school-based surveillance can allow accurate tracking of emerging infectious diseases -evidence from serial cross-sectional surveys of febrile respiratory illness during the h n influenza pandemic in singapore the work was supported by a singapore ministry of defence funded operational research program and the centre for infectious disease epidemiology and research in the saw swee hock school of public health of the national university of singapore and national university health system. the funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. biodefence centre, ministry of defence, singapore, singapore. yale-nus college, national university of singapore, singapore, singapore. program in health services and systems research, duke-nus graduate medical school, singapore, singapore. department of statistics and applied probability, national university of singapore, singapore, singapore. department of medicine, national university of singapore, singapore, singapore.received: january accepted: july the authors declare that they have no competing interests, except for vl who had previously received unrelated research grants from gsk.authors' contribution jp wrote the manuscript and analysed the data. jj analysed the data. lyh, arc and mic revised the manuscript and assisted with data analysis. vjl conceptualized the study and revised the manuscript. bht, jpl, whvk, shn were involved in the laboratory testing of the specimens. mh and qg were involved in subject recruitment and screening. all authors read and approved the final manuscript. key: cord- -nzptmdbe authors: neske, florian; prifert, christiane; scheiner, barbara; ewald, moritz; schubert, jörg; opitz, andreas; weissbrich, benedikt title: high prevalence of antibodies against polyomavirus wu, polyomavirus ki, and human bocavirus in german blood donors date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: nzptmdbe background: dna of the polyomaviruses wu (wupyv) and ki (kipyv) and of human bocavirus (hbov) has been detected with varying frequency in respiratory tract samples of children. however, only little is known about the humoral immune response against these viruses. our aim was to establish virus-specific serological assays and to determine the prevalence of immunoglobulin g (igg) against these three viruses in the general population. methods: the capsid proteins vp of wupyv and kipyv and vp of hbov were cloned into baculovirus vectors and expressed in sf insect cells. igg antibodies against wupyv vp , kipyv vp , and hbov vp were determined by immunofluorescence assays in plasma samples of blood donors. results: the median age of the blood donors was years (range - yrs), % were male. % of the samples were positive for wupyv igg (median age yrs, . % male), % were positive for kipyv igg (median age yrs, . % male), and % were positive for hbov igg (median age yrs, . % male). for wupyv and hbov, there were no significant differences of the seropositivity rates with respect to age groups or gender. for kipyv, the seropositivity rate increased significantly from % in the age group - years to % in the age group > years. conclusions: high prevalences of antibodies against wupyv, kipyv, and hbov were found in plasma samples of healthy adults. the results indicate that primary infection with these viruses occurs during childhood or youth. for kipyv, the seropositivity appears to increase further during adulthood. infections of the respiratory tract are a major cause of human morbidity. they are most often caused by respiratory viruses, which include the well-known pathogens respiratory syncytial virus, influenza viruses a and b, adenoviruses, parainfluenza viruses, rhinoviruses, and coronaviruses. in recent years, a number of unknown viruses have been identified in the respiratory tract by molecular methods, including the human metapneumovirus, several corona viruses, the human bocavirus (hbov), and most recently the polyomaviruses wu (wupyv) and ki (kipyv) [ ] [ ] [ ] [ ] . initial studies on wupyv and kipyv have looked at genome detection rates using polymerase chain reaction (pcr) methods. the genoprevalences for wupyv and kipyv in respiratory samples from children with acute respiratory tract diseases were found to range from . to . % [ ] [ ] [ ] [ ] [ ] [ ] [ ] and . to . % [ , [ ] [ ] [ ] ] , respectively. however, wupyv and kipyv dna were found at similar frequencies in control groups without respiratory tract disease [ , , ] . therefore, the clinical relevance of wupyv and kipyv infections is currently unclear. in contrast to pcr assays, serological assays for antibody detection against hbov are more complex to establish. however, determining immunoglobulin (ig) m and igg antibodies in appropriate serum or plasma samples allows to define the point in time of primary infection as well as exposure rates. one study describing the seroepidemiology of polyomaviruses including wupyv and kipyv in adults has recently been published [ ] . hbov is a virus of worldwide distribution. its dna has been found in . % to % of respiratory secretions from children with acute respiratory tract diseases using pcr [ ] . elucidation of the clinical relevance of hbov has been difficult because of a high co-infection rate of hbov dna positive samples with other respiratory viruses. based on the combination of a high hbov load in nasopharyngeal samples (> copies/ml) and concomitant hbov dna detection in sera, a model has been proposed in which hbov is associated with acute respiratory tract diseases but persists in the respiratory tract for a longer period of time than other respiratory viruses after primary infection [ ] . recent serological studies have demonstrated evidence of primary hbov infection in children with acute respiratory tract diseases, strongly indicating that hbov is indeed a respiratory pathogen [ , ] . in seroprevalence studies from japan, the united states, china, and germany the proportions of hbov igg-positive samples increased with age during infancy until reaching levels of > % at the age of > years [ , [ ] [ ] [ ] . in order to expand the epidemiological knowledge about hbov, wupyv, and kipyv, we expressed capsid proteins in the baculovirus system and established an immunofluorescence assay (ifa) for the detection of igg antibodies against these three viruses. we used this system to determine the prevalence of antibodies against hbov, wupyv, and kipyv in healthy adults. the specimens tested for wupyv, kipyv, and hbov serology consisted of consecutive plasma samples of healthy blood donors received in from the institute of transfusion medicine and hemotherapy at the university clinic of würzburg. the median age of the blood donors was . years (range . - . years) and % were male. the samples were screened routinely for infectious diseases transmitted by blood (human immunodeficiency virus, hepatitis b virus, hepatitis c virus, syphilis). remaining material was stored at - °c until use. the study was carried out in compliance with the helsinki declaration. informed consent of the blood donors was obtained. according to the ethics committee of the medical faculty at the university of würzburg, formal approval of the study was not necessary because the samples were tested in an anonymised fashion. the vp gene of hbov was amplified from a nasopharyngeal aspirate using the primers bov s and bov a and pfu polymerase (fermentas, st. leon rot, germany) resulting in a bp pcr product (table ). similarly, the vp genes of wupyv and kipyv were amplified from nasopharyngeal aspirates with the primer pairs wu s/wu a and ki s/ki a resulting in amplicons of bp and bp, respectively (table ). sequences were submitted to genbank with the accession numbers fj (hbov), eu (wupyv), and fj (kipyv). for production of the recombinant baculoviruses bacwuvp , backivp , and bacbovp the lr recombination system (invitrogen, karlsruhe, germany) was used according to the recommendations of the manufacturer. briefly, the amplicons were inserted by topo cloning into the pentr vector (invitrogen) and subsequently transformed in chemically competent e.coli top cells (invitrogen). the resulting plasmids pbovp , pwuvp , and pkivp were extracted from overnight culture with the qiaprepspin miniprep kit (qiagen, hilden, germany) and sequenced by standard techniques to confirm orientation and sequence identity of the plasmid insert. the plasmid inserts were then transferred into baculoviral dna by homologous recombination based on gateway technology and the baculodirect system (invitrogen). the resulting recombinant baculovirus dna was directly transfected to adherent sf cells in one well of a six-well-plate using cellfectin reagent (invitrogen). the cells were kept in tc- growth medium (lonza, basel, switzerland) supplemented with % fetal calf serum, u/ml penicillin, μg/ml streptomycin, . μg/ml amphotericin b, and μm ganciclovir as selective reagent. after five days, the cell culture supernatant was collected and used to infect fresh sf cells. this procedure was repeated twice, resulting in the supernatant of passage (p ), which was finally collected and stored at °c for further use. protein expression was confirmed by sodium dodecyl sulfate polyacrylamide gel electrophoresis (sds-page) and immunoblotting. to this end, sf cells at approximately % confluence were inoculated with recombinant virus of p . the cells were observed daily until at least % of the cells displayed a cytopathic effect, which typically happened after seven days. subsequently, the cell culture supernatant was collected and centrifuged with g at °c for min to remove cells and large debris. the samples were separated by % sds-page and blotted on a nitrocellulose membrane by standard procedures. after blocking, immunodetection was performed with hrp-conjugated mouse-anti-v -antibody (invitrogen) at a concentration of ng/ml. bands were visualised by super-signal west pico chemiluminescence substrate (pierce, rockford, usa). uninfected sf cells were used as negative control. sf cells were inoculated with recombinant baculoviruses and observed daily until approximately % of the cells displayed a cytopathic effect. the cells were harvested and centrifuged for min at g. the supernatant was discarded and the cell pellet was washed three times in phosphate buffered saline (pbs). the sf cells were spotted on glass slides, air dried, fixed with cold acetone and stored at - °c until use. slides with uninfected sf cells were prepared in the same manner and were used for control staining to detect anticellular antibodies. one spot each with sf cells expressing wupyv vp , kipyv vp , or hbov vp and one spot with uninfected sf cells were incubated with a : dilution of each plasma sample for h at °c. subsequently, the slides were washed twice for min with pbs containing . % tween followed by a short rinse in pbs without tween . next, the slides were incubated with a : dilution of fluoresceine-conjugated goat-anti-human-igg (invitrogen) and a : dilution of evans blue (mast diagnostik, reinfeld, germany) for h at °c. after another washing step as described above, coverslips were mounted for immunofluorescence microscopy. the slides were independently read by two experienced investigators. plasma samples, which exhibited a vesicular fluorescence adjacent to the membrane of sf cells, were recorded as antibody positive, if no staining of the control cells was detected. for the absorption test, three plasma samples were selected that were positive for antibodies against wupyv, kipyv, and hbov. sf cells infected with each of the recombinant baculoviruses (bacwuvp , back-ivp , bacbovp ) were harvested from small flasks and resuspended in μl pbs. the suspensions were sonicated for s on ice with a sonicator (branson sonifier ). each of the three plasma samples was diluted : in each of the cell lysates. after a h of incubation on ice, the samples were centrifuged at g for min and the supernatants were diluted in pbs to a final sample concentration of : . twofold dilution series ranging from : to : of the absorbed and unabsorbed plasma samples were tested in parallel by ifa as described above. statistical analysis was carried out using graphpad prism version . c for mac (graphpad software, san diego, usa) and spss version for windows (spss, chicago, usa). capsid proteins of wupyv, kipyv, and hbov were expressed by infection of sf cells with the recombinant baculoviruses bacwuvp , backivp , and bacbovp . as antibodies against these proteins are not yet available, the correct size of the expressed proteins was confirmed by sds-page and immunoblotting using an anti-v antibody. the v -epitope is located upstream of the inserted gene in the baculovirus dna. a band of the expected size (~ kda for wupyv and kipyv vp ;~ kda for hbov vp ) was observed for all recombinant baculoviruses, whereas no band was observed in the uninfected control ( figure ) . to determine the frequency of past exposure with wupyv, kipyv, and hbov in healthy adults, blood donor plasma samples were tested for the presence of igg against wupyv vp , kipyv vp , and hbov vp using an ifa based on sf cells infected with the recombinant baculoviruses (figure ) . all samples were also tested by ifa using uninfected sf cells. no positive reaction with cellular antigens was observed in any of the samples. in order to further confirm the specificity of positive ifa results, three plasma samples were studied in an absorption assay. antibody titers were determined before and after absorption with lysates of sf cells infected with recombinant baculoviruses. a significant titer decrease was only observed after absorption with the matching sf cell lysate (table ). there was no indication of antibody cross-reaction between sf cells infected with bacwuvp , backivp , and bacbovp . of all plasma samples, % were positive for anti-wupyv vp igg, % were positive for anti-kipyv vp igg, and % were positive for anti-hbov vp igg. neither the median age nor the gender distribution of the antibody positive samples were significantly different from the total population for any of the three viruses tested (mann-whitney test and fisher's exact test, respectively; table ). for wupyv and hbov, there were no significant differences of the seropositivity rates with respect to age groups. for kipyv, the seropositivity rate increased significantly from , % in the age group - years to % in the age group > years (p = . ; chi-square test for trend; figure ). table shows the number of samples grouped by individual results for wupyv, kipyv, and hbov igg. one percent of the samples were negative for all three viruses and % were positive for all three viruses. no obvious cross-reaction between the three antibody specificities was apparent. using ifa based on insect cells expressing capsid proteins of wupyv, kipyv, and hbov, we studied the seroprevalence of these viruses in plasma samples of healthy adults. we found high rates of positive igg antibodies against all three viruses: % for wupyv, % for kipyv, and % for hbov. our data on seroprevalence for wupyv and kipyv are somewhat higher than reported in a recent study from the united states, which found seroprevalences of . % to . % for kipyv and . % to . % for wupyv in the age groups from to years of north american blood donors [ ] . in contrast to our findings, a rising prevalence for kipyv igg in adults was not observed. further studies are necessary in order to determine whether there may be regional differences in the kipyv epidemiology. taken together, the data of kean et al. and of our study indicate that wupyv and kipyv have a widespread distribution similar to the well-known polyomaviruses bkv and jcv. also in agreement with bkv and jcv epidemiology, primary exposure seems to occur mainly in childhood and youth. primary bkv infection has been reported to occur mainly in the first decade of life leading to a seroprevalence of % to % at the age of (reviewed in [ ] ). the jcv seroprevalence in adults has been found between % and %. whereas some studies reported a time frame for primary jcv infection similar to bkv infections, others showed a continuing rise of the jcv seroprevalence during adulthood (reviewed by [ ] ). vp is the major capsid protein of polyomaviruses. several studies on bkv and jcv serology have used this protein successfully in enzyme immunoassays, immunoblots, or ifa [ ] [ ] [ ] [ ] . therefore, we decided to use expression of vp proteins to establish ifas for the detection of wupyv and kipyv igg. in the study by kean et al., wupyv and kipyv antibodies were determined by an enzyme immunoassay based on bacterially expressed vp capsomeres [ ] . validation of these ifa was limited by the lack of defined serum samples that could be used to determine assay sensitivity and specificity. by testing selected study samples before and after absorption with lysates of sf cells that were infected with different recombinant baculoviruses, we were able to exclude general cross reactivity between wupyv and kipyv. furthermore, we excluded anticellular reactivity of the plasma samples by performing an ifa with uninfected sf cells. therefore, we are confident that our serological data are reliable. comparison of different serological methods and antigen preparations as well as sample exchange will be useful to further validate the assays for wupyv and kipyv antibody determination. as to hbov, the adult seroprevalence of % in our study is slightly lower than in previous studies from japan, the united states, and germany, which reported hbov igg antibody prevalence of %, %, and %, respectively [ , , ] . the total number of adult samples tested was small in two of these reports ( , , and , respectively). whether methodological issues or differences in the study populations account for the lower seroprevalence observed in this study, could be addressed by method comparisons and sample exchange. we chose the hbov vp gene for recombinant expression on the basis of current knowledge about immunodominant antigens of parvovirus b , which belongs to the same virus family as hbov. previous studies on hbov serology have used ifa based on vp expressing insect cells [ ] , enzyme immunoassay based on vp purified from insect cells [ , ] , immunoblot based on vp and the unique region of vp expressed in e. coli [ ] , and enzyme immunoassay based on vp virus-like particles [ ] . in the study of kantola et al., a higher immunoreactivity was demonstrated for hbov vp than for the unique part of hbov vp . overall, the vp protein seems to be suitable for use in serological assays. however, a comparison of the vp and vp protein has not yet been performed. limitations of the hbov ifa validation are similar to the limitations described for the wupyv and kipyv ifa. results of a control ifa using uninfected sf cells and of pre-absorption experiments suggest that the positive hbov igg results are specific. recently, novel bocaviruses related to hbov have been described in humans [ , ] . if there are cross-reactions between antibodies against the different hbov types is not known at present and needs to be further investigated. in this seroepidemiological study, only igg antibodies were determined. evidently, the ifas used in this study may easily be modified for the determination of igm antibodies. igg and igm serology applied on appropriate sample collections will allow to determine the point in time of primary infection. information on this issue is figure age distribution of virus-specific igg antibodies. the seroprevalence for wupyv and hbov did not differ significantly between age groups (p = . and p = . , respectively). for kipyv, a significant rise of seroprevalence rates was observed with increasing age (p = . ). antibodies against hbov, wupyv, and kipyv were found at high rates in sera of healthy german adults. these results suggest that primary infections with these viruses occur mainly during childhood and youth. for kipyv, the seropositivity appears to increase further during adulthood. newer respiratory virus infections: human metapneumovirus, avian influenza virus, and human coronaviruses cloning of a human parvovirus by molecular screening of respiratory tract samples identification of a third human polyomavirus identification of a novel polyomavirus from patients with acute respiratory tract infections wu polyomavirus in children agerelated pattern of ki and wu polyomavirus infection presence of the newly discovered human polyomaviruses ki and wu in australian patients with acute respiratory tract infection wu polyomavirus in children with acute lower respiratory tract infections, south korea. emerg infect dis clinical and epidemiologic characterization of wu polyomavirus infection no evidence for an association between infections with wu and ki polyomaviruses and respiratory disease seroepidemiology of human polyomaviruses allander t: human bocavirus human bocavirus and acute wheezing in children seroepidemiology of human bocavirus in hokkaido prefecture serodiagnosis of human bocavirus infection seroepidemiology of human bocavirus defined using recombinant virus-like particles elisas using human bocavirus vp virus-like particles for detection of antibodies against hbov humoral immune response against human bocavirus vp virus-like particles discovery and epidemiology of the human polyomaviruses bk virus (bkv) and jc virus (jcv) comparison of antibody titers determined by hemagglutination inhibition and enzyme immunoassay for jc virus and bk virus serological cross-reactivities between antibodies to simian virus , bk virus, and jc virus assessed by virus-like-particle-based enzyme immunoassays seroepidemiology of the human polyomaviruses prevalence of polyomavirus bk and jc infection and replication in healthy blood donors a newly identified bocavirus species in human stool a novel bocavirus associated with acute gastroenteritis in australian children we thank the technicians of the viral diagnostic lab for skillful and dedicated assistance. authors' contributions bw and fn designed and coordinated the study. fn and me produced the recombinant baculoviruses. fn, cp, bs, me, and js established and performed the ifa testing. ao collected the blood donor samples. all authors participated in the data analysis. bw and fn drafted the manuscript. all authors read and approved the final version of the manuscript. the authors declare that they have no competing interests. key: cord- -botshfa authors: abolfotouh, mostafa a.; almutairi, adel f.; banimustafa, ala’a a.; hussein, mohamed a. title: perception and attitude of healthcare workers in saudi arabia with regard to covid- pandemic and potential associated predictors date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: botshfa background: healthcare workers (hcws) face considerable mental and physical stress caring for patients with covid- . they are at higher risk of acquiring and transmitting this virus. this study aims to assess perception and attitude of hcws in saudi arabia with regard to covid- , and to identify potential associated predictors. methods: in a cross-sectional study, hcws at three tertiary hospitals in saudi arabia were surveyed via email with an anonymous link, by a concern scale about covid- pandemic during – april, . concerns of disease severity, governmental efforts to contain it and disease outcomes were assessed using concern statements in five distinct domains. multiple regression analysis was used to identify predictors of high concern scores. results: a total of hcw responded to the survey. their average age was . ± . years, . % were nurses, . % had direct patient contact, and . % were living with others. the majority of participants ( . %) had overall concern scores of or less out of a maximum score of points, with an overall mean score of . ± . reflecting moderate level of concern. three-fourth of respondents felt at risk of contracting covid- infection at work, . % felt threatened if a colleague contracted covid- , . % felt obliged to care for patients infected with covid- while . % did not feel safe at work using the standard precautions available. nearly all hcws believed that the government should isolate patients with covid- in specialized hospitals ( . %), agreed with travel restriction to and/or from areas affected by covid- ( . %) and felt safe the government implemented curfew and movement restriction periods ( . %). predictors of high concern scores were; hcws of saudi nationality (p < . ), younger age (p = . ), undergraduate education (p = . ), living with others (p = . ) working in the western region (p = . ) and direct contact with patients (p = . ). conclusions: this study highlights the high concern among hcws about covid- and identifies the predictors of those with highest concern levels. to minimize the potential negative impact of those concerns on the performance of hcws during pandemics, measures are necessary to enhance their protection and to minimize the psychological effect of the perceived risk of infection. in december , a cluster of patients with pneumonia was linked to a seafood wholesale market in wuhan, china, which lead to the discovery of a new betacoronavirus [ ] , on january, , named severe acute respiratory syndrome coronavirus- (sars-cov- ) [ ] that causes coronavirus disease . with its novelty and rapid national and international spread on jan , the world health organization (who) international health regulation (ihr) emergency committee declared the disease a public health emergency of international concern (pheic). it was declared by who [ ] as a worldwide pandemic on march . at the time of this writing, it has infected , , individuals, with , , recoveries and , deaths, with an overall estimated case fatality rate (cfr) of . % [ ] . on the nd of march , a saudi citizen coming from iran through bahrain was tested positive for covid- and reported by the ministry of health as the first case in saudi arabia [ ] . as of th june, , saudi arabia had , infected cases, with , recoveries and deaths [ ] . health care workers face considerable mental and physical stress caring for patients with covid- . several reports around the world suggest that this stress has led some physicians to take their own life [ , ] . furthermore, others were overstressed and died from exhaustion [ ] [ ] [ ] [ ] . one approach to minimize such stresses during pandemics is for hospitals to organize physician shifts with mandatory rest and meal breaks. professional societies can also play a significant role by offering online networking to keep doctors connected to provide some level of social support. the government can also play a role by improving the benefits for hcws and their families [ ] . these initiatives can be further enhanced by understanding the level of concerns and worries among healthcare workers and provide targeted strategies that address those concerns. along this line, several studies have investigated the self-satisfaction of hcw and their personal feelings across several important domains [ ] [ ] [ ] [ ] [ ] . these domains cover concerns around risks posed to family members, perception of risk at the work place, and perception of the response of government to the epidemic management [ ] .. understanding the concern level across these different domains can be of importance to targeted mitigation strategies. in saudi arabia, a previous study has shown that hcws had, in general, a negative attitude toward mers-cov infection [ ] . in this study, the majority of the respondents felt that the work environment poses a high risk for contracting the infection and did not feel safe using the standard infection-control measures. one reason for the observed low attitude score might have been the lack of hcw experience with exposure to such outbreaks. due to the potential rapid dissemination of covid- within the public and a large probability of a countrywide outbreak, along with the country's experience in battling this similar coronavirus (mers-cov), the ksa was amongst the leading bodies in the world for its swift community action and hospital preparedness. this study aims to assess perception and attitude of hcws in saudi arabia with regard to covid- , and to identify potential associated predictors. this is a cross sectional study of hcw working at the medical cities of the saudi ministry of national guard health affairs (mng-ha). mng-ha provide healthcare services to national guard service members and their dependents through large medical cities located in the most densely populated regions of ksa, namely the central, western and eastern regions. all facilities have been joint commission international (jci) accredited since . during the covid- , and following the first reported case in ksa, mng-ha has taken drastic infection control measures that included the reduction of elective surgeries, stopping in person outpatient services, and introducing er workflow to minimize covid- cases flow through the main er. the target population of the current study was all hcw employed by the mng-ha at all three regions. an email with an anonymous link to an electronic survey was sent to all hcws who were on duty during the data collection period (~ hcws), across all departments and specialties. this electronic survey was structured using the option that allowed for every participant to participate only once. the target sample size for the survey was estimated assuming a prevalence of high concern among hcw of . % which was observed in another study in the same setting [ ] . we estimated the sample needed for the survey to be participants, assuming % confidence limits and % precision. those who agreed to participate and who responded with completed questionnaires totaled hcws, with a response rate of . %. a structured, self-administered survey of hcws was conducted via email, using a concern scale to assess their concern about covid- pandemic. this survey was designed based on a validated concern scale previously used in a study of the concerns of hcws with regard to mers-cov . the scale consists of statements that cover domains; self-satisfaction, social status, work environment, infection control measures, government action and activities [ ] . the scale was modified to also include a statement about the perception of hcws towards curfew: "i feel safe that government implemented the curfew and the movement restriction periods". a copy of the revised concern scale was attached as a supplementary material. data on gender, age, nationality, marital status, level of education, living status, professional characteristics and contact with patients were collected. hcws were categorized according to their direct contact with covid- patients to "direct contact group", or "non direct contact group". the direct contact group included all subjects caring directly for patients in the er, ward, or icu. all statements were coded using points likert scale, taking values from ("strongly disagree") to ("strongly agree") resulting in a total concern score that ranges from to . participants were further classified into one of three groups based on their total concern score. the first group included subjects below the first quartile of the concern score (score of and below), the second group included subjects with concern score between the th percentile (concern score of ) and th percentile (concern score of ) and the third group included subjects above the th percentile (score of and above) [ ] . the survey was distributed in the english language, as an electronic survey, to all hcws via a link attached to a mass e-mail distribution, with no identifiers. a cover letter was attached to an email as a link sent to hcws in their office emails, during the period between and of april, . study participants were expected to complete the survey and return it back without identifiers. participation in this study was voluntary. hcws were assured in a written informed consent that their responses would remain anonymous and would not affect their performance evaluations, work status or compensations. hcws were asked to respond to the survey if they agree on the informed consent. this study was approved by the institutional review board of the mng-ha in riyadh, saudi arabia (april , ; rc / /r). all categorical variables including age, gender and occupation status were summarized and reported using frequency and proportions. the total concern score was summarized and reported using mean and standard deviation. association of categorical variables with the different levels of concern was analyzed using the chi square test for homogeneity. all continuous variables were compared across the different concern levels using the student-t test and one-way anova. multiple linear regression analysis was used to determine significant predictors of high concern scores to covid- pandemic. for all statistical analyses, significance was considered at a p value of ≤ . . all analyses were performed in the statistical package for the social sciences software (spss version . ; ibm corporation, armonk, ny, usa). a total of mng-ha hcws responded to the survey ( males and females). they had an average age of . ± . years, ( . %) were from the central region, ( . %) from the eastern region and ( . %) from the western region. a total of . % were nurses, . % had direct patient contact, and . % were living with family members and/or others, table . the majority of participants ( . %) had an overall concern score of or less out of a maximum score of points. the responses to the items in the questionnaire varied considerably. with regard to selfsatisfaction domain, responses of concern varied from a high of . % who expressed fear of getting infected from an infected colleague, to a low of . % who felt unconfident a colleague would care for them if they contract the disease. in social status-related domain, concern varied from a high of . % agreeing that they should limit their social activities due to covid- to a low of . % not feeling satisfied of telling their family if they get infected. in workplace-related domain, responses ranged from a high of . % preferring to be absent from work to lower the chance of getting infected to a low of . % agreeing they would feel ashamed telling their managers/colleagues if contracting covid- . in infection control-related domain, responses varied from a high of . % not feeling there was a plan for covid- outbreak in their area to a low of . % did not feel an ic specialist is accessible to respond to their concerns and . % did not feel safe at work when using the standard precautions. in the government-related domain, responses varied from a high of . % agreeing with travel restrictions implemented by the government to a low of . % agreeing that covid- was not discussed efficiently in the media, table . overall, . % of hcws had high concern, . % moderate concern and . % low concern. the average concern score was . ± . , out of a maximum possible concern score of . level of concern was significantly associated with age (χ = . ; p = . ), marital status (χ = . ; p = . ), nationality (χ = . ; p < . ), level of education (χ = . ; p = . ), occupation (χ = . ; p < . ), geographical region of table . in multiple regression analysis ( table ) , predictors of high concern scores were; hcws of younger age (p = . ), saudi nationality (p < . ), undergraduate education (p = . ), and those working in the western region (p = . ), living with others (p = . ) and in direct contact with patients (p = . ). this study aimed to assess perception and attitude of hcws in saudi arabia with regard to covid- , and to table comparison between the levels of concern about covid- and personal characteristics of healthcare workers in saudi arabia characteristics low concern (score = - ) moderate concern (score = - ) high concern (score = - ) total ( identify potential associated predictors. an overall average concern score of . ± . out of a maximum possible score of points was observed, with a negative range of attitude, indicating a moderate level of concern. in comparison with the results of a previous survey in the same settings using the same data collection tool, to assess the concern of hcws about mers outbreak in saudi arabia [ ] , hcws reported significantly higher mean concern scores about covid- pandemic. this may reflect the impact and role of mass media and social media marketing on the way we perceive our world and our everyday lives on individual, social and societal levels, during these critical times. even with the help of the media, this pandemic has had worldwide repercussions and is not yet controlled in some countries. a study was carried out on hcws at king khalid university hospital (kkuh), riyadh, saudi arabia, showed that the majority of hcws had mild anxiety from covid- [ ] . however; the survey was conducted before registering any case of covid- in saudi arabia. an important finding in the present study was that a high level of concern about covid- pandemic was prevalent across the different concern domains. the highest level of concern was observed in the hcws' responses to questions regarding fears of infection of a family member, fears of being in public places that may result in infection, the closure of schools and workplaces in the event of an epidemic and risks associated with dealing with a febrile patient, obligation of care provision for patients infected with covid- and government's action to implement the curfew and the movement restriction periods. it was interesting that in the present study, % agreed that school and shopping markets need to be closed, while only % during the previous mers outbreak [ ] . this finding may reflect the perception of hcws in our study of the magnitude of covid- pandemic. however, it is important to note that this perception of fear might differ from country to another. for example in japan with the absence for an epidemic during the sars-cov outbreak, more than % reported having a high level of fear and an anxiety of infection [ ] , while in thai study, nearly all hcws reported acceptance to take the risk of caring for h n patients [ ] . in line with the who recommendations for institutional preparedness to reduce the impact of potential outbreaks, mng-ha has developed a comprehensive plan of medical and public health response for covid- epidemic [ ] . this plan aimed at the protection of hcws through the implementation of strict infection control measures and personal protection practices. despite these efforts, hcws in our study did not feel safe at the workplace and felt at risk of contracting the infection. this finding is similar to a study in the uk in table comparison between the levels of concern about covid- and personal characteristics of healthcare workers in saudi arabia (continued) low concern (score = - ) moderate concern (score = - ) high concern (score = - ) living condition β beta coefficient, se standard error, t t statistics, *---reference category, **---significant association which % of the hcws did not feel confident in the healthcare system's ability to cope with bird flu epidemic [ ] .. the exact reasons of such high concern among hcws, despite the existence of a preparedness plan, cannot be determined from the current study and further studies are needed. our study shows that hcws who were in direct contact with patients had significantly higher concern scores than those who were not in direct contact. this finding was in agreement with the results of a study in china [ ] to compare the average values of fear, anxiety and depression due to covid- pandemic between medical and admin staff, where medical staff reported greater fear, anxiety and depression than administrative staff. this finding is not surprising given the higher perceived risk by those hcw due to the condition of the work environment. however it is important to pay special attention to those hcws to manage their perception of risk by ensuring that they have access to proper personal protective equipment (ppe) and safe patients' handling procedures [ ] . saudi hcws, in the present study, reported higher concern to covid- pandemic as compared to non-saudis. this can be explained by the culture norms and the difference in living conditions between saudis and non saudi hcw. the majority of non saudi hcw are expats who are likely to live alone with their family memebrs living in their home countries. therfore expats are less likely to worry about the risk of infecting their family members and loved one compared to saudi hcw who live with their families and tend to have a very active social life [ ] .. the present study also showed that living with others was an independent predictor of high level of concern about covid- infection, most likely due to their fear of transmitting the infection to others if they get infected. an interesting but a little counterintuitive finding of our study is the fact that older hcws were less concerned about covid- than the younger ones. this is especially true given that risk factors for severe disease and death in covid- include older age among many other factors [ ] . however, this finding could be attributed to the fact that oldest hcw's could not be working in direct contact to patients, due to the higher risk of severe disease. further, there was a significant association between higher concern score and lower education level. in a survey on the undergraduate medical students in medical institutes of karachi, the majority of students found worrisome of getting infected with covid- during medical rotations, dreaded insufficient care and inappropriate treatment if they acquire infection and thought their institute-associated hospital won't be able to handle the situation in case of an uncontrolled outbreak [ ] . one possible explanation can be inferred from the theory of reasoned action of a causal relationship between knowledge and experience and the subsequent positive perception and intention to change behavior [ ] attitudes and behavioral intent. in the current study hcws of western region had significantly higher concern score compared to other regions. this was different than the study during mers where the hcws of central region had higher concern than other regions [ ] . we believe that these differences are likely due to the perception of hcws of the magnitude of the pandemic in the different regions. during covid- , the western region had shown much rapid increase of confirmed cases compared to the other regions [ ] . additionally, the government has implemented complete lockdown of the western region prior to other regions. however during mers, the largest outbreak has taken place in the central region. the large magnitude of the epidemic the western region compared to other regions in the country could have contributed to the observed level of concern of hcws in this region. our study is not without limitation. our survey was based on self-reported information which might suffer from a recall bias. moreover, all study participants were hcws in tertiary hospitals, and therefore could limit the generalizability of the findings to other settings. finally, all identified predictors of concerns cannot be interpreted beyond general association. despite these limitations, our study addresses a major problem faced by hcws in many countries around the world during this pandemic. the current study highlights the high concern among healthcare workers about covid- and identifies the predictors of those with the highest level of concern. high level of concern could lead to suboptimal healthcare service as well as less effective management of covid- cases. this could be mitigated by implementing strategies designed to minimize perceived risk of infection by hcws. these strategies should be part of the early planning for a response to an epidemic and it should cover a wide range of programs that focus on financial incentives, education, personal counseling and education. supplementary information accompanies this paper at https://doi.org/ . /s - - - . additional file . a novel coronavirus from patients with pneumonia in china clinical features of patients infected with novel coronavirus in wuhan who director-general's opening remarks at the media briefing on covid- - coronavirus covid- global cases by the center for systems science and engineering clinical characteristics of covid- in saudi arabia: a national retrospective study saudi ministry of health dashboard. covid- . accessed th french doctor commits suicide after covid- diagnosis nyc emergency room doctor dies by suicide after treating covid- patients doctors in hubei received more than , consultations in one month. sudden death at home was not identified as a work-related injury hero's award honored for medics who passed away to covid- coronavirus doctor dies of heart attack after working days straight physician deaths from corona virus (covid- ) disease the psychological impact of covid- pandemic on health care workers in a mers-cov endemic country global alert and response: novel mers-cov virus infections state of knowledge and data gaps of middle east respiratory syndrome mers-cov virus (mers-cov) in humans an assessment of the level of concern among hospital-based healthcare workers regarding mers outbreaks in saudi arabia concerns, perceived impact and preparedness in an avian influenza pandemic--a comparative study between healthcare workers in primary and tertiary care awareness, attitudes, and practices related to the swine influenza pandemic among the saudi public sars risk perceptions in healthcare workers impact of knowledge and positive attitudes about avian influenza (h n virus infection) on infection control and influenza vaccination practices of thai healthcare workers world health organization. the world health report -working together for health two thirds of doctors in uk say the nhs could not cope with bird flu epidemic psychological status of medical workforce during the covid- pandemic: a cross-sectional study european centre for disease prevention and control (ecdc)-technical report-infection prevention and control and preparedness for covid- in healthcare settings -third update a closer look at the middle eastern respiratory syndrome (mers-cov) outbreak in saudi arabia covid- : risk factors for severe disease and death concerns of undergraduate medical students towards an outbreak of covid- understanding and promoting aids-preventive behavior: insights from the theory of reasoned action publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations authors' contributions maa contributed to concept development, manuscript preparation and final writing, afa and aab contriputed to concept development and data collection, mah contributed to concept development statistical analysis and manuscript finalization, and afa and aab contributed to research proposal writing, data collection, analysis and interpretation, and manuscript drafting. all authors read and approved the final manuscript. none. most of the data supporting our findings is contained within the manuscript, and all others, excluding identifying/confidential patient data should, will be shared upon request. this study was approved by the institutional review board of the mng-ha in riyadh, saudi arabia (april , ; rc / /r). participation in this study was voluntary. those who agreed to participate signed a written consent form. hcws were assured in a written informed consent that their responses would remain anonymous and would not affect their performance evaluations, work status or compensations. not applicable. the authors declare that they have no competing interests.author details key: cord- -d jrn ip authors: van gageldonk-lafeber, arianne b; van der sande, marianne ab; heijnen, marie-louise a; peeters, marcel f; bartelds, aad im; wilbrink, berry title: risk factors for acute respiratory tract infections in general practitioner patients in the netherlands: a case-control study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: d jrn ip background: acute respiratory tract infections (arti) are an important public health problem. improved identification of risk factors might enable targeted intervention. therefore we carried out a case-control study with the aim of identifying environmental risk factors for arti consultations in the dutch general population. methods: a subset of patients visiting their gp in the period of – with an arti (cases) and age-matched controls (visiting for other complaints) were included in a case-control study. they were asked to complete a questionnaire about potential risk factors. conditional logistic regression was used to calculate odds ratio's (or) and % confidence intervals (ci) to estimate the independent effect of potential risk factors. results: a total of matched pairs of case and control subjects were enrolled. exposure to persons with respiratory complaints, both inside and outside the household, was found to be an independent risk factor for visiting a gp with an arti (respectively or(adj )= . and or(adj )= . ). participants exposed to dampness or mould at home (or(adj)= . ) were significantly less likely to visit their gp. in accordance with the general risk of consultations for arti, participants with a laboratory-confirmed arti who were exposed to persons with respiratory complaints outside the household were also significantly more likely to visit their gp (or(adj)= . ). conclusion: this study confirmed that heterogeneity in the general population as well as in pathogens causing arti makes it complicated to detect associations between potential risk factors and respiratory infections. whereas it may be difficult to intervene on the risk posed by exposure to persons with respiratory complaints, transmission of arti in the general population might be reduced by improved hygienic conditions. acute respiratory tract infections (arti) are an important public health problem. worldwide they are responsible for considerable morbidity and mortality, and lead to an increase in absence from work and school and an increased number of consultations with clinicians [ ] [ ] [ ] [ ] [ ] [ ] . based on a population of million individuals, it is estimated that, in the netherlands, almost , persons annually visit their gp for an arti [ ] . although antibiotics are effective against bacteria, many arti are caused by a multitude of other micro-organisms, mainly viruses [ , , [ ] [ ] [ ] [ ] . the development of an effective universal treatment for arti is hampered by this great number of different pathogens causing arti as well as a diagnostic deficit of around % [ ] . symptomatic management therefore remains the only therapeutic option. the development of preventive initiatives to reduce morbidity and mortality, like vaccines or antiviral agents, is also hindered by the mixed pathogenesis [ , ] . another approach towards prevention is to intervene in risk factors contributing to respiratory infections. identification of these risk factors may be useful for efforts to interrupt transmission. a multitude of studies have identified various risk factors for arti. studies in developing countries have identified risk factors to be among others crowding, nutritional factors, and parental smoking [ ] [ ] [ ] [ ] . because of major differences in living conditions and environmental circumstances these study outcomes can not directly be extrapolated to industrialised countries. studies in industrialised countries can roughly be divided into studies addressing children, and studies addressing specific respiratory diseases. known risk factors for children are for instance young age [ ] , environmental tobacco smoke [ , ] , home-dampness [ , ] , and attending day-care centres [ , , ] . risk factors noted in relation with specific respiratory diseases, like asthma, copd and tuberculosis, are active smoking, low socioeconomic status, occupational exposure and exposure to air pollution [ ] [ ] [ ] [ ] [ ] [ ] . however, little is known about the role of risk factors for arti in the general population in industrialised countries. to contribute to the present knowledge of artis we have carried out a case-control study with the aim of identifying environmental risk factors in the dutch general population. this study was performed in collaboration with the network of general practices in the continuous morbidity registration of the netherlands institute of primary health care (nivel). this network constitutes a representative group of about general practitioners (gps) in practices. their patient population accounts for approximately % of the dutch population and is representative with regard to age, sex, regional distribution, and degree of urbanization. from october through october all patients who presented with arti were classified by week of consultation and age to obtain stratified incidence rates. in addition in this period, almost half of the practices ( in - ; in - ; and in - ) , consented to participate in a case-control study [ ] . patients were classified by the gp's. inclusion criteria for cases were: consulting gp for acute respiratory complaints, and being diagnosed as influenza-like illnesses (ili) or another arti, and consulting gp for the first time in that episode. patients were not eligible for inclusion when they reported use of antibiotics or anti-viral medication in the last two weeks. for each case patient a control patient, matched by age group ( - , - , - , - , - and ≥ year), was recruited within week of consultation. inclusion criterion for controls was a consultation of the gp for complaints other than respiratory. exclusion criteria were: complaints of an arti in the last two weeks, belonging to the same household as the case, and the use of antibiotics or anti-viral medication in the last two weeks. from all participants, nose and throat swabs were obtained during the visit. furthermore, both cases and controls were asked to complete a detailed questionnaire about potential risk factors. these questionnaires extracted information about exposure to persons with respiratory complaints within or outside their household in the week before consulting the gp, family composition (number of children and adults, presence of children attending day-care, primary school or secondary education), working outside the home and kind of job, use of public transport, type of heating system, exposure to mechanical ventilation system, exposure to dampness or mould at home, keeping pets or cattle, smoking behaviour, and exposure to passive smoking (see additional file ). parents or guardians were asked to complete questionnaires for young children. to assess the burden of the arti, the questionnaires of case patients also extracted information about restriction of daily activities, bed rest, and absence from school or work. moreover, after the first and second year of study all gps participating in the casecontrol study were asked to complete a questionnaire for each reported case patient. these follow-up questionnaires obtained information about additional consultations, referral to specialists and hospitalisation for the reported episode of arti. viral culture and polymerase chain reaction (pcr) were performed at the national institute of public health and the environment. both nose and throat swabs were tested for adeno-, corona-, entero-, human metapneumo-(hmpv), influenza-, para-influenza-, rhino-, and respiratory syncytial (rs) virus, and also for m. pneumoniae, c. pneumoniae and c. psittaci [ ] [ ] [ ] [ ] . bacteriological cultures were performed at the regional public health laboratory in tilburg using the throat swabs to detect bacterial pathogens known to cause 'community acquired' respiratory infections [ ] . apart from the original matching at the selection of cases and controls, a secondary matching was done to match single cases and controls. first of all for every single case a control was sought in the same age group, the same practice and within a period of three months. if no control could be selected within the same practice, a control from a different practice was selected. associations with potential environmental risk factors were assessed by comparing all case patients with control patients matched by age. a sub analysis addressed only these case patients in whom at least pathogen was detected and their matched control patients. conditional logistic regression was used to calculate odds ratio's (or) and % confidence intervals (ci) to estimate the independent effect of potential risk factors. sex was included in the multivariate regression model in order to adjust for confounding. variables were excluded from the regression model if the crude or was not significant (p > . ). an exception was made for variables with respect to smoking because of the existing data on of adverse health effects of (passive) smoking [ ] [ ] [ ] . in total, nose and throat swabs were obtained from case patients and control patients. three hundred and thirty two ( ) originally matched pairs of case patients and control subjects could be included. after the secondary matching, an additional pairs could be formed, thus pairs were included in the matched analyses. characteristics of case patients and control subjects are summarized in table . viruses were detected in of the case patients ( %), β-haemolytic streptococci in ( %), and mixed infections in case patients ( %). influenza and rhino viruses were the most common pathogens. no pathogens were detected in of the case patients ( %). moreover, pathogens were detected in of the control subjects ( %). the percentage of nose and throat swabs obtained from case patients with arti and from control patients that tested positive for one or more pathogens, according to age group, is shown in figure . in general, the proportion of positive nose/throat swabs decreased with increasing age group for both case patients and control subjects. these data have been described in more detail previously [ ] . perceived restrictions in daily activities were reported by % of the case patients with a median duration of days. both bed rest and absence from school or work were reported by approximately % of the cases patients (median duration respectively and days). these findings apply for all case patients as well as for those with a laboratory-confirmed arti. a total of respectively and follow-up questionnaires were received after the first and second year of study. all questionnaires were included in the analyses, whether or not cases fulfilled the in-/exclusion criteria. in the first year of study of the case patients ( %) consulted their gp more than once for the same episode. for of these ( %) medication, like antibiotics, codeine, and prednisone, was prescribed at a later stage. three patients were referred to an ent specialist, two to a lung specialist and two to a paediatrician. three patients were hospitalized for an arti, of which two with tonsillitis and one with pneumonia. in the second year of study of the case patients ( %) consulted their gp more than once, of which ( %) were prescribed medication. two patients were referred to an ent specialist and three to a lung specialist. five patients were hospitalized for an arti ( tonsillitis and pneumonia). table presents the results of the univariate and multivariate risk factor analyses for all case patients consulting their gp for an arti compared with their matched control subjects. in the univariate risk factor analyses no significant association was observed for number of adults in the household, belonging to families with children attending day-care centres, working outside the home, use of public transport, type of heating system, keeping pets or cattle, and educational level. therefore these potential risk factors were not included in table . exposure to persons with respiratory complaints, both inside and outside the household, was found to be an independent risk factor (respectively or = . and or = . after adjustment for other relevant factors). persons who reported to be uncertain about their exposure were significantly more prone to visit their gp with complaints due to arti then persons without such exposure (or adj = . ). participants belonging to families with children in secondary education had a significantly lower risk of consulting a gp with arti (or adj = . ). in addition, participants exposed to dampness or mould at home were significantly less likely to visit their gp with an arti (or adj = . ). apart from the overall smoking behaviour we also looked at the extent of reported smoking. the participants were divided into five classes based on the daily number of smoked items (including cigarettes, cigars and/of pipes). we compared non-smokers with participants smoking < , - , - , and ≥ items daily. participants reporting smoking less than items a day had a significantly higher risk of consulting a gp (or adj = . [ . - . ]) compared with non-smokers. in contrast, among participants smoking of or more items a day, the risk of a consultation for arti was not significantly different from that of nonsmokers (or = . [ . - . ], . [ . - . ] and . [ . - . ] when the daily number of smoked items is respectively - , - and ≥ ). table presents the results of the univariate and multivariate risk factor analyses for a sub analysis of case patients with a laboratory-confirmed arti (i.e. ≥ pathogen detected) and their matched control patients. in the univariate risk factor analyses no significant association was observed for number of adults in the household, belong-ing to families with children attending day-care centres, primary or secondary school, working outside the home, use of public transport, type of heating system, keeping pets or cattle, and educational level. therefore these potential risk factors were not included in table . in line with the general risks of consultations for arti, participants with a laboratory-confirmed arti who were exposed to persons with respiratory complaints outside the household were also significantly more likely to visit their gp (or adj = . ). also persons with a laboratory-confirmed who reported to be uncertain about their exposure were significantly more prone to visit their gp then persons without such exposure (or adj = . ). in contrast, exposure to persons with respiratory complaints inside the household and exposure to passive smoking as well as to dampness or mould at home were not significantly associated with laboratory-confirmed arti, while being associated with consultations for complaints due to any arti. with respect to the extent of smoking we again found that participants smoking less than items a day were at higher risk for arti with a microbiological pathogen identified (or adj = . [ . - . ]) compared with non-smokers. for participants smoking or more items a day this was not significantly different from that of nonsmokers (or = . to our knowledge, this is the first case-control study investigating the role of risk factors for arti in the general population in an industrialised country. our study showed that exposure to persons with respiratory complaints, both inside and outside the household, is a risk factor for consulting a gp with an arti. only exposure outside the household appears to be a risk factor for a laboratory-confirmed arti. while it may be difficult to intervene on this risk factor, transmission of respiratory infections might be reduced by improved hygienic conditions. considering the substantial morbidity it is worthwhile to investigate number of nose and throat swabs in case patients and con-trol subjects that tested positive for one or more pathogens, by age group figure number of nose and throat swabs in case patients and control subjects that tested positive for one or more pathogens, by age group. * number of matched case-control pairs. the feasibility and effect of intensified hygiene on illness transmission in the general population. this study adds that risk factors found in specific study populations can not be extrapolated to the general population. we demonstrated that in the general population (passive) smoking, dampness or mould at home and having family members attending day-care were not associated with a higher risk for arti, which is in contrast with studies carried out in children or patients with specific respiratory diseases. the pathogenesis of arti involves a complex interplay between pathogens and the host's inflammatory response [ , ] . this complicated mechanism in combination with a diagnostic deficit of over % [ ] might account for differences between risk factors found in specific study populations and in the general population. in contrast with our findings in the general population, many studies in children concluded home dampness to be associated with increased respiratory symptoms [ , , , ] . knowledge about the mechanisms behind the association between dampness and health effects and the effect of moulds on the immune system is still limited [ , ] . it cannot be excluded that the presence of dampness or mould at home in our study, based on selfreported questionnaire-data, is not observed in enough detail to assess the relation with arti in the general population. several studies describe an association of cigarette smoking or exposure to environmental tobacco smoke with the occurrence and severity of arti [ , , [ ] [ ] [ ] . smoking is believed to exacerbate respiratory diseases by harming respiratory defence mechanisms [ ] . nevertheless, in this study smoking or exposure to passive smoking was not a risk factor for consulting a gp with an arti nor for consulting with a laboratory-confirmed arti. it is possible that, selection bias is part of the explanation for this finding. smokers might be less inclined to visit a gp with respiratory complaints as compared with non-smokers. moreover, our findings are based on questionnaire-data about exposure, which are often prone to recall bias. questionnaires are relatively cheap and easily used, but are likely to be less valid and reliable then the measurement of biomarkers in body fluids for true exposure [ ] . furthermore, awareness of the adverse health effects of passive smoking has increased substantially in the past years because of extensive information. this might have resulted in lower exposure levels, explaining the lack of association with arti in the general population. a recent study in school children also concluded that smoking by a care-provider was not significantly associated with respiratory infections [ ] . looking at the intensity of reported smoking, only moderate smoking (less than items a day) was a risk factor for consulting a gp with a (laboratory-confirmed) arti in our study. the intensity of smoking is only a rough indication based on questionnaire-data, without taking into account the number of years smoking. recall bias with regard to the extent of exposure may certainly play a part as well. besides, it cannot be ruled out that the extent of smoking is related to care-seeking behaviour. therefore, this finding must be interpreted with caution. several studies noted attendance to day-care centres to increase the risk of upper respiratory symptoms in young children [ , ] . it is plausible that day-care centre children would transmit respiratory infections to their family members [ , ] . nevertheless, we find that having family members attending day-care, was not associated with a higher risk in the general population for consulting a gp with an arti. this might be an underestimation of the real number of patients with an arti, because an unknown proportion of subjects with an arti will not visit a gp. selection bias could also play a role, persons with family members attending day-care might be less inclined to visit a gp with respiratory complaints. some limitations of our study should be mentioned. first the study was carried out in a period of moderate influenza-activity, affecting the number of consultations for ili as well as other arti [ ] . as a consequence of a limited sample size, risk factors could not be analyzed in relation to the separate pathogens, but only to the entity of arti. secondly, questionnaire data were used to measure exposure to potential risk factors. this could be less reliable compared with observational data. moreover, heightened attention to the cause of their complaints by patients with an arti may have caused recall bias. thirdly, we cannot rule out the possibility that controls were in the incubation period for an arti, even though they had no respiratory complaints. we expect the number of controls developing those infections to be low, because the gp's actively asked for the presence of respiratory symptoms at the moment the nose and throat swabs were taken. nevertheless, this might have diluted the investigated relations between potential risk factors and arti. this study showed that heterogeneity in the general population as well as in the pathogens causing arti in combination with the diagnostic deficit makes it complicated to observe associations between potential risk factors and respiratory infections. the risk factor found in this study, exposure to persons with respiratory complaints, can hardly be avoided. yet better understanding of disease transmission might result in improved hygienic conditions, like hand washing more frequently, and so affect the transmission of arti in the general population. increasing awareness of the importance of hygienic measures with regard to prevention and control of arti will also be useful facing a potential pandemic threat, assuming that the route of transmission of the pandemic virus is similar to that of other respiratory viruses. the 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and wales, and in the netherlands the author(s) declare that they have no competing interests. ag participated in the coordination of the study, performed the statistical analyses and drafted the manuscript. ms participated performing the statistical analyses and drafting the manuscript. m-lh participated in the design and coordination of the study. mp participated in the design and coordination of the study, and was responsible for the bacteriological assays. ab participated in the design and coordination of the study, and headed the network of general practices. bw participated in the design and coordination of the study, and was responsible for the virological assays. all authors read and approved the final manuscript. the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -zfvzxlj authors: yu, jianxing; xie, zhengde; zhang, tiegang; lu, yanqin; fan, hongwei; yang, donghong; bénet, thomas; vanhems, philippe; shen, kunling; huang, fang; han, jinxiang; li, taisheng; gao, zhancheng; ren, lili; wang, jianwei title: comparison of the prevalence of respiratory viruses in patients with acute respiratory infections at different hospital settings in north china, – date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: zfvzxlj background: acute respiratory infections (aris) are a great public health challenge globally. the prevalence of respiratory viruses in patients with aris attending at different hospital settings is fully undetermined. methods: laboratory-based surveillance for aris was conducted at inpatient and outpatient settings of hospitals in north china. the first – patients with aris were recruited in each hospital weekly from through . the presence of respiratory viruses was screened by pcr assays. the prevalence of respiratory viruses was determined and compared between patients at different hospital settings. results: a total of hospitalized cases and outpatients/emergency department (ed) patients were enrolled. the most commonly detected viruses in the hospitalized cases were respiratory syncytial virus (rsv, . %) in children less than two years old, adenoviruses ( . %) in patients – years old, and influenza viruses (ifvs, . %) in patients ≥ years. ifvs were the most common virus in outpatient/ed patients across all age groups ( . %). after controlling for the confounders caused by other viruses and covariates, adenoviruses (adjusted odds ratio [aor]: . , % confidence interval [ % ci]: . – . ) and rsv (aor: . , % ci: . – . ) were independently associated with increased hospitalization in children, as well as adenoviruses in adults (aor: . , % ci: . – . ). additionally, co-infection of rsv with ifvs was associated with increased hospitalization in children (aor: . , % ci: . – . ). conclusions: a substantial proportion of aris was associated with respiratory viruses in north china. rsv, adenoviruses, and co-infection of rsv and ifvs were more frequent in hospitalized children (or adenoviruses in adults), which might predict the severity of aris. attending clinicians should be more vigilant of these infections. electronic supplementary material: the online version of this article ( . /s - - - ) contains supplementary material, which is available to authorized users. acute respiratory infections (aris) are a major global public health problem because of their high morbidity and mortality [ ] . they represented . billion upper respiratory tract infections (urtis), million lower respiratory tract infections (lrtis) and . million deaths in the global burden of diseases estimates in [ , ] . in a substantial proportion of patients, aris are found to be associated with respiratory viruses (rvs) [ , [ ] [ ] [ ] . in addition to their high frequency, infection with rvs can lead to severe outcomes, including hospitalization and death [ ] . notably, respiratory syncytial virus (rsv) causes most of the severe lrtis in young children [ ] , while influenza viruses (ifvs) and human rhinoviruses (hrvs) are the predominant causative agents in hospitalized adults with pneumonia [ ] . shortly after the sars events in [ ] , regional and nationwide laboratory-based surveillance studies for aris were conducted in china to help clarify the epidemiological feature of rvs by employing the highly sensitive modern molecular techniques, such as polymerase chain reaction (pcr) [ ] [ ] [ ] [ ] [ ] [ ] [ ] . these studies are very helpful for characterizing the prevalence and burden of specific rvs in pre-vaccine chinese populations (i.e., vaccines against streptococcus pneumoniae, haemophilus influenzae type b, and influenza viruses are currently not incorporated into the national immunization program [ ] ). in addition, these studies are useful to identify new emerging viral infections [ ] [ ] [ ] [ ] . however, few previous studies have studied rvs across different hospital settings (e.g., outpatient, emergency room, inpatient wards and intensive care units) and explored etiological, environmental and host factors that predisposed patients to severe disease presentation, such as hospitalization or intensive care admission. it is very important to clarify the most common viral agents causing aris and their relationship with the severe disease presentation for clinicians who treated their patients with aris at different hospital settings. the purpose of the study was to determine the prevalence of rvs in patients with aris at different hospital settings and to identify factors that were most likely associated with hospitalization due to aris. the patients were enrolled from january , through december , at hospitals (additional file ) in north china according to the following criteria: ( ) had symptoms of acute infection, defined as fever (a body temperature > . °c) or hypothermia (a body temperature < . °c), chill, or leukocytosis (a white blood cell count > , /ml) or leukopenia (a white blood cell count < /ml); ( ) had signs/symptoms of acute respiratory illness, defined as sore throat, runny nose, cough, sputum-production, shortness of breath, wheezing or crackles, or chest pain; and ( ) with or without radiograph abnormality. patients were screened for enrollment eligibility by attending physicians of the hospitals in the following settings: emergency department (ed), outpatient (internal medicine, pediatric, respiratory or febrile illness clinics) and/or inpatient (the respiratory medicine ward or the intensive care unit). the first two to five eligible patients (children and adults) in each hospital were enrolled each week via convenience sampling. enrollment was conducted simultaneously in both inpatient and outpatient/ed settings in four hospitals. five hospitals enrolled patients in outpatient/ed settings and two hospitals enrolled patients in inpatient settings. to avoid duplicated inclusion, patients who were referred from other hospitals or not initially diagnosed in our hospitals were excluded. the study is observational and did not intervene with the choice of clinical management, namely hospitalization or ambulatory care. respiratory specimens (nasopharyngeal swab or aspirate, and sputum), regardless of the clinical severity, were regularly collected by physicians as quick as possible after patient enrollment. bronchoalveolar lavage or pleural puncture fluid were collected upon physicians' orders. the collected respiratory specimens were stored immediately in viral transportation media (vtm, copan, brescia, italy) at − °c at the hospitals before being transported to the central laboratory for viral screening. demographic and clinical information were collected from each enrolled patient via a standardized case reporting form. total nucleic acids (dna and rna) were extracted from respiratory specimens using rna/dna mini-kits (qiagen, valencia, ca) or nucliens easmag (biomérieux, marcy i′ etoile, france), according to the manufacturers' instructions. the presence of ifvs (a, b and c), hrvs, parainfluenza viruses (pivs - ), rsv (a and b), human adenoviruses (hadvs), human bocaviruses (hbov), human metapneumovirus (hmpv), and human coronaviruses (hcov- e, oc , nl and hku ) were screened by reverse-transcriptase polymerase chain reaction (rt-pcr) and pcr assays as described previously [ ] . the prevalence (or detection rate) of viruses was calculated by dividing the number of positive cases by the total case numbers tested for that virus. the % binomial confidence interval ( % ci) for detection rates were calculated by the pearson-klopper method. to compare variables between subgroups of patients, we used chi-square tests or fisher's exact tests for categorical variables and wilcoxon rank-sum or kruskal-wallis tests for continuous variables as appropriate. two-sided p value of < . was considered statistically significant. to explore factors that predispose patients to hospitalization, multivariable logistic-regression modeling was used. the dependent variable was hospitalization (hospitalized = , outpatient/ed patient = ). the independent variables were the presence or absence of specific viral pathogen (identified = , unidentified = ). to assess and quantify the contribution of a specific virus without regard to the presence of other pathogens, this model includes all tested respiratory viruses as well as their two-way interactions. in this case, the effect of co-infection upon hospitalization of aris, assigned as interaction term of pairs of viruses, could be explained as the excessive increment/decrement of hospitalization that could not be explained by additive effects or confounding of either composite virus. variables (e.g., age, gender, surveillance year, and season of illness onset), were also included in multivariable model to account for potential confounding if significant at p < . in bivariate analysis. since we hypothesized that the prevalence of rvs was disproportionately affected by the age of patients, multivariable models were constructed for patients of all ages, children aged - years, adults aged - years and elderly aged ≥ years. the results of multivariable analyses were exhibited as adjusted odds ratio (aor), namely the exponential of model coefficients. as we included a large number of cross-product terms in our model, type i errors and over-fitting will be significant problems. we used % ci for aors instead in the multivariable analyses. all statistical analyses were conducted using r version . . (r foundation for statistical computing, vienna, austria) [ ] . in total, patients were enrolled from the hospitals. of these patients, ( . %) were hospitalized cases and ( . %) were outpatient/ed patients. the median age of hospitalized cases was . years (interquartile range [iqr]: . - . years), which was much lower than the median age of outpatient/ed patients ( . years, iqr: . - . years) (p < . ). compared with outpatient/ed patients, the hospitalized cases were more frequently male ( . % vs. . %, p < . ), children less than five years old ( . % vs. . %, p < . ) and elderly patients ≥ years ( . % vs. . %, p < . ) ( table ) . of all patients, ( . %) were urtis and ( . %) were lrtis. compared with outpatient/ed patients, the hospitalized cases were diagnosed more often with lrtis ( . % vs. . %, p < . ) and pneumonia ( . % vs. . %, p < . ), but less often with urtis ( . % vs. . %, p < . ). moreover, some of clinical sign/symptoms were more frequent among the hospitalized cases than the outpatient/ed patients, including cough, wheezing/crackles, tachypnea, dyspnea, anemia, and leukocytosis (p < . for each symptom). in contrast, fever of > . °c, malaise, headache, rhinorrhea, and sore throat (p < . for each symptom) were more frequent among the outpatient/ed patients. at least one virus was detected in ( . %) hospitalized cases and ( . %) outpatient/ed patients (p = . ). the overall detection rates of rvs varied disproportionately according to age of patients and hospital settings. the highest rates of rv infection were in children under two years of age ( . % and . % in hospitalized and outpatient/ed patients, respectively; p < . ) and the lowest rates of rv infection were in patients ≥ years ( . % and . % in hospitalized and outpatient/ed patients, respectively; p < . ) ( table ). the prevalence of specific rvs was significantly higher (p < . ) in hospitalized patients compared to outpatient/ed patients for specific ages. for instance, rsv, hrvs, hadvs, pivs and hbov in young children and hadvs, pivs, hmpv and hcovs in young adults were more frequent in the hospitalized patients. the exception was ifvs, which was more frequent in outpatient/ ed patients ( fig. & table ). among the hospitalized patients, the viruses detected most frequently were rsv ( . %, n = / ) in children under two years, hadvs ( . %, n = / ) in patients - years, and ifvs ( . %, n = / ) in elderly patients ≥ years. however, among outpatient/ed patients the most common virus detected was ifvs across all age groups ( . %, n = / ). viral co-infection was detected in ( . %) cases, of which ( . %) were dual-infection, ( . %) were triple-infection, and ( . %) were co-infected by more than four different rvs. patients with more viruses detected were more likely to be hospitalized. specifically, patients infected by one, two, three, or more than four types of rvs were hospitalized . %, . %, . % and . %, respectively (p < . ). the hospitalized cases generally had more co-infections than outpatient/ed patients ( . % vs. . %, p < . ) ( table ). among the hospitalized cases, rsv co-infected with hrvs (rsv:hrvs) was the most common combination identified in children less than five years old ( . %, n = / ) and hadvs:pivs in patients five years and older ( . %, n = / ). among outpatient/ed patients, the most common combinations were ifvs:hrvs in children (fig. ). early acquisition of viral diagnosis is helpful for clinicians when treating patients with aris by decreasing unnecessary prescription of antibiotics, guiding appropriate use of antiviral agents, and preventing virus transmission in both outpatient and inpatient settings [ ] . the wide use of modern molecular techniques enables rapid diagnosis and greatly improves our understanding on the clinical role of respiratory viruses [ ] . the high sensitivity of these testing methods reveals multiple pathogens in respiratory specimens (about %- % of hospitalized children with aris had two or more viruses identified [ , , ] ). however, these results were often clinically confusing as it is difficult to distinguish whether the viruses were colonized or prolonged shedding from infection [ ] . a better understanding of the distribution of a specific rvs in patients with aris in different hospital settings and their interactive relationship with other microbial agents can help clinicians make a better assessment of the role of rvs in the current infections. toward this end, we simultaneously screened for the presence of eight common respiratory viruses in patients with aris in outpatient and inpatient settings at hospitals in north china. after adjusting for other viruses and significant confounding factors, the detection of rsv, hadvs, and co-infection of rsv with ifvs was still significantly higher in hospitalized children (or hadvs in adults) with aris than in non-hospitalized children. these data suggest that these infections might predict the severity of aris. rsv is a major viral agent identified in severe lrtis in young children [ ] . in our study, . % of hospitalized cases were diagnosed with lrtis, of which one third ( %) were young children. of these hospitalized young children, one third ( %) had rsv detected. these results are highly consistent with previous studies, in which rsv infections were implicated in about %- % of hospitalized children [ , , [ ] [ ] [ ] . in addition, in our study rsv was found more frequently among hospitalized young children than outpatients and was a significant predicator of hospitalization among young children with aris. this pattern is consistent with that of previous studies [ , ] . our results confirmed that rsv was still an important health threat to pediatric populations in north china. these data highlight the need to develop effective vaccines and new therapeutics to better treat rsv in north china [ ] . ifvs are important causing agents of aris [ , ] , and were reported to be the most common identified agent in hospitalized adults [ , , ] . in our study, ifvs was also the most common agent in hospitalized patient ≥ years. interestingly, co-infection of rsv with ifvs was a significant predicator of increased hospitalization among young children in our study. although ifvs were found more frequently among outpatient/ed patients ( . %) than among hospitalized cases ( . %), . % of children co-infected with rsv and ifvs were admitted to the hospital. this was significantly higher than children hospitalized with the single infection of rsv ( . %, n = / ) or ifvs ( . %, n = / ). based on these observations, we hypothesized that co-infection of ifvs with other microbial agents, like rsv in our study, might have played an important role in the hospitalization of aris in young children as has been described previously [ ] . both rsv and ifvs have caused substantial burden worldwide [ , , ] , and the epidemic season of rsv and ifvs are partly overlapping for winter months in china [ ] . if our hypothesis that co-infection of ifvs with other microbial agents will cause more severe aris among young children is true, the immunization of young children with seasonal influenza vaccine would certainly offer additional benefits by preventing not only ifvs-associated hospitalizations, but also the excessive hospitalizations caused by co-infected rsv or other microbial agents. since currently no influenza vaccines have been introduced into china's routine immunization program, vaccination of the elderly people and young children with seasonal influenza vaccine should be considered a high priority to reduce the large burden of aris among these population groups at high risk of more severe disease. in our study, viral infection was more common in inpatients than outpatients among younger age group but not older age group. this observation suggests that rvs infection is common among young children and is more likely to be associated with severe aris when compared with other age groups, while in adults and elderly people, though rvs infection is not uncommon, rvs are less likely to cause severe aris in this age group. moreover, we also found co-infection of two or more viruses in %- % of hospitalized young children as opposed to %- % of hospitalized adults. higher proportions of co-infection were observed in hospitalized young children than in non-hospitalized children ( %- %). these results suggest that viral co-infection might predict hospitalization of young children but not of adults. aging is a protecting factor for rvs infections in young children. as the age of children increased, infection and co-infection of rvs became less frequent and were less likely to be associated with hospitalization. this observation suggested that the frequent exposure of children to rvs could lower their risk of rvs-associated infection and/or severe infection, and that the utility of vaccines, when available, could be used to control and prevent the infection of rvs. the most frequent combination of viral co-infection among hospitalized children was rsv with hrvs, which is consistent with that of previous studies [ , , , , ] . in studies by papadopoulos et al. and aberle et al., co-infection of rsv with hrvs was associated with prolonged stay in the hospital in patients with bronchiolitis [ , ] . in our study, however, the strength of association with hospitalization for rsv co-infected with hrvs was at a marginal value (significant at p < . but not p < . ), which warrants investigation in future studies. in our study, hadvs were the most common viral agents among adolescents and young adults hospitalized with aris, which is consistent with previous studies [ , ] . in addition, hadvs in our study were more frequent in hospitalized children and young adults than in outpatient/ed patients. hadvs are important agents associated with severe aris, for several serotypes of hadvs (e.g., hadv- , , and ), might induce severe and fatal necrotizing pneumonia [ , [ ] [ ] [ ] . the host response to hadvs infection is very similar to that of invasive bacterial infections [ ] . in our previous study of the same population in north china, we found that % of identified hadvs were hadv- [ ] . we believe that certain serotypes of hadvs, e.g. hadv- , were more prevalent in this population, which played an important role in the hospitalization of children and young adults in our study. future studies which sought to sequence the identified hadvs strains are warranted. our study had limitations. first, we did not include an asymptomatic control in our study but rather made comparisons between patient subgroups at different hospital settings which might make the results difficult to interpret. yet finding no difference does not mean that there is no association of specific virus with the hospitalization of patients. however, the heterogeneous distribution of rvs in patient subgroups at different hospital settings deserved attention from clinicians when treating these patients. second, we only investigated way interactions of viruses. the interactions and bacterial-viral infections were not explored. third, other host factors were not examined in our study, such as those underlying diseases (e.g. exacerbation of chronic obstructive pulmonary disease, asthma, or cardiovascular diseases), and the previous usage of antibiotics or steroids. the issue of viral-viral co-infection associated with severity of aris still remains controversial as laid down by others [ , , ] . future studies that test multi-pathogens using common standards at multi-sites, employ appropriate community controls and use death as clinical endpoints are warranted. in conclusion, a substantial proportion of aris are associated with the infection or co-infection of rvs in north china. rsv, hadvs, and co-infection of rsv and ifvs were more frequent in hospitalized children (or adenoviruses in adults), which might predict the severity of aris. thus, clinicians treating patients with aris should be vigilant 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chinese adults with acute respiratory tract infection differential seroprevalence of human bocavirus species - in beijing prevalence and clinical characteristics of human respiratory syncytial virus in chinese adults with acute respiratory tract infection to save children's lives, china should adopt an initiative to speed introduction of pneumonia vaccines coxsackievirus a , enterovirus , and acute respiratory tract infection respiratory infection with enterovirus genotype c , china and mongolia human enterovirus genotype c wu and ki polyomavirus present in the respiratory tract of children, but not in immunocompetent adults r: a language and environment for statistical computing the management of communityacquired pneumonia in infants and children older than months of age: clinical practice guidelines by the pediatric infectious diseases society and the infectious diseases society of america comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory virus infections in children admitted to hospital with an acute respiratory illness single versus dual respiratory virus infections in hospitalized infants: impact on clinical course of disease and interferon-gamma response viral pneumonia the burden of respiratory syncytial virus infection in young children viral etiology of severe pneumonia among kenyan infants and children community-acquired pneumonia requiring hospitalization among u.s. children respiratory syncytial virus: coinfection and paediatric lower respiratory tract infections lower respiratory tract infection caused by respiratory syncytial virus: current management and new therapeutics influenza-associated hospitalizations in the united states beijing network for adult community-acquired p: viral etiology of communityacquired pneumonia among adolescents and adults with mild or moderate severity and its relation to age and severity the role of influenza in the severity and transmission of respiratory bacterial disease global burden of respiratory infections due to seasonal influenza in young children: a systematic review and metaanalysis the underrecognized burden of influenza in young children association of rhinovirus infection with increased disease severity in acute bronchiolitis the impact of dual viral infection in infants admitted to a pediatric intensive care unit associated with severe bronchiolitis detection of three human adenovirus species in adults with acute respiratory infection in china adenovirus serotype associated with a severe lower respiratory tract disease outbreak in infants in shaanxi province emergent severe acute respiratory distress syndrome caused by adenovirus type in immunocompetent adults in : a prospective observational study molecular identification and epidemiological features of human adenoviruses associated with acute respiratory infections in hospitalized children in southern china british thoracic society of standards of care committee. bts guidelines for the management of community acquired pneumonia in childhood adenovirus infection in children with acute lower respiratory tract infections in beijing, china does viral co-infection influence the severity of acute respiratory infection in children? clinical disease severity of respiratory viral co-infection versus single viral infection: a systematic review and meta-analysis we wish to thank all the patients, nurses, clinicians, and the laboratory, research, and administrative staff who took part in our study. the datasets used and/or analyzed during the current study are available from the corresponding authors on reasonable request.authors' contributions jy, zx, tz, yl, hf and dy contributed equally to this work. jw, lr, zx, zg, hf, jh, and tl designed the study. zx, tz, yl, hf, dy, ks, fh, jh, tl and zg collected samples and clinical information, and analyzed the data. jy performed statistical analysis. jy, lr and jw drafted the initial manuscript. lr and jw obtained funding and conceived the study. tb and pv commented and critically revised the manuscript for important intellectual content. all authors read and approved the final manuscript. not applicable. the authors have no conflicts of interest to disclose.• we accept pre-submission inquiries • our selector tool helps you to find the most relevant journal submit your next manuscript to biomed central and we will help you at every step: key: cord- -zn urrxc authors: chi, qiong; dai, xinjian; jiang, xiangao; zhu, lefei; du, junyan; chen, yuxi; zheng, jiyang; huang, jianping title: differential diagnosis for suspected cases of coronavirus disease : a retrospective study date: - - journal: bmc infect dis doi: . /s - - -y sha: doc_id: cord_uid: zn urrxc background: since december , the coronavirus disease (covid- ) has infected more than , , people and killed over , people worldwide. however, differential diagnosis remains difficult for suspected cases of covid- and need to be improved to reduce misdiagnosis. methods: sixty-eight cases of suspected covid- treated in wenzhou central hospital from january to february , were divided into confirmed and covid- -negative groups based on the results of real-time reverse transcriptase polymerase chain reaction (rt-pcr) nucleic acid testing of the novel coronavirus in throat swab specimens to compare the clinical symptoms and laboratory and imaging results between the groups. results: among suspected patients, were confirmed to covid- -positive group and were distinguished to covid- -negative group. patients with reduced white blood cell (wbc) count were more common in the covid- -positive group than in the covid- -negative group ( . % vs . %, p = . ). subsequently, correlation analysis indicated that there was a significant inverse correlation existed between wbc count and temperature in the covid- -positive patients (r = − . , p = . ), instead of the covid- -negative group. but reduced lymphocyte count was no different between the two groups ( . % vs . %, p = . ). more common chest imaging characteristics of the confirmed covid- cases by high-resolution computed tomography (hrct) included ground-glass opacities (ggos), multiple patchy shadows, and consolidation with bilateral involvement than covid- -negative group ( . % vs . %, p = . ; . % vs . % vs p = . ; . % vs . %, p = . ; respectively). the rate of clustered infection was higher in covid- -positive group than covid- -negative group ( . % vs . %, p = . ). through multiplex pcr nucleic acid testing, cases of influenza a, cases of influenza b, cases of adenovirus, cases of chlamydia pneumonia, and cases of mycoplasma pneumoniae were diagnosed in the covid- -negative group. conclusions: wbc count inversely correlated with the severity of fever, ggos, multiple patchy shadows, and consolidation in chest hrct and clustered infection are common but not specific features in the confirmed covid- group. multiplex pcr nucleic acid testing helped differential diagnosis for suspected covid- cases. since december , the epidemic of pneumonia caused by novel coronavirus in china, has continued to progress [ ] , having now infected more than , , people and killed over , people worldwide [ ] . on february , , the international committee on taxonomy of viruses officially named this severe acute respiratory syndrome coronavirus (sars-cov- ) and the world health organization (who) named the disease coronavirus disease (covid- ) [ ] . phylogenetic analysis revealed that sars-cov- falls into the genus betacoronavirus, which includes coronaviruses (sars-cov, bat sars-like cov, and others) discovered in humans, bats, and other wild animals [ ] . on march , , the who also designated covid- a pandemic [ ] . according to epidemiological investigations, the general population is susceptible to sars-cov- , which has the possible route of transmission via droplets, fecal matter, and contact [ ] . because symptoms overlap significantly with other respiratory infections like influenza, diagnosis remains difficult. wenzhou had hundreds of confirmed imported cases of covid- and even more suspected cases. measures to more rapidly and accurately diagnose suspected cases of covid- are challenges that urgently need to be addressed by clinicians. we therefore conducted this study to investigate the clinical characteristics of suspected cases of covid- and to improve the differential diagnosis for covid- , thus reduce misdiagnosis. we retrospectively collected the clinical data, including demographics, clinical manifestations, laboratory and radiological findings and contact history of suspected covid- cases in isolation ward (single rooms) of wenzhou central hospital from january , to february , . the diagnostic criteria [ ] of suspected cases were: individuals matching any one of the criteria for epidemiological history and any of the clinical manifestations, or individuals matching any of the clinical manifestations when there was no definitive epidemiological history. epidemiological history included ( ) history of travel or residence in wuhan within days before the disease onset; ( ) history of contact with patients confirmed with covid- within days before the disease onset; ( ) history of contact with individuals with respiratory symptoms who came from wuhan or communities with reported covid- cases within days before the disease onset; and ( ) clustered disease, meaning ≥ cases with fever and/or respiratory symptoms. clinical manifestations included ( ) fever, ( ) chest imaging showing multiple small patchy shadows and interstitial changes, particularly in the lung periphery, during the early stages, which progressed to multiple ground-glass opacities (ggos), infiltrates, and consolidation; and ( ) normal or reduced total white blood cell (wbc) count or reduced lymphocyte count in the early stages. criteria to confirm or rule out the diagnosis of covid- was as follows [ ] : confirmed covid- cases: positive real-time reverse transcriptase polymerase chain reaction (rt-pcr) sars-cov- nucleic acid testing or viral gene sequencing showing high sequence homology to known gene sequences of sars-cov- ; covid- -negative cases: suspected cases with consecutive negative results of respiratory pathogen nucleic acid testing (sampling time interval at least day). in the same period, these samples were analyzed by multiplex pcr named gexp assay (multiplex pcr combined with automated capillary electrophoresis) for common respiratory pathogens including influenza a (flu a), influenza b (flu b), influenza a h n pdm ( h ), influenza h n (h ), human para-influenza virus (hpiv), respiratory syncytial virus (rsv), rhinovirus (hrv), adenovirus (adv), human metapneumovirus (hmpv), human bocavirus (hbov), human coronavirus (hcov), chlamydia (ch) and mycoplasma pneumoniae (mp) [ ] . characteristics of patients were summarized using descriptive statistics. the continuous variables were presented as mean ± standard deviation (mean ± sd), and the comparison between groups was analyzed by independent sample t-test. categorical variables were expressed as the counts and percentages of patients in each category, and the group comparisons were performed using chi-square test or fisher's exact test or chi-square. p < . was considered statistically significant. the spss . software (ibm spss inc., chicago, il) was used for statistical analysis in this study. sixty-eight suspected covid- cases were recruited retrospectively to our study from january to february , . among them, were confirmed to be covid- positive and were covid- negative. the clinical symptoms were no difference between two groups (table ) . laboratory tests, chest imaging, and nucleic acid testing are shown in table . patients with reduced wbc count were more common in the confirmed covid- group than in the covid- -negative group ( . % vs . %, p = . ). subsequently, correlation analysis indicated that there was a significant inverse correlation existed between wbc count and temperature in the covid- -positive patients (r = − . , p = . ), instead of the covid- -negative group (fig. ) . but reduced lymphocyte count was not found to be significantly different between the two groups ( . % vs . %, p = . ). more common chest imaging characteristics of the confirmed covid- cases by high-resolution computed tomography (hrct) included ggos, multiple patchy shadows, and consolidation with bilateral involvement than covid- -negative group ( . % vs . %, p = . ; . % vs . % vs p = . ; . % vs . %, p = . ; respectively). bronchial wall thickening ( . %) and reversed halo signs ( . %) only saw in chest hrct of the covid- -negative group. ( . %) sars-cov- nucleic positive were identified in the first test of rt-pcr. . % patients appeared negative results in the first round of test but turned to positive in the second round of test. among the covid- -negative cases, patient ( . %) had a weakly positive result in the first viral nucleic acid test, but had negative results in the two following re-tests. among the patients in the covid- -negative group, multiplex pcr testing showed ( . %) cases of influenza a with characteristic scattered and patchy shadows and nodular shadows in both lungs ( fig. h & i) . no co-infections was observed in the covid- -positive or covid- -negative patients. the rate of clustered infection was higher in covid- -positive group than covid- -negative group ( . % vs . %, p = . ). the first familial cluster of covid- involved transmission from a wife (who visited a physician due to days of fever; she was confirmed to be sars-cov- positive in the second round of nucleic acid testing) to her husband (fatigue for a week and a day of fever; positive result on first sars-cov- nucleic acid test). chest hrcts showed a grid images in the inferior lobes of both lungs, especially obvious in the lung periphery ( fig. a & b) . in the second cluster, the wife had a positive result in the first sars-cov- nucleic acid testing after days of fever and her chest computed tomography (ct) showed multiple ggos near the bilateral pleura. her husband had a negative result in the first sars-cov- nucleic acid testing but a positive result upon the re-test on the first day of his fever and his chest ct showed a single ggo in the left lower lung near the pleura (fig. c & d) . the third familial cluster involved transmission from husband (fever and cough for days; positive result in the first sars-cov- nucleic acid testing) to his wife who remained asymptomatic (weak positive result in the first sars-cov- nucleic acid testing, and a positive result upon re-testing). the husband's chest ct showed multiple ggos and consolidation near the pleura of the right lung, while the wife's chest ct showed patchy shadows near the pleura in the right lung ( fig. e & f) . however, their son had no symptoms and normal wbc and lymphocyte counts with multiple ggos and patchy shadows in his left lung (fig. g) . after three negative viral nucleic testing of throat swabs or sputum, he was diagnosed covid- -negative. one familial cluster occurred in the covid- -negative group (father and son). both were diagnosed with mycoplasma pneumonia after multiplex pcr nucleic acid testing with hyperpyrexia and cough. there were two noninfectious cases in covid- negative group. one is -year-old previously healthy male patient who was diagnosed suspected case of covid- due to cough, fever, increasing chest tightness gradually and ggos and consolidation images in anterior basal segment of right lower lung of chest ct. finally, deep venous ultrasound showed right femoral vein thrombosis and computed tomography pulmonary angiogram (ctpa) showed multiple pulmonary embolisms in both lungs (fig. a & b) . his medical history showed often long-term sedentary position in last months for a test, and intermittent pain in his right lower extremity. the other suspected case had cough, (covid- ) negative group. a pneumonia caused by influenza a virus: scattered and patchy shadows and nodular shadows, with some of the nodular shadows surrounding the bronchovascular bundles; b pneumonia caused by influenza b virus: subpleural patchy shadows in the right lower lung; c pneumonia caused by adenovirus: consolidation near pleura of the right lower lung; and d chlamydia pneumonia: multiple groundglass opacities (ggos) and consolidations in both lungs fever, dyspnea and rashes symptoms with interstitial abnormalities in his both lungs (fig. c & d) . this patient was eventually diagnosed as dermatomyositis with pulmonary involvement through testing of the spectrum of idiopathic inflammatory myopathies as. as cases of covid- increase in number worldwide, clinicians are struggling to diagnose new cases quickly enough to implement appropriate isolation measures. this is particularly difficult given how closely symptoms of covid- match other common viral respiratory infections, including influenza. the aim of this study was to summarize the diagnostic features of suspected cases of covid- in our hospital to help improve differential diagnosis, reduce misdiagnosis in future. results of our study suggest that pneumonia in covid- patients and pneumonia caused by other pathogens (eg, influenza viruses, adenovirus, and mycoplasma) are difficult to distinguish based on their clinical manifestations, which included fever, cough, and fatigue in our study. rarer clinical manifestations, such as expectoration, sore throat, intolerance of cold, shivering, chest tightness, dyspnea, palpitation, and diarrhea, were also common to both covid- and other respiratory pathogens, which was similar to the results from previous studies [ , ] . routine blood tests of covid- -positive patients showed that the wbc count was reduced, inversely correlating with the severity of fever, instead of covid- negative patients. this may contribute to the differential diagnosis of suspected cases. approximately half ( . %) of covid- -positive patients had reduced lymphocyte count; therefore, a reduced lymphocyte count in suspected cases of covid- suggests the possibility of covid- . c-reactive protein of the covid- -positive patients was elevated, but was not significant for differential diagnosis. most covid- -positive cases had bilateral pulmonary involvement with ggos, multiple patchy shadows, and consolidation in their chest upon hrct imaging, which may be helpful for differential diagnosis. however, these chest hrct imaging including patchy shadows, nodular shadows and grid-like also seen in the pneumonia caused by influenza a virus, influenza b virus and adenovirus, consistent with the previous reports [ , ] . clustered occurrence is one of the epidemiological criteria for the diagnosis of covid- [ , ] . our study also shows out of cases were clustered, but clustering is not unique to covid- . mycoplasma pneumonia can also occur in cluster with ggos in chest ct. in this study, the patients diagnosed with mycoplasma pneumonia had a mean age of . years. three of the patients were younger than years old. bronchial wall thickening, characteristic change of mycoplasma pneumonia, in the chest hrct of young adults may help distinguish mycoplasma pneumonia from covid- . additionally, we recommend performing multiplex pcr nucleic acid testing using throat swabs or sputum. it should be noted that these results may be related to factors such as sampling quality, specimen preservation, and different nasopharyngeal virus concentrations at different stages of the disease [ ] . using multiplex pcr, we distinguish influenza a virus, influenza b virus, adenovirus, chlamydia pneumoniae, mycoplasma pneumoniae and so on from suspected cases easily. in this study, no patient with covid- was found coinfection with other respiratory pathogen(s). recent report showed that rate of co-infection between sars-cov- and other respiratory pathogens reached . % in northern california, usa. so, testing of sars-cov- should been done for patients with non-sars-cov- respiratory pathogens in high incidence of covid- region [ ] . the detection of non-sars-cov- respiratory pathogens by multiplex pcr may help to assess individual the risk of covid- in areas of low transmission [ ] . because of the highly infectious nature of sars-cov , the suspected covid- cases were all isolated and monitored in a single-person single-room isolation ward. although communication with healthcare professionals was limited, a detailed medical history should not be neglected. therefore, it is important to remain open to all causes of lung pathology, including non-infectious causes like pulmonary embolism. for suspected covid- cases, a comprehensive multidisciplinary collaborative diagnosis and treatment (mdt) mechanism should be established. relevant departments including respiratory medicine, infectious diseases, and radiology should collaborate closely when covid- is suspected to avoid misdiagnosis. positive result of sars-cov- nucleic acid testing remains the gold standard for the diagnosis of covid- . however, highly suspicious cases with false negative viral nucleic acid testing results should have chest ct and consecutive viral nucleic acid testing in different specimens collected from multiple regions of the body (eg, sputum, throat swabs, blood, urine, and feces) [ ] . these patients should also have the tests of serum sars-cov- specific-igm and igg antibodies [ ] to improve the diagnosis rate. this study has some limitations. because covid- was managed as class a infectious disease, this study only performed routine blood tests, c-reactive protein, chest hrct, throat swab sars-cov- nucleic acid testing, but not blood biochemical tests in the patients. as a result, we cannot comment on co-morbidities in our population. additionally, the number of cases in this study was limited by the fact that covid- is an emerging disease, and our findings need to be further verified by a large-scale study. the clinical characteristics of patients with confirmed diagnosis of covid- were similar to those negative cases. however, wbc count inversely correlated with the severity of fever, ggos, multiple patchy shadows, and consolidation in chest hrct and clustered infection are common but not specific features in the confirmed covid- group. multiplex pcr nucleic acid testing helped differential diagnosis for suspected covid- cases. clinical features of patients infected with novel coronavirus in wuhan who. situation report - : coronavirus disease (covid- who. situation report - : novel coronavirus ( -ncov a novel coronavirus genome identified in a cluster of pneumonia cases -wuhan clinical characteristics of coronavirus disease in china national health commission of the people's republic of china, diagnosis and treatment program of the novel coronavirus pneumonia a comparison study between gexp-based multiplex-pcr and serology assay for mycoplasma pneumoniae detection in children with community acquired pneumonia clinical findings in a group of patients infected with the novel coronavirus (sars-cov- ) outside of wuhan, china: retrospective case series clinical characteristics of patients with coronavirus disease in a non-wuhan area of hubei province, china: a retrospective study radiographic and ct features of viral pneumonia radiographics update: radiographic and ct features of viral pneumonia a novel coronavirus from patients with pneumonia in china a familial cluster of pneumonia associated with the novel coronavirus indicating person-to-person transmission: a study of a family cluster thoughts on the name and clinical diagnosis of the respiratory tract infection caused by the novel coronavirus rates of co-infection between sars-cov- and other respiratory pathogens trend of respiratory pathogens during the covid- epidemic zhejiang da xue xue bao yi xue ban antibody detection and dynamic characteristics in patients with covid- publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we would like to thank the medical staffs in the isolation ward of wenzhou central hospital for their providing information about patients. we also thank letpub (www.letpub.com) for its linguistic assistance during the preparation of this manuscript. authors' contributions qc, jz and jh conceived and designed the research. qc and xj analyzed data and wrote the manuscript. qc, xd and jz analyzed data and modified the paper. lz, xd, jd, yc, and jz collected patient samples. all authors read and approved the final manuscript. this work was supported by the wenzhou science and technology key problem program [grant number zy ]. the funders had no role in study design; in the collection, analysis, or interpretation of data; in the writing of the manuscript, or in the decision to submit the article for publication. the datasets of the current study are not publicly available due individual privacy of patients could be involved, are available from the corresponding author on request. this study was approved by the institutional review board (irb) of wenzhou central hospital (no. l - - ). written consent was waived by the irb as described previously. this study has been approved by the ethics committee of wenzhou central hospital. the data used in this study was anonymised before analysis. not applicable. the authors declare that they have no competing interests to disclose. key: cord- -anqvdi k authors: fischer, doris; schlößer, rolf l.; kempf, volkhard a. j.; wichelhaus, thomas a.; klingebiel, thomas; philippi, sabine; falgenhauer, linda; imirzalioglu, can; dahl, udo; brandt, christian; reinheimer, claudia title: overcrowding in a neonatal intermediate care unit: impact on the incidence of multidrug-resistant gram-negative organisms date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: anqvdi k background: overcrowding, reduced nurse to patient ratio, limited distance between incubators and absence of microbiological surveillance have been shown to promote spread of multidrug-resistant gram-negative organisms (mdrgn) in patients with birthweight < g. patients > g treated on an intermediate care unit are unrepresented in recent literature. we therefore intended to present data obtained from a short-term overcrowded neonatal intermediate care unit (nimcu) at a level iii (international categorization) perinatal center at university hospital frankfurt, germany. methods: during a day overcrowding (ov) and day post-overcrowding period (post-ov) on nimcu, epidemiological data obtained from continuously hold microbiological surveillance were investigated and compared to the last months of ward-regular bed occupancy preceding ov (prae-ov). results: during ov, the number of patients simultaneously treated at the nimcu increased from to , resulting in a reduced bed-to-bed space. nurse: patient ratio was : during ov compared to : during prae-ov. cumulative incidence of mdrgn was . % in ov and . % post-ov compared to . % to prae-ov, respectively, without any significant variations. during ov and post-ov, septic episodes due to mdrgn were not observed. in one case, potential nosocomial transmission of enterobacter cloacae resistant to piperacillin and rd/ th generation cephalosporins was observed. conclusions: prevention of nosocomial spread of mdrgn in an overcrowded nimcu is based on staff’s diligent training and adequate staffing. concise microbiological surveillance should be guaranteed to escort through overcrowding periods. in our setting, impact of bed-to-bed distance on mdrgn transmission seemed to be less strong. the rapid global spread of multidrug-resistant gramnegative organisms (mdrgn) is a serious global health risk, associated to several factors, e.g. traveling to high-prevalence countries (hpc) for mdrgn or contact to healthcare-systems in hpc [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . since mdrgn frequently cause community and healthcare-associated infections in almost all countries and medical disciplines [ ] [ ] [ ] , efforts to prevent infections caused by mdrgn should therefore not only address adequate usage of antibiotics and the development of strategies to prevent spread of mdrgn, but also on enhancement of the knowledge of mdrgn transmission, especially in hospital settings. overcrowding has been shown to promote the spread of the middle east respiratory syndrome coronavirus (mers-cov) in medical wards [ ] [ ] [ ] . overcrowding was mentioned as well as one major factor in the spread of severe acute respiratory syndrome (sars) due to sars-coronavirus (cov) in healthcare workers [ ] . transmission of methicillin-resistant staphylococcus aureus (mrsa) is also promoted by overcrowding [ , ] . in neonatal units overcrowding has also been shown to foster the spread of gram-negative bacteria and central venous lines (cvc) associated bloodstream infections [ ] . overcrowding therefore should be considered to promote transmission and spread of mdrgn on neonatal units. although overcrowding in medical wards should be impeded in order to prevent transmission of multidrug-resistant pathogens, it however might be inevitable sometimes [ , ] . due to inalienable reconstruction in june / july , number of simultaneously treated patients at the neonatal intermediate care unit (nimcu) at the university hospital frankfurt (uhf) increased under identical spatial conditions, resulting in an overcrowed nimcu. this study highlights epidemiological, microbiological and neonatological management in order to give insight of infection control in a short-term overcrowded nimcu. increase of hospital mortality has been attributed to overcrowding [ ] [ ] [ ] [ ] , however, no detailed definition has been formulated. we followed the definition by gordon et al [ ] . prae-ov was defined as the one-year-period prior to the overcrowding period with nimcu running under regular bed-occupancy. prae-ov was introduced to calculate the average incidence of mdrgn on nimcu which is the epidemiological benchmark for ov and the post-overcrowding observational period (post-ov), the latter being defined as the -day-period following ov. nosocomial transmission was given, if neonatal as well as the corresponding mother's mdrgn screening was negative on day of admission and the following neonatal mdrgn screening turned positive. vertical transmission was given, if baby and mother were tested positive for identical mdrgn species and phenotypical resistance pattern at any location. mdrgn have previously been defined as enterobacteriaceae with extended spectrum beta-lactamase (esbl)phenotype, and enterobacteriaceae, acinetobacter baumannii and pseudomonas aeruginosa resistant against piperacillin, any rd/ th generation cephalosporin, and fluoroquinolones +/− carbapenems [ , ] . the pediatric intensive care unit (picu) at uhf had been under construction from calendar week (cw) to cw in . given the nearly continuous maximally occupancy of nicu beds at any paediatric hospital in the rhine-main area, transferring neonates to other nicu/nimcus was less likely. therefore, all neonates meeting the entry criteria for nimcu were transferred from picu to nimcu at uhf. this period took days under which the ward was scheduled under overcrowded conditions. during ov, number of beds increased from n = to n = ; bed to bed-distance decreased to . - . m. considering that distance between nicu and nimcu beds has been recommended to be m at least [ ] [ ] [ ] [ ] , ov resulted in a infectiologically risky setting. detection of multidrug-resistant gram-negative organisms and molecular resistance analysis all laboratory testing procedures were performed under strict quality-controlled criteria (iso : standards) and as previsouly described [ ] . detection of genes encoding carbapenemases are routinely performed via pcr analysis and subsequent sequencing from carbapenem resistant enterobacteriaceae including the genes for carbapenemases genes ndm, vim, imp, oxa- , and kpc [ ] . identification of acinetobacter baumannii within the acinetobacter calcoaceticus -acinetobacter baumannii (acb) complex was done by a molecular in house procedure for detection of the species-specific carbapenemase oxa- . patients collective on the nimcu includes neonates needing special neonatal care as defined in recent literature [ ] and neonates needing standard paediatric care. on the herein described nimcu, nurse:patient ratio was intended to follow present recommendations. for patients, the shift teams were composed of at least board certificated neonatal experienced nurses and mostly trainee nurse. during ov, nurse:patient ratio was increased as described below. patient's entry criteria for nimcu were a stable circulatory constitution and respiratory status without the need of invasive ventilation neither non-invasive continuous positive airway pressure (cpap). at nimcu, continuous monitoring of heart-and breathing rate and oxygen saturation was performed. supplementary (high flow) oxygen was available. alimentation was performed with total or partial parenteral, nutrition via nasogastric tube, baby bottle or breast feeding. parenteral nutrition was given via central or peripheral venous lines. feeding of mothermilk and breastfeeding was preferred and supported. during ov and post-ov, no patient had either external liquor drainage or arterial inserted cannulas. breast feeding policy on the neonatal intermediate care unit nutrition with mother milk and breast feeding is a definitive goal at nimcu. staff team members and mothers were instructed and supported by an international board-certificated lactation nurse. in case of mdrgn positive mother, the mother was instructed to use a separate milk pump; her milk was stored in a separate fridge with the intention to avoid mdrgn transmission of potential mdrgn containing mother milk via external contamination of the mothermilk containing baby bottles. if the baby's condition allowed breastfeeding, it was performed like in mdrgn negative mothers. scheduled mdrgn screening of mothermilk was not done. all neonatal staff team members are being trained in hygiene management of neonates at least twice a year. additionally, one senior pediatrician at nimcu is designated as duty hygiene officer (dho) to supervise hygiene procedures. in any case of suspected increase of mdrgn incidence on the ward, an additional ward round was immediately held with staff of the institute for medical microbiology and infection control (immic) of uhf in order to evaluate hygiene procedures on the ward,. further advice or assistance was constantly given by staff of the immic. during ov and post-ov, no additional staff team training was provided. care of mdrgn colonized patient was provided by staff wearing gloves and gowns according to the current recommendations by the commission for hospital hygiene and infection prevention of the robert koch institute, berlin, germany (krinko) [ ] . in case of detection of enterobacteriaceae with extended spectrum beta-lactamase (esbl)-phenotype, and enterobacteriaceae, acinetobacter baumannii and pseudomonas aeruginosa resistant to piperacillin and any rd/ th generation cephalosporin or mrgn, the patient was not separated in single room. parents need not wear gloves and gowns but were trained in hands hygiene, repeatedly. in case of detection of an aforementioned species with additional resistance to fluoroquinolones and / or to carbapenems, patient was separated in single room or bed-to-bed distance was arranged to m at least. in these cases, parents were strictly advised to wear gloves and gowns. parents were encouraged to visit their neonate h a day. the total amount of visitors/baby was limited to two persons. at their first visit, all visitors received a short instruction of the hygienic rules performed by the attending nurse. microbiological surveillance for multidrug-resistant gramnegative organisms on the neonatal intermediate care unit as announced by the krinko in , microbiological screening for detection of multidrug-resistant organisms is recommended for patients on day of admission to nicu or nimcu as well as weekly routine screening for patients treated at nicu or nimcu in germany [ , ] . in order to intervene in case of any epidemiological shift exceeding the average mdrgn incidence on the ward, we furthermore set these findings in relation to the mdrgn incidence monthly calculated for the nimcu since january . patients admitted to the department of neonatology at uhf were screened for mdrgn, methicillin-resistant staphylococcus aureus (mrsa) as well as vancomycinresistant enterococcus faecalis/faecium (vre) by rectal and throat swabs. weekly regular infectious disease interdisciplinary rounds were co-hosted by staff of the immic as well as of department of neonatology in order to ensure continuous active surveillance on the nimcu. for whole genome sequencing (wgs), dna was isolated using the purelink genome dna mini kit (thermo fisher scientific, darmstadt, germany) from overnight cultures according to the manufacturer's instruction. wgs was carried out using an illumina nextera xt library with x bp paired-end reads on an illumina miseq instrument (illumina, san diego, ca, usa). the raw data was assembled using spades (version . ) ( ). the assembled contigs were analysed for multilocus sequence types using mlst . ( ) and acquired antibiotic resistance genes using resfinder ( ). average incidence of nosocomial mdrgn transmissions in the nimcu was calculated based on the monthly nosocomial mdrgn incidence of the last months of ward-regular bed occupancy (prae-ov). chi squared test was performed for statistical analysis. % confidence intervals ( % ci) for frequencies were calculated based on binomial distribution and used to confirm statistical significance. p-value calculations were not used to evaluate statistical significance as it has been criticized for low reliability [ ] . this study was a prospective observational study escorting a high risk period on a neonatal intermediate care unit. data were obtained and compared to pre-and post high risk period surveillance data. major endpoint was incidence of mdrgn; variables included nurse to patient ratio and bed-to bed-distance. based on the months period prior to ov, mean number of patients admitted to the nimcu was per month, defined as the basic occupancy. during prae-ov, the average number of nosocomial transmission of mdrgn amounted to per month, resulting in a cumulative incidence of . % ( %ci = . - . ). bed capacity was , nurse:patient ratio : . - (numeri integri - : ). under these conditions, bed-to-bed distance amounted to m. parent-child contact was supported with more than h of kangarooing/day. number of nimcu beds increased from to . this resulted in a decrease of bed-to-bed distance to . - . m; kangarooing-time could not be continuously provided. considering that the distance between bed-to-bed-distance is as been recommended to be m at least [ ] [ ] [ ] [ ] , ov was suggested to be a risk setting for transmission of mdrgn. in contrast to prae-ov, these patients were merged in one room. nurse:patient ratio was increased to : . - . ( - : ) in terms of an increased number of board certificated neonatal nurses taking care for all patients with special neonatal care. one additional neonatal trainee nurse was implemented into patients care for babies without the need for special neonatal care. in the following, only the numbers of experienced nurses are mentioned. numbers of ward physicians or consultants remained unchanged as well as teaching schedules for nurses or students. in total, patients with a mean birthweight of g (mean; range - g) were treated on the nimcu during ov. the ward occupancy therefore increased by almost % compared to the basic occupancy during prae-ov. treatment of / patients extended into post-ov. patients were tested positive for mdrgn (table ). in two patients (b and e; table ) potential nosocomial transmission of mdrgn was observed. nosocomial transmission of patient e is a particular case. mother of baby c was tested positive for enterobacter cloacae with resistance to rd/ th generation cephalosporins (e. cloacae ceph) prior to birth of her baby. she vertically transmitted the strain to her own child and additionally was involved in nosocomial transmission of e. cloacae ceph to baby e. this nosocomial transmission of e. cloacae ceph to child e was most likely associated to misbehavior of mother c as she provoked direct close skin-to-skin contact to baby e for several times. one week after skin contact between mother c and baby e, baby e was tested positive for e. cloacae ceph for the first time. resistance pattern of the e. cloacae ceph detected in baby e were identical to the e. cloacae ceph strains detected in mother c and baby c which might indicate bacterial transmission from mother c to baby e. in total, cumulative incidence of nosocomial mdrgn transmissions amounted to n = in patients, resulting in . % ( . - . ) in ov. this did not significantly differ from the cumulative incidence of nosocomial mdrgn transmissions during prae-ov ( . %; . - . ). in total, patients were treated on nimcu during post-ov. ward occupancy was almost %, slightly exceeding the basic occupancy during prae-ov. patients were tested positive for mdrgn (table ) , with two of them being residuals from ov (a and f), one pair of twins (h twin and h twin ) and two vertical transmissions (i and j) -similarly. nosocomial transmission of e. cloacae ceph (patient g; table ) occurred in one case. number of nosocomial mdrgn transmissions amounted to n = in patients, resulting in a cumulative incidence of . % ( . - . ). this did not significantly differ from the cumulative incidence of nosocomial mdrgn transmissions during prae-ov ( . %; . - . ) and ov ( . %; . - . ) . the enterobacter cloacae isolates from the transmission event were whole genome sequenced to investigate their identity. they depicted an identical new multilocus sequence type (allele variants depicted in table ), and carried both the ampc beta-lactamase bla act- and the fosfomycin resistance gene fosa, indicating a clonal spread. in this study, we implemented the available recommendations concerning neonatal mdrgn transmission into the daily life settings on nimcu in the scenario of a short-term and high risk overcrowding period. overcrowding has formerly been described as a promotor of nosocomial transmission for several pathogens [ ] [ ] [ ] [ ] [ ] [ ] ] . considering that the nimcu at uhf was facing a -day-overcrowding-period, we prepared in several ways for this precarious period. prior to ov the hygiene risk associated with this situation was diligently discussed with the nimcu staff. parents received fundamental information as well. nimcu staff underwent scheduled infection control training on e.g. correct hands hygiene during prae-ov, ov and post-ov. this training was held repeatedly and multidisciplinary by the dho and by staff of the immic of uhf. thus, these training units did not exceed the scheduled ones. low-threshold and broad access to all issues regarding aspects of infection control arising from ov and post-ov was provided. feedback was given immediately, precisely and constructively in any case, either in a positive or negative respect. incidence of mdrgn on our nimcu did not increase during the ov. we therefore assume our management consisting of active surveillance, staff training as well as adherence to infection control demands has proven to be a successful procedure to escort through overcrowding period and prevent the spread of multidrug-resistant organisms in hospital setting. nevertheless, according to other influencing factors like staff turnover and to maintain continuous alertness; training, bed side supervision and active surveillance have to be implemented as a substantial part in the care of this challenging patient group not only in special risk situations like overcrowding. recently published recommendations for perinatal centers [ ] suggest a nurse:patient ratio of : for neonatal intensive treatment patients, : for neonatal intensive monitored patients (e.g. those with non-invasive ventilation, cvc, pleural drainage) and : for neonatal special care. in nicus, caring of more than two patients by the same nurse increases the risk of nosocomial infections (ni) clearly [ , ] . ventilatory or circulatory support as well as necessity of cvcs or other invasive catheters is less likely in nimcu than in nicu patients implicating fewer patients to staff contact and less risk for ni. however, patients' immunological maturation is still ongoing and host defence against bacteria remains fragile [ ] . prematurity related apnoea-bradycardia-syndrome with the need of frequent tactile stimuli is one of the predominant diseases of the nimcu patients and regular care procedures are performed to times per day. moreover, ultrasound examinations are regularly provided and additional patient to staff contact is common e.g. because of teaching issues. the number of patient to staff contacts of an nimcu patient might therefore exceed the number during his nicu period. given this characteristics of a nimcu patient, the takeover of nicu recommendations for nimcu seems reasonable, but does not represent daily routine on nimcu. in our setting, we aimed to reach the nurse:patient ratio of : at least in the high risk overcrowding period. though we did not reach the recommended ratio regarding the total amount of nimcu patients, we were able to provide the : ratio in terms of one experienced board certificated neonatal nurse for four neonates receiving special care. as this staff management together with the microbiological surveillance mentioned above resulted in an unaltered mdrgn-transmission rate during ov, we were able to underline the pivotal role of adequate staffing on nimcus or nicus. an appropriate bed to bed distance seems reasonable to allow easy staff movements around the incubator without contaminating the opposite patient with non-airborne mre. the question, how narrow would be enough to prevent transmission is not examined, however, the literature recommend m bed to bed distance for nicus [ ] [ ] [ ] . moreover, if kangarooing should be provided, m space is needed to place deckchairs and to allow parents enough space. during ov, kangarooing was less likely and parental presence between two beds was limited. in this special setting, bed-to-bed distance of - . m seemed to be sufficient in terms of preventing mre transmission. our results also point out the impact of parental carriage of mdrgn. considering that to % of the adult rhine-main population is colonized with mdrgn [ , ] , we suggest that parents might serve a risk in terms of infection control. although pointed out by several information boards and easy accessible dispensers for hand disinfection, hospital visitors seem to be still less aware on the impact of hand hygienic discipline [ ] . limiting access for visitors but not for parents therefore seems reasonable. regular visits of parents with intense skin to skin contact (e.g. kangooroing) to their newborn child are inevitable for adequate bacterial skin and gut colonization [ ] . though, paternal mdrgn colonization will not be recognized as fathers are not screened and therefore serve as microbiological 'black box'. nevertheless, even if maternal mdrgn colonization is emerged, many questions may arise in terms of adequate preventive policy, e.g. the management of mother milk remain. data on incidence, time course, and outcome of mdrgn transmission by breast milk are not available and prospective studies will not be performed due to ethical reasons. thus, individual adjusted risk management, weighing the benefits of mother milk nutrition for the individual patient (e.g. reduced risk for necrotizing enterocolitis (nec)) against the probability of mdrgn transmission (either vertical or nosocomial) and potential biological hazard, especially in case of colonization with mdrgn, is recommended [ , , ] . the consideration whether mother milk and breast feeding could be the key incident in materno-fetal (and potential nosocomial) mdrgn transmission cannot be answered up to now and remains critical, particularly in high risk situations such as overcrowding. impressingly, our study demonstrated that language skills, intellectual or cultural causes or simple lack of time might lead to a violence of hygienic rules; however,, the final result might be nosocomial mdrgn transmission. the transmission of e. cloacae ceph between baby c and baby e has been verified via molecularbiological techniques. this transmission event shows that unexpected parental misbehaviore.g. due to language barrierscan counteract any attempt of infection control. this, however, is an immutable factor but shows all the more that infection control is a team game being never at rest [ ] [ ] [ ] . our results might be biased by the fact, that child-to-parent contact including kangooroing was reduced due to less space between beds. close skin-to-skin contact might increase the mdrgn transmission from parents to their newborn baby. given the baseline incidence of mdrgn carrier in germany, there is a theoretical risk for paternal-child bacterial transmission that was probably reduced by limitation of kangarooing during our ov setting. the key issues in prevention of nosocomial spread of mdrgn on nimcus during overcrowded periods of a nimcu seems to be an adequate nurse:patient ratio, staff training and concise microbiological surveillance which should be guaranteed to escort through overcrowding periods. in our setting, bed-to-bed distance might not have played a key role. we think that the role of parental mrdgn transmission, transmission via breast milk in particular, and their impact on infection control management on neonatal departments should be in the focus of future investigations. colonization with extended-spectrum beta-lactamase-producing and carbapenemase-producing enterobacteriaceae in international travelers returning to germany 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nosokomialer infektionen bei neonatologischen intensivpflegepatienten mit einem geburtsgewicht unter . g laboratory detection of enterobacteriaceae that produce carbapenemases strukturelle voraussetzungen der perinatologischen versorgung in deutschland risikocharakterisierung intensivmedizinisch behandelter frühund neugeborener und daten zur ist-situation in deutschen neonatologischen intensivpflegestationen spades: a new genome assembly algorithm and its applications to singlecell sequencing multilocus sequence typing of total-genome-sequenced bacteria identification of acquired antimicrobial resistance genes statistical errors high rate of transfer of staphylococcus aureus from parental skin to infant gut flora icu ward design and nosocomial infection rates: a cross-sectional study in germany recommended standards for newborn icu design recommended nicu design standards and the physical environment of the nicu prevalence of multidrug-resistant organisms in refugee patients, medical tourists and domestic patients admitted to a german university hospital handwashing adherence by visitors is poor: is there a simple solution? we thank christina gerstmann for excellent technical assistance. availability of data and materials the datasets used and analysed during the current study are available from the corresponding author on reasonable request. authors' contributions df and cr designed and conducted the study and wrote the manuscript; df served as principal investigator. cr, df, sp and ud carried out the study bed-side. cr, vk and ta were involved in the microbiological analysis. lf and ci did the molecular characterization of transmitted bacteria.rs, tk and cb did manuscript-proofreading and substantively revised it. all authors read and approved the submitted version of the manuscript. the study was performed in accordance with the ethical standards laid down in the declaration of helsinki and its later amendments. ethical approval was given by the ethics committee of goethe university frankfurt (file number: / ). not applicable. on behalf of all authors, the corresponding author states that there is no competing interest. key: cord- -qfvm x authors: maponga, brian a; chirundu, daniel; gombe, notion t; tshimanga, mufuta; shambira, gerald; takundwa, lucia title: risk factors for contracting watery diarrhoea in kadoma city, zimbabwe, : a case control study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: qfvm x background: kadoma city experienced an increase in watery diarrhoea from cases during week beginning (th) september, to cases during week beginning (th) september . the weekly diarrhoea cases crossed the threshold action line during week beginning (th) september at the children’s clinic in rimuka township, and the remaining four clinics reported cases crossing threshold action lines between week beginning (th) september and week beginning (th) september. eighty-two percent of the cases were children less than years old. we conducted a case controlstudy to determine risk factorsfor contracting watery diarrhoea in children less than years in kadoma city. methods: an unmatched : case control study was conducted in ngezi and rimuka townships in kadoma city, zimbabwe. a case was a child less than years old, who developed acute watery diarrhoea between (th) september and (st) october . a control was a child less than years old who stayed in the same township and did not suffer from diarrhoea. a structured questionnaire was administered to caregivers of cases and controls.laboratory water quality tests and stool test results were reviewed.epi info™ statistical software was used to analyse data. results: a total of cases and controls were enrolled. independent protective factors were: having been exclusively breastfed for six months [aor = . ; % ci ( . - . )]; using municipal water [aor = . ; % ci ( . - . )]; using aqua tablets, [aor = . ; % ci ( . – . )] and; storing water in closed containers, [aor = . ; % ci ( . – . . ). the only independent risk factor for contracting watery diarrhoea was hand washing in a single bowl, [aor = . ; % ci ( . – . )]. salmonella, shigella, rotavirus, and enteropathogenic escherichia coli were isolated in the stool specimens. none of the municipal water samples tested showed contamination with escherichia coli, whilst of ( %) shallow well water samples and of ( %) borehole water samples tested were positive for escherichia coli. conclusions: the outbreak resulted from inadequate clean water and use of contaminated water. evidence from this study was used to guide public health response to the outbreak. acute diarrhoea is defined as the passage of three or more loose or liquid stools per day, in a period not exceeding days. other types of diarrhoea include dysentery; and persistent diarrhoea which lasts days or longer. patients presenting with diarrhoea can also present with other symptoms such as vomiting, fever, and body weakness. diarrhoea causes loss of body fluid and electrolytes, which can result in dehydration. if dehydration is not corrected, death can result [ ] . the causes of diarrhoea can be bacterial, viral or parasitic. bacteria causes of diarrhoea include vibrio cholerae, escherichia coli, campylobacter jejuni, salmonellae and shigella species. viral causes of diarrhoea include rotavirus, adenovirus, and corona viruses. parasitic causes of diarrhoea include giardia, entamoeba, cryptosporidium and the helminthes (strongyloides, schistosoma) [ ] . diarrheal disease is a leading cause of child mortality and morbidity in the world, and mostly results from contaminated food and water sources.in developing countries, children below years experience on average episodes of diarrhoea every year. globally, there are about billion cases of diarrhoeal disease every year [ ] . fifteen percent of child deaths are directly attributable to diarrhoeal diseases [ ] . according to the world health organization (who), key measures to prevent diarrhoea include access to safe drinking-water, improved sanitation, exclusive breastfeeding for the first six months of life, good personal and food hygiene and health education about how infections are spread [ ] . in , kadoma city had an estimated , inhabitants. of these , were children less than years old based on the census projections [ ] . more than half the population resides in the city's rimuka township. nearly the whole population in kadoma city has access to piped water. however, demand for water is very high, reaching as high as mega litres per day, yet the city can only pump at best mega litres per day [ ] . the quantity of water pumped is further compromised by recurrent power cuts to the water treatment plants. in the absence of municipal water, residents use shallow wells and boreholes among others as water sources [ ] . kadoma city has had recurrent problems with watery diarrhoeal outbreaks since . in , a rotavirus outbreak killed children [ ] . a cholera outbreak in and affected , people, with deaths [ ] . another cholera outbreak in affected people and caused deaths [ ] . most of the problems, such as shortage of water, and recurrent sewer blockages have not had permanent solution due to lack of financial resources. in , kadoma city experienced an upsurge of watery diarrhoeal cases during week (week beginning th september), in ( figure ). the total cases reported by the city's five clinics doubled from to cases between week and . the cases further doubled from to between week and week . the weekly watery diarrhoeal cases at the under clinic crossed the action threshold line during week ( figure ). the action threshold was defined using the c cusum method (defined as the sum of the mean plus three standard deviations for preceding weekly diarrhea recordings, skipping two most recent weeks) [ ] . the diarrhoeal cases at the children's clinic increased fold, from to cases, between weeks and . the remaining four clinics (for all age groups) also experienced more than doubling of cases between week and week . initial analysis showed that about % of the cases were children less than five years old. the study was conducted to determine factors associated with contracting watery diarrhoea in children less than five years old. an unmatched : case-control study was conducted in kadoma city's rimuka and ngezi townships. the study was conducted among children less than years old. a case was defined as a child less than five years old who presented to a health facility with acute watery diarrhoea, with or without vomiting and other symptoms, between th september and st october , who had been resident in kadoma city for one week prior to onset of symptoms. a control was defined asa child less than five years old, who did not develop diarrhoeabetween th september and st october , and had been resident in kadoma city, one week prior to the interview. children whose caregivers agreed to participate were recruited into the study. children who had passed diarrhoea for more than days were excluded from participating in the study. epi info™ statistical software was used to calculate sample size. assuming % exposure in controls and % exposure in cases, using unchlorinated water, odds ratio of . [ ] , % confidence level, % power, and % refusal rate, we calculated a minimum sample size of ( cases and controls). multistage sampling of cases was done. rimuka and ngezi townships were selected because they were the most affected. using proportionate sampling, rimuka was to provide ( %) cases whilst ngezi was to provide ( %) cases. cases were selected from clinic line lists; using random numbers generated using microsoft™ excel . a control was selected from a household houses away from a case. where there was more than one child less than years old at the household, a child whose age was closest to the age of the case was selected. if there was no child less than years old, or if the caregiver declined to participate, the next house was selected till a less than years old was obtained. a pre-tested, interviewer administered questionnaire, was used to collect data from caregivers of cases and controls. the information collected included caregiver and case/control demographic information, symptoms, knowledge of caregiver on diarrhoea illness and treatment, duration of exclusive breast feeding, immunization status, the source of water, water treatment and storage, and sanitation facilities at the family home. the questions were in english and local shona languages. record review of water surveillance reports was done. the water quality parameters tested were bacteriological and chemical (chlorine content for municipal water, and ph). bacteriological water quality tests had been done using a delagua field testing machine. water samples that were found with zero colonies after incubation for hours were considered satisfactory. residual chlorine content was tested using the lovibond comparator using dpd tablets (n,n diethyl- . phenylenediamine sulphate) to check colour changes. specimens within the range . to . parts per million (ppm) were considered satisfactory. the ph was measured with the same equipment using phenol red tablets. specimens with ph ranging between . and . were considered satisfactory. the results were reported as proportions. data were analyzed using epi info™. means, proportions, frequencies, odds ratios and chi square tests at % significant levels were generated using the software. stratified analysis and forward step-wise logistic regression analysis was used to control for confounding and effect modification. permission to conduct the study was obtained from the local health authorities. ethical review was done at the health studies office, university of zimbabwe. written informed consent was obtained from all study participants caregivers. children or relative with diarrhoea found during interviews were referred to the nearest health facility for free treatment. a total of , cases of watery diarrhoea were attended to between th september and nd november . of these, ( %) were residents of kadoma city. the crude attack rate was per , people. five hundred and fifty two cases ( %) were females. six hundred and ninety three ( %) cases were children less than years old. the attack rate among children less than years old was per , people, whilst for those above years old, the attack rate was per , people. five cases, all children less than five years old died of diarrhoea. the crude case fatality rate was . %. one child died in hospital, and four children died at home after having sought treatment at health facilities. all the residential areas in kadoma city were affected. the highest risk of developing diarrhoea was in ngezi township, with an attack rate of per , people, followed by rimuka township, with an attack rate of per , people. however, during the early part of the outbreak, the highest risk of contracting diarrhoea was in rimuka township. figure shows the epidemic curve for kadoma city for the period st september to rd november . the outbreak commenced during the week beginning th september. the epidemic curve shows multiple waves with progressively taller peaks that are - days apart up to the st of october. the peaks became progressively shorter, until less than cases were attended to per day from rd october . a total of stool specimens were submitted to the local laboratory. thirty specimens yielded e.coli, yielded shigella species, yielded salmonella species and one yielded rotavirus. one out of the sixteen specimens submitted to the national virology reference laboratory tested positive for rotavirus. two of the specimens/isolates submitted to the national microbiology reference laboratory (nmrl) were confirmed as enteropathogenic escherichia coli. no cholera or cryptosporidium was isolated. all municipal water samples tested were below the threshold limit for contamination with escherichia coli. three of the ( %) borehole water samples and of the ( %) shallow well samples were above the threshold for contamination with escherichia coli. thirty-two ( %) of the municipal water samples tested for chlorine had chlorine levels within normal range. all the samples had ph levels within normal range. the samples were collected and analyzed between st september and nd october . a total of cases and controls were recruited into the study. there were no significant differences in the demographic characteristics of cases and controls as shown in table . less than % of cases and controls caregivers had received health education on diarrhoea six months prior to the outbreak. the knowledge on prevention and home treatment of diarrhoea was fair, and did not differ significantly between cases and controls as shown in table . table . statistically significant protective factors against contracting diarrhoea were: using municipal water source table . hand washing in a single bowl [aor = . , % ci ( . - . )] was an independent risk factor for contracting diarrhoea. independent protective factors against contracting diarrhoea were: using municipal water [aor = . , % ci ( . table . this study sought to establish risk factors for contracting watery diarrhoea among children less than five years old in kadoma city. the epidemic curve is typical of a propagated outbreak, highly suggestive of person-toperson transmission. the multiple peaks, to days apart, suggest the incubation period of the causative organisms to be between to days. the causative organisms that were isolated, (rotavirus, salmonella, shigella and escherichia coli) fit into the average incubation period of to days which is typical of faecal contamination [ ] . the importance of hand washing practices in preventing diarrheal illness is highlighted in this study. hand washing in a single bowl was found to be a risk factor for contracting watery diarrhoea. it is biologically plausible for infection to be spread from one person into the water, then to the next person. having a hand washing facility at home was found to be protective as the facility encourages run to waste method of hand washing, especially after using the toilet. these findings are supported by several studies. a systematic review of several studies, estimated that appropriate hand washing with soap, could reduce the risks of severe intestinal infections and of shigellosis by up to % and %, respectively [ ] . the water could have been contaminated by blocked sewer. the problem of blocked sewer had earlier been highlighted by mangizvo r.v et al., [ ] . this could explain the contamination of shallow wells and boreholes in kadoma city, most probably through seepage. in karachi, pakistan, khan et al. demonstrated that % of sewer influent samples were positive for rotavirus, which was also identified in one of the cases in the outbreak in kadoma city [ ] . contamination of water can occur at any stage from the source, to the point of use. the biological plausibility of storing water in a closed container, boiling and use of aqua tablets was demonstrated in this study. the use of chlorine containing materials have been made use of in situations of acute water shortage, where people tend to use unsafe sources of water, as reported by lantagne d s, et al., in [ ] . in kadoma city, in this epidemic, the use of municipal water was protective, compared to water from other sources (aor = . , p = . ). similar findings were obtained in harare by gonese g, et al., in . (risk factors associated with salmonella outbreak in budiriro suburb, harare city, zimbabwe, -unpublished). the laboratory tests on municipal water which was of satisfactory bacteriological and residual chlorine levels support the findings. at the same time, families who accessed water from municipal supply are more likely to access water in the family home. this study demonstrated that distance of the water source from home increased risk of developing diarrhoea. this results in families accessing less than the required amount of water per day, compromising personal hygiene practices such as hand washing. exclusive breast feeding for months was protective against developing diarrhoea. exclusively breastfed children were protected as they were unlikely to take any other foods or fluids likely to be contaminated. this protective effect was less for children on mixed breastfeeding and weaned from their mother's milk.the protective effect of exclusive breastfeeding had been demonstrated using different study designs, in different settings. in belarus, kramer m.s. et al., , reported that exclusive breast feeding reduced risk of diarrhoea by % [ ] . in guinea bissau, mølbalk k, et al., , reported that partial breast feeding, rr = . ( . - . ), and no breast feeding rr = . ( . - . ) increased risk of diarrhoea [ ] . in zimbabwe, a nutritional survey in reported that . % of children were [ ] . thus, efforts to increase the prevalence of exclusive breast feeding could reduce the impact of diarrhoea on child morbidity and mortality, in zimbabwe. limitations of this study included the following: controls might have contracted the disease but not developed clinical symptoms leading to case ascertainment bias. this could have under estimated the strength of some associations. the observed protective effect of the water treatment tablets is self reported, thus may lead to bias. some of the caregivers were interviewed more than two weeks after treatment, which could have resulted in recall bias. the study was conducted in children less than five years old, thus the results may not be generalizable to residents older than five years. some agents isolated in the local laboratory, salmonella and shigella could not be confirmed by the national microbiology reference laboratory. the outbreak was propagated and affected all the residential areas in kadoma city. mixed etiological agents were responsible for the outbreak. knowledge on prevention and home treatment of diarrhoea was fair and did not differ between caregivers of cases and controls. the importance of providing adequate safe water, appropriate hand washing, exclusive breast feeding and good environmental hygiene were demonstrated to be protective against contractingdiarrhoea. the results of the study provided guidance for policy makers in responding to the outbreak, and formulating strategies to improve child health. based on the evidence from this study water purification tablets, municipal water trucking to residential areas, disinfection and repair of boreholes, and sewer unblocking was conducted in the affected communities. health education on importance of practicing good personal hygiene, boiling and treating water with water purification tablets before drinking and long term benefits of exclusive breast feeding was conducted, and is ongoing. all municipal clinics now have weekly updated diarrhoea reports. the national electricity distribution company installed dedicated electricity power lines to the municipal water pumping stations to maintain continuous water pumping. world health organization: diarrhea, factsheet number world gastroenterology organization practice guideline: acute diarrhoea where and why are million children dying every year? lancet zimbabwe government printers (now renamed printflow) kadoma city council offices kadoma city health department: cholera outbreak report kadoma city health department: cholera outbreak report assessing the performance of the early aberration reporting system (ears) syndromic surveillance algorithms. united states of america: naval postgraduate school a nationwide case-control study of escherichia coli o : h infection in the united states water: monitoring and assessment. available on url effect of washing hands with soap on diarrhoea risk in the community: a systematic review the problem of burst sewage pipes and sewerage outflows in east view suburb in kadoma city diarrhea due to rotavirus and probability of sewage contamination household water treatment and safe storage options in developing countries: a review of current implementation practices promotion of breastfeeding intervention trial (probit): a randomized trial in the republic of belarus risk factors for diarrheal disease incidence in early childhood: a community cohort study from guinea-bissau zimbabwe launches nutritional survey report. health harare risk factors for contracting watery diarrhoea in kadoma city i would like to express my sincere gratitude to my field supervisor, mr. d chirundu for his guidance and support, and to the staff and management at kadoma city council, and sanyati district for their support. special thanks go to the staff in the department of community medicine and mph field office for all the help they rendered. we would also like to express our profound gratitude to centres for disease control (cdc) zimbabwe for funding and technical input.many thanks go to all the caregivers for their consent to allow me to interview them. last, but not least, i would like to thank my family for social support throughout the project. the authors declare that they have no competing interests.authors' contributions bam: conception, design, acquisition, analysis and interpretation of data and drafting the manuscript. dc: conception, design, acquisition, analysis and interpretation of data and drafting the manuscript. ntg: conception, design, data collection, analysis, interpretation and reviewing of several drafts of the manuscript for important intellectual content. gs and lt :conception, design, key: cord- -ywb krdp authors: barr, margo; raphael, beverley; taylor, melanie; stevens, garry; jorm, louisa; giffin, michael; lujic, sanja title: pandemic influenza in australia: using telephone surveys to measure perceptions of threat and willingness to comply date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ywb krdp background: baseline data is necessary for monitoring how a population perceives the threat of pandemic influenza, and perceives how it would behave in the event of pandemic influenza. our aim was to develop a module of questions for use in telephone health surveys on perceptions of threat of pandemic influenza, and on preparedness to comply with specific public health behaviours in the event of pandemic influenza. methods: a module of questions was developed and field tested on adults using the new south wales department of health's in-house computer assisted telephone interviewing (cati) facility. the questions were then modified and re field tested on adults. the module was then incorporated into the new south wales population health survey in the first quarter of . a representative sample of , adults completed the module. their responses were weighted against the state population. results: the reliability of the questions was acceptable with kappa ranging between . and . . overall . % of the state population thought pandemic influenza was very or extremely likely to occur; . % were very or extremely concerned that they or their family would be affected by pandemic influenza if it occurred; and . % had made some level of change to the way they live their life because of the possibility of pandemic influenza. in the event of pandemic influenza, the majority of the population were willing to: be vaccinated ( . %), be isolated ( . %), and wear a face mask ( . %). people with higher levels of threat perception are significantly more likely to be willing to comply with specific public health behaviours. conclusion: while only . % of the state population thought pandemic influenza was very or extremely likely to occur, a significantly higher proportion were concerned for self and family should a pandemic actually occur. the baseline data collected in this survey will be useful for monitoring changes over time in the population's perceptions of threat, and preparedness to comply with specific public health behaviours. if an outbreak of pandemic influenza should occur, it is essential that public health authorities are prepared to act. while resources have been prepared to educate the population about the nature of a threat and planned government actions, [ ] it is necessary to understand the potential response of a population. most of the existing information about a population's response to the threat of pandemics comes from research on outbreaks of the sars coronavirus, most notably in hong kong, singapore, and canada, [ ] [ ] [ ] [ ] and on studies of risk perception and anticipated behaviours in a potential pandemic in humans from the avian influenza virus (especially the h n subtype). [ ] [ ] [ ] [ ] to date, australia has been relatively unaffected by sars or h n ; however, some of australia's neighbours have experienced limited outbreaks: for example, sars in hong kong and singapore; and h n in indonesia and hong kong and china. globally, the threat of a pandemic of h n is high. a key component of a population's response is the perception of risk or threat. research shows that in a sars outbreak willingness to comply with risk-reducing behaviours is linked to the perceived immediacy and seriousness of the threat. [ , , ] three risk perception studies on potential avian influenza outbreaks were conducted in . in the first study, lau et al. surveyed residents of hong kong on a potential outbreak of h n . [ ] their study focussed on protective behaviours and likely compliance with them; however, the researchers also asked respondents about the perceived threat of h n and the likelihood of it occurring within the next months. it was found that % of respondents felt the chance of an outbreak was high or very high. lau's study also asked respondents how worried they would be about oneself or a family member contracting the virus in the event of a local outbreak; % said they would be very worried. in the second study, de zwart et al. compared the risk perceptions of european and asian respondents to the threat of avian influenza, [ ] and measured self-efficacy beliefs to assess the likely compliance with protective health measures. overall the study found that % of respondents thought they were likely or very likely to become infected should an outbreak of avian influenza occur. this figure varied from % (denmark and singapore) to % (poland and spain). the researchers took a composite measure of risk perception and found that higher scores were observed in europe rather than asia. they found higher risk perceptions in females and older respondents; while lower self-efficacy beliefs in europe suggested that adherence to protective measures would be lower in europe. in the third study, di giuseppe et al. surveyed the knowledge and attitudes of an italian population to avian influenza. [ ] they found that around % of respondents had a high risk perception and felt very much at risk of contracting avian influenza. in this study lower socioeconomic status and lower education levels were associated with higher risk perception, and those with a higher risk perception were more likely to comply with hygiene practices to avoid the spread of disease. our aim was to develop a module of questions for use in telephone health surveys on perceptions of threat of pandemic influenza, and on preparedness to comply with specific public health behaviours in the event of pandemic influenza. [ ] [ ] [ ] as such, our literature search identified no relevant studies on response to pandemic influenza specifically, although other studies have been published on general threat perception and compliance with protective behaviours in the context of infectious diseases or other emergencies. the primary reference was a study by canadian researchers on anticipated public response to terrorism. [ ] questions on the threat likelihood, effect on family, and behavioural compliance, were adapted with permission by subject matter experts and survey methodologists. each proposed question was considered for clarity, ease of administration, and possible biases. a set of questions was developed for field-testing (table ) , as well as an additional open question: "do you have any comments you would like to make on any of the questions or any other issues?" the pandemic influenza questions were field tested for test-retest reliability using the protocol of the new south wales health survey program. [ ] the questions were then modified based on the results from the field testing and were re field tested. for both field tests the target sample was persons living in the state aged years and over stratified by geographical region. this sample size ensures that a kappa of . (good or excellent) is able to be detected at a significance level of % and a power of % when compared to a kappa of . or less (fair or poor) for response frequencies greater than %. [ ] additional context added before question to provide better context. likelihood of being affected was changed to concern about being affected, to tap a sense of vulnerability rather than probability. responses altered to reflect concern and increase to options. households were contacted using random digit dialling. one person aged years and over from each household was randomly selected for field testing. trained interviewers conducted the interviews. up to calls were made to establish initial contact with a household, and at least calls were made to contact a selected respondent. when the respondent completed the first field test, an appointment was made for a retest at least a week later but within weeks of the initial field test. if a respondent was unable to be contacted during this week window they were deemed to be unavailable and their initial field test was deleted. [ ] test-retest reliability and validity were estimated by cohen's kappa statistic for binary variables, and weighted kappa with cicchetti-allison weights for ordinal variables. unbalanced tables were corrected using the method described by crewson. [ ] since erroneously low values of kappa can arise from skewed data, per cent agreement was also presented for categorical variables, calculated as the proportion of respondents in the same category at test and retest. responses for don't know and refused are also reviewed. [ ] data manipulation and analysis were conducted using sas version . . [ ] the survey the new south wales population health survey is a continuous telephone survey of the health of the state population using the in-house cati facility of the new south wales department of health. [ ] only residential phone numbers were used in the sample, as residential phone coverage in australia still remains high, [ ] and results from persons who only have mobile phones has been shown to be comparable in the united states. [ , ] the pandemic influenza module was administered as part of the survey between january and march . the pandemic influenza questions were submitted to a lead ethics committee for approval prior to use. the survey also includes other modules on health behaviours, health status (including psychological distress, using the kessler k measure, and self-rated health status), and access to health services, as well as the demographics of respondents and households. the target population for the survey is all state residents living in households with private telephones. up to calls were made to establish initial contact with a household, and calls were made in order to contact a selected respondent. response categories were dichotomised into indicators of interest and don't knows and refused were removed. for the hypothetical questions -that is, likelihood of pandemic influenza, likelihood that family or self affected, willingness to comply with vaccination, isolation or wearing a face mask -the responses of extremely likely and very likely were combined into the indicator of interest. for the non-hypothetical question "changed way live because of the possibility of an influenza pandemic" responses a little, moderately, very much and extremely were combined into the indicator of interest: that is, changed life. the survey data were weighted to adjust for probability of selection and for differing non-response rates among males and females and different age groups. [ ] data were manipulated and analysed using sas version . . [ ] the surveyfreq procedure in sas was used to analyse the data and calculate point estimates and per cent confidence intervals for the prevalence estimates. for pairwise comparisons of subgroup estimates, the p-value for a two-tailed test was calculated using the normal distribution probability function probnorm in sas, assuming approximate normal distribution of each individual subgroup estimates with the estimated standard errors, and approximate normal distribution for the estimated difference. in total, residents aged years and over completed the first field test and residents completed the second field test. estimates of test-retest reliability for the first and second field tests are shown in second field test. kappa values for the indicators derived from the questions ranged between . and . in the second field test. there were low don't know response rates ( - . %) and no respondent refused to answer any question. in response to the open question "do you have any comments you would like to make on any of the questions or any other issues?": % made positive comments about the questions, . % found the question wording easy to understand and answer, and . % found the subject matter relevant and interesting. of the respondents who had difficulty answering the questions, the main issues were: the questions were too vague ( . %), response options were not descriptive enough ( . %), or the topic area was difficult ( . %). table shows the responses to each question, including don't know and refused. the percentage of don't know or refused responses was low. table shows the indicators for pandemic influenza likely, concern for self and family, and changed life by sex, age group, demographic characteristics, and the indicators of level of psychological distress and general self-rated health status. overall . % of the population thought pandemic influenza was very or extremely likely, . % were very or extremely concerned that they or their family would be affected by pandemic influenza, and . % had made some (small to extreme) level of change to the way they live their life because of the possibility of pandemic influenza. when the indicators for pandemic influenza likelihood, concern for self and family and changed life were combined, as shown in figure , the greatest proportion of the population ( . %) thought pandemic influenza was unlikely to occur, would not be concerned for themselves or their family, and had not changed the way they lived their life because of the possibility of pandemic influenza. a quarter of the population ( . %) thought pandemic influenza was unlikely to occur and had not made any changes to their lives, but would be concerned for themselves and their family in the event of pandemic influenza. table also shows the combined indicators pandemic influenza likely and concern for self and family as well as pandemic influenza likely and concern for self and family and changed life by sex, age group, demographic characteristics, and the indicators of level of psychological distress and general self-rated health status. table shows the indicators willing to receive vaccination, isolate themselves, or wear a face mask by sex, age group, demographic characteristics, and the indicators of level of psychological distress and general self-rated health status. overall, the majority of the population would be willing to receive vaccination ( . %), willing to be isolated ( . %), and willing to wear a mask ( . %), if pandemic influenza were to occur. when the indicators for willing to receive vaccination, isolate themselves, and wear a face mask were combined, as shown in figure , . % reported being willing to receive vaccination, isolate themselves, and wear a face mask if pandemic influenza were to occur; . % would not be willing to receive vaccination, isolate themselves and wear a face mask; . % would be willing to receive vaccination, isolate themselves but not wear a face mask; and . % would be willing to receive vaccination and wear a face mask but not isolate themselves. table also shows the combined indicator for willing to receive vaccination, isolate themselves, and wear a face mask by sex, age group, demographic characteristics, and the indicators of level of psychological distress and general self-rated health status. table shows the indicators for willing to receive vaccination, isolate themselves, or wear a face mask as well as complying with all the specific public health behaviours: that is, willing to receive vaccination, isolate themselves, and wear a face in people who think a pandemic influenza is very or extremely likely, and who are also very or extremely concerned for themselves and their family. this study shows it is possible to construct a small set of questions about threat perception for a general population, which can be used for health surveillance. field testing identifies improvements that can be made to the questions and the response structure, and highlights the population's interest in surveys of this nature. kappa values for the indicators ranged from . - . , which is acceptable for hypothetical questions. the items had low don't know response rates ( - . %); no respondents refused to answer any of the questions; and the majority of respondents made positive comments about the questions. those reporting the highest levels of threat perception are older people, those with fair or poor self-rated health status, no formal qualifications, low household incomes, and those living in rural areas. perhaps surprisingly, there were no differences noted between the perceptions of men and women, or between those persons with or without children. overall, the majority of the population has taken no action, at this point, to change the way they live their life because of the possibility of pandemic influenza. the only two subgroups reporting moderate changes are those born overseas and those who speak a language other than english in the home. although direct comparisons with other studies are difficult to make, these findings suggest that the threat perceptions of the new south wales population are similar to those reported by residents of hong kong, even though australia has not been exposed directly to sars or h n . willingness to comply with specific public health behaviours is generally high ( - %), with willingness to be vaccinated greater than being willing to be isolated, which in turn is greater than being willing to wear a face mask. there is clearly a lower level of willingness to comply with wearing a face mask, especially in younger people, those living in urban areas, and those who speak a language other than english in the home. current findings on compliance with protective behaviours are comparable with findings from studies con- ducted in hong kong in relation to anticipated sars and h n . [ , ] a study about sars in hong kong indicates that those with higher risk perception and moderate levels of anxiety were more likely to take comprehensive precautionary measures against infection, and younger less educated males were least likely to adopt preventative measures. [ ] our data suggest that younger people are less likely to comply with protective behaviours, while a higher level of formal education (a university degree or equivalent) is associated with higher willingness to comply with all protective behaviours, but especially wearing a face mask. a study of this nature has a number of limitations. first, people are being asked about a hypothetical event of which they have no experience. however, comparisons [ ] and reported mask wearing rising from % in the early stages to % in the later stages of the outbreak. clearly data in that study support the increased likelihood of protective behaviours being adopted with increased risk perception; and, in our study, those with higher levels of threat perception were significantly more likely to be willing to comply with specific public health behaviours. prevalence estimates and % confidence intervals for response combinations to the three questions on health protection behaviours for pandemic influenza willing to be vaccinated, isolated, and wear a face mask willing to be vaccinated and isolated, but not willing to wear a face mask willing to be vaccinated and wear a face mask, but not willing to be isolated willing to be vaccinated, but not willing to be isolated or wear a face mask willing to be isolated and to wear a face mask, but not willing to be vaccinated willing to be isolated, but not willing to be vaccinated or to wear a face mask willing to wear a face mask, but not wiling to be vaccinated or isolated not willing to be vaccinated, isolated, or wear a face mask % our data indicate that while most respondents are very or extremely willing to perform a behaviour; the remaining respondents are expressing varying, but lower, degrees of willingness to perform these behaviours, with - % indicating they would be moderately or a little willing, and - % indicating they would be not at all willing to perform these behaviours. however, evidence such as data indicating very high levels of compliance with quarantine and minimal requirement for enforceable quarantine orders during sars in canada suggests that, in the event of a serious and immediate threat, the majority of those who are indecisive would shift their position and comply. [ ] it is likely, however, that even with such a compliance 'shift' the relative compliance of sub groups within the population noted in our study will be upheld; as these patterns of compliance have been supported consistently by studies of actual protective behaviours. [ , ] this study of the response of the new south wales population to the threat of pandemic influenza is part of a broader study of perceptions and behaviours around adverse events, including terrorism and global warming. as post-disaster studies generally report a lack of baseline data as a major handicap to understanding the trajectory for psychosocial recovery, [ , ] our study takes the first steps in establishing baseline for data vital for emergency planning, against which impact and recovery can be monitored. australian health management plan for pandemic influenza canberra: australian government department of health and ageing infectious disease and risk: lessons from sars london: the nuffield trust the impact of community psychological responses on outbreak control for severe acute respiratory syndrome in hong kong monitoring community psychological responses to the sars epidemic in hong kong: from day to day risk perception and compliance with quarantine during the sars outbreak avian influenza risk perception: hong kong anticipated and current preventative behaviours in response to an anticipated human-to-human h n epidemic in hong kong chinese general population notes: level of statistical significance: * p < ‡ population level frequencies do not agree with table as don't know/ refused responses were excluded from this analysis. § for pairwise comparison testing in subgroups with more than two categories comparisons are made between each subgroup prevalence and the overall population prevalence. ψ psychological distress was measured using the k . values range from - , with 'high' psychological distress considered as being ≥ avian influenza risk perception a survey of knowledge, attitudes and practices towards avian influenza in an adult population of italy national public survey of perceived cbrn terrorism threat and preparedness. in university of ottawa in partnership with health canada and the canadian food inspection agency ottawa: institute of population health nsw population health survey: description of methods australian bureau of statistics: population survey monitor. catalogue no. . canberra: abs estimation issues in dual frame sample of cell and landline numbers surveying households on cell phones: results and lessons. paper presented at the annual conference of the american association for public opinion research nsw population health survey: review of weighting procedures australian bureau of statistics: census of population and housing effects of fear and anger on perceived risks of terrorism: a national field experiment comparison of post-disaster psychiatric disorders after terrorist bombings in nairobi and oklahoma city public health measures to control the spread of the severe acute respiratory syndrome during the outbreak in toronto this study was funded by emergency management australia and the new south wales department of health. the following staff of the centre for epidemiology and research, new south wales department of health, assisted with the study: matthew gorringe, question development and data collection; raymond ferguson, sas programming and infrastructure; frances garden, comparing weighted survey data against census data. the authors declare that they have no competing interests. the authors contributed equally to this work.publish with bio med central and every scientist can read your work free of charge the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /pre pub key: cord- - f kmw authors: tang, jialiang; chen, jinkun; he, tingting; jiang, zhuojing; zhou, jiale; hu, bin; yang, shangxin title: diversity of upper respiratory tract infections and prevalence of streptococcus pneumoniae colonization among patients with fever and flu-like symptoms date: - - journal: bmc infect dis doi: . /s - - -z sha: doc_id: cord_uid: f kmw background: many upper respiratory pathogens cause similar symptoms. in china, routine molecular tests for upper respiratory pathogens are not widely performed and antibiotics abuse in treating upper respiratory tract infections (urtis) is a major public health concern. methods: we performed qualitative real-time pcr tests to detect common upper respiratory tract pathogens including viruses and bacteria in nasopharyngeal swabs from patients with fever and influenza-like symptoms in a chinese city. a quantitative real-time pcr was also performed to measure the bacterial density of the colonizing streptococcus pneumoniae in these samples. results: we found very diverse pathogens including . % viruses, . % bacteria and . % mixed viruses and bacteria. s. pneumoniae colonization was found in . % of the cases but most of them had low bacterial density (mean = . log cfu/ml). we also discovered an increase of s. pneumoniae colonization frequency (but not the density) in patients with detectable upper respiratory tract pathogens, in a pathogen variety-dependent manner. conclusions: our study provided strong evidence against empiric antibiotic use for treating urtis, and highlighted a strong need for improving the diagnostic capacity for urtis by using more molecular testing in china. it has been well recognized that many pathogens, mainly viruses and some bacteria, cause urtis but present with almost indistinguishable clinical symptoms [ ] [ ] [ ] . differentiation of viral urtis versus bacterial urtis has the direct implication of antibiotics use. however, a lack of timely diagnosis by using rapid and accurate tests had contributed to overuse and abuse of antibiotics around the world [ ] . in china, antibiotics abuse is very common due to a culture of self-medication (antibiotics are largely available over the counter) and over-prescription by clinicians [ , ] . antibiotics are mainly prescribed empirically, not based on microbiological investigation [ , ] . this is particularly problematic for patients with urtis, for which most clinics and many hospitals in china only offer limited routine tests for the influenza viruses (flu). common upper respiratory tract pathogens other than flu, such as human rhinovirus (hrv), respiratory syncytial virus (rsv), parainfluenza viruses (pivs), adenovirus (adv), human metapneumovirus (hmpv) and bacterial pathogens that are difficult to culture including mycoplasma penumoniae, chlamydophila pneumoniae and bordetella pertussis are generally not tested in china, except for a few high-ranking academic medical centers (facts based on personal observations and communication with colleagues in china). therefore, improving the accurate diagnosis of pathogens causing the urtis (abbreviated as urti pathogens in this article) is of great significance for rational selection of antibiotics and reduction of antibiotics abuse in china. as one of the most densely populated provinces in china, zhejiang has been the center of attention for emerging infectious diseases since the h n bird flu outbreak first started there in early [ ] . since then, chinese state and local government had implemented surveillance programs to actively monitor the circulating flu viruses. in one of such programs, hospitals and clinics routinely submit nasopharyngeal swabs of patients with fever and flu-like symptoms to the local public health laboratories for flu viruses testing by pcr and then genotyping by sanger sequencing if positive. however, even during the peak of flu season, there are still many patients with fever and flu-like symptoms but tested negative for flu viruses. the question remains as what other respiratory pathogens are circulating and causing the similar flu-like symptoms, and what pathogens co-infect with flu virus in the chinese community. another important question is about the frequencies of bacterial pathogens such as m. pneumoniae, c. pneumoniae and b. pertussis that do require antibiotics treatment and how often they co-infect with viral pathogens. in addition, upper respiratory tract colonization of streptococcus pneumoniae, the most common cause of bacterial pneumonia, was considered prerequisite for its infections in the lower respiratory tract [ ] . both the density and frequency of s. pneumoniae in the upper respiratory tract had been shown to increase during viral infections [ ] , but very limited data exist regarding the prevalence and density of s. pneumoniae colonization in patients with flu-like symptoms. a better understanding of the epidemiology of s. pneumoniae colonization and its relationship with the pathogens causing urtis may help solve the controversy over using antibiotic prophylaxis to prevent possible secondary lower respiratory tract infections (lrtis) caused by s. pneumoniae, which is considered to be another inappropriate antibiotic prescription practice that could contribute to antibiotic resistance [ , ] . to help answer these questions, we carried out a study based on nasopharyngeal swabs collected from patients of all ages (majority adults) with urtis in shaoxing, the third largest city in zhejiang province in eastern china, in a full year ( ). we tested these upper respiratory samples for common viral urti pathogens including flua, flub, rsv, hrv, pivs ( - ), adv, and hmpv, and common bacterial urti pathogens including m. pneumoniae, c. pneumoniae and b. pertussis, as well as colonizing s. pneumoniae to study its relationship with these urti pathogens. from january , to december , , nasopharyngeal swabs were collected from outpatients (age ranged from months to years) with fever (≥ °c) and influenza-like symptoms such as cough, runny or stuffy nose, sore throat, muscle aches, chills and fatigue. all samples were de-identified to protect patients' private information except for the age. no clinical information such as x-ray results, severity of the illness, diagnosis, or use of antibiotics were available. the nasopharyngeal swabs were collected using the flock swabs (copan, italy) and stored in the universal transportation medium (copan, italy) provided in the same collection kit. the samples were refrigerated for up to week until being tested. total nucleic acids were extracted using ingenigen total nucleic acids extraction kit, following the manufacturer's instruction (ingenigen xunminkang biotechnology inc., shaoxing, china), and the real-time pcr assays were performed using the ingenigen respiratory pathogen multiplex pcr kits and abi system (thermofisher, boston, ma, usa). ms phage and plasmids containing the human albumin gene were added to the samples as internal controls for rna virus detection, and dna virus and bacteria detection, respectively. quantitative real-time pcr for s. pneumoniae the s. pneumoniae quantitative real-time pcr kit was purchased from ingenigen xunminkang biotechnology inc., shaoxing, china. included in the kit, a set of calibrators, which were dna extracted from -fold serial dilutions of a laboratory strain s. pneumoniae (atcc ) with the highest concentration of × colony forming units per milliliter (cfu/ml) and the lowest concentration of × cfu/ml, were tested to create a standard curve and a formula for the bacterial load quantification. the lower limit of detection (lod) and the lower limit of quantification (loq) of the test were both cfu/ml according to the manufacturer's package insert. the quantification formula was determined to be log cfu/ml = ( . -ct value) / . . all data were statistically processed using spss . and graphpad prism . the frequencies of urti pathogens and s. pneumoniae colonization were analyzed using pearson's chi-square test (χ ) to identify any difference between a specific age group vs. all other age groups. the colonizing s. pneumoniae density were analyzed by one-way analysis of variance (anova) to identify any difference between a specific age group vs. all other age groups, and by t-test to compare the samples with or without urti pathogens detected. all the s. pneumoniae density results were summarized using means and standard deviation (sd). p < . was considered as statistically significant. there were participants recruited into the study, with - recruited each month. all participants agreed to contribute the residual nasopharyngeal swab samples for this study after the routine flu a/b pcr test was done. the additional test results were only for epidemiological research purpose and not provided to the patients. among all age groups, the overall positive rate for the urti pathogens was . % ( / ). the most frequently detected urti viruses were flua ( . %), flub ( . %), rsv ( . %) and hrv ( . %) and the most frequently detected urti bacteria was m. pneumoniae ( . %). the other urti pathogens including adv, pivs, hmpv, c. pneumoniae and b. pertussis were also detected but their positive rates were much lower (table ) . no statistically significant difference was found in the detection rates of overall urti pathogens among the different age groups. notably, flua rate was significantly higher in the older adults of - years old ( . %) but was significantly lower in the young children < years old ( . %) compared to other age groups ( . - . %). children < years old also had much higher detection rates of hrv ( . %), piv ( . %) and adv ( . %) ( table ). in addition, we found rsv detection rate was significantly lower in the young adults of - years ( . %) compared to other age groups ( . - . %, excluding age group of - years due to only positive case) ( table ) . among all the cases positive for at least one urti pathogens, the majority ( . %) were of viral only infections. only . % of cases were of bacterial-only (m. pneumoniae, c. pneumoniae, or b. pertussis) infections and . % of cases were of viral + bacterial co-infections ( table ) . co-infections were quite common and found in . % of the positive cases with the majority double co-infections and only cases of triple-infections. no statistically significant difference was found among different age groups regarding the detection rates of different pathogen classification (viral vs bacterial, double vs triple infections) ( table ) . during our -months study period in shaoxing in , an average of around respiratory samples in each month were collected ( fig. a) . the prevalence of overall urti pathogens was significantly higher in the months of january to march ( . - . %) than the rest months of the year ( . - . %) (fig. a) . interestingly, the frequency of s. pneumoniae colonization also peaked in the months of january and february ( . - . %) but stayed low in the rest of the months ( . - . %) except november ( . %). the overlapped peaks of the overall urti pathogens and s. pneumoniae colonization frequency suggested there might be a positive correlation between the two. co-infection rate also peak from january to march, overlapping with the peak of overall urti pathogens (fig. b) . the seasonal pattern of the overall urti pathogens was mainly attributed to the seasonal pattern of a few high prevalent urti pathogens ( fig. c) including flua, flub, rsv ( fig. d ) and m. pneumoniae (fig. e ), whose prevalence generally peaked from january to march. other urti pathogens seemed to have less obvious seasonal patterns, except for adv and pivs which appeared to both peak in the month of august (fig. f ). respiratory infection is often characterized by mixed infection of various pathogens. in the urti table prevalence of urti pathogens among different age groups among the patients with fever and flu-like symptoms, although the overall s. pneumoniae colonization frequency was . % ( / ) (table ), in most of the months except for january, february, march and november, the frequency was much lower ( . - . %) (fig. a) . we found significantly increased s. pneumoniae colonization frequencies in both young children < years old ( . %) and the elderly > years old ( . %) compared to other age groups ( . - . %) ( table ). to investigate the relationship between s. pneumoniae colonization and urti pathogens, we found there is a dose-dependent relationship between how many urti pathogens were detected and the s. pneumoniae colonization frequency (fig. a) . in cases without urti pathogen detected, the s. pneumoniae colonization frequency was . %, which increased to . % when only one urti pathogen was detected. this difference was with only borderline statistical significance (p = . ). however, in cases when more than one urti pathogens were detected, the s. pneumoniae colonization frequency increased to . % with a statistical significance (p = . ) (fig. a) . the increase of s. pneumoniae colonization frequency was found to be unrelated to the classification of the urti pathogens since no statistically significant differences of s. pneumoniae colonization frequency were found among the case groups with viral-only, bacterial-only or bacterial + viral infections (fig. b ). to single out which urti pathogen may contribute to this increase, we found although most urti pathogens (except flub, b. pertussis and c. pneumoniae) seem to be associated with higher s. pneumoniae colonization, the only statistically significant contributor was rsv infection (p = . ) (fig. c ). without rsv infection, the s. pneumoniae colonization frequency was only . %. with rsv infection, the frequency increased to . %, more than folds compared to the average s. pneumoniae colonization frequency ( . %). the bacterial density of s. pneumoniae was found to be generally low (mean = . log cfu/ml) in our patient population. the majority ( . %, / ) had s. pneumoniae density lower than . log cfu/ml (fig. a ). only one sample had a very high s. pneumoniae density ( . log cfu/ml), which is higher than . log cfu/ml, a density cut-off shown to be able to predict streptococcal pneumonia in children aged < years by a recent study [ ] . the patient, however, was a -year-old elderly who was co-infected with rsv and hmpv. due to the unavailability of clinical information, whether this patient had later developed streptococcal pneumonia was unknown. no significant difference of s. pneumoniae colonization density was identified among different months of the year (fig. d) . the correlation between s. pneumoniae colonization density and age as continuous intervals only showed a slight uptrend without statistical significance (pearson correlation coefficient r = . ) (fig. e) . however, when the s. pneumoniae density was compared among different age groups, we found a statistically significant higher density ( . log cfu/ml) in the elderly (> years) compared to other age groups ( . - . log cfu/ml, excluding age group of - due to only positive cases), with more than -log difference ( table ). the relationship between s. pneumoniae colonization density and urti pathogens, however, was not observed since we did not find any difference in the s. pneumoniae density between the urti pathogen positive cases vs. negative cases (fig. f) . only two cases ( . %) of pertussis were found among the samples in . interestingly, both samples were also positive for other urti pathogens. one sample was positive for b. pertussis and adv, and the other sample was positive for b. pertussis, flua and hrv. both samples were from relatively young adults with age and , who are in the child-bearing age and may transmit b. pertussis to their newborn babies. since b. pertussis infection alone generally do not cause fever [ ] , our sample collection criteria might have resulted in a significant underestimation of the b. pertussis infection prevalence among the community in shaoxing. several molecular epidemiological studies with the focus on hospitalized children have been reported in china, which demonstrated the complexity and diversity of pathogens involved in urtis [ ] [ ] [ ] [ ] . although our overall findings were similar, our study was unique in a few aspects: ) we did not set age limit, which provided a better representation of the whole community; ) we only included outpatients with fever and flu-like symptoms which largely limited the cases to urtis only; ) . ± . *significant difference between a specefec age group vs. all other age groups was identified using pearson's x test for s. pneumoniae frequency density *p < . ; **p < . ; ***p < . the age group of - is excluded for the analysis due to very low number of cases we tested both viral pathogens and bacterial pathogens that cause urtis; ) we also investigated s. pneumoniae frequency and density in the upper respiratory tract in order to study the relationship between the urti pathogens and s. pneumoniae colonization. consistent with other studies [ ] [ ] [ ] [ ] , we found flua ( . %), flub ( . %), rsv ( . %), hrv ( . %), and m. pneumoniae ( . %) to be the most common urti pathogens in patients with fever and flu-like symptoms. the majority ( . %) of the urtis were of exclusive viral etiology, and only . % were of bacterial etiology (bacteria alone or bacteria + viral co-infections). these findings added to a large body of evidence supporting the guidelines that recommend against antibiotics prescription to treat urtis without accurate diagnosis [ ] . the seasonality pattern of most high-prevalence pathogens including flua, flub, rsv, and m. pneumoniae largely overlapped, with much higher prevalence in winter and spring (january -april). the mechanisms for the seasonality of the respiratory pathogens remain unclear but a few possible driven factors have been suspected such as enhanced wintertime survival of pathogens due to lower temperature and lower humidity, increased travel and social gathering due to more holidays in winter, as well as weakened immunity associated with a lack of vitamin d [ ] . interestingly, the prevalence of the overall urti pathogens and the s. pneumoniae colonization frequency both peaked in the months of january and february, suggesting a possible positive correlation between urti infections and s. pneumoniae colonization frequency. the most significant finding in this study is that we discovered a pathogen variety-dependent positive correlation between how many urti pathogens were detected and s. pneumoniae colonization frequency. mixed infections with more than one urti pathogens seemed to be associated with a significant increase of s. pneumoniae colonization frequency (from . to . %). in addition, we identified rsv infection as the most significant contributor to the increased s. pneumoniae colonization frequency. the relationship between rsv infection and s. pneumoniae colonization have been recognized before. studies have shown that rsv could enhance both the infectivity and virulence of s. pneumoniae [ , ] . on the other hand, s. pneumoniae was found to enhance rsv infection both in vivo and in vitro [ ] . s. pneumoniae colonization has also been linked to increased severity in children with rsv infections [ ] . it should be noted that in our study, rsv is also associated with a higher percentage of co-infections ( . %) compared to other urti pathogens, which might be a confounding factor since we have demonstrated mixed infections itself could be associated with increased s. pneumoniae colonization frequency. notably, our findings are novel as most other studies about the relationship between s. pneumoniae colonization and urtis were limited to pediatric patients and viral infections, while our study included both viral and bacterial pathogens in patients of all ages. the biological mechanisms for the positive correlation between the s. pneumoniae colonization frequency and urti pathogens require further investigation, which may help better understand the disease progression of urtis and provide useful information for developing proactive monitoring protocols for high-risk patients. the density (bacterial load) of the colonizing s. pneumoniae, however, was almost the same in our patients with or without detectable urti pathogens, and there was no seasonal difference. these results indicate a lack of relationship between the s. pneumoniae colonization density and the urti pathogens in patients only with urtis. this is consistent with other studies showing that heavy s. pneumoniae colonization density is usually associated with more advanced lower respiratory streptococcal infections [ , ] . however, we did find heavier density of s. pneumoniae colonization among elderly patients (> years) regardless of detectable urti pathogens, which combined with our finding that the s. pneumoniae frequency was also increased in this age group, provided consistent evidence to help explain why elderly patients are at higher risk of developing streptococcal pneumonia. importantly, we detected b. pertussis in samples, both of which were also positive for other urti viruses. this is particularly interesting because we only included patients with fever, which is not typically associated with b. pertussis infection [ ] . therefore, we might detect only a small fraction of b. pertussis cases with viral co-infections that presented with fever. the true prevalence of b. pertussis infection in this chinese community is most likely underestimated. this is alarming since both b. pertussis positive cases were young adults in their childbearing age, who might transmit to their newborns. although the vaccination coverage rate in china is estimated to be as high as %, surprisingly, % of the . million pertussis cases globally in were from china [ ] . this is probably attributed to the shorter duration of protection by the acellular pertussis vaccines, which were introduced to china in [ ] and had largely replaced the whole-cell pertussis vaccines since [ ] . recent studies have shown that the pertussis toxin igg (a marker for the immunity) seropositive rate among the chinese populations was as low as only % [ ] and the misdiagnosis rate for pertussis in some areas was as high as . % [ ] , highlighting the underrecognized severity of pertussis infections in china, and an urgency for improved diagnostic capacity, better surveillance system and vaccination strategies [ ] . we plan to expand our molecular epidemiological study by using more appropriate criteria to include afebrile patients with prolonged non-productive cough [ , ] . our studies have several limitations. first, we did not detect coronaviruses, which are an important group of urti pathogens and their prevalence can be as high as % in china [ ] . however, this is not expected to change the conclusions that viral etiology dominated the urtis, and s. pneumoniae colonization frequency increased in patients with detectable urti pathogens. second, we only included patients with fever, which may not be presented in many urtis. the exclusion of afebrile patients could potentially lead to an underestimation of the true prevalence. third, we did not test other pathogens including enterovirus, epstein-barr virus, cytomegalovirus and group a streptococcus that could also present with fever and flu-like symptoms [ , ] , which may partially explain the low positive rate during summer and fall. fourth, we did not acquire the information about patient's antibiotic usage, which may impact the prevalence of bacterial pathogens as well as the density of s. pneumoniae in the upper respiratory tract. last, we did not include a control group of healthy people as a comparison. it has been shown that asymptomatic carriage of respiratory viruses and m. pneumoniae are not uncommon, especially in children [ ] [ ] [ ] . we plan to expand our study to survey more people in the community including both symptomatic and asymptomatic individuals. in summary, our molecular epidemiological study in a medium-sized city in eastern china demonstrated that the pathogens causing the urtis in the community were very diverse, complex, and dominated by viral infections. co-infections were common and mainly involved the high frequency pathogens (flua, rsv, hrv and m. pneumoniae). the seasonal patterns of the four most frequent pathogens (flua, flub, rsv and m. pneumoniae) overlapped and peaked during winter and spring (january to april), which also overlapped with the seasonal pattern of s. pneumoniae colonization in the upper respiratory tract. we also found a positive correlation between the s. pneumoniae colonization frequency (but not the density) and the number of urti pathogens detected, in a pathogen variety-dependent manner. we observed higher s. pneumoniae colonization frequency in both the young children and the elderly, and higher s. pneumoniae colonization density in the elderly, regardless of whether urti pathogens were detected or not. in addition, we found the majority of patients with urtis had low s. pneumoniae colonization frequency and density, which is not indicative of streptococcal lower respiratory infections. our results strongly support the recommendation by the guidelines not to treat urtis without accurate diagnosis, nor to use antibiotics for prophylaxis in patients with urtis but without the signs and symptoms of pneumonia [ , ] . alarmingly we found cases of pertussis in two young adults both of which had viral co-infections. our fever criteria could potentially lead to a serious underestimation of the true pertussis prevalence in the studied community. a better pertussis epidemiological study aiming at afebrile patient with prolonged non-productive cough should be taken to assess the true prevalence of pertussis, which have been shown to be on the rise in china and requires immediately attention. the diversity and complexity of the urti pathogens detected in this chinese community also highlighted the need to improve the diagnostic capacity for urtis, particularly by using more molecular testing, to encourage a more evidence-based antibiotics prescription practice and to alleviate the drug resistance burden caused by a massive scale of antibiotic abuse and misuse in china. abbreviations adv: adenovirus; anova: analysis of variance; b. pertussis: bordetella pertussis; c. pneumoniae: chlamydophila pneumoniae; flu: influenza viruses; hmpv: human metapneumovirus; hrv: human rhinovirus; lod: lower limit of detection; loq: lower limit of quantification; lrtis: lower respiratory tract infections; m. pneumoniae: mycoplasma pneumoniae; pivs: parainfluenza viruses; rsv: respiratory syncytial virus; s. pneumoniae: streptococcus pneumoniae; urtis: upper respiratory tract infections multiplex pcr and emerging technologies for the detection of respiratory pathogens the common cold high value care task force of the american college of p, for the centers for disease c, prevention: appropriate antibiotic use for acute respiratory tract infection in adults: advice for high-value care from the american college of physicians and the centers for disease control and prevention antibiotic resistance-the need for global solutions a systematic review of antibiotic prescription associated with upper respiratory tract infections in china antibiotic resistance amongst 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upper respiratory colonization with streptococcus pneumoniae and its role in the diagnosis of pneumococcal pneumonia among children aged < years in the perch study molecular pathogenesis, epidemiology, and clinical manifestations of respiratory infections due to bordetella pertussis and other bordetella subspecies prevalence of respiratory viruses among children hospitalized from respiratory infections in shenzhen new epidemiological and clinical signatures of pathogens from respiratory tract infections based on a -year study epidemiology of acute respiratory infections in children in guangzhou: a three-year study prevalence and correlation of infectious agents in hospitalized children with acute respiratory tract infections in central china seasonality of viral infections: mechanisms and unknowns enhanced adherence of streptococcus pneumoniae to human epithelial cells infected with respiratory syncytial virus respiratory syncytial virus increases the virulence of streptococcus pneumoniae by binding to penicillin binding protein a. a new paradigm in respiratory infection streptococcus pneumoniae enhances human respiratory syncytial virus infection in vitro and in vivo streptococcus pneumoniae colonization of the nasopharynx is associated with increased severity during respiratory syncytial virus infection in young children pneumococcal bacterial load colonization as a marker of mixed infection in children with alveolar community-acquired pneumonia and respiratory syncytial virus or rhinovirus infection an update of the global burden of pertussis in children younger than years: a modelling study acellular pertussis vaccines in china pertussis booster vaccine in china is pertussis infection neglected in china? evidence from a seroepidemiology survey in zhejiang, an eastern province of china epidemiological features of pertussis resurgence based on community populations with high vaccination coverage in china seroprevalence of pertussis in china: need to improve vaccination strategies prolonged afebrile nonproductive cough illnesses in american soldiers in korea: a serological search for causation clinical and laboratory features of pertussis in hospitalized infants with confirmed versus probable pertussis cases infectious diseases society of a: clinical practice guideline for the diagnosis and management of group a streptococcal pharyngitis: update by the infectious diseases society of america upper airway viruses and bacteria in urban aboriginal and torres strait islander children in brisbane, australia: a cross-sectional study seasonal variation of respiratory pathogen colonization in asymptomatic health care professionals: a singlecenter, cross-sectional, -season observational study carriage of mycoplasma pneumoniae in the upper respiratory tract of symptomatic and asymptomatic children: an observational study guidelines for the use of antibiotics in acute upper respiratory tract infections we would like to acknowledge linlin pan, shifang tao, yifang wang, yuanyuan cai, weifeng chu, and lin zheng from ingenigen xunminkang biotechnology inc., shaoxing, for providing technical support in this study. this study was funded by ingenigen xunminkang biotechnology inc., shaoxing, zhejiang.availability of data and materials not applicable. jlt and sxy designed the study. jlt, jkc, tth, zjj and bh performed the sample collection, processing and real-time pcr tests. jlt, jlz, and sxy analyzed the data and wrote the manuscript. sxy edited the draft.ethics approval and consent to participate this research project had been approved by the research ethics committee of shaoxing center for disease control and prevention (project # sxcdc - ). because no patient information was collected except for the age, and the patients' test results were de-identified and not provided to either patients or providers, the committee had deemed it's not necessary to acquire written patient consent. all authors have read and approved the final manuscript for publication. the authors declare that they have no competing interests. springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations. key: cord- -taj swwc authors: lu, guilan; peng, xiaomin; li, renqing; liu, yimeng; wu, zhanguo; wang, xifeng; zhang, daitao; zhao, jiachen; sun, ying; zhang, li; yang, peng; wang, quanyi title: an outbreak of acute respiratory infection at a training base in beijing, china due to human adenovirus type b date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: taj swwc background: twelve students experienced symptoms of acute respiratory infection (ari) at a training base in beijing from august to august , . we investigated the cause of this ari outbreak. methods: in partnership with the local center for disease control, we collected a total of twelve pharyngeal swab specimens as well as demographic information for the affected patients. we used multiplex real-time pcr to screen for sixteen common respiratory viruses in these samples. to isolate hadv, we inoculated hep- cells with the human adenovirus (hadv)-positive samples and then carried out sequencing and phylogenetic analysis of the hexon, fiber, and penton genes of the isolated adenoviruses. in addition, we analyzed the entire genome of one strain isolated from the index case to identify single-nucleotide substitutions. results: we identified ten hadv-positive students using multiplex real-time pcr. none of the students were co-infected with other viruses. we successfully isolated seven hadv strains from the pharyngeal swab specimens. the coding sequences of the hexon, fiber, and penton genes of these seven hadv strains were identical, suggesting that they represented seven strains from a single virus clone. one hadv isolate obtained from the index case, bjdx- - , was selected for whole genome analysis. from this isolate, we obtained a , -nucleotide sequence. the genome of bjdx- - clustered with hadv-b in phylogenetic analyses and had . % identity with human adenovirus isolate hadv-b/chn/bj / / (genbank accession no. jx ). conclusions: we identified hadv-b as the strain associated with the august ari outbreak at a training base in beijing. this was the first reported outbreak in beijing due to hadv-b . continuous surveillance of respiratory adenoviruses is urgently needed to understand the epidemiological and evolutionary features of hadv-b , and an epidemiological modeling approach may provide further insights into this emerging public health threat. furthermore, the clinical laboratory data from this outbreak provides important reference for the clinical diagnosis and may ultimately aid in informing the development of strategies to control and prevent respiratory tract infections caused by hadv-b . human adenoviruses (hadvs) cause a wide variety of clinical manifestations, including respiratory tract infection, gastroenteritis, kerato-conjunctivitis, acute hemorrhagic cystitis, nephritis, hepatitis, and encephalitis [ ] [ ] [ ] . hadvs are responsible for - % of all respiratory illnesses and for - % of pneumonias in children [ , ] . most hadv infections are mild, self-limiting, and indistinguishable from other viral infections. however, the illnesses caused by hadvs can be severe or even fatal and can result in substantial morbidity [ , ] . outbreaks of hadv-associated acute respiratory infection (ari) usually occur in healthy children or in adults in enclosed or crowded settings [ , ] . hadv was first reported as a viral pathogen in [ ] . since this initial identification, hadvs have been classified into seven species (a to g), and the human adenovirus working group has identified hadv types as of july (http://hadvwg.gmu.edu/) [ ] [ ] [ ] [ ] [ ] [ ] . adenoviruses are non-enveloped icosahedral particles that contain linear double-stranded dna genomes with sizes ranging from to kb. adenovirus genomes are characterized by inverted terminal repeat sequences (itrs) with sizes ranging from to over bp [ ] . the hadv viral capsid, which surrounds the genome, is composed of three major proteins: hexon, penton base, and fiber [ ] . different hadv species display various tissue tropisms that correlate with the different clinical symptoms of infection [ ] . hadv species a has often been associated with the gastrointestinal tract, whereas species b (hadv- , , , and ), c (hadv- , , , and ), and e (hadv- ) are known to cause respiratory tract infections. species d (hadv- , , and ) commonly causes adenoviral kerato-conjunctivitis. species f variants, including hadv-f and-f , and species g variant hadv-g are mainly associated with gastrointestinal tract infections [ ] . hadvs have been associated with previous outbreaks of ari. hadv-b and hadv-b cause frequent outbreaks in the united states [ ] . in asia, the prevalence of hadvs in patients with aris has ranged from . to . % [ ] [ ] [ ] [ ] [ ] . recently, guo et al. ( ) reported that hadv-b , hadv -b , hadv -b , and hadv -b were the most frequently detected virus strains among patients with acute adult adenovirus infections in beijing from may to july [ ] . in china, hadv-b and hadv-b , two hadv species b subtypes, are common causes of epidemic ari outbreaks [ ] [ ] [ ] [ ] . in , an ari outbreak occurred in qishan, shaanxi province, china. a re-emergent isolate of hadv-b (qs-dll), originally described as hadv- a and fully characterized in , was reported to be the cause of this outbreak [ ] . this re-emergent hadv-b was shown to have evolved from a hexon recombination between hadv-b and hadv-b [ , ] . since its characterization, hadv-b has been associated with several respiratory infection outbreaks and is known to be responsible for severe respiratory diseases [ , [ ] [ ] [ ] [ ] . here we describe an outbreak of ari caused by hadv-b at a training base in the daxing district of beijing, china. to help identify the causative pathogen, we collected pharyngeal swab specimens from the affected students and carried out molecular detection and typing, phylogenic analysis, and whole-genome sequencing. this is the first reported outbreak of ari in beijing due to hadv-b . the training base where the hadv-b outbreak took place on august , , local public health authorities were informed about an outbreak of ari among young students at a training base located in the daxing district of beijing. the training base consisted of two three-floor buildings for teaching and three three-floor dormitories with eight persons to a room. the training base recruits only male middle school graduates. approximately students majoring in mathematics, chinese, and english were enrolled in a total of sixty classes. the training base employs full-time staff members. on august , , one student reported symptoms of an ari and had a body temperature of . °c. by august , a total of ari patients from the same class were reported by the local hospital. for the purposes of our analysis, we defined ari cases as individuals with a body temperature over . °c and with at least one symptom of a respiratory tract infection, such as cough or sore throat. on august , the daxing district center for disease prevention and control (cdc) began an epidemiological investigation, collecting demographic, clinical and laboratory data. under the guidance of the cdc, the training base took precautionary measures, including quarantining the affected students, carrying out a routine cleaning and disinfection of living quarters, and morning body temperature checks. no further new cases were reported by september , . pharyngeal swab samples were obtained from each of the twelve students infected in this ari outbreak. the specimens were collected in ml vials containing viral transport medium and quickly transported on ice to the laboratory of the daxing district cdc. the specimens were stored at − °c until further use. patient information and laboratory results are shown in table . nucleic acids were extracted from μl of each of the clinical samples using qiaamp viral rna mini kits (qiagen, hilden, germany) according to the manufacturer's instructions. pharyngeal swab specimens were screened for common respiratory pathogens via realtime pcr multiplex assays using commercial kits (uninovo biological technology, zhenjiang, china) as described by shi w., et al. [ ] . the pathogens assayed using this approach were influenza virus a (h ), pandemic influenza virus a (h n ), influenza viruses a and b (flu a and b), parainfluenza viruses , , , and (piv , , , and ), human metapneumovirus (hmpv), human bocavirus (hbov), human coronavirus oc / nl , e/ hku , human respiratory syncytial virus (hrsv), human rhinovirus (hrhv), and hadv. hep- cells were inoculated with hadv pcr-positive specimens and cultured in high-glucose dulbecco's modified eagle medium (gibco, ny, usa) containing % fetal bovine serum (gibco, ny, usa), u/ml penicillin (gibco, ny, usa), and mg/ml streptomycin (gibco, ny, usa) at °c in a % co incubator for week following standard protocols [ ] . the cultured cells were checked regularly for cytopathic effects (cpe) and harvested when cytopathic effects (cpe) were observed. cultures with cpe were screened for specific hadvs as described by kim c et al. [ ] . molecular typing of hadvs was performed via conventional pcr using specific primers targeting the complete coding sequences of the hexon, fiber, and penton genes [ ] . viral dna was extracted from cultured medium using qiaamp rna mini kits (qiagen, hilden, germany) according to the manufacturer's instructions [ ] . conventional pcr was conducted using highfidelity dna polymerase (takara, dalian, china) according to the manufacturer's instructions. the hexon, fiber, and penton genes of hadv were amplified as described previously [ ] . for the hexon and penton genes, the pcr protocol was: °c for min., followed by cycles of s. at °c, s. at °c, min. at °c, and a final extension step of °c for min. the pcr protocol for amplification of fiber gene fragments was identical, except for the annealing temperature, which was °c instead of °c. the amplified pcr products were excised from agarose gels, purified using an axyprep dna gel extraction kit (axygen, hangzhou, china), and bidirectionally sequenced using the sanger sequencing method by invitrogen biotechnology co., ltd. (invitrogen, beijing, china) with an abi dna analyzer (applied biosystems, austin, tx, usa). to further analyze mutations in the genome sequences of the viruses isolated in this ari outbreak, we sequenced the whole genome of one isolate from the index case using the sanger method. targeted - kb segments that covered the entire genome with overlapping sequences of about bp were amplified by pcr. the ′ and ′ itrs of the genome were amplified and cloned into a plasmid t-vector and then sequenced. a set of pairs of primers was designed in-house to amplify the whole genome according to the reference sequence (genbank accession no. fj ) and then used for separate pcrs. primer sequences are available upon request. whole-genome sequencing segments were amplified using high-fidelity polymerase (takara, dalian, china) using . mm of each primer. pcrs were carried out using a biorad thermocycler (applied biosystems, austin, tx, usa) with the following protocol: °c for min., followed by cycles of s. at °c, s. at appropriate annealing temperature for separate primers, min. at °c, and a final extension step of °c for min. the amplified segments were purified and bi-directionally sequenced. gaps and ambiguous sequences were pcramplified using new primers and re-sequenced. dna sequence fragments were assembled using the seqman program implemented in dnastar lasergene . (dnas tar, inc. madison, wi) into a single contig. the genomic sequence determined in this study was deposited in gen-bank under accession number mk . nucleotide sequence homologies were identified using the basic local alignment search tool (blast, https://blast. ncbi.nlm.nih.gov/). multiple nucleotide sequence alignments were performed using the clustalw program implemented in bioedit. comparisons between the whole genome sequence of the bjdx- - virus strain and those of other types of hadvs were generated using clc genomics workbench (qiagen, hilden, germany). phylogenetic trees were constructed using the maximum likelihood method in the mega program (version . ). one thousand bootstrap replications were used to estimate distances. bootstrap values greater than % are shown for selected nodes in fig. (a-d) . wholegenome sequences and hexon, fiber, and penton gene sequences from other hadvs were downloaded from genbank on april , . on august , , a -year old male student developed a case of ari, with a peak body temperature of . °c. by august , a total of male students were infected. no females were infected. the mean age of the infected students was . years (median, years; range, - years). the distribution of daily cases is shown in fig. . reported students were from the same class but living in six different dormitory rooms. the outbreak lasted for days. the clinical symptoms of the infected students are described in table . all infected individuals had a fever; ( %) students had a sore throat, and ( . %) students had a headache. other symptoms reported by the patients were cough, body ache, stuffy nose, and diarrhea. all affected students had normal white blood counts. most of the infected students were treated in outpatient clinics; only one patient, who had non-severe pneumonia, was hospitalized. the index case (id no. ) was diagnosed as having an upper respiratory infection with a peak temperature of . °c, a neutrophil count of . % (normal range, - %), percentage of lymphocytes of . % (normal range, - %), and white blood cell count of . × / l (normal range, . - . × /l). the hospitalized student (id no. ) had a peak temperature of . °c accompanied by a cough, headache, sore throat, and diarrhea. he had a neutrophil count of . % (normal range, - %), a percentage of lymphocytes of . % (normal range, - %), and a white blood cell count of . × /l (normal range, . - . × /l). a chest x-ray showed patchy shadows on the right lung of the hospitalized student. a total of respiratory samples were obtained from the students. ten specimens were shown to be hadvpositive using multiplex pcr (uninovo biological technology, jiangshu, china). none of the hadv-positive patients were co-infected with other respiratory viruses. to isolate viruses, we inoculated hep- cells with the hadv-positive samples and isolated seven hadv virus strains. using typing primers, which allow for the determination of viral type, we sequenced the hexon, fiber, and penton genes in the seven hadv strains [ ] . the hexon ( bp), fiber ( bp), and penton ( bp) sequences from the seven hadv isolates were % identical, suggesting that this outbreak was caused by a single viral strain. we then compared the hexon, fiber, and penton sequences of one of the viral strains isolated from the index case (id no. ; referred to as bjdx- - ), with other hadv-b , hadv-b and hadv-b strains. based on blast analysis, the hexon, fiber, and penton genes were . , , and % identical to the genes of the hadv-b reference strain qs-dll in china (genbank accession number fj ), respectively,. the hexon, fiber, and penton genes were each %, identical to the genes of the isolate hadv-b/chn/bj / / (genbank accession no. jx ; table ), which was identified from a single patient in beijing with severe community-acquired pneumonia (cap) who was infected with hadv-b . one of the seven strains, bjdx- - , was selected for further whole-genome studies. we obtained and assembled the full genome of the index case isolate, bjdx- - . the sequence of this genome has been deposited in genbank (accession number mk ). the complete genome of bjdx- - was , nucleotides in length and had -bp inverted terminal repeat sequences in the ′-and ′untranslated regions ( ′-utr and ′-utr). to investigate the genetic relationships between isolate bjdx- - and other hadv strains, we constructed phylogenetic trees (fig. (a-d) ) using the maximum likelihood method based on the complete hexon, fiber, and penton gene sequences of strain bjdx- - and other hadv strains. all phylogenetic trees showed that bjdx- - , associated with this outbreak, clustered with hadv-b . the bjdx- - hexon gene also clustered with hadv-b (genbank accession number af ), while the fiber and penton genes and the full bjdx- - genome clustered with hadv-b (gen-bank accession number ay and jx ). comparisons of the whole genome sequence of the bjdx- - virus strain with other hadv-b , hadv-b and hadv-b strains are shown in fig. . fig. ). we then generated alignments between the genome sequence of strain bjdx- - and those of strains hadv-b , hadv-b , and hadv-b . most of the nucleotide differences in bjdx- - relative to hadv-b/chn/bj / / (genbank accession number jx ) were observed in the coding region of protein vi (fig. a) , where we found seven nucleotide changes and six amino acid substitutions. we also found different numbers of poly "t" and ploy "a" tracts (fig. b) . this study describes an ari outbreak with students infected at a training base in the daxing district of beijing, china, in august . based on epidemiological and laboratory investigation, we confirmed that the etiologic pathogen of this ari outbreak was hadv-b . hadv-b has received additional attention in recent years. hadv-b is a recombinant virus, and it is associated with more severe acute respiratory diseases than other types of hadvs [ ] . hadv-b was originally described as hadv-b a based on earlier putative, sporadic occurrences in spain ( ) [ ] , turkey ( ) fig. percent nucleotide identity and differences in the genome of bjdx- - relative to strains of hadv-b , hadv-b , and hadv-b . the numbers above the white grids represent the percent nucleotide identity, while those below the grids represent the nucleotide difference values calculated by clc genomic workbench fig. (a) alignment analysis on the coding sequence of protein vi among hadvs. '.' represents identical bases in the sequence alignment. (b) different numbers of poly "t" and poly "a" tracts were observed in the aligned genomes of the hadv-b , hadv-b , and hadv-b strains. '.' and '-' represent identical bases and deletions in the sequence alignment, respectively [ ] , and singapore ( ) [ ] . hadv-b was also identified as a re-emergent acute respiratory disease pathogen after a recent outbreak in qishan county, shaanxi province, china in [ , ] . subsequent analysis revealed that hadv-b consists of an hadv-b backbone and a partial hadv-b hexon gene. this re-emergent virus exhibited a neutralizing antigen epitope of hadv-b and the pathogenic properties of hadv- . therefore, this virus was renamed hadv-b [ , ] . in china, an increasing number of outbreaks of hadv-b have been reported since march [ ] . epidemic outbreaks of hadv-b have occurred in military camps, schools, and even hospitals in hebei province (february ) [ ] , tianjin city (january ) [ ] , beijing ( ; this study), guangzhou city ( ) [ ] , tibet, sichuan and yunnan provinces ( ) [ ] . in the hadv-b outbreak in china, a total of patients were infected, and one died [ ] . although the virus spread quickly in the outbreak reported here, only individuals were infected, including one student who developed pneumonia and was hospitalized. this suggests that the hadv-b strain associated with this outbreak is not as virulent as those reported previously [ , ] , yet the strain should still be considered an urgent public health threat that necessitates measures to contain or control it in order to prevent epidemics. unlike other outbreaks caused by hadvs in the northern regions of china [ , , ] that occurred in the winter or spring, this outbreak in beijing took place at the end of summer. studies by yu j., et al. and liu t., et al. have reported that, although infections of hadvs occur throughout the year, hadv outbreak prevalence often peaks in winter and spring in the north of china and in summer and spring in the south of china [ , ] . it is possible that hadv outbreaks differ in their seasonality based on relative humidity and temperature. the fact that the hadv outbreak reported here occurred in summer is thus significant because it could indicate this hadv-b virus strain is capable of circulating in the climate and environmental reservoir. the hexon, fiber, and penton genes of virus strain bjdx- - isolated in this study shared % sequence identity with those of the hadv-b strain hadv-b/chn/bj / / (genbank accession number jx ), which was isolated from a patient with severe cap in a previous study by bin cao et al. at beijing in [ , ] . the genome of bjdx- - is most similar to that of hadv-b/chn/bj / / . in our study, the strain of bjdx- - was obtained from the index case (id# ), who presented with an upper respiratory infection. furthermore most of the infected students were treated in outpatient clinics, and only one patient with non-severe pneumonia was hospitalized. the isolate of hadv-b/chn/bj / / was obtained from a patient with severe cap in a previous study by bin cao et al. at beijing [ ] , in which a total of cases with laboratory-confirmed adenovirus infections, including the mentioned case with hadv-b/ chn/bj / / , were detected in cap cases. furthermore, hadv-b was most frequently detected in the cases of adenovirus pneumonia ( / ), and six of the patients ultimately developed acute respiratory distress syndrome. based on the information of the two cases, two genetic viral samples, and the two independent studies that reported laboratory-confirmed aris with hadv-b in beijing, the isolate of bjdx- - led to milder aris. these findings imply that severe aris were not simply caused by hadv-b virus strains themselves but also depend on the conditions of the hosts, such as the individual's age, general state of health, and the presence of co-morbidities or additional infections. in this outbreak, we also found that for both the index case and the hospitalized case neutrophils increased and /or lymphocytes in the peripheral blood decreased. studies on other respiratory viruses, such as influenza virus, respiratory syncytial virus, or human rhinovirus, have shown that an increase in neutrophils has a significant role in limiting virus replication [ , ] . thus, the increase in neutrophils observed here may have limited and resulted in milder clinical symptoms in this outbreak. most of the mutations at the amino acid level in bjdx- - were observed in the coding regions of protein vi. during the replication of hadvs, protein vi functions as an adaptor to shuttle the hexon protein to the nucleus, where virus assembly occurs [ ] . whether these variations affect the virulence of bjdx- - and are responsible for the milder clinical symptoms observed in this study requires further investigation. we also found that bjdx- - has different numbers of poly "t" and poly "a" tracts compared with other hadv-b , hadv-b , and hadv-b strains. the role that poly "t" and poly "a" tracts play in the evolution of hadvs remains unclear and requires further research. the increasing frequency of ari outbreaks due to hadv-b suggests that this re-emergent virus poses a serious threat to public health. it is therefore urgent that the local cdc improve epidemiological and virological surveillance of hadv-b . we identified hadv-b as the cause of a recent localized ari outbreak. this incident was the first reported outbreak in beijing that can be attributed to this reemergent virus. our findings show that the risk of an hadv-b epidemic should be paid more attention in beijing, the capital of china, with a population of more than million. continuous surveillance of respiratory adenoviruses is an urgent need to understand the epidemiological and evolutionary features of hadv-b and could also find value in an epidemiological modeling approach. we also found that the clinical laboratory data from this outbreak provides important reference for clinical diagnosis and may ultimately aid in informing the development of strategies to control and prevent respiratory tract infections caused by hadv-b . abbreviations pcr: polymerase chain reaction; ari: acute respiratory infection; hadv: human adenovirus; itr: inverted terminal repeat; cdc: center for disease prevention and control; cap: community-acquired pneumonia; utr: untranslated region adenovirus: epidemiology, global spread of novel serotypes, and advances in treatment and prevention new adenovirus species found in a patient presenting with gastroenteritis an outbreak of epidemic keratoconjunctivitis caused by a new intermediate adenovirus /h identified by molecular typing epidemiology of severe pediatric adenovirus lower respiratory tract infections in manitoba, canada community-acquired pneumonia requiring hospitalization among u.s. adults isolation of a cytopathogenic agent from human adenoids undergoing spontaneous degeneration in tissue culture human adenovirus: viral pathogen with increasing importance emergence and re-emergence of respiratory adenoviruses in the united states computational analysis identifies human adenovirus type as a 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adenovirus in china whole-genome analyses of human adenovirus type emerged in tibet, sichuan and yunnan in china comparison of the prevalence of respiratory viruses in patients with acute respiratory infections at different hospital settings in north china viral etiology of acute respiratory tract infections in hospitalized children and adults in shandong province severe community-acquired pneumonia caused by adenovirus type in immunocompetent adults in beijing genome sequence of human adenovirus type , a re-emergent acute respiratory disease pathogen in china the role of neutrophils in the upper and lower respiratory tract during influenza virus infection of mice respiratory syncytial virus and neutrophil activation a single maturation cleavage site in adenovirus impacts cell entry and capsid assembly publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors thank all colleagues in the beijing cdc for their help in molecular detection and whole-genome analysis. they also thank the technicians of the daxing cdc of beijing for their excellent technical assistance with the epidemiological investigation and with the initial laboratory diagnosis. all authors made significant contributions to the data, analysis, and drafting of this manuscript and approved the final submitted version. gl wrote the manuscript. gl, xp and rl performed genomic analysis. yl, dz and jz conducted the diagnostic experiments. zw, xw, ys and lz participated in collecting information of this outbreak. py and qw helped review this manuscript. all authors have read and approved the manuscript. this study was supported by the research on early detection, genetic evolution and risk assessment for novel influenza virus by capital's funds for health improvement and research ( - - ). the datasets used and/or analyzed in the current study are available from the corresponding author upon reasonable request. this study was approved by the ethics committee of beijing cdc. written informed consent was obtained from all the participants or the guardians if participants were under years of age. not applicable. the authors declare no competing interests. key: cord- -ystkjdwk authors: gao, yi-jie; ye, lei; zhang, jia-shuo; yin, yang-xue; liu, min; yu, hong-biao; zhou, rong title: clinical features and outcomes of pregnant women with covid- : a systematic review and meta-analysis date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: ystkjdwk background: the recent covid- outbreak in wuhan, china, has quickly spread throughout the world. in this study, we systematically reviewed the clinical features and outcomes of pregnant women with covid- . methods: pubmed, web of science, embase and medline were searched from january , , to april , . case reports and case series of pregnant women infected with sars-cov- were included. two reviewers screened studies and studies were included. four reviewers independently extracted the features from the studies. we used a random-effects model to analyse the incidence (p) and % confidence interval ( % ci). heterogeneity was assessed using the i( ) statistic. results: the meta-analysis included pregnant women with covid- . the results were as follows: positive ct findings ( %; % ci, . – . ), caesarean section ( %; % ci, . – . ), fever ( %; % ci, . – . ), lymphopenia ( %; % ci, . – . ), coexisting disorders ( %; % ci, . – . ), cough ( %; % ci, . – . ), fetal distress ( %; % ci, . – . ), preterm labor ( %; % ci, . – . ), and severe case or death ( %; % ci, . – . ). the subgroup analysis showed that compared with non-pregnant patients, pregnant women with covid- had significantly lower incidences of fever (pregnant women, %; non-pregnant patients, %; p < . ) and cough (pregnant women, %; non-pregnant patients, %; p < . ). conclusions: the incidences of fever, cough and positive ct findings in pregnant women with covid- are less than those in the normal population with covid- , but the rate of preterm labor is higher among pregnant with covid- than among normal pregnant women. there is currently no evidence that covid- can spread through vertical transmission. unexplained clusters of pneumonia cases related to the south china seafood wholesale market were reported in wuhan city, hubei province, china, in december [ ] . chinese scientists isolated the novel coronavirus from patients, sequenced the genome, and found that the genetic sequence of the virus was at least % similar to that of human severe acute respiratory syndrome coronavirus (sars-cov). the world health organization (who) named the novel coronavirus -ncov [ ] , also called severe acute respiratory syndrome coronavirus (sars-cov- ), which causes coronavirus disease (covid- ) [ ] . as of june , , more than , cases have been confirmed in china, and , , cases have been confirmed worldwide [ ] . both sars-cov- and sars-cov are βcoronaviruses. the mortality rate of sars-cov infection is %, including a mortality rate of % for maternal infection [ ] . the clinical outcomes of pregnant women are worse than those of non-pregnant women. to date, clinical data on pregnant women with sars-cov- are very limited. therefore, we conducted this systematic review and meta-analysis to assess the clinical features and pregnancy outcomes of pregnant women with covid- to help formulate clinical treatment strategies. the protocol for the meta-analysis was based on the moose (meta-analysis of observational studies in epidemiology) checklist [ ] and equator reporting guidelines (preferred reporting items for systematic reviews and meta-analyses) [ ] . we only conducted a literature review; thus, ethics approval was not required. we systematically searched the literature in the pubmed, web of science, embase, and medline databases. the retrieval period was from january , , to april , we hand-searched the bibliographies of the retrieved papers for additional references. inclusion criteria ) case reports, case series and observational studies of pregnant women with covid- . ) description of the clinical features and/or outcomes of the patient and the foetus/new-born. ) literature that has been republished; ) article types including authors' replies, editorials, guidelines; ) case reports, case series and observational studies that have a number of cases less than ; ) literature with incomplete or missing data. the two reviewers (ly and js.z) independently screened the literature based on the search strategy, inclusion criteria and exclusion criteria and extracted relevant data. when the reviewers' opinions were inconsistent, they sought the opinion of the third reviewer (yj.g) or negotiated solutions. four reviewers (yj.g, yx.y, ml, and hb. y) independently extracted the following features of the literature, listed in the study characteristics section: first author, publication date, study date, the number and age of patients and the number of severe cases or deaths, fever, cough, lymphopenia, positive ct findings, coexisting disorders, preterm labor, caesarean section, fetal distress, neonatal asphyxia or neonatal death or stillbirth, neonatal infection, and virus in the breast milk. they also evaluated the quality of the literature using the institute of health economics (ihe) case series methodological quality evaluation tool [ ] , which evaluated areas of the literature: ( ) research purpose, ( ) research population, ( ) intervention and joint intervention, ( ) outcome measures, ( ) statistical analysis, ( ) results and conclusions, ( ) conflict of interest and funding sources, and ( ) new entry. of the aforementioned items that were extracted and evaluated for each study, studies that provided information related to ( %) or more of the items were considered to be of acceptable quality. all calculations were performed with review manager software (version . , nordic cochrane centre) and were guided by the previous work, implement meta-analysis with non-comparative binary data in revman software [ ] . the i statistic was used to assess heterogeneity among the studies. an i of less than % indicated low heterogeneity, - % indicated medium heterogeneity, and more than % indicated high heterogeneity. because of the high heterogeneity of this study, we used a random effects model to pool the studyspecific frequencies and % confidence intervals ( % cis) of the clinical features or outcomes. a p < . using the cochran's q test was considered statistically significant. funnel plots were used to assess the publication bias. a subgroup analysis was used to assess the sensitivity. a total of relevant documents were retrieved by the search methods above, including articles from pubmed, articles from web of science, articles from embase, and articles from medline. we hand-searched the bibliographies of the retrieved papers, and additional articles were included. after the removal of duplicate documents, papers were deemed ineligible after the title and abstract screening, and papers were excluded after further screening through reading the full text. after the exclusion of all unqualified studies, a total of retrospective case analyses were included in this meta-analysis [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the process of the study selection is illustrated in fig. . we extracted the features of the literature above. the study included pregnant women with laboratoryconfirmed covid- from december , , to april , , of whom were in china and were in america. the characteristics of the included literature are presented in table . we evaluated the quality of the fourteen included documents according to the ihe case series methodological quality evaluation tool. thirteen articles had quality values ranging from to %, all of which were of low quality because the values were lower than %. only one article had a quality value that reached % and was considered to be of acceptable quality. these articles were all retrospective studies with few cases and without case was neonatal death control groups, interventions or blind methods, so they were rated as low quality. however, these are the only published documents at present, which necessitated their inclusion. the literature quality assessment is shown in table . because of the high heterogeneity of this study, we used a random effects model. the meta-analysis showed the following results: the incidence of severe case or death was , % ci: . - . , i = %, p = . ; the incidence of fever was , % ci: . - . , i = %, p < . ; the incidence of cough was , % ci: . - . , i = %, p < . ; the incidence of lymphopenia was , % ci: . - . , i = %, p < . ; the incidence of positive ct findings was , % ci: . - . , i = %, p < . ; the incidence of coexisting disorders was , % ci: . - . , i = %, p < . ; the incidence of preterm labor was , % ci: . - . , i = %, p < . ; the incidence of caesarean section was , % ci: . - . , i = %, p < . ; the incidence of fetal distress was , % ci: . - . , i = %, p = . ; the incidence of neonatal asphyxia or neonatal death or stillbirth was , % ci: − . - . , i = %, p = . ; the incidence of neonatal infection was , % ci: − . - . , i = %, p = . ; and sars-cov- testing of breast milk was only mentioned in the study by chen h ( . . ), and the incidence was , which cannot be calculated by metaanalysis. in summary, the p values of neonatal asphyxia or neonatal death or stillbirth and neonatal infection were both greater than . , which were not statistically significant. we also could not calculate the incidence of a positive sars-cov- testing in breast milk. otherwise, the p values in the remaining indicators were all less than . and were statistically significant. the most common clinical features were positive ct findings ( %), caesarean section ( %), and fever ( %), followed by lymphopenia ( %), cough ( %) and severe case or death ( %). adverse pregnancy outcomes included coexisting disorders ( %), fetal distress ( %) and preterm labor ( %), in descending order. among these indicators, the i value of severe cases or deaths was %, which indicated low heterogeneity. although the indicators mentioned above refer to studies, the incidences in eight documents were all , and there were only two non-zero indicator data points. the i value of preterm labor was %, which indicated low heterogeneity. the i value of cough was %, which indicated medium heterogeneity, and the remaining i values of indicators ranged from to %, which indicated high heterogeneity. furthermore, we carried out a subgroup analysis based on the data from the fourteen retrospective analyses of covid- infection in the pregnant women above and one meta-analysis of the epidemiology of all the patients covid- [ ] . all the patients were divided into two subgroups, namely, pregnant women and non-pregnant patients. in the fifteen articles, only two indices, i.e., fever and cough, were coincident, and were analyzed in subgroups. the results were as follows. the incidence of fever in the pregnant women was %, which was significantly lower than the % fever incidence in the nonpregnant patients (p < . ). the incidence of cough in the pregnant women was also significantly lower than that in the non-pregnant patients ( % vs %, p < . ). the forest plot of the subgroup analysis is illustrated in figs. and . the funnel plots of fever, cough, positive ct findings and coexisting disorders were symmetric, which meant that these indicators may not have publication bias. in contrast, the funnel plots of severe case or death, lymphopenia, preterm labor, caesarean section, fetal distress, neonatal asphyxia or neonatal death or stillbirth and neonatal infection were asymmetric, which meant that the indicators above may have publication bias. since there was only article about sars-cov- testing of breast milk, it was meaningless to draw a funnel plot; therefore, publication bias was not evaluated. the funnel plots of fever and cough are shown in figs. and . the cases discussed in this article involved pregnant women with covid- in china and pregnant women with covid- in america. the pooled results of this meta-analysis showed that among the pregnant women with covid- , % had positive ct findings, % had a caesarean section, % had fever, % had lymphopenia in laboratory examination, % had coexisting disorders, % had cough, % had fetal distress, % had preterm labor and % had severe cases or died. pregnant women with covid- had significantly lower rates of fever and cough than non-pregnant patients with covid- . currently, there are only meta-analyses of the epidemiology of typical patients infected with covid- , and there are few meta-analyses to explore the clinical features and outcomes of pregnant women with covid- . this study is helpful to formulate clinical treatment strategies for pregnant women with covid- . the disadvantage of this study was the small sample size and the general quality of the included documents, which lowered the credibility of the meta-analysis results. in addition, all the included articles were retrospective case analyses without control groups, which fig. the forest plot of subgroup analysis of fever also biased the results. furthermore, the funnel plots showed that most indicators may have publication bias. most of the included patients were chinese, and the others were american. there are few reports describing the cases outside the two regions. further research on pregnant women with covid- worldwide is needed. we referred to the normal population the non-pregnant group to distinguish it from the pregnant group in the subgroup analysis. the subgroup analysis between pregnant women with covid- and non-pregnant patients with covid- showed that the incidences of fever and cough in pregnant women with covid- ( , %) were lower than those in normal people ( , %), which may be due to the changes in the immune system of pregnant women, and further research is needed. the available data do show no increased or even lower maternal mortality rate after infection [ ] , but it does not mean that pregnancy is a protective factor for severe infection. it was reported that severe infection mostly occurred in the elderly (> years old), patients with basic diseases, such as diabetes, obesity, hypertension, coronary heart disease, cerebrovascular diseases, and other chronic diseases [ ] [ ] [ ] [ ] [ ] , as well as those who did not receive timely treatment or delay treatment [ ] . pregnant women are usually younger without primary diseases. besides, pregnant women are usually more likely to receive attention after the onset of the disease (pregnant women themselves, family members, and medical staff), with fewer delays in treatment. all these can explain the low fatality rate of infected pregnant women. there is no evidence that pregnancy can alleviate the disease yet, which needs further study. aya mohr-sasson et al. compared clinical characteristics between pregnant women and non-pregnant women. the study showed that there were no statistical differences in clinical features such as respiratory symptoms and fever between the two groups [ ] . it was reported that male patients were likely to develop more severe symptoms and have higher prevalence rates and mortality rates than female patients [ ] [ ] [ ] . zeng f et al. showed that compared with male patients, more female patients were generating a relatively high level of sars-cov- igg antibody in severe cases, and the igg antibody presented a stronger production in female patients in disease early phase [ ] . it may be the reason why the clinical characteristics of the pregnant group in this study are better than those of the non-pregnant group. a study from the china cdc showed that . % of chinese patients were considered to be asymptomatic or to have mild pneumonia [ ] . desmond sutton et al. showed that of the pregnant women who gave birth at the new york-presbyterian allen hospital and columbia university irving medical center, ( . %) of the patients who were positive for sars-cov- testing had no symptoms of covid- at the time of admission [ ] . the fact that the asymptomatic rate in the infected general population in china is lower than the rate in infected pregnant women in the new york medical center seems to support this conclusion in this study. these findings suggest that the sars-cov- testing should be universally administered in high-risk areas to improve the isolation of asymptomatic infected individuals. this result is different from the finding that pregnant women infected with sars-cov have a worse prognosis than ordinary people infected with sars-cov [ ] . it is possible that in pregnant women, the clinical outcome of covid- infection is better than that of sars-cov. yan et al. confirmed that the current mortality rate of covid- ( %) is significantly lower than that of sars ( . %), which may indicate that sars is more pathogenic and lethal than covid- ; thus, pregnant women with covid- infection had better outcomes than those with sars-cov [ ] . however, our finding that pregnant women with covid- had better clinical features might be biased owing to the relatively small sample included in this meta-analysis. a meta-analysis showed that the ct positive rate of covid- in the normal population was . % [ ] , which was more than the % positive rate in this paper. this finding also corresponded to the conclusion above that the clinical features of pregnant women with covid- were superior to those of the general population. the incidence of positive ct findings was the highest among the selected indicators. shital j. patel et al. confirmed that chest ct was considered a low-dose examination, provided the foetus was excluded from the primary beam, and the estimated radiation doses were too low to induce foetus neurologic deficits during any trimester of pregnancy [ ] . it seemed that chest ct was suitable for routine screening of patients. however, there was a large percentage of pregnant women with asymptomatic infections ( . %) [ ] . if chest ct is used for routine screening, it means that almost all pregnant women need to undergo chest ct. in addition, the who defines screening as the presumptive identification of unrecognized disease in an apparently healthy, asymptomatic population by means of tests, examinations or other procedures that can be applied rapidly and easily to the target population [ ] . consequently, it is not appropriate to perform chest ct as a screening tool for pregnant women with covid- . we recommend using chest ct as the routine examination for suspected cases. the rate of preterm labor in normal pregnant women who are healthy and not infected with any virus worldwide is approximately % [ ] , which is lower than the result in this article ( %). the possible reason for the higher rate is that women in the third trimester of pregnancy induce delivery early after becoming ill with covid- to proceed with further treatment. most of these women choose early delivery by caesarean section to avoid a prolonged labor, which may worsen covid- for pregnant women [ ] and increase the risk of infection for the medical staff [ ] . chen r et al. confirmed that both epidural anesthesia and general anesthesia were safe and effective for women with covid- during caesarean section [ ] . because the p value was greater than . , the rate of neonatal covid- infection should not be considered. wang s et al. reported the first case in china in which a mother with covid- gave birth to an infected baby on february , [ ] , and the instant sars-cov- nucleic acid tests of the umbilical cord blood and placenta were both negative. there were infected neonates in the included literature. khan s. et al. reported that the swab samples tested within h after delivery were positive in two neonates, and intrauterine tissue samples such as placenta, cord blood or amniotic fluid were not tested [ ] . yu n et al. reported that the nucleic acid test for the throat swab of one neonate was positive at h after birth [ ] . without testing the intrauterine tissue samples, we could not confirm whether the sars-cov- infection in the neonate was the result of intrauterine transmission. two studies also showed that the test for sars-cov- -specific antibodies (igg and igm) in neonatal serum samples could be evidence of vertical transmission [ , ] . other literature revealed that almost all the other new-borns from infected women tested negative for sars-cov- [ - , - , , - ] . wang c et al. summarized that there was currently no evidence for intrauterine infection caused by vertical transmission in women with covid- during the third trimester of pregnancy, but it was uncertain whether there could be a risk of vertical transmission when the covid- infection occurs in the first or second trimester or when there was a long clinical manifestation-todelivery interval [ ] . therefore, we must remain alert to the possibility of vertical transmission. the incidence of fever, cough and positive ct findings in pregnant women with covid- is less than that in the normal population with covid- . the rate of preterm labor in normal pregnant women worldwide who are healthy and not infected with any virus is lower than that in pregnant women with covid- . there is currently no evidence that covid- can spread through vertical transmission. the conclusions above are possibly helpful to formulate clinical treatment strategies for pregnant women with covid- . abbreviations sars-cov: .; who: .; -ncov: .; sars-cov- : .; covid- : .; the moose checklist: .; equator reporting guidelines: .; ihe case series methodological quality evaluation tool: .; % ci: . the continuing -ncov epidemic threat of novel coronaviruses to global health -the latest novel coronavirus outbreak in wuhan, china director-general's remarks at the media briefing on accessed dxy.dx doctor covid- global 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literature importance of timely management of patients in reducing fatality rate of coronavirus disease laboratory characteristics of pregnant compared to non-pregnant women infected with sars-cov- clinical features of patients infected with novel coronavirus in wuhan clinical characteristics of coronavirus disease in china clinical course and risk factors for mortality of adult inpatients with covid- in wuhan, china: a retrospective cohort study a comparison study of sars-cov- igg antibody between male and female covid- patients: a possible reason underlying different outcome between sex epidemiology working group for ncip epidemic response, chinese center for disease control and prevention. the epidemiological characteristics of an outbreak of novel coronavirus diseases (covid- ) in china a case-controlled study comparing clinical course and outcomes of pregnant and non-pregnant women with severe acute respiratory syndrome the first days of novel coronavirus (sars-cov- ) outbreak: recent advances, prevention, and treatment coronavirus disease (covid- ) ct findings: a systematic review and meta-analysis imaging the pregnant patient for nonobstetric conditions: algorithms and radiation dose considerations who. cancer: screening accessed the global epidemiology of preterm birth expert consensus for managing pregnant women and neonates born to mothers with suspected or confirmed novel coronavirus (covid- ) infection. international journal of gynaecology and obstetrics: the official organ of the international federation of gynaecology and obstetrics obstetric anesthesia during the covid- pandemic a case report of neonatal covid- infection in china possible vertical transmission of sars-cov- from an infected mother to her newborn antibodies in infants born to mothers with covid- pneumonia pregnant women with new coronavirus infection: a clinical characteristics and placental pathological analysis of three cases perinatal transmission of covid- associated sars-cov- : should we worry? clin infect dis a case of novel coronavirus in a pregnant woman with preterm delivery journal of microbiology, immunology, and infection = wei mian yu gan ran za zhi intrauterine vertical transmission of sars-cov- : what we know so far springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations not applicable. authors' contributions ly and js.z conducted the literature search and selected studies. yx.y assessed the methodologic quality of the studies. ml and hb.y extracted data. yj.g conceived and planned the review, assessed the methodologic quality of the studies, verified the data, and drafted and revised the manuscript. rz provided methodologic advice, content expertise, and revised the manuscript. all authors contributed to writing the protocol. all authors read and approved the final manuscript. not applicable. this work was supported by the national natural science foundation of china (no. , no. ). the mentioned foundation had no rule in the study design, data analysis, drafting the manuscript, or decision to submit this article for publication. the datasets used and/or analyzed during the current study are available from the corresponding author on reasonable request.ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests.received: april accepted: july key: cord- -nr fwc q authors: porten, klaudia; rissland, jürgen; tigges, almira; broll, susanne; hopp, wilfried; lunemann, mechthild; van treeck, ulrich; kimmig, peter; brockmann, stefan o; wagner-wiening, christiane; hellenbrand, wiebke; buchholz, udo title: a super-spreading ewe infects hundreds with q fever at a farmers' market in germany date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: nr fwc q background: in may the soest county health department was informed of an unusually large number of patients hospitalized with atypical pneumonia. methods: in exploratory interviews patients mentioned having visited a farmers' market where a sheep had lambed. serologic testing confirmed the diagnosis of q fever. we asked local health departments in germany to identiy notified q fever patients who had visited the farmers market. to investigate risk factors for infection we conducted a case control study (cases were q fever patients, controls were randomly selected soest citizens) and a cohort study among vendors at the market. the sheep exhibited at the market, the herd from which it originated as well as sheep from herds held in the vicinity of soest were tested for coxiella burnetii (c. burnetii). results: a total of reported q fever cases was linked to this outbreak. the mean incubation period was days, with an interquartile range of – days. the case control study identified close proximity to and stopping for at least a few seconds at the sheep's pen as significant risk factors. vendors within approximately meters of the sheep's pen were at increased risk for disease compared to those located farther away. wind played no significant role. the clinical attack rate of adults and children was estimated as % and %, respectively, % of cases were hospitalized. the ewe that had lambed as well as % of its herd tested positive for c. burnetii antibodies. conclusion: due to its size and point source nature this outbreak permitted assessment of fundamental, but seldom studied epidemiological parameters. as a consequence of this outbreak, it was recommended that pregnant sheep not be displayed in public during the (rd )trimester and to test animals in petting zoos regularly for c. burnetii. q fever is a worldwide zoonosis caused by coxiella burnetii (c. burnetii), a small, gram-negative obligate intracellular bacterium. c. burnetii displays antigenic variation with an infectious phase i and less infectious phase ii. the primary reservoir from which human infection occurs consists of sheep, goat and cattle. although c. burnetii infections in animals are usually asymptomatic, they may cause abortions in sheep and goats [ ] . high concentrations of c. burnetii can be found in birth products of infected mammals [ ] . humans frequently acquire infection through inhalation of contaminated aerosols from parturient fluids, placenta or wool [ ] . because the infectious dose is very low [ ] and c. burnetii is able to survive in a spore-like state for months to years, outbreaks among humans have also occurred through contaminated dust carried by wind over large distances [ ] [ ] [ ] . c. burnetii infection in humans is asymptomatic in approximately % of cases. approximately % of cases are hospitalized, and fatal cases are rare [ ] . the clinical presentation of acute q fever is variable and can resemble many other infectious diseases [ ] . however, the most frequent clinical manifestation of acute q fever is a self-limited febrile illness associated with severe headache. atypical pneumonia and hepatitis are the major clinical manifestations of more severe disease. acute q fever may be complicated by meningoencephalitis or myocarditis. rarely a chronic form of q fever develops months after the acute illness, most commonly in the form of endocarditis [ ] . children develop clinical disease less frequently [ , ] . because of its non-specific presentation q fever can only be suspected on clinical grounds and requires serologic confirmation. while the indirect immunofluorescence assay (ifa) is considered to be the reference method, complement fixation (cf), elisa and microagglutination (ma) can also be used [ ] . acute infections are diagnosed by elevated igg and/or igm anti-phase ii antibodies, while raised anti-phase i igg antibodies are characteristic for chronic infections [ ] . in germany, acute q fever is a notifiable disease. between and the annual number of cases varied from to cases per year [ ] . in and , and cases were notified, respectively [ , ] . on may , the health department of soest was informed by a local hospital of an unusually large number of patients with atypical pneumonia. some patients reported having visited a farmers' market that took place on may and , in a spa town near soest. since the etiology was unclear, pathogens such as sars coronavirus were considered and strict infection control measures implemented until the diagnosis of q fever was confirmed. an outbreak investigation team was formed and included public health professionals from the local health department, the local veterinary health department, the state health department, the national consulting laboratory (ncl) for coxiellae and the robert koch-institute (rki), the federal public health institute. because of the size and point source appearance of the outbreak the objective of the investigation was to identify etiologic factors relevant to the prevention and control of q fever as well as to assess epidemiological parameters that can be rarely studied otherwise. on may and , we conducted exploratory interviews with patients in soest hospitalized due to atypical pneumonia. attending physicians were requested to test serum of patients with atypical pneumonia for mycoplasma pneumoniae, chlamydia pneumoniae, legionella pneumophila, coxiella burnetii, influenza a and b, parainfluenza - , adenovirus and enterovirus. throat swabs were tested for influenza virus, adenovirus and sars-coronavirus. laboratory confirmation of an acute q fever infection was defined as the presence of igm antibodies against phase ii c. burnetii antigens (elisa or ifa), a -fold increase in anti-phase ii igg antibody titer (elisa or ifa) or in anti phase ii antibody titer by cf between acute and convalescent sera. a chronic infection was confirmed when both anti-phase i igg and anti-phase ii igg antibody titers were raised. because patients with valvular heart defects and pregnant women are at high risk of developing chronic infection [ , ] we alerted internists and gynaecologists through the journal of the german medical association and asked them to send serum samples to the ncl if they identified patients from these risk groups who had been at the farmers' market during the outbreak. the objective of the first case control study was to establish whether there was a link between the farmers' market and the outbreak and to identify other potential risk factors. we conducted telephone interviews using a standardised questionnaire that asked about attendance at the farmers' market, having been within km distance of one of sheep flocks in the area, tick bites and consumption of unpasteurized milk, sheep or goat cheese. for the purpose of ccs we defined a case (ccs case) as an adult resident of the town of soest notified to the statutory sur-veillance system with q fever, having symptom onset between may and june , . exclusion criterion was a negative igm-titer against phase ii antigens. two controls per case were recruited from soest inhabitants by random digit dialing. we calculated the attributable fraction of cases exposed to the farmers' market on may (afe) as (or- )/or and the attributable fraction for all cases due to this exposure as: the farmers' market was held in a spa town near soest with many visitors from other areas of the state and even the entire country. to determine the outbreak size we therefore asked local public health departments in germany to ascertain a possible link to the farmers' market in soest for all patients notified with q-fever. a case in this context ("notified case") was defined as any person with a clinical diagnosis compatible with q fever with or without laboratory confirmation and history of exposure to the farmers' market. local health departments also reported whether a notified case was hospitalized. to obtain an independent, second estimate of the proportion of hospitalizations among symptomatic patients beyond that reported through the statutory surveillance system we calculated the proportion of hospitalized patients among those persons fulfilling the clinical case definition (as used in the vendors' study (s.b.)) identified through random sampling of the soest population (within ccs (s.b.)) as well as in two cohorts (vendors' study and the sailor friends (see below)). the objective of ccs was to identify risk factors associated with attendance of the farmers' market on the second day. we used the same case definition as in ccs , but included only persons that had visited the farmers' market on may , the second day of the market. we selected controls again randomly from the telephone registry of soest and included only those persons who had visited the farmers' market on may and had not been ill with fever afterwards. potential controls who became ill were excluded for analysis in ccs , but were still fully interviewed. this permitted calculation of the attack rate among visitors to the market (see below "estimation of the overall attack rate") and gave an estimate of the proportion of clinically ill cases that were hospitalized (s.a.). in the vendors' study we investigated whether the distance of the vendor stands from the sheep pen or dispersion of c. burnetii by wind were relevant risk factors for acquiring q fever. we obtained a list of all vendors including the approximate location of the stands from the organizer. in addition we asked the local weather station for the predominant wind direction on may , . telephone interviews were performed using standardized questionnaires. a case was defined as a person with onset of fever between may and june , and at least three of the following symptoms: headache, cough, dyspnea, joint pain, muscle pain, weight loss of more than kg, fatigue, nausea or vomiting. the relative distance of the stands to the sheep pen was estimated by counting the stands between the sheep pen and the stand in question. each stand was considered to be one stand unit (approximately meters). larger stands were counted as units. the direction of the wind in relation to the sheep pen was defined by dividing the wind rose ( °) in equal parts of °. the predominant wind direction during the market was south-south-east ( figure ). for the purpose of the analysis we divided the market area into sections with the sheep pen at its center. in section the wind was blowing towards the sheep pen (plus minus °). section was on the opposite side, i.e. where the wind blew from the sheep pen towards the stands, and sections and were east and west with respect to the wind direction, respectively. location of the stands in reference to the sheep pen was thus defined in two ways: as the absolute distance to the sheep pen (in stand units or meters) and in reference to the wind direction. we identified a small cohort of sailor friends who visited the farmers' market on may , . all of these were serologically tested independently of symptoms. we could therefore calculate the proportion of laboratory confirmed persons who met the clinical case definition (as defined in the cohort study on vendors). the overall attack rate among adults was estimated based on the following sources: ( ) interviews undertaken for recruitment of controls for ccs allowed the proportion of adults that acquired symptomatic q fever among those who visited the farmers' market on the second day; attributable fraction afe number of cases exposed all cases = * ( ) interviews of cases and controls in ccs yielded information about accompanying adults and how many of these became later "ill with fever"; ( ) results of the small cohort of sailor friends (s.a.); ( ) results from the cohort study on vendors. local health departments that identified outbreak cases of q fever (s.a. "determination of outbreak size and descriptive epidemiology") interviewed patients about the number of persons that had accompanied them to the farmers' market and whether any of these had become ill with fever afterwards. however, as there was no differentiation between adults and children, calculations to estimate the attack rate among adults were performed both with and without this source. to count cases in ( ), ( ) and ( ) we used the clinical case definition as defined in the cohort study on vendors. for the calculation of the attack rate among children elicited in ccs was the same for all visitors. the number of children that visited the market could then be estimated from the total number of visitors as estimated by the organizers. we then estimated the number of symptomatic children (numerator). for this we assumed that the proportion of children with q fever that were seen by physicians and were consequently notified was the same as that of adults. it was calculated as: thus the true number of children with q fever was estimated by the number of reported children divided by the estimated proportion reported. then the attack rate among children could be estimated as follows: because this calculation was based on several assumptions (number of visitors, proportion of adult visitors and clinical attack rate among adults) we performed a sensitivity analysis where the values of these variables varied. serum was collected from all sheep and cows displayed in the farmers' market as well as from all sheep of the respective home flocks ( animals). samples of sheep from five other flocks in the soest area were also tested for c. burnetii. tests were performed by elisa with a phase i and phase ii antigen mixture. we conducted statistical analysis with epi info, version . (cdc, atlanta, usa). dichotomous variables in the case control and cohort studies were compared using the chi-square test and numerical variables using the kruskal-wallis test. p-values smaller than . were considered statistically significant. the outbreak investigation was conducted within the framework of the communicable diseases law reform act of germany. mandatory regulations were observed. patients at the local hospital in soest reported that a farmers' market had taken place on may and , in a spa town close to the town of soest. it was located in a park along the main promenade, spanning a distance of approximately meters. the market attracted mainly three groups of people: locals, inhabitants of the greater soest region, patients from the spa sanatoria and their visiting family or friends. initial interviewees mentioned also that they had spent time at the sheep pen watching new-born lambs that had been born in the early morning hours of may , . the ewe had eaten the placenta but the parturient fluid on the ground had merely been covered with fresh straw. overall ( %) of serum samples submitted to the ncl were positive for igm anti-phase ii antibodies by elisa. results of throat swabs and serum were negative for other infectious agents. (figure ). if we assume that symptom onset in cases was normally distributed with a mean of days, % of cases (mean +/- standard deviations) had their onset between day and . the two notified cases with early onset on may and , respectively, were laboratory confirmed and additional interviews did not reveal any additional risk factors. of the cases with known gender, ( %) were male and ( %) were female. of the notified cases, ( %) were from the county of soest, ( %) were porportion reported number of notified adults number of vis = i iting adults attack rate among adults * attack rate among children estimated true number of childr = e en with q fever estimated number of children at the market from other counties in the same federal state (northrhine westphalia) and ( %) were from five other federal states in germany (figure ). only eight ( %) cases were less than years of age, the mean and median age was and years, respectively ( figure ). ( %) of notified cases were hospitalized, none died. calculation of the proportion of cases hospitalized through other information sources revealed that of ( %; % ci = - %; ( / (ccs ), / (vendors study) and / (sailor friends)) clinically ill cases were hospitalized. laboratory confirmation was reported in ( %) outbreak cases; ( %) were confirmed by an increase in anti-phase ii antibody titer (cf), ( %) had igm antibodies against phase ii antigens, ( %) were positive in both tests and one was confirmed by culture. no information was available as to whether the ( %) cases without laboratory confirmation were laboratory tested. patients with valvular heart defects and eleven pregnant women were examined. none of them had clinical signs of q fever. two ( %) of cardiological patients and four ( %) of pregnant women had an acute q fever infection. during childbirth strict hygienic measures were implemented. lochia and colostrum of all infected women were tested by polymerase chain reaction and were positive in only one woman (case ; table ). serological follow-up of the mothers detected chronic infection in the same woman (case ) weeks after delivery. one year follow-up of two newborn children (of cases and ) identified neither acute nor chronic q fever infections. we recruited cases and controls who visited the farmers' market on may for the second case control study. they did not differ significantly in age and gender (or for male sex = . ; %ci = . - . ; p = . ; p-value for age = . ). seventeen ( %) of cases indicated that they had seen the cow (that also was on display at the market next to the sheep) compared to ( %) of geographical location of q fever outbreak cases notified to the statutory surveillance system figure geographical location of q fever outbreak cases notified to the statutory surveillance system. or directly at the gate of the sheep pen compared to ( %) of controls (or = . ; %ci = . - . ; p = . ). touching the sheep was also significantly more common among cases ( / ( %) ccs cases vs. / ( %) controls; or undefined; lower % ci = . ; p = . ). ( %) of ccs cases, but only ( %) of controls stopped for at least a few seconds at or in the sheep pen, the reference for this variable was "having passed by the pen without stopping" (or = . ; %ci = . - . ; p < . ). among ccs cases, self-reported proximity to or time spent with/close to the sheep was not associated with a shorter incubation period. we were able to contact and interview ( %) of vendors, and received second hand information about more (overall response rate: %). fourty-five ( %) were male and ( %) were female. ( %) met the clinical case definition. of the vendors who worked within two stand units of the sheep pen, ( %) became cases compared to only ( %) of persons who worked in a stand at a greater distance (relative risk (rr) = . ( %ci = . - . ; p = . ); figure ). of these vendors, had spent time within meters of the pen on may , one had been near the pen, but at a distance of more than meters, and no information on this variable was available for the remaining . in the section of the market facing the wind coming from the pen (section , figure ), ( %) of vendors became cases, compared to ( %) of persons who worked in section (p = . ). among persons who worked in stands that were perpendicular to the wind direction, ( %) became cases. (table ). in all scenarios the ar among adults was significantly higher than that among children ( figure ). in total, lambs and ewes were displayed on the market, one of them was pregnant and gave birth to twin lambs at : a.m. on may , . of these, ewes including the one that had lambed tested positive for c. burnetii. the animals came from a flock of ewes, of which had given birth between february and june. the majority of the births ( ( %)) had occurred in february and march, usually inside a stable or on a meadow located away from the town. six ewes aborted, had stillbirths or abnormally weak lambs. among all ewes, / ( %) tested positive for c. burnetii. the percentage of sheep that tested positive in the other sheep flocks in the region ranged from % to % ( %; %; %; %; %). we have described one of the largest q fever outbreaks in germany which, due to its point-source nature, provided the opportunity to assess many epidemiological features of the disease that can be rarely studied otherwise. in , more than cases of q fever were, similar to this outbreak, linked to the abortion of an infected cow at a farmers' market [ ] . more recently a large outbreak occurred in jena (thuringia) in with reported cases [ ] associated with exposure to a herd of sheep kept on a meadow close to the housing area in which the cases occurred. the first case control study served to confirm the hypothesis of an association between the outbreak and the farmers' market. the fact that only attendance on the second, but not the first day was strongly associated with illness pointed towards the role of the ewe that had given birth persons accompanying notified cases (source ) were a mixture of adults and children and are therefore listed separately. in the early morning hours of may , . this strong association and the very high attributable fraction among all cases suggested a point source and justified defining cases notified through the reporting system as outbreak cases if they were clinically compatible with q fever and gave a history of having visited the farmers' market. the point-source nature of the outbreak permitted calculation of the incubation period of cases which averaged days and ranged from to days with an interquartile range of to days. this is compatible with the literature [ ] . an additional interview with the two cases with early onset ( and days after attending the market on may , attack rates among adults and children in a most likely scenario and other scenarios figure attack rates among adults and children in a most likely scenario and other scenarios. most likely scenario: visitors, % adult visitors and % clinical attack rate among adults. scenarios - varied in the assumptions made for "number of visitors", "proportion of adult visitors" and "attack rate among adults" (see table ). displayed are attack rates and % confidence intervals. respectively) could not identify any other source of infection. a short incubation period was recently observed in another q fever outbreak in which the infectious dose was likely very high [ ] . the second case control study among persons who visited the market on may demonstrated that both close proximity to the ewe and duration of exposure were important risk factors. this finding was confirmed by the cohort study on vendors which showed that those who worked in a stand close to (within meters) the sheep pen were at significantly higher risk of acquiring q fever. the study failed to show a significant role of the location of the stand in reference to the wind direction, although we must take into account that the wind was likely not always and exactly as reported by the weather station. however, if the wind had been important at all more cases might have been expected to have occurred among vendors situated at a greater distance to the sheep. according to statutory surveillance system data, the proportion of clinical cases hospitalized was %, similar to the proportion of % found in persons pooled from the other studies conducted. several publications report lower proportions than that found in this investigation: % ( / ) [ ] , % [ ] and % ( / ) [ ] ), and there was at least one study with a much higher proportion ( % ( / )) [ ] . it is unlikely that hospitals reported cases with q fever more frequently than private physicians because the proportion hospitalized among q fever patients identified through random telephone calls in the soest population or those in the two cohorts was similar to that of notified cases. thus reporting bias is an unlikely explanation for the relatively high proportion of cases hospitalized. alternative explanations include overly cautious referral practices on the part of attending physicians or the presumably high infectious dose of the organism in this outbreak, e.g. in those cases that spent time in the sheep pen. the estimated attack rate among adults in the four studies varied between % and %. the estimate of % based on the random sample of persons visiting the market on the second day would seem most immune to recall bias, even if this cannot be entirely ruled out. the estimation based on information about persons accompanying the cases may be subject to an overestimation because these individuals presumably had a higher probability of being close to the sheep pen, similar to the cases. on the other hand the estimate from the cohort study on vendors might be an underestimate, since the vendors obviously had a different purpose for being at the market and may have been less interested in having a look at the sheep. nevertheless, all estimates were independent from each other and considering the various possible biases, they were remarkably similar. in comparison, in a different outbreak in germany, in which inhabitants of a village were exposed to a large herd of sheep (n = - ) [ , ] the attack rate was estimated as %. in a similar outbreak in switzerland several villages were exposed to approximately sheep [ ] . in the most severely affected village, the clinical attack rate was % (estimated from the data provided) [ ] . it is remarkable that in the outbreak described here, the infectious potential of one pregnant ewe -upon lambing -was comparable to that of entire herds, albeit in different settings. our estimate of the proportion of serologically confirmed cases that became symptomatic ( % ( / )) is based on a very small sample, but consistent with the international literature. in the above mentioned swiss outbreak, % of serologically positive patients developed clinical disease [ ] . only approximately half of all symptomatic cases were reported to the statutory surveillance system. patients who did not seek health care due to mild disease as well as underdiagnosis or underreporting may have contributed to the missing other half. our estimated % attack rate among children is based on a number of successive assumptions and must therefore be interpreted with caution. nevertheless, sensitivity analysis confirmed that adults had a significantly elevated attack rate compared to children. while it has been suggested that children are at lower risk than adults for developing symptomatic illness [ , ] few data have been published regarding attack rates of children in comparison to adults. the estimated c. burnetii seroprevalence in the sheep flocks in the area varied from % to %. the % seroprevalence in the flock of the exhibited animals together with a positive polymerase chain reaction in an afterbirth in june suggested a recent infection of the flock [ ] . seroprevalence among sheep flocks related to human outbreaks tend to be substantially higher than those in flocks not related to human outbreaks. the median seroprevalence in a number of relevant studies performed in the context of human outbreaks [ , , ] , was % compared to % in sheep flocks not linked to human outbreaks [ ] . this outbreak shows the dramatic consequences of putting a large number of susceptible individuals in close contact to a single infected ewe that (in such a setting) can turn into a super-spreader upon lambing. there is always a cultural component in the interaction between people and animals, and these may contribute to outbreaks or changing patterns of incidence. during the past decades urbanization of rural areas and changes in animal husbandry have occurred [ ] , with more recent attempts to put a "deprived" urban population "in touch" with farm animals. petting zoos, family farm vacations or the display of (farm) animals at a market such as this may lead to new avenues for the transmission of zoonotic infectious agents [ , [ ] [ ] [ ] . while not all eventualities can be foreseen, it is important to raise awareness in pet and livestock owners as well as to strengthen recommendations where necessary. this outbreak led to the amendment and extension of existing recommendations [ ] which now forbid the display of sheep in the latter third of their pregnancy and require regular testing of animals for c. burnetii in petting zoos, where there is close contact between humans and animals. due to the size and point source nature this outbreak permitted reassessment of fundamental, but seldom studied epidemiological parameters of q fever. it also served to revise public health recommendations to account for the changing type and frequency of contact of susceptible humans with potentially infectious animals. abbreviations afe = attributable fraction of cases exposed the author(s) declare that they have no competing interests. q fever: current concepts a large outbreak of q fever in the west midlands: windborne spread into a metropolitan area? an outbreak of sheep-associated q fever in a rural hyperendemic focus of q fever related to sheep and wind q fever in children diagnosis of q fever rki: meldepflichtige zoonosen annual summary for infectious diseases rki: infektionsepidemiologisches jahrbuch für [annual summary for infectious diseases risks factors and prevention of q fever endocarditis q fever during pregnancy: diagnosis, treatment, and follow-up archiv für hygiene und bakteriologie ausbruch in jena rki: aufklärung eines q-fieber ausbruchs durch erkrankung eines film-teams investigation of a slaughterhouse-related outbreak of q fever in the french alps an important outbreak of human q fever in a swiss alpine valley changing epidemiology of q fever in germany q fever outbreak in cdc: outbreaks of escherichia coli o :h associated with petting zoos -north carolina, florida, and arizona salmonella typhimurium infection in domesticated fowl in a children's zoo ratgeber infektionskrankheiten -merkblätter für Ärzte: q-fieber [physicians' advisor for infectious diseases: q fever we would like to thank the veterinary state laboratory arnsberg (head: franz holling), katharina alpers, andrea ammon and walter haas from the robert koch institute, brunhilde schweiger at the nrl for influenza, colleagues in the institute of public health, state of northrhine westfalia, münster, and local health departments in germany for their assistance in this investigation. kp headed the investigation, analysed data and wrote the first version of the manuscript. jr and uvt assisted in the design of the study and co-ordinated reporting of cases. at co-ordinated activities on the local level and conducted exploratory interviews. sb assisted in the design of the study and co-ordinated collection of veterinary data. wh collected veterinary data and assisted with advice on veterinary issues. ml collected data from reporting health departments. pk, sb and cw co-ordinated laboratory confirmatory testing and followed up patients with valvular heart defects and pregnant women. wh assisted with the study design, literature review and writing of the manuscript. ub conceived of the study, analysed data and wrote later versions of the manuscript. all authors read and approved the final manuscript.publish with bio med central and every scientist can read your work free of charge the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /pre pub key: cord- - njgmdd authors: leecaster, molly; gesteland, per; greene, tom; walton, nephi; gundlapalli, adi; rolfs, robert; byington, carrie; samore, matthew title: modeling the variations in pediatric respiratory syncytial virus seasonal epidemics date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: njgmdd background: seasonal respiratory syncytial virus (rsv) epidemics occur annually in temperate climates and result in significant pediatric morbidity and increased health care costs. although rsv epidemics generally occur between october and april, the size and timing vary across epidemic seasons and are difficult to predict accurately. prediction of epidemic characteristics would support management of resources and treatment. methods: the goals of this research were to examine the empirical relationships among early exponential growth rate, total epidemic size, and timing, and the utility of specific parameters in compartmental models of transmission in accounting for variation among seasonal rsv epidemic curves. rsv testing data from primary children's medical center were collected on children under two years of age (july -june ). simple linear regression was used explore the relationship between three epidemic characteristics (final epidemic size, days to peak, and epidemic length) and exponential growth calculated from four weeks of daily case data. a compartmental model of transmission was fit to the data and parameter estimated used to help describe the variation among seasonal rsv epidemic curves. results: the regression results indicated that exponential growth was correlated to epidemic characteristics. the transmission modeling results indicated that start time for the epidemic and the transmission parameter co-varied with the epidemic season. conclusions: the conclusions were that exponential growth was somewhat empirically related to seasonal epidemic characteristics and that variation in epidemic start date as well as the transmission parameter over epidemic years could explain variation in seasonal epidemic size. these relationships are useful for public health, health care providers, and infectious disease researchers. respiratory syncytial virus (rsv) has long been recognized as a substantial public health threat [ ] with annual epidemics exacting an enormous toll on vulnerable populations and health care delivery systems. rsv is associated with substantial morbidity in children in both the hospitalized and outpatient setting [ ] [ ] [ ] [ ] . in addition to the toll on the health of the population, this disease imposes a large burden on the health care system in terms of human and material resources. although no rsv vaccine exists, infants and children with risk factors for severe rsv infection (eg, lung disease or prematurity) can receive monthly doses of palivizumab, a humanized murine anti-rsv monoclonal antibody, during the rsv season. palivizumab treatment is extremely costly; the cost-effectiveness of this therapy could be improved if treatment is given only during times of high rsv activity. treatment of vulnerable individuals also improves overall health in the population. prediction of seasonal epidemic characteristics including times of high activity and total size would support efficient management of resources and delivery of palivizumab. health care facilities could forecast requirements for beds, staffing, testing, treatment, and other resources needed to care for sick children. for greatest effectiveness, these predictions should be made early in the rsv season; the authors, including public health practitioners and physicians, hold the expert opinion that these predictions would be useful within the first month of the observed start of the rsv seasonal epidemic. in some regions, total epidemic size generally follows a biennial cycle from year to year with smaller epidemic seasons followed by larger epidemic seasons [ ] . this cycle is currently used to gauge upcoming rsv seasonal epidemic size based on total size of the previous epidemic season. the centers for disease control and prevention (cdc) researchers using the national respiratory and enteric virus surveillance system found that the prior epidemic season's data were a relatively imprecise predictor of the epidemic season onset in a given community and that timing of the rsv epidemic season may vary substantially in the same year among communities in close proximity [ ] . one goal of this research was to explore year-to-year variation in epidemic seasons using local data. the biennial variation in our seasonal epidemic data was seen in the early exponential growth rates (slope of the cumulative case curves, figure ) as well as total epidemic size. we explored the relationship between exponential growth of rsv epidemics and the seasonal epidemic characteristics of total epidemic size, days to peak, and epidemic length to assess predictions made early in the epidemic season. knowledge about viral transmission characteristics and the data derived from surveillance systems can be used to inform novel approaches for estimating characteristics of rsv epidemics through the application of methods rooted in epidemiological models of infectious disease transmission [ , ] . these methods are being increasingly applied to emerging threats like sars [ ] [ ] [ ] and pandemic influenza, but their application to routine epidemics of common respiratory viruses like seasonal influenza and rsv has only begun to be explored. weber et al. [ ] model rsv transmission to examine how climate and social factors influence transmission in a population. they consider compartmental models using susceptible-infected-recovered-susceptible (sirs) with additions to include latency and stages of susceptibility. they find no single best model for rsv epidemics; many "competing" models fit the observed data well. we further explored the variation in seasonal epidemics using compartmental models. the variation in exponential growth could potentially be related to variation in transmission rates, epidemic start dates, or proportions susceptible as well as a host of other factors. the second goal of this research was to evaluate the ability of a compartmental model based on epidemiologic principles to fit observed data from a series of epidemics and examine the extent to which seasonal variations in epidemics can be accounted for by variation in specific model parameters. for these analyses, we used daily laboratory data from the major pediatric health care facility in utah where routine viral testing is a fixture of standard clinical care for children presenting to regional emergency departments. the utility of the data from these surveillance systems for relating final epidemic size and modeling the epidemic curve has not been fully evaluated. we investigated the estimation of seasonal epidemic characteristics using regression of exponential growth across seven epidemic seasons. we also modified the model of weber et al. to explore the model fits and estimates of epidemic size using variation of parameters within a susceptible-exposed-infected-infected/detected-recovered (seidr) model. primary children's medical center (pcmc) is a -bed children's hospital that serves both as a community pediatric hospital for salt lake county, utah ( population million [ ] ), and as a tertiary referral center for five states in the intermountain west (utah, idaho, wyoming, nevada, and montana, total population . million [ ] ). eighty percent of pediatric hospital admissions occurring in salt lake county and % occurring in the state of utah are at pcmc. during the study period, july through june , direct respiratory sampling (mainly saline-assisted nasopharyngeal aspiration) for respiratory viral testing was performed for about % of children evaluated in the pcmc emergency department for respiratory complaints (unpublished data) and was required for all hospitalized children with respiratory symptoms (eg, upper or lower respiratory tract infection, bronchiolitis, asthma, or bacterial or viral pneumonia). in addition, respiratory viral testing was recommended for all febrile infants one to days of age. test results were used to inform patient cohorting and isolation procedures and to assist with medical management. all samples were initially tested by direct fluorescent antibody staining (dfa). dfa testing was performed three to five times daily depending on the season, with a mean turnaround time of four hours. for all dfa negative specimens, multiplex polymerase chain reaction (pcr) or viral culture was performed. the data included in our analyses were all positive test results from the above sampling protocols from any of the testing methods during the study period. the practice of testing and test methods did not change appreciably during the study period (unpublished data on percentage of children tested and methods used). the data were used as daily counts by age group, under two and over two years old. the rsv epidemic year was defined to be from july of one year through june of the following year. this time period was chosen to place the beginning date close to the middle of the inter-epidemic period, approximately six months from the average historical peak of the seasonal epidemic. this study was reviewed by the institutional review boards of intermountain healthcare and the university of utah and determined by both organizations to be exempt. regression analysis was used to explore the relationship between the initial exponential growth rate and the epidemic season characteristics of size, days to peak, and length using the seven epidemic seasons of rsv data from pcmc. the exponential growth rate, λ t , t , for time interval t to t was calculated as , where x t i denotes the cumulative number of cases at time t i , i = , . the exponential growth rate was calculated at four weeks to assess regression predictions made early in the season. for comparison, exponential growth rate was also calculated at weeks one through six. the total epidemic size was the sum of cases over the epidemic year, including sporadic interepidemic cases. an observable seasonal epidemic start date of t was defined as the start of the first week of the epidemic year with at least five confirmed rsv cases. this was the definition used by the hospital epidemiologists at pcmc to declare the start of rsv outbreaks during the study period. the term seasonal epidemic refers to the period from the epidemic start date until the epidemic end date, defined as the end of the last week of the epidemic year with at least five confirmed rsv cases. the number of days until the peak for the epidemic seasons was calculated as the midpoint day of the largest seven-day moving average window minus the epidemic season start day. the length of the epidemic season was calculated as the epidemic season end day minus the epidemic season start day. relationships between the initial exponential growth rate and seasonal epidemic characteristics were described using the pearson correlation coefficient and assessed using standard regression statistics. the fits of the regression models were assessed using the percent error of the model fits from the observed values. to combine across seasons, the absolute values of the percent errors were averaged providing the mean absolute percent error for the model. we modeled the observed rsv cases using an extension of the sir model that included individuals (c for children and a for adults) that were susceptible (s c and s a ), exposed (e c and e a ), infectious(i c and i a ), infectious and subsequently detected children (d), and recovered combined across children and adults (r). this seidr model was applied to a series of seven epidemic years. the population was split into children less than two years old (children) and those older than two (adults). it has been shown that the initial rsv infection is the most severe and occurs in almost every child in their first two years of life. transmission is modeled as a function of time using a cosine function to mirror the cyclic nature of epidemics [ ] . there is an offset to this cycle (α), which we estimate along with transmission parameter (β). births and deaths (μ) are accounted for in the susceptible class only. achievement of age two is accounted for in all age-separated classes (η). assumptions of simple compartmental models that we made were as presented in koopman [ ] . our seidr transmission model ( figure ) was defined using the following system of non-linear differential here β was the transmission parameter, l the latency period, f the under-two detection fraction, and γ the recovery parameter. all parameters are presented in the next subsection with descriptions, ranges, and reference values from the literature. solution to the set of differential equations is addressed below. to fit the seidr model to the empiric epidemic data, three parameters-latency period, birth and death rate, and recovery period-were specified based on the literature. three parameters associated with variation across epidemic years were estimated: ) the temporal offset of the epidemic cycle (α), ) detection fraction (f), and ) transmission parameter (β). different models were specified to explore the effect of these three parameters. all combinations of these were considered: models with one parameter allowed to vary across seasons, models with two parameters allowed to vary across seasons, and a model with all parameters allowed to vary across seasons. each parameter is described below. birth and death rate (μ) the number of daily births and deaths were entered in the model based on census data for salt lake county. it was assumed that / th of the children in each ageseparated compartment reached the age of two each day. detection fraction (f) the detection fraction parameter reflected the fraction of the rsv epidemic in children under two years old that was captured in our data set. the detection fraction parameter was estimated as a constant parameter across years and also allowed to vary by epidemic year. the latency period is the time between exposure resulting in transmission and time of infectiousness. the latency period was specified using the median value from crowcroft [ ] , five days. the transmission parameter determined the rate of transmission from contacts between infectious and susceptible individuals. we assumed a homogeneous, uniformly mixing population. the transmission parameter was estimated as a constant parameter across years and also allowed to vary by epidemic year. the recovery parameter specifies the time from infectiousness to recovery. this was specified as . , which translates to a ten-day recovery period, following the work by weber [ ] and in the range of one to reported by hall [ ] . the final model parameter was the offset of the annual epidemic cycle. a regular annual cycle is thought to vary due to weather and climate conditions. the seidr model captures the entire epidemic, detected and not detected. prior to observing rsv cases, the epidemic cycle started within the undetected population. this offset parameter was estimated as a constant parameter across years and also allowed to vary by epidemic year. the nonlinear equations were solved using the lsoda function from the odesolve library [ ] in r statistical software [ ] . the parameters were estimated using a grid search. two fitting statistics were used. the estimates were the values that minimized the square root of the sum of standardized squared errors (rse) and/or the square root of the sum of squared standardized errors (rmse the denominator from these measures adjusted for the magnitude of the epidemic curve to avoid fitting the model mainly to the peak, where differences could over-inflate the fitting statistic and under-value differences during the early and late stages of the epidemic. the rmse reduces the effect of fit to the peak more than does the rse. a grid search was used starting with an initial wide range of values for f, β, and α. the search grid was repeated with successively narrowing ranges to minimize the rse. the grid started with the range of reasonable values, - for β and f and one to days for α. the range was reduced and resolution increased iteratively around minimal rse and rmse values. the minimum grid resolution was . for β, . for f, and one day for α. the rses and rmses from the grid search results were used to select the best parameter estimates within each model type (eg, one model type had only transmission rates that varied by epidemic year). the model with all three parameters allowed to vary by epidemic year was fit as a saturated model to provide a benchmark for rse and rmse, along with the schwarz criteria described below, and percent error in estimating epidemic size when evaluating more parsimonious models in which only one of the parameters was allowed to vary by epidemic year. multiple measures were used to compare the models, in part because the schwarz criteria assumed the residuals were independent and identically distributed, which was not the case; they are, in fact, autocorrelated. the schwarz information criterion [ ] were calculated based on the weighted least squares method used for parameter estimation. there were n = data points, days of case data for each of seven years, and k, the number of parameters estimated was in the full model (four parameters for seven years) and in each other model (two parameters for seven years and two parameters overall). the schwarz criteria were calculated as: bic = × ln j= m j + k ln( ) where m represents either the rse or rmse fit statistic [ ] . the absolute values of the percent error in estimating total epidemic size were summed across seasons for comparison of models. the number of children with test-positive rsv infection ranged from cases in - to cases in - ( table ). the median size of the annual epidemic was cases. overall, % of cases were detected between the months of october and april. larger epidemics alternated with smaller epidemics. the amplitude of this biennial cycle was approximately cases. the total number of children (under years of age) tested per epidemic year ranged from approximately to , with numbers of tests increasing over time. overall, % percent of these were positive for rsv, varying according to the biennial cycle. of children tested, % were less than three years old and % were less than years old. of children with positive tests, % were less than three years old and % were less than years old. of the children tested, % were from salt lake county and % of children with positive tests were from salt lake county. exponential growth rates calculated from cases accumulated for four weeks from the observed epidemic season start ranged from . to . (table ) across the epidemic seasons. the effective reproductive numbers ranged from . to . using a serial interval of seven days [ ] . in regression analyses (table ) , the fourweek exponential growth rate exhibited a substantial positive correlation with epidemic size (r = . , p = . ), and was negatively correlated with start day (r = - . , p-value = . ), days to peak (r = - . , p-value = . ), and length of the epidemic (r = - . , p-value = . ). the regression models provided estimates of epidemic season characteristics that were on average within % of observed epidemic season size, % of observed days to peak, and % of observed epidemic length. using exponential growth rates calculated from weeks one through six provided, in general, increasing correlation (table ) . the saturated seidr model was fit to seven epidemic years of observed rsv data with epidemic year-specific rse values that ranged from to , rmse values that ranged from . to . and percent error of total cases that ranged from % to %. the fit statistics for the models with either transmission parameter or table observed rsv epidemic size, start date, days to peak, duration, and -week exponential growth detection fraction estimated as a constant across epidemic year did not differ substantially from those from the saturated model (table ) . the minimum rse model with detection fraction held constant across epidemic years had the smallest % error, smallest schwarz rse criterion, and had other fit statistics nearly equal to the saturated model. the minimum rmse models were, in general, fitting to the tails of the epidemic and resulted in large errors in estimating epidemic size. the pattern of variation in estimates of offset from all models matched the biennial cycle variation in total epidemic size across epidemic years (figure ) . the variation in estimates of the transmission parameter and detection fraction did not necessarily match this cycle for all epidemic years. the parameter estimates for the transmission parameter were negatively correlated with total epidemic size. the seidr model we presented made assumptions that simplified the reality of rsv transmission. we have identified three limitations to the seidr modeling effort. first, the population age separation does not take full advantage of differences in interaction among a non-homogenous population. second, related to this, the parameter values were not allowed to vary within the population. transmission, for instance, could be age-dependent (due, eg, to hand-washing habits). third, the grid search method of parameter estimation did not provide estimated standard errors for parameter estimates, which limited the ability to compare models and seasons. despite these limitations, this seidr model was useful; it modeled the observed rsv cases from pcmc as part of larger unobserved epidemic seasons and provided a framework for investigating the model parameters. the parameters offset and transmission may not be completely identifiable within this framework but more likely represent combined other forces unmeasured here. our future work includes addressing these limitations and expanding the complexity of the models. rsv is carried by all age groups but is, in general, only a concern for infants. thus, an age-stratified model, possibly with different mixing mechanisms, would more closely resemble the true transmission. the biennial cycle of large, early, and short seasonal epidemics followed by smaller, later, and longer seasonal epidemics the next year observed in utah is similar to other published studies of seasonal rsv epidemics in temperate climates. the theories for this phenomenon include the existence and switching of two rsv disease strains, climate patterns, and waning immunity after infection [ , , , [ ] [ ] [ ] . these and other theories could be investigated in more complex models. it is understood that immunity after infection of rsv is partial, at best. this incomplete immunity and severity of re-infections could be incorporated into more complex models [ , ] . finally, future modeling efforts will involve approaches that include measures of uncertainty in parameter estimates, including bayesian methods [ , ] and likelihood and other methods [ , ] . the first main conclusion of this work was that exponential growth was somewhat empirically related to seasonal epidemic characteristics. the variations in epidemic seasons from data collected at pcmc during the seven years of the study can be partially explained by the variation in exponential growth, especially characteristics of epidemic size, peak day, and length of the epidemic. the seven years of data were not sufficient to make conclusive statements on the nature of the relationships. these early findings based on just seven data points can be built upon to explore early prediction of table results of regression analysis using exponential growth to predict epidemic size, days to peak, and length the upcoming rsv epidemic season. these early predictions could be used by hospitals to budget and allocate resources and to coordinate the timing of palivizumab treatment. they can be used by public health to advise clinicians and the public and also to help identify nonstandard epidemics earlier in the season. for example, health departments might take specific actions if the number of observed cases during the season greatly exceeds early predictions. the second main conclusion of this work was that variation of the transmission parameter and the start of the epidemic (offset) over epidemic years could explain the variation in seasonal epidemic size. the three model parameters allowed to vary by epidemic year (detection fraction, transmission parameter, and offset) provided possible rationale for the variation in seasonal epidemic size. the model with detection fraction held constant across epidemic year fits the observed data well with the fewest parameters. the parameter estimates from this model also match the expected biennial pattern of the epidemic years. from the models considered in this study, this one performs best overall (figure ). write the introduction and discussion sections of the text, providing public health perspective to the study. cb helped conduct the literature review and write the introduction and discussion sections of the text. ms conceived the study and directed its implementation, including contributions to all sections of the text. all authors read and approved the final manuscript. respiratory syncytial virus epidemics: the ups and downs of a seasonal virus prospective population-based study of viral lower respiratory tract infections in children under years of age (the pride study) recent trends in severe respiratory syncytial virus (rsv) among us infants economic impact of respiratory syncytial virus-related illness in the us: an analysis of national databases bronchiolitisassociated hospitalizations among us children defining the timing of respiratory syncytial virus (rsv) outbreaks: an epidemiological study variation in timing of respiratory syncytial virus outbreaks: lessons from national surveillance modeling epidemics caused by respiratory syncytial virus (rsv) understanding the transmission dynamics of respiratory syncytial virus using multiple time series and nested models transmission dynamics and control of severe acute respiratory syndrome invited commentary: real-time tracking of control measures for emerging infections different epidemic curves for severe acute respiratory syndrome reveal similar impacts of control measures census bureau population division-counties census bureau population division-states modeling infection transmission respiratory syncytial virus infection in infants admitted to paediatric intensive care units in london, and in their families respiratory syncytial virus infections in infants: quantitation and duration of shedding solvers for ordinary differential equations. r package version . - edn vienna, austria: r foundation for statistical computing development core team: a language and environment for statistical computing generalizing the derivation of the schwarz information criterion multiexponential, multicompartmental and noncompartmental modeling, ii: data analysis and statistical considerations pattern of respiratory syncytial virus epidemics in finland: twoyear cycles with alternating prevalence of groups a and b occurrence of groups a and b of respiratory syncytial virus over years: associated epidemiologic and clinical characteristics in hospitalized and ambulatory children the incidence of infectious diseases under the influence of seasonal fluctuation a stochastic method for solving inverse problems in epidemic modeling bayesian inference for partially observed stochastic epidemics predicting case numbers during infectious disease outbreaks when some cases are undiagnosed inference for nonlinear dynamical systems statistical challenges of epidemic data partial support for this work was provided by the public health services research grant ul -rr from the national center for research resources, nih/niaid u ai and u -a , us cdc # po cd , and the nih/eunice kennedy shriver nichd k -hd . author details division of epidemiology, university of utah school of medicine, salt lake city, usa. department of pediatrics, university of utah school of medicine, salt lake city, usa. division of disease control and prevention, utah department of health, salt lake city, usa.authors' contributions ml performed the analysis and wrote the bulk of the manuscript. pg helped to conceive the study and prepare the data and also wrote a large part of the introduction, methods, and discussion sections of the text. tg advised on the design of the study's analysis and helped prepare the methods and results sections of the text. nw acquired and managed the data. ag provided clinical insight and helped conduct the literature review. rr helped the authors declare that they have no competing interests. key: cord- -str r a authors: al ghamdi, mohammed; alghamdi, khalid m.; ghandoora, yasmeen; alzahrani, ameera; salah, fatmah; alsulami, abdulmoatani; bawayan, mayada f.; vaidya, dhananjay; perl, trish m.; sood, geeta title: treatment outcomes for patients with middle eastern respiratory syndrome coronavirus (mers cov) infection at a coronavirus referral center in the kingdom of saudi arabia date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: str r a background: middle eastern respiratory syndrome coronavirus (mers-cov) is a poorly understood disease with no known treatments. we describe the clinical features and treatment outcomes of patients with laboratory confirmed mers-cov at a regional referral center in the kingdom of saudi arabia. methods: in , a retrospective chart review was performed on patients with a laboratory confirmed diagnosis of mers-cov to determine clinical and treatment characteristics associated with death. confounding was evaluated and a multivariate logistic regression was performed to assess the independent effect of treatments administered. results: fifty-one patients had an overall mortality of %. most patients were male ( %) with a mean age of years. almost a quarter of the patients were healthcare workers ( . %) and % had a known exposure to another person with mers-cov. survival was associated with male gender, working as a healthcare worker, history of hypertension, vomiting on admission, elevated respiratory rate, abnormal lung exam, elevated alanine transaminase (alt), clearance of mers-cov on repeat pcr polymerase chain reaction (pcr) testing, and mycophenolate mofetil treatment. survival was reduced in the presence of coronary artery disease, hypotension, hypoxemia, cxr (chest x-ray) abnormalities, leukocytosis, creatinine > · mg/dl, thrombocytopenia, anemia, and renal failure. in a multivariate analysis of treatments administered, severity of illness was the greatest predictor of reduced survival. conclusions: care for patients with mers-cov remains a challenge. in this retrospective cohort, interferon beta and mycophenolate mofetil treatment were predictors of increased survival in the univariate analysis. severity of illness was the greatest predictor of reduced survival in the multivariate analysis. larger randomized trials are needed to better evaluate the efficacy of these treatment regimens for mers-cov. coronaviruses cause a spectrum of illness from asymptomatic disease to respiratory failure. early reports of coronavirus infections suggested that most infections were mild until the sars epidemic that was associated with significant morbidity and mortality [ ] . in september , a novel coronavirus was identified in a -year old man in saudi arabia [ ] . a second case was identified in a qatari patient hospitalized in the united kingdom [ ] . the two coronaviruses were genetically identical and similar to isolates obtained from bats [ ] . in july , the coronavirus study group named this new virus middle east respiratory syndrome coronavirus (mers-cov) [ ] . as of december , , there have been cases worldwide with deaths [ ] . the epidemiology and clinical manifestations of this disease have described a spectrum of illness from asymptomatic infection to severe respiratory failure and death. the overall mortality rate remains at % [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . importantly, there are no known effective treatments. in there was an increase in mers-cov cases reported from the jeddah region of saudi arabia. to describe the changing epidemiology and outcomes, we report the clinical features and treatment outcomes of patients admitted to a regional referral hospital in jeddah, saudi arabia. king fahd general hospital is an -bed hospital in jeddah, kingdom of saudi arabia and is a regional coronavirus referral center. there are icu beds and one infectious disease physician that serves the hospital. between january through december , all patients admitted or transferred to king fahd hospital with a positive mers coronavirus pcr from clinical nasal swabs or nasopharyngeal aspirates were included. all pcr testing was performed at the ministry of health regional lab in jeddah. the magna pure compact/ magna pure (roche) automated system was used to extract rna from samples. primers and probes for upe and orf a targets of mers-cov were used from tib molbiol (germany) along with master mix from roche for the light cycler ii (roche) were used to amplify upe and orf a gene targets. samples that tested positive for both upe and orf a gene targets with a cycle threshold time of less than were considered confirmed cases. positive and negative controls were used to monitor the amplification process & to check for any inhibition of amplification. medical charts for all patients were reviewed and data abstracted on standardized data collection forms by an infectious disease trained physician. demographic, clinical and laboratory data were entered into a database. to understand the epidemiology, age was categorized as < , - and > . hypotension was defined as blood pressure < / mm hg, tachypnea as a respiratory rate greater than , hypoxia as an oxygen saturation < %, thrombocytopenia as platelets < , / cubic millimeter, leukopenia was defined as a white blood cell count < cells/cubic millimeter and leukocytosis as a white blood cell count > , cells/ cubic millimeter. renal insufficiency was defined as a creatinine > . mg/dl. liver function abnormalities were defined as a lactate dehydrogenase (ldh) > u/liter, alanine transaminase (alt) > u/liter and aspartate aminotransferase (ast) > u/liter. immunosuppression was defined as aids, history of organ transplant, neutropenia, known malignancy, taking immunosuppressive medication and congenital immunodeficiency. pregnancy was considered an immunosuppressed state. a modified acute physiologic and chronic health evaluation (apache ) score was calculated using age, temperature, mean arterial blood pressure, respiratory rate, potassium, creatinine, acute renal failure, and comorbid conditions to estimate severity of illness [ ] . pao was estimated using pulse oximetry oxygen saturation results and hematocrit was calculated by multiplying the hemoglobin times three. all statistical analyses were performed using stata software (version . , college station, tx). the percent distribution of clinical variables among patients who survived and those who died were compared using the fisher exact test. a multivariate logistic regression was done on treatments administered and severity of illness to determine which treatments were associated with survival. mycophenolate mofetil was not included in this logistic regression analysis because % of patients receiving mycophenolate mofetil survived. the association between severity of illness and treatments administered was assessed by performing a linear regression of treatments administered onto the modified apache score. there were a total of cases, thirty patients ( . %) of whom were saudi nationals, and ( . %) were foreign nationals. the median age was years old (iqr . - ). most were male (n = , . %). twenty-one patients ( . %) had exposure to a known patient with mers coronavirus and ( . %) were healthcare workers. none of the patients had animal exposure. two patients ( . %) were on pilgrimage to mecca. overall, % of patient had at least one co-morbid condition. seventeen patients had diabetes ( . %), had hypertension ( %), ( . %) had end stage renal disease, eight ( . %) had coronary artery disease and six ( . %) patients were immunosuppressed, two of whom were pregnant. patients received a variety of novel treatments including immunosuppressants and antivirals. forty-two ( . %) patients received broad-spectrum antibiotics and five ( . %) received hydrocortisone. thirty one patients received antiviral treatment. twenty-three patients ( . %) were treated with interferon beta, eight ( . %) were treated with interferon alpha. a variety of anti-viral combinations were used. eight patients ( . %) received mycophenolate mofetil, seven of these patients received it in combination with interferon beta. nineteen ( . %) patients required intensive care unit (icu) care, and patients received extracorporeal membrane oxygenation (ecmo). all patients treated in the icu and all patients receiving ecmo died. in this recent cohort, when comparing survivors to nonsurvivors, survival was associated with male gender, vomiting on admission, elevated respiratory rate, abnormal lung exam on physical exam, working as a healthcare worker, history of hypertension, elevated alt, clearance of mers cov on repeat pcr testing, and receiving mycophenolate mofetil or beta interferon (table ). in contrast, markers of severe disease like hypotension, hypoxemia, chest radiographic abnormalities, leukocytosis, elevated creatinine, thrombocytopenia, anemia, renal failure were associated with death. treatments given were based as indicated based on the clinical assessment of the infectious disease consult team. thirty-one patients received antivirals, ribavirin or alpha or beta interferon, and patients received immunosuppressive medication. most patients received a combination of alpha interferon and ribavirin ( , . %), beta interferon and ribavirin ( , . %) or beta interferon alone ( , . %). two patients received alpha interferon alone ( . %). eight patients received mycophenolate mofetil ( . %) and seven of them received this in combination with beta-interferon. five patients received hydrocortisone; two in combination with beta interferon and ribavirin and in combination with alpha interferon and ribavirin. all eight patients given mycophenolate mofetil survived therefore mycophenolate mofetil could not be evaluated in this model. while the results of the univariable analysis demonstrated improved survival in patients treated with betainterferon and mycophenolate mofetil, the multivariable analysis which included a marker of severity of illness, demonstrated a strong association between severity of illness and reduced survival, and no association between treatment with beta interferon and survival. mycophenolate mofetil was not evaluable in this model ( table ). in analyzing the relationship between severity of illness and treatments administered, beta interferon and mycophenolate mofetil were given to less severely ill patients (table ) discussion mers-cov is an emerging disease for which the initial epidemiology has been described, but in-depth clinical studies and the role of therapy in incompletely understood. while the clinical features for mers-cov have been described in several large case series [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] , there is a paucity of literature on therapy. our results from a relatively large number of patients demonstrate similar clinical features and mortality to previous studies [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . in our cohort, treatment with beta interferon and mycophenolate mofetil may be predictive of survival, but the greatest predictor of survival is the severity of illness on presentation. improved diagnostics have demonstrated an expanded spectrum of disease that includes less severe cases than previously reported. we now understand that mers-cov causes an acute respiratory disease syndrome and [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . this may be partially related to the epidemiology of increased disease transmission in healthcare settings rather than a true host risk factor. laboratory findings have been nonspecific and consistent with other viral infections. thrombocytopenia ( %) and lymphopenia ( %) have been commonly described in these patients [ , [ ] [ ] [ ] [ ] [ ] ] . forty three percent had acute kidney injury [ , [ ] [ ] [ ] ] and - % had cxr abnormalities with bibasilar infiltrates as the most common finding [ - , , ] . the outcomes in these more severely ill patients remain poor. between - % required icu care [ , , , ] and - % in the icu setting required invasive ventilation for a median of - days [ , , ] . in addition to mechanical ventilation, several patients have received extracorpeal membrane oxygenation (ecmo) to support ventilation. from non-randomized data from the world health organization, five out of six patients receiving ecmo died [ ] . fifty-eight to % required renal replacement therapy [ , , ] and - % of hospitalized patients died [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] [ ] . the severity of illness can be partially explained by the widespread lung disease caused by mers-cov and it appears that mortality in those patients requiring intensive care is extremely high. although no autopsy data is available, in explanted lung, infection with mers-cov causes widespread infection and alveolar disease [ , ] . the clinical features in our cohort similarly also show a high proportion of patients with fever ( %) and cough ( . %) shortness of breath ( %), and almost one third of patients ( . %) with gastrointestinal symptoms. our cohort consisted of ill patients with hypotension ( . %), tachypnea ( . %) and hypoxia ( %). thirty seven percent required icu care and patients received ecmo. similar to previous results, all of the patients who received ecmo died [ ] . there is no known effective treatment for mers cov. many compounds have been screened in vitro for possible activity against this coronavirus [ ] [ ] [ ] [ ] , however, the in vivo efficacy has not been subjected to clinical investigation. in vitro data suggests that mers-cov inhibits host interferon production through various molecular pathways [ ] [ ] [ ] [ ] [ ] [ ] mycophenic acid, the active agent of prodrug mycophenolate mofetil, and cyclosporine strongly inhibit mers coronavirus in human and monkey cell lines even more so than they inhibit sars coronavirus [ , [ ] [ ] [ ] . interferon alpha and interferon beta reduce mers coronavirus replication in explanted lung tissue [ ] . in vivo, comparing host response in two patients with mers coronavirus and differing outcomes, the patient who was able to clear mers cov infection was able to mount an interferon response and the patient who died had low levels of interferon alpha suggesting a therapeutic role for interferon [ ] . the combination of interferon alpha and ribavirin has been used successfully in rhesus monkeys infected with mers coronavirus [ ] , and in a few small case series [ ] [ ] [ ] . beta-interferon seems to be an even more potent inhibitor of mers coronavirus in vitro [ ] [ , [ ] [ ] [ ] . one small study with exceptionally high mortality rates using interferon beta for treatment found no difference in mortality between interferon beta use and interferon alpha use [ ] . our data, albeit from a retrospective cohort support the findings that interferon beta is associated with a decrease in mortality. there are limited data on the efficacy of treatment regiments for this virulent disease. we present data from a retrospective cohort of ill patients with mers-cov and the results of the evaluation of the clinical efficacy of beta interferon beta, alpha interferon, ribavirin and mycophenolate mofetil in addition to routine supportive care. forty five percent of patents ( patients) received interferon beta and in this cohort, sixteen percent of patients received interferon alpha ( patients) and % of patients ( patients) received ribavirin, either in conjunction with interferon alpha or interferon beta, and patient received mycophenolate mofetil. patients receiving beta interferon and mofetil had improved survival, however this was confounded by the severity of illness on presentation for beta interferon. all of the patients who received mycophenolate mofetil survived however because of the small number, we could not analyze the independent efficacy of mycophenolate mofetil. while this is a relatively large series of mers-cov cases, the primary limitation of our study is that it is a retrospective review of cases and not a randomized trial and thus subject to confounding as seen in our cohort. we used a modified apache score without all of the clinical variables, which may have underestimated the association of severity of illness with reduced survival. importantly, the mortality in patients receiving additional therapies that modulate the immune response was low. all of the eight patients who received mycophenolate mofetil in our study survived. hence, it may be reasonable to further study this agent in controlled trials. this observational study investigates novel treatment options like beta interferon and mycophenolate mofetil for mers-cov in humans which have in vitro activity. our cohort demonstrated severity of illness is an important effect modifier and needs to be considered in evaluating novel agents. to better assess the efficacy of these therapies, international prospective randomized trials with adequate numbers of patients are needed to further evaluate the impact of these treatments in addition to routine supportive care when compared to other treatment options. this study was reviewed and approved by johns hopkins university institutional review board and the directorate of health affairs. data supporting the findings are in the manuscript, additional data available upon request. abbreviations aids: acquired immune deficiency syndrome; alt: alanine transaminase; apache : acute physiologic and chronic health evaluation; ast: aspartate aminotransferase; cxr: chest x ray; ecmo: extracorporeal membrane oxygenation; icu: intensive care unit; ldh: lactate dehydrogenase; mers co-v: middle eastern respiratory syndrome coronavirus; pcr: polymerase chain reaction. the authors declare that they have no competing interests. authors' contributions ma conceived of the study, participated in its design and helped draft the manuscript. ka participated in data collection and analysis, and reviewed the manuscript. yg participated in data collection and analysis, and reviewed the manuscript. aa participated in data collection and analysis, and reviewed the manuscript. fs participated in data collection and analysis, and reviewed the manuscript. aa participated in data collection and analysis, and reviewed the manuscript. mb participated in data collection and analysis, and reviewed the manuscript. tmp participated in the design and analysis as well as the writing of the manuscript. dv participated in the statistical analysis of the study. gs helped analyze the data and write the manuscript. all authors read and approved the final manuscript. the severe acute respiratory syndrome isolation of a novel coronavirus from a man with pneumonia in saudi arabia patient with new strain of coronavirus is treated in intensive care at london hospital latest outbreak news from promed-mail: novel coronavirus -middle east middle east respiratory syndrome coronavirus (mers-cov): announcement of the coronavirus study group who summary 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epithelium highlights its zoonotic potential mers-coronavirus replication induces severe in vitro cytopathology and is strongly inhibited by cyclosporin a or interferon-α treatment interferon-β and mycophenolic acid are potent inhibitors of middle east respiratory syndrome coronavirus in cell-based assays distinct immune response in two mers-cov-infected patients: can we go from bench to bedside? treatment with interferon-α b and ribavirin improves outcome in mers-cov-infected rhesus macaques ribavirin and interferon alfa- a for severe middle east respiratory syndrome coronavirus infection: a retrospective cohort study ribavirin and interferon (ifn)-alpha- b as primary and preventive treatment for middle east respiratory syndrome coronavirus (mers-cov): a preliminary report of two cases ribavirin and interferon therapy in patients infected with the middle east respiratory syndrome coronavirus: an observational study ifn-α a or ifn-β a in combination with ribavirin to treat middle east respiratory syndrome coronavirus pneumonia: a retrospective study there are no acknowledgements. there was no external funds provided for this project. key: cord- - kntd t authors: radonovich, lewis j.; bessesen, mary t.; cummings, derek a.; eagan, aaron; gaydos, charlotte; gibert, cynthia; gorse, geoffrey j.; nyquist, ann-christine; reich, nicholas g.; rodrigues-barradas, maria; savor-price, connie; shaffer, ronald e.; simberkoff, michael s.; perl, trish m. title: the respiratory protection effectiveness clinical trial (respect): a cluster-randomized comparison of respirator and medical mask effectiveness against respiratory infections in healthcare personnel date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: kntd t background: although n filtering facepiece respirators and medical masks are commonly used for protection against respiratory infections in healthcare settings, more clinical evidence is needed to understand the optimal settings and exposure circumstances for healthcare personnel to use these devices. a lack of clinically germane research has led to equivocal, and occasionally conflicting, healthcare respiratory protection recommendations from public health organizations, professional societies, and experts. methods: the respiratory protection effectiveness clinical trial (respect) is a prospective comparison of respiratory protective equipment to be conducted at multiple u.s. study sites. healthcare personnel who work in outpatient settings will be cluster-randomized to wear n respirators or medical masks for protection against infections during respiratory virus season. outcome measures will include laboratory-confirmed viral respiratory infections, acute respiratory illness, and influenza-like illness. participant exposures to patients, coworkers, and others with symptoms and signs of respiratory infection, both within and beyond the workplace, will be recorded in daily diaries. adherence to study protocols will be monitored by the study team. discussion: respect is designed to better understand the extent to which n s and mms reduce clinical illness among healthcare personnel. a fully successful study would produce clinically relevant results that help clinician-leaders make reasoned decisions about protection of healthcare personnel against occupationally acquired respiratory infections and prevention of spread within healthcare systems. trial registration: the trial is registered at clinicaltrials.gov, number nct ( / / ). healthcare personnel (hcp) are exposed to respiratory pathogens in many clinical settings [ ] . infected hcp may spread infection to their patients [ ] [ ] [ ] [ ] or coworkers [ ] [ ] [ ] [ ] , to family members [ , ] , or to other community members [ , ] . respiratory viral infections among healthcare workers can negatively impact delivery of healthcare services [ ] [ ] [ ] . united states national guidelines call for modes of transmission to dictate infection control measures [ ] . for most human respiratory viruses, the precise mode(s) of person-to-person transmission is incompletely understood [ , ] . the predominant mode of transmission for some human respiratory pathogens, such as influenza virus, respiratory syncytial virus, and coronavirus is believed to be droplet transmission. airborne transmission plays a role with some human respiratory pathogens via small aerosol particles, often called droplet nuclei [ ] . airborne transmission is the predominant mode of transmission for mycobacterium tuberculosis [ , ] and recent evidence has suggested a larger role than previously thought for influenza a and b viruses [ , ] . disposable respiratory protective devices (rpd) that fit tightly to the wearer's face, sometimes called airpurifying respirators or filtering facepiece respirators, are primarily designed to protect the wearer against infection spread by ill patients. n filtering facepiece respirators (commonly known as "n respirators") are one type of rpd capable, with proper facial fit and usage, of reducing inhalation of airborne particulates by a factor of or greater [ ] . medical masks (mm), typically called surgical masks in operative settings, are primarily devised to protect patients against infection spread by the wearer [ ] . both types of devices also serve as a physical barrier keeping sprays and splashes of infectious materials and contaminated hands and objects away from oronasal region of wearer. although rpd and mm are capable of filtering particulates [ ] , rpd are designed to filter smaller particulates that may remain airborne for long periods. a tight seal between the respirator and the wearer's face is designed to prevent leakage of particulates, a feature not provided by loose-fitting mm. the u.s. department of labor's occupational safety and health administration (osha) requires employers to ensure each hcp, who may be exposed to airbornetransmissible infections in the workplace, receives an rpd with an adequate respirator-to-face seal that is determined during a mandated annual "fit-test". however, evidence is inconclusive that rpd are better than mm at protecting hcps from respiratory infections in clinical settings [ ] [ ] [ ] [ ] [ ] [ ] , despite tight-fitting rpd produced by manufacturers, with higher levels of exposure reduction validated by numerous laboratory studies [ , [ ] [ ] [ ] , and the use of a complete respiratory protection program (e.g., training, initial and annual fit test) as defined by osha to protect hcp. intuitively, rpd should better protect hcp against airborne infections than mm, but objective evidence has not validated this supposition. one possibility that may explain this discrepancy between expectations and observations is pragmatic: hcp, in general, do not tolerate n respirators as well as medical masks [ , ] , perhaps prompting them to remove respirators more frequently and/or for longer periods, increasing the likelihood of exposure to infections. models have shown that % or more non-wear time during exposure negates any significant differences in protective ability between types of rpds [ , ] . given the difficulty with hcp adherence to guidelines [ ] and general dissatisfaction [ , [ ] [ ] [ ] with rpd, medical masks worn more consistently may provide similar levels of reduction in respiratory viral disease transmission as n respirators. this key gap in knowledge has contributed to discrepant clinical and public health recommendations about respiratory protection for hcp [ , ] . needed are additional well-designed clinical trials conducted in patientcare settings during outbreaks of respiratory infections. the following is an abridged version of the full research protocol for the respiratory protection effectiveness clinical trial (respect). to compare the effectiveness of n respirators and medical masks at protecting hcp from acquiring viral respiratory illnesses in the workplace. null hypothesis: the incidence of laboratory confirmed influenza (primary), influenza-like illness (ili), acute respiratory illness (ari) and other respiratory infections will not be different between hcps who practice guidelines (medical masks) or guidelines (n respirators). alternative hypothesis: the incidence of laboratory confirmed influenza (primary), influenza-like illness (ili), acute respiratory illness (ari) and other respiratory infections will be different between hcps who practice the cdc's guidelines for influenza protection (medical masks) versus guidelines for influenza protection (n respirators). respect is a prospective comparison of respiratory protective equipment to be conducted at multiple, geographically distributed u.s. study sites. hcp who work in outpatient settings will be cluster-randomized to wear n respirators [ ] or mm [ ] for protection against infections during respiratory virus season, the "intervention" period. the null hypothesis assumes n and mm intervention groups will have no differences in outcomes, including ( ) laboratory confirmed influenza or ( ) influenza-like illness (ili), ( ) acute respiratory illness (ari), and ( ) laboratory confirmed respiratory illness (lcri). the alternative hypothesis asserts the incidence of at least one outcome would be different between intervention groups. because respiratory virus season varies year-to-year in onset, severity, and duration, multiple season-years of the study will be necessary to account for expected variance and optimally generalize the resulting knowledge. the beginning of each season's data collection will be independently determined for each study site using an epidemiologic predictive tool designed for respect to capture the largest possible number of respiratory infections. these data will be collected for twelve weeks each season. participant exposures to patients, coworkers, and others with symptoms and signs of respiratory infection, both within and beyond the workplace, will be recorded in daily diaries. adherence to study protocols will be measured by the study team at each site. since periodic changes in infection control guidance and practice may occur over the study years, participants will be expected to adhere to the most up-to-date guidance issued by the centers for disease control and prevention (cdc) and local policies at each study institution, at a minimum. for example, a participant randomized to the mm arm will be expected to don an n when participating in an aerosol-generating procedure, assuming no further changes in pertinent national guidance [ ] . the study participants will be recruited from outpatient settings where patients are relatively likely to present with symptoms and signs of acute respiratory infection. participants will be eligible to enroll for multiple study seasons, yet each will be provided with informed consent and complete enrollment procedures prior to each study season. clinical study sites will be distributed geographically: participants will be cluster-randomized to one of the following n respirators or mm models, selected because they are commonly used in u.s. medical facilities, including the respect study sites. participants who participate in more than one of the study years will be cluster-randomized anew each year. n respirators: ( ) precept or ( ) kimberly clark technol fluidshield . all subjects participating in the study will be required to pass an osha-accepted respirator fit test for the n respirator model(s) available at the study site. no fit testing of medical masks will be performed as these devices are not designed to be tight-fitting to the face and studies [ , ] have shown that their fit capabilities are generally low. filter performance although medical masks are loose-fitting, they create a physical barrier that helps prevent splashes and sprays from reaching the wearer's mucous membranes. in addition to passage around the mask, some of the small particle aerosols are able to pass through the mask's filter media. therefore, in addition to rpds, filtration testing was done on medical masks prior to enrollment of subjects to ensure consistency between models across study locations. the filtration performance of the n respirators and medical mask models in the study were tested in a manner similar to that used by the national institute for occupational safety and health (niosh). devices were attached to a test fixture and placed in a tsi automated filter tester operated with an air flow rate of liters per minute. the tsi uses a photometer to measure the flux of light scattering from aerosol particles. polydispersed particles (mass median diameter of~ . microns) were generated from a % nacl solution and passed through each device being tested for min. each test was repeated times with a fresh n respirator or medical mask. to be certified as an n respirator, filter penetration needs to be less than % (or % efficient). as shown in table , the average penetration percentages for the niosh certified n respirators were an order of magnitude lower than those of medical masks, which are not niosh certified. filter results between n respirator models and between medical mask models were comparable. filter airflow resistance was measured simultaneously using the tsi . as filter airflow resistance increases, more energy expenditure is required for ventilation during device wear and the greater potential for perception of discomfort [ ] . the medical mask models selected for this study have filter airflow resistance levels about half of that of the n respirators. however, one study [ ] found that subjective and physiological responses were not different among subjects exercising while wearing devices purposely made with different filter airflow resistance levels ( mm h o, mm h o, and mm h o) in the range similar to those of the devices in this study (table ) . participants will be instructed to don a new n /mm with each patient interaction, every time a participant encounter occurs within feet of a patient who has suspected or confirmed respiratory infection. hand hygiene will be recommended to all participants in accordance with cdc guidelines [ ] and policies at each study institution. trained research assistants will observe participants during study periods to assess adherence to their assigned intervention arms and hand hygiene. a portable computer equipped with data recording software (handyaudit; toronto, canada) will be used to document adherence. participants will be expected to complete surveys about their attitudes and opinions concerning personal protective equipment before and after each seasonal study period. during the twelve week data collection period each year, participants will self-document (a) perceived occupational exposures to patients or coworkers who have symptoms or signs of respiratory infection, (b) perceived anterior nasal and pharyngeal swabs [ ] [ ] [ ] [ ] [floqswabs utm ( - ), diagnostic hybrids; athens, oh] will be collected by research assistants when symptomatic with study defined respiratory symptoms, as well as two, randomized asymptomatic swabs during each seasonal study period. swabs will be collected when (a) participants self-report respiratory symptoms within a h period, and again if participants remain symptomatic after days; and (b) randomly, on all participants, twice during the active intervention period. the primary outcome measures will be the incidence of: laboratory-confirmed influenza (lci) a or b infection in participants, defined as a) detection of influenza virus by reverse-transcription polymerase chain reaction (rt-pcr) in an upper respiratory specimen swab collected within seven days of symptom onset, or b) influenza seroconversion defined as at least a -fold rise in hemagglutination inhibition antibody (hai) titers to influenza a or b virus from the pre-to post-season serological samples that is not deemed attributable to vaccine. the secondary outcome measures will be the incidence of: ( ) acute respiratory illness (ari) defined as the occurrence of one sign or two symptoms ( the incidence rate ratios between participants randomly assigned to wear n respirators or medical masks will be estimated for each of the primary and secondary outcomes. investigators will be paired and provided with blinded information about clinical and laboratory data to determine if a participant meets a primary or secondary outcome. if the paired investigators do not agree, a principal investigator will adjudicate the outcome. assays will be performed at johns hopkins university. collected respiratory specimens will be stored at − °c until analyzed using multiplex pcr (plex-id, abbott labs, chicago il). automated extraction of nucleic acid (na) from respiratory specimens will be performed utilizing nordiag's arrow instrument and the magna pure robotic system (roche indianapolis, in) per manufacturer instructions. each extraction run will include a quality control (natrol respiratory validation panel , zeptometrix inc., buffalo ny); runs with control failures will be repeated. purified na will be amplified via rt-pcr using a broad respiratory virus identification kit (plex-id rvs . , abbott molecular, des plaines, il). desalting of rt-pcr product and electrospray mass spectrometry-based na analysis will be performed on the plex-id analyzer instrument. if funding is sufficient, samples will also be assayed by rt-pcr for bordetella pertussis, mycoplasma pneumoniae, and for chlamydophila pneumoniae. each study season, blood samples will be collected twice from each participant; one sample will be collected within two weeks of the beginning of the intervention period and a second within two weeks of the end of the intervention period. hemagglutination inhibition (hai) antibody assays will be performed on serum for influenza a and b virus strains, dependent on the antigens in each annual trivalent vaccine using standard methods [ , ] . in brief, serial -fold dilutions of serum samples will be incubated with hemagglutinin units of influenza antigen and a turkey red blood cell suspension. the serum hai antibody titer will be defined as the dilution factor of the highest serum dilution that completely inhibits agglutination of turkey red blood cells in the presence of type-specific hemagglutinin antigen. assays will be performed at the immunology core lab for the study at the va saint louis veterans affairs healthcare system. to optimize compliance and generalizability, a clusterrandomized design will be utilized. all participants working in the same clinical unit will be assigned to wear the same respiratory protective equipment (i.e., an n or mm) during patient interactions for the entire week seasonal study period. clusters will be pairmatched within each study site based on the characteristics of each clinical cluster, including the (a) number of participants (b) occupational location, such as an emergency department, urgent care or primary care, (c) patient population served, such as children or adults, and (d) requirements for participants to wear additional protective equipment, such as goggles donned by dental hygienists. for each study season, the clinics in each matched pair will be randomly assigned to opposing study arms. for matched pairs participating in multiple study seasons, random sequences of arm assignments will ensure each is assigned to both study arms during the multi-year study. each study season, an individual not involved in the study implementation and data analyses will perform the randomization scheme for each study site, using a random number generator in microsoft excel. the principal investigators will be blinded to the randomization scheme prior to assignment. incidence rates of lci, ari, ili, and lcri among cluster-randomized participants will be compared. the relationships between incidence of clinically diagnosed and laboratory-confirmed illnesses will be analyzed with attention to potential confounders, such as participants' demographics, study arm compliance, attitudes and opinions about infection control, receipt of influenza vaccination, and infectious exposures within and beyond the workplace. standard statistics will describe baseline characteristics and follow-up measures, summarized by treatment arm and stratified by study site. to assess the primary outcome, a logistic regression model will be fit using a dichotomous variable to indicate whether a participant became infected with a respiratory pathogen. the odds of infection between the two treatment groups will be reported with a % confidence interval. for secondary outcomes, poisson loglinear mixed effects regression models will assess the difference in seasonal respiratory infection rates between intervention groups. cluster-and individual-level random effects will be considered to account for clustered observations. additional covariates may be added to the models to adjust for confounding. participants will be encouraged to complete the study. those who withdraw from an intervention arm will be encouraged to complete follow-up laboratory specimen collection. an intent-to-treat analysis, in which all available data on all randomized participants are included, will be used for the primary comparison of interventions. a per-protocol secondary analysis will compare treatment effectiveness, accompanied by a planned sensitivity analysis that accounts for participants from whom researchers were not able to obtain a second serological sample. to detect a % reduction (i.e., a relative risk of . ) in the incidence of laboratory confirmed influenza or laboratory confirmed respiratory illness among participants wearing an n respirator, compared to participants wearing a medical mask, respect will need to accumulate approximately , or personseasons of data over four seasons respectively. sample size calculations are based on several assumptions about the incidence rate and levels of withincluster correlation. the attack rate laboratory-confirmed influenza during a single study season is assumed to be % among unvaccinated individuals in the medical mask group. we assume % of our population will be administered a vaccine that is % effective in preventing influenza infection. vaccine effectiveness at the higher end of published reports ( % in health care workers) will lead to a reduction in the yearly attack rate to approximately . %, and effectiveness at the lower end of published reports ( % in the general population) would lead to an increased yearly attack rate of approximately . %. importantly, the anticipated effect on the needed sample size of annual variations in influenza incidence is larger than the expected impact of variation in vaccine effectiveness. the respect study will need independent clusters with a median size of participants each to achieve % power to detect a relative risk of . between n and surgical masks at preventing laboratory-confirmed influenza infection, with a type-i error rate of . . the total number of individuals participating each season will need to be approximately , with , total person-seasons accumulating over the multi-year study. for the secondary outcome of laboratory confirmed respiratory illness, the estimated total number of clinics will need to be , the total number of individuals participating each season will need to be , and total person-seasons accumulated need to be (table ) over the multi-year study. the sample size are made using the clusterpower software package for r [ ] . power is estimated using an expected annual attack rate of % { % = . * . + . *( - . )* . } [ ] . this yearly attack rate translates into a -year attack rate of % { % = -( -( . * . + . * . * . )) . accounting for correlation of outcomes within clusters by assuming the correlation coefficient is . , leads to a design effect of . . for scenarios representing the lower and higher ends of anticipated attack rates in the medical mask group, two quantities were calculated (a) the power to detect a relative-risk of . between the n group and the medical mask group and (b) the relative-risk that can be detected with % power (table ). for all of these calculations the two-sided type i error probability is . . potential outcome analysis for laboratory-confirmed influenza some data on the primary outcome may be missing due to participants withdrawing from the study early and missing the second serological sample. to account for the unavoidable uncertainty posed by missing primary outcome data, due to participant withdrawal or loss of follow-up, a sensitivity analysis will be conducted that randomly assigns binary outcomes to participants who did not complete the study. a two-dimensional grid will be created that varies the influenza attack rates among participants who withdraw. withdrawal attack rates in both arms will be fixed between half and twice the observed attack rates, based on complete data. by varying these two parameters across the grid, and for each combination, the adjusted odds ratio will be calculated by averaging across n = imputed datasets for each point on the grid. analysis of differential withdrawal the characteristics at the time of randomization for participants without complete follow-up will be examined. to assess the potential biases introduced by differential withdrawal among different n respirators, a comparison of withdrawal rates and time to withdrawal will be included as an ancillary analysis to the analyses of the primary and secondary outcomes. respect will be approved by the institutional review board at each participating study site and the centers for disease control and prevention, prior to study initiation. (an unabridged version of the respect protocol was approved by the intitutional review board at each study site and the centers for disease control and prevention). viral respiratory infections cause a wide range of illnesses, varying from mild to severe, in hcps who may spread infection to their patients, family members, and other community members. healthcare-associated infections cost $ b annually in u.s [ ] . factors influencing transmission of respiratory infections in healthcare facilities include the population density of ill patients in healthcare settings, the types of exposures within healthcare settings, the administrative and physical structures of healthcare facilities, and intrinsic characteristics of virulence [ ] . measures to prevent transmission within healthcare facilities include hcp vaccination, handhygiene, cleaning and disinfection of inanimate surfaces, pre-and post-exposure antiviral chemoprophylaxis, patient isolation, and personal protective equipment [ , ] . respect is designed to better understand the extent to which ppe, specifically represented by differences in exposure reduction afforded by n s and mms, reduces clinical illness among hcps. while it may seem that n respirators should better protect hcps than mm against airborne infections in the workplace, this notion has not been validated by objective clinical evidence. low tolerance to respirator wear among hcps may prompt more frequent or longer periods of removal, compared to mm, to an extent that the benefits of higher levels of filtration and lower levels of leakage around the facial seal afforded by respirators is offset or subjugated. key sources of variability in hcp health outcomes are difficult to control for, even in a rigorously designed clinical study such as respect. for example, the inability to prevent hcp community exposures to respiratory infections and the inherent year-to-year variation of viral respiratory infections provide a challenging setting in which to evaluate the effectiveness of personal protective equipment. while community-acquired infections may pose a significant source of exposure for hcps, this type of exposure, if occurring non-differentially between study arms, would bias the results from respect towards the null hypothesis. key reasons for choosing a cluster-randomized design are (a) to increase compliance by equipping all members of a healthcare team with the same equipment and (b) to capture indirect effects of the intervention at the cluster-level, such as herd immunity [ ] . a fully successful study would produce clinically relevant results that help clinician-leaders make reasoned decisions about protection of hcps against occupationally acquired respiratory infections and prevention of spread within healthcare systems. abbreviations ari, acute respiratory illness; cdc, centers for disease control and prevention; dsmb, data safety monitoring board; hai, hemagglutination inhibition antibody; hcp, healthcare personnel; ili, influenza like illness; lcri, laboratory confirmed respiratory illness; mm, medical mask; n , n respirator; niosh, national institute for occupational safety and health; osha, occupational safety and health administration; ppe, occupational protective equipment; respect, respiratory protection effectiveness clinical trial; rpd, respiratory protective devices; rt-pcr, reverse-transcriptase polymerase chain reaction; us, united states. occupationally acquired infections in health care workers. part i healthcare providers as sources of vaccine-preventable diseases guideline for isolation precautions: preventing transmission of infectious agents in health care settings preparing for and influenza pandemic: personal protective equipment for healthcare workers health care-associated infection outbreak investigations by the centers for disease control and prevention transmission of influenza: implications for control in health care settings risk of transmission of severe acute respiratory syndrome to household contacts by infected health care workers and patients surveillance network of catalonia spain tm. implication of health care personnel in measles transmission disruption of services in an internal medicine unit due to a nosocomial influenza outbreak the potential influence of common viral infections diagnosed during hospitalization among critically ill patients in the united states outbreak of pandemic influenza a/h n infection in the hematology ward: fatal clinical outcome of hematopoietic stem cell transplant recipients and emergence of the h y neuraminidase mutation epidemiology of viral respiratory infections how contagious are common respiratory tract infections? aerodynamics of droplet nuclei aerosol transmission is an important mode of influenza a virus spread modes of transmission of influenza b virus in households commentary: the use of respirators to reduce inhalation of airborne biological agents disposable surgical face masks for preventing surgical wound infection in clean surgery. the cochrane library surgical mask filter and fit performance surgical mask vs n respirator for preventing influenza among health care workers: a randomized trial a randomized clinical trial of three options for n respirators and medical masks in health workers efficacy of face masks and respirators in preventing upper respiratory tract bacterial colonization and co-infection in hospital healthcare workers n respirators or surgical masks to protect healthcare workers against respiratory infections: are we there yet? simulated workplace performance of n respirators respiratory protection for healthcare workers in the workplace against novel h n influenza a: a letter report simulated workplace protection factors for half-facepiece respiratory protective devices performance of an n filtering facepiece particulate respirator and a surgical mask during human breathing: two pathways for particle penetration professional and home-made face masks reduce exposure to respiratory infections among the general population respirator tolerance in health care workers discomfort and exertion associated with prolonged wear of respiratory protection in a health care setting b : a new respirator for health care personnel reusability of facemasks during an influenza pandemic: facing the flu health care workers' views about respirator use and features that should be included in the next generation of respirators differences in the compliance with hospital infection control practices during the influenza h n pandemic in three countries institute of medicine committee on respiratory protection for healthcare workers in the workplace against novel h n influenza a letter to president obama on federal ppe guidance interim recommendations for facemask and respirator use to reduce influenza a (h n ) virus transmission guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings prevention strategies for seasonal influenza in healthcare settings impact of low filter resistances on subjective and physiological responses to filtering facepiece respirators hand hygiene task force: guideline for hand hygiene in health-care settings: recommendations of the healthcare infection control practices advisory committee and the hicpac/shea/apic/idsa hand hygiene task force comparison of combined nose-throat swabs with nasopharyngeal aspirates for detection of pandemic influenza a/h n virus by real-time reverse transcriptase pcr nasal swab versus nasopharyngeal aspirate for isolation of respiratory viruses comparative study of nasopharyngeal aspirate and nasal swab specimens for detection of influenza comparing nose-throat swabs and nasopharyngeal aspirates collected from children with symptoms for respiratory virus identification using real-time polymerase chain reaction diagnostic procedures for viral, rickettsial, and chlamydial infections concepts and procedures for laboratory based influenza surveillance. atlanta: world health organization collaborating centers for reference and research in influenza clusterpower: power calculations for cluster-randomized and cluster-randomized crossover trials health care-associated infections: a meta-analysis of costs and financial impact on the us health care system design and analysis issues in cluster-randomized trials of interventions against infectious diseases we wish to thank the members of the respect team (complete list to be provided prior to publication). respect is jointly funded by the u.s. all authors read and approved the final manuscript. all authors meet icmje guidelines. lr and tp conceived and designed the study, coordinated and supervised the study and drafted the manuscript. mb, ae, cn, mr, cs, and ms designed the study, coordinated and supervised the study and drafted the manuscript. dc designed the study, conceived and designed the epidemiologic and statistical analyses and drafted the manuscript. gg and cg designed the study, conceived and designed laboratory analyses and drafted the manuscript. nr designed the study, conceived and designed epidemiologic and statistical analyses, coordinated and supervised the study and drafted the manuscript. rs designed the study and drafted the manuscript. the authors declare that they have no competing interests. the findings and conclusions in this manuscript are the authors' own and do not necessarily represent the views of the national institute for occupational safety and health, the u.s. department of veterans affairs, or other affiliates. mention of product names does not imply endorsement. submit your next manuscript to biomed central and we will help you at every step: key: cord- - n m authors: wang, shixin; wei, maoti; han, yi; zhang, keju; he, li; yang, zhen; su, bing; zhang, zhilun; hu, yilan; hui, wuli title: roles of tnf-α gene polymorphisms in the occurrence and progress of sars-cov infection: a case-control study date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: n m background: host genetic factors may play a role in the occurrence and progress of sars-cov infection. this study was to investigate the relationship between tumor necrosis factor (tnf)-α gene polymorphisms with the occurrence of sars-cov infection and its role in prognosis of patients with lung interstitial fibrosis and femoral head osteonecrosis. methods: the association between genetic polymorphisms of tnf-α gene and susceptibility to severe acute respiratory syndromes (sars) was conducted in a hospital-based case-control study including sars patients, health care workers and healthy controls. relationships of tnf-α gene polymorphisms with interstitial lung fibrosis and femoral head osteonecrosis were carried out in two case-case studies in discharged sars patients. pcr sequencing based typing (pcr-sbt) method was used to determine the polymorphisms of tnf-α gene in locus of the promoter region and univariate logistic analysis was conducted in analyzing the collected data. results: compared to tt genotype, the ct genotype at the - locus was found associated with a protective effect on sars with or( %ci) of . ( . – . ). also, tt genotype, ct and cc were found associated with a risk effect on femoral head necrosis with ors( %ci) of . ( . – . ) and . ( . – . ), respectively and the glucocorticoid adjusted or of ct was . ( %ci . – . ) and the combined (ct and cc) genotype or was . ( %ci . – . ) at - site of tnf-α gene. at the same time, the - ac genotype was manifested as another risk effect associated with femoral head necrosis with or( %ci) of . ( . – . ) and the adjusted or was . ( %ci . – . ) in cured sars patients compared to cc genotype. conclusion: snps of tnf-α gene of promoter region may not associate with sars-cov infection. and these snps may not affect interstitial lung fibrosis in cured sars patients. however, the - ct/cc and - ac genotypes may be risk factors of femoral head necrosis in discharged sars patients. tnf, the gene encoding tumour necrosis factor (tnf), resides in the central part (class iii region) of the major histocompatibility complex (mhc) surrounded by a large number of other immunological genes [ ] . because of the special locus of this gene, it can be deduced that this gene may associate with many diseases, and this hypothesis was confirmed by many research results [ , ] . tnf-α is a key mediator of the inflammatory response and is critical for host defense against a wide variety of pathogenic microbes. however, the over-expression of this cytokine may lead to badness in disease recovery. the dual role of tnf, acting as an agent of both innate immunity and inflammatory pathology, poses a considerable challenge for gene regulation [ ] , and this regulation mainly located on promoter region of this gene. the capacity for cytokine production in an individual has a major genetic component, and striking differences existed among individuals in terms of their ability to produce cytokines. several biallelic polymorphisms had been described within the tnfα gene, including seven in the promoter region at positions - t→c, - c→a, - c→t, - g→a, - g→a, - g→a and - g→a base pairs from the transcription start site [ , ] . moreover, a number of studies had shown that the tnf-α promoter polymorphism had a significant effect on its transcriptional activity [ , ] . severe acute respiratory syndrome (sars) is a newly described human infectious disease caused by a novel coronavirus-sars-cov. sars-cov infection is important because of its high infectivity and unpredictable clinical course, which is characterized by a high mortality rate [ ] . till now, many researchers had reported that susceptibilities to infection sars-cov may associated with hla, mxa, oas- and clec m gene polymorphisms [ ] [ ] [ ] [ ] [ ] , yet these results were variable in different populations. for example, ng reported that sars-cov infection was associated with hla-b* and hla-drb * in hongkong population [ ] , however, lin's results showed that hla-b* and hla-b* were closely related to sars-cov infection [ ] . chan reported that clec m was attributed to sars-cov infection [ ] , but zhi's results failed to support this conclusion. these differences may be attributed to the study population used in each report, also the complex mechanism infection to sars-cov should be considered as another factor of these differences. in order to explore more host factors influencing the occurrence of sars-cov infection, we studied the polymorphisms of tnf-α gene at the promoter region, which had been ascribed to polymorphisms within the regulatory regions or signal sequences of cytokine genes [ ] . after discharging from hospital, interstitial lung fibrosis was observed in sars patients. clinical data showed that the prevalence rate of this change was %( / ) in cured sars patients nine months from the discharge [ ] . tnf-α was one of the earliest cytokines implicated in the pathogenesis of lung fibrosis diseases and, together with il- , has been found to over-expressed in regenerated type ii pneumocytes in human lung, thus enhanced fibroblast proliferation [ ] tnf-α polymorphisms have been discovered significantly associated with increased risk of developing pulmonary fibrosis [ , ] . given that genetic variation may potentially alter inflammation and fibrosis in the lung, the aim of this case-case control study was to examine the tnf-α polymorphisms with interstitial lung fibrosis in sars patients. in spite of interstitial lung fibrosis in cured sars patients, another sequela -femoral head necrosis was also observed in this population and the prevalence rate was . %( / ) and . %( / ) in tianjin and beijing patients respectively [ , ] . the cause of this disease was still unknown and there were arguments about it. for example, some author considered sars-cov as the cause of femoral head necrosis, yet other authors disagreed with this view [ , ] . previous studies showed that femoral head necrosis may caused by hormone usage [ ] , yet our data failed to agree with this point. so, it need further study to explore the cause of this sequela and tnf-α polymorphisms were considered first in this report. in this paper, we aimed to study whether polymorphisms in tnf-α promoter region were associated with sars-cov infection, development, and progression of interstitial lung fibrosis and femoral head necrosis in cure sars patients. this study was reviewed and approved by ethics committees in the medical college of cpafp. the study population comprised sars patients in pingjin hospital, tianjin, china, health care workers of the same hospital, who had come into contact with sars patients but had not developed into sars, and individuals having no contact history with sars patients. among sars patients, could be classified into severe and light sars according to their clinical condition history during the hospitalized period and this population also had the history of hormone therapy by reviewing the clinical treatment. anti-sars-cov antibodies of the serum samples were tested by sars elisa kits (huada diagnostics ltd, beijing, china). considered that the progression of interstitial lung fibrosis or femoral head necrosis may be affected by hormone therapy, hormone using dosage, method and lasting period were considered in this study when analyzing the associations between gene polymorphisms with disease. three kinds of hormone were used in sars patients including methylprednisolone, deltadehydrocortisone and dexamethasone. in order to simplify analyzing, deltadehydrocortisone and dexamethasone dosage were calculated into methylprednisolone using the following equation: mg methylprednisolone = mg deltadehydrocortisone = . mg dexamethasone. lash therapy means more than mg methylprednisolone were used in a single day. cured sars patients with interstitial lung fibrosis were diagnosed by respiratory experts according to ct results following the standard proposal for therapy and diagnosis of sars patients issued by chinese ministry of health in [ ] . interstitial lung fibrosis of sars patients manifested as irregular patch and strip shadow or high density strip shadow and honeycomb interstitial lung fibrosis, these changes could combine with the bronchiectasis. femoral head necrosis was diagnosed using magnetic resonance imaging (mri). an mri scan of a normal femoral head would show uniformly high signal intensity on t and t -weighting throughout the femoral head. agree with one of the following image could be diagnosed as femoral head necrosis in sars patients: abnormal signal with clear margin in cartilage of femoral head, or double thread image, or fracture or joint dent under cartilage, or t wi low signal, t wi and stir high signal of the marrow cavity edema with blur edge [ ] . leucocytes were isolated within h of blood collection using percoll reagent. then genomic dna was extracted using cell dna extraction kit (tiangen biotec co, beijing, china, patch number: - - ) according to the manufacturer's instructions. primers were designed according to gewaltig [ ] . standard -μl polymerase chain reactions (pcrs) contained μl( . μm) forward primer '-gatggactcaccaggtgag- ', μl( . μm) reverse primer '-ctcatggtgtcctttccagg- ', μl buffer [ mm (nh ) so , mm tris-cl(ph = . ), μm edta-na , mm mgcl , mm β-mercaptoethanol], . μl dna polymerase (tiangen biotec co, beijing, china), μl dna template. amplification was carried out in a thermal cycler tc (techne, duxford cambridge, uk) with cycle parameters of min at °c (initial dena-turation), rounds of °c s, °c s and °c s, and a final extension for min at °c. the reactions were carried out in molecular bioproducts μl capped tubes, as these gave optimal heat transfer in the thermal cycler. the tnf-α gene bp fragments in this paper were sequenced in double directions with forward primer '-gatggactcaccaggtgag- ' and reverse primer '-ctcatggtgtcctttccagg- ' and invitrogen company (invitrogen co, shanghai, china) using abi thermal cycler carried out this job. the homozygote genotype of each snp site manifested as a single peak, yet the heterozygote with an ambiguous nucleotide position of a double color peak in the big dye chemistry pictures. according to reading the sequence graphs, the genotype was determined. the differences in values between two groups were evaluated by chi analysis for frequencies or student t test for quantitative index and binary logistic regression was done using spss . software (spss inc, chicago, illinois, usa). a total of sars patients, health care workers and individuals were included in this study. all the populations were chinese han ethnic. the mean age was . years for sars, . for health care workers and . for individual controls (sars vs hcw, p > . ; sars vs individual control, p > . ). the proportion of male was . % in sars, . % in health care workers and . % in individual controls (sars vs hcw, p > . ; sars vs individual control, p < . ). the sera positive rate anti-sars-cov antibody was . % in sars, significantly higher than that of health care workers and individual controls (sars vs hcw, p < . ; sars vs individual control, p < . ) ( table ) . tnf-α genotype frequencies were variable in sars, health care workers and individual controls. there were no differences of tnf-α genotype distribution at the - (t→c), - (c→a), - (a→c), - (g→a) and - according to the clinical history, symptoms of sars patients were classified into light and severe. because of the complicated clinical condition during sars outbreak, some patients' histories were incomplete and could not be classified following the severity standard [ ] . the severe sars referred to those with one or more of the following: ( ) dyspnea, more than times per min respiratory frequencies in still condition;( ) oxygenation index less than mmhg; ( ) shock or multiple organ dysfunction syndrome. among all patients, fifty-four were classified into light and severe. and there were no association of tnf-α polymorphisms and sars severity (table ) . glucocorticoid using dosage, method and lasting period were not associated with interstitial lung fibrosis or femoral head necrosis in binary logistic analysis in sars patients (table ) . and there was no difference of hormone using dosage between the interstitial lung fibrosis and non-interstitial lung fibrosis group(t = . , p = . ) and this trend was also observed in the femoral head necrosis and non-femoral head necrosis group(t = . , p = . ) ( table ) . allele frequencies of tnf-α polymorphisms were listed in table and there were no significant differences between interstitial lung fibrosis and non-interstitial lung fibrosis in sars patients at promoter region of tnf-α gene. allele frequencies of tnf-α gene were compared in sars patients between femoral head necrosis and non-femoral head necrosis ( four years after sars occurrence, many problems still remained unknown to us. till now, many researchers have reported that susceptibility to infection sars-cov may associate with hla, mxa, oas- and clec m gene polymorphisms, yet the results are variable in different populations [ ] [ ] [ ] [ ] [ ] . these differences may be attributed to the study population used in each report, also the complex mechanism infection to sars-cov should be considered as another factor of these differences. in order to explore more host factor influence the occurrence of sars-cov infection, we studied the polymorphisms of tnf-α gene at the promoter region, which have been ascribed to polymorphisms within the regulatory regions or signal sequences of cytokine genes [ ] . allele distributions at - , - , - , - , - and - were almost the same among the sars, the health care workers and individual controls, but a higher a allele frequency in sars population when compared with the control at the - locus(x = . , p = . ). though previous study showed that tnf-α - ag genotype was associated with the clearance of hepatitis b virus and the infection of helicobacter pylori caga subtype infection [ , ] , our results failed to show the role of this locus in sars-cov infection and this conclusion agreed with that of chong wp et al [ ] . we found that there was a weak protective effect of ct genotype at - locus of tnf-α gene against sars-cov infection. the - locus was a new discovered snp site of tnf-α promoter region and its role in infectious diseases might need further study. at the same time, no obvious association between the polymorphisms of tnfα promoter region with the severity of sars was observed. however, lu reported that the - g/a polymorphism of tnf-α associated with the outcomes of hepatitis b virus infection [ ] . thus, the roles of tnf-α gene in infectious diseases should be further studied. tnf-α, a key mediator of the inflammatory response, is critical for host defense against a wide variety of pathogenic microbes, but a higher concentration of this cytokine may cause severe pathology. the capacity for cytokine production in an individual has a major genetic component, and striking differences existed among individuals in terms of their ability to produce cytokines. a number of studies had shown that the tnf-α promoter polymorphism has a significant effect on transcriptional activity [ , ] . during the process of sars-cov infection, there was a cytokine storm in patients including il- , il- , il- , il- , il- , il- , ifn-γ, tnf-α and tgf-β [ ] . the elevated levels of pro-inflammatory cytokines which may cause immuno-mediated damage to lung and other organs, resulting in acute lung injury and, subsequently, multi-organ dysfunction [ ] . so, tnf-α genetic variation may potentially alter inflammation and fibrosis in the lung. after discharging from hospital, interstitial lung fibrosis was observed in cured sars patients and the prevalence rate was %( / ). tnf-α was one of the earliest cytokines implicated in the pathogenesis of lung fibrosis disease and, together with il- , has been found to over-express in regenerating type ii pneumocytes in human lung, thus enhancing fibroblast proliferation [ ] .tnf-α genetic polymorphisms have been found significantly associated with increased risk of developing pulmonary fibrosis [ , ] . during the progress of idiopathic pulmonary fibrosis, activated epithelial cells are thought to release potent fibrogenic molecules and cytokines, such as tnf-α and tgf-β , which in turn foster the transformation of fibroblasts into myofibroblasts and promote their production of extracellular matrix molecules and a vicious cycle of injury and abnormal epithelial healing sets the stage for progressive fibrosis and architectural distortion of the lung parenchyma [ ] . however, our data failed to show that alleles at - (t→c), - (c→a), - (c→t), - (a→c), - (g→a) and - (g→a) were related to interstitial lung fibrosis when compared with non-interstitial lung fibrosis in sars patients. at the same time, - and - were homozygote genotype of tt and gg respectively in sars patients and no relationship of genotype with interstitial lung fibrosis could be calculated. this result implicated that there maybe a different mechanism of interstitial lung fibrosis of sars compared with idiopathic pulmonary fibrosis. femoral head necrosis, another sequela of discharged sars patients, prevailed with a rate of . %( / ) in tianjin [ ] . however, the cause of this sequela was still unknown and there were arguments about it. for example, some author considered sars-cov as the cause of femoral head necrosis, yet other authors disagree with this view [ , ] . previous studies showed that femoral head necrosis may caused by hormone usage, our data was far to agree with this conclusion. there was no obvious association between hormone using including hormone dosage, method and lasting period with femoral head necrosis in binary logistic analysis in sars patients. we found that the - ct and cc genotypes were more frequent in sars patients with femoral head necrosis( . % and . %, respectively) than in non-femoral head necrosis( . % and . %, respectively). and ct and cc were related with a risk effect on femoral head necrosis with ors ( %ci) of . ( . - . ) and . ( . - . ), respectively when compared to tt genotype. the hormone using adjusted or of ct was . ( %ci . - . ) and the combined (ct and cc) genotype or was . ( %ci . - . ). also, the - ac genotype accounted for . % of femoral head necrosis group but . % of non-femoral head necrosis. compared to cc genotype, the ac genotype was manifested as another risk effect associated with femoral head necrosis with or( %ci) of . ( . - . ) and the adjusted or was . ( %ci . - . ) in cured sars patients. tnf-α can activate activity of osteoclasts and accelerate absorption of the bone and cartilage and induce occurrence of oxygen free radicals an d lipid peroxidation, which can induce ischemic necrosis of the femoral head [ ] . so, the ploymophism tnf-α gene may be directly or indirectly attributed to the occurrence of femoral head necrosis in sars patients. in conclusion, there may be no association of tnf-α polymorphisms in promoter region with sars-cov infection. also, tnf-α gene polymorphisms may no affect the occurrence of interstitial lung fibrosis in cured sars patients. however, the polymorphisms may relate with femoral head necrosis. note:*:or was calculated using non-interstitial lung fibrosis as control. &: odds ratio replaced with interstitial lung fibrosis group; ‡: odds ratio replaced with non-interstitial lung fibrosis group sxw conceived of the study, and participated in its design and coordination. mtw, co-first author, carried out the molecular genetic studies and drafted the manuscript. yh, kjz and lh carried out the molecular genetic studies zy, bs and zlz participated in field investigation and samples collection of the study ylh and wlh participated in the design of the study and performed the statistical analysis all authors contributed to writing of the final manuscript all authors read and approved the final manuscript the mhc sequencing consortium: complete sequence and gene map of a human major histocompatibility complex. the mhc sequencing consortium transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes nucleotide diversity of the tnf gene region in an african village polymorphism of the '-flanking region of the human tumor necrosis factor (tnf)-alpha gene in japanese human leukocyte antigens class ii and tumor necrosis factor genetic polymorphisms are independent predictors of non-hodgkin lymphoma outcome the - tumor necrosis factor-alpha promoter polymorphism effects transcription idiopathic pulmonary fibrosis-new insights a major outbreak of severe acute respiratory syndrome in hong kong association of human-leukocyte-antigen class i (b* ) and class ii (drb * ) genotypes with susceptibility and resistance to the development of severe acute respiratory syndrome association of hla class i with severe acute respiratory syndrome coronavirus infection association of sars susceptibility with single nucleic acid polymorphism of oas- and mxa gene: a case-control study polymorphisms of interferon-inducible genes oas- and mxa associated with sars in the vietnamese population homozygous l-sign (clec m) plays a protective role in sars coronavirus infection cytokine production by normal human monocytes: inter-subject variation and relationship to an il- receptor antagonist (il- ra) gene polymorphism the follow-up study on the health status of convalescent patienrs recovered from sars in tianjin co-expression of tnf alpha and il- beta in human acute pulmonary fibrotic diseases: an immunohistochemical analysis increased risk of fibrosing alveolitis associated with interleukin- receptor antagonist and tumor necrosis factor-a gene polymorphisms analysis of tumor necrosis factor-a, lymphotoxin-a, tumor necrosis factor receptor ii and interleukin- polymorphisms in patients with idiopathic pulmonary fibrosis dynamic changes of serum sars-coronavirus igg, pulmonary function and radiography in patients recovering from sars after hospital discharge nested case-control study of avascular necrosis of femoral head during sars patients' convalescence factors of avascular necrosis of femoral head and osteoporosis in sars patients' convalescence chinese ministry of health: proposal for therapy and diagnosis of sars association of polymorphisms of the transforming growth factor-beta gene with the rate of progression of hcv-induced liver fibrosis association of tnf-alpha promoter polymorphisms with the clearance of hepatitis b virus infection association between tnf-α promoter polymorphism and helicobacter pylori caga subtype infection the interferon gamma gene polymorphism + a/t is associated with severe acute respiratory syndrome association of - g/a polymorphism of tumor necrosis factoralpha gene promoter region with outcomes of hepatitis b virus infection in chinese han population analysis of serum cytokines in patients with severe acute respiratory syndrome expression of elevated levels of pro-inflammatory cytokines in sars-cov-infected ace + cells in sars patients: relation to the acute lung injury and pathogenesis of sars experimental study on gufusheng in treatment of steroid-induced ischemic necrosis of femoral head in rabbits this paper was supported by the tianjin science fund (no. yfszsf ) and the science fund of medical college of cpafp(no. wy- - ). the author(s) declare that they have no competing interests. key: cord- -aoi xq authors: bialasiewicz, seweryn; mcvernon, jodie; nolan, terry; lambert, stephen b; zhao, guoyan; wang, david; nissen, michael d; sloots, theo p title: detection of a divergent parainfluenza virus in an adult patient with influenza like illness using next-generation sequencing date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: aoi xq background: human parainfluenza viruses are a common cause of both upper and lower respiratory tract infections, particularly in children. of the four parainfluenza virus serotypes, parainfluenza is least well characterised from both the clinical, epidemiological and genetic perspectives. methods: flocked nose or throat swabs from a previous study investigating viral prevalence in community-based adults suffering from influenza like illness were used as the basis for this study. samples in which no virus was detected using a viral respiratory pathogen real-time pcr panel were barcoded and pyrosequenced using the roche gs flx titanium chemistry. the sequences were analysed using the virushunter bioinformatic pipeline. sanger sequencing was used to complete the detected parainfluenza coding region. results: a variant parainfluenza subtype b strain (qld- ) was discovered in an otherwise healthy adult who presented with influenza like illness. strain qld- shared genomic similarities with both a and b subtypes. the extent of divergence of this genome from the available whole parainfluenza genomes impacted the predicted binding efficiencies of the majority of published parainfluenza pcr assays. conclusions: these findings further support a possible role for parainfluenza in the aetiology of adult respiratory disease within the community setting, and highlight the caution needed to be used in designing pcr assays from limited sequence information or in using proprietary commercial pcr assays. human parainfluenza viruses are a common cause of both upper and lower respiratory tract infections, particularly in children [ ] [ ] [ ] [ ] [ ] . four serotypes are known, but most epidemiological and clinical research has been focused on parainfluenza serotypes - . this has been primarily due to the poor growth characteristics in cell culture of parainfluenza (piv ), the lack of commercial diagnostic reagents, and historical exclusion from routine diagnostic testing [ ] . two antigenically distinct piv subtypes, piv a and piv b, exist [ ] . functionally and epidemiologically, little is known about the two piv subtypes, however both are capable of co-circulating within the same population [ ] . with the advent of reverse transcription pcr (rt-pcr), it has become easier to screen for an expanded range of rna viruses, leading to a re-examination of piv 's epidemiology and role in human disease [ , , , ] . the majority of research into piv has focused on children within the hospital setting, however little information is available on the role of piv in disease within the broader community. despite the resurgent interest in piv , a dearth of publically available piv sequences, and in particular, whole genomes, still exists. the lack of appreciable sequence information hampers the design and evaluation of sensitive research and diagnostic rt-pcr assays, since these tests are reliant on oligomer homology to the target sequence. thus it is imperative to increase the number of publically available sequences for clinically relevant pathogens where little information is currently available, particularly when variant genomes are observed. powerful new techniques such as next-generation sequencing have been applied to clinical samples over the last five years with the aims of discovering novel pathogens. numerous viruses and variant strains have been identified using this approach, including a divergent piv subtype a isolate late in [ ] . unlike insensitive traditional virological methods and highly specific rt-pcr, nextgeneration sequencing methods have the advantage of being able to sequence total or targeted dna and rna from samples in an unbiased way, without a priori knowledge of the possible viral agent(s) present, thus making them the ideal tool for novel and divergent viral genome discovery. in this study, we used a combination of rt-pcr and next-generation sequencing to identify and characterise the full coding sequence of a novel piv variant from an adult participating in a community-based cohort study of respiratory illness. furthermore we compared the primer sequences of existing rt-pcr assays to the genome of this variant strain. samples used for this study were derived from a previous study [ ] investigating viral prevalence in a sub-population of participants in a community-based, randomised control trial assessing influenza vaccine effectiveness. briefly, flocked nose or throat swabs were collected from otherwise healthy adults aged from - years who presented with influenza-like illness (ili). ili was defined as cough, sore throat, runny nose or nasal congestion and at least one systemic symptom (fever greater/equal than . °c, feverishness, chills or myalgia). in total, samples were screened for adenovirus, human metapneumovirus, parainfluenza viruses , & , respiratory syncytial virus, influenza a and b, picornaviruses, bocavirus, coronaviruses (oc , e, nl and hku ) and wu and ki polyomaviruses using real-time pcr [ ] . study samples from which no viral pathogen was detected were used as templates for novel virus discovery. written consent was obtained from all study participants. the original and current studies were approved by the royal children's hospital human research ethics committee (melbourne) and the human research protection office of washington university, respectively. total nucleic acid was extracted from each sample, subjected to sample-specific barcoded random-priming cdna synthesis and then pcr amplified using barcodespecific primers. standard library construction and gs flx titanium pyrosequencing was performed as previously described [ ] . the sequences were analysed using the virushunter bioinformatic pipeline [ ] . in brief, high quality reads with similarity to viruses at the nucleotide level or amino acid level were identified using blastn and blastx, respectively. individual reads were assembled and mapped against piv subtype a and piv subtype b reference genomes m- (ab ) and skpiv (eu ), respectively. walking primers for amplifying and sequencing the remainder of the genome were designed based on the assembled contigs and reference genomes (see additional file ). sanger sequencing was performed on the overlapping amplified cdna bidirectionally. contig assembly and genome characterisation was performed using clc bio genomics workbench . software. (clc bio, denmark) phylogenetic analysis was performed using mega . software (http://www.megasoftware.net/) [ ] . recombination event analyses were performed using the recombination analyses tool software (https://github.com/ethering/ rat) [ ] , with widow sizes of , , and applied to a whole genome alignment using qld- as the reference. a sample collected in melbourne (australia) from a year old male produced reads (see additional file ) with highest similarity to piv ( . - . %). apart from the presentation with ili meeting the study case definition, no other clinical information was recorded in the subject's symptom diary. the reads mapped to six singletons and five contigs of , , , , and nt in length, and showed highest similarity to skpiv , against which the genome walking primers were designed. final assembly of the sequenced amplicons yielded a near-complete genome of , nt in length, and included the entire coding region. the sequence was submitted to genbank as isolate qld- (kf ). during the study period, one other piv detection was observed within the study population. overall qld- sequence similarity to the existing whole piv genomes' concatenated coding regions showed a similarity ranging from . - . %. phylogenetic analyses confirmed qld- was a divergent member of the b subtype clade (figure ), while the second piv clustered within the a subtype (figure , kf ) . predicted protein similarities indicated qld- 's closest homology to skpiv in most, but not all proteins, with the greatest variation being found in the v protein ( . - . %). no evidence of recombination between qld- and other isolates was found (see additional files , , and ). strain qld- and the recently described divergent piv a isolate dk( ) shared several structural features identified by alquezar-planas et al. [ ] ; an extended c-terminal end and a residue stretch within the globular head of the predicted hn protein, as well as a nt insertion at the ′ leader non-coding sequence. these features were not unique to the two isolates, and in the case of the hn protein, were more common than not across both genotypes. conversely, qld- contained the most divergent residue sequence ( . - . %) of the piv b genotype within the variable c terminal end (residues - ) of the nucleocapsid protein. due to the variant nature of qld- , a literature search was undertaken to assess the compatibility of published pcr primer/probe targets with the isolate. of the six assays evaluated [ , , [ ] [ ] [ ] [ ] , four contained mismatches in their primer/probe sequences, in particular at the ′ end (table ). improvements in sequencing and detection technologies over the past years have led to increasing detection rates of existing, neglected, and unknown pathogens. the existence of piv has been known since ; however only recently has piv been more appreciated as a respiratory pathogen in its own right through the use of modern molecular methods [ ] [ ] [ ] [ ] [ ] ] . modern molecular diagnostics heavily rely on pcr-based techniques. because of their high specificity, these same methods are susceptible to decreased sensitivity or even false negative results when confronted with even minor changes in target sequences. this limitation is particularly relevant to clinically important pathogens for which little sequence data are available to guide pcr assay development and ongoing evaluation. the variant piv isolate described in this study is the most divergent of the five available whole genome sequences. thus it is not surprising that four of the six evaluated published pcr assays contained potentially deleterious mismatches with the isolate. in particular, all four assays contain mismatches at or near their primers' ′ ends, which are especially sensitive to incorrect base pairing and would potentially lead to decreased primer binding efficiency, and in conjunction with the other primer mismatches, false negative results. these mismatches illustrate the difficulty in designing sensitive pcr assays based on very limited sequence information. additionally, the use of commercial assays for which primer sequences are not readily available, such as those used in recent piv epidemiological studies [ , ] , should be used with the understanding that there is no capacity to evaluate the assays' target sequence conservation as new data on emerging variant viral strains becomes available. isolate qld- was found in the upper respiratory tract of an adult with ili but no other known conditions. it was the sole virus detected, despite extensive screening for other known and unknown respiratory pathogens, thus supporting a potential role as the aetiological agent of the subject's ili symptoms. recent studies have reported piv infections associated with both lower and upper respiratory tract symptoms within the hospital setting [ ] [ ] [ ] [ ] [ ] ] . this study provides further evidence of piv 's possible involvement in upper respiratory tract infections in otherwise healthy adults within the community setting. in regard to its genomic structure, qld- 's highest similarity was to isolate skpiv . however its overall genomic and np c-terminal end divergence separate it from skpiv and other piv b genomes. in other parainfluenza viruses, the np c terminus binds the proteinassociated viral rna to the rna polymerase [ ] , thus qld- 's variant c-terminal end may alter the isolate's viral rna synthesis kinetics. isolate qld- 's shared lpw gtgtctgatcccata agcagc [ ] piv- reverse gcatgttctgcatctctgga [ ] piv forward caaatgatccacagcaaagattc [ ] piv reverse atgtggcctgtaaggaaagca [ ] piv probe gtatcatcatctgccaaatcggcaattaaaca [ ] published piv primer and probe sequences with mismatches to isolate qld- underlined and in bold. genomic features with both its closest piv b homologue and the divergent piv a raises the possibility of recombination events occurring between the viral subtypes, however no conclusive evidence was observed to support this hypothesis. in this study, the utility of a combined rt-pcr and next-generation sequencing approach to identifying novel viral pathogen was demonstrated with the discovery of a variant strain of piv . the whole coding region of the variant strain was sequenced and showed that the majority of publically available piv pcr assays contained mismatches when aligned to this variant, which may lead to decreased sensitivity and false negative results, thereby underestimating the prevalence of piv . additional file : genome walking primers. pcr primers used to amplify and sequence piv strain qld- . all sequences are shown in the ′- ′ orientation. additional file : gs flx qld- reads. table showing positions of the original reads mapped to the skpiv genome. additional file : recombination analyses of piv genomes; nt window. rat analyses of piv genomes using a nt scanning window. qld- is used as the reference genome. nucleotide position is shown on the x-axis, and relative percentage identity is shown on the y-axis. additional file : recombination analyses of piv genomes; nt window. rat analyses of piv genomes using a nt scanning window. qld- is used as the reference genome. nucleotide position is shown on the x-axis, and relative percentage identity is shown on the y-axis. additional file : recombination analyses of piv genomes; nt window. rat analyses of piv genomes using a nt scanning window. qld- is used as the reference genome. nucleotide position is shown on the x-axis, and relative percentage identity is shown on the y-axis. epidemiology and clinical presentation of parainfluenza type in children: a -year comparative study to parainfluenza types - human parainfluenza virus type infection in chinese children with lower respiratory tract infections: a comparison study detection and identification of human parainfluenza viruses , , , and in clinical samples of pediatric patients by multiplex reverse transcription-pcr microarray detection of human parainfluenzavirus infection associated with respiratory failure in an immunocompetent adult human parainfluenza virus outbreak and the role of diagnostic tests antigenic variation among newly isolated strains of parainfluenza type virus clinical and molecular epidemiology of human parainfluenza virus infections in hong kong: subtype b as common as subtype a epidemiology and clinical presentation of the four human parainfluenza virus types discovery of a divergent hpiv from respiratory secretions using second and third generation metagenomic sequencing virus detection and its association with symptoms during influenza-like illness in a sample of healthy adults enrolled in a randomised controlled vaccine trial klassevirus , a previously undescribed member of the family picornaviridae, is globally widespread identification of novel viruses using virushunter -an automated data analysis pipeline mega : molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods recombination analysis tool (rat): a program for the high-throughput detection of recombination a novel duplex real-time pcr for hpiv- detects co-circulation of both viral subtypes among ill children during respifinder: a new multiparameter test to differentially identify fifteen respiratory viruses rapid and sensitive method using multiplex real-time pcr for diagnosis of infections by influenza a and influenza b viruses, respiratory syncytial virus, and parainfluenza viruses , , , and increased detection of respiratory syncytial virus, influenza viruses, parainfluenza viruses, and adenoviruses with real-time pcr in samples from patients with respiratory symptoms an amino acid of human parainfluenza virus type nucleoprotein is critical for template function and cytoplasmic inclusion body formation submit your next manuscript to biomed central and take full advantage of: • convenient online submission • thorough peer review • no space constraints or color figure charges • immediate publication on acceptance • inclusion in pubmed, cas, scopus and google scholar • research which is freely available for redistribution this work was supported in part by: nih grant u ai to the midwest regional center of excellence for biodefense and emerging infectious disease research, arc linkage grant lp , and qcmri program grant . sb is supported by the qcmri early career research fellowship . the authors declare that they have no competing interests.authors' contributions sb: sample sequencing, assay design, genome assembly, data analyses, manuscript drafting and revision. jm, tn, & sbl: project conception, manuscript drafting and revision. gz: pyrosequencing & data analyses. dw: project conception, data analyses, manuscript drafting and revision. mwd: project conception, manuscript revision. tps: project conception and supervision, manuscript drafting and revision. all authors read and approved the final manuscript. key: cord- -r vxz vu authors: mukherjee, pranab k.; esper, frank; buchheit, ken; arters, karen; adkins, ina; ghannoum, mahmoud a.; salata, robert a. title: randomized, double-blind, placebo-controlled clinical trial to assess the safety and effectiveness of a novel dual-action oral topical formulation against upper respiratory infections date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: r vxz vu background: current prevention options for upper respiratory infections (uris) are not optimal. we conducted a randomized, double-blinded, placebo-controlled pilot clinical trial to evaluate the safety and efficacy of arms-i™ (currently marketed as halo™) in the prevention of uris. methods: arms-i is patented novel formulation for the prevention and treatment of influenza, comprising a broad-spectrum antimicrobial agent (cetylpyridinium chloride, cpc) and components (glycerin and xanthan gum) that form a barrier on the host mucosa, thus preventing viral contact and invasion. healthy adults ( – years of age) were randomized into arms-i or placebo group ( subjects each). the drug was sprayed intra-orally ( × daily) for days. the primary objectives were to establish whether arms-i decreased the frequency, severity or duration of uris. secondary objectives were to evaluate safety, tolerability, rate of virus detection, acceptability and adherence; effect on uri-associated absenteeism and medical visits; and effect of prior influenza vaccination on study outcomes. results: of the individuals who completed the study (placebo: n = , arms-i: n = ), six presented with confirmed uri (placebo: , arms-i: ), representing a % relative reduction, albeit this was statistically not significant). influenza, coronavirus or rhinovirus were detected in three participants; all in the placebo group. moreover, frequency of post-treatment exit visits was reduced by % in arms-i compared to the placebo group (n = and , respectively). fever was reported only in the placebo group. arms-i significantly reduced the frequency and severity of cough and sore throat, and duration of cough (p ≤ . for all comparisons). arms-i was safe, well tolerated, had high acceptability and high adherence to medication use. medical visits occurred only in the placebo group while absenteeism did not differ between the two arms. prior influenza vaccination had no effect on study outcome. conclusions: this randomized proof-of-concept clinical trial demonstrated that arms-i tended to provide protection against uris in the enrolled study participants, while reducing severity and duration of cough and sore throat. a clinical trial with a larger number of study participants is warranted. trial registration: clinicaltrials.gov nct (retrospectively registered). electronic supplementary material: the online version of this article (doi: . /s - - - ) contains supplementary material, which is available to authorized users. upper respiratory infections (uris) are associated with significant morbidity and mortality, particularly in children, the elderly and those with underlying medical conditions (e.g. cancer, cardiopulmonary disease, diabetes and immunosuppression) [ ] [ ] [ ] [ ] [ ] . the centers for disease control (cdc) conducted a review of influenza cases over influenza seasons and reported that the annual rate of influenza-associated death in the us during this period ranged from . to . deaths per , persons [ ] . moreover, influenza is associated with million hospital visits and > , hospitalizations annually [ , , ] . infections associated with non-influenza viruses are known to cause million lost work and school days annually, and yearly economic burden due to viral uris ranges between $ and $ billion [ ] [ ] [ ] . uris are caused by respiratory viral pathogens including influenza, respiratory syncytial virus (rsv), human metapneumovirus, rhinovirus and adenovirus [ , , ] . the current prevention strategies for influenza involve the use of vaccines and antiviral medications. although vaccines are generally effective, their coverage and effectiveness vary ( - %) [ , [ ] [ ] [ ] [ ] . other limitations of vaccinations include vaccine/strain mismatch and "vaccine hesitancy" [ , ] . while neuraminidase inhibitors (nais, e.g. oseltamivir, zanamivir) are approved in the us to prevent and treat influenza [ , ] , these agents often induce only a modest decrease in symptom duration in people with uncomplicated illness [ ] [ ] [ ] . nais can also be associated with resistance development, side effects, high cost and limited effectiveness [ , [ ] [ ] [ ] [ ] [ ] [ ] [ ] . therefore, an unmet need exists for the development of an effective therapeutic approach to prevent uris. arms-i (currently marketed as halo™) was developed as a "first-in-class" novel dual-action formulation that can prevent viral uris by killing the virus (by disrupting the host-derived viral lipid membrane) while forming a protective barrier on the host mucosa. recently, our group demonstrated that the arms-i formulation exhibits novel potent activity against respiratory viruses in vitro [ ] , and reduced influenza-associated mortality and morbidity in an influenza infection murine model [ ] . in the current study, we report on the safety and effectiveness of arms-i in preventing uris in a randomized, double-blind, placebo-controlled pilot clinical trial. arms-i is a single-stream oral spray that targets the oral oropharynx mucosal surfaces, and comprises a broad-spectrum antimicrobial agent (cetylpyridinium chloride, cpc) that disrupts the viral lipid envelope through physicochemical interactions, and components (glycerin and xanthan gum) that form a barrier on the host mucosa, thus preventing viral contact and invasion. the current study was a randomized, double-blinded, placebo-controlled pilot clinical trial. the hypothesis of the study was that the use of the active product (arms-i) sprayed intra-orally × daily is associated with fewer episodes and a lower duration and symptom severity of acute uris. acute uris were defined as a combination of three of any of the following symptoms: fever (≥ . °c), non-productive cough, sore throat, rhinorrhea (runny nose), sinus congestion (stuffy nose) and malaise [ ] . the enrollment target was healthy men and women ( - years old, inclusive). health of study participants was assessed based on patient's recall of symptoms and clinical assessment (inclusion criteria: bmi of - kg/m , no tobacco/nicotine use for at least months, and nonpregnant or breast-feeding; for all inclusion/exclusion criteria and study design details, see protocol in additional file ). participants were enrolled into the study after informed consent following a clinical trial protocol approved by the university hospitals case medical center institutional review board for human investigation, cleveland, oh (protocol number - - , approval date: / / , for details regarding background information about the eligible participants and eligibility criteria, see full protocol in additional file ). all subjects provided written consent, obtained in accordance with federal regulations, and were compensated monetarily for their participation. the written document embodied the elements of informed consent as described in the declaration of helsinki and adhered to the ich harmonized guideline for good clinical practice. the clinical trial protocol went through a rigorous review at our institutional review board, and adhered to all components necessary for a pilot clinical study of this nature. moreover, the protocol was also carefully vetted by clinical-trials.gov during registration process (nct ), which did not indicate any deficiencies in trial design. there are no additional currently ongoing clinical trials with this product. study participants were randomized with equal proportion ( each) into two groups: (a) active product (arms-i) administered intra-orally by spray three times daily (dosing regimen selected based on a pilot clinical study evaluating the ability of arms-i to reduce oral microbial load [ , , ] ) or (b) placebo (purified sterile water containing the same flavor as the active but without neither the active antimicrobial agent (cpc) nor the barrier forming components) administered intra-orally by spray three times daily. randomization lists were generated using the website https://www.randomizer.org/, and all study personnel except the pharmacist were blinded. the active or placebo agents were selfadministered daily by participants for days. an exit visit occurred within weeks post-treatment. the study was conducted during the respiratory virus season in northeastern ohio (date range for patient recruitment was january , through march , ), completed on june , and unblinded on july , . the primary objectives were: ( ) to determine whether arms-i decreases the frequency of acute uris, and ( ) to assess whether arms-i decreases the duration and severity of uri-related symptoms. secondary objectives were: ( ) to assess the tolerability, acceptability and adherence to arms-i medications vs. placebo, ( ) to compare whether acute uris in those receiving arms-i compared to placebo are associated with differences in absenteeism (from work or school) and visits to physicians' offices, emergency departments and urgent care centers, ( ) to determine whether arms-i decreases the detection of respiratory viruses by polymerase chain reaction (pcr) [ ] [ ] [ ] [ ] , and ( ) to evaluate the effect of arms-i on those who did or did not receive the influenza vaccine. the endpoints were: ( ) frequency and duration of clinical respiratory disease at study visits and as assisted by electronic patient diaries, ( ) intraand extra-oral exams, ( ) solicited and unsolicited adverse events, ( ) respiratory virus multiplex pcr, and ( ) self-report for adherence to medication usage. subjectlevel characteristics were summarized per study group, age, gender, prior influenza vaccine status and medications taken for symptom relief. the study length and number of study surveys completed were used to summarize the information on the frequency, duration and severity of symptoms. severity of uri-related symptoms was scored on a -point scale ( = none, = minor, = mild, = moderate, = severe), based on diary entries from study participants with at least three symptoms using the validated wisconsin upper respiratory symptom survey (wurss- ) [ ] . determination of duration of uris-related symptoms was performed by assessment of self-reported diaries of study participants (with at least three symptoms) to identify instances where the symptoms were present for at least two consecutive days. frequency of symptoms occurring at least week apart were recorded as distinct occurrences (since multiple uri events can occur per individual in a season). participants with a diary-based uri event were asked to present at the clinic, where the study physicians examined them to confirm clinical uri. we used the research electronic data capture (red-cap) method to collect, store and disseminate trialspecific clinical data [ ] . electronic diaries were created using the redcap system, and participants recorded their symptoms and addressed the study-related questionnaire using these electronic diaries. data analysis was performed to address the primary and secondary objectives described in the study design. frequency of uri was assessed based on: ( ) visits to the clinic where the study participant had at least three uri-related symptoms ("sick visits", confirmed clinically by study staff ), ( ) interviews conducted by study nurses with the study participants within weeks of treatment completion and ( ) analysis of daily diaries electronically completed by study participants, describing the presence of at least three symptoms. sample size calculations were carried out assuming that the two groups would be of equal size and that the random assignment would be balanced. further, it was assumed that an average of two events would occur in the control group, compared to an average of . events in the treated group (mean ± sd = . ± . ), and that the average duration of illness would be days and days in the control and treated groups, respectively (mean ± sd = ± and ± , respectively). taking an alpha to be . , a sample size of per group would allow the detection of a % difference in primary outcomes between the two groups with % power. the sample size was increased to per group to account for potential losses to follow-up. each symptom of uris was investigated separately. for each endpoint, the total number of days for which there was an event was recorded. then, the number of days for which there was an event per days of person-time follow-up (related to the study duration per subject) was recorded in each group. since this is a prevention study conducted during the flu season of , we selected days so that we covered the entire season. next a logistic regression model was constructed. the data were taken at the day level, so the endpoint is yes/no for an event on that day. furthermore, the data included every day for which there was a completed survey. the endpoints were assessed for each treatment group (placebo vs. arms-i). because there were multiple daily observations for each individual in the study (nominally repeated measures per subject, but different for each subject) an ordinary logistic regression model was inappropriate because the observations within a subject from day-to-day would be expected to be correlated. therefore, generalized estimating equations were used to fit the regression model. medical visits (an indicator of whether a subject went to an emergency department, an urgent care center or a doctor's office due to uris symptoms on each day) and absenteeism (an indicator of whether a subject missed school or work or would have missed school or work if it were scheduled on each day) were analyzed the same way as the individual symptom analyses. the effect of vaccine status on the outcomes was assessed by fitting a multivariable logistic regression model with the treatment arms and vaccine status as the explanatory variables. a total of individuals were enrolled and randomly assigned to the two treatment arms, of which five did not begin the study. one subject (in placebo group) did not return for follow-up visit, and was excluded from the analyses. thus, analysis of results was performed for participants, of whom were in the placebo group and were in the arms-i group (see fig. and additional file for consort checklist). table summarizes the demographics of study participants. the age of study participants ranged between and years in both groups, with the mean age of . ± . years in the placebo group and . ± . years in the active group, with no significant difference observed between the two groups (p = . ). the gender distribution was also similar in the two study groups, with males and females in the placebo group ( . and . %, respectively) and males and females in the active group ( and %, respectively). the study duration (number of days from enrollment until the rd follow-up visit) was similar with . ± . for the placebo group and . ± . for the arms-i group. there were a total of surveys completed in the study ( in the placebo group and in the active group). moreover, percent surveys completed (number of surveys completed divided by study duration) were similar in both groups; . ± . % and . ± . % for the placebo and active group, respectively. among the enrolled individuals, there were six participants who presented to the clinic for clinical confirmation and collection of oral and nasal swabs related to the development of uris symptoms (confirmed uris episode). of the participants who presented with a confirmed uri episode, four ( %) belonged to the placebo and two ( %) belonged to the active group ( % ci . , . , p = . ), indicating a relative reduction of % in the latter. moreover, six (additional) subjects reported uri-related symptoms at their post-treatment visit (within weeks of study completion); among these individuals, four were in the placebo group and two were in the active group. analyses of diary entries showed that one additional subject in the active group and four additional subjects in the placebo group recorded symptom-based uris. these analyses showed that the cumulative frequency of uris tended to be lower in individuals using arms-i than those using the placebo. analyses of the symptoms reported by study participants in their daily diaries showed a total of occurrences of uris, observed in individuals. among these, occurred in the placebo group (in individuals) while occurred in the active group (in nine individuals). the frequency of uris tended to be higher in individuals in the placebo group than those in the active group ( % vs. %, respectively, or = . , % ci: . - . ), indicating a % lower relative frequency of uris in the active group. analysis of the data based on daily surveys (events) of symptoms also revealed a similar pattern with a % relative reduction in the frequency of uris in the active group, although this difference was not significant (or = . , % ci: . - . ). analysis of severity of uris showed that while fever was reported only in the placebo group ( . %), frequency of cough and sore throat were significantly reduced in the active group (table , p ≤ . ). moreover, severity of cough and sore throat were also significantly reduced in the active group compared to placebo group, while frequency of stuffy nose was significantly increased in the active group (p ≤ . , table ). chi-square analysis of symptoms in individuals with uris showed that the relative risk of cough in the placebo group was -times that of people in the active group, while the relative risk of sore throat was . -times that of people in the active group (table , p ≤ . ). furthermore, multivariable logistic regression analysis indicated that cough was the only symptom that associated significantly with uri, with less cough in the active group (placebo vs. active, p = . ; % ci: . - . ). we assessed the duration of symptoms in individuals who reported uris-related symptoms for at least two consecutive days. fever was reported only in the placebo group with duration of days. the median duration of cough, sore throat or runny nose was . days for each in the placebo group, while the median duration of these symptoms was , or days, respectively, in the arms-i group. the median duration of stuffy nose and malaise was days in both study groups. the maximum duration for all the non-fever symptoms was between and days in the placebo group, while this duration was lower ( - days) in the active group (p = . for cough, > . for all other comparisons). safety, tolerability, acceptability and adherence to use of arms-i the safety, tolerability, acceptability and adherence were evaluated by oral exams, solicited and unsolicited adverse events (aes), end-of-study acceptability surveys and self-reported use of sprays. as part of the study protocol, oral exams were conducted on all study participants. among the enrolled participants, abnormal oral exams were reported for four individuals, of which three belonged to the placebo group (cheek biting for two, and labial mucosal injury in one participant) and one was in the active group (enlarged tonsils at enrollment, not noted at subsequent visits or at end of study). none of these oral events were considered related to the study drug. a total of nine adverse events (aes) were reported in the study (with a -day duration), of which five occurred in the placebo group (headache, two; anxiety, labial mucosal injury and muscle strain, one each), while four occurred in the active group (headache, two; anxiety and extremity rash, one each). none of the aes were considered related to the study medication. participants were asked to complete an exit questionnaire with questions related to acceptability of the active product at the end of the study. we found that % of the respondents "strongly liked" or "liked" the taste of the active product, while . % were "neutral". moreover, % of the respondents had a favorable opinion about the smell ( % "strongly liked" or "liked", % were "neutral") of the product. in addition, . % of the participants stated that they would recommend the product to others, while a majority ( . %) expressed willingness to continue to use the product after the study ended. these results demonstrated that arms-i had high acceptability among the study participants. our analysis showed that the single-stream spray bottle was used as indicated in ≥ % of the days in the placebo and ≥ . % in the active group. these results indicate that study participants exhibited a high degree of compliance applying the study drug times a day. there were a total of surveys completed in the study ( in placebo and in active group). the medical care question was left blank on surveys, thus data is only available for surveys ( in placebo and in active group). among individuals with uris, there were two medical visits, both in the placebo arm, and nine absenteeism of which five ( . %) were in the placebo group, and four ( . %) in the active group. these results showed that medical visits occurred only in the placebo group while absenteeism did not differ between the two arms. pcr analysis performed on the oral and nasal swabs collected from individuals with uris showed the presence of influenza b, coronavirus or rhinovirus (oc ) in three participants (detected in february, march and april, respectively). all three infected participants belonged to the placebo arm. among the enrolled individuals, reported receiving influenza vaccine previously, of which ( . %) belonged to the placebo group while ( %) belonged to the active group. multivariable logistic regression analysis revealed the vaccine status had no significant effect on uris (p = . ). these results showed that vaccination status did not influence the uris between the two arms. in the current study, we evaluated the safety and effectiveness of arms-i, a novel intra-oral formulation in the prevention of uris in a randomized, double-blind, placebo-controlled proof-of-concept clinical trial in healthy adults. our data showed that the product is safe and well tolerated, and it reduces symptoms associated with influenza. use of arms-i was associated with a trend to reduced frequency of uris. our study demonstrated that arms-i was safe and had no drug-related adverse effects. this is to be expected, based on the known safety profile of the active ingredients and their long history of use in humans [ ] [ ] [ ] [ ] [ ] [ ] . the novel, patented arms-i formulation contains cetylpyridinium chloride (cpc) as an antimicrobial, and xanthan gum and glycerin as the barrier forming agents. these ingredients have been used since the s as components of various drug products including oral sprays, tablets, lozenges and capsules at concentrations similar to those present in arms-i [ ] . two randomized, double-blind clinical trials have reported the efficacy of orally administered agents in the prevention of uris. o'neil et al. [ ] compared the efficacy of commercially available echinacea capsules in preventing uris symptoms compared to placebo over a period of weeks during the winter months, and reported that echinacea capsules did not significantly alter the frequency of uris symptoms. bennett et al. [ ] determined the efficacy of low dose interferon alpha (ifnα) lozenges in the prevention of uris in healthy adults (n = , aged - years), based on weekly health data questionnaires. these investigators reported that lowdose oral ifn-α prophylaxis did not affect the incidence of uri, but did reduce the severity and duration of symptoms. our study showed that oral topical administration of the active agent was associated with a trend of frequency reduction, and significantly reduced severity and duration of cough and sore throat associated with uris. interestingly, severity of runny nose increased significantly in the active group, as did frequency and severity of stuffy nose, which could be linked to the fact that the product is applied orally and not intranasally. in this regard, lakdawala et al. [ ] identified soft palate of the oropharynx as an important site of isolation of transmissible virus and an initial site of infection. thus, drugs like arms-i, that target the oropharynx, could represent an novel approach for the prevention of viral respiratory infections. arms-i possesses a dual mechanism of action that: (a) targets the host by forming a barrier that prevents contact between the virus and the host mucosa, and (b) exerts direct virucidal activity that disrupts the outer viral membrane [ , ] . since cpc, the antiviral component of arms-i, targets host-derived lipid membrane through physicochemical interactions and does not target a viral protein, activity of arms-i is unlikely to be affected by mutations in the viral genome. thus, arms-i has the additional advantage of having a low potential for the development of resistance. limitations of the current study include being underpowered, and the low incidence of uris in the cohort, which may be due to the seasonal nature of uris, as well as participants who recorded uris in their diaries but did not present at the clinic. other potentially confounding variables include ethnicity, occupational status and co-morbidity of chronic respiratory diseases. in future planned investigations, we intend to power the clinical trial based on the low incidence of uris as well as conduct the study over multiple sites, and multiple seasons. arms-i is safe and well-tolerated, and it reduces influenza symptoms. this product has the potential to prevent viral upper respiratory tract infections. further clinical development of this novel product is warranted. seasonal influenza in adults and children-diagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america infections of the respiratory system characteristics and outcome of respiratory syncytial virus infection in patients with leukemia role of colonization of the upper 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and composition of the - influenza vaccine science and society: vaccines and public health vaccine hesitancy, vaccine refusal and the anti-vaccine movement: influence, impact and implications prevention and treatment of seasonal influenza antiviral agents for the treatment and chemoprophylaxis of influenza: recommendations of the advisory committee on immunization practices (acip) neuraminidase inhibitors for preventing and treating influenza in healthy adults and children review: oseltamivir relieves symptoms but does not reduce hospitalizations in influenza the value of neuraminidase inhibitors for the prevention and treatment of seasonal influenza: a systematic review of systematic reviews influenza virus resistance to neuraminidase inhibitors community transmission of oseltamivir-resistant a (h n ) pdm influenza association between adverse clinical outcome in human disease caused by novel influenza a h n virus and sustained viral shedding and emergence of antiviral resistance neuropsychiatric adverse effects of oseltamivir in the fda adverse event reporting system oseltamivir treatment for influenza in adults: a meta-analysis of randomised controlled trials influenza neuraminidase inhibitors: antiviral action and mechanisms of resistance. influenza other respir viruses barrier-forming formulation containing cetylpyridinium chloride (fcpc) possesses anti-infective activity against influenza virus novel antiviral prevents viral upper respiratory infections in vitro and in vivo validation of a short form wisconsin upper respiratory symptom survey (wurss- ) a barrier-forming oral formulation exhibits sustained post-antimicrobial effect barrier-forming oral formulation containing cetylpyridinium chloride (fcpc) exhibits potent activity against oral pathogens the frequency and seasonality of influenza and other respiratory viruses in tennessee: two influenza seasons of surveillance data influenza a virus isolation, culture and identification prospective evaluation of a novel multiplex real-time pcr assay for detection of fifteen respiratory pathogens-duration of symptoms significantly affects detection rate multiplex real-time pcr for detection of respiratory tract infections research electronic data capture (redcap)-a metadata-driven methodology and workflow process for providing translational research informatics support fda dailymed database accessed xanthan gum: safety evaluation by -year feeding studies in rats and dogs and a three-generation reproduction study in rats sprays and lozenges for sore throats scientific opinion on the evaluation of the safety and efficacy of cecure® for the removal of microbial surface contamination of raw poultry products british industrial biological research association (bibra) safety evaluation of cosmetics ingredients effects of echinacea on the frequency of upper respiratory tract symptoms: a randomized, double-blind, placebocontrolled trial low-dose oral interferon alpha as prophylaxis against viral respiratory illness: a double-blind, parallel controlled trial during an influenza pandemic year the soft palate is an important site of adaptation for transmissible influenza viruses the authors want to thank mr. raymond webber for assistance in conduct of the study. key: cord- -d lfktf authors: kofman, aaron d.; sizemore, emma k.; detelich, joshua f.; albrecht, benjamin; piantadosi, anne l. title: a young adult with covid- and multisystem inflammatory syndrome in children (mis-c)-like illness: a case report date: - - journal: bmc infect dis doi: . /s - - -z sha: doc_id: cord_uid: d lfktf background: a healthy -year-old woman developed covid- disease with clinical characteristics resembling multisystem inflammatory syndrome in children (mis-c), a rare form of covid- described primarily in children under years of age. case presentation: the patient presented with week of weakness, dyspnea, and low-grade fevers, followed by mild cough, sore throat, vomiting, diarrhea, and lymph node swelling. she was otherwise healthy, with no prior medical history. her hospital course was notable for profound acute kidney injury, leukocytosis, hypotension, and cardiac dysfunction requiring icu admission and vasopressor support. mis-c-like illness secondary to covid- was suspected due to physical exam findings of conjunctivitis, mucositis, and shock. she improved following ivig, aspirin, and supportive care, and was discharged on hospital day . conclusion: mis-c-like illness should be considered in adults presenting with atypical clinical findings and concern for covid- . further research is needed to support the role of ivig and aspirin in this patient population. background covid- is increasingly recognized to have a protean range of clinical manifestations in adults, from respiratory illness to hyper-inflammatory and coagulopathic complications, as well as a broad spectrum of disease severity. when the epidemic began in china in late december , case reports of pediatric illness were relatively rare, and almost all children had mild clinical courses. however, a growing number of reports from the united kingdom, italy, the united states, and elsewhere has now described a severe inflammatory syndrome in children similar to kawasaki's disease, a vasculitic illness of unclear etiology originally described in japan in [ ] [ ] [ ] . this syndrome has been named multisystem inflammatory syndrome in children (mis-c). to date, case series of mis-c have described multisystem organ involvement including the mucocutaneous, cardiac, gastrointestinal, and respiratory systems [ ] . the mortality rate of mis-c appears to be low, though severe illness is common, and a number of fatalities in children have been reported. anecdotal reports of mis-c-like illness have been reported in young adults in their early twenties, raising concern that this rare presentation of covid- may also have some penetrance into younger adult age groups [ ] . herein we describe a unique case report of mis-c-like illness in a young adult with covid- . a -year-old previously healthy woman presented to an emergency department (ed) in atlanta, georgia in june with a chief complaint of fatigue. she reported week of weakness, dyspnea, and low-grade fevers, followed by mild cough, sore throat, vomiting, diarrhea, and lymph node swelling. she lived at home with family and had no recent travel or known sick contacts. she was a nonsmoker, drank alcohol socially, and did not use recreational drugs. she was not on any chronic medications and had no known allergies. she endorsed taking ibuprofen and acetaminophen over the prior week for symptomatic relief. on presentation, she was afebrile, with mild hypotension (blood pressure / mmhg) and normal oxygen saturation on room air. she appeared ill, with tender cervical lymphadenopathy; significant conjunctival injection without perilimbal sparing; injected, erythematous, and cracked lips; and tenderness to palpation in the left lower abdominal quadrant. she had no rash, splenomegaly, or swelling of the extremities. laboratory work-up was notable for profound acute kidney injury and leukocytosis (table ) . sars-cov- pcr from nasopharyngeal swab and sars-cov- igg from serum were both positive. blood cultures and legionella urine antigen was negative. the patient's urine culture grew escherichia coli, which was treated with ceftriaxone switched to piperacillin-tazobactam due to ampc-type resistance of the isolate. chest x-ray and ct without contrast were unremarkable. point of care echocardiogram revealed a dilated inferior vena cava. ct abdomen/pelvis demonstrated mild peripancreatic fat stranding, felt to possibly represent acute uncomplicated pancreatitis, as well as nonspecific bilateral perinephric fat stranding. the patient was admitted to the intensive care unit (icu) for hypotension, with diagnosis of covid- and concern for possible mis-c due to mucocutaneous, renal, gi and cardiac system involvement. the patient's blood pressure initially normalized and her creatinine improved to . mg/dl with aggressive fluid resuscitation. she was transferred to the floor on hospital day , however, within h she experienced recurrent hypotension requiring transfer to the icu for the initiation of vasopressors. workup for the new shock revealed evidence of worsening cardiac dysfunction. an electrocardiogram demonstration right axis deviation, troponin-i was newly detectable at . ng/ml and bnatriuretic peptide (bnp) increased to pg/ml. she due to concern for inflammatory multi-system organ involvement similar to that seen in mis-c, and risk of progression to more florid cardiac involvement, a riskbenefit discussion was held with the patient regarding treatment with intravenous immunoglobulin (ivig), including potential risk of hypercoagulability [ ] . she was treated with ivig g/kg split equally between hospital days and to reduce risk for thromboembolic and renal toxicities, along with aspirin mg daily for days, based on treatment courses suggested for pediatric patients with mis-c or kawasaki disease [ ] [ ] [ ] . additionally, the patient was offered remdesivir under an emergency use authorization (eua) basis, but declined. her leukocytosis began to downtrend on hospital day , and clinical symptoms improved including conjunctivitis. she was discharged on hospital day with pulmonary clinic follow-up for pulmonary hypertension, and she was treated with a -day course of apixaban for covid- -associated coagulopathy per emory university hospital covid- treatment guidelines. the centers for disease control and prevention (cdc)'s case definition for mis-c is ( ) an individual less than years of age presenting with fever, ( ) laboratory evidence of inflammation by one or more markers (such as crp, esr, fibrinogen), ( ) evidence of clinically severe illness requiring hospitalization, with greater than organ systems involved (cardiac, renal, respiratory, hematologic, gi, mucocutaneous, or neurological), ( ) no other plausible alternative diagnosis, and ( ) sars-cov- infection confirmed by rt-pcr, serology, or antigen testing (or, absent a positive sars-cov- test, exposure to a suspected or confirmed covid- case within weeks prior to symptom onset). our patient, a previously healthy young adult woman in her mid- 's, met these criteria with the exception of age. several features of our patient's presentation raised concern for mis-c-like illness. first, she was noted to have conjunctivitis and mucositis upon evaluation in the ed, and the cracked lips in particular were suggestive of the mucositis seen in kawasaki's disease. conjunctivitis has very rarely been reported in adults with covid- [ ] , but multiple case series of mis-c in the pediatric population have noted this clinical feature [ , , ] . additionally, our patient had profound gi symptoms leading to hypovolemia and aki to a creatinine of . , which was fluid-responsive. while gi symptoms do occur in adults with covid- , they are typically less severe; by contrast, prominent gi symptoms are seen in many patients with mis-c [ , ] . finally, our patient's stable respiratory status was itself a feature shared by patients with mis-c, who often lack intrinsic respiratory disease [ ] . other features were potentially compatible with mis-c-like illness, including shock and cardiac dysfunction. like many patients with mis-c, our patient required treatment with vasopressors in the icu; her shock was thought to be multifactorial including hypovolemic and cardiogenic. she had elevated troponin and bnp, but unlike many patients with mis-c, her cardiac dysfunction was primarily right-sided. echocardiogram showed severe right ventricular dysfunction and ct showed evidence of pulmonary vascular disease by enlarged pa without evidence of thrombus. it is interesting to speculate whether she had a covid- related vasculitic process or diffuse microthrombi leading to elevated pulmonary vascular resistance and subsequent right ventricular strain. she did not have lv dysfunction, coronary aneurysms, or valvular dysfunction, as have been described in pediatric patients with mis-c [ , ] . several other features of our patient's clinical presentation were less consistent with mis-c as it has been described in the pediatric population. her profound acute kidney injury and leukocytosis were not features described in the majority of mis-c cases described to date [ ] . additionally, her neutrophilia and lymphopenia were more consistent with typical covid- findings in adults, though they have been described in cases of mis-c as well. in conclusion, we describe an unusual case of misclike illness in a young adult with covid- . mis-c is an emerging and poorly understood clinical entity associated that has been described in children with covid- and has features similar to kawasaki's disease. children with mis-c are increasingly treated with ivig, aspirin, and steroids; it is not clear what if any clinical features in adults may warrant similar treatment approaches. our patient was treated with ivig and aspirin and improved without further cardiac involvement, but this is obviously anecdotal. further research into covid- in the young adult population is needed to better characterize the full range of clinical manifestations, and to identify potential opportunities for targeted treatment of inflammatory processes. authors' contributions ak and ap analyzed and interpreted the patient data regarding clinical infectious diseases presentation. jd analyzed and interpreted the patient data regarding critical care portion of hospitalization. ba provided interpretation regarding pharmacological treatment for the patient. es provided interpretation regarding the patient's initial clinical presentation to the emergency department. all authors read and approved the final manuscript. there was no funding source for this report. data sharing is not applicable to this article as no datasets were generated or analysed as a part of this case report. ethics approval and consent to participate ethics approval and consent to participate was not applicable for this case report. written informed consent was obtained from the patient for publication of this case report and any accompanying images. a copy of the written consent is available for review by the editor of this journal. acute febrile mucocutaneous syndrome with lymphoid involvement with specific desquamation of the fingers and toes in children hyperinflammatory shock in children during covid- pandemic an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov- epidemic: an observational cohort study multisystem inflammatory syndrome in u.s. children and adolescents young adults are also affected by kawasakilike disease linked coronavirus, doctors say association of immune globulin intravenous and thromboembolic adverse events covid- associated multisystem inflammatory syndrome in children (mis-c) guidelines; a western new york approach aspirin dose and prevention of coronary abnormalities in kawasaki disease comparison of risk of recrudescent fever in children with kawasaki disease treated with intravenous immunoglobulin and low-dose vs high-dose aspirin evaluation of coronavirus in tears and conjunctival secretions of patients with sars-cov- infection multisystem inflammatory syndrome in children in new york state gastrointestinal symptoms as a major presentation component of a novel multisystem inflammatory syndrome in children (mis-c) that is related to covid- : a single center experience of cases acute heart failure in multisystem inflammatory syndrome in children (mis-c) in the context of global sars-cov- pandemic springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations the authors declare that they have no competing interests.received: august accepted: september key: cord- -ey vnddu authors: fusco, francesco m; schilling, stefan; de iaco, giuseppina; brodt, hans-reinhard; brouqui, philippe; maltezou, helena c; bannister, barbara; gottschalk, rené; thomson, gail; puro, vincenzo; ippolito, giuseppe title: infection control management of patients with suspected highly infectious diseases in emergency departments: data from a survey in facilities in european countries date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ey vnddu background: in emergency and medical admission departments (eds and mads), prompt recognition and appropriate infection control management of patients with highly infectious diseases (hids, e.g. viral hemorrhagic fevers and sars) are fundamental for avoiding nosocomial outbreaks. methods: the euronhid (european network for highly infectious diseases) project collected data from eds and mads in european countries, located in the same facility as a national/regional referral centre for hids, using specifically developed checklists, during on-site visits from february to november . results: isolation rooms were available in facilities ( , %): these rooms had anteroom in , dedicated entrance in , negative pressure in , and hepa filtration of exhausting air in . only centres ( , %) had isolation rooms with all characteristics. personnel trained for the recognition of hids was available in facilities; management protocols for hids were available in . conclusions: preparedness level for the safe and appropriate management of hids is partially adequate in the surveyed eds and mads. emergency departments (eds) and medical admission departments (mads) are high-risk areas for disease transmission in hospitals, since they are often overcrowded, and potentially infectious patients and susceptible individuals may wait in close proximity for several hours. moreover, the identification and isolation of potentially infectious patients may be delayed, because of high work burden, lack of specific training and skills, or unavailability of adequate isolation procedures or areas [ ] . in particular, highly infectious diseases (hids, see definition in additional file : annex ) pose a special risk for nosocomial outbreaks, if not adequately isolated and appropriately managed [ , ] . the european network for highly infectious diseases (euronhid) project, a -month (july -december ) european commission co-funded network, aims to enhance and maintain co-operation, and exchange of information and experiences on hids management among infectious disease clinicians, and to enhance preparedness and response to health threats from these diseases within europe, whether naturally occurring, or deliberately released. euronhid includes european countries (austria, bulgaria, denmark, finland, france, germany, greece, ireland, italy, luxembourg, malta, norway, poland, slovenia, spain, and united kingdom), is managed by a coordination team, based at the national institute for infectious diseases "lazzaro spallanzani", rome, italy. from february to november , euronhid performed a survey in isolation facilities identified by national health authorities as referral centres for the management of imported or autochthonous cases of hids [ ] . among these, in countries reported to have an ed or a mad operating in the same hospital. the aim of this paper is to present data about logistic and infrastructures, infection control procedures, and availability of staff for the appropriate management of hids in these eds and mads. moreover, indications for the adequate management of hids in these settings are given. a cross-sectional study has been performed, in order to investigate resources and capabilities for the management of hids in eds and mads in countries. national health authorities in all european countries were contacted by the coordination team and by the european commission, in order to suggest a physician with expertise in hid management as project partner. this process led to the inclusion of countries, while a norwegian isolation facility later joined the group after direct request from the coordination team. most partners are clinicians working in isolation facilities designated for referral of patients with hids. their areas of expertise include infectious diseases, intensive care, infection control, pulmonary medicine, occupational health, epidemiology and public health. in order to survey only isolation facilities identified by national health authorities for the referral and management of hids, we asked partners to provide official documents in which these hospitals are clearly indicated. this process led to the identification of facilities, which represent all identified centres for all participating countries except spain, from which centres from catalonia only were identified. forty-one isolation facilities in countries reported to have an ed or a mad operating in the same medical centre. we define an ed as the department of the hospital responsible for the provision of medical and surgical care to patients in need of immediate care arriving at the hospital. the mad is the department, usually open round the clock, through which patients are admitted to the hospital. mads can operate for planned admissions only, or for self-referring patients also, but usually are not able to provide immediate care. among the centres surveyed, some had an ed or a mad only, and some had both departments: in these cases, we surveyed the department serving self-referring patients mainly, because our goal was to identify settings where patients with hids were most likely to be unrecognized. data were collected during on-site visits, using a set of checklists specifically developed. three checklists were developed, including main issues and specific questions. management of hids in eds and mads represents one of the main issues in checklist . the checklists are available on the website http://www.eunid. eu, after registration. all on-site visits were performed by the project coordinator together with a representative of the surveyed facility, during the period february-november . all eds and mads were visited, except : in these cases, only fulfilled checklists were available. in order to assess the status of each surveyed facility, a standard evaluation form was developed, on the basis of a literature review and the partners' expert opinion. for the literature review data published up to june, , were obtained by searches of pubmed and medline, and from review of the references listed in retrieved articles, using as search term "emergency services, hospital" as mesh, coupled with general terms such as "civil defense", "bioterrorism" and "hospital preparedness" and with the name of each hid included as mesh term. no data restrictions were placed on our searches. in the evaluation form all data were summarized in topics: availability and adequacy of isolation room(s), of infection control procedures, and of strategies for early recognition of hids. the level of adequacy for each topic is assessed by the euronhid expert panel: in particular about isolation room(s), the panel defined as adequate the availability of at least one isolation room equipped with at least one logistic or technical feature as listed in the table ; as partially adequate the availability of at least one isolation room without specific logistic/technical features; as inadequate the lack of an isolation room. about infection control procedures, the presence of all explored features and procedures as listed in the table was defined as adequate; the presence of at least explored features and/or procedures as partially adequate; and or less features and procedures as not adequate. finally, early recognition strategies were defined as adequate if trained triage staff or other procedures are in place on a hour-basis, partially adequate if these staff/procedures are not continuously in place, not adequate if not in place at all. the evaluation form is available at http://www.eunid.eu, after registration. two members of the coordination team, including the project coordinator, applied this evaluation form for each surveyed facilities, in order to identify all critical points, and suggest affordable solutions. these evaluation forms were sent to the contact persons at the surveyed facilities, asking for feedback. on the basis of the selected literature, partners' expert opinion, and data collected during the surveys, euronhid developed indications for the adequate management of hids in eds and mads. these recommendations were discussed with all partners, and a consensus agreement was reached during the final meeting, in rome in may . european countries were surveyed. in the centres included in our study, had a general ed only, while only had a mad. in the remaining , where both departments were present, self-referring patients with suspected infectious diseases are referred to the mad in cases, while in the remaining self-referring patients are referred to the general eds. consequently, our study includes eds and mads. given the few mads, data are not presented separately. thirty-four facilities had at least one dedicated room for the rapid isolation and evaluation of patients with suspected hids. in facilities these were standard rooms. in the remaining facilities the rooms were equipped with at least one specific logistic feature or at least one technical feature. the availability of these if yes, the transport is performed by: stretcher isolator ( , ) special ambulance through an external pathway ( , ) different procedures depending on risk assessment ( , ) with a normal stretcher, without special procedures ( , ) * not routinely used in all surveyed eds/mads features is reported in table , and the level of adequacy in the explored eds and mads is showed in the figure . the availability of infection control procedures and equipments were addressed in the checklists, results are summarized in table . the figure shows the level of adequacy of infection control in the surveyed eds and mads. the project explored the availability of triage staff specifically trained for the early recognition of suspected hid patients, or alternatively the existence of other procedures for the early identification of these patients, such as a syndromic approach. out of the facilities, have triage personnel with a specific training and background for the early identifying of suspected patients, or other procedures in place for this purpose. in facilities, only some of triage staff have this expertise, thus the early recognition capability is not available on -h basis. finally, the remaining eds and mads have not specific strategies for the early recognition of suspected hid patients. the figure shows the level of adequacy of this topic in surveyed eds and mads. after september and the "anthrax letter" attacks in the us, plans and strategies for the early recognition of hid patients (such as those with suspected vhfs or with smallpox-like symptoms), and for the management of infectious diseases outbreaks (e.g. due to bioterrorism attack) have been promoted in eds and mads, both in usa and in many european countries. all surveys conducted in the aftermath of these events demonstrated severe shortcomings in preparedness [ , ] . a study addressing the availability of preparedness plans before and after september , showed significant improvements [ ] . the sars outbreak in also dramatically changed the approach to isolation and infection control in eds and mads [ ] , and after its emerging the centers for disease prevention and control, and afterward the world health organization, issued new infection control guidelines, introducing the respiratory hygiene/ cough etiquette measures as part of standard precautions [ , ] . some years after these events, the influenza a(h n ) pandemic in became a test-bed, with encouraging results. indeed, many eds and mads reported the adoption of interventions for the management of surge, including rapid systems for triage, logistic modifications of waiting and evaluation area, revised infection control procedures, and modification of staff number and roles [ ] [ ] [ ] [ ] . it is, however, likely that there may be reporting bias of successful experiences. in real life, it is likely that the presence of plans did not assure their consistent application: a survey conducted in atlanta, georgia, after the pandemic, in eds revealed that, despite most ( %) of the facilities having pandemic influenza plans, reported "overcrowding," reported "severe overcrowding," and pediatric eds reported "dangerous overcrowding". moreover, many reported various space limitations including an insufficient number of treatment rooms, insufficient waiting areas, or lack of space to designate a separate waiting room [ ] . after these experiences, it is now clear that eds and mads represent a key setting for the management of infectious diseases emergencies. indeed, they serve as the frontline for patients acutely entering the health care system, and their personnel are the guardians at the gate. it is widely recognized, also, that an important role of eds and mads during an infectious diseases emergencies would be to identify sentinel cases involved in the event, or the isolated case suspected to be affected by an hid; while other tasks include an important infection control role, the appropriate triage, the staff protection, the initial diagnostic and therapeutic approaches, and the coordination with external emergency response and public health authorities [ ] . a limit of our study is represented by the fact that surveys mainly were performed before august . this means that we collected most of data before that eds and mads experienced the surge of cases due to the influenza a (h n ) pandemic, that peaked in november . the experience gained during the pandemic may have caused modifications and improvements of procedures and capabilities, not registered in our data. as the main target of the project were isolation facilities for hid cases, the eds and mads surveyed are operating within, or in the same hospital compound, as a regional/national reference hospital for infectious diseases: thus, we believe that the occurrence of hids is more likely in these facilities, due to referral of suspected patients from other medical facilities or to selfreferral of patient returning from endemic countries for hid. this may affect the external validity of our results as the surveyed facilities probably have put more emphasis on isolation and infection control issues than ordinary hospitals which may also have to handle cases with a hid. moreover, we only collected data about the availability of procedures, but we didn't assess their appropriateness or their application in the real-life. similarly, we didn't collected data about the contents, and the completeness, of staff training. the indications for adequate management have some limits, also. given the infrequency of suspected and confirmed hids, no high-quality studies exist, or in some cases no studies at all. consequently, no evidence-based recommendations, neither any system of ranking of recommendations, is possible. therefore our indications are based on experiences reported in the literature or revealed during the surveys, and on the partners' expert opinion. despite these limits, some comments are possible. in most of surveyed eds and mads at least one isolation room for the isolation and evaluation of suspected hid patients is available, but their logistic and technical level is generally not adequate. indeed, only have rooms with all explored items, and rooms with a minimal technical requirement for isolation according to modern standards (negative pressure, anteroom and hepa filtration of exhausting air) are present in facilities only. in the remaining , isolation rooms are not present, or not fully adequate. based on international guidelines [ , , [ ] [ ] [ ] [ ] [ ] , it is our opinion that the following features are essential for safe and effective isolation: negative pressure is necessary for the isolation of patients with confirmed or suspected diseases with obligate airborne transmission (such as xdr-tb), as well as for the effective isolation of patients with suspected or confirmed diseases with opportunistic airborne transmission, such as sars, human-adapted highly pathogenic strains of influenza virus and smallpox; the presence of an anteroom increases the efficiency of the system, providing an obstacle against pressure loss, and provides a controlled environment in which donning and removal of ppe and other procedures can be done safely; finally the use of hepa filtration for exhausting air is important in order to protect the environment and the persons around the room. infection control procedures are generally available in the surveyed eds and mads. the majority of surveyed eds and mads have logistically adequate waiting areas, or procedures for surge capacity. indeed, in order to reduce the risk of spreading of infectious diseases, adequate distancing among waiting persons, or the use of a dedicated area for coughing and sneezing patients, is very important. we also explored the availability of procedures for the early management of patients suspected to be affected by hids: these procedures, which are not in place in , % of surveyed eds and mads, are mainly focused on early recognition, isolation and infection control, and on steps for alerting and notifying the case. however, most of these procedures do not include strategies for initial diagnostic work-up and treatments, that are not considered by eds and mads their responsibility. however, we believe that certain diagnostic tests and treatments can be performed by the eds and mads. these include tests to rapidly exclude the most common causes of fever, such as malaria, in patients coming from endemic areas. conveniently, the vast majority of eds and mads have easy access to specific ppe, such as ffp respirators. all explored features and procedures are not enough for a safe management of these patients, if the staff is not sufficiently trained and skilled. indeed, the effectiveness of protocols for the early recognition relies upon their correct application, or upon the staff awareness of potentially infected patients. according to our data, specifically trained triage staff are lacking in , % of surveyed eds and mads, and in % these staff are not continuously available. thus, despite that fact that these facilities are located in the same centre as a regional/national reference centre for infectious diseases, in about the half of them a patient with an hid could be unrecognized. overall, the preparedness status of the eds and mads surveyed is only partially adequate, and this is more surprising considering their location. different interventions should be promoted for an appropriate infection control management in eds and mads. basically, standard precautions, including respiratory hygiene and cough etiquette measures, plus transmission-based precautions, should be implemented as completely as possible. the triage procedures should not only include an assessment of disease severity/ urgency, but should consider, wherever possible, also the risk of disease transmission posed by the patient. a brief epidemiological investigation of patients with symptoms consistent with an hid may help in the rapid identification of suspected patients. simple standardized forms should be available for rapid use by triage personnel, and should include (i) a brief travel history, (ii) an occupational history (e.g. the patient is an hcw, a veterinarian, a laboratory worker, a farmer), (iii) a contact history of exposure to other persons with similar illness; and (iv) the history of being part of a cluster. these patients should be placed in separate waiting/evaluation areas, if available, or removed as soon as possible from common areas. once identified as a suspected patient, detailed procedures should be available and rigorously applied. these procedures should include at least the basic steps for the infection control measures to be applied (isolation, ppe to be used, disinfection issues if needed), and the actions for the activation and alerting of the response chain. these procedures could also include, if appropriate, the basic diagnostic work-up to be applied, and therapeutic interventions. the availability of technically well-equipped and logistically adequate isolation rooms is fundamental. these rooms should have a separate access directly from outside, or be logistically isolated from other common areas, and should be equipped with an anteroom. ideally, these rooms should have negative pressure, hepa filtration of exhausting air, sealing of windows and door, and surfaces inside should be easy to decontaminate. finally, all hcws, or at least dedicated personnel depending on eds policies, should be familiar with ppe use, donning and removal, isolation procedures and disinfection issues, as well as with the alert and command chain. recommendations about core-curriculum for hcws dealing with hids have been proposed [ ] , and should be adapted by responsible persons to ed and mad settings. triage hcws, in particular, should be specifically trained in the recognition of suspected patients. this also implies a continuous updating on outbreaks ongoing in the world. operatively, one hcw (from ed/ mad or from infectious diseases department according to local policies) should be responsible to monitor the major on-line epidemiological alerts sites and bulletins, and to disseminate the news to the triage staff. the application of optimal requirements summarized in the additional file : annex , including the availability of a technically and logistically adequate isolation room, should be reserved to those eds and mads where it is more likely to have patients with hids (such as the eds with an high average number of patients located in the capitals or nearby international airports/ ports, those located in specialist infectious diseases hospitals or in the same centre as an isolation facilities, those located in endemic areas for diseases of interest), because of the cost of some of these interventions. on the opposite, the minimal requirements suggested in the additional file : annex should be applied in all eds/ mads. despite the fact that health threats due to hids are constantly present, our survey reveals that the general preparedness level in surveyed eds and mads is only partially adequate. this is a cause for concern as the surveyed facilities are located in the same hospital as the respective regional/national reference centers for hids. interventions to improve the capacity for early recognition and appropriate management if hids in eds and mads are strongly advised. additional file : annex . definition and list of highly infectious diseases (hids). annex indications for the safe and appropriate management of suspected hid patients in eds and mads. work was supported by the ec grant euronhid ( ), and by the ministero della salute, italia-ricerca corrente, istituti di ricovero e cura a carattere scientifico. author details public health committee of the american college of emergency physicians: respiratory hygiene in the emergency department group: framework for the design and operation of high-level isolation units: consensus of the european network of infectious diseases eunid working group: infection control in the management of highly pathogenic infectious diseases: consensus of the european network of infectious disease euronhid study group: euronhid checklists for the assessment of high-level isolation units and referral centres for highly infectious diseases: results from the pilot phase of a european survey hospital preparedness for victims of chemical or biological terrorism bioterrorism preparedness. i: the emergency department and hospital hospital bioterrorism preparedness linkages with the community: improvements over time guideline for isolation precautions: preventing transmission of infectious agents in healthcare settings world health organization: infection prevention and control of epidemicand pandemic-prone acute respiratory diseases in health care boston medical center pediatric emergency response to h n ed syndromic surveillance for novel h n spring h n : one pediatric emergency department's experience azziz-baumgartner e, de guadalajara hospital civil, fray antonio alcalde emerging respiratory infections response team: hospital triage system for adult patients using an influenza-like illness scoring system during the pandemic-mexico a survey of emergency department pandemic influenza a (h n ) surge preparedness weapons of mass destruction events with contaminated casualties: effective planning for health care facilities designing a biocontainment unit to care for patients with serious communicable diseases: a consensus statement guidelines for preventing the transmission of mycobacterium tuberculosis in healthcare settings guidelines for environmental infection control in health-care facilities. recommendations of cdc and the healthcare infection control practices advisory committee (hicpac) updated guidelines for design and construction of hospital and health care facilities detection of airborne severe acute respiratory syndrome (sars) coronavirus and environmental contamination in sars outbreak units european network of infectious diseases: a curriculum for training healthcare workers in the management of highly infectious diseases authors' contributions fmf drafted the manuscript, substantial contributed to design the study, participated in the acquisition and analysis of data, and gave the final approval of the version to be published; ss, gdi, hrb, pb, hcm, bb, rg, gt and vp substantial contributed to design the study, participated in the acquisition and interpretation of data, and gave the final approval of the version to be published; gi substantial contributed to design the study, and gave the final approval of the version to be published. all authors read and approved the final manuscript. the authors declare that they have no competing interests. key: cord- -f dq j authors: chong, wai po; ip, wk eddie; tso, gloria hoi wan; ng, man wai; wong, wilfred hing sang; law, helen ka wai; yung, raymond wh; chow, eudora y; au, kl; chan, eric yt; lim, wilina; peiris, js malik; lau, yu lung title: the interferon gamma gene polymorphism + a/t is associated with severe acute respiratory syndrome date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: f dq j background: cytokines play important roles in antiviral action. we examined whether polymorphisms of ifn-γ,tnf-α and il- affect the susceptibility to and outcome of severe acute respiratory syndrome (sars). methods: a case-control study was carried out in chinese sars patients and healthy controls. we tested the polymorphisms of ifn-γ,tnf-α and il- for their associations with sars. results: ifn-γ + a allele was associated with susceptibility to sars in a dose-dependent manner (p < . ). individuals with ifn-γ + aa and at genotype had a . -fold ( % confidence interval [ci], . - . ) and . -fold ( % ci, . - . ) increased risk of developing sars respectively. the polymorphisms of il- and tnf-α were not associated with sars susceptibility. conclusion: ifn-γ + a allele was shown to be a risk factor in sars susceptibility. severe acute respiratory syndrome (sars) is an infectious disease caused by sars coronavirus [ ] with > cases and deaths reported in [ ] . much progress has been made in understanding sars coronavirus but the pathogenesis is still unclear [ ] . it was reported that old age, diabetes mellitus and heart disease were risk factors for adverse prognosis of sars [ ] [ ] [ ] , however, little is known about the contribution of genetic factors. we have demonstrated that genetic haplotypes associated with low serum mannose-binding lectin (mbl) were associated with sars [ ] and our findings were recently replicated [ ] . recently, homozygotes for clec m tandem repeats were reported to be less susceptible to sars in hong kong chinese [ ] . cytokines are known to be important in antiviral action. interferon (ifn)-γ from t and natural killer (nk) cells is important in driving the t helper cell type (th ) responses. it also activates monocytes and macrophages, which in turn take part in antiviral responses by producing free radicals and pro-inflammatory cytokines like tumor necrosis factor (tnf)-α. [ ] . tnf-α then regulates expression of neutrophil-endothelial cell adhesion molecules and chemokines, which recruit leukocytes to the site of infection [ ] [ ] [ ] . thus, ifn-γand tnf-α play important role in antiviral response and inflammation. interleukin (il- ) is an antiinflammatory cytokine that inhibits the activation and effector function of th cells, monocytes, and macrophages [ ] . il- appears to limit and ultimately terminate inflammatory responses by blocking the expression of a number of pro-inflammatory cytokines and chemokines [ ] . in animal model, il counteracts the inflammatory response by inhibiting tnfα production and neutrophil activation, and leads to a reduction of the lung tissue injury [ ] . thus, il- plays an important role in regulating many immune and inflammatory processes. various studies showed that a high il- level would result in suppression of innate host defense and lead to increasing susceptibility of the host to various microbes and death [ ] [ ] [ ] . in this study, we hypothesized that the polymorphisms of the cytokine genes, i.e. ifn-γ + a/t, tnf-α - g/a, il- - g/a and - a/c, might be associated with sars. these genes were chosen based on their functions in antiviral response and inflammation regulation that may be involved in sars pathogenesis and their polymorphisms based on their potential regulation on gene expression (table ) . we tested our hypotheses in sars patients and healthy controls and found that polymorphism of ifn-γ + a allele was associated with susceptibility to sars in a dose-dependent manner. genotyping ifn-γ + a/t, il- - g/a and - a/c were genotyped by taqman system (applied biosystems, foster city, ca, usa) as described previously [ ] . tnf-α - g/a was also genotyped by taqman system with same condition. the sequences of the primers were '-cct ggt ccc caa aag aaa tg- ' and '-tct tct ggg cca ctg act ga- ' and the probes were -fam-ttg agg ggc atg ggg acg g-tamra and vic-ttg agg ggc atg agg acg gg-tamra. the frequencies of genotypes and alleles of the single nucleotide polymorphisms (snps) were compared between the sars patients and healthy controls by × and × chi square test respectively. in case of significance, logistic regression was used for calculating or with % ci and corresponding p-values between groups by controlling age and sex as covariables. the genotypes of all snps were tested for hardy-weinberg equilibrium (hwe) by chi square test. our case-control study genotyped the snps ifn-γ + a/t, tnf-α - g/a, il- - g/a and - a/c in chinese patients with sars and healthy controls. the genotype distributions and allele frequencies of these snps were shown in table . the ifn-γ + a allele was overrepresented in sars patients ( . %) when compared with the controls ( . %) (p < . ). it was also significantly associated with susceptibility to sars in a dose-dependent manner (p < . ), i.e. individuals with ifn-γ + aa and at genotype had an odds ratio (or) of . ( % ci, . - . ) and . ( % ci, . - . ) in developing sars respectively. however, no significant correlation was observed in snps of il- and tnf-α. all snps were in hardy-weinberg equilibrium (hwe) (p > . ) in sars patients and controls by chi square test, except il- - a/c. ifn-γ + a allele has been previously reported to be associated with infectious diseases such as tuberculosis, hepatitis b virus infection, and parvovirus infection [ ] [ ] [ ] , revealing its potential role of function in host defense against microbial infections. the mechanism by which the ifn-γ + a/t allele influences the susceptibility to sars may depend on its role in the regulation of ifn-γ production. the t allele of ifn-γ + a/t provides a binding site for the transcription factor nuclear factor-κb (nf-κb), which is able to regulate ifn-γ expression [ ] . it is possible that low ifn-γ production may impair their anti-viral response against sars-cov, rendering these individuals more susceptible to this virus infection. our observation that ifn-γ + a allele was significantly associated with sars-cov infection suggests a genetic risk factor for sars. the role of ifn-γ in antiviral response against sars-cov has also been supported by recent studies showing that ifn-γ can inhibit the replication of sars-cov in combination with ifn-β in vitro [ , ] . il- and tnf-α snps were also included in this study. they were chosen due to their potential regulation on protein expression level [ ] [ ] [ ] . however, our present data did not show any significant association of these snps with sars (table ) . nevertheless, we cannot exclude the role of il- and tnf-α as the susceptibility genes for sars, because other snps in these genes may also be involved in gene expression regulation. further association studies on other snps, which could alter the gene expression level are required to ascertain the relationship of il- and tnf-α in sars. we have also compared the genotype and allele frequencies of all the polymorphisms between the death group and survival group of the sars patients (table ) . however, no significant association was established. we demonstrated that ifn-γ + a allele was significantly associated with sars susceptibility in a dose dependent manner. due to its role in regulating ifn-γ expression [ ] , this allele may be involved in the pathogenesis of sars by altering the ifn-γ production. the author(s) declare that they have no competing interests. wpc and wkei: genotyping, data analyses, drafting the manuscript ghwt: genotyping mwn and whsw: data analyses, drafting the manuscript .. ns = not significant. *p-value and or ( % ci) were calculated with the use of logistic regression models, adjusted with sex and age. sars study group: coronavirus as a possible cause of severe acute respiratory syndrome severe acute respiratory syndrome pathogenesis of severe acute respiratory syndrome clinical features and short-term outcomes of patients with sars in the greater toronto area short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) hong kong epidemic: an analysis of all patients summary for patients in mannose-binding lectin in severe acute respiratory syndrome coronavirus infection association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection homozygous l-sign (clec m) plays a protective role in sars coronavirus infection natural killer cells in antiviral defense: function and regulation by innate cytokines tumor necrosis factor locus: genetic organisation and biological implications role of endothelial-leukocyte adhesion molecule (elam- ) in neutrophil-mediated lung injury in rats tumor necrosis factor alpha regulates in vivo intrapulmonary expression of icam- two types of mouse t helper cell iv. th clones secrete a factor that inhibits cytokine production interleukin- and the interleukin- receptor effect of interleukin- (il- ) on experimental lpsinduced acute lung injury respiratory syncytial virus induces interleukin- by human alveolar macrophages. suppression of early cytokine production and implications for incomplete immunity neutralization of il- increases lethality in endotoxemia. cooperative effects of macrophage inflammatory protein- and tumor necrosis factor anti-il- therapeutic strategy using the immunomodulator as in protecting mice from sepsis-induced death: dependence on timing of immunomodulating intervention association of interferon gamma and interleukin genes with tuberculosis in hong kong chinese cytokine gene polymorphisms in patients infected with hepatitis b virus cytokine gene polymorphisms associated with symptomatic parvovirus b infection a single nucleotide polymorphism in the first intron of the human ifn-gamma gene: absolute correlation with a polymorphic ca microsatellite marker of high ifn-gamma production increased sensitivity of sars-coronavirus to a combination of human type i and type ii interferons interferon-beta and interferon-gamma synergistically inhibit the replication of severe acute respiratory syndrome-associated coronavirus (sars-cov) hutchinson iv: an investigation of polymorphism in the interleukin- gene promoter polymorphic haplotypes of the interleukin- ' flanking region determine variable interleukin- transcription and are associated with particular phenotypes of juvenile rheumatoid arthritis effects of a polymorphism in the human tumor necrosis factor alpha promoter on transcriptional activation the pre-publication history for this paper can be accessed here:http://www.biomedcentral.com/ - / / /prepub key: cord- -jniqriu authors: xu, xinyin; zeng, jing; liu, runyou; liu, yang; zhou, xiaobo; zhou, lijun; dong, ting; cha, yuxin; wang, zhuo; deng, ying; zhang, yu; feng, liao; pu, chen; wu, xianping; zhong, bo title: should we remain hopeful? the key weeks: spatiotemporal epidemic characteristics of covid- in sichuan province and its comparative analysis with other provinces in china and global epidemic trends date: - - journal: bmc infect dis doi: . /s - - - sha: doc_id: cord_uid: jniqriu background: the covid- spread worldwide quickly. exploring the epidemiological characteristics could provide a basis for responding to imported cases abroad and to formulate prevention and control strategies in areas where covid- is still spreading rapidly. methods: the number of confirmed cases, daily growth, incidence and length of time from the first reported case to the end of the local cases (i.e., non-overseas imported cases) were compared by spatial (geographical) and temporal classification and visualization of the development and changes of the epidemic situation by layers through maps. results: in the first wave, a total of cases were reported in sichuan, with an incidence rate of . / , . the closer to hubei the population centres were, the more pronounced the epidemic was. the peak in sichuan province occurred in the second week. eight weeks after the wuhan lockdown, the health crisis had eased. the longest epidemic length at the city level in china (except wuhan, taiwan, and hong kong) was days, with a median of days. spatial autocorrelation analysis of china showed positive spatial correlation (moran’s index > , p < . ). most countries outside china began to experience a rapid rise in infection rates weeks after their first case. some european countries experienced that rise earlier than the usa. the pandemic in germany, spain, italy, and china took , , , and days, respectively, to reach the peak of daily infections, after their daily increase of up to cases. during this time, countries in the african region and southeast asian region were at an early stage of infections, those in the eastern mediterranean region and region of the americas were in a rapid growth phase. conclusions: after the closure of the outbreak city, appropriate isolation and control measures in the next weeks were key to control the outbreak, which reduced the peak value and length of the outbreak. some countries with improved epidemic situations need to develop a continuous “local strategy at entry checkpoints” to to fend off imported covid- . faced with unknown infectious diseases, policymakers are always looking for the best point of prevention and control, balancing between underestimating and overestimating the risk. in december , there were reports of unexplained pneumonia infections in wuhan, china [ ]. on december , experts from the chinese center for disease control and prevention (cdc) went to wuhan to learn about the situation and collected samples from patients [ ] . on january , , scientists from the national institute for viral disease control and prevention identified the sequence of novel β-genus coronaviruses from specimens taken from patients in wuhan and later published this information to the public. the study found that the patients were infected with β-genus coronaviruses with genetic characteristics different from sars-cov and mers-cov that was temporarily named the novel coronavirus ( -ncov) and later renamed severe acute respiratory syndrome coronavirus (sars-cov- ). the disease caused by it is called coronavirus disease [ , ] . subsequently, cases of covid- were reported in provinces of china, hong kong, macao, taiwan and other countries. facing the grim situation of the global covid- outbreaks, this study focuses on analysing the overall epidemic characteristics of sichuan province, which is close to wuhan in the east. this study also analysed the overall epidemic characteristics of china, selected key dates for comparison, considered the epidemic laws, and analysed the global epidemic trends to provide a basis for china to cope with imported cases abroad and for other countries to grasp the timing of prevention and control to formulate appropriate strategies. to reflect the actual geographical distribution risk of confirmed cases in sichuan province, the current residential addresses of confirmed cases from the sichuan covid- surveillance system were selected for distribution analysis. for comparison with neighbouring provinces, the situation of confirmed cases reported by the national health commission [ ] , hubei province [ ] , and the health commissions of other provinces were analysed using the address of the reporting unit (usually the hospital or cdc). data from various countries were taken from the who covid- daily report published since january [ ] . the number of cases in china before that first daily report and the first case information in thailand, japan, the republic of korea, and the usa, mentioned in the previous report of who, was taken from their national government websites [ ] [ ] [ ] [ ] . the number of counties and districts of the cities in sichuan province were taken from the statistical yearbook of sichuan province [ ] . the population of various cities in china were taken from the statistical yearbook of kinds of provinces. the characteristics of cumulative confirmed cases, daily new cases, morbidity, etc. were compared based on temporal and spatial factors; the length of time from the first case report to the end of the local cases (i.e., nonforeign imported cases), abbreviated hereafter as "epidemic length", was analysed to explore the influencing factors of prevention and control of the outbreaks. key analysis times and units were set based on key dates of policy and disease incubation periods. the epidemic maps of sichuan and china were drawn at different times and spaces by sas software (sas studio https://welcome.oda.sas.com/) to directly display the development and changes of the epidemic situation and examine the epidemic law by layers. confirmed case scatter plots, column diagrams and line diagrams were also used to display the time trend. spatial stratified heterogeneity of incidence rate in china was analysed by r software using the package "geodetector" [ , ] and was described through q statistic. the city incidence rate was considered as a dependent variable and the province was considered as an explanatory variable in this study. furthermore, the spatial autocorrelation was analysed by arcgis software and was described through the global moran's index [ , ] and hot spot analysis. global moran's index was proposed by the australian statistician patrick alfred pierce moran [ ] to assess the overall pattern and spatial agglomeration. the hot spot analysis could identify statistically significant spatial clusters of high values (hot spots). prevalence in sichuan province (non-foreign imported cases) geographical distribution on march , sichuan province ended the increase in confirmed cases on the mainland (the first wave). each city of sichuan province ( in total) reported confirmed cases, with a total mainland case number of and an incidence rate of . / , . analysis of confirmed cases was based on their current address, with . % in urban and . % in rural areas. the top five cities with confirmed case numbers were chengdu, ganzi, dazhou, nanchong, and guang'an. the cities with top five incidence rate were ganzi, panzhihua, guang'an, chengdu, and dazhou (table ) . moreover, the confirmed cases were distributed in counties, accounting for . % of sichuan province ( in total). among them, cities (out of cities) found cases reported in all their counties. geographically, the regions with the highest number of cases and counties were located in the eastern part of sichuan province, which is directly adjacent to chongqing and closer to hubei province. although the number of confirmed cases in the eastern counties was relatively high, a small number of counties in the western part also had a high incidence (table , figs. and ) . the time of the first confirmed case report in the cities of sichuan province mainly came one week after the wuhan lockdown (january -january ) [ ] . at this stage, the current addresses of the first confirmed cases were distributed in counties, accounting for % of the reported counties ( in total) in sichuan province on march . the maximum number of cases was on the third and fourth days after the wuhan lockdown (january - ). during those two days, counties ( %) reported their first case. the rapid increase in the confirmed the concentrated outbreak time of confirmed cases was the first weeks after the implementation of the lockdown measures (fig. ) . on december , cases were only reported in wuhan. later, on january , the first confirmed case was reported outside wuhan in shenzhen of guangdong province. from december to january (i.e., cities reported cases on the day of wuhan lockdown), the number of confirmed cases increased by a factor of . in the next four weeks after the wuhan lockdown, % of the cities nationwide were affected, and the number of cases increased by a factor fig. time distribution of the number of confirmed cases and counties with the first diagnosed cases in sichuan province (mainland cases). *the maximum number was the third and fourth days after the wuhan lockdown. the rapid increase was particularly obvious in the first two weeks. january , with the first confirmed case, the sichuan cdc launched an emergency response. january , sichuan government launched the first-level response to major public health incidents. february , commencement of district classification prevention and control at the city level. february , commencement of district classification prevention and control at the county level. february , sichuan government reduced the response to level . march , the last local cases in the first wave of the epidemic. march , establishment of a thematic working group on overseas imported cases. march reduced the response to level . from march to april , a total of overseas imported cases were reported in sichuan of , compared with january . in the fifth and sixth weeks, the number of newly confirmed cases and the number of reported areas slowed down significantly, with an increase of only a factor of . ( cities except wuhan increased with a total of cases, and the average daily increase was . cases/city). in the th week (march -march ), there was only a small increase of cases in cities of china except for wuhan. the q statistics values of spatial stratified heterogeneity of city incidence among provinces in china (excluding hong kong, macao and taiwan) on january , february , march and march were . , . , . and . (all the p value < . ), which revealed that the heterogeneity existed and the province as a space factor explained , , and % of the city incidence. the global moran's index [ ] of spatial autocorrelation analysis on january , february , march and march were . , . , . and . (all the p value < . ). all the global moran's index values were positive expressed that the epidemic in china tending to cluster spatially. the results also showed that from january to february , the epidemic situation was rapidly gathering in space. besides, the spread of the epidemic slowed down after february , and the spatial concentration decreased slightly. hot spot analysis further demonstrated that the epidemic gathered in hubei, hunan, jiangxi provinces on february and did not expand, which suggested that the spread of the epidemic was under effective control after february (fig. ). on march , in the seventh week after the closure of wuhan, the growth of local cases in the first wave ended in all the cities in china (excluding wuhan, taiwan and hong kong). based on statistics at the city level (the districts of municipalities were analysed as a city unit according to the high population density of municipalities), the length calculated by the date of occurrence and the end of local cases in a total of cities were used for analysis. the study shows that the longest date length from the first case confirmed to zero growth in each city was days, and the average and median lengths were and days, respectively. the st quartile and rd quartile were and days, respectively. when the length was days, the frequency of the cities was highest at approximately cities ( . % in cities). further analysis of the epidemic length at the county level (regional unit) in sichuan province included a total of counties that had epidemics. the results showed that there was no local case increase after march . the largest date length from the first confirmed case to zero growth was days, with a mean and median of days. the st quartile and rd quartile were and days, respectively. the highest city frequency of the date length was day for approximately cities ( % in cities) (fig. ) . taking the migration from hubei during the two weeks before its lockdown as a possible highrisk factor, this study compared the provinces for which the migration rank was close to sichuan province and found that the peak time in sichuan province was earlier (occured in the second week according to the weekly analysis, ranked the first in common) and the peak value was lower ( . cases average daily increment in that week, only higher than shaanxi) (fig. ) . according to the daily analysis, the peak of sichuan appeared only one day in part b, all the original level hot spots turned into level . after february , the hot spots did not expand, which reflected that the spread of the epidemic was effectively controlled fig. analysis of the epidemic length (local cases) in china and sichuan province. *here, zero growth excludes overseas imported cases as well as their associated cases and a very few cases who leave wuhan after it was unsealed on april . briefly, the length was calculated by the information of occurrence and the end of local cases in the first wave fig. the comparison of average daily increments between sichuan and the other provinces. *one unit of the abscissa was a week ( days). it set the day that the first cases reported in each province as the starting point. during the two weeks before the hubei lockdown, the top destination of hubei's emigration population was henan province. sichuan province ranked seventh. taking the migration from hubei at this stage as a possible high-risk factor, this chart selected the provinces that rank close to sichuan province (rank to ) for comparison. the peak time in sichuan province was earlier (occured in the second week according to the weekly analysis, ranked the first in common), and the peak was lower ( . cases average daily increment in that week, only higher than shaanxi). the fluctuation after the eighth week of each province mainly came from imported cases abroad. the data go up to april in this figure later than zhejiang among the provinces, but its peak value was much lower than zhejiang province ( cases compared to cases). the earliest confirmed cases outside china were reported in thailand on january , followed by japan (january ), the republic of korea (january ), the usa (january ), singapore and vietnam (january ). on february , four weeks after the first reported case in korea, the number of confirmed cases began to rise rapidly. in addition, cases in italy began to rise rapidly two weeks after the first case was reported there, and cases in iran increased quickly in the second week after the first case report. furthermore, the usa ( weeks after the first case) and other european countries such as spain ( weeks after the first case), france ( weeks after the first case), germany ( weeks after the first case), and switzerland ( week after the first case) began to see a rapid rise in infections. overall, many european countries experienced that rise earlier than the usa. the length of time from the first case reported to the period of rapid rise in different countries varied. most of them entered the rapid growth phase within month (fig. ) . the confirmed cases reported in the usa increased sharply in weeks to . from the scatter plot of confirmed cases (top countries), it was found that when the daily increase exceeded - cases, most countries entered an obvious increase stage (fig. ). from the time point of the first daily increase of cases, germany, spain, and italy reached the peak of daily incremental rising after , , and days, respectively, and then the daily increment showed a downward trend. the average daily increment of a day rolling timeframe also showed an inflection point in italy. the countries of france, the uk, and the usa appeared to peak at days, days, and days, respectively. according to the regional division in the who report, many countries in the african region and southeast asia region were closer to the equator with slow growth or only at the early stage of infection spread. this study also analysed the top countries of confirmed cases in each region (fig. , data up to april ) . firstly, dividing the average daily increment value of the final week by the previous week, to get the change coefficient for each country here. the change coefficient was used to express the growing speed of a country. the average change coefficient value of the countries in a region was used to evaluate the growing speed of a region. the average change coefficient was . in the eastern mediterranean region and was . in the region of the americas. besides, the change coefficient was . in russian. secondly, most european countries presented an inflection point or were at a plateau period in the fourth to fifth weeks but were falling slowly. the curve of cases in china dropped rapidly after the fifth week. on february - , hubei province in china reported the clinical diagnosis. the number of clinical diagnosis cases was counted in the who covid- situation report- with a statement that this was only applicable to hubei province. if the clinical diagnosis cases were not taken into account, china reached the peak of the daily increment of the epidemic in days, and if such cases were taken into account, china peaked in days. china has been subjected to imported cases since the eighth week, with basically a disappearance of local cases and the rapid rise of epidemics in other countries abroad (fig. ) . the overseas imported cases were first reported in ningxia province in china on february and cumulatively included as of april , of which were reported by sichuan province. the eastern region of sichuan province is adjacent to chongqing and hubei provinces. many counties there have reported cases, which is related to the fact that imported cases were the main cases in sichuan province in the early stage. combined with the "baidu migration data" analysis [ ] , the national migration scale index [ ] on january and earlier was slightly higher than that of the same period last year (january was the chinese new year's eve), but after january , the national migration scale index fell slightly until march , which was lower than the same time of last year. on january , for the proportion of migrants in cities across china (the ratio of the population that moved to a city to the total population that moved into different kinds of cities in whole country), the top were chongqin, chengdu, and zhoukou city. twelve of the top cities were in sichuan province and all of these are located in the eastern part. among them, chengdu ranked first in the - -day timeframe. the proportion of emigration place (the ratio of the emigration to a certain city to the total emigration for the whole country), of the top were in sichuan province, and were also located in the east. this result suggested that the population flow in eastern sichuan was relatively large. in addition, the overall low incidence in western sichuan was also associated with a lower population density in the western region than in the east. in a few of these areas, the incidence was high because a few people, who went back from chengdu in the incubation period, participated many aggregation activities there [ ] . the first reports of the kinds of cities in sichuan province were concentrated in the first week after wuhan instituted a lockdown. this reflected the rapid response and quarantining of sichuan province. the number of cases increased significantly in the first weeks, which was in line with the usual incubation period [ , ] . this suggested that the first two weeks after the occurrence of the first case was a critical stage for establishing quarantine, prevention and control measures. the spatiotemporal distribution of the country suggests that the virus spreads rapidly during the two incubation periods. the analysis of the length of the epidemic indicates that after china adopted strong prevention and control measures, most regions can control the growth rate of the epidemic in approximately weeks. the average and median of the epidemic length of county-level regional units are significantly lower than the epidemic length of (see figure on previous page.) fig. the analysis of the average daily increment, per days, in different countries and regions. *one unit of the abscissa was a week ( days). it set the day when the new cases exceeded as the starting point. according to the regional division in the who report, this figure included the top - countries of each region, for which the daily increase exceeded , to show the trends of confirmed cases. countries meeting this condition in the southeast asia region and western pacific region were just and , respectively, both less than . the countries in parts a and e were closer to the equator with slow growth or at the early stage of infections. in parts b and d, most countries were in the rapid growth phase, as well as russian in part c. dividing the average daily increment value of the final week by the previous week, to get the change coefficient for each country here. the average change coefficient value of the countries in a region was used to evaluate the growing speed of a region. the average change coefficient was . in the eastern mediterranean region and . in the region of the americas. besides, the change coefficient was . in russian. most european countries in part c as well as the usa in part d presented an inflection point or at a plateau period in the fourth to fifth weeks but declined slowly. germany, spain, italy, and china (a) took , , , and days, respectively, to reach their peak daily increments. china city-state regional units. it suggested that regional units of different levels can combine actual conditions to formulate prevention and control measures, duration of prevention and control, and resumption time. especially for areas where there is no community outbreak in the local area, production can be resumed early to reduce economic losses. the increased number of covid- case in spatiotemporal distribution showed that the epidemic in china went through mainly two stages of release. in the first stage, the weeks since the wuhan reported, the confirmed case increased and the epidemic spread rapidly (part a of fig. ). in the second stage, the next four weeks after the wuhan lockdown, the release was accelerated and was then quickly close to the end of the first epidemic wave (part b, c and d of fig. ) . results indicated that on march , the case incidence in major countries worldwide was still in the first stage, and from march onward, most countries entered the second stage of rising cases. after comparing and analysing the morbidity, we found that the length of time from the first case to the rapid rise of cases varies in different countries. most countries entered the period of rapid growth after weeks. the delayed entry of rapid growth in a few countries was related to the late launch of extensive testing. china has taken many effective measures to address the epidemic. very important activities included well-coordinated prevention, control and treatment and reflected ) centralized and efficient command, ) a tight prevention and control system involving all sectors of society, ) an all-out effort to treat patients and save lives, ) information that was released in an open and transparent manner, and ) science and technology underpinnings. in addition, the government assembled a powerful effort to beat the virus. this fight mobilized the whole country with a message that lives are precious, which let the people unite as one. during the battle against the virus, the government did much to coordinate prevention and control with social and economic development. moreover, the whole process of fighting the epidemic could not be separated from the support of the international community and chinese citizens overseas. covid- is one of the most significant public health emergencies that has occurred in china since the founding of the people's republic of china. in the early stage of the epidemic, due to insufficient resources for medical treatment and the failure to connect quickly with the disease prevention and control information system in a few newly designated hospitals, as well as the overloaded operations in hospitals and the very busy treatment schedule for medical staff late reports, missed reports and false reports occurred. on march , the newly confirmed growth was reported to be zero in wuhan for the first time. since then, the spread of the epidemic has been basically blocked, and the control of the travel from wuhan to other cities has been lifted, creating favourable conditions for the comprehensive and detailed verification and revision of the epidemic data. the revised types included reductions and additions. among them, cases were removed after verification (some patients had been treated in more than one district or in multiple hospitals, resulting in duplicate reporting), and cases were added (previous cases not reported in time due to late reporting and omission). the total number of confirmed cases was revised from , to , on april based on the principles of objectivity and the transparency of data [ ] . this part of the newly confirmed number accounted for a small proportion ( . %) of the total confirmed number in wuhan. the limitation of this study was that the . % confirmed number was presented centrally on april in figs. , and , because the data released by health commission of hubei province were not assigned to the date of previous diagnosis. considering the history of the epidemic in china, it is suggested that even if countries take effective and powerful measures, the number of confirmed cases will increase rapidly in the first month, which will inevitably challenge the medical resources and population health of those countries. if no measures are taken, it is difficult to estimate the number of infections and the overall economic losses that would be caused. combined with the severity of the illness and mortality rates, the health costs of the population would be substantial. in addition, upon entering the period of zero growth and the gradual cure of local cases in china, high vigilance must be maintained. from february , , the different provinces of china began to report imported cases from abroad. with the change in the epidemic situation in different countries and the different measures taken by governments, the cases imported into china have soared. a small number of overseas associated cases have appeared. in response, from march , , the entry of foreigners holding valid chinese visas and residence permits were suspended. some countries with improved epidemic situations also need to quickly formulate a continuous "local strategy of entry checkpoints" to fend off imported covid- in the context of the global epidemic. until vaccines are widely available, these governments need to be highly vigilant about localized outbreaks. republic of korea; mers-cov: middle east respiratory syndrome coronavirus; sars-cov: severe acute respiratory syndrome coronavirus; sars-cov- : severe acute respiratory syndrome corona virus who: world health organization; -ncov: novel coronavirus, tentatively named sars-cov- references . wuhan municipal health commission. briefing on the pneumonia epidemic situation the -ncov outbreak joint field epidemiology investigation team, li q. notes from the field: an outbreak of ncip ( -ncov) infection in china -wuhan naming the coronavirus disease (covid- ) and the virus that causes it severe acute respiratory syndromerelated coronavirus -the species and its viruses, a statement of the coronavirus study group china national health commission. notification on the pneumonia epidemic situation press statement by hubei province health commission coronavirus disease (covid- ) situation reports thailand ministry of public health situation press statementon of novel coronavirus kcdc.press statement of novel coronavirus a measure of spatial stratified heterogeneity the interpretation of statistical maps new approaches for calculating moran's index of spatial autocorrelation hubei province health commission hubei province health commission. a letter to the the people of hubei province baidu migration data sichuan province health commission. the th press statement of covid- chinese clinnical guidance for covid- pneumonia diagnosis and treatment chinese prevention and control guidance for covid- pneumonia wuhan revised the number of covid- confirmed cases and death cases novel coronavirus (covid- ) cases publisher's note springer nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations we thank dr. zhao xing and dr. zhang linghua for the help. thanks to all the cdc staff and healthcare workers for collecting information on the frontlines.authors' contributions xx and bz conceived of and designed the study. the data collection was arranged and carried out by xx, jz, rl, yl, lz, td, yc and zw. yz, lf and cp provided support for the interpretation of the data. xx analysed the data and used sas as well as arcgis software to draw the map and drafted the work. xz, yd and xw provided substantive revisions. all authors contributed to the manuscript preparation and read and approved the manuscript. the current position of xinyin xu is an associate research fellow, working in the sichuan centers for disease control and prevention, chengdu, china. her primary research interests are the analysis of public health emergencies, as well as the prevention and control of chronic diseases with some nutrition factors. during the period of fighting covid- , she worked on the covid- situation analysis task force of sichuan province. this study was supported by the sichuan province covid- science and technology emergency project [grant no. yfs ] and the tianfu famous doctor and the national natural science foundation of project (the epidemiologic study of covid- asymptomatic infection) [grant no. ]. the authors disclose no other sources of funding. the funding body did not participate in the design, collection, analysis, interpretation, and writing of this study. the city-level covid- confirmed case number information for sichuan was made available from the health commission of sichuan province website [ ] . the county level information of sichuan was obtained by applying it to the surveillance system of sichuan cdc. the confirmed case number information of other provinces was available from the national and province level health commission website [ , ] . data from various countries were from the who covid- daily report published since january , , which also contained the confirmed information of the different provinces in china before the date of march . the case number information before the first who reports came from different national government websites [ ] [ ] [ ] [ ] . the confirmed number data in the who reports could also be downloaded from the github website, collated as a public database continuously updated by the johns hopkins university center for systems science and engineering [ ] . the population of sichuan province was available from the statistical yearbook of sichuan province [ ] .ethics approval and consent to participate not applicable. not applicable. the authors declare that they have no competing interests. received: april accepted: october key: cord- -ipg vwmv authors: baker, stephen; favorov, michael; dougan, gordon title: searching for the elusive typhoid diagnostic date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: ipg vwmv typhoid (enteric) fever is still a common disease in many developing countries but current diagnostic tests are inadequate. studies on pathogenesis and genomics have provided new insight into the organisms that cause enteric fever. better understanding of the microorganisms explains, in part, why our current typhoid methodologies are limited in their diagnostic information and why developing new strategies may be a considerable challenge. here we discuss the current position of typhoid diagnostics, highlight the need for technological improvements and suggest potential ways of advancing this area. enteric (typhoid) fever remains a common disease in many parts of the world where access to clean water is limited. in places such as india, nepal, pakistan, indonesia and parts of sub-saharan africa typhoid is still a substantial public health problem [ ] [ ] [ ] [ ] . in these areas, febrile disease is common, so accurate diagnosis facilitates treatment selection, particularly as antimicrobial resistance is emerging [ ] . enteric fever is an all encompassing term for the disease caused by several serovars of salmonella enterica including (s.) typhi and (s.)paratyphi a. although globally s. typhi is the most common cause, s. paratyphi a infections occur in significant numbers in some parts of the world and is often associated with travelers [ ] [ ] [ ] [ ] . in contrast, s. paratyphi b and c are relatively uncommon. this article focuses specifically on s. typhi and the disease it causes; typhoid. with respect to other invasive salmonella, s. typhi causes a greater disease burden and there is a superior level of understanding of this organism. however, all of the arguments presented here are poignant for the diseases caused by other human invasive salmonella pathovars. despite who recommendations, few countries have taken on typhoid immunization [ ], this is in part related to uncertainties about disease burden. the best incidence assessment is based on available, sparse surveillance information, estimated that in there were , , illnesses and , deaths due to typhoid and that paratyphoid caused , , illnesses [ ] . these data is extrapolated from limited studies and such figures, therefore, may be imprecise, this is compounded by a lack of accurate diagnosis. therefore, new diagnostics will play a key role in decreasing the incidence of typhoid fever, by permitting governments to accurately assess the particular burden of disease and apply vaccination regimes accordingly. the development of cheap and reliable enteric fever diagnostics would undoubtedly benefit long term disease control and treatment. presently, direct blood culture, followed by microbiological identification is the gold standard, any potential new test needs to offer a higher diagnostic rate than this procedure [ ] . blood culturing of s. typhi, whilst considered "routine", is expensive and requires specialist facilities and personnel. furthermore, s. typhi and s. paratyphi a are not always culturable even if good microbiological facilities are available. diagnostics based on serology, antigen detection or dna are available but have limitations. in the document entitled 'the diagnosis, treatment and prevention of typhoid', the who state that 'the method used as the gold standard for the laboratory diagnosis of typhoid should approach % each for sensitivity, specificity, and positive and negative predictive values' [ ] . however, current tests need significant improvement to reach such rigorous standards. in view of these problems, is the goal set by who achievable and what are the barriers? significant advances have been made in our understanding of the biology and genomics of both s. typhi and s. paratyphi a [ ] [ ] [ ] [ ] . using this information we can reassess typhoid diagnostics and consider the potential and the limitations of different approaches ( figure ). s. typhi causes typhoid, a fecal-oral infection leading to systemic disease ( figure ). tissue invasion drives a potentially close encounter with the immune system. however, s. typhi is an immuno-modulatory pathogen which goes to great lengths to avoid detection by the immune host defenses. the pathogenesis of typhoid fever in man has received only limited attention. this is mainly because both s. typhi and s. paratyphi a are host-restricted to humans and there is no known zoonotic reservoir. experimentation using surrogate hosts and s. enterica serotypes (e.g. s. typhimurium) suggests that tissue invasion occurs predominantly through m cells on peyer's patches in the terminal ileum ( figure ) [ , ] . whilst these mechanisms have never been directly proven for typhoid, it is clear that s. typhi has predominantly forsaken ongoing transmission in the habitat of the mammalian gastrointestinal tract of most enteric bacteria, in the favor of systemic dissemination. the infection eventually localizes to the bone marrow and ultimately the gall bladder where the internal transmission cycle is completed as organisms are shed in bile, potentially in high numbers ( figure ). s. typhi (and s. paratyphi a) is highly clonal, exhibiting limited genome variation, suggesting this organism is recently evolved [ , ] . the genetic basis of the switch from an enteric to a systemic lifestyle is imprinted in its genetic makeup. many genes associated with intestinal persistence (e.g. shda, ratb) or interaction with host surfaces (e.g. fimbria, pili etc.) are inactivated, limiting potential mechanisms for colonizing within and between hosts. for example, genes that contribute to fluid release (e.g. sopa) or intracellular survival (e.g. sope , ssej,) are inactivated [ , ] . thus, invading s. typhi may follow a simple default pathway favoring limiting activation of the inflammatory response. s. typhi (but not s. paratyphi a) also expresses the vi capsular polysaccharide, that possesses immuno-modulatory properties, potentially further dampening the immune response [ , ] . one of the consequences of minimal early inflammation is a lack of the classical gastroenteritis associated with other gastrointestinal pathogens. additionally, humans do not react clinically to the initial invasion step and there is an incubation period before disease symptoms emerge, which occurs during the systemic phase of infection. this stage is one of the early confounders to typhoid diagnostics, the temporal distance between infection and disease hinders the detection of the organism. the presence of bacteria in any tissue may be transient, as cells traffic or become activated. thus, trafficking in blood may only occur during a limited window, making a positive blood culture challenging. this situation may be exacerbated as many patients reach microbiological facilities at a later stage of infection or may be "self treated" with antimicrobials. thus, there are a number of characteristics of the pathogenesis of typhoid that impinge on diagnostics [ , ] . an obvious caveat is the low number of bacteria in the blood and other tissues. it is theoretically possible that there are actually high levels of organisms in the blood but that these are present in an 'unculturable' form. for example, the rapid transfer of bacteria in a semi-quiescent form from within an intracellular vacuole to laboratory media may kill this fastidious and delicate organism. s. typhi is certainly less robust than many other salmonella. bone marrow is the most sensitive culture route but this is an invasive procedure and is seldom performed outside specialist hospitals. volunteers challenged with virulent and attenuated s. typhi strains only shed the organism sporadically in stools, potentially compromising the stool culturing approach [ , ] . many systemic pathogens exhibit the ability to undergo antigenic variation, thus allowing the organism to divert the immune response. analysis of multiple s. typhi genomes shows a lack of obvious evidence for any amount of immune selection on the organism. furthermore, no known s. typhi antigens exhibit significant evidence for variation, a fact highlighted by comparing gene sequences between phylogenetically representative s. typhi [ ] . these data provide further evidence of the ability of s. typhi to cause a systemic infection without stimulating a significant inflammatory response and transfer from the gastrointestinal lumen to the reticuloendothelial system in a relatively undetected fashion. indeed, one may argue that the ability of s. typhi to avoid immune detection constitutes the organism as a "stealth" pathogen and this has significant implications for diagnostics. the lack of immune selection on the organism suggests that s. typhi predominantly occupies an privileged niche within the host, a predominantly intracellular pathogen that can survive for long periods in this state. indeed, frequent relapses associated with the recrudescence of the pathogen and the lack of immune protection in typhoid patients to re-infection further supports a dampening of immunity [ ] . thus, s. typhi may induce only weak immunity, including a muted antibody response. it is worth noting that in typhoid endemic areas many individuals who have never reported typhoid exhibit serological evidence that they have been infected by the pathogen [ ] . thus, it is highly feasible that many people who get exposed by s. typhi do not progress to develop the recognized disease syndrome, or individuals have a small amount of constant boosting due to prolonged exposure. an additional caveat for an effective and appropriate diagnostic test is the cross section of organisms that can cause a disease syndrome that is, clinically, indistinguishable from typhoid fever. depending on the location, a number of viral, parasitic and bacterial pathogens can mimic the basic features of typhoid thus confounding the issues of sample collection, clinical management and efficient diagnosis. the limitations of microbiological culture s. typhi are ordinarily cultured from - ml of blood in - ml of broth. the probability of recovering typhi is transferred to monocytic cells and is trafficked to the reticulo-endothelial system, potentially in a semidormant state. e; s. typhi re-emerges at an unknown time from the reticulo-endothelial system, possibly as the acquired immune response is activated, and re-enters the blood stream in low numbers. f; s. typhi seeds into the liver, the gall bladder and the bone marrow where it can reside and may be detected for months or years. g; s. typhi can enter into the bile duct and be shed sporadically, potentially in high numbers into the environment via the intestine. organisms is increased at greater blood volumes, compromising diagnosis in children. in the developed world, blood culture is semi-automated, exploiting sophisticated culture apparatus. blood taken from patients is inoculated into vessels which are designed to fit in specific machines and contain specialized media, often there is minimal or no dilution of the sample into this media. in this way, the whole process can be captured by an integrated system and a particular laboratory may become dependent on the same supplier, which has particular financial constraints. the advantage of this approach is that it improves specificity and standardization. the main limitation to the wide spread distribution of semi-automated blood culture systems is cost. blood culture facilities are rare in many developing countries, often limited only to major hospitals in large cities. access to receiving a blood culture becomes, therefore, the limiting factor to typhoid diagnosis. it is worth speculating that alternative culture systems, made available at a lower cost and less dependent on expensive consumables, could encourage more facilities to be established in poorer regions. it is also worth noting that in a single tropical setting blood infections may be caused by a wide range of other gram negative and gram positive organisms (e.g. s. typhi, streptococci, leptospira, etc.), parasites (e.g. plasmodium) and viruses (e.g. dengue) [ , ] . blood culture may or may not be a suitable assay for a specific infection, depending on the pathogen and the location. taken that there are only low levels of s. typhi present in blood, how might we improve approaches to direct culture? could s. typhi culture be further optimized, by taking advantage of some atypical biochemical properties of the organism? examination of the s. typhi genome highlights metabolic and scavenging pathways inactivated by the accumulation of pseudogenes [ ] . examples include the cobalamin pathway, many metabolic transporters and iron uptake systems. understanding specific biochemical pathways that are up-regulated under defined conditions may permit some modeling of conditions in which s. typhi can be grown more efficiently. in short, could we use a method that we define as "metabolomic modeling" to design recovery media to enrich for s. typhi? this approach is certainly worth considering but may only have a marginal effect on bacterial recovery. ultimately, the low level of bacteria in the sample may set an impenetrable practical barrier which may only be circumvented by purification or enrichment technology. the culturing of bone marrow biopsies is more sensitive than that of blood culture and a modified technique to take bone marrow in a more straightforward and somewhat less brutal manner would be desirable [ ] . the first typhoid diagnostic, the widal test, was developed in . the methodology is dependent on agglutination; s. typhi cells are used to detect antibodies in blood. this crude assay is a visual test that monitors agglutinating antibodies that react with s. typhi [ ] . problems associated with the use of widal are somewhat obvious and may apply to other serologically based assays for typhoid. s. typhi is a relatively invariant pathogen so antigenic variation per se should not be a significant confounder. however, s. typhi is a member of the enterobacteriaceae. many of the surface antigens of the enterobacteriaceae demonstrate significant conservation and induce antibodies that are cross-reactive. consequently, as humans mature they accumulate antibodies that are cross-reactive with s. typhi. thus, it may be impossible to develop a specific diagnostic kit for typhoid using semi-purified antigens. indeed, any such kit would likely yield significant false positives. s. typhi expresses a number of immunogenic structures on the surface, some of which provide a basis for serology identification. these include o (lipopolysaccharide), h (flagella) and the somewhat less immunogenic vi capsule. s. typhi exhibiting variation in these antigens are uncommon, with notable exceptions. s. typhi found in indonesia express variant h antigens including h:j and h:z [ ] [ ] [ ] [ ] . vi-negative s. typhi isolates have been reported in pakistan but are rare [ , ] . therefore, s. typhi expressing o (o , o ), vi and h:d are ubiquitous in most endemic areas. seroprevalence studies have been performed in endemic regions to determine antibody titers to o, h and vi in the general population [ , ] . many individuals in endemic areas have cross-reactive antibodies even though they have no clinical record of typhoid. additionally, such raised antibody levels frequently cannot be detected in patients with culture confirmed typhoid. problems have also been encountered during the testing of commercial serological tests, including typhidot and tubex [ , ] . these assays were assessed in population-based typhoid surveillance studies in several countries and in all locations the sensitivity and specificity for tubex and typhidot was only around % and % respectively [ , ] . clearly the abundance and avidity of anti-s. typhi antibodies varies and it is difficult to imagine how a clean diagnostic assay with high specificity could be produced targeting these classical antigens. can other antigen/antibody complexes be used as more accurate diagnostics? this is an under-studied area with few s. typhi specific antigens being investigated in any detail. experiments utilizing convalescent serum from typhoid patients, indicates that individuals can respond to a range of s. typhi antigens [ ] . however, such responses appear to be variable and no obvious immuno-dominant antigens have been identified. studies may be confounded by the fact that in vitro grown s. typhi are used to measure responses, a factor that would eliminate the detection of any antigen exclusively expressed in the host. this could be an important consideration as many surface structures, e.g. pili, have such properties. a potentially productive area may be to search for novel antigens which are specific for s. typhi. candidate targets could be identified initially by bioinformatics. novel candidates could be expressed in systems such as yeast to minimize contamination with cross-reactive antigens. a pool of highly purified specific antigens could be screened using serum from typhoid patients and appropriate controls. protein microarrays could be exploited in the screening [ ] . testing in a cohort of patients could reveal specific patterns or quantities of antibodies which would be indicative of typhoid infection. ultimately, novel antigen(s) could be placed onto membrane to form the basis of a low cost rapid test. this is an open and uninvestigated area and with a suitable assay and patient material, it may be one of the most straightforward ways to initially develop a low cost and highly specific test. the detection of specific dna sequences within the genome of s. typhi would appear to be an attractive proposition. is a robust dna-based test a real option for routine typhoid diagnostics? many s. typhi pcrbased assays have targeted the flic gene, utilizing nested primers to improve sensitivity [ ] [ ] [ ] [ ] [ ] . there is an additional sensitivity benefit of pcr, in that it can theoretically amplify dna from dead or unculturable bacteria. various pcr-based studies on typhoid suggest that the assay is specific and sensitive and relatively straight forward to perform. indeed, such studies have yielded sensitivities > %. however, we believe pcr offers only limited potential for typhoid diagnostics. currently there is no validated pcr test in common use, only in-house systems which are open to differing interpretation and none would meet the rigors of quality control to make this assay used worldwide. massi et al. utilized a real-time system based on flic to detect s. typhi in patients with clinically diagnosed typhoid [ ] . they were able to amplify flic from all culture-positive and negative blood samples tested but reported a higher gene copy number in culture positives ( , - , ), compared to negatives (< ). however, this real-time pcr data is somewhat contradictory with the microbiological data, which demonstrates that bacteria/ml of blood is generally low with the majority of patients having < organism/ml of blood [ ] . it is somewhat surprising that typhoid patients may have between a , to , times more dead bacteria than live bacteria in the blood. we recently found disappointingly poor pcr sensitivity using a three color real-time pcr assay that was capable of detecting s. typhi, s. paratyphi a and an incorporated internal control [ ] . when tested on spiked and control samples the assay demonstrated high specificity and sensitivity. however, when tested on dna extracted from ml of blood taken from culture confirmed typhoid patients the sensitivity rate was less that %. thus confirming that pcr results are related to the actual colony forming units found in the blood. the assay did, however, demonstrate % sensitivity on culture positive bone marrow samples, which are known to harbor significantly more bacteria [ ] . for these reasons we believe that dna amplification may not be an easy route towards developing a robust diagnostic. collecting and then extracting dna from a large volume of blood is not a straightforward option, due to large concentrations of human dna. analyzing stool or urine samples may be an alternative approach. a dna or bacterial capture system or even a culture enrichment step prior to amplification may improve molecular sensitivity. however, molecular diagnostics are not a cost effective or a straightforward to perform as other methods, not every diagnostic laboratory in an endemic setting would be able to perform such an assay. however, if simplified and new technology is applied it an area that warrants further independent studies. is it possible to identify host specific responses to typhoid that are distinct from other febrile diseases such as malaria or dengue? if so, what sort of responses should we look for? typhoid patients display a number of symptoms including fever and mount a number of immune and physiological responses. such responses can be examined by simple stimulation assays, exploiting whole blood, cell fractions or serum. currently, there has been no precise correlate of infection or biomarker for typhoid identified. an expansive, yet costly option would be to take an approach based on human microarrays [ ] . transcriptional analysis of rna extracted from the blood of typhoid patients could be performed to identify specific genes, pathways, interactions or transcriptional regulatory hubs that are activated in the host during infection. microarray data is often publicly available and comparative analysis with the transcriptional profile from patients with other diseases could be studied at databases such as innatedb http://www.innatedb.ca/. such analysis may highlight suitable targets that could be tracked in patients [ ] . mass spectrometry, proteomics or similar expression monitoring technologies could be applied to identify particular genes or pathways that are functionally activated during typhoid. once a gene or transcriptional pathway is identified, expression could be monitored using dna or protein probes. this approach may be a long term aim and comparative analysis with similar materials from other diseases would be an essential requirement. blood would most likely be the assay material of choice and this in itself may present limitations if responses are localized to deeper tissues. however, this approach is highly novel, powerful and worthy of further investigation and investment. an further alternative approach would be to identify potential biomarkers, i.e. discover a physiological signature or metabolic product associated with typhoid. the signature could be of host or bacterial origin or a combination of both that is/are produced in real time during infection. the science of host metabolomics is growing with the development of applications such as nmr and mass spectrometric technologies. metabolomics could, theoretically, work on a range of bodily fluids, including blood and urine and may detect specific small or complex macromolecules. some research groups have developed systems for identifying biomarkers in biological material from patients infected with various pathogens such as tuberculosis [ , ] . surface-enhanced laser desorption/ionization time-of-flight (seldi-tof) mass spectroscopy has also been utilized in studying sars protein biomarkers, as reviewed by mazzulli et al. [ ] . seldi-tof may add insight into those proteins that are expressed in serum, blood, saliva, urine or any other biological material that may harbor specific markers for typhoid infections. some early studies on pathogenesis and diagnostics did focus on the detection of s. typhi antigens such as vi in the urine of patients and this is worth revisiting in view of a substantial increase in the sensitivity of detection technologies [ , ] . what about the diagnosis of typhoid in carriers infected with s. typhi or s. paratyphi a [ , ] ? clearly, such individuals warrant special consideration as they are a silent threat to others in the population. monitoring s. typhi in the stool is one option but shedding may be low level or sporadic. further, stool sampling at a routine level is expensive, time consuming and unpopular, although improved bacterial recovery methods could be one approach. we know of no obvious signature that can be used to categorically identify s. typhi carriers. however, important studies have indicated that typhoid carriers may produce higher levels of vi antibodies over extended periods compared to acutely infected patients [ , ] . this may be in part because vi is a polysaccharide and the immune response to vi is t cell-independent, stimulating poor memory. however, carriers may receive continual, natural boosting when the organisms are reseeded, potentially in high numbers (figure ) , back into the intestinal tract. if we could develop simple, cheap and none invasive vi antibody assays these may prove valuable in identifying carriers. the ultimate question is which direction do we follow in terms of developing typhoid diagnostics and how can these be applied to location with limited resources? in the short term, it appears that whilst current techniques are limited there is no real alternative without extensive research and culturing remains the inadequate gold standard. however, laboratories in developing countries with typhoid should be prepared to evaluate new diagnostics as they evolve. as a way forward for culture, it may be prudent to investigate specialized growth media that would favor the regeneration of s. typhi from blood. simple methods for enriching the small population of bacteria present in blood using simple direct enrichment procedures that do not rely on growth could be considered. dna methodology has specific limitations that are similar to those presented with bacterial culture. advancement in this field would require the capture and amplification from a smaller number (maybe even a single organism) from blood or other bodily fluids. such a task is not insurmountable but it will be a challenge to make it cost effective. serological advancements will rely on the identification of novel s. typhi-specific antigens that are conserved and highly immunogenic in the human host. we will need simple methods to prepare highly purified antigens free of potentially cross reacting materials and antigen pools may be needed to increase sensitivity. serological approaches may be more tractable to convert into a simple, cheap and rapid test. host response assays will have to be developed through the application of genomics and highly sensitive mass spectrometric, nmr or similar sensitive physical assays. looking for host or pathogen material in biological samples is an area that clearly warrants further investigation. once targets have been identified, the next limiting step, with respect to locations with limited resources is developing a reliable test that is affordable. with the identification of novel targets is should be feasible to create simple point of care assays aimed at these specific targets. however, making such tests that can be manufactured at a reasonable cost that can aid typhoid diagnostics in the locations where they are required most may add an additional hurdle. typhoid and paratyphoid fever the global burden of typhoid fever still an enteric fever capital of the world typhoid fever 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fever in the faisalabad region of pakistan vi antigen expression in salmonella enterica serovar typhi clinical isolates from pakistan use of paired serum samples for serodiagnosis of typhoid fever one-step -minute test to detect typhoid-specific antibodies based on particle separation in tubes dot enzyme immunoassay (typhidot) in diagnosis of typhoid fever in children evaluation of new-generation serologic tests for the diagnosis of typhoid fever: data from a community-based surveillance in calcutta a study of typhoid fever in five asian countries: disease burden and implications for controls a genome-wide proteome array reveals a limited set of immunogens in natural infections of humans and white-footed mice with borrelia burgdorferi multiplex pcr for differential diagnosis of emerging typhoidal pathogens directly from blood samples development of an ultra rapid and simple multiplex polymerase chain reaction technique for detection of salmonella typhi pcr method to identify salmonella enterica serovars typhi, paratyphi a, and paratyphi b among salmonella isolates from the blood of patients with clinical enteric fever rapid diagnosis of typhoid fever by pcr assay using one pair of primers from flagellin gene of salmonella typhi diagnosis of typhoid fever by polymerase chain reaction quantitative detection of salmonella enterica serovar typhi from blood of suspected typhoid fever patients by real-time pcr comparison of blood culture and realtime pcr for the detection of invasive salmonella serovars in blood and bone marrow specimens from enteric fever patients using dna microarrays to study host-microbe interactions innatedb: facilitating systems-level analyses of the mammalian innate immune response biomarker discovery in infectious diseases using seldi identification and characterization of mycobacterium tuberculosis antigens in urine of patients with active pulmonary tuberculosis: an innovative and alternative approach of antigen discovery of useful microbial molecules proteomics and severe acute respiratory syndrome (sars): emerging technology meets emerging pathogen enzyme-linked immunosorbent assay for detection of salmonella typhi vi antigen in urine from typhoid patients rapid diagnosis of typhoid fever by enzyme-linked immunosorbent assay detection of salmonella serotype typhi antigens in urine precise estimation of the numbers of chronic carriers of salmonella typhi in santiago, chile, an endemic area gallbladder carriage of salmonella paratyphi a may be an important factor in the increasing incidence of this infection in south asia vi serology in detection of chronic salmonella typhi carriers in an endemic area pre-publication history the pre-publication history for this paper can be accessed here searching for the elusive typhoid diagnostic this was supported by the wellcome trust and the international vaccine institute, seoul, korea. sb is supported by an oak foundation fellowship through oxford university. authors' contributions sb, mf and gd were responsible for the concept, the content and the writing of the manuscript. all authors have read and approved this manuscript. the authors declare that they have no competing interests. key: cord- -x bzrpcu authors: faires, meredith c; pearl, david l; ciccotelli, william a; berke, olaf; reid-smith, richard j; weese, j scott title: the use of the temporal scan statistic to detect methicillin-resistant staphylococcus aureus clusters in a community hospital date: - - journal: bmc infect dis doi: . / - - - sha: doc_id: cord_uid: x bzrpcu background: in healthcare facilities, conventional surveillance techniques using rule-based guidelines may result in under- or over-reporting of methicillin-resistant staphylococcus aureus (mrsa) outbreaks, as these guidelines are generally unvalidated. the objectives of this study were to investigate the utility of the temporal scan statistic for detecting mrsa clusters, validate clusters using molecular techniques and hospital records, and determine significant differences in the rate of mrsa cases using regression models. methods: patients admitted to a community hospital between august and february , and identified with mrsa > hours following hospital admission, were included in this study. between march and february , mrsa specimens were obtained for spa typing. mrsa clusters were investigated using a retrospective temporal scan statistic. tests were conducted on a monthly scale and significant clusters were compared to mrsa outbreaks identified by hospital personnel. associations between the rate of mrsa cases and the variables year, month, and season were investigated using a negative binomial regression model. results: during the study period, mrsa cases were identified and mrsa isolates were spa typed. nine different spa types were identified with spa type /t ( . %) the most prevalent. the temporal scan statistic identified significant mrsa clusters at the hospital (n = ), service (n = ), and ward (n = ) levels (p ≤ . ). seven clusters were concordant with nine mrsa outbreaks identified by hospital staff. for the remaining clusters, seven events may have been equivalent to true outbreaks and six clusters demonstrated possible transmission events. the regression analysis indicated years – , compared to , and months march and april, compared to january, were associated with an increase in the rate of mrsa cases (p ≤ . ). conclusions: the application of the temporal scan statistic identified several mrsa clusters that were not detected by hospital personnel. the identification of specific years and months with increased mrsa rates may be attributable to several hospital level factors including the presence of other pathogens. within hospitals, the incorporation of the temporal scan statistic to standard surveillance techniques is a valuable tool for healthcare workers to evaluate surveillance strategies and aid in the identification of mrsa clusters. in the healthcare setting, the timely identification of healthcare-associated infections (hais) and the institution of infection control measures are crucial steps for the prevention and reduction of transmission events and outbreaks in the patient population. however, the detection of transmission events is based on limited evidence [ ] , and detection of specific pathogen clusters is generally subjective in nature [ ] . furthermore, even well-established infection prevention strategies can be often disregarded [ ] at various levels. overall, these factors may lead to the transmission of pathogens within the hospital, a missed opportunity to investigate a disease cluster, or false ascertainment of a cluster resulting in the misuse of hospital resources for investigational purposes [ ] . statistical methods, such as the scan statistic, may enhance the identification of disease clusters and/or outbreaks in the hospital setting [ , ] . the scan statistic [ ] can be used to detect and evaluate clusters of infectious diseases in space, time, and/or space-time [ ] [ ] [ ] . by understanding the clustering of infectious diseases spatially and/or temporally, potential risk factors can be identified [ ] , detailed investigations determining the association between exposures and disease interventions can be facilitated [ ] , and outbreaks can be detected [ ] . methicillin-resistant staphylococcus aureus (mrsa) outbreaks in healthcare facilities represent a significant burden to public health in terms of ward closures, infection control measures, increased patient morbidity and mortality rates, and healthcare costs [ , ] . in hospitals, identification of outbreaks is routinely based on the examination of microbiological test results and patients' charts [ ] , with the definition of an outbreak generally relying on rule-based criteria [ ] . however, these types of criteria are prone to error since they fail to address changes in population size or random variation [ ] . with the increasing availability of timely surveillance data within the hospital setting, the use of analytical methods may lead to the earlier detection of disease clusters or outbreaks [ ] . in hospitals, the incorporation of the scan statistic for the detection of mrsa disease clusters, spatially and/or temporally, has been limited. in one study, a space-time permutation scan statistic was utilized to detect clusters throughout the hospital [ ] . however, molecular data were not collected to validate mrsa clusters. in another investigation, a temporal scan statistic, incorporating molecular data, was employed to identify mrsa clusters which were used as a gold standard to evaluate other algorithms for cluster detection [ ] . for both investigations, the application of a scan statistic to hospital data resulted in the identification of several significant mrsa clusters that were not identified by hospital personnel. however, as both studies were conducted in academic medical centres, further investigation of the scan statistic for identifying mrsa clusters under different healthcare settings is required [ , ] . the objectives of this study were to investigate the utility of the temporal scan statistic for detecting mrsa clusters in a community hospital and to validate significant clusters using molecular techniques and hospital records concerning known mrsa outbreaks; and to determine if there were significant differences in the rate of mrsa infection and colonization cases by month, season, and year using regression models. located in southern ontario, canada, the participating study hospital has beds, over , in-and outpatient visits annually, and provides an array of services including internal medicine, surgery, emergency, pediatrics, oncology, rehabilitation, intensive care, and psychiatry. both urban and rural populations are served by this facility. this study was approved by the research ethics boards of the university of guelph and the participating hospital. the research ethics approval covered all aspects of the study including the collection of de-identified isolates from the hospital's microbiology laboratory. in the study hospital, targeted surveillance for mrsa is conducted based on recommendations provided by a provincial infectious diseases advisory committee [ ] . briefly, during the study period, at the time of hospital admission, patients identified as having an increased risk for mrsa acquisition are screened. these risk factors include [ ] : previous colonization or infection with mrsa; time spent in any healthcare facility in the previous twelve months (time defined as > continuous hours); recent exposure to a unit/ward/area of a healthcare facility identified with an mrsa outbreak; or individuals receiving home healthcare or treatment with an indwelling medical device. for mrsa detection, specimens are obtained from the anterior nares and the perineal area (surveillance sample); and from any skin lesions, wounds, incisions, ulcers, and exit sites of indwelling devices if present (surveillance and/or clinical sample) [ ] . during an outbreak or for patients located on high risk units (e.g., intensive care unit), universal admission screening is conducted. for patients hospitalized for an extended period of time, weekly mrsa surveillance cultures are performed. in addition, for patients identified with indwelling medical devices or wounds, these sites are monitored by weekly culture. if a patient is identified as an mrsa contact (i.e., roommate of a patient who is found to be mrsa positive), the mrsa contact will have two sets of surveillance cultures taken with at least one set taken seven days following their last exposure to the mrsa patient [ ] . in the participating hospital, mrsa cases were classified as healthcareassociated (ha) if the patient was newly identified with mrsa (infection or colonization) > hours following hospital admission. for a case to be included in this investigation, mrsa was newly identified between august , and february , and > hours following hospital admission. patients that were identified as being either infected or colonized with mrsa were included. only one mrsa isolation event per patient per admissiondischarge period was included in the analyses. the admission-discharge period was defined as the time interval from when a patient was admitted to, and discharged from, the hospital. transfer to another ward within the hospital was not considered a discharge. for a patient to be admitted ≥ times to the hospital, > hours between the discharge and admission dates was required. data from the first bacteriology report per patient per admission-discharge period were obtained. bacteriology reports from mrsa cases located in the emergency and hemodialysis wards were excluded as these departments specifically support outpatients. for mrsa cases identified between march , and february , , the hospital's microbiology laboratory collected and submitted patient mrsa isolates for molecular typing. in the participating facility, mrsa was confirmed from surveillance samples using chromogenic agar (bbl chromagar, mrsa, becton, dickinson and company, sparks, maryland, usa), the penicillin-binding protein a latex agglutination test (mrsa latex agglutination test, oxoid ltd., hants, uk), and vitek® (biomérieux, marcy-l'Étolie, france) according to the manufacturers' instructions. for clinical samples, s. aureus was confirmed using the gram stain and the tube coagulase test and mrsa was confirmed using vitek® . for surveillance and clinical mrsa isolates, a standard panel of antimicrobials was used for susceptibility testing and included rifampin, clindamycin, trimethoprim and sulfamethoxazole, and vancomycin. testing was performed using vitek® and results were based on the standards set by the clinical and laboratory standards institute [ ] . at the hospital level, all specimens submitted for mrsa testing were collected at the discretion of medical personnel. only one mrsa isolate per patient was collected for molecular typing. data collected from the bacteriology report included a unique patient identifier, dates pertaining to patient admission, discharge, and when a specimen was collected for mrsa testing, the ward location of the patient when the specimen was obtained, and the antimicrobial susceptibility profile of the mrsa isolate. for ward location, bacteriology reports provided both service and ward designations. services were defined as specific departments (e.g., internal medicine, surgery) whereas wards were characterized as specific, physically distinct units that comprised a service (e.g., wards s and s constituted the surgery service). information pertaining to the number of patient days per month for each service was obtained. for wards, data on patient days were collected only from those wards that were operational and provided the same service for the complete study period (i.e., months). for descriptive statistics, incidence rates for mrsa were expressed as the number of mrsa cases per , patient days. data pertaining to known mrsa outbreaks that occurred during the study period (e.g., start and end date, ward location, and number of patients involved) were collected from the hospital's infection prevention and control (ipc) department. the molecular identity of mrsa strains involved in previous outbreaks was not available as this healthcare facility does not routinely analyze mrsa strains at the molecular level. in the study hospital, standardized rule-based criteria to identify mrsa outbreaks are not employed. due to the lack of a formal definition, mrsa outbreaks were identified by hospital personnel based on an increase in the baseline mrsa rate over a rapid time period, especially when localized to one patient care area. patient isolates were obtained from the hospital's microbiology laboratory following mrsa confirmation. isolates were collected from culture plates using a sterile culture swab with stuart's media and forwarded to the laboratory at the university of guelph. upon arrival, culture swabs were streaked onto blood agar (oxoid, nepean, ontario, canada) and incubated at °c, aerobically, for hours. colonies were identified as s. aureus by gram stain, catalase test, tube coagulase test, and the s. aureus latex agglutination assay (pastorex staph-plus, bio-rad laboratories ltd., mississauga, ontario, canada). the presence of methicillin-resistance was confirmed by the penicillin-binding protein a latex agglutination test (mrsa latex agglutination test, oxoid ltd., hants, uk). molecular typing of mrsa was conducted using sequence analysis of the x region of the staphylococcal protein a gene (spa typing) [ ] . sequences were then analyzed using two different methodologies; egenomics software [ ] and the ridom system [ ] . based on egenomics, spa types are reported using a numerical system (e.g., spa type ) whereas ridom spa types are reported using a numerical system preceded by a 't' (e.g., t ). the spa types were compared to epidemic mrsa strains that are frequently found in canada [ ] . in addition, all mrsa isolates were investigated for the lukf-pv gene encoding the panton-valentine leukocidin (pvl) toxin by real-time pcr [ ] . to determine the clustering of spa types, all spa typing data were imported into bionumerics (version . ; applied maths, ghent, belgium) and were analyzed using the spa typing plug-in tool. a minimum spanning tree (mst) was constructed using the default distance bin size of %. only spa types that differed by ≤ repeats were considered to be closely related [ ] . all bacteriology reports were provided by the hospital in electronic format. the temporal scan statistic was performed using satscan version . [ ] and all descriptive statistics and model building were conducted using stata . (statacorp, college station, texas, usa). for all hypothesis tests, a % significance level was applied (α ≤ . ), if not stated otherwise. to evaluate the association between the rate of mrsa cases in the hospital and the independent variables year, month, and season, a poisson regression analysis was conducted. for the independent variable season, months were categorized in the following groupings: winter (january -march), spring (april -june), summer (july -september), and fall (october -december). the dependent variable and offset were the number of mrsa cases and the natural log of the population at risk (i.e., patient days), respectively, for a particular month. due to the hierarchical structure of the data, mrsa cases nested in wards, a multilevel poisson model including a random intercept for ward and a fixed effect for service, was used to adjust for clustering. specifically, the variable service was categorized as medicine (intensive care, oncology, pediatrics, internal medicine), surgery, and other (psychiatry, rehabilitation, hospice, childbirth, nursery). the spearman's rank correlation coefficient was used to identify correlations between all independent variables. variables with a correlation above . were investigated for collinearity and the biologically more plausible variable was retained in the model [ ] . univariable multilevel poisson models were fit using marginal likelihood estimation via the adaptive quadrature algorithm (as implemented in the xtmepoisson procedure in stata) to screen each independent variable with the dependent variable using a liberal significance level (α ≤ . ). manual backwards step-wise modeling was applied to fit a multivariable multilevel poisson model to all previously identified significant co-variables. to assess the significance of the independent variables, the likelihood ratio test was utilized. confounding was evaluated by examining the effect of the removed variables on the coefficients of the remaining variables. a variable was considered to be a confounder if it changed the model coefficients by ≥ % [ ] . interaction terms were examined for all independent variables. due to concerns regarding unexplained overdispersion, the poisson random effects model was compared to a negative binomial random effects model using akaike's information criteria (aic). the random effects negative binomial model allowed the overdispersion parameter to vary randomly by cluster based on a beta distribution (using the xtnbreg command in stata) [ ] . the model with the lowest aic was selected. based on the final multivariable model, contrasts for independent variables with > categories were examined to investigate significant differences between any two categories. to identify mrsa clusters, the temporal scan statistic employing a poisson model was used. the scan statistic involves a flexible scanning window that gradually moves across time. the number of observed and expected observations inside the window is compared to outside the window, at each time period, with the greatest excess of observed cases noted [ , ] . under the null hypothesis, the expected number of cases in each time period covered by the scanning window is proportional to its population size; whereas under the alternative hypothesis, there is an elevated risk within the window as compared to outside the window [ ] . for this investigation, the population size was defined as the number of patient-days for each service and ward on a monthly basis. the window identified as least likely due to chance, is subsequently evaluated by a maximum likelihood test with a test decision based on a monte-carlo simulated p-value [ ] . monte carlo replications were set at for this analysis. to detect mrsa clusters, only periods with high rates (i.e., a one-tailed test) were scanned. the maximum temporal window size was set to % of the study period. in addition, the scan test was adjusted for more likely clusters via an iterative test procedure with the identified clusters from previous iterations removed from the data set and a new analysis performed using the remaining data [ ] . data were analyzed on a monthly scale. a cluster was defined as a period where the rate of mrsa cases was statistically higher than expected inside a window compared to outside a window. retrospective monthly scan tests were conducted for the entire study period (i.e., august, to february, ) as well as annually (january st -december st ) from to . for , the time interval was restricted to august st -december st and for , the time interval was limited to january st -february th . for each time interval, temporal scan tests were conducted to identify mrsa clusters at three different levels including hospital wide, services, and wards. for this analysis, services were identified and included acute care, complex care, hospice, the intensive care unit, internal medicine, oncology, pediatrics, psychiatry, rehabilitation, and surgery. five wards were identified and included m (internal medicine), s (surgery), s (surgery), c (complex care), and c (complex care). significant (p ≤ . ) clusters that were identified by the temporal scan statistic were compared to outbreaks identified by the ipc department. for clusters that were characterized with molecular data, mrsa cases that comprised significant clusters were evaluated based on spa type. characteristics of significant clusters (e.g., time frame, observed and expected case numbers, p-value, and spa type) are reported. data on mrsa cases, from patients, were obtained during the study period. a total of ( . %) patients were identified with mrsa during one hospital admission-discharge period and ( . %) patients were identified with mrsa during two or more hospital admission-discharge periods. for the patients, . % (n = ) were male and . % (n = ) were female. for male patients, ages ranged from months to years (mean = . years) and for female patients, ages ranged from to years (mean = . years). the monthly incidence rate of mrsa fluctuated over the study period ( figure ) ranging from . to . mrsa cases (colonization and infection)/ , patient days with a mean of . mrsa cases/ , patient days. summary characteristics of the mrsa incidence rate per month, year, season, service, and ward are presented in table . overall, the highest incidence rates of mrsa occurred during and in (during the first two months in which surveillance data were available). on average, march, april, and may reported the highest mrsa rates on a monthly basis. at the service level, the highest mrsa rates occurred in the surgery, internal medicine, and hospice departments, whereas, the pediatric department reported the lowest mrsa incidence rate in the hospital. from march , to february , , mrsa cases were identified and ( . %) patient isolates were obtained for spa typing (table ) . overall, nine different spa types were identified with spa type /t ( . %) the most prevalent. when spa types were categorized according to the epidemic cmrsa type, the majority ( . %) of spa types were classified as cmrsa- ( table ). only one spa type, /t , was classified as cmrsa- . all isolates were negative for the pvl toxin gene. based on the mst that was constructed for clustering spa types (figure ), one major group was observed with seven different spa types reported as being closely related (i.e., difference is ≤ repeats) to spa type /t . these closely related spa types all corresponded to cmrsa- . for two or more mrsa isolates found with identical spa types, antimicrobial susceptibility profiles of spa types were examined to determine if the molecular findings could be further characterized (table ). for spa types /t , /t , /t , and /t , the antimicrobial susceptibility profiles were identical between spa types. for spa type /t , three different antimicrobial susceptibility profiles were identified. a random effects negative binomial model was chosen over the random effects poisson model based on the aic value. results from the univariable multilevel negative binomial models indicated that year and month were all significantly associated with the rate of mrsa cases in the hospital [see additional file ] . the final multivariable multilevel negative binomial model indicated that year and month were significantly associated with the rate of mrsa cases (table ). interactions between the variables year and month could not be assessed due to the number of categories for each independent variable and the resulting small number of observations per interaction term. for the independent variable year, years - had mrsa rates that were significantly higher than the mrsa rates in . results from model-based contrasts indicate that years - also had significantly higher mrsa rates than years and (table ). in the final model, the rates of mrsa were also significantly higher in months march and april compared to january. results from model-based contrasts demonstrated that significant increased mrsa rates were also noted in march and april compared to june, and for april compared to february. over the study period, the temporal scan statistic identified statistically significant mrsa clusters at the hospital (n = ), service (n = ), and ward (n = ) levels ( table ) . as separate scan tests were performed at various levels, it was observed that several clusters overlapped in time and location (i.e., service and wards). of the clusters identified at the service and ward levels, were classified as unique events. overall, clusters ranged in duration from to months (mean = . months) and involved to patients incidence rate presented is based on an average for that specific period. incidence rate presented is the total for that specific year. incidence rate presented is the total for that service or ward from august , to february , . (mean = . ) per cluster. using routine surveillance, ipc personnel identified nine mrsa outbreaks during the study period. these outbreaks occurred in five wards (equivalent to three services), ranged from weeks to months in duration, and involved to patients (mean = ) per outbreak. seven ( . %) of these previously known outbreaks were identified as significant clusters (cluster id , , , - ) based on the temporal scan statistic. of the seven events that were identified by both ipc personnel and the temporal scan statistic, three clusters (cluster id , , ) were further characterized using molecular data. for all three clusters, more than one spa type was identified; however, spa type /t was the most prevalent mrsa strain in all three events. for the cluster identified in ward s from july to february , further investigation revealed that spa type /t was identified in two different patients in november, . for the remaining mrsa clusters, ( . %) events were considered to be of short duration (i.e., - months in length) and ( . %) events were considered to be of long duration (i.e., - months in length). examination of short duration clusters revealed that seven of the events (cluster id , , , , , , ) may have been potential outbreaks as there were considerably large numbers of mrsa cases detected during a short time span. for two of these clusters, typing data indicated that spa type /t was the prevalent mrsa strain identified in the patient population. for long duration clusters, seven (cluster id , , , , , , ) were further characterized with molecular data. for six of these events (cluster id , , , , , ), several spa types were identified circulating in the patient population. however, like the short duration clusters, spa type /t was the predominant mrsa strain in all seven events. further analysis of the long duration clusters revealed potential transmission of non-spa type mrsa strains. for the complex care cluster (cluster id ), spa type /t was identified in two different patients, both admitted to the same ward, in december . in the internal medicine cluster (cluster id ), two patients were identified with spa type /t , seven weeks apart. for the surgery cluster (cluster id ), two patients, located in the same ward, were identified with spa type /t in november . in the present investigation, a cluster was defined as a statistically significant high rate of mrsa cases within a time period. using the temporal scan statistic, several significant mrsa clusters were identified, during the - surveillance period, in a community hospital. during the same time period, nine mrsa outbreaks were identified by ipc personnel using standardized surveillance techniques. however, only seven of these outbreaks were identified by the temporal scan statistic as significant clusters. this was not surprising, as others have reported similar results regarding the discordance between events identified by hospital staff and the identification of clusters employing a scan statistic [ , ] . investigation of the seven outbreaks identified by both hospital staff and the temporal scan statistic indicated that five (cluster id , , - ) were identified by the scan test with a starting and/or end date that were different from the dates provided by ipc personnel. specifically, clusters identified by the temporal scan statistic were one to seven months longer in duration; consequently, a greater number of mrsa cases per cluster were identified. for four of the clusters (cluster id , - ), the temporal scan statistic indicated that these events occurred prior to dates reported by ipc personnel. this is an important finding as these events demonstrate that there may have been a delay in the institution of infection control strategies. furthermore, two of the clusters (cluster id , ) were identified as being longer in duration by the temporal scan statistic; therefore, premature discontinuation of infection control measures by ipc personnel may have occurred. for the two mrsa outbreaks identified by ipc staff only, one outbreak was located in the intensive care unit (n = patients, month in duration) and the other outbreak was located in a ward in the internal medicine department (n = patients, weeks duration). the outbreak in the intensive care unit was identified by the temporal scan statistic; however, the cluster was not significant. for the outbreak identified in the ward of the internal medicine department, this particular ward was only operational for of the months that the study was conducted, and therefore, did not meet the inclusion criteria for a temporal scan test to be conducted at the ward level. however, temporal scan tests conducted at the hospital and service (e.g., internal medicine) levels did not result in the identification of a cluster that corresponded in time to the mrsa outbreak. in the participating healthcare facility, specific rule-based criteria (e.g., ≥ ha-mrsa cases in a week period) are not used to establish mrsa outbreaks. rather, data pertaining to the number of mrsa cases and the time period (i.e., number of days) are used to ascertain if an outbreak exists in the patient population and if an investigation should be initiated. this type of surveillance is subjective in nature and may be prone to under-reporting or overreporting of mrsa outbreaks. of the short duration (e.g., - months) clusters that were identified during this investigation, seven events were labelled by the investigators as potential outbreaks due to the large number of mrsa cases identified over a short time period. however, only two of these events could be analyzed at the molecular level, with spa type /t as the predominant mrsa strain in each cluster. these molecular findings indicate that transmission events may have occurred and that these clusters may have been equivalent to true outbreaks. for the long duration (e.g., - months) clusters identified, their biological relevance is difficult to discern. these clusters may represent extended outbreaks, temporal trends, changes in pathogen characteristics, or the representation of systematic changes (e.g., cleaning policies) at the hospital level during the surveillance period. for many of the long duration clusters, several non-spa type /t mrsa strains were identified in the patient population. these particular mrsa strains may indicate unique transmission events between patients or be part of an outbreak, as researchers have documented the existence of more than one mrsa strain during an outbreak investigation [ , ] . alternatively, these non-spa type /t strains may represent genetic changes within the spa gene, such as deletions or duplications of repeats and point mutations [ ] , resulting in different spa types. based on the mst that was constructed (figure ) , six of the spa complexes were closely related to spa type /t by a difference in one repeat. for mrsa and s. aureus, research exploring the time required for a genetic event to occur has been conducted [ ] [ ] [ ] ; however, results of these investigations are based on specific strains and specific locations of the genetic event. other plausible explanations for observing diverse spa types in these clusters include the introduction of spa types into the hospital via staff, visitors, and patients which resulted in transmission events, and healthcare workers and patients that may have been colonized with variant mrsa strains. in the study hospital, molecular typing of mrsa isolates is not routinely performed. this was a major limitation of this study, as there was no prior knowledge of the endemic mrsa strains in this facility. furthermore, not all mrsa isolates were collected from the hospital's microbiology laboratory for spa typing. consequently, the true molecular composition for some clusters is not known, and it could not be determined if all cases within clusters were a result of unique transmission events or part of a true outbreak. lastly, with the identification of a predominant spa type circulating in the patient population, the application of spa typing provided very little benefit for elucidating transmission events or recognizing potential outbreaks, especially for long duration clusters. although it was not conducted in this study, the incorporation of whole-genome sequencing may be a viable tool for further elucidating possible transmission events and identify potential/true outbreaks in the hospital setting. whole-genome sequencing provides an inventory of the microevolutionary changes of a bacterium and can be used to map genome-wide single-nucleotide polymorphisms, insertions, and deletions to a reference sequence [ ] . furthermore, this typing technique provides the best discrimination between closely related bacterial isolates in a timely manner [ , ] . for spa types identified with ≥ mrsa isolates, antimicrobial susceptibility profiles were examined to long duration cluster ( - months in length). clusters are potential outbreaks. short duration cluster ( - months in length). cluster was identified as part of a mrsa outbreak identified by infection prevention and control personnel. cluster was also identified in the rehabilitation department for the annual analysis. a-g indicates a cluster identified by > temporal scan at the service and ward levels. determine if differences in antibiograms could provide further characterization of clusters. for spa type /t , nine mrsa isolates were identified as being susceptible to clindamycin whereas all other spa type /t isolates were resistant. further investigation of these nine mrsa isolates revealed that three isolates were identified in the same internal medicine cluster (cluster id ; n = in may, ; n = in december, ) and three isolates were identified in the same surgery cluster (cluster id ; n = in july, ; n = in august, ; n = in september, ). the identification of identical spa types and antibiograms in two long duration clusters, suggests that transmission events among patients, hospital staff, or possibly even the contaminated environment, may have occurred. for spa types /t , / t , /t , and /t , it was noted that these particular spa types had indistinguishable antimicrobial profiles. this demonstrates that for the identification of transmission events and the determination of cases as outbreak or non-outbreak, relying on antibiogram data may result in inaccurate findings and the misclassification of cases as mrsa strains that differ genotypically may display identical antimicrobial susceptibility profiles. during the study period, the incidence rate of mrsa fluctuated considerably, with significant increases noted in years - . starting in december , ontario hospitals were required to report the number of newly acquired ha-mrsa bacteremias on a quarterly basis, to be posted on a web site that is accessible to the public [ ] . although the figures are affiliated with bacteremias only, the public reporting of mrsa did not result in a dramatic decrease in the overall incidence of mrsa in this facility, which is in contrast to c. difficile. in september , monthly data on c. difficile infections (cdis) from ontario hospitals were also posted on a publicly accessed web site [ ] . at the participating hospital, a separate analysis of cdis demonstrated that in and , there were significantly more cases of cdi, compared to and (data not shown). it is theorized that the decrease in c. difficile case rates may be attributable to hospitals adhering to best practices in c. difficile prevention due to the mandatory public reporting of rates [ ] . it was anticipated that this would also apply to mrsa; however, this was not observed. the significant increases in the incidence rate of mrsa cases in years - , based on the final multivariable model and model-based contrasts, are concordant with the findings from the temporal scan statistic as approximately % of the clusters identified spanned - . the increase in the mrsa case rate during this time period may have been due to the presence of respiratory viruses (e.g., influenza) within the hospital. reports of increased hospital mrsa rates during outbreaks of respiratory pathogens have been published [ , ] . in the northern hemisphere, influenza season occurs from october to march [ ] . for - , in addition to the regular influenza season, the h n influenza pandemic was identified in canada [ ] and h n patients were admitted to the participating facility. an increase in the mrsa case rate may have occurred as infection control activities and surveillance were focused on influenza and away from mrsa [ ] , reduced staffing as a result of illness, antimicrobial prescribing practices [ ] especially with fluoroquinolones which may have led to an increase in the risk for acquiring mrsa [ ] , and environmental contamination of mrsa which may have resulted in transmission events among staff and patients. the independent variable season was not found to be significantly associated with the number of mrsa cases in this hospital. seasonal variation in mrsa is debatable with some studies documenting spring [ ] or summer [ , ] with increased mrsa rates. however, in these studies, specific aspects of mrsa infections (e.g., community-acquired, healthcare-acquired, severe only) were investigated. a significant increase in the rate of mrsa cases was observed specifically in the months of march and april. as previously discussed, an increase in the number of mrsa cases in march may have been a result of the presence of influenza, or other respiratory viruses, in the hospital. the application of a temporal scan statistic to historical data from a community hospital resulted in the identification of several significant mrsa clusters. further examination of these clusters revealed several events that may be equivalent to mrsa outbreaks or transmission events that were not recognized by hospital personnel. by adopting a comprehensive approach for mrsa surveillance, clusters were identified at the hospital, service, and ward levels. infection control efforts can be focused at one or more levels to identify risk factors for mrsa acquisition and transmission, establish interventions, and evaluate control measures. the identification of specific time periods that corresponded to significant increases in the rate of mrsa cases in the patient population may have been correlated with other determinants at the hospital level, including the presence of other pathogens. in this investigation, spa typing provided very little information due to the presence of a predominant spa type. therefore, the use of a different typing technique (e.g., whole-genome sequencing) or additional supplementary information may be warranted to decipher transmission events and clusters. application of scan statistics for hospital surveillance of mrsa would probably be most rewarding in facilities with access to higher resolution molecular typing data. future research utilizing the temporal scan statistic, prospectively, with the application of molecular typing, especially whole-genome sequencing, to identify mrsa clusters in real-time and elucidate transmission events in the hospital setting, is warranted. additional file : univariable multilevel* negative binomial regression analyses of variables associated with the rate of mrsa cases. the authors declare that they have no competing interests. authors' contributions mcf contributed to the design of the study, statistical and molecular analyses, and drafting of the manuscript. dlp and ob contributed to study design and statistical analysis. wac contributed to study design and data collection. rrs contributed to study design. jsw contributed to study design and molecular analysis. all authors contributed to the editing and final version of the manuscript. rapid whole-genome sequencing for investigation of a neonatal mrsa outbreak automated detection of infectious disease outbreaks in 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