key: cord-010540-0zkc5w74 authors: lei, pinggui; fan, bing; mao, jujiang; wang, pingxian title: multiple parameters required for diagnosis of covid-19 in clinical practice date: 2020-03-19 journal: j infect doi: 10.1016/j.jinf.2020.03.016 sha: doc_id: 10540 cord_uid: 0zkc5w74 nan we read with interest the recent papers in this journal by hao who described clinical features of atypical 2019 novel coronavirus pneumonia with an initially negative rt-pcr assay. 1 we would like to share our opinions for diagnostic approach of covid-19 infection, the diagnostic approach for covid-19 infection should be made comprehensive analysis. since december 2019, a series of patients with unknown cause pneumonia had been reported in wuhan of china. recently, study on the early transmission dynamics had been reported that human-to-human was the epidemiologic characteristics for covid-19 infection. 2 therefore, it is very essential to diagnose precisely the patients suspected with covid-19 infection for opportune isolation or treatment. currently, the real-time reverse transcriptase polymerase chain reaction (rt-pcr) amplification of the viral dna is considered as the "gold standard". however, initial rt-pcr is not always positive in the patients with covid-19 infection. 3 , 4 in that situation, chest computed tomographic (ct) images could be played an important role to detect the lesions in the pulmonary parenchyma in the patients suspected with covid-19 infection. but it doesn't mean that the abnormalities of ct images could be observed in the covid-19 infection while the initial rt-pcr is positive or negative. 3-6 therefore, even though chest ct plays a key role in detection or diagnosis of covid-19 infection, however, chest ct examination and rt-pcr results should be mutual verification for precise diagnosis in the patient suspected covid-19 infection. clinical characteristics. beside ct examination results, initial symptoms were helpful in screening. for clinical manifestation, fever, dyspnea, chest tightness, cough, sputum, weakness, vomiting, diarrhea, etc. were observed in the patients with covid-19 infection. 5 , 7 of these clinical characteristics, fever and cough were the most frequency percentage of initial symptoms, and the other manifestations should be taken consideration. laboratory results. laboratory results were considered as auxiliary information to diagnose covid-19 infection. 5 , 7 in our hospital, there were 14 cases confirmed covid-19 infection. of 14 patients, white-cell count was tended to be normal in 12 patients (85.71%), lymphocyte (8/14, 57.14%) and monocyte (9/14, 64.29%) percentage had a tendency to decrease. significant statistical differences were observed in lymphocyte percentage decreased and c-reactive protein elevated (all p = 0.015) in the patients with covid-19 infection between initial positive chest ct results (10/14) and negative chest ct results (4/14). thus, knowing these clinical laboratory results was helpful for the doctor to diagnose the current novel coronavirus infection. exposure history. recent studies reported that most of the patients with covid-19 infection had the exposure history to the source of transmission within past 14 days. approximately 13 patients (2%) had the history of direct contact with wildlife, 75% cases in the epidemic area or contact with resident of epidemic area. 5 , 7 however, 28 cases (35%) with covid-19 infections were no obvious history of exposure, 5 which needs to pay more attention in this situation. in conclusion, even though chest ct has played a key role in detection or diagnosis of covid-19 infection with some typical ct features while the initial rt-pcr result is negative. however, not all the cases had the initial abnormality chest ct results or positive rt-pcr in the patients with covid-19 infection. consequently, rt-pcr results, chest ct features, clinical manifestation, laboratory results, and exposure history should be made a comprehensive analysis to diagnose covid-19 infection for the clinical decisions beyond clinical and radiological features. clinical features of atypical 2019 novel coronavirus pneumonia with an initially negative rt-pcr assay early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases sensitivity of chest ct for covid-19: comparison to rt-pcr radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia clinical characteristics of coronavirus disease 2019 in china key: cord-025482-9iy4fxd5 authors: zhong, yueyang; wang, kai; zhu, yanan; lyu, danni; yao, ke title: covid-19: evidence of the eye date: 2020-05-28 journal: j infect doi: 10.1016/j.jinf.2020.05.054 sha: doc_id: 25482 cord_uid: 9iy4fxd5 nan we read with interest of the article by huang et al. 1 in your journal about the nonspecific and atypical manifestations of covid-19 patients. as the initial epicenter of the outbreak, china has gained much clinical knowledge and experience in response to the disease. based on 13 case series and 9 case reports, we would like to share five key points of the ocular manifestations of covid-19 patients, hoping to provide a new perspective and broader view of the disease. first, severe acute respiratory syndrome coronavirus 2 (sars-cov-2) can cause ocular manifestations. patients mainly present with conjunctivitis that is similar to other types of viral infection. the first large epidemiological study reported 9 cases of conjunctival congestion among 1099 patients in china 2 . other symptoms include conjunctival secretion, epiphora, itching, foreign body sensation, and dry eye, with the prevalence ranging from 0.5% to 32% 3 . however, a recent case report observed retinal lesions of microhemorrhages and cotton wool spots among four patients, suggesting potential neurological manifestations 4 . 3 second, the characteristics of ocular involvements are atypical. ocular manifestations may present as the initial and the only symptoms of infection. the first case concerns a chinese expert, who got infected in wuhan and presented with conjunctival congestion before the onset of pneumonia 5 . ocular involvements are more likely to present in severe covid-19 cases, and there is no age or gender preference. to our knowledge, the youngest case was a 34-month-old boy, who had conjunctival congestion and eyelid dermatitis as the only symptoms 6 although there is no uniform standard for conjunctival sars-cov-2 detection, it is speculated that conjunctival swab technique has yielded higher sensitivity over schirmer's test. however, further studies are warranted to reach a definitive conclusion. fifth, appropriate use of qualified personal protective equipment is necessary. no evidence has shown the protective effect of contact lenses and personal eyeglasses. clinical workers should wear protective goggles, masks, and face shields. for the authors declare no conflicts of interest. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. rapid asymptomatic transmission of covid-19 during the incubation period demonstrating strong infectivity in a cluster of youngsters aged 16-23 years outside wuhan and characteristics of young patients with covid-19: a prospective contact-tracing study clinical characteristics of coronavirus disease 2019 in china characteristics of ocular findings of patients with coronavirus disease 2019 (covid-19 retinal findings in patients with covid-19 peking university hospital wang guangfa disclosed treatment status on weibo and suspected infection without wearing goggles 2020 a child confirmed covid-19 with only symptoms of conjunctivitis and eyelid dermatitis sars-cov-2 isolation from ocular secretions of a patient with covid-19 in italy with prolonged viral rna detection jia zhi-fang. 2019-ncov transmission through the ocular surface must not be ignored tumpey terrence m. ocular tropism of respiratory viruses sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor key: cord-276991-gv1k7u7j authors: zhang, xu; chen, xiaoyuan; zhang, zhipeng; roy, ayan; shen, yongyi title: strategies to trace back the origin of covid-19 date: 2020-04-08 journal: j infect doi: 10.1016/j.jinf.2020.03.032 sha: doc_id: 276991 cord_uid: gv1k7u7j nan the recent outbreak of coronavirus disease 2019 (covid-19), caused by the sars-cov-2, had raised great concern. 1 although the virus appears to be less pathogenic than mers-cov and sars-cov, it shows a better efficacy of human-to-human transmission. 2 -4 who raised the global covid-19 risk to its highest level on feb 28, 2020. due to the strict effort s t aken by the chinese government, there has been considerable descent in newly diagnosed cases in the country. however, the sharp rise in number of new cases outside china signifies that it is still a challenging task to control the virus. in spite of considerable research advancements on sars-cov-2, the origin of the virus is yet ambiguous. chinese authorities originally announced that the first infection case was reported on december 31, 2019, and many of the initial cases were linked directly to huanan seafood market in wuhan, in the hubei province. 5 apart from fish and seafood, the market also sold domestic and wild animals. thirty-three of the 585 environmental samples collected from the market were found to contain the novel coronavirus, according to china cdc. the hypothesis that the outbreak originated at the market, with its initial transmission from live animals to human beings followed by rapid human-to-human transmission, is suggested to be most likely and convincing. 6 however, another recent study claimed that the first patient, diagnosed on december 1, 2019, was never exposed to the market. 7 thus, origin of the disease still remains a puzzle unresolved. identification and subsequent elimination of the zoonotic source appears to be the most imperative and demanding task at present to prevent further events of viral spillover at the animal-human interface. 8 the seafood market was closed on january 1, 2020, and existing animals had been cleaned up, making it very difficult to identify the intermediate hosts. however, tracing the source of the virus might be targeted from the following perspectives: (1) tracing back the viral emergence at the huanan seafood market. it has been more than two months that the market has been closed and thus, it seems impossible to collect animal samples at present. however, the government can list all merchants in the market and clarify which animals they sold, and pertaining purchase channels of the animals. thus, sampling the animals from their purchase channels, aimed at detection of viral population in animal samples, promises to be a feasible way of catering the current problem. it appears crucial to specifically identify as who is "patient zero" for the outbreak. proper identification of "patient zero" might be a valuable know-how to address the complex riddles as how, when and why the virus emerged. it might be possible that the virus has been circulating in the human population before the outbreak in december 2019. detection and profiling of antibodies against the novel coronavirus, in the sera of human individuals before december 2019, present in wuhan hospital (the sera kept in the hospital during physical examination or medical treatment, especially fever clinic), might confer scopes to determine as when the event of viral transmission to human population had originally occurred, thus, unraveling its enigmatic origin. emergence of sars-like coronavirus poses new challenge in china a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster a family cluster of sars-cov-2 infection involving 11 patients in nanjing, china novel coronavirus disease (covid-19): the first two patients in the uk with person to person transmission a novel coronavirus outbreak of global health concern genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding clinical features of patients infected with 2019 novel coronavirus in wuhan, china the british infection association emergence of sars-like coronavirus in china: an update a pneumonia outbreak associated with a new coronavirus of probable bat origin isolation and characterization of 2019-ncov-like coronavirus from malayan pangolins this work was supported by the national natural science foundation of china (grant no. 31822056 ), key program of the department of education of guangdong province ( 2019kzdxm004 ), the guangdong science and technology innovation leading talent program (2019tx05n098), and the 111 project (d20 0 08). key: cord-255174-h1izji2g authors: wei, yuan-yuan; wang, rui-rui; zhang, da-wei; tu, youhui; chen, changshan; ji, shuang; li, chun-xi; li, xiu-yong; zhou, meng-xi; cao, wensheng; han, mingfeng; fei, guang-he title: risk factors for severe covid-19: evidence from 167 hospitalized patients in anhui, china date: 2020-04-17 journal: j infect doi: 10.1016/j.jinf.2020.04.010 sha: doc_id: 255174 cord_uid: h1izji2g nan dysfunction (1, (3) (4) (5) . due to a previous lack of in-depth research on the characteristics of the disease, the mortality of severe illness is high(4). it is very important to analyse the clinical characteristics of covid-19 in international regions and identify risk factors to reduce the incidence of severe and critical illness in the early stage. in this letter, we present discrepancies of patients with different disease severities and risk factors for severe covid-19 by comparing and analysing epidemiological and clinical data of 167 confirmed patients in anhui, china. in the present study, the rate of severely ill patients was as high as 17.9%. comparisons of demographics and clinical characteristics between 30 severe and 137 non-severe patients are shown in table 1. the mean age was 49.03 years in severe patients and 40.83 years in non-severe patients, with a significant difference (p=0.007). there were 95 males (56.89%) and 72 females (43.11%); however, there was no significant differences in sex between the two groups. among 167 patients, 146 had fever (87.43%), 132 had cough (79.04%) and 61 had shortness of breath (36.53%). the prevalence of shortness of breath was 73.33% in severe patients, which was significantly greater than the 28.47% prevalence in non-severe patients (p<0.001). there were 44 patients (26.35%) with comorbidities, of which 9 had multiple comorbidities (5.39%). among patients with diabetes, severe cases were significantly more common than in non-severe patients (p<0.001). compared to non-severe patients, fingertip oxygen saturation decreased significantly in severe patients (p<0.001), which predisposed patients with chronic obstructive pulmonary disease to acute exacerbation. since sars-cov-2 affected multiple organs by binding angiotensin converting enzyme 2 (ace2) receptor(6) and many severe covid-19 patients had comorbidities, multidisciplinary team (mdt) consultation played an important role in reducing the mortality of severe infection. there were significant differences in the use of mechanical ventilation, glucocorticoids and immunoglobulin between severe and non-severe patients (all p<0.05). table 2 presents comparisons of laboratory parameters between severe and non-severe patients. lymphocyte, cd4 and cd8 cell counts were decreased significantly in severe patients compared to non-severe patients (p=0.004, 0.021 and 0.002), suggesting that t lymphocytes were seriously destroyed. the increased level of c-reactive protein (crp) in severe patients was significantly higher than that in non-severe patients (p=0.001). interleukin-6 (il-6) levels increased in 122 patients (73.05%); the increase was more significant in severe patients than in non-severe (p=0.001). by clearing or blocking inflammatory factors(7), artificial liver therapy and tocilizumab, a monoclonal antibody of il-6 receptor, may prevent serious injuries in the lungs in severe patients. the lactate dehydrogenase (ldh) concentration was higher and the albumin concentration was lower in severe patients, with significant differences (p=0.002 and p<0.001). in severe patients, the fibrinogen concentration was significantly higher (p=0.008) than in non-severe patients, suggesting that severe patients were more likely to experience myocardial infarction or sudden death. between the two groups, there was a significant difference in the neutrophil to lymphocyte ratio (nlr), a predictor for severe infection(8) (p=0.033). other laboratory parameters that changed in covid-19 patients were not significantly different between the two groups (all p>0.05). there are still no specific therapies for covid-19(1); nevertheless, assessing risk factors and symptomatic treatment in the early stage of the disease can improve the prognosis. of 167 patients, 99.40% received initial antiviral therapy using lopinavir and ritonavir (400 mg/100 mg, bid) for no more than 10 days and/or atomized inhalation of interferon-α except for a 2-year-old patient, who was given interferon-α only, and 25.15% of patients received 40 mg methylprednisolone for 3 to 5 days. a total of 16.17% of patients were supported with immunoglobulins, and 13.18% of patients were managed with suitable oxygen therapy. all severe patients received mdt consultation, and three critical patients were treated with artificial liver therapy every other day, three times consecutively. finally, all patients in our study, both severe or non-severe, improved and were discharged without death. the clinical characteristics of 167 covid-19 patients are summarized in tables 1 and 2. the similarities and differences between severe and non-severe patients in this letter suggested that elderly patients with multiple comorbidities, hypoxia, decreased cd4 and cd8 cell counts and increased levels of crp and il-6 are all closely associated with disease severity and prognosis, which should be assessed seriously during diagnosis and treatment. a rapid decline in t lymphocytes and significant increases in the levels of inflammatory factors, including crp and il-6, can be clinical warnings of severe infection. mdt consultation and artificial liver therapy are very effective methods for severe patients with covid-19. with these integrated prevention and treatment strategies, there will be a good prognosis for covid-19. a. mono-factor logistic regression analysis was used to analyse risk factors for severe illness using r software; b. multi-factor logistic regression analysis was used to analyse independent risk factors for severe illness using r software. notes: p<0.05 indicates a risk factor in figure 1a and an independent risk factor in figure 1b for severe patients. coronavirus 2019-ncov: a brief perspective from the front line we thank the medical workers fighting against covid-19 for their hard work and sincere responses to our information requests. funding. this work was funded by the key technology research and development program of anhui province (no: 1804h08020237). key: cord-282053-ftjx29lw authors: luis garcía de guadianaromualdo; mulero, maría dolores rodríguez; olivo, marta hernández; rojas, carlos rodríguez; arenas, verónica ramos; morales, mercedes gonzález; abellán, ana blazquez; zamora, pablo conesa; garcía, josefina; hernández, andrés conesa; morell-garcía, daniel; otón, maría dolores albaladejo; consuegra-sánchez, luciano title: circulating levels of gdf-15 and calprotectin for prediction of in-hospital mortality in covid-19 patients: a case series date: 2020-08-12 journal: j infect doi: 10.1016/j.jinf.2020.08.010 sha: doc_id: 282053 cord_uid: ftjx29lw nan we read the recent articles by li and colleagues and lin and colleagues about the role of two inflammatory biomarkers, serum amyloid a 1 and ferritin 2 , in the evaluation of severity in coronavirus disease 2019 (covid-19) patients. we fully agree with authors that early identification of patients with a higher risk for severity remains crucial to define an optimal strategy for the management of covid-19 patients. in severe cases, the interaction of sars-cov-2 with the immune system contributes to a dysfunctional immune response, which triggers a cytokine storm that mediates widespread inflammation and multi-organ damage, major cause of disease severity and death in infected patients 3, 4 . higher blood levels of inflammatory biomarkers in blood, such as c-reactive protein (crp), ferritin and d-dimer, have been reported as predictors of a poor outcome in covid-19 patients 5 . the role of biomarkers associated to inflammation other than those above mentioned, such as calprotectin or growth differentiation factor 15 (gdf-15), is less known. the association of inflammation and calprotectin and gdf-15 has been previously reported [6] [7] [8] . during the inflammatory response, neutrophils and monocytes quickly arrive to the site of inflammation. the heterodimeric protein s100a8/a9, named as calprotectin, is an alarmin mainly derived by both cell types which play a critical role in inflammatory response, exerting its functions by binding to two patterns recognition receptors: toll-like receptor and receptor of advanced glycation endproducts and activating pro-inflammatory signaling pathways leading to further recruitment and activation of immune cells 6 . infection-induced inflammation is one of the main triggers that leads to calprotectin liberation 8 . in a recent study, calprotectin levels were significantly higher in covid-19 patients who required mechanical ventilation, similarly to crp 9 . gdf-15 is a member of the tumor growth factor-beta (tgf-) family, primarily expressed under conditions of inflammation and oxidative stress. recently, an experimental study showed a novel function of gdf-15 in the promotion of lung human rhinovirus and virus-associated inflammation, contributing to the severity of respiratory viral infection 10 . in the present observational study, we aimed to explore a potential role of in our cohort, the mortality rate was 12.1% (8/66). median time from symptoms onset to ed admission was 7 (5-10) days. table 1 shows the differences in demographics and comorbidities between survivors and non-survivors. table 2 ), as assessed by the analysis of the auc of roc curve for in-hospital mortality, similar to both d-dimer and crp but numerically higher than ferritin ( figure 1 and table 2 ). table 2 shows the unadjusted odds ratio to each of the studied biomarkers stratified by a cut off obtained by means of the youden index for the prediction of in-hospital mortality. we have shown that circulating levels of two emerging inflammatory biomarkers, calprotectin and gdf-15, are significantly higher in covid-19 patients who died, suggesting a potential role in the evaluation of prognosis in these patients. our study is the first, to our knowledge, exploring a potential prognostic value of both in covid-19 patients. this early report has some limitations, namely the small sample size. hence, we did not perform multivariable analysis, due to the small number of included patients and outcomes. however, the ipw method allowed us to adjust for the sofa score, that was shown to be a powerful predictor of mortality. the aim was not to generate a predictive model, but rather to explore the potential role of these novel biomarkers. our findings suggest that calprotectin and gdf-15 might have a potential role in the assessment of prognosis in covid-19 patients. the authors declare no competing interests note: data are expressed as frequency (percentage) or mean (standard deviation). weighted by inverse probability of dying during admission (obtained from a propensity score model using sofa sca 95% ci: 0.919-1) 2 for the calculation the youden index derived cutoffs for each of the biomarkers were used. serum amyloid a is a biomarker of severe coronavirus disease and poor prognosis serum ferritin as an independent risk factor for severity in covid-19 patients pathological inflammation in patients with covid-19: a key role for monocytes and macrophages the trinity of covid-19: immunity, inflammation and intervention molecular, serological, and biochemical diagnosis and monitoring of covid-19: ifcc taskforce evaluation of the latest evidence s100a8/a9: from basic science to clinical application growth differentiation factor-15 (gdf-15): from biomarker to novel targetable immune checkpoint s100a8/a9 in inflammation neutrophil calprotectin identifies severe pulmonary disease in covid-19 overproduction of growth differentiation factor 15 promotes human rhinovirus infection and virus-induced inflammation in the lung key: cord-007926-um2khqhn authors: zhang, jiahao; ma, kaixiong; li, huanan; liao, ming; qi, wenbao title: the continuous evolution and dissemination of 2019 novel human coronavirus date: 2020-02-22 journal: j infect doi: 10.1016/j.jinf.2020.02.001 sha: doc_id: 7926 cord_uid: um2khqhn nan -s3). the overall genome identity of the 2019-ncov and sarsr-cov was ranging from 82% to 89%. with rapid dissemination of the 2019-ncov, the viruses had been transmitted rapidly in more than 25 countries, and a steep increase in human infection with the 2019-ncov occurred in china ( fig. 1 ) . to further explore the genetic evolution of the 2019-ncov, phylogenic relationships of 31 isolates examined in this study were explored and divided into four genotypes, including g1, g2, g3, and g4. we found that 25 isolates clustered together and had been circulating in thailand and multiple provinces (e.g. taiwan, guangdong, zhejiang, and hubei) of china, suggestive of the ongoing co-circulation of the viruses. the 2019-ncov circulating in usa and wuhan, china clustered into an independent cluster. however, interestingly, the szth-0 02, 20sf013, and szth-0 01 strains in guangdong province were located at basal branch of the 2019-ncov ( fig. 2 ) , indicative of a single origin. the genetic diversity of rna-dependent rna polymerase of the 2019-ncov was very small (supplementary figure s1 ). by contrast, the spike (s) genes of some 2019-ncov prevailing in guangdong province were found at the root of the 2019-ncov in wuhan (supplementary figure s2) . these findings indicated that the 2019-ncov were infected from different regions in wuhan and had been undergoing continuous evolution and dissemination in different regions. compared with the rapid mutation of the influenza a viruses, the degree of diversification of the 2019-ncov was much smaller. nevertheless, we cannot rule out if the 2019-ncov continue evolving to become more transmissible and virulent in humans in the near future. the s protein mediates receptor binding and membrane fusion, and of particular note, it is of great importance to determine host tropism and transmission capacity. 5 zhou et al. demonstrated that the 2019-ncov use the same cell entry receptor, ace2, as sars-cov. 6 the receptor binding region of the 2019-ncov was more similar to that of sars-cov; however, we found that four amino acid substitutions in the receptor binding region of the 2019-ncov, including s486q, f485y, n493g, and y501v substitutions (2019-ncov numbering), were different from that of sars-cov ( fig. 2 ) , which might affect the receptor binding ability. bats provide a rich "gene pool" for interspecies exchange of genetic fragments of cov. 7 continuous surveillance in bats provide us a clue to the correlation between the 2019-ncov and the animal origin cov. despite the shared cluster between the 2019-ncov and bat sarsr-cov, we cannot infer that the reservoir of the 2019-ncov was originated from bats. most of the patients infected with novel 2019-ncov had a history to the seafood and live animal markets, and the vendor used to sale wild animal species, including marmot, snake, leopard cat, bamboo rat, badger, and hedgehog in huanan seafood wholesale market (supplementary figure s4 ), all of which were susceptible to the novel cov in nature, indicating that it remains likely there was intermediate hosts in the transmission cascade from bats to humans ( fig. 2 ) . however, a question of a public health interest is which intermediate hosts harbor the 2019-ncov that could infect humans, which should be examined in greater detail. recently, the continuous interspecies transmission events of cov occurred, including the emergence of mers-cov from camels to humans and swine acute diarrhea syndrome cov from bat to swine, posing serious threats to public health. 8 , 9 with the tradition of feeding wild animals for food or use in traditional medicine in china, wild birds, mammals, and reptiles carrying the novel zoonotic viruses flowed frequently in trading center, which had considerable potential to transmit to humans of emerging viruses. in 2018, china had participated a global virome project to identify unknown viruses from wildlife to better prepare for the epidemics of infectious diseases in humans. 10 with multiple species of cov circulating in different animal species that could be transmitted to humans, no one knows when or where the next outbreak will occur. nevertheless, decreasing the risk for the spread of novel viruses including reducing contact among humans and wild animal species and stopping novel viruses at their origins is urgently needed. we call upon wildlife biologists, ecologists, doctors, and veterinarians should promote exchange and share data across disciplines as a mean to minimize the potential for pandemics of the 2019-ncov. all authors have no potential conflicts of interest to disclose. emergence of sars-like coronavirus poses new challenge in china epidemiology, genetic recombination, and pathogenesis of coronaviruses clinical features of patients infected with 2019 novel coronavirus in wuhan a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-toperson transmission: a study of a family cluster bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond a pneumonia outbreak associated with a new coronavirus of probable bat origin discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus vaccine against middle east respiratory syndrome coronavirus fatal swine acute diarrhoea syndrome caused by an hku2-related coronavirus of bat origin the global virome project we sincerely thank the authors of the human 2019 coronavirus from gisaid epiflu tm database. this work was supported by the national natural science foundation of china ( 31941014 , 31830097 , supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.02.001 . key: cord-027498-cfzfgzqi authors: hattori, takeshi; amishima, masaru; morinaga, daisuke; kamada, keisuke; nakakubo, sho; yamashita, yu; shichinohe, yasuo; fujisawa, shinichi; nishida, mutsumi; nasuhara, yasuyuki; teshima, takanori; konno, satoshi title: older age is associated with sustained detection of sars-cov-2 in nasopharyngeal swab samples date: 2020-06-21 journal: j infect doi: 10.1016/j.jinf.2020.06.046 sha: doc_id: 27498 cord_uid: cfzfgzqi nan in this journal, iwasaki and colleagues compared the quality of pcr from saliva and nasopharyngeal swabs as a diagnostic measure of sars-cov-2 infection (1). currently, the standard for diagnosis of severe acute respiratory syndrome-coronavirus-2 (sarsinfection is a positive result based on a polymerase chain reaction (pcr) test from nasopharyngeal swab samples. pcr test is also used as a guide for patient discharge from designated hospitals and medical institutions. we recently experienced a case of a 97-year-old female who was diagnosed with coronavirus disease 2019 (covid-19). although her clinical symptoms and radiological findings resolved within a few days, pcr results from nasopharyngeal swab samples remained positive for 50 days after the onset. this case prompted us to conduct a retrospective study of the association of age the duration of positive pcr testing. we specifically hypothesized that old age could be a risk for prolonged duration of positive pcr results from nasopharyngeal swab samples. this study was approved by the ethics committees of national hospital organization hokkaido medical center and hokkaido university hospital. nasopharyngeal swab sample was collected and quantitative real-time reverse transcription-pcr (rt-pcr) was conducted as described before (1). in a rt-pcr assay, cycle threshold (ct) value is defined as the number of cycles required for the fluorescent signal to cross a baseline threshold. the test results of sars-cov-2 were reported as negative in tests in which ct＞45). this enabled us to determine the precise day that each covid-19 patient tested "negative" by pcr. we defined "negative pcr" as confirmed negative results over two sequential days. characteristics of the 66 patients diagnosed with mild covid-19 are presented in table 1 . forty-two subjects were mild cases, who did not require supplemental oxygen treatment. eighteen subjects were moderate cases who needed oxygen treatment. six subjects were severe cases who needed ventilator or/and ecmo. we found that older age was significantly associated with prolonged positive pcr tests (p=0.0053; figure 1a ). this relationship remained unchanged when the findings were adjusted for the potential impact of severity of the disease (mild, moderate, severe) and the used of medication (p=0.026). when we analyzed only mild cases of covid-19 in order to exclude the influence of disease severity, the result remained significant (p=0.036; figure 1b ). in our analysis, older age is significantly associated with prolonged duration of positive pcr tests from nasopharyngeal swab samples, irrespective of the disease severity and the used of medication ( figure 1 ). the reasons underlying these observations remain unclear. of note, we recently reported that quality of pcr from saliva as a diagnostic measure of sars-cov-2 infection was equivalent to that of the samples from nasopharyngeal swabs (1). we also found that findings from pcr tests reverted from positive to negative much more quickly when using saliva than nasopharyngeal samples (1). we speculate that in older individuals, cell turnover is less robust and as such, clearance of virus from the nasopharynx is prolonged; these factors may lead to positive pcr tests that persist after acute disease has resolved. one group from taiwan has already discussed the possibility that covid-19 may no longer be contagious at two weeks after the onset of symptoms (3) . it is possible the positive pcr tests reflect the presence of that inactive virions remaining within nose. in a linked study, zheng and colleagues evaluated viral loads in respiratory samples, stool, serum, and urine using pcr: they found that more than half the respiratory samples remained positive for sars-cov-2 as did a full one third of the stool samples at the end of the four week trial period (4) . as such, we propose that there should be a change in the strategy currently in use for determining time of discharge to one that relies on other clinical tests or/and patients' condition, which could be helpful to inhibit the development of patients' flail and dementia and to reduce the burden of ongoing and potentially unnecessary prolonged hospitalization. in summary, we demonstrated that old age is significantly associated with prolonged duration of positive pcr results from nasopharyngeal swab samples; this is the case regardless of disease severity. further studies will be needed in order to clarify how long these patients are actually contagious. comparison of sars-cov-2 detection in nasopharyngeal swab and saliva world health organization. home care for patients with covid-19 presenting with mild symptoms and management of their contacts contact tracing assessment of covid-19 transmission dynamics in taiwan and risk at different exposure periods persistence of viral rna in stool samples from patients recovering from covid-19 pcr has limitations, and isolating patients for a month or more may not be feasible isolation the authors declare that they have no competing interests. key: cord-266564-imj1lcy9 authors: liu, yangli; chen, haihong; tang, kejing; guo, yubiao title: clinical manifestations and outcome of sars-cov-2 infection during pregnancy date: 2020-03-05 journal: j infect doi: 10.1016/j.jinf.2020.02.028 sha: doc_id: 266564 cord_uid: imj1lcy9 nan tang and colleagues, in this journal, drew readers attention to emerging covid19[1]. we focused on the pregnant covid19 patients. given the maternal physiologic and immune function changes in pregnancy [2] , pregnant individuals might face greater risk of getting infected by sars-cov-2 and might have more complicated clinical events. we described epidemiological, clinical characteristics, pregnancy and perinatal outcomes of all hospitalized pregnant patients diagnosed with covid-19 in china. we identified all hospitalized pregnant patients with laboratory-confirmed sars-cov-2 infection between december 8, 2019, and february 25, 2020 officially reported by the central government, in areas outside wuhan, china. information including age, geographic location, epidemiological history, prenatal course, maternal and newborn hospital course, discharge data and outcome were obtained by centers for disease control and prevention and local health commission. when necessary, we attempted to contact local hospital or patients by telephone to supply missing information. this investigation was part of an emergency public health outbreak investigation and therefore not subject to institutional review board. there were a total of 13 chinese patients with sars-cov-2 admitted to hospitals outside of wuhan (table) . there were 3 patients from zhejiang, 3 from other cities of hubei and 1 each from fujian, shanxi, beijing, guangdong, jiangxi, heilongjiang and anhui. the maternal age ranged between 22 to 36 years. two women were less than 28 weeks of gestation and the other 11 patients were in their third trimesters at presentation. none of the patients had underlying medical disease. previous studies suggested that covid-19 is more likely to affect older males with comorbidities [3] . we reported 13 pregnant covid-19 patients in china, indicating pregnant women also susceptible to sars-cov-2. clinical manifestations of the pregnant covid-19 patients in this study varied widely from asymptomatic to very severe, similar to previous report in non-pregnant patients [4] . most of the pregnant patients had mild to moderate symptoms. fever and fatigue were the principal symptoms, and less common symptoms were sore throat and shortness of breath. almost all the patients had a clear epidemiologic history. one of the 13 patients (7.6%) developed severe pneumonia requiring icu care with multiple organ dysfunction syndrome in the third trimester in our study, similarity with the general population reported to be with critical rate of 5% [5] . cytokine storm might be the reason for very severe cases since chaolin huang et al [6] found that compared with non-icu patients, icu patients had higher plasma levels of various cytokines. five patients of thirteen (38%) were delivered by emergency cesarean section because of pregnancy complications including fetal distress, premature rupture of the membrane and stillbirth. six patients (46%) had preterm labour. these perinatal complications could be ascribed to the virus infection as well as the physiologic changes that reducing the woman intolerant to hypoxia during late pregnancy [7] . fortunately, no severe neonatal asphyxia was observed in the nine livebirths and no vertical transmission was found. in conclusion, our report showed pregnant women are also susceptible to sars-cov-2 infection. sars-cov-2 may increase health risks to both mothers and infants during pregnancy. efforts should be taken to reduce the infection rate of sars-cov-2 both in pregnant and perinatal period, and more intensive attention should be paid to pregnant patients. all authors declare that they have no competing interests. emergence of a novel coronavirus causing respiratory illness from wuhan emerging infections and pregnancy epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention clinical features of patients infected with 2019 novel coronavirus in wuhan, china middle east respiratory syndrome coronavirus infection during pregnancy: a report of 5 cases from saudi arabia key: cord-010536-9ea7vvsz authors: chu, yanan; li, tong; fang, qiang; wang, xingxiang title: clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (covid-19): a multi-center study in wenzhou city, zhejiang, china date: 2020-04-28 journal: j infect doi: 10.1016/j.jinf.2020.03.023 sha: doc_id: 10536 cord_uid: 9ea7vvsz nan we read with great interest the article by wenjie yang and colleagues1, accepted for publication in the journal of infection. the authors performed a retrospective multi-center cohort study and presented important data regarding the observation that most patients of 2019 novel coronavirus disease (covid-19) from wenzhou city, zhejiang, exhibited mild infection. however, the information of critically ill patients, especially with icu care and extracorporeal membrane oxygenation (ecmo) treatment, were scare. no study to date has provided evidence that the clinical features of critically ill patients with confirmed covid-19 from zhejiang province. we performed a single-centered, retrospective, observational study to investigate the clinical characteristics and ventilation conditions of critically ill patients infected with sars-cov-2. from late january, 2020, to february 23, 2020, 33 critically ill patients in the icu of the first affiliated hospital of zhejiang university who were diagnosed as covid-19 in accordance with the diagnosis and treatment guidance published by the chinese government were enrolled in the study2. we obtained patients demographics, epidemiology data, and details of laboratory tests, treatments, and ecmo implantation. the baseline epidemiological characteristics and clinical features of 33 studied patients as classified by with or without ecmo treatment, were shown in table 1 . most of the patients admitted to the icu were older and had several common comorbid conditions, which demonstrated that age and comorbidities might be the indicators for severely ill one and poor prognosis. of all patients, the mean age was 65.2±16.6 years, and most of the patients were aged 65 years and older. of the seven patients who received ecmo, the mean age was 67.0±17.7 years. observed man probably had more complicated clinical conditions and worse inhospital outcomes as compared to women in severe covid-19 patients. the median time from onset of symptoms to hospital admission was 10 days (iqr 5-13 days) which was longer than wenjie yang and colleagues' study. in terms of baseline laboratory data of severely confirmed covid-19 patients, three (9%) and 22 (66.7%) of 33 patients exhibited leucopenia and lymphopenia, respectively. platelets levels on admission were lower in patients with ecmo treatment than non-ecmo patients. also, a recent case report verified the counts of peripheral cd4 and cd8 t cells were both decreased in a 50-year-old man with sars-cov-2 infection through the technology of flow cytometric analysis4. specifically, the levels of aspartate aminotransferase (ast) and alanine aminotransferase (alt) on admission were higher in ecmo treated patients (median ast 38. table 1 ). besides, admission levels of total bilirubin were increased substantially in ecmo treated patients. these abnormalities suggested that sars-cov-2 might be related to hepatic injury. however, almost all of the included patients received antivirus treatment, the drug induced liver injury could not be excluded. huang et al. reported that increased level of ast was found in about 62% of the icu patients in their study5. therefore, damaged liver function is more common in serious covid-19 patients. up to now, there was no sufficient evidence to clarify sars-cov-2 as the main reason of damaged liver function. further studies should concentrate on the reasons of liver function damage in patients with covid-19. the level of procalcitonin increased in more than 70% of included patients, and most of patients in our study received antibacterial and antifungal agents. one possible explanation for the results may be that many of the critically ill patients were associated with combined infection of bacterial or fungal before the commencement of ecmo. ecmo has been increasingly being used as a rescue treatment for refractory hypoxemia in patients with severe acute respiratory distress syndrome6. the initial mode was veno-venous (vv) ecmo in the 7 patients. initiation of ecmo was accompanied by a significant improvement in pao2/fio2 ratio, and a significant decreases in paco2, fio2 (table 2 ). research showed too high level of fio2 was related to increased production of reactive oxygen-derived free radicals which are noxious to the humans health7. in summary, our data indicate that sars-cov-2 infection might cause damage to the immune and liver function of covid-19 patients. ecmo support was associated with improved ventilation conditions in covid-19 patients with refractory hypoxemia. the study may be helpful to providing evidence of the appropriate time to initiate ecmo for critically ill patients with covid-19, and add further evidence for critically ill patients characteristics by wenjie yang et al. clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (covid-19):a multi-center study in wenzhou city national administration of traditional chinese medicine. guidelines for the diagnosis and treatment of novel coronavirus pneumonia (trial version sixth) pathological findings of covid-19 associated with acute respiratory distress syndrome clinical features of patients infected with efficacy and economic assessment of conventional ventilatory support versus extracorporeal membrane oxygenation for severe adult respiratory failure (cesar): a multicentre randomised controlled trial bench-to-bedside review: the effects of hyperoxia during critical illness the authors would like to thank all participants of the study , the nurses and clinical staff who are providing care for the patients, and thanked for the guidance and help from hdh, wyk. this work was supported by the department of science and technology of zhejiang province. the authors of this study declared no conflict of interest. key: cord-030636-wpjmwwpu authors: nan title: dear editor, date: 2020-08-19 journal: j infect doi: 10.1016/j.jinf.2020.08.023 sha: doc_id: 30636 cord_uid: wpjmwwpu nan since december 2019, some hospitals in wuhan city, hubei province, have successively found multiple cases of unexplained pneumonia which have now been confirmed as a new type of acute respiratory infection caused by a coronavirus infection. the coronavirus isolated from the lower respiratory tract has been named as covid-19; it has presented an unprecedented challenge for the healthcare community across the world. based on the rapid increase in the rate of human infection, the world health organization (who) has classified the covid-19 outbreak as a pandemic. respiratory involvement, presenting as mild flu-like illness to potentially lethal acute respiratory distress syndrome or fulminant pneumonia, is the dominant clinical manifestation of covid-19. unlike other respiratory diseases, mortality of covid-19 increased with age while children were observed less susceptible to death. despite the observation that elderly subjects are more susceptible to severe illness, probably due to underlying co-morbidities such as diabetes, hypertension, cardiovascular and cerebro-vascular diseases, 1,2 literature concerning geriatric patients with covid-19 pneumonia remained very scant. most of the studies are editorial commentaries 3 and the clinical studies including patients of varying ages admitted to hospital have only a slight highlight on the association between age and severity of clinical manifestation. [4] [5] [6] further, the majority of data at the moment available often originating from chinese surveys where elderly patients accounted only for a very limited part of the total. in particular, a paper recently published into your journal by wang et al. 2 observed a high fatality rate in the very first days after hospitalization (a median survival of 5 days), and another paper by liu et al. 6 found a higher mortality rate in elderly than in young and middle-aged patients. however, both these studies considered as elderly patients those aged > 60 years old which is a very different population from that observed in europe and in particular in italy, where the elderly (aged > 80 years old) accounted for a large proportion of individuals with severe covid-19 pneumonia. in our cohort we included elderly subjects (> 80 years old) hospitalized for covid-19 pneumonia at two north-italy district hospitals from march 9 th to april 30 th 2020. we included in this analysis 118 consecutive patients; data on clinical and demographic characteristics, blood test results and covid-19-related treatments were collected. survival status and clinical data were compared with a control group of covid-19 patients aged < 70 years (n=109). survival analysis was done using a multilevel mixed-effects parametric survival model. sixty-eight patients aged > 80 years died (57.6%) after a median time of 4.5 days from admission to emergency department compared to 4 patients (3.7%) in the control group (median time to death in control group: 16.4 days). table 1 summarizes demographic and clinical characteristics and serum biomarkers of inflammation of the two groups. older patients were more likely to have lower bmi (24.4 vs 27.3, p<0.001), copd (18.6% vs 3%, p<0.0001), earlier access to emergency department from disease presentation (4 vs 6.2 days, p=0.02) and prolonged hospital stay (26 vs 14 days, p<0.0001). on the contrary, gastrointestinal symptoms and fever at admission were significantly more frequent in younger subjects. furthermore, elderly patients had higher white blood cells (11000/mm 3 vs 6700/mm 3 , p<0.0001), c-reactive protein (9.8 mg/dl vs 6.5 mg/dl, p=0.001), procalcitonin (0.27 md/dl vs 0.1 mg/dl, p<0.0001), ldh (416.5 mg/dl vs 297.6, p=0.01) and d-dimer levels (1100 mg/dl vs 386 mg/dl, p<0.0001) at admission. at survival analysis, higher d-dimer levels [hr 1.11 (1.03-1.2), p=0.004] at admission in the emergency department and the combined use of antivirals and hydroxichloroquine [hr 8.42 (2.53-27.98), p<0.0001] were independently associated to a higher risk of death. at our knowledge, this is the first report that evaluated the outcome of covid-19 pneumonia in subjects > 80 years old. in this subset, we observed a high mortality rate especially in the very first days after hospitalization, probably due to a more rapid disease progression. these findings confirmed those by wang et al. 2 who observed a median survival of 5 days after admission. elderly patients have higher levels of inflammatory blood tests at the time of admission in the emergency department; in particular, elevated d-dimer levels was an independent predictor of mortality, confirming the close correlation between this parameter and the severity of covid-19 disease. also the use of a treatment including both antivirals and hydroxychloroquine was associated with a higher risk of death. this detrimental effect of the combined treatment could be possibly due to drug-related side effects in the elderly population, but this hypothesis needs to be further confirmed in prospective studies. in conclusion, our study confirms that the majority of elderly subjects with covid-19 pneumonia have an unfavorable outcome, especially in the very first days after admission. we also confirm the high importance of d-dimer levels as predictors of mortality and that the treatment with antivirals and hidroxychloroquine is ineffective if not harmful in elderly individuals. analysis of epidemiological and clinical features in older patients with coronavirus disease 2019 (covid-19) outside wuhan coronavirus diseases 2019 in elderly patients: characteristics and prognostic factors based on 4 week follow-up covid-19 through the lens of gerontology epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with novel coronavirus-infected pneumonia in wuhan, china clinical features of covid-19 in elederly patients: a comparison with young and middle-aged patients table 1. demographic and clinical characteristics and serum biomarkers of inflammation in subjects > 80 and < 70 years old authors are grateful to john tremamondo and aldo bellini for their support. our special thanks go to all nursing and medical colleagues in asst rhodense hospitals. key: cord-287823-avi14ee5 authors: wong, martin cs; teoh, jeremy yc; huang, junjie; wong, sunny h title: the potential impact of vulnerability and coping capacity on the pandemic control of covid-19 date: 2020-05-28 journal: j infect doi: 10.1016/j.jinf.2020.05.060 sha: doc_id: 287823 cord_uid: avi14ee5 • the european commission has developed an index for risk management named inform; • two dimensions (vulnerability and lack of coping capacity) are relevant to covid-19; • we examined if these dimensions were associated with covid-19 pandemic control; • higher vulnerability and poorer coping capacity were associated with poorer control; • modifying these two dimensions might potentially mitigate covid-19 pandemic control.  we examined if these dimensions were associated with covid-19 pandemic control  higher vulnerability and poorer coping capacity were associated with poorer control  modifying these two dimensions might potentially mitigate covid-19 pandemic control dear editor, worldwide, the coronavirus disease 2019 (covid-19) has induced a substantial global burden. since its first diagnosis in wuhan, china, its spread has affected 216 countries. 1 as of 16 may, 2020 , there were more than 4.4 million cases and greater than 302,000 confirmed deaths among patients with covid-19. arguably, some nations with lower capacity to cope with the pandemic, especially in low and middle-income countries, might have poorer control of the disease. however, no previous study has proven this association. on the contrary, a recent study published in the journal of infection examined the association between country-specific global health security index (ghsi) and the burden of covid-19, but the findings showed that countries with higher ghsi did not have higher covid-19 rate and had greater number of covid-19 cases and deaths. 2 hence, further exploration of the association between country capacity and covid-19 burden is needed based on other indicators. ) and vulnerable groups (uprooted people or other groups). it represents the economic, political and social features of the populations that can be destabilised in the event of a hazardous incident. 3 the lack of coping capacity measures if a country is unable to cope with disasters through the government's effort and existing infrastructure. it could be institutional (disaster risk reduction and governance) or infrastructure-related (communication, physical infrastructure, and access to health systems). we aimed to evaluate if countries with lower vulnerability and higher coping capacity were associated with better control of the covid-19 pandemic, as measured by incidence and mortality outcomes. we established a panel of experts consisting of epidemiologists, physicians, and public health professionals who were tasked to determine the outcomes used in this study based on literature review. after discussion the panel determined the following outcome variables: the maximum 14-day cumulative incidence rate per 100,000 population since the first case (22 january to 30 april, 2020); and the incidence and mortality per 100,000 population within 30 days since the first covid-19 diagnosis and first covid-19 related death, respectively, from the johns hopkins centre for systems science and engineering (csse). 4 the variables tested for association with these outcomes included the covid-19 vulnerability and the covid-19 lack of coping capacity as of 2018. three linear regression models were constructed for the three outcomes whilst adjusting for gross domestic product (gdp) of the same year for each nation; 5 and the population density of each country from the world population review. 6 the study was approved by the survey and behavioral research ethics committee of the chinese university of hong kong (sbre-19-592). all p values ≤ 0.05 were considered statistically significant. the distribution of vulnerability and coping capacity scores was shown in figure 1 . the covid-19 vulnerability score was the highest in italy (score 8.2 out of 10), japan (8.2), croatia (8.1) and latvia (8.1). countries with the severest lack of coping capacity included central african republic (9.4), comoros (9.1), equatorial guinea (7.7), and burundi (7.6). from multivariate regression analysis ( table 1) , countries with higher vulnerability were significantly associated with higher maximum 14-day cumulative incidence since the first case ( coefficient our findings imply that reducing vulnerability and enhancing capacity to cope could potentially mitigate the covid-19 pandemic. since the components of the two predictor variables are modifiable, countries that aim to increase their capability to combat the covid-19 pandemics could make reference to the detailed subcategories under these two dimensions. the government could consider to take active steps in enhancing the resilience of the society and availability of 6 measures that could protect the vulnerable population. nevertheless, there are limitations of our study. firstly, there may be other confounders that could not be controlled for, including personal behaviour and the stringency of governmental policies, 7 such as measures related to social distancing, school closure, supply of personal protective equipment (ppe), as well as quarantine and containment strategies. [8] [9] [10] in addition, the covid-19 vulnerability used was developed in 2018, and we assumed that the index of each country did not change before the beginning of the pandemic in 2019. also, we should emphasize that these are preliminary findings, and the cause-and-effect relationships are yet to be further examined by larger-scale studies. in conclusion, we identified vulnerability and ability to cope as two important aspects in the face of an infectious disease pandemic, and they bear a potential impact to mitigate the covid-19 pandemic. future studies should evaluate the specific components of these indices that exert the greatest impact on pandemic control. table 1 the association between vulnerability index, ability to cope score and the incidence/mortality outcomes related to covid table 1 the association between vulnerability index, ability to cope score and the incidence/mortality outcomes related to covid covid-19) outbreak situation rethinking pandemic preparation: global health security index (ghsi) is predictive of covid-19 burden, but in the opposite direction online ahead of print index for risk management inform concept and methodology report -version the 2019 novel coronavirus covid-19 (2019-ncov) data repository by johns hopkins centre for systems science and engineering (csse). available at countries by density by population 2020 variation in government responses to covid-19 the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study association of public health interventions with the epidemiology of the covid-19 outbreak in wuhan how will country-based mitigation measures influence the course of the covid-19 epidemic? index for risk management inform concept and methodology report -version key: cord-026603-h4zy3244 authors: gallo, oreste; giovanni, locatello luca; orlando, pietro; martelli, federica; piccica, matteo; lagi, filippo; trotta, michele title: “is really the cancer population at risk for more severe covid-19? some hints from the cytokine profile” date: 2020-06-10 journal: j infect doi: 10.1016/j.jinf.2020.06.011 sha: doc_id: 26603 cord_uid: h4zy3244 nan were in accordance with the ethical standards of the institutional and/or national research committee and with the 1964 helsinki declaration and its later amendments or comparable ethical standards. informed consent: informed consent was obtained from all individual participants included in the study. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. dear editor, we read with great interest the meta-analysis by afshar and colleagues regarding the very high case-fatality rate in cancer patients infected with sars-cov-2. [1] the authors suggest that such findings can be explained by the "systemic immunosuppression" because of cancer and its associated treatments. [1] the official death toll of the covid-19 pandemic has reached, as of may 27th, 350 000 and it is now recognised that severe outcomes of this infection are associated to a complex dysregulated immune response to sars-cov-2 which clinically translates into acute respiratory distress syndrome, the cytokine release syndrome, the secondary hemophagocytic lymphohistiocytosis, and the disseminated intravascular coagulation. [2] large series from wuhan, china have identified several risk factors for death in the covid-19 population: for instance, li and colleagues found at survival analysis that male sex, older age, leukocytosis, high lactate dehydrogenase level, cardiac injury, hyperglycemia, and the use of systemic steroids were all significant predictors. [3] in such early reports, cancer patients who were infected with this novel coronavirus were considered to have an incredibly high mortality rate (almost 30%). [4] however, these data were subsequently criticised because of several methodological issues such as the many non-cancer comorbidities or other confounding factors that were not controlled in the analysis of the cohort; in addition, a clear definition of some of the variables (e.g., cancer type or stage) was not always fully specified. [5] on the contrary, other authors have subsequently suggested that cancer patients, because of their impaired immune system due to the tumour itself and its therapies, are expected to have a reduced systemic inflammatory response to the virus and, thus, non-inferior mortality rates. [6] in the face of these conflicting results, more data are needed. we therefore (table 1) . overall, 33.3% in the cancer group died from covid-19, while among non-cancer patients, fatal evolution was reported in a lower yet statistically not significant rate (13.9%; p=0.289). by univariate logistic regression analysis, cancer patients seem to have an increased risk of death (or=4.77, p=0.013). however, when performing multivariate analysis accounting for age, smoking status, and cardiovascular disease, the result is no longer significant ( table 2 ). looking at laboratory parameters, white blood cell count and several inflammatory markers were recorded on admission (ie, before beginning any possibly modifying treatment such as steroids or anti-il6 drugs). interestingly, no significant differences were found in terms of the mean concentration of the most important cytokines between the two groups. it is assumed that the development of cancer is associated with a blunted immunestatus characterized by an altered production of cytokines, an impaired dendritic cell activation, and a dysfunctional lymphocyte population; such impairment is also enhanced by the unavoidable surgical and non-surgical treatments. [7] on the contrary, covid-19 patients with adverse clinical outcomes usually show an aggressive inflammatory response, and the levels of il-6 were recently shown to be directly associated with the risk of adverse outcomes. [8] overall, our findings seem to confirm the role of age as one of the strongest prognostic factors; in addition, we suggest that cancer patients are not necessarily at higher risk for covid-19 associated death because their impaired immune responsiveness might act as a protective factor from the cytokine storm. [5] this apparently paradoxical situation in the immune impaired populations that have a higher risk to be infected but a higher probability of a benign clinical course was also recently reported for a cohort of patients under immunosuppressive treatment because of several rheumatological disorders. [9] even in the case of hiv-sars-cov-2 coinfection, the preliminary results seem to confirm that the prognosis is not worse than the general population. [10] while waiting for more robust evidence, a feared worse outcome for cancer patients affected by covid-19 appears, at the moment, not justified. our analysis was also the first, to the best of our knowledge, to directly compared covid-19 cancer and non-cancer patients from both a clinical and immunological point of view. however, it does suffer from some limitations that are in common with the aforementioned studies: the cancer type and clinical/pathological stage, and the type and time elapsed since the last treatment should be separately evaluated. reasonably, only future studies in larger cohorts accounting for all the complex interactions between the host response to this novel coronavirus and the consequences of cancer and its treatment on the immune system will solve this issue. fatality rate of covid-19 in patients with malignancies: a systematic review and meta-analysis covid-19: unanswered questions on immune response and pathogenesis risk factors for severity and mortality in adult covid-19 inpatients in wuhan clinical characteristics of covid-19-infected cancer patients: a retrospective case study in three hospitals within wuhan challenges faced by medical journals during the covid-19 pandemic do patients with cancer have a poorer prognosis of covid-19? an experience in new york city elevated levels of il-6 and cpr predict the need for mechanical ventilation in covid-19 risk of covid-19 for patients with cancer covid-19 in immune-mediated inflammatory diseases covid-19 in patients with hiv: clinical case series key: cord-025481-ljs80v45 authors: hu, jianhua; zhang, xiaoli; zhang, xuan; zhao, hong; lian, jiangshan; hao, shaorui; jia, hongyu; yang, meifang; lu, yingfeng; xiang, dairong; cai, huan; zhang, shanyan; gu, jueqing; ye, chanyuan; yu, guodong; jin, ciliang; zheng, lin; yang, yida; sheng, jifang title: covid-19 patients with hypertension have more severity condition, and acei/arb treatment have no infulence on the clinical severity and outcome date: 2020-05-28 journal: j infect doi: 10.1016/j.jinf.2020.05.056 sha: doc_id: 25481 cord_uid: ljs80v45 nan severe acute respiratory syndrome coronavirus-2 (sars-cov-2); coronavirus disease 2019 (covid-19); hypertention; angiotensin-converting enzyme inhibitor (acei); angiotensin receptor blocker (arb); outcome a number of pneumonia cases of unknown causes have emerged in wuhan, hubei, china since december 2019. (1) after sequencing analysis of samples from the lower respiratory tract, a coronavirus, (2) which was last named as severe acute respiratory syndrome coronavirus-2 (sars-cov-2). (3) on february 11, 2020, the world health organization (who) announced a new name for the disease caused by 2019-ncov: coronavirus disease 2019 (covid-19). (4) with the arrival of the spring festival, an epidemic sars-cov-2 infection has spread rapidly. it has swept across china and all over the world, and became a major global health concern. chinese scientists found that sars-cov-2, like the sars virus in 2003, enters human cells by recognizing angiotensin-converting enzyme 2 (ace2) protein, which is the key to the invasion of the "new coronavirus" into the body. (5) decreased ace2 expression is a cause of hypertension because ace2 is identified as a major angiotensin 1-7 (ang1-7)-forming enzyme. (6) based on studies of covid-19, we found that hypertension initially occurs in many complications in covid-19 patients. (7) however, limited reports on covid-19 patients with hypertension are available in literature. whether patients with hypertension who undergo angiotensin-converting enzyme inhibitor (acei)/angiotensin receptor blocker (arb) therapy are more likely to suffer sars-cov-2 infection and whether acei/arb therapy would have an influence on the clinical outcomes of patients with covid-19 are controversy. (8, 9) moreover, the epidemiologic and clinical features of covid-19 patients with hypertension are also not completely elucidated. thus, in this study, we describe the demographic, epidemiologic, and clinical characteristics of covid-19 patients with hypertension. and we also attempted to analyze whether acei/arb treatment would have an influence on the clinical severity and outcomes of covid-19 patients. altogether, 884 covid-19 patients between january 17, 2020 and february 8, 2020, who confirmed with sars-cov-2 infection in zhejiang province, diagnosed as having covid-19 according to who interim guidance (10) were enrolled in this study. among various coexisting conditions, the proportion of patients with hypertension (149 patients, 16.86%) was higher than that of others. compared with covid-19 patients without hypertension, those patients with hypertension had a higher percentage of male sex (59.06% vs 49.93%, p=0.042), were older (57.00 years vs 43.00 years, p=0.000) and had a higher percentage of age ≥60 years (43.62% vs 13.88%, p=0.000). in this study, 723 patients were diagnosed to have a mild type; 123 patients, severe type; and 37 patients, critical type. patients with hypertension had a lower rate of mild type (59.06% vs 86.39%, p=0.000), but had a higher rate of severe (26.17% vs 11.43%, p=0.001) and critical types (14.77% vs 2.04%, p=0.000) than patients without hypertension. compared with patients without hypertension, patients with hypertension had a higher incidence of acute respiratory distress syndrome(ards) (24.16% vs 6.67%, p=0.000), were more likely to use glucocorticoids (31.54% vs 12.79%, p=0.000), antibiotic (50.33% vs 39.32%, p=0.013), and intravenous immune globulin therapy (21.48% vs 6.67%, p=0.000) and more likely to need mechanical ventilation (14.77% vs 2.04%, p=0.000) and intensive care unit (icu) admission (16.11% vs 2.31%, p=0.000), extracorporeal membrane oxygenation (ecmo) (4.03% vs 0.82%, p=0.007) and continuous renal replacement therapy (crrt) (2.01%vs 0.14%, p=0.016) therapy. the time intervals from illness onset to discharge and from admission to discharge in patients with hypertension (median 25.00 days and 20.00 days, respectively) were longer than those in patients without hypertension (median 22.00 days and 18.00 days, respectively) (p=0.000, p=0.002) ( table 1 ). we found that the level of leukocyte count (median 5.40×10 9 /l vs 4.70×10 9 /l, p=0.000) and neutrophil count (median 3.60×10 9 /l vs 2.90×10 9 /l, p=0.000) was higher, but the level of lymphocyte count (median 1.00×10 9 /l vs 1. in summary, we reported the largest cases of covid-19 patients with hypertension. this study showed that patients with hypertension might have more severe respiratory symptoms, more abnormality laboratory indication, and more proportion of severe/critical type of covid-19. moreover, they may need more antibiotic, hormone, and intravenous immune globulin therapy and intensive care unit admission and have a longer hospital stay. treatment with acei/arb have no influence on the severity and the clinical outcome of covid-19 patients. updated understanding of the outbreak of 2019 novel coronavirus (2019-ncov) in wuhan a novel coronavirus from patients with pneumonia in china severe acute respiratory syndrome-related coronavirus: the species and its viruses -a statement of the coronavirus study group severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and corona virus disease-2019 (covid-19): the epidemic and the challenges a pneumonia outbreak associated with a new coronavirus of probable bat origin multiple functions of angiotensin-converting enzyme 2 and its relevance in cardiovascular diseases clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet association of inpatient use of angiotensin converting enzyme inhibitors and angiotensin ii receptor blockers with mortality among patients with hypertension hospitalized with covid-19. circulation research chronic use of angiotensin-converting enzyme inhibitors and angiotensin ii receptor blockers is high among intensive care unit patients with non-covid-19 sepsis but carry a 9 clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance (ncov)-infection-is-suspected note 1: *, p value of comparison between acei/arb and non-acei/arb note 2: alb, albumin; alt, alanine aminotransferase; ast: aspartate aminotransferase; bun, blood urea nitrogen; ck, creatine kinase; copd, chronic obstructive pulmonary disease extracorporeal membrane oxygenation; inr: international normalized ratio; ldh: lactate dehydrogenase we would like to thank editage (www.editage.cn) for english language editing. none. key: cord-252980-1e28zj1d authors: zhang, jiahao; jia, weixin; zhu, junhai; li, bo; xing, jinchao; liao, ming; qi, wenbao title: insights into the cross-species evolution of 2019 novel coronavirus date: 2020-03-04 journal: j infect doi: 10.1016/j.jinf.2020.02.025 sha: doc_id: 252980 cord_uid: 1e28zj1d nan recent study reported in this journal that the threats of continuous evolution and dissemination of 2019 novel coronaviruses. 1 since its emergence in december 2019, a "seventh" member of the family of human coronavirus named "sars-cov-2" was responsible for an outbreak of coronavirus disease in wuhan, china. 2 as of march 7, 2020, china had reported more than 80,815 confirmed cases of sars-cov-2, with 3,073 fatalities and counting ( http://www.nhc.gov.cn ). strikingly, sars-cov-2 had been transmitted rapidly in more than 90 countries to date ( https://www.who.int ), including asia, europe, north america, south america, africa, and oceania, posing serious concerns about its pandemic potential. despite of droplet and contact transmissions of sars-cov-2, recent studies demonstrated that sars-cov-2 might be transmitted via aerosol and fecal-oral routes 3 ( fig. 1 ) , which needs to be paid attention in particular. the close phylogenetic relationship to bat-origin coronaviruses provided evidence for a bat origin of sars-cov-2. 4 bats provided a rich "gene pool" for interspecies exchange of genetic fragments of coronaviruses, which were established as mixing vessels of different coronaviruses. 5 although humans and bats live in different environments, some wildlife species were susceptible to the novel coronaviruses in nature, highlighting that the need of tracing its origin of sars-cov-2 in wild animals. previously, researchers had demonstrated that coronaviruses had been detected in pangolins. 6 here, we explored the phylogenic relationship of the human sars-cov-2 together with pangolin-and bat-origin coronaviruses. the similarity analysis of sars-cov-2 and the animal-origin coronaviruses demonstrated that recombination events were likely to occur in bat-and pangolin-origin coronaviruses (supplementary figure s1) . a blast search of the compete genome sequences of sars-cov-2 suggested that the closely related coronaviruses were the betacov/bat/yunnan/ratg13/2013 (bat/ratg13) and betacov/pangolin/guangdong/1/2020 (pangolin/1), with ∼96% and ∼ 90.5% overall genome sequences identity, respectively. in the 1ab, s, e, m, and n genes, the bat/ratg13 coronavirus exhibited 96.2%, 97.3%, 100%, 99.6%, and 99.0% amino acid identical to that of sar-cov-2, respectively, while the pangolin/1 coronavirus showed the 96.3%, 92.4%, 100%, 98.7%, and 97.9% amino acid identical to that of sars-cov-2, respectively ( fig. 2 ) . however, it was notably that 1b gene sequence identity of pangolin/1 coronavirus was greater that bat-origin ratg13 coronavirus, with the highest being 99.3%. the spike (s) protein mediates receptor binding and membrane fusion. 7 the amino acids of the spike 2 protein of pangolin-origin coronaviruses and sars-cov-2 were more conserved than that of the spike 1 protein, and only a few minor deletions of amino acids of s protein were found in pangolin-origin coronavirus compared with the sars-cov-2 (supplementary figure s4) . interestingly, the receptor-binding domain (rbd) of sars-cov-2 was more similar to that of the bat/ratg13 strain and pangolin/1 coronavirus. although the s amino acid identities of pangolin-origin coronavirus exhibited lower amino acid identities with bat/ratg13, it was noteworthy that six amino acids associated with the receptor binding preference of human receptor angiotensin converting enzyme ii-464 l, 495f, 502q, 503s, 510 n, and 514y (sars-cov-2 numbering)-in the pangolin/1 coronavirus were the same as that of sars-cov-2 ( fig. 2 ), but were distinct from that of the bat-origin coronaviruses. besides, the prra-motif insertion was occurred in the s1/s2 junction of sars-cov-2; however, the prra-motif insertion in the pangolin-and bat-origin coronaviruses was missing (supplementary figure s4 ), suggesting that the convergent cross-species evolution of sars-cov-2-related coronaviruses. the phylogenic tree of full-genome of sars-cov-2 related coronaviruses could be classified into four clades, including clade 1, clade 2, clade 3, and clade 4 ( fig. 1 ) . the two bat-origin sars-like strains (bat-sl-covzc45 and bat-sl-covzxc21) formed clade 1, and pangolin-derived pangolin/1, betacov/ pangolin/guangxi/p2v/2017 (pangolin/p2v), and be-tacov/pangolin/guangxi/p4l/2020 (pangolin/p4l) coronaviruses formed newly independent clade 2 and clade 3, which were notable for the long branch separating the bat/ratg13 strain and sars-cov-2 ( fig. 1 ) . of note, we found that the full genome and rna-dependent rna polymerase genome of pangolin/1 coronavirus were genetically closely related to the bat/ratg13 and sars-cov-2 strains ( fig. 1 and supplementary figure s3 phylogenic tree of s gene, the pangolin/p2v and pangolin/p4l coronaviruses were more closely related to that of the bat/ratg13 and sars-cov-2 strains (supplementary figure s2) , indicative of the continuous evolution and genetic recombination of pangolinand bat-derived coronaviruses. there was clearly a genetic gap between sars-cov-2 and the nearest bat-and pangolin-origin coronaviruses, and the phylogenic relationship of pangolin-origin coronaviruses were far from that of bat/ratg13 ( fig. 1 ) . given the use of pangolins use in traditional medicine and for food, what kind of role do the pangolins play in the cross-species evolution and transmission of the novel coronavirus? further details needed to be sought in the future. frequent human-animal interface had been recognized the major cause for viral cross-species transmission. it is speculated that the coronaviruses circulating in pangolin, bat, and other animal species are likely perceived to be a "gene pool" for the generation of new recombinants ( fig. 1 the continuous evolution and dissemination of 2019 novel human coronavirus severe acute respiratory-related coronavirus-the species and its viruses, a statement of the coronavirus study group clinical characteristics of 2019 novel coronavirus infection in china a pneumonia outbreak associated with a new coronavirus of probable bat origin discovery of a rich gene pool of bat sars-related coronaviruses provides new insights into the origin of sars coronavirus viral metagenomics revealed sendai virus and coronavirus infection of malayan pangolins (manis javanica) bat-to-human: spike features determining 'host jump' of coronaviruses sars-cov, mers-cov, and beyond we sincerely thank the authors of the human 2019 coronavirus from gisaid epiflu tm database. supplementary material associated with this article can be found, in the online version, at doi:10.1016/j.jinf.2020.02.025 . key: cord-264285-1sne3ng1 authors: sze, shirley; pan, daniel; williams, caroline m.l.; wong, nicholas; sahota, amandip; bell, david; tang, julian w.; wiselka, martin; stephenson, iain; pareek, manish title: letter to the editor: variability but not admission or trends in news2 score predicts clinical outcome in elderly hospitalised patients with covid-19 date: 2020-05-29 journal: j infect doi: 10.1016/j.jinf.2020.05.063 sha: doc_id: 264285 cord_uid: 1sne3ng1 • in elderly covid-19 inpatients, admission news2 scores did not predict mortality. • of components of new2 score, only systolic blood pressure predicted mortality. • a high variability in new2 score predicted mortality. • news2 score does not consider the degree of supplemental oxygen patients require. • a more sensitive early warning score for covid-19 is needed. in a recent article in the journal, bruno and colleagues present short-term outcomes in elderly patients with severe covid-19 disease admitted to a single italian infectious disease unit. (1) the study found that elderly patients are at increased risk of adverse outcomes due to high number of comorbidities and emphasises the need to improve clinical management in these patients. in particular, elderly patients who are likely to deteriorate will need to be rapidly identified. (2) existing prognostic models for covid-19 based on clinical, laboratory and radiological variables are at high risk of bias. (3) in the uk, the national early warning score (news) and its updated version news2an a priori weighted composition of the patient's observations -is used routinely to monitor patients in hospital and identify early those who may deteriorate. (4) compared to other early warning scores, the news score has a greater ability to discriminate patients at risk of cardiac arrest, unanticipated intensive care unit admission or death. (5) currently, guidance from the national institute of clinical excellence (nice) supports the use of the news2 score to risk assess patients with covid-19 in the community, who may require hospitalisation. (6) in a recent rapid review, greenhalgh and colleagues do not recommend using the news2 score alone for risk assessment of patients with covid-19 in the community. (7) blood pressure and oxygen saturation measurements are difficult to take remotely. the score also does not include age or comorbidities, strong independent predictors of survival in patients with covid-19. the value of the news2 score in predicting outcome in patients admitted to hospital with covdi-19 remains uncertain. we therefore undertook, as part of service evaluation, a prospective pilot assessment of patients with confirmed covid-19 admitted to a tertiary infectious diseases unit, in the first month of the pandemic reaching the uk. we studied all patients who had a clinical outcome (either discharged from hospital or died) between 12 th march and 2 nd april 2020. clinical (presenting symptoms, comorbidity and the news2 score throughout hospital stay), laboratory (routine blood tests) and radiological (chest x-ray reports) findings on admission were collated. our main aim was to examine the utility of the news2 score in predicting the clinical deterioration of hospitalised covid-19 patients. continuous data are expressed as a median (25 th -75 th percentiles) and categorical data are expressed as n (%). independent t-tests and mann-whitney u tests were used to compare two continuous variables for normally and non-normally distributed data. the chi-squared test was used to compare proportions between groups. overall, 17 patients with covid-19 had an outcome by 2 nd april 2020. the median age of our cohort was 85 years (iqr 83-88 years); 53% were male and 82% were caucasian. all patients who were unsuitable for escalation to intensive care and received ward-based care as per nice rapid guidance for covid-19. (5) the majority of patients died (n=10, 59%). compared to patients who survived, those who died were more likely to be male, with bilateral consolidation on chest radiographs on admission. admission sars-cov-2 quantitative pcr ct values on nasopharyngeal swab did not seem to relate to survival. all patients who died required some form of oxygen therapy, ranging from nasal canulae to non-invasive ventilation through continuous positive airway pressure. less than half of those who survived required oxygen therapy, all of which were delivered via nasal canulae. figure 1 shows the trend in the national early warning score2 (news2) throughout hospitalisation, stratified by severity of news2 on admission and clinical outcome. first, we found that the initial news2 score did not predict mortality. for example, four out of the ten patients (40%) who died presented with a news2 score of 0-3 while three out of seven patients (43%) who survived presented with a new2 score of 5 or above. secondly, there was no significant difference in the admission news2 score and its components, between patients who died and those who survived, apart from systolic blood pressure. (table 1) . thirdly, examining the news2 scores over time, patients who died had a higher variability in their scores compared to those who survived. seven out of ten patients (70%) who died had a maximum daily change in news2 score of over 5, while none of those who survived had such dramatic fluctuations. (figure 1 ) in our small pilot of elderly patients admitted to hospital with covid-19, admission news2 scores did not seem to be useful in predicting clinical outcomes. for some patients, death occurred regardless of admission news scores and without a prior deteriorating trend. originally, the news score was developed using data from 35585 acute hospital admissions, most of whom would have had an underlying diagnosis of sepsis. however, covid-19 is caused by sars-cov-2, a coronavirus which predominately appears to affect the respiratory system as an initial viral pneumonitis. in china, a fifth of all covid-19 inpatients rapidly became critically ill with hypoxia and respiratory failure. (9) the weighting of the news2 score does not account for the degree of supplemental oxygen (fio2) a patient may require, thus limiting its utility to identify early deterioration in patients with covid-19. in our cohort, patient 8 had a news2 score of 2 on day 2 and 3 despite requiring a large increase in fio2 (from room air to 60%). a more sensitive early warning score for covid-19 needs to be urgently developed and validated. mp is supported by the nihr and has received grants and personal fees from gilead sciences outside of this work. all other authors have nothing to declare. short-term outcomes in individuals aged 75 or older with severe coronavirus disease (covid-19): first observations from an infectious diseases unit in southern italy impact of non-pharmaceutical interventions (npis) to reduce covid19 mortality and healthcare demand prediction models for diagnosis and prognosis of covid-19 infection: systematic review and critical appraisal the ability of the national early warning score (news) to discriminate patients at risk of early cardiac arrest, unanticipated intensive care unit admission, and death covid-19 rapid guideline: critical care in adults should we use news2 to assess possible covid-19 patients in primary care? centre for evidence-based medicine views--towards a national early warning score for detecting adult inpatient deterioration characteristics of and important lessons from the coronavirus disease covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention total number during admission 2 (1-3) 6 (4-13) 9 (7-10) 0.13 key: cord-260925-puuqv6zk authors: wen, feng; yu, hai; guo, jinyue; li, yong; luo, kaijian; huang, shujian title: identification of the hyper-variable genomic hotspot for the novel coronavirus sars-cov-2 date: 2020-03-05 journal: j infect doi: 10.1016/j.jinf.2020.02.027 sha: doc_id: 260925 cord_uid: puuqv6zk nan a recent study in this journal studied the genomes of the novel sars-like coronavirus (sars-cov-2) in china and suggested that the sars-cov-2 had undergone genetic recombination with sars-related cov 1 . by february 14, 2020, a total of 66,576 confirmed cases of covid-19, people infected with sars-cov-2, were reported in china, leading to 1524 deaths, per the chinese cdc ( http://2019ncov.chinacdc.cn/2019-ncov/ ). several full genomic sequences of this virus have been released for the study of its evolutionary origin and molecular characteristics [2] [3] [4] . here, we analyzed the potential mutations that may have evolved after the virus became epidemic among humans and also the mutations resulting in the human adaptation. the sequences of betacov were downloaded on february 3, 2020 from the gisaid platform 5 . a total of 58 accessions were available, among which betacov/bat/yunnan/ratg13/2013 is a known close relative of sars-cov-2. four accessions, namely, betacov/italy/inm1/2020, betacov/italy/inm2/2020, beta-cov/kanagawa/1/2020, and betacov/usa/il1/2020, were excluded because of the short-truncated sequences or multiple ambiguous nucleotides. a total of 54 accessions (supplementary table 1 ) isolated from humans were utilized in the following analysis. the sequences nc_004718.3 of sars coronavirus 6 genes were utilized to define the protein products of sars-cov-2. the protein sequences of orf1ab, s, e, m, and n genes were translated, and all of the loci without experimental evidences were excluded. first, the protein sequences of sars-cov-2 were compared with ratg13, human sars (nc_004718.3), bat sars (dq022305.2), and human mers (nc_019843.3) by calculating the similarity in a given sliding window ( fig. 1 a) . the sliding window was set to 500 for orf1ab and s, and to 50 for proteins e, m, and n considering their short length. sars-cov-2 were highly similar to ratg13 isolated from bats, showing 96% identity based on the whole-nucleotide sequences and 83% based on the protein sequences, suggesting a bat zoonotic origin of sars-cov-2. orf1a, and the head of s seemed to have diverged from other beta coronaviruses. the molecular phylogenetic tree ( fig. 1 b) was built by using the maximum likelihood method based on the jtt matrix-based model 7 . it hinted that the protein sequences of sars-cov-2 had over 99% similarity. twenty-eight viruses had shared the same protein sequences, and could be the original strain circulated in the humans. the other viruses had only a few mutations from it. this indicates that the virus could have evolved for only a very short time after gaining the efficient human to human transmissibility, as expected. next, we analyzed the mutations that occurred after infecting humans ( fig. 1 c) in order to identify mutations associated with more severe infection. here, two accessions (betacov/shenzhen/szth-001/2020 and betacov/shenzhen/szth-004/2020) from shenzhen, which had 5 and 16 mutations, respectively, were excluded, considering the possible experimental issues. all of the mutations only occurred once, so it is possible that all of these mutations occur naturally and are associated with viral survival and infection. several mutations were clustered in peptides nsp3 and nsp4 of orf1ab and in the header of s. these results suggested that there had probably been no hyper-variable genomic hotspot in the sars-cov-2 population until now. we compared these results with those of the work of ceraolo and giorgi 8 , who reported at least two hyper-variable genomic hotspots based on the shannon entropy of nucleotide sequences. they utilized all of the sequences, while we merged all of the fully identical sequences into one during our shannon entropy calculation. as shown in fig. 1 b, 28 sequences were merged into one in present study because they had been collected in such a short time, so collection time and location could not have produced any large bias. if those identical sequences were calculated individually, any mutations on these 28 sequences would have sharply increased shannon entropy. the protein sequences were used to exclude any unimportant silent mutations. finally, the sequences of earliest sars-cov-2 were compared with ratg13 from bats ( fig. 1 d) . fisher's exact test with post hoc test suggested that nsp1, nsp3, and nsp15 of orf1ab and gene s had significantly more mutations than other genes, which might facilitate human adaptation and infection. s gene encodes spike glycoprotein, which binds host ace2 receptors and is required for initiation of the infection 9 . they reported that a 193-amino acid fragment was able to bind ace2 more efficiently than its unmutated counterpart. this region in which spike glycoprotein binds to ace2 had 21 mutations not found in ratg13, suggesting their role in the adaptation to human hosts. peptide nsp1 facilitated viral gene expression and evasion from the host immune response 10 . peptide nsp3, named papainlike proteinase, was found to be associated with the cleavages, viral replication, and antagonization of innate immune. these two peptides are probably associated with the latent period after infection in humans. peptide nsp15 acted as uridylate-specific endoribonuclease. these results collectively suggest that peptides nsp1, nsp3, and nsp15 might have unclear but critical roles in this outbreak of sars-cov-2. to summarize, this study confirmed the relationship of sars-cov-2 with other beta coronaviruses on the amino acid level. the hyper-variable genomic hotspot has been established in the sars-cov-2 population at the nucleotide but not the amino acid level, suggesting that there have been no beneficial mutations. mutations in nsp1, nsp3, nsp15, and gene s that identified in this study would be associated with the sars-cov-2 epidemic and was worthy of further study. none. this study was supported by key laboratory for preventive research of emerging animal diseases in foshan university ( klpread201801-06 ), ( klpread201801-10 ), youth innovative talents project of guangdong province ( 2018kqncx280 ), and national key research and development project (grant no. 2017yfd050 080 0 ). the continuous evolution and dissemination of 2019 novel human coronavirus genome composition and divergence of the novel coronavirus (2019-ncov) originating in china a new coronavirus associated with human respiratory disease in china a pneumonia outbreak associated with a new coronavirus of probable bat origin gisaid: global initiative on sharing all influenza data -from vision to reality the genome sequence of the sars-associated coronavirus the rapid generation of mutation data matrices from protein sequences genomic variance of the 2019-ncov coronavirus a 193-amino acid fragment of the sars coronavirus s protein efficiently binds angiotensin-converting enzyme 2 severe acute respiratory syndrome coronavirus protein nsp1 is a novel eukaryotic translation inhibitor that represses multiple steps of translation initiation we thank the researchers who deposited the sars-cov-2 sequences in the gisaid. we thank letpub for its linguistic assistance during the preparation of this manuscript. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.02.027 . key: cord-261405-n05wjimk authors: lui, grace; ling, lowell; lai, christopher kc; tso, eugene yk; fung, kitty sc; chan, veronica; ho, tracy hy; luk, fion; chen, zigui; ng, joyce kc; chow, kai-ming; cheng, peter kc; chan, rickjason cw; tsang, dominic nc; gomersall, charles; hui, david sc; chan, paul ks title: viral dynamics of sars-cov-2 across a spectrum of disease severity in covid-19 date: 2020-04-18 journal: j infect doi: 10.1016/j.jinf.2020.04.014 sha: doc_id: 261405 cord_uid: n05wjimk nan dear editor, fang et al. reported in this journal that viral shedding of sars-cov-2 in nasal swabs was longer in intensive care unit (icu) patients compared with non-icu patients with coronavirus disease 2019 (covid-19). 1 covid-19 encompasses a heterogeneous spectrum of illness, ranging from asymptomatic and mild infections, to severely ill cases in 4-16%. 2, 3 here, we report the findings of a prospective cohort study to determine viral dynamics of sars-cov-2 in mild to critical severity of illness, and the temporal viral burden at different body sites. we included the first eleven laboratory-confirmed covid-19 patients hospitalized in two hospitals in hong kong in february 2020. disease severity was categorized as previously defined. 3 we collected serial upper (pooled nasopharyngeal and throat swabs, n=75) and lower respiratory tract samples (sputum and tracheal aspirate, n=43), peripheral blood plasma (n=50), urine (n=43) and stool (n=43) samples from all participants, and monitored sars-cov-2 viral loads in these samples. 4 one participant had mild, 5 moderate, 3 severe, and 2 critical disease. all patients were discharged; no one died. their clinical and epidemiological features are shown in table 1. viral loads for each sample are shown in supplementary table 1. nine participants, including 4 participants with mild-moderate disease, had viral shedding lasting longer than 14 days in the respiratory tract. in four (36%) participants, return to pcr positivity in the respiratory tract was observed after ≥1 negative test, without worsening of symptoms. in patient a, pcr positivity occurred in upper and lower respiratory tract samples after a 7-day "apparent clearance" where 3 upper and 3 lower respiratory samples were negative (figure) . 2 viral loads in respiratory tract samples did not correlate with disease severity. however, the timing of peak viral burden differed between participants with different severity. in all five participants with severe/critical and three with moderate disease, viral loads in respiratory tract samples continued to rise and peaked in the second week of illness (range 5.57-9.66 log copies/ml). in the remaining three with mild/moderate disease, viral load peaked in the first week of illness (range 3.25-6.40 log copies/ml) table 2 ), but did not differ between mild-moderate and severe-critical diseases. sars-cov-2 was not detected in any of the 43 urine samples. in particular, all 8 urine samples from patient b were negative despite the need for renal replacement therapy for acute kidney injury. his renal biopsy showed features of acute tubular injury; electron microscopy did not reveal viral particles in proximal tubules. one patient with moderate and one with critical disease had transient viremia, lasting 1 and 5 days, with peak viral loads 4.00 and 3.65 log copies/ml respectively. in this study of patients with covid-19 across a wide spectrum of severity, we observed that viral shedding in the respiratory tract lasting longer than 14 days was common. viral load peaked later in the second week of illness in more severe disease. extrapulmonary detection of rt-pcr positivity other than the gut was uncommon. we observe two patterns of viral dynamics trajectory in the respiratory tract. in more severe disease, viral load appeared to peak in the second week of illness in both upper and lower respiratory tract. a more heterogeneous pattern was seen in milder disease. time to dyspnoea and intensive care was around 8-10 days after illness onset. 5 therefore, continued viral replication in the respiratory tract, correlated with this timing of clinical deterioration, as observed in other studies. 6, 7 this observation implied that antiviral therapy, rather than immunomodulatory agents, might be more effective as treatment for severe disease. 7 viral load was significantly higher and peaked later in lower than upper respiratory tract samples. in severe/critical disease, monitoring should be performed using lower respiratory tract samples. firstly, viral shedding was more prolonged in lower than upper respiratory tract samples, thus serves as better guidance for the duration of infection prevention measures. 8 secondly, viral loads in lower respiratory tract better reflected the temporal course of clinical progression in severe disease than upper respiratory tract samples. 6, 9 all patients demonstrated stool rt-pcr positivity at some stage of their disease, regardless of gastrointestinal symptoms. while other series had positivity rates of around 50%, 6, 9, 10 repeated testing in our study allowed detection of transient positivity lasting 1-2 days. there is widespread presence of ace2 as cellular receptors and viral particles in epithelial cells along different parts of the gastrointestinal tract. 10 while the clinical role of testing sars-cov-2 from stool as monitoring or 4 discharge criteria remains uncertain, stool as an additional specimen to assist diagnosis, particularly in asymptomatic contacts of confirmed patients, is worth exploring. our study was limited by the small sample size. asymptomatic patients were not studied, and the effect of antiviral therapy could not be determined. viral dynamics at different sites should be further explored in larger populations of covid-19 patients. in conclusion, viral shedding in respiratory tract lasting longer than 14 days was common in all spectrum of covid-19 severity. clinical deterioration correlated temporally with viral replication in severe disease, especially in the lower respiratory tract, highlighting the importance of effective antiviral therapy. extrapulmonary detection of sars-cov-2 pcr other than the gut was uncommon. death 0 (0%) 0 (0%) 0 (0%) comparisons of nucleic acid conversion time of sars-cov-2 of different samples in icu and non-icu patients china medical treatment expert group for c. clinical characteristics of coronavirus disease 2019 in china clinical characteristics of imported cases of covid-19 in jiangsu province: a multicenter descriptive study 2019-novel coronavirus (2019-ncov) real-time rrt-pcr panel primers and probes clinical features of patients infected with 2019 novel coronavirus in viral load of sars-cov-2 in clinical samples. the lancet infectious diseases clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study duration of quarantine in hospitalized patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection: a question needing an answer singapore novel coronavirus outbreak research t. epidemiologic features and clinical course of patients infected with sars-cov-2 in singapore evidence for gastrointestinal infection of sars-cov-2 we would like to thank ms iris wong, ms rity wong and ms vickie li for their clerical support in the preparation of the manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or notfor-profit sectors. key: cord-007562-4hcs0z65 authors: bijlenga, g. title: proposal for vaccination against sars coronavirus using avian infectious bronchitis virus strain h from the netherlands date: 2005-07-19 journal: j infect doi: 10.1016/j.jinf.2005.04.010 sha: doc_id: 7562 cord_uid: 4hcs0z65 nan occult hepatitis b virus infection among anti-hbc positive blood donors: necessitates substitution of screening by hbv nat safe blood transfusion still remains a major concern and so far all the efforts in this direction have failed to achieve zero residual risk of transfusion transmitted hepatitis b virus (hbv) infection. in this direction the recently published work by silva et al. in journal of infection has revealed remarkable observations. 1 this report shows 3.3% hbv dna positivity of the blood donor's samples that were anti-hbc positive, more enlightening finding is the hbv dna positivity among the high level anti-hbs positive donors. at this tertiary care centre of saudi arabia out of 26 606 blood units collected during 2000-2003, isolated anti-hbc positivity was 3.2% and hbsag positivity 1.9%, where as 10.1% of the blood units were anti-hbc and anti-hbs positive. as per policy of health ministry, the anti-hbc and anti-hbs positive blood units were utilized and the isolated anti-hbc blood units were rejected. 2 the blood units which are anti-hbc and anti-hbs positive do not appear to transmit hbv infection and there is inverse correlation between anti-hbs level and infectivity, only 10% of the blood units with low level (!0.1 iu/ml) anti-hbs are infectious. 3 the observation by silva et al. that hbv dna positivity among anti-hbc and high level anti-hbs positive blood donors is a pointer towards the transfusion transmitted risk involved by transfusion of anti-hbc and anti-hbs positive blood units. though the viral load in these samples was low (!1000 copies/ml) but this can be highly infectious if transfused to an immunocomprised patient. considering the volume of infectious blood transfused any amount of hbv dna will be infectious as the minimum infecting dose of hbv in chimpanzees is only 100 virus particles. 4 in many of the developed countries and most of the developing countries the blood units collected are still being screened for hbsag, anti-hbc and anti-hbs by enzyme immuno assay. on many occasions the results are indeterminate and has to be repeated leading to higher per unit cost of blood screening and lot of rejection of the invaluable units of collected blood or exclusion of the generous donor because of isolated anti-hbc positivity and still the safety of transfusion transmitted hbv is compromised. this high rate of rejection of collected blood units and the exclusion of the anti-hbc positive blood donors leads to the unceasing blood shortage in the blood banks. the hbv screening policy for the collected units of blood needs reassessment in light of the present report 1 and hbv dna testing should be preferred instead of three enzyme immuno assay tests. hbv dna testing by nat of all the collected units of blood should be adopted by all the blood banks, in order to possibly achieve zero risk of transfusion transmitted hbv infection and also to reduce the rejection rate of the precious units of collected blood by testing for anti hbc. the outbreak of severe acute respiratory syndrome (sars) in 2003 has resulted in a number of infections and deaths among healthcare workers (hcws) and those in contact with sars-infected persons. the virus, now classified provisionally as a coronavirus in group 4, is highly contagious and treatment of infected persons has so far been disappointing. the first evidence of successful treatment in monkeys (cynomolgus macaques) was reported recently using alpha-interferon (ifn-alpha) 4 administered from 1 to 3 days after experimental exposure. this gave only some success, whereas the drug given at 3 days before experimental infection significantly reduced viral replication and excretion from their throats. lung damage was also reduced by 80% as compared with non-treated monkeys. in a review article on avian infectious bronchitis (ib) vaccine strain h, 1 various characteristics of this vaccine were outlined. here i shall mention the most valuable properties of this ib vaccine so far known to underline the hypothesis that it may be beneficial in people at risk from sars coronavirus. (1) it has been observed that the ib vaccine h is able to protect against a broad spectrum of different heterologous serotypes of ib challenge viruses. 12 these serotypes differ in their surface proteins (spikes-s1) which are responsible for the induction of neutralizing antibody. differences in s1 of only 2-3% can change the serotype of an ib virus. 3 therefore, it can be concluded that the protection provided by the vaccine strain h is not only dependant on the production of neutralizing antibody, but is also due to the induction of other immunological reactions. (2) the role of the nucleocapsid protein (n) is still not well understood but it may play an important role in protection, inducing specific cytotoxic t lymphocytes. 2,7-10 thus, the vaccine strain h may be responsible for the induction of protection through its nucleocapsid protein. 13 in order to evaluate the importance of cellular mediated immunity (cmi) in protecting against ibv infections more studies would be necessary to explain all the mechanisms of cross-protection of the vaccine strain h, for instance the induction of interleukine 2 (il 2). (3) the observation that interferon (if) is poorly induced by ibv and may not be induced by the vaccine strain at passage level 52, could be an indication that if plays a limited role in heterologous protection. 5 (4) in a study by marra et al. 6 it was concluded that the sars coronavirus is a novel coronavirus. stavrinides and guttman 11 concluded recently that the sars coronavirus is mammalian-like through the replicase protein, and avian-like through the m and n proteins. they also observed a mammalian-avian mosaic in the s protein. these observations are of extreme importance to the consideration of an avian coronavirus as a possible candidate for a vaccine against sars coronavirus. in adequately equipped laboratory facilities (p4): (a) it is proposed to use passage 52 of the h strain of vaccine in preliminary experimental studies in monkeys. this passage level has been chosen for its retention of cross-protective characteristics. the vaccine strain h at passage 120 induces only a low level of interferon 5 but has lost its heterologous protection characteristics due to the attenuation of the virus. (b) in order to produce a valuable immunological reaction in monkeys with the ib h52 vaccine it will be necessary to inoculate a high dose of live virus vaccine, for example 10 8 median embryo infectious doses (100.000.000 eid 50 ) intranasally, intramuscularly and/or subcutaneously. it is not expected that the virus will be infectious for macaques, therefore, a high dose will be required in order to achieve an adequate response of the immune system. for more than 50 years avian ib infections have occurred worldwide and there are no reports of infection among human beings, including in poultry farmers or other people who have had direct contact with highly contagious ib viruses of chickens. (c) in the study using alpha if in macaques the amount of sars coronavirus virus (scv) used for challenge was 1!10 6 median tissue culture infectious dose (tcid 50 ) in 5 ml of pbs administered intratracheally. 4 however, it was not mentioned in that publication whether or not a prechallenge titration of this virus was performed. it is very important to establish the amount of challenge virus, which will provoke disease and eventually death. therefore, before starting the experiment titration of the challenge virus in these monkeys should be performed in order to determine the amount of virus, which will produce clinical symptoms in not more than 90% of the infected animals. if an overdose is applied no real effect of the treatment will be demonstrable and if insufficient challenge virus is used no results will become available. (d) it is of extreme importance that the h52 vaccine virus should be free of all microorganisms other than ib live vaccine virus, therefore, its production and passage in specific pathogen free (spf) embryonated eggs is a prerequisite. (e) it is proposed to challenge the vaccinated monkeys at 2 and 14 days after vaccination with a challenge scv which has been titrated in macaques (see point c). this proposal is based on the likely immediate effect of the vaccine at 2 days through immunostimulation mechanisms and at 2 weeks, if protection is observed, through the heterologous cross-protective activity of the vaccine virus. it is without question that careful consideration by the relevant official health authorities must be given before an animal live virus vaccine is applied to human beings. the application of the ib vaccine strain h in humans should be restricted and only hcws and other persons at risk but not yet showing any signs of the disease will be considered as candidates for vaccination. if clinical symptoms are observed other methods of treatment, such as administration of alpha if are recommended. low rate of occult hepatitis b virus infection among anti-hbc positive blood donors living in a low prevalence region in brazil epidemiology of antibody to hepatitis b core antigen screening among blood donors in eastern saudi arabia: need to replace the test by hbv dna testing occult hepatitis b virus infection: implications in transfusion b-propiolactone irradiation: a review of its effectiveness for inactivation of viruses in blood derivatives development and use of the h strain of avian infectious bronchitis virus from the netherlands as a vaccine: a review induction of anti-viral immune responses by immunization with recombinant-dna encoded avian coronavirus nucleocapsid protein review article. severe acute respiratory syndrome vaccine development: experiences of vaccination against avian infectious bronchitis coronavirus pegylated interferon-alpha protects type 1 pneumocytes against sars coronavirus infection in macaques induction of chicken interferon by avian infectious bronchitis virus the genome sequence of the sarsassociated coronavirus specific cytotoxic t lymphocytes are involved in in vitro clearance of infectious bronchitis virus the carboxyl-terminal 120-residue polypeptide of infectious bronchitis virus nucleocapsid induces cytotoxic t lymphocytes and protect chickens from acute infection cytotoxic t lymphocytes responses to infectious bronchitis virus infection adoptive transfer of infectious bronchitis virus primed alphabeta t cells bearing cd8 antigen protects chicks from acute infection mosaic evolution of the severe acute respiratory syndrome coronavirus potential for polyvalent infectious bronchitis vaccines study of protection by recombinant fowlpox expressing c-terminal nucleocapsid protein of infectious bronchitis virus against challenge 74250 la tour-en-faucigny, france q 2005 the british infection society key: cord-287030-xzothuf7 authors: pigott, david c. title: emergency department evaluation of the febrile traveler date: 2006-05-23 journal: j infect doi: 10.1016/j.jinf.2006.03.028 sha: doc_id: 287030 cord_uid: xzothuf7 the emergency department evaluation of the febrile traveler presents the emergency physician with a set of unique and often challenging circumstances. in addition to evaluating and managing the usual array of community-acquired infections, the clinician must be prepared to diagnose and treat a host of uncommon and potentially life-threatening pathogens. this diseases range from widespread tropical diseases such as malaria to the more exotic and lethal viral hemorrhagic fevers. a thoughtful approach guided by geographic patterns of illness offers a reliable method for determining the most likely sources of fever in the returned traveler as well as a focused diagnostic and treatment strategy. while the presentation of a patient with undifferentiated febrile illness to the emergency department is rarely a cause for alarm, the additional factor of recent international travel suggests a host of unusual pathogens, some with the potential for rapid and devastating transmission among both patients and health care workers. other infectious diseases may lead to significant morbidity and mortality if not rapidly identified and treatment initiated. with the current ease and rapidity of global travel, the likelihood that exotic infectious agents may appear at any given hospital has increased significantly. the search for these rare diseases should not, however, come at the expense of a ''common sense'' approach to the evaluation of the febrile patient. additional information about specific international travel destinations can help guide the evaluation while suggesting more likely infectious agents that may be endemic to that region. common things being common, febrile patientsd even those returning from destinations in developing nationsdare still likely to present with routine viral and bacterial illnesses. 1,2 a prospective review of 195 febrile patients requiring admission after returning to the united kingdom from the tropics found that while malaria (42%) was the most common cause for hospital admission, non-specific viral illness and bacterial infections (including urinary tract infection, community-acquired pneumonia and pharyngitis) accounted for 34% of patients. 2 in this issue, woodrow et al., demonstrate in their work on viral hemorrhagic fever (vhf) screening that a more comprehensive strategy that accounts for even rare pathogens is likely the most appropriate means of evaluating the febrile returned traveler. 3 in their article, they develop a risk-stratification strategy for patients presenting to the london hospital for tropical diseases with fever in an effort to identify those patients at moderate or high risk for vhf. through a simple triage tool that uses criteria including recent fever, travel to a country endemic for vhf, travel outside urban areas or contact with animals, they demonstrate that patients with moderate or high risk for vhf can be identified without the need for unnecessary hospitalization or extensive use of viral polymerase chain reaction (pcr) assays. expanding this methodology to account for other potential infectious agents that may present to a given hospital or emergency department can provide some guidance for clinicians who may encounter febrile patients who have recently returned from any geographic region, unwittingly harboring undiagnosed viral, bacterial or parasitic infection. rather than a scattershot approach to evaluation of the febrile returned traveler where all patients are tested for virtually any etiology, a focused strategy is more likely to be effective. a concise, specific, travel history is essential, including names of visited countries and regions, duration and purpose of visit, as well as trips to non-urban areas or areas where infectious diseases are known to be endemic. freedman et al., in a recent review of geosentinel infectious disease surveillance data gathered on more than 17,000 patients from 30 sites worldwide, found that significant regional differences were noted among ill returned travelers. 4 specific destinations were associated with increased probability of certain diseases, potentially guiding diagnostic and empiric treatment strategies. after an initial travel history is obtained, a list of the most likely etiologies for a patient's illness can be developed based on the relative risk of various illnesses according to geographic region. for example, in patients returning from recent travel to sub-saharan africa, malaria is generally the most common cause of systemic febrile illness, while among recent visitors to central and south america, both dengue fever and malaria are seen in equal numbers. table 1 , from the us centers for disease control and prevention (cdc) ''yellow book,'' health information for international travel, 2005e2006, provides a list of these endemic pathogens as well as the geographic regions where travelers may potentially encounter these illnesses. 5 travelers to geographic areas where there is elevated risk for infection with endemic pathogens should be viewed with high suspicion if a history of fever or other systemic symptoms is elicited. in addition, information regarding specific exposures, such as contaminated food or water, insect bite or ill contacts can help narrow the differential diagnosis for the febrile traveler (see table 2 ). consumption of untreated water or unpasteurized dairy products can lead to hepatitis a (in an unvaccinated traveler), salmonellosis, shigellosis, as well as parasitic infections such as giardiasis. 6 although substantially less common, rare food-borne illnesses such as cholera may also occur. the incidence of traveler's diarrhea has been estimated to be as high as 20e60% in visitors to underdeveloped nations and the geosentinel data also confirm this finding, describing a high frequency of all types of diarrheal illness among returned travelers. 7 a history of insect bite or unexplained soft tissue infection or abscess should prompt the clinician to consider vector-borne illness. malaria, as previously noted, has historically been the most common cause of febrile illness in returned travelers requiring hospitalization. the diagnosis of malaria should be considered even in patients without a clear history of mosquito exposure and in those who have taken appropriate malaria chemoprophylaxis. a missed or delayed diagnosis of malaria can lead to significant morbidity and mortality. because of its high incidence among returned travelers, thick and thin blood smears for malaria should be obtained for any febrile patient who has had recent travel to areas where malaria is endemic, especially, sub-saharan africa, central and south america and southeast asia. other vector-borne diseases which may present with fever include dengue, yellow fever, rickettsial diseases such as african tick fever, and trypanosomiasis. a history of yellow fever vaccination, required by many countries prior to travel to endemic areas, makes the disease much less likely, although it cannot be entirely excluded. also, vaccination histories may be inaccurate. 8 information regarding the time course of the patient's fever is also helpful in excluding certain diseases. incubation periods shorter than 21 days are typical for dengue, viral hemorrhagic fevers, typhoid fever and yellow fever. incubation times greater than 21 days are seen in tuberculosis, amebic liver abscess, rabies and leishmaniasis. while an incubation period of 21 days is generally seen in acute hiv infection, the onset of hiv seroconversion illness may vary considerably. 9 similarly, malaria can present with fever of variable onset, with more delayed presentations seen in patients taking ineffective prophylaxis. fever patterns, although often non-specific, may suggest certain etiologies. periodic fever spikes, long considered characteristic of malaria, may take some time to become established, limiting their utility in the diagnosis of early disease. continuous presence of fever is more common in rickettsial infections or typhoid fever. 6 the presence of specific physical examination findings in the returned traveler can provide additional diagnostic clues. the presence of a maculopapular rash, while seen in many relatively innocuous clinical entities such as drug eruption or viral exanthem, may also indicate the presence of more serious pathology, including dengue fever, leptospirosis or typhus. the initial diagnostic evaluation for these patients should focus on identifying routine, treatable illnesses as well as high risk occult infections. complete blood count with differential, chemistry panel, hepatic enzyme profile, blood cultures, urinalysis and urine culture should be part of the initial screening tests. while these basic laboratory tests are unlikely to yield a definitive diagnosis, they may be useful in detecting more subtle abnormalities such as eosinophilia in parasitic infection or underlying metabolic disorders. thick and thin blood smears for malaria should be obtained for travelers to tropical regions, and stool studies including stool culture, fecal leukocytes, ova and parasite assays should be ordered for patients complaining of diarrheal illness. serum samples should be drawn and set aside for subsequent testing if rickettsial or arboviral diseases are a consideration. 6 comparison of acute phase antibody titers to subsequent, convalescent phase assays can be useful in tracking the course of a disease. in the rare situation where patients are suspected of harboring highly pathogenic illnesses such as hemorrhagic fever viruses, plague or anthrax, all specimens from such patients should be regarded as high risk. laboratory personnel who may be handling these specimens should be notified before processing any sample so that tests may be carried out without risk of transmission of infection to laboratory personnel. refer definitive testing for suspected high risk pathogens such as hemorrhagic fever viruses, anthrax or tularemia to specialized reference laboratories equipped to handle these agents. particularly for high risk infectious agents, the subject of decontamination and isolation becomes increasingly important. while most diseases that may be present in returned travelers require only the implementation of routine universal precautions, some of the more virulent pathogens may require the clinician and treating health care team to adopt more strict infection control measures. in patients with suspected vhf infection, the use of impermeable gowns, n-95 masks, face shields or goggles, as well as negative-pressure isolation rooms with dedicated medical equipment for each patient (such as stethoscopes and blood pressure cuffs) is imperative, as is the restriction of access by nonessential staff and visitors. 10 these precautions also apply to other airborne illnesses that carry significant potential risk for staff or patientepatient transmission such as severe acute respiratory syndrome (sars) coronavirus. patients with active pulmonary tuberculosis infection may be managed with standard airborne precautions. treatment regimens for ill returned travelers should initially center on high risk tropical diseases such as malaria, as well as common clinical entities including pneumonia, upper respiratory tract infection, and urinary tract infection. failure to consider the diagnosis of malaria or to institute prompt therapy may be a potentially fatal error. empiric antibiotic coverage with fluoroquinolones should be instituted for travelers presenting with fever and diarrheal illness, particularly if bloody diarrhea is present. bloody diarrhea and the presence of fecal leukocytes are more associated with invasive infections such as salmonellosis or shigellosis. more specific anti-infective therapies for such illnesses as malaria, rickettsial infection or typhoid fever should be instituted only after initial investigations or clinical findings suggest these etiologies. while patients with mild illness may be managed as outpatients, suspected infection with highly virulent or rare tropical diseases should mandate inpatient hospitalization to allow further investigation and consultation with infectious disease specialists. patients presenting to an emergency department or other health care setting with febrile illness after recent travel may harbor a daunting array of illnesses, some of which may have even predated their travel, while others may be acquired in virtually any geographic region worldwide. knowing the most likely infectious diseases that a traveler might encounter on a given trip to africa, central or south america or the middle east is an important first step in determining the proper evaluation and management strategy for a given febrile returned traveler. screening questions that identify time of onset of fever, specific locations and durations of travel as well as high risk exposures, such as encounters with ill individuals or rural settings, can rapidly exclude certain diseases from the differential diagnosis. subsequent diagnostic efforts should attempt to exclude the most common tropical diseases such as malaria, dengue, and food-borne illnesses along with more routine viral and bacterial illnesses. patients who are suspected of carrying highly pathogenic organisms such as viral hemorrhagic fevers, anthrax, plague, and tularemia should be managed with special attention paid to issues of decontamination, isolation and biocontainment. a concise evaluation and management strategy for the ill traveler based on careful consideration of possible diagnoses and the prompt institution of appropriate therapies for tropical illnesses is essential, as the potential for morbidity and mortality in ill travelers who suffer missed or delayed diagnosis may be significant. fever in returned travelers: review of hospital admissions for a 3-year period fever as the presenting complaint of travellers returning from the tropics early risk assessment for viral haemorrhagic fever: experience at the hospital for tropical diseases geosentinel surveillance network. spectrum of disease and relation to place of exposure among ill returned travelers atlanta: us department of health and human services evaluation of fever in the returned traveler the burden of illness in international travelers a haemorrhagic fever from the cote d'ivoire incubation time of acute human immunodeficiency virus (hiv) infection and duration of acute hiv infection are independent prognostic factors of progression to aids update: management of patients with suspected viral hemorrhagic feverdunited states. mmwr morb mortal wkly rep key: cord-008678-zi3aunqz authors: piñana, josé luis; albert, eliseo; gómez, maría dolores; pérez, ariadna; hernández-boluda, juan carlos; montoro, juan; salavert, miguel; gonzález, eva maría; tormo, mar; giménez, estela; villalba, marta; balaguer-roselló, aitana; hernani, rafael; bueno, felipe; borrás, rafael; sanz, jaime; solano, carlos; navarro, david title: clinical significance of pneumocystis jirovecii dna detection by real-time pcr in hematological patient respiratory specimens date: 2020-01-10 journal: j infect doi: 10.1016/j.jinf.2020.01.001 sha: doc_id: 8678 cord_uid: zi3aunqz nan we read with interest a recent paper in this journal by luzatti and colleagues, 1 who explored the significance of the presence of herpes simplex virus (hsv) dna in lower respiratory tract (lrt) specimens for the diagnosis of hsv pneumonia in immunocompromised patients. the authors underlined the difficulty in gauging the clinical relevance of such a laboratory finding in the absence of histopathological data, as hsv shedding in the lrt may occur in the absence of disease. the interpretation of real-time pcr results for diagnosis of pneumocystis jirovecii (pj) pneumonia (pjp) faces an analogous challenge, since the presence of pj dna in lrt may reflect colonization (carriage) rather than infection. 2 there is limited information on the clinical value of pj real-time pcr in diagnosing pjp in patients with hematological diseases; [3] [4] [5] [6] this is exceedingly challenging as the sensitivity of direct examination procedures is suboptimal due to low fungal burdens. 3 here, we report on our experience on this matter. a total of 219 episodes of pneumonia occurring in 192 consecutive patients with hematological disorders in which pjp was considered in the differential etiological diagnosis were included. of these, 127 episodes developed in patients undergoing either allogeneic hematopoietic stem cell transplantation-allo-hsct-( n = 86) or autologous-hsct ( n = 19), and 92 in non-transplant patients (acute leukemia, n = 61; lymphoma, n = 16; chronic leukemia, n = 8; others, n = 2). the patients were attended at the hospital clínico universitario-hcu-( n = 100) or at the hospital universitario politécnico "la fe" -hlf-( n = 92) between june 2014 and august 2019. no patients in the cohort tested positive for hiv. this study was approved by the respective hospital ethics committee and informed consent was obtained from all patients. a single specimen per episode was available for diagnosis (bal fluids, n = 179; sputa, n = 22; ta, n = 17 and bronchial biopsy, n = 1). the realcycler pjir kit r (progenie molecular, spain) was used at hcu, and the pneumocystis jirovecii real time pcr detection (certest biotech; zaragoza, spain) was employed at hlf (see footnote in table 1 ). both assays target the large sub-unit of ribosomal (mtlsu) rna gene. preliminary experiments using 5 bal specimens indicated that both assays yield comparable pcr cycle thresholds (c t s) (median, 28.2, range, 26.4-32.3 vs. median 27.5; range, 26.3-33.1, respectively; p = 0.89). all specimens tested negative by direct examination for pj, whereas 27 were positive by real-time pcr (bal, n = 18; sputa, n = 7, and ta, n = 2); following stringent clinical, microbiological and imaging criteria ( table 1 ) , pjp was deemed to be the most probable diagnosis in 12 episodes occurring in unique patients. no histopathological confirmation of pjp was available for any patient. pcr c t values inversely correlate with fungal burden in the sample. 6 which is higher in patients with pjp than in colonized individuals. 2 here, overall, pj pcr c t s in specimens from patients with pjp tended to be lower than in pj carriers ( p = 0.39); when only bal fluid specimens were considered, the difference reached statistical significance (median, 29.0; range, 26.4-34.7 vs. median 34.6; range, 30.0-41.0; p = 0.04). this finding is likely related to use of more standardized procedures for bal fluid sampling. receiver operating characteristic (roc) curve analysis showed that a threshold c t value of 30.0 in bal specimens displayed a sensitivity of 85.7% (95% ci, 45.0-100%) and a specificity of 80% (95% ci, 40.8-100%) for pjp diagnosis. a number of studies have established different c t s cut-offs for that purpose, [6] [7] [8] [9] . in our view, however, the variability in the performance of different pcr assays and sampling conditions, heterogeneity of patient populations, and in particular the lack of a pj international standard material for pcr result normalization precludes defining a consensus universal threshold nowadays. the absence of anti-pj prophylaxis, treatment with corticosteroids and serum ldh levels ≥400 u/l have been shown to be associated with pjp. 3 here, patients not undergoing anti-pj prophylaxis were more likely to display a clinically significant pj pcr result ( table 1 ). in turn, roc curve analysis indicated that a cut-off ldh value ≥400 u/l had a sensitivity of 81.8% (ci 95%, 59.0-100%) and specificity of 67% (95% ci, 34.0-99.3%) for pjp diagnosis. in univariate regression logistic models, serum ldh values ≥400 u/l were associated with a clinically significant positive pcr pj result (or, 9.0; 95% ci, 1.2-63.8; p = 0.02). in contrast, corticosteroid use within the month before sampling was not different between patients with clinically significant pj detection and pj carriers ( table 1 ) . detection or recovery of other microbial agents (one or more) was documented in 17 of the 27 specimens testing positive by pj pcr ( table 2 ). in line with a previous report, 9 this microbiological finding was significantly less frequent ( p = 0.001) in specimens from patients with pjp than in colonized patients; in fact, microbial co-detection was inversely associated with pjp in univariate logistic regression models (or, 0.024; 95% ci, 0.002-0.26; p = 0.002). strengths of the current study are the following: (i) clinical categorization of pjp was based upon stringent criteria defined by a multidisciplinary team; (ii) only hematological patients were included; (iii) a comprehensive routine investigation of microbial causes of pneumonia other than pj was conducted; (iv) the experience of two centers was collected. in addition to its retrospective nature, our study also has some limitations: (i) we cannot completely rule out that some patients categorized as being pj carriers the probability of pneumocystis jirovecii (pj) pneumonia (pjp) for each patient was retrospectively evaluated by an expert committee including infectious diseases and microbiology specialists at both centers, on the basis of (i) documented pj presence in respiratory specimens by microscopy; (ii) compatibility of clinical signs and symptoms (at least 2 of the following: subtle onset of progressive dyspnea, pyrexia, nonproductive cough, hypoxaemia and chest pain), (iii) compatible (suggestive) radiological findings (chest radiograph and/or high-resolution computed tomographic scan detection of interstitial opacities and/or diffuse infiltration infiltrates); (iv) complete resolution of symptoms after a full course of anti-pjp treatment; (v) absence of alternative diagnosis. the efficacy of therapy was assessed on a daily basis. pjp was ruled out if real-time pcr for pj tested negative, or if clinical recovery occurred in the absence of pj-targeted antimicrobial therapy. pj colonization (carriage) was the most likely possibility when patients did not meet the above criteria and an alternate diagnosis was made. b frequencies were compared using the χ 2 test (fisher exact test) for categorical variables. two-sided exact p values were reported and p values ≤ 0.05 were considered statistically significant. the data were analyzed with the spss (version 20.0) statistical package. c respiratory tract specimens were obtained following conventional procedures. specimens were examined for presence of ascus or trophic forms of pj by microscopy following blue toluidine, calcofluor white or grocott's methenamine silver staining. cytospin preparations were prepared from bal specimens for direct examination. sputa and ta samples were mixed v/v with sputasol (oxoid, uk) and vortexed for 5 min. all samples were centrifuged at 30 0 0 g for 10 min, and the pellets were resuspended 1/10 in 0.9% nacl for further processing. for real-time pcr, dna was extracted from 200 μl of specimens using the qiaamp dna blood mini kit (qiagen, hilden, germany) on either qia symphony or ez-1 platforms (qiagen), following the manufacturer's instructions. at hcu, a commercially-available real-time pcr assay previously evaluated by others, the realcycler pjir kit r (progenie molecular, spain), which targets the mitochondrial large sub-unit of ribosomal (mtlsu) rna gene, was used according to the manufacturer's instructions ( http://www.progenie-molecular.com/pjir-u-in.pdf ). at hlf, the commercially-available pneumocystis jirovecii real time pcr detection. (certest biotech; zaragoza, spain), which also targets the large sub-unit of ribosomal (mtlsu) rna gene, was employed following the manufacturer instructions ( https://www.certest. es/wpontent/uploads/2019/02/viasure _ real _ time _ pcr _ pneumocystis _ jirovecii _ sp1.pdf ). at both centers pcr were performed in the applied biosystems 7500 fast real-time pcr platform (applied biosystems, ca, usa). pcr results were reported as positive or negative. for positive samples, threshold cycle (c t ) values were also recorded. no standard curve was generated with a positive control for quantitative estimations. d antimicrobial prophylaxis for pjp was performed with trimethoprim-sulfamethoxazole (tmp/smx), one double-strength tablet (160 mg tmp/800 mg smx) given 2 (in allogeneic hsct patients) or 3 times a week with oral folic acid (15,16). patients with suspicion of pjp according to the attending physician were treated with tmp/smx 15-20 mg/kg (tmp) 75-100 mg/kg (smx) per day for 2-3 weeks. e in all these cases, death was attributable to pjp. did in fact have pjp, as most of these patients received full courses of tmp/smx in combination with antimicrobials targeting other microbial agents. the lack of standardized criteria for pjp diagnosis makes clinical misclassification of patients a potential drawback in studies such as ours, particularly when no positive microscopy or histopathology findings are available; (ii) although we evaluated over 200 patients, only 12 presumptively had pjp; (iii) two different commercially-available pcr assays were used across centers. nevertheless, we found them to yield rather comparable c t s. in summary, we found that a positive pj pcr result in respiratory specimens from transplant and non-transplant hematological patients with pneumonia frequently reflects colonization rather bal, bronchoalveolar lavage; pjp, pneumocysis jirovecii pneumonia; ta, tracheal aspirate. a as per our routine protocol, all specimens were examined by gram and acid-fast bacilli stain. samples were also examined for presence of respiratory viruses (rvs) using either the luminex xtag rvp fast assay (luminex molecular diagnostics, austin, tx,usa) at hcu, or the clart® pneumovir assay (genomica, coslada, spain) at both centers, as previously reported. 10 semiquantitative (sputa) and quantitative (bal and ta) cultures for bacteria were performed on conventional media: bacterial loads > 10 4 cfu/ml or > 10 5 cfu/ml were deemed to be clinically relevant on bal fluids and ta samples, respectively. specimens were cultured on bcye-alpha agar, bd (becton dickinson) mgit® ( mycobacteria growth indicator tube)/lowenstein-jensen agar slants and sabouraud agar for recovery of legionella pneumophila, mycobacterium spp., and other fungal organisms, respectively. the platelia tm aspergillus ag kit (bio-rad, hercules, ca, usa) was used for quantitation of aspergillus spp. galactomannan in bal fluid and serum specimens. all bal fluid specimens underwent cytomegalovirus (cmv) pcr testing using the realtime cmv pcr assay (abbott molecular) at hcu or the cmv r-gene® assay (biomerieux) at hlf, as previously reported. we have no conflict of interest to declare. herpes simplex virus (hsv) pneumonia in the non-ventilated immunocompromised host: burden and predictors colonization by pneumocystis jirovecii and its role in disease ecil guidelines for the diagnosis of pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients pcr diagnosis of pneumocystis pneumonia: a bivariate meta-analysis evaluation of pcr in bronchoalveolar lavage fluid for diagnosis of pneumocystis jirovecii pneumonia: a bivariate meta-analysis and systematic review molecular diagnosis of pneumocystis pneumonia in immunocompromised patients realtime pcr assay-based strategy for differentiation between active pneumocystis jirovecii pneumonia and colonization in immunocompromised patients detection of pneumocystis jirovecii by quantitative pcr to differentiate colonization and pneumonia in immunocompromised hiv-positive and hiv-negative patients diagnosis of pneumocystis jirovecii pneumonia in immunocompromised patients by real-time pcr: a 4-year prospective study pulmonary cytomegalovirus (cmv) dna shedding in allogeneic hematopoietic stem cell transplant recipients: implications for the diagnosis of cmv pneumonia no public or private funds were used for the current study. key: cord-256808-lxlerb13 authors: lim, w.s; anderson, s.r; read, r.c title: hospital management of adults with severe acute respiratory syndrome (sars) if sars re-emerges—updated 10 february 2004 date: 2004-06-02 journal: j infect doi: 10.1016/j.jinf.2004.04.001 sha: doc_id: 256808 cord_uid: lxlerb13 severe acute respiratory syndrome (sars) is a potentially severe and highly infectious disease to which healthcare workers involved in the management of cases are particularly vulnerable. these guidelines briefly summarise optimal and safe practice for clinicians involved in the emergency care of patients with probable or confirmed sars. during 2003 severe acute respiratory syndrome caused by a novel coronavirus (sars-cov) emerged as an infectious disease with a significant inhospital mortality and posed a considerable occupational risk for healthcare workers. 1 -5 the initial sars outbreak ended in july 2003 when the world health organisation (who) announced that all known person-to-person transmission of sars-cov had ceased. at the time of preparation of these guidelines, there have been a further two laboratory-acquired cases of sars and further community-acquired cases. these cases emphasise the potential for sars to re-emerge and spread unpredictably. these guidelines document the hospital management of adults with probable or confirmed sars. they are meant only as a brief summary for clinicians. these guidelines do not cover the management in the community of a person under investigation (pui) (see case definitions). guidelines for the management of paediatrics cases have not yet been developed. as more information about sars becomes available, guidance will be appropriately updated. please consult the latest guidance available on the websites of the british thoracic society (http:// www.brit-thoracic.org.uk/) and health protection agency (http://www.hpa.org.uk/infections/ topics_az/sars/menu.htm). the following case definitions (see tables 1 -4 ) are designed for use during an outbreak of sars, once the re-emergence of sars has been verified by the world health organisation (who). it is anticipated that patients with sars will have a respiratory illness severe enough to warrant hospital admission. management of such cases is covered in sections 2 -4. a person with a mild respiratory illness and a potential epidemiological link to sars should be defined as a pui (see table 4 ) and should be assessed in primary care and reviewed within 72 h. a pui does not require routine hospitalisation nor do they require a chest radiograph (cxr) or laboratory investigation for sars cov as part of their assessment. a pui should only be hospitalised if his or her condition deteriorates. the management of such patients is covered in section 5. if these patients are subsequently found to have radiographic evidence consistent with sars, they should be reclassified as a 'probable sars' case unless an alternative diagnosis is made. a pui should be reported to local health protection units but does not need to be reported to cdsc colindale. please discuss the classification of sars patients with the health protection agency's communicable disease surveillance centre (cdsc) duty doctor (tel.: 0208-200-6868) and complete a standard sars report form and fax to your local consultant in communicable disease control (ccdc) and cdsc (details at: http://www.hpa.org.uk/infections/ topics_az/sars/forms.htm). † negative antibody test on acute serum followed by positive antibody test on convalescent phase serum tested in parallel or † four-fold or greater rise in antibody titre between the acute and convalescent phase sera tested in parallel (c) virus isolation † isolation in cell culture of sars-cov from any specimen; plus pcr confirmation using a validated method patients are likely to present initially with a clinical picture of pneumonia which may be consistent with sars. therefore, other causes of pneumonia should be considered. confirmation that a patient has sars may occur following further investigation. detailed guidance regarding the infection control issues during hospital management of a patient, or patients, presenting with sars can be found at the hpa website: http://www.hpa.org.uk/infections/ topics_az/sars/hops_infect_cont.htm. briefly, the key recommendations are: (a) give the patient a surgical mask to wear continuously (unless requiring face mask for oxygen confirm the travel history and/or history of contact with a patient with sars. explore other possible causes of pneumonia. assess pneumonia disease severity according to the bts guidelines on the management of community acquired pneumonia (cap) in adults (http:// www.brit-thoracic.org.uk/guide/guidelines.html). 10 in addition, determine whether the patient has any medical history of illness associated with a more severe outcome of sars, i.e. diabetes and cardiopulmonary disease. 1 obtain investigations as listed below (observe high risk infection control measures for all samples). for full details, please see the hpa website at http://www.hpa.org.uk/infections/topics_az/ sars/micro.htm. only send specimens once cdsc have been informed of a case via their standard report form. please observe strict infection control procedures. all specimens should be double bagged and labelled as a biohazard. do not obtain a nasopharyngeal aspirate as this is likely to generate aerosols. admit the patient to a designated isolation unit (see section 2.1). manage as for severe cap according to bts guidelines. 10 administer fluids and oxygen as required. commence intravenous co-amoxiclav 1.2 g tds or cefuroxime 1.5 g tds plus erythromycin 500 mg qds or clarithromycin 500 mg bd. please refer to the bts guidelines for alternative recommended regimens. oxygen supplementation should be administered according to standard/local guidelines. however, in order to reduce the risk of aerosol generation and hence spread of infection, high flow oxygen is not recommended, i.e. avoid oxygen flow rates of . 6 l/min. it should be possible to provide 30 -40% oxygen supplementation using a standard low flow oxygen system and an air-entrainer together with a ventimask. procedures and practices that promote the generation of aerosols (table 5) should be avoided wherever possible to reduce the risk of infection to healthcare workers. 11, 12 if such procedures need to be performed, e.g. tracheal intubation, it is advised that experienced operators only should undertake these procedures. these should, where possible, be planned and controlled. these procedures should ideally be undertaken in a negative pressure room. only a minimum number of staff should be present and all must wear gowns, gloves, goggles/visors and respirators as described under infection control issues (see section 2.1). entry and exit from the room should be minimised during the procedure. the use of powered air purifying respirators (paprs) during aerosol generating procedures is not recommended. this is because there are concerns over the removal, disposal, cleaning and decontamination of this equipment, which may increase the potential risk of self-contamination and at this time there is inadequate evidence to determine whether paprs further reduce the transmission of sars. if paprs are used, staff must be properly trained in their safe use. in studies from canada and singapore, approximately 20% of patients with suspected or probable sars, according to the prevailing who case definition from march to june 2003, required icu admission. 3, 4 of these patients, 66 -76% required mechanical ventilation. average length of icu stay was 10 days. preplanning and early consultation with local critical care providers is recommended. 13 patients who are likely to require intubation, should be identified early and the procedure should be undertaken electively. in order to avoid the use of cpap or niv, early intubation and invasive positive pressure ventilation (ippv) may be required in some patients with impending respiratory failure. the following issues need to be carefully considered: further guidance for the management of critically ill patients is being developed. the use of high-dose steroids has been anecdotally reported to contribute to decrease in fever and need for oxygen supplementation. 14 a study from guangzhou, china has suggested that the early administration of high-dose steroids together with cpap ventilation is associated with a lower mortality. 15 however, these findings are not based on adequately controlled data and there remain concerns regarding the use of high-dose steroids. the use of cpap is certainly no longer recommended (see section 2.5.2). in a retrospective analysis of hong kong patients who had received ribavirin in combination with different steroid regimens, patients who received initial high dose pulsed methylprednisolone intravenously had less oxygen requirement, better radiological improvement and less likelihood to require rescue pulse steroid use than patients who received non-pulse steroid therapy. however, the overall mortality rate, and requirement for mechanical ventilation or admission to the intensive care unit was the same for both regimens. 16 recommendation the current recommendation is to consider moderate doses of steroid (prednisolone 30 -40 mg/day or iv equivalent) in severely ill patients with sars with increasing oxygen requirements who have a pao 2 , 10 kpa or o 2 sats , 90% on air. currently there is no convincing evidence that ribavirin alters clinical outcome. in laboratory studies, no in vitro activity against sars-associated coronavirus (sars-cov) has been consistently demonstrated either. in addition, use of ribavirin is associated with significant toxicity including haemolysis (in , 76%) and decrease in haemoglobin of 2 g/dl or more (in , 49%). 1 the routine use of ribavirin in patients with sars is not recommended. the antiviral activity of interferons against sars coronavirus has been measured in vitro and interferon beta appears to be particularly active. 17 the world health organisation is currently coordinating plans for clinical trials of interferons in the event of re-emergence of the disease. recommendation none can be given at this time. generate a list of all close contacts. this should be initiated by the attending physician at the time of first contact with the patient. the local hospital infection control and occupational health teams may need to be involved if any healthcare workers are identified as close contacts. record the date on which all close contacts last had contact with the case and inform them about sars. inform the local ccdc/health protection team of any contacts and their details to ensure follow-up. these contacts may continue with everyday activities, as long as they remain well. the local health protection team will contact them on a regular basis to review their health. these contacts should be isolated at home. please refer to the health protection agency's guidelines on voluntary home isolation at http://www.hpa. org.uk/infections/topics_az/sars/homeiso.htm. if a contact becomes unwell within 10 days of their contact with a probable or confirmed sars case they should phone a doctor urgently. for more information please refer to: http://www.hpa.org. uk/infections/topics_az/sars/guidelines.htm. guidelines for the safe discharge of patients recovering from sars have been published by who. please refer to the who website at http:// www.who.int/csr/sars/discharge/en/. briefly, the following criteria should be considered before discharge: (a) afebrile for 48 h (b) resolving cough (c) laboratory tests, if previously abnormal, returning to normal (d) chest x-ray improved. patients should monitor and record their temperature twice daily. if they have an elevated temperature of 38 8c or above on two consecutive occasions they should inform (by telephone) the healthcare facility from which they were discharged. patients should remain at home for 7 days after discharge, keeping contact with others at a minimum. this is to reduce the risk of transmission until more is known regarding the potential for continued carriage in convalescent cases. additional home confinement may need to be considered, particularly in patients who are immunosuppressed. inform the local health protection team/ccdc regarding the hospital discharge of patients to ensure follow-up in the community. a standard follow-up form should be completed and faxed to the local ccdc and cdsc on day 2, day 10, and/or once the patient is asymptomatic. forms are available from http://www.hpa.org.uk/infections/ topics_az/sars/forms.htm. follow-up post-discharge will be the responsibility of the local infection control team. convalescent serology should be obtained at 21 days after the date of disease onset. puis who have symptoms and signs consistent with a lower respiratory tract infection (lrti) but have a normal cxr do not fulfil the sars case definition (see table 1 ). patients should be discharged and followed up in primary care unless their symptoms or social circumstances warrant continued hospital care. up to 30% of patients with probable sars may initially present with normal chest radiographs. therefore, puis who need ongoing hospitalisation require careful medical review in the first 48 h following admission. infection control measures as for patients with probable sars should apply (see section 2.1) until it is clinically clear that the pui does not have probable sars. such patients should be treated as for non-pneumonic lrti. if the patient improves with treatment in the first 48 h following admission, the likelihood of sars is small. infection control measures may be relaxed and the patient discharged if this is clinically appropriate. if the patient does not improve with initial treatment (either no change or deteriorates), a repeat cxr should be obtained. an abnormal cxr with changes consistent with sars would require the patient to be re-classified as having probable sars and be managed accordingly (see section 2). if the repeat cxr remains normal, the patient remains a pui. further repeat cxrs may be required at 1 -2 days intervals depending on clinical circumstances. a pui should be reported to the local health protection unit but does not need to be reported to cdsc colindale. these guidelines were produced as a joint initiative between the british thoracic society, the british infection society and the health protection agency. membership of the guideline development group: lead clinical features and short-term outcomes of 144 patients with sars in the greater toronto area coronavirus as a possible cause of severe acute respiratory syndrome acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome critically ill patients with severe acute respiratory syndrome koch's postulates fulfilled for sars virus effectiveness of precautions against droplets and contact in prevention of nosocomial transmission of severe acute respiratory syndrome (sars) clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study identification of severe acute respiratory syndrome in canada a major outbreak of severe acute respiratory syndrome in hong kong bts guidelines for the management of community acquired pneumonia in adults severe acute respiratory syndrome (sars): infection control sars: experience at prince of wales hospital, hong kong anaesthesia and sars development of a standard treatment protocol for severe acute respiratory syndrome description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china high dose pulse versus nonpulse corticosteroid regimens in severe acute respiratory syndrome treatment of sars with human interferons useful contact details: scottish centre for infection and environmental health (scieh) telephone: 0141-300-1100 e-mail: jim.mcmenamin@scieh.csa.scot.nhs.uk key: cord-273751-61eeykj1 authors: yang, zhenwei; liu, jialong; zhou, yunjiao; zhao, xixian; zhao, qiu; liu, jing title: the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis date: 2020-04-10 journal: j infect doi: 10.1016/j.jinf.2020.03.062 sha: doc_id: 273751 cord_uid: 61eeykj1 objectives: an outbreak of novel coronavirus in 2019 threatens the health of people, and there is no proven pharmacological treatment. although corticosteroids were widely used during outbreaks of severe acute respiratory syndrome and middle east respiratory syndrome, their efficacy remainedhighly controversial. we aimed to further evaluate the influence of corticosteroids on patients with coronavirus infection. methods: we conducted a comprehensive search of literature published in pubmed, embase, cochrane library, and china national knowledge infrastructure (cnki) from january 1, 2002 to march 15, 2020. all statistical analyses in this study were performed on stata14.0. results: a total of 5270 patients from 15 studies were included in this meta-analysis. the result indicated that critical patients were more likely to require corticosteroids therapy (risk ratio [rr] = 1.56, 95% confidence interval [ci] = 1.28-1.90, p<0.001). however, corticosteroid treatment was associated with higher mortality (rr = 2.11, 95%ci = 1.13-3.94, p = 0.019), longer length of stay (weighted mean difference [wmd] = 6.31, 95%ci = 5.26–7.37, p<0.001), a higher rate of bacterial infection (rr = 2.08, 95%ci = 1.54–2.81, p<0.001), and hypokalemia (rr = 2.21, 95%ci = 1.07–4.55, p = 0.032) but not hyperglycemia (rr = 1.37, 95%ci=0.68–2.76, p = 0.376) or hypocalcemia (rr = 1.35, 95%ci = 0.77–2.37, p = 0.302). conclusions: patients with severe conditions are more likely to require corticosteroids. corticosteroid use is associated with increased mortality in patients with coronavirus pneumonia. in december 2019, the pneumonia caused by a new coronavirus spread in wuhan, china. unbiased sequencing of samples from patients with pneumonia reveals a previously unknown type of beta-coronavirus which is similar to the severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). 1 the causative agent was named severe acute respiratory syndrome coronavirus 2 (sars-cov-2) by coronavirus study group, and the disease it caused was named coronavirus disease 2019 (covid-19) by the world health organization (who). 2 , 3 sars-cov-2 is a new type of highly diverse enveloped positive single stranded rna virus, which can cause a range of symptoms including self-reported fever, fatigue, dry cough, myalgia, and diffi* corresponding author. e-mail address: liujing_gi@whu.edu.cn (j. liu). 1 the authors contributed equally to this work. culty breathing. 4 there is evidence that the transmission pattern of sars-cov-2 is human-to-human which is spread by respiratory droplets caused by coughing or sneezing. 5 , 6 as of march 19, there are now more than 20 0,0 0 0 covid-19 cases and more than 8,0 0 0 deaths in the world. 7 nevertheless, there is no vaccine or antiviral treatment for human coronavirus. therefore, it is crucial to determine the drug treatment plan as soon as possible to deal with the outbreak of covid-19. 8 corticosteroids have a good inhibitory effect on inflammatory factors and are often used as an auxiliary treatment for viral pneumonia. the main anti-inflammatory effect of glucocorticoids is to inhibit a large number of pro-inflammatory genes that encode cytokines, chemokines, cell adhesion molecules, inflammatory enzymes, and receptors to address the inflammatory process and restore homeostasis. 9 however, the results of clinical studies on the role of corticosteroids remain controversial. a retrospective study showed that the vast majority of sars patients received satisfactory results from the use of corticosteroids. 10 but in a retrospective observational study of mers patients, the result showed that patients who were given corticosteroids were more likely to require the inclusion criteria in this meta-analysis were as follows: (1) subjects in each study were patients with coronavirus infection; (2) the patients were divided into the experimental group using corticosteroids and the control group not using corticosteroids; (3) the outcomes included the use of corticosteroids in critical and noncritical patients, mortality, length of stay (los) and adverse reactions to corticosteroids. exclusion criteria: (1) the same patients were enrolled in different articles; (2) commentaries, editorials, case reports, letters and family-based studies; and (3) patients in studies were under 18 years old. the two researchers (zhenwei yang and jialong liu) who performed the inclusion and exclusion of the literature also independently extracted data from the included studies. differences were resolved with a third investigator (xixian zhao) or by consensus. we extracted the following variables: the authors, the publication year, the study design, viral type, population, treatment details (including corticosteroid use, types and doses of corticosteroids, and other treatments), and outcome measures such as the use of corticosteroids in critical and non-critical patients, mortality, los and adverse reactions to corticosteroids (including bacterial infection, hyperglycemia, hypocalcemia and hypokalemia). we used the newcastle-ottawa scale (nos), 12 which includes patient selection, study comparability and outcome assessment three components, to evaluate the quality of the original study. the work was done by two authors (zhenwei yang and jialong liu) and agreed upon through discussion. all statistical analyses in this study were performed on stata14.0 (stata, college station, tx, usa). for dichotomized data, we calculated the risk ratio (rr) and the 95% confidence interval (ci), while for continuous data, we calculated the weighted mean difference (wmd) and the 95% ci. heterogeneity among the studies was assessed by the chi squared and i 2 tests. a random-effects model was used when either p < 0.10 or i 2 > 50% defined significant heterogeneity across the articles. otherwise, the fixed-effects model was used. we carried out a sensitivity analysis on the stability of the combined results. in addition, we also performed a subgroup analysis by virus type to explore the source of heterogeneity. publication bias was assessed by funnel plots. as shown in fig. 1 , the total number of records initially determined based on the search strategy was 1685. after removing 412 duplicates, we deleted another 1203 articles by reading the title and abstract of the article. we eliminated 55 articles by reading the full-text articles of the remaining 70 studies, four of which enrolled the same patients, two of which were non-adult patients, and 49 of which did not have a control group not using corticosteroids. finally, there were 15 articles included in our metaanalysis. 5 , 10 , 11 , 13-24 study characteristics 5270 patients from 15 articles were included in our systematic review and meta-analysis. due to one article did not give the number of people treated with corticosteroids, 14 patients with sars-cov infection, two included patients with mers-cov infection, and the remaining two included patients with sars-cov-2 infection. there were 7 articles describing the use of corticosteroids in critical and non-critical patients, 5 , 13 , 15 , 16 , 19 , 22 , 23 9 articles recorded the mortality, 10 , 11 , 14-18 , 20 , 22 three studies reported the los, 11 , 18 , 22 2 articles described the adverse reactions to corticosteroids. 21 , 24 in addition, all studies had nos scores ≥6. the details of each included study are presented in table 1 . the results showed that patients with severe conditions were more likely to require corticosteroids therapy (rr = 1.56, 95% ci = 1.28-1.90, p < 0.001; fig. 2 ). there was significant heterogeneity among the studies ( i 2 = 90.2%, p < 0.001), the random effects model was adopted. similar results were also observed in the subgroup analysis of patients with sars-cov-2 infection (rr = 2.36, 95%ci = 1.31-4.28, p = 0.004, i 2 = 29.1%, p = 0.235) and patients with sars-cov infection (rr = 1.46, 95%ci = 1.18-1.80, p < 0.001, i 2 = 92.7%, p < 0.001). sensitivity analysis showed that the result was stable. the pooled rr from the nine studies revealed that the mortality was higher in patients who received corticosteroids therapy (rr = 2.11, 95%ci = 1.13-3.94, p = 0.019, i 2 = 80.9%, p < 0.001; fig. 3 ). when we performed subgroup analysis, we found that the mortality of neither sars-cov (rr = 2.56, 95%ci = 0.99-6.63, p = 0.053, i 2 = 77.4%, p < 0.001) nor mers-cov (rr = 2.06, 95%ci = 0.66-6.44, p = 0.213, i 2 = 89.4%, p = 0.002) was correlated with corticosteroids therapy. sensitivity analysis showed that the result was not stable. when we excluded a study, 14 the result was different from the previous conclusion. los was longer in the corticosteroid group (wmd = 6.31, 95%ci = 5.26-7.37, p < 0.001, i 2 = 1.8%, p = 0.361; fig. 4 ), and the same result was found in the subgroup analysis of patients with sars-cov infection (wmd = 6.34, 95%ci = 5.24-7.44, p < 0.001, i 2 = 50.3%, p = 0.156). as shown in table 2 we examined the publication bias of the included literature on the use of corticosteroids in critical and non-critical patients and mortality. funnel plots showed that there was no publication bias on the use of corticosteroids in critical and non-critical patients ( fig. 5 a) , while there might be publication bias on mortality ( fig. 5 b) . as sars-cov-2 is an emerging virus, there is no effective antiviral treatment at present. covid-19 patients were mainly treated with symptomatic therapy. in clinic, corticosteroids are widely used in symptomatic treatment of severe pneumonia. however, there has been considerable controversy as to whether covid-19 patients should be treated with corticosteroids. russell and colleagues recommend that corticosteroids should not be used in sars-cov-2-induced lung injury or shock outside of a clinical trial. 25 but a team of front-line physicians from china had a different perspective, they recommended short courses of corticosteroids at low-to-moderate dose, used prudently, for critical patients with covid-19 pneumonia. 26 so it is very important to provide evidence for corticosteroid treatment of in patients with coronavirus. in this systematic review and meta-analysis, the result indicated that patients with severe conditions were more likely to require corticosteroids therapy. the similar results were also observed in the subgroup of patients with sars-cov-2 infection and patients with sars-cov infection. a study showed that the concentrations of cytokines (such as interleukin 7 [il7], il8, il9, il10 and so on) in serum in the covid-19 patients were higher than in healthy adults. in addition, cytokines (such as il2, il7, il10 and so on) concentrations in intensive care unit (icu) patients were higher than non-icu patients. these revealed that patients with covid-19 were usually accompanied by increased immune factors and inflammatory responses, and the concentrations of immune factors were associated with the severity of the disease. 5 further autopsy revealed bilateral diffuse alveolar injury with fibrous mucinous exudate and interstitial mononuclear inflammatory infiltration dominated by lymphocytes, which were very similar to sars-cov and mers-cov infections. 27 as we all known, corticosteroids do not directly inhibit virus replication, their main role is anti-inflammatory and suppress immune response. 28 in the early stage of inflammation, glucocorticoids reduce capillary dilation, inflammatory cell exudation, leukocyte infiltration, and phagocytosis. in the late stage, glucocorticoids can inhibit the excessive proliferation of capillaries and fibroblasts. furthermore, by binding to their receptors, glucocorticoids inhibit nuclear transcription factor-κb (nf-κb) signaling and further inhibit the transcription and translation of inflammatory factors. 9 these explained why corticosteroids therapy was more needed in severely ill patients with coronavirus infection. our analysis demonstrated that patients treated with corticosteroids had a higher mortality rate, and longer los. there might be multiple mechanisms that contributed to these outcomes. there is a study shows that glucocorticoids inhibit the production of il-2 and interferon-γ (ifn-γ ) in t lymphocytes, shift t cell responses from the th1 to the th2 type, induce programmed cell death in a variety of different immunologically relevant cells, including immature t and b cell precursors and mature t cells. 29 another study find that preexisting cd4 + t cells are associated with lower viral shedding and less severe disease. 30 there is evidence that the use of corticosteroids may lead to prolonged removal of viral rna from the airways, 11 blood, 31 and feces of patients, 32 resulting in longer hospital stays, and ultimately increasing the risk of mortality. in addition, our analysis found that patients receiving corticosteroid therapy were more likely to develop bacterial infection due to immunosuppression. this could make the disease worse and lead to death. we also performed subgroup analysis, the result indicated that the mortality of neither sars-cov nor mers-cov was associated with corticosteroids therapy. sensitivity analysis showed that the use of corticosteroids was not associated with mortality when we excluded a study. 14 therefore, we need to treat this result with caution. our analysis found that patients receiving corticosteroids therapy might cause some serious adverse reactions such as bacterial infection and hypokalemia. however, only two studies in our analysis reported data on adverse reactions to corticosteroids, bias might have occurred due to the limited number of patients. there were several meta-analyses explored the role of corticosteroids in viral pneumonia, most of which shown adverse consequences. in a meta-analysis of corticosteroid use in patients with sars, a total of 29 studies on corticosteroids were included, of which 25 were inconclusive, and only 4 provided conclusive data on the harms of corticosteroids. 33 in a meta-analysis of corticosteroid use in patients with influenza pneumonia, the results showed that compared with placebo, corticosteroids were associated with higher mortality, longer icu los, and a higher rate of secondary infection but not mechanical ventilation days. 34 in addition, a meta-analysis included ten studies with 1137 recovered sars patients showed that patients who received higher cumulative doses and longer treatment durations of steroids were more likely to develop osteonecrosis. 35 these meta-analyses indicated that patients with coronavirus pneumonia could not benefit from corticosteroid treatment. however, there are some limitations in this meta-analysis. first, most of the included studies are retrospective cohort studies, historical control studies, etc., with a low level of evidence and a lack of randomized controlled trials with optimized design. second, there is no uniform standard for the time and dosage of hormones used in various studies. third, the effects of corticosteroids may be influenced by other therapeutic options, such as antiviral drugs. finally, due to the rapid evolution of the sars-cov-2 situation, some studies have not been published, while other developments are not intended to be reported for reasons of confidentiality, which will lead to publication bias. patients with severe conditions were more likely to require corticosteroids. corticosteroids could lead to higher mortality, longer los, a higher rate of bacterial infection and hypokalemia. therefore, corticosteroid should be used with caution in the treatment of covid-19 patients: corticosteroids are not recommended for pa-tients with mild conditions, and moderate corticosteroids can be used in patients with severe conditions to suppress the immune response and reduce symptoms. nevertheless, more multicenter clinical trials are needed to further verify this conclusion. all the authors designed the study. zhenwei yang, jialong liu and yunjiao zhou designed the literature search and searched the articles. zhenwei yang, jialong liu and xixian zhao contributed to the data extraction process. all the authors analysed the data. zhenwei yang wrote the first draft of article. all the authors revised the article and approved the final version. no authors have competing interests in this research. the work was supported by a research grant from the national natural science foundation of china (jing liu, grant no. 81472735 ) and the wuhan university (jing liu, grant no. 2042019kf0206 ). a novel coronavirus from patients with pneumonia in china severe acute respiratory syndrome-related coronavirus: the species and its viruses -a statement of the coronavirus study group who. who director-general's remarks at the media briefing on 2019-ncov on 11 coronavirus disease 2019 (covid-19): what we know clinical features of patients infected with 2019 novel coronavirus in wuhan a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster drug treatment options for the 2019-new coronavirus (2019-ncov) one hormone, two actions: anti-and pro-inflammatory effects of glucocorticoids pubmed central pmcid corticosteroid treatment of severe acute respiratory syndrome in hong kong corticosteroid therapy for critically ill patients with middle east respiratory syndrome critical evaluation of the newcastle-ottawa scale for the assessment of the quality of nonrandomized studies in meta-analyses clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china clinical predictors of mortality of middle east respiratory syndrome coronavirus (mers-cov) infection: a cohort study yjinf [m5g dose of glucocorticosteroids in the treatment of severe acute respiratory syndrome 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 effects and adverse drug reactions of mtrisone in the treatment of patients with severe acute respiratory syndrome a clinical investigation in 83 cases with severe acute respiratory syndrome clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury on the use of corticosteroids for 2019-n-cov pneumonia pathological findings of covid-19 associated with acute respiratory distress syndrome. the lancet respiratory medicine role of glucocorticoids on inflammatory response in nonimmunosuppressed patients with pneumonia: a pilot study glucocorticoid-mediated control of the activation and clonal deletion of peripheral t cells in vivo preexisting influenza-specific cd4 + t cells correlate with disease protection against influenza challenge in humans effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients persistence and clearance of viral rna in 2019 novel coronavirus disease rehabilitation patients sars: systematic review of treatment effects the effect of corticosteroids on mortality of patients with influenza pneumonia: a systematic review and metaanalysis steroid therapy and the risk of osteonecrosis in sars patients: a dose-response meta-analysis none. key: cord-280188-gir0y1m1 authors: wang, yanqun; li, yamin; liu, jun; zhao, yanjie; xie, zhengde; shen, jun; tan, wenjie title: genetic characterization of human bocavirus among children with severe acute respiratory infection in china date: 2016-06-13 journal: j infect doi: 10.1016/j.jinf.2016.05.014 sha: doc_id: 280188 cord_uid: gir0y1m1 objectives: to investigate the genetic character of human bocavirus (hbov) among children with severe acute respiratory infection (sari) in china. methods: we screened 993 respiratory samples for hbov by pcr among hospitalized children with sari between september 2007 and march 2014. four of hbov1 samples were selected for complete genomes analysis by next-generation sequencing. results: the results show that 200 (20.1%) out of 993 samples were hbov-positive, most of these hbov belong to hbov1 subtype (n = 197), hbov2 (n = 1) and hbov3 (n = 2) were also detected. fifty (5.04%) of 993 sari patient were detected as hbov-positive only. four hbov1 genomes in this study were conserved and showed no significant difference among the nucleotide diversity from different regions. analyses of evolutionary rates showed that ns1 exhibited the highest degree of conservation while the vp1 gene exhibited the fastest rate of evolution at 4.20 × 10(−4) substitutions/site/year. the nucleotide deletions and substitutions occurred in np1 and vp1 represented novel molecular signatures enabling subtype differentiation between hbovs. conclusions: we described some new characteristics in the epidemiology of hbov among children with sari, these data will significantly expand the current knowledge of hbov epidemic and genomic characterization among children with sari. human bocavirus1e4 (hbov) represents a novel pathogen associated with gastrointestinal and respiratory tract illnesses. 1e3 according to the latest ictv classification of parvovirus, hbov1 and hbov3 belonged to primate bocaparvovirus 1 species; hbov2 and hbov4 are part of p. bocaparvovirus 2 species. 4 the genome of hbov is w5.3 kb in length, and is divided into four partially overlapping genes, namely ns1, np1, vp1, and vp2, vp2 is totally included within vp1. 5, 6 while the prototype hbov (hbov1) were first discovered from nasopharyngeal samples, 7 three additional viruses (hbov2e4) have since been discovered in stool specimens, classified based upon their close phylogenetic relationship with hbov1. 8, 9 hbov1 is the most commonly reported genotype and occurring primarily in pediatric patients with respiratory tract infection, but also gastrointestinal symptoms are often observed. 10, 11 in contrast, hbov2 are preferentially detected in stool samples and appear to be more strongly associated with enteric disease, hbov3 and hbov4 are occasionally detected in faeces and too rare for any associations. 2 since the discovery of hbovs, numerous epidemiological surveillance efforts examining hbov prevalence in children have been performed across multiple regions, comprising thailand, united states, france, jordan and brazil. 10e14 severe acute respiratory infection (sari) is among the leading causes of morbidity and mortality among children globally. 15 hbov infection in children has been reported associated with respiratory tract infection, a few cases reported that hbov as the cause of sari among children. 16e18 however, prolonged shed periods of hbov and high coinfection detection resulted in the on-going debate of the hbov as the agent of sari 19 ; in addition, the comprehensive research of hbov genome among children with sari were limited, especially in china. to better understand the molecular epidemiology and characterization of hbov genome in children with sari, we investigated the prevalence of hbov among 993 inpatient children with sari in china. analyses of genome characterization were also performed. from sep 2007 to mar 2014, a total of 993 nasopharyngeal aspirates (npas) or induced sputum (is) were randomly collected from hospitalized children with sari in beijing (n z 259), shanghai (n z 441) and zhejiang (n z 293) area. the case of sari was defined according to the world health organization case definition for all hospitalized children in whom the onset of illness occurred within seven days of admission. most of the patients had received clinical diagnosis of respiratory tract infection, including pneumonia, acute bronchitis/bronchiolitis, asthma exacerbation and acute pharyngitis. the most common respiratory symptoms included fever (temperature !38 c), cough, sore throat, shortness of breath, vomit, dyspnea and so on. 15 in addition, none of the samples come from the patients in pediatric intensive care unit (picu) and most of the children have no co-morbidities, such as heart and liver diseases. all the samples were collected by medical professionals and placed in a tube containing of viral transportation medium and stored at à80 c. this project was approved by the research ethics committee of beijing children's hospital, children's hospital of fudan university, wenzhou medical college, and the institutional review board at the china cdc. written informed consent was obtained on the participants' behalf from their parents or guardians. viral nucleic acids were extracted from virus transport medium by the qiaamp minelute virus spin kit (qiagen, germany), according to the instructions provided by the manufacturer. as previously described, 8 partial vp1/vp2 gene fragment was amplified by nested pcr to screen and type hbov infection. the first round-pcr primers were f1 (5 0 -cgccgtggctcctgctct-3 0 ) and r1 (5 0 -tgttcgccatca-caaaagatgtg-3 0 ) with 609 bp product, the second-round pcr primers were f2 (5 0 -ggctcctgctctaggaaataaa-gag-3 0 ) and r2 (5 0 -cctgctgttaggtcgttgttgtatgt-3 0 ) with 576 bp pcr product. positive products were cloned into pmdt-18 vector and sequenced by abi 3730xl automated sequencer. hbov co-infection with other respiratory viruses, including hrsv, hrv/ev, hadv, hmpv, hpiv1e4, influenza a/b virus and hcovs (-oc43, -229e, -nl63, -hku1), was also screened as described previously. 20, 21 sequencing and phylogenetic analysis of hbov1 genome four samples of hbov1 infection only in this study were used for complete genomes sequencing by next-generation sequencing. samples were pretreated as previously, 22 and the amplified dna was used as a template for illumina hiseq 2500 sequencing, paired-end reads (2 â 125 bp reads) were assembled into contigs by clc genomic workbench. to analyze genetic variation of hbov detected, nucleotide sequences were compared to strains available from genbank. nucleotide sequence alignment was conducted through mafft version 5. 23 phylogenetic and molecular evolutionary analyses were constructed by neighbor-joining method using mega 5.0 24 with the bootstrap value of 1000. evolutionary rates were calculated using the bayesian markov chain monte carlo approach employed by beast version 1.7. 25 fragments of the ns1, np1 and vp1 genes of hbov were aligned separately and used to calculate the rate of nucleotide substitutions/site/year, under an uncorrelated lognormal-relaxed clock model of rate variation among lineages. the best-fit models were selected by jmo-deltest, with the following models: hky þ i þ g for np1, gtr þ g for ns1 and gtr þ i þ g for vp1. the evolutionary rates of individual genes were then compared to identify the differences in conservation throughout the genome. dna sp5 software 26 was used to analyze the nucleic acid sequence diversity of the hbov genomes. the model of hbov1 vp1 (nc_007455) was based on the crystal structure of a capsid viral protein of adenoassociated virus (pdb code: 4iov) 27 which shared a close genetic relationship with hbovs. molecular modeling was performed manually using the coot software under the guidance of fo-fc and 2fo-fc electron density maps. consequently, we refined the initial rigid body, and performed a series of restrained tls refinements using refmac5. additional rounds of refinements were performed using the phenix refine program implemented in the phenix package with isotropic adp refinement and bulk solvent modeling. we assessed the stereochemical quality of the final models with the program procheck. eventually, the molecular model was generated using pymol (http://www.pymol.org/). the nucleotide sequences generated in this study have been deposited in genbank under the accession numbers km378039wkm378094, km877548wkm877592, km8877594wkm877614 and km464728wkm464730. data were analyzed by the chi-squared test using sas software version 9.2. p < 0.05 was considered statistically significant. all the respiratory samples were collected from hospitalized children with sari in beijing, shanghai and zhejiang from 2007 to 2014. the sample information was provided in table 1 . the median age of the population in each of the three regions studied is 7 months, 7.5 months and 1 year, respectively. no significant differences were observed regarding gender and age distribution (p > 0.05) and the characteristics of three regions' population was matched well. in all, 200 (20.1%) of 993 sari patient were positive for hbov and the infection rates of hbov among inpatient in beijing, zhejiang and shanghai area were 21.6% (56/ 259), 22.5% (66/293) and 17.7% (78/441), respectively. no significant difference was observed regarding infection rate (p > 0.05). phylogenetic analysis based on partial vp1/vp2 sequences indicated that hbov1 was the most prevalent in china. the strains isolated from beijing were identified as hbov1 (n z 54), hbov2 (n z 1) and hbov3 (n z 1). similarly, of the 66 hbov-positive samples collected from zhejiang province, 65 were hbov1, one was hbov3 (fig. 1) . in addition, all the hbov-positive samples from shanghai city belonged to hbov1. combined with the three cohorts, the most frequently detected strains in china was hbov1 (98.5%, [197/200] ), followed by hbov3 (1%, 2/200) hbov2 (table s1 ). through high-throughput sequencing, four complete genomes of hbov were obtained (km464728, km464729, figure 1 phylogenetic analysis of hbov detected among children with sari. the phylogenetic tree was constructed based on partial vp1/vp2 gene sequence. all the sequences presented here were indicated in black solid ball and reference strains of hbov were indicated in red solid triangle. sequences were aligned by neighbor-joining method with 1000 bootstrap replicates using mega 5.0. km464730 and km464731), which all belong to hbov1 (fig. 2) . all four genomes of hbov1 sequenced in this study were shown to be closely related to each other at the nucleotide (nt) level (99.47%e99.74% nt identity for each genome) and to be closely related (99.60%e99.87%) to prototypes hbov1 (nc_007455.1). a comparative analysis of the four hbov1 genomes presented here and all available other hbov genomes identified a total of three deletions within np1 and vp1 together, which could be used to distinguishing hbov1 from hbov2e4. these differences include a 3-bp deletion in vp1 of hbov1 at nucleotides 419e421, a 12-bp deletion in vp1 of hbov2e4 at nucleotides 993e996 and 1004e1011, and a 3-bp deletion in np1 of hbov2e4 at nucleotides 88e90 (fig. 3) . to further investigate the potential implications of vp1 variations in hbov1, relative to hbov2e4, we generated a 3d model of hbov1 vp1 based on the crystal structure of a capsid viral protein of adeno-associated virus (pdb code: 4iov) (fig. 4) . according to the previous report, 27 the structure of vp1 contains two parts: the capsid exterior and capsid interior. the unique substitutions and insertions (residues 334e337) within hbov1 compared to hbov2e4 mapped primarily to the capsid exterior. interestingly, among the 32 unique substitutions, 19 were related with serine or threonine (substitutions of s/t with other amino acids), which has a dramatic influence on the hydrophobicity of the protein. these consensus nucleotide deletions or substitutions can be used to distinguish hbov1 from hbov2e4 and may be associated with pathogenicity of hbov. to determine the differences in gene-specific mutation rates, we calculated and compared the mutation rates of ns1, np1 and vp1 of hbov1 prevalent in china. the evolutionary rates of ns1, np1 and vp1 of hbov1 were 2.840 â 10 à5 , 3.917 â 10 à4 and 4.204 â 10 à4 substitutions/ site/year with the ess values >200, respectively ( table 2 ). comparative analysis showed that the evolutionary rate of vp1 is much faster than that of ns1 for hbov1, and hbov evolved relatively slowly in china. no genetic recombination event was found among hbov1 from china. in addition, analysis of dna polymorphisms within hbov1 and hbov2 was performed using the dna sp5 software. the overall mean of diversities (pi) were 0.00371(hbov1 in china), 0.00395(hbov1 out of china), 0.00377 (hbov1 in this study) and 0.03728(hbov2), respectively. there was no significant difference among the nucleotide diversity of hbov1 from different regions and the nucleotide sequences diversity of vp1 gene was greater than others. in addition, the high degree of nucleotide sequence diversity in hbov2 is greater than that of hbov1 as expected (fig. 5 ). hbov is a novel parvovirus associated with respiratory tract infections in infants or children, including four genotypes (hbov1-hbov4) . 8, 28 among the four recognized hbov genotypes, hbov1 have more often been associated with sari. the prevalence of different hbov genotype varies among the same region. only limited data is available on the prevalence of hbov infection in sari children. here, we performed a comprehensive molecular epidemiological study of hbov in china between 2007 and 2014 and made genomic characterization analyses. our study indicated that hbov was frequently detected in sari children from 2007 to 2014 in china; the frequency of hbov1 was much higher than that of other hbov subtypes. however, the infection rates of hbov among hospitalized children with sari in three different regions were almost consistent (21.6%, 22.5% and 17.7%). previous reports demonstrate the prevalence of hbov varies considerably between 1.5% and 19% in children with acute respiratory tract infections (artis). 29, 30 however, the prevalence of hbov among children with sari was more common (200/993, 20.1%) in this study. furthermore, fifty (5.04%) of 993 sari patient were detected as hbov positive only, about 25% among all 200 hbov positive patients with sari. these data indicated that the hbov infection may play an important role among children with sari, although co-infection of hbov with other viruses was much higher than that of hbov infection alone. in addition other factors may also affect the infection rate of hbov, including assay sensitivity, specificity, sampling time and sampling locations, more respiratory samples were needed to provide more detailed information about the prevalence of hbov among the children with sari. we calculated the evolutionary rates of individual hbov1 genes prevalent in china, identifying strong conservation in ns1 (2.840 â 10 à5 substitutions/site/year), compared with vp1, which exhibited substantially higher mutation rates (4.204 â 10 à4 substitutions/site/year). furthermore, the evolutionary rate of hbov1 vp1 is more than 10 time than that of ns1, indicating the strongest degree of conservation in non-structural protein ns1; while vp1, which encodes the capsid protein, mutated significantly more rapidly than other genes. however, we only calculated the evolutionary rates of hbov1 genes, rather than hbov2e4 genes, due to genomic recombinant 31, 32 and limited number of genome sequence. more extensive studies will be needed to address the evolution of hbovs. comparative analysis of the hbov1 genome sequences presented here revealed consistent and reproducible nucleotide deletions and substitution. three sets of deletions within np1 and vp1 were shown to be diagnostic for hbov1, clearly differentiating these from those of hbov2e4. this difference is consistent with phenotypic analyses, which show hbov1 to be the most commonly occurring in respiratory tract samples, while hbov2e4 are detected mainly in gastrointestinal samples and are presumably enteric. furthermore, the unique substitutions within hbov1 relatively to hbov2e4 mapped primarily to the capsid exterior. these otherwise uncommon substitutions and insertions within hbov1 vp1 are likely to play a major role in the antigenicity of hbov species and may account for differences in tissue tropism between hbov1 and hbov2e4. further studies are necessary to validate these findings and to confirm the effects of these nucleotide differences on tissue tropism and pathogenicity. in summary, we first reported the circulating hbov genotype and their genome character among children with sari in china, providing more evidence for a causal role of hbov1 in sari. novel molecular signatures were identified for distinguishing hbov1 from other hbov subtypes. this study complements and significantly expands upon the current knowledge of hbov infection and sari among children in china. nil. human bocavirus and acute wheezing in children a novel bocavirus associated with acute gastroenteritis in australian children human bocavirus, a respiratory and enteric virus the family parvoviridae genomic features of the human bocaviruses human bocavirus capsid structure: insights into the structural repertoire of the parvoviridae cloning of a human parvovirus by molecular screening of respiratory tract samples human bocaviruses are highly diverse, dispersed, recombination prone, and prevalent in enteric infections a newly identified bocavirus species in human stool circulating of human bocavirus 1, 2, 3, and 4 in pediatric patients with acute gastroenteritis in thailand human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus human bocavirus in french children human bocavirus infection among children human bocavirus species 2 and 3 in brazil global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis human bocavirus (hbov) in thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization human bocavirus infection as a cause of severe acute respiratory tract infection in children genetic variability of human metapneumo-and bocaviruses in children with respiratory tract infections correlation of viral load of respiratory pathogens and co-infections with disease severity in children hospitalized for lower respiratory tract infection viral etiology and clinical profiles of children with severe acute respiratory infections in china prevalence of human parvovirus b19, bocavirus, and parv4 in blood samples from the general population of china and lack of a correlation between parvovirus and hepatitis b co-infection identification of diverse alphacoronaviruses and genomic characterization of a novel severe acute respiratory syndrome-like coronavirus from bats in china mafft multiple sequence alignment software version 7: improvements in performance and usability mega4: molecular evolutionary genetics analysis (mega) software version 4.0 beast: bayesian evolutionary analysis by sampling trees dnasp v5: a software for comprehensive analysis of dna polymorphism data the structure of aavrh32. 33, a novel gene delivery vector human bocavirus-1 primary infection and shedding in infants surveillance and genome analysis of human bocavirus in patients with respiratory infection in human bocavirus: prevalence and clinical spectrum at a children's hospital recombination analysis based on the complete genome of bocavirus prevalence analysis of different human bocavirus genotypes in pediatric patients revealed intra-genotype recombination supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2016.05.014. key: cord-257732-3xuy6tbn authors: azzi, lorenzo; carcano, giulio; gianfagna, francesco; grossi, paolo; gasperina, daniela dalla; genoni, angelo; fasano, mauro; sessa, fausto; tettamanti, lucia; carinci, francesco; maurino, vittorio; rossi, agostino; tagliabue, angelo; baj, andreina title: saliva is a reliable tool to detect sars-cov-2 date: 2020-04-14 journal: j infect doi: 10.1016/j.jinf.2020.04.005 sha: doc_id: 257732 cord_uid: 3xuy6tbn objectives: this study analyzed salivary samples of covid-19 patients and compared the results with their clinical and laboratory data. methods: salivary samples of 25 covid-19 patients were analyzed by rrt-pcr. the following data were collected: age, sex, comorbidities, drugs. lactate dehydrogenase (ldh) and ultrasensitive reactive c protein (usrcp) values were registered on the same day when a salivary swab was collected. prevalence of positivity in saliva and association between clinical data and the cycle threshold as a semiquantitative indicator of viral load were considered. results: twenty-five subjects were recruited into this study, 17 males and 8 females. the mean age was 61.5 +/− 11.2 years. cardiovascular and/or dysmetabolic disorders were observed in 65.22% of cases. all the samples tested positive for the presence of sars-cov-2, while there was an inverse association between ldh and ct values. two patients showed positive salivary results on the same days when their pharyngeal or respiratory swabs showed conversion. conclusions: saliva is a reliable tool to detect sars-cov-2. the role of saliva in covid-19 diagnosis could not be limited to a qualitative detection of the virus, but it may also provide information about the clinical evolution of the disease. it was december 31st, 2019 when chinese health officials informed the world health organization (who) about the cluster of a mysterious pneumonia in 41 patients in the city of wuhan and in the chinese province of hubei. 1 one week later a new coronavirus, currently known as sars-cov-2, was identified as the etiologic agent of the severe acute respiratory syndrome, and soon after the first death was recorded. 2 since then, the infection has rapidly spread worldwide due to the fact that sars-cov-2, despite sharing an 80% of sequence homology with the virus responsible of 2003 sars epidemic, 3 has a highly increased contagiousness. 4 on march 11th, 2020 the who declared coron-avirus disease 2019 ( covid-19 ) a global pandemic, for the second time in the 21st century after the influenza pandemic caused by h1n1. 5 currently, a massive viral spread is hitting 205 countries, more than 1,0 0 0,0 0 0 people are positive for sars-cov-2 infection and more than 58,0 0 0 have died. on april 3rd, italy is the second country, after the united states of america, with the largest outbreak (more than 110,0 0 0 cases and 14,0 0 0 deaths). 7 during the month of march, the emergency decrees and regulations of the government, regions and city councils have de facto quarantined the country, urging citizens to home self-isolation, in order to drastically reduce the source of contagion. the government's regulations have had the difficult task of striking a balance between health needs (the necessity of preventing contagion through social isolation) and economic issues, resulting from the lockdown of factories, businesses and other commercial activities. 8 these drastic measures have been necessary, since it has not been possible, so far, a mass screening test to identify the infected people. the diagnosis of covid-19 is made through a nasopharyngeal swab. initially, the test was carried out on patients with severe symptoms and on the subjects who had come into contact with them in the previous days. today, only patients with severe symptoms undergo the test, while asymptomatic patients go completely undetected. at present, real time reverse transcription polymerase chain reaction (rrt-pcr) on respiratory specimens represents the gold standard test for detection of sars-cov-2 infection. 9 rrt-pcr, however, is not an ideal screening procedure to be adopted for massive screening, as it implies the patient's stay at home or in hospital until diagnosis, thus causing the crowding of the centers appointed to collect specimens. for these reasons, some companies are trying to develop new diagnostic testing solutions, which allow rapid assessment of infection in central facilities dedicated to the diagnosis of covid-19. among them, more rapid pcr-based assays or immunochromatography-based in vitro assays to detect specific antibodies on blood specimens have been proposed. although these techniques have advantages, including setup and faster time for results, the major limitation for their suitability in a mass screening is represented by the collection of blood samples at a medical point-of-care. 10 , 11 sputum and oropharyngeal secretions have recently been suggested as a possible target for the molecular diagnosis of covid-19, 12 and salivary droplets represent the main source of the human-to-human transmission of the sars-cov-2 infection when social distance is less than 2 m. 13 to date, there are not any studies regarding the possible role of oral fluids and saliva in the detection of sars-cov-2. the use of saliva as a diagnostic sample has several advantages: since saliva can be easily provided by the patient, 14 it does not require specialized personnel for its collection. in addition, the comfort of the procedure is significantly higher if compared with the nasopharyngeal swab or sputum procedure. however, before considering saliva a promising tool to detect sars-cov-2, it is imperative to confirm the presence of the virus in this fluid. the aim of this study was to analyze samples of saliva collected from patients already diagnosed with covid-19 and compare the results compared the results with their clinical data and laboratory data. a group of 25 sars-cov-2 infected patients with severe or very severe disease were recruited. patients were admitted to our hospital (asst dei sette laghi -ospedale di circolo e fondazione mac-chi) after the diagnosis of covid-19 provided by rrt-pcr on nasopharyngeal swabs. this study was carried out in agreement with the helsinki declaration and authorized by the hospital direction, due to the situation of emergency. saliva was collected through the drooling technique. this technique allows to collect only oral fluids, thus excluding mucous secretions from oropharynx or lower respiratory tract (i.e., sputum). 15 patients' clinical situation was classified according to the diagnosis and treatment plan of covid-19 issued by the chinese national health commission. 16 when a patient underwent endotracheal intubation and mechanical ventilation, saliva was collected intraorally by a physician with the use of a pipette. when it was possible, a second salivary swab was collected after 4 days. the following data were collected for each patient: age, sex, comorbidities (with special attention to hypertension, diabetes, dyslipidemia and obesity, and previous lung or mediastinal diseases), drugs, inflammatory indices or tissue damage biomarkers at the moment of salivary swab, thus ultrasensitive reactive c protein (us-rcp) and lactate dehydrogenase (ldh). saliva specimens were resuspended in 2 ml of pbs, 140 μl were subjected to rna extraction by qiamp viral rna mini kit (qiagen) and eluted in 60 μl. one step rrt-pcr was performed using luna universal qpcr master mix (new england biolab) from μl of extracted rna. forward (5 -accttcccaggtaacaaacca-3 ) and reverse (5 -ttacctttcggtcacacccg-3 ) primers targeting the 5 utr region of sars-cov-2 were used. all samples were run in four replicates, together with a previous known positive control, with saliva from healthy people as a negative control, and with water molecular grade using abi prism 70 0 0 sequence detection system (applied biosystems). in the same run, samples were amplified with beta-actin primers in order both to control amplification and normalize their account. the ct values were considered 'highly positive' when below the ct median or 'low positive' when above the ct median. distribution of continuous variables was assessed using the kolmogorov-smirnov test, and the characteristics of participants were reported by sex and comparisons between males and females were performed using the mann-whitney u test and fisher's exact test. to analyze the potential association between the continuous variables (i.e., age, uscrp and ldh levels) and positivity levels, we performed a regression analysis using age and sex as covariates. to analyze the potential association between the categorical variables and positivity levels, we firstly categorized the positivity level according to the cycle threshold (ct, the number of cycles required for the fluorescent signal to exceed background level) observed in the rt-pcr. "low positive" or "highly positive" signals were then defined for ct values below or above the mean value. due to the low number of subjects in these groups, we used the non-parametric fisher's exact test. a p value (pfdr) < 0.05 was considered as significant. the analyses were conducted with sas (v9.4, sas institute inc., cary, nc). a total number of 25 subjects were analyzed in this study, 17 males and 8 females. age values ranged from 39 to 85 years, with a mean age of 61.5 + / − 11.2 years. all patients were affected by severe or very severe covid-19 and were selected among those subjects hospitalized in the intensive care unit or in the unit of infectious and tropical diseases. on admission, the nasopharyngeal swab followed by rt-qpcr confirmed the diagnosis of sars-cov-2 infection. the main clinical and anamnestic data are summarized in table 1 . most of these patients (i.e., 65.22%) were affected by cardiovascular and/or dysmetabolic disorders, especially hypertension, dyslipidemia and obesity. about 20% of the subjects had previous lung, mediastinal or upper airways diseases, like thymoma or obstructive sleep apnea syndrome (osas). as regards chronic medication intake, 40% of the patients reported the intake of at least one drug, primarily statins (i.e., 25%) and ace-inhibitors or angiotensin ii receptor blockers (arbs) (i.e., 20%). there were not significant differences regarding the clinical and anamnestic history between males and females, with the only exception of the values of serum ldh, which were higher in the female patients' haematochemical analyses carried out on the day of saliva collection ( p = 0.025). sars-cov-2 was detected in all 25 patients' first salivary swab, with different ct values (range 18.12-32.23, mean value 27.16 + / − 3.07), but all of them were under the ct value of 33. there were not any differences in the ct values with regards to the period elapsed after the onset of symptoms ( p = 0.25). interestingly, there was an inverse correlation between the ldh values recorded in the haematochemical analyses and the ct values, thus the viral load detected in the saliva was correlated to the tissue damage reported by biomarkers ( p = 0.04) ( table 2 ) ( fig. 1 a and b ). in contrast, there was not a significant correlation between usrct and the ct values ( p = 0.07), but an inverse tendency between this inflammatory index and the viral load detected in saliva ( fig. 1 c and d) was observed. the ct values were not influenced by the patients' age ( p = 0.34), sex ( p = 0.31) or comorbidities ( table 3 ) . eight patients underwent a second salivary swab after 4 days, and the results were consistent with the first analysis, without relevant differences in the ct values. a striking feature was highlighted in two patients who showed positive salivary results on the same days when their pharyngeal or bronchoalveolar swabs proved to be negative. in the first patient, the salivary specimen was positive on the same day when a nasopharyngeal swab converted to negative, and this result was also confirmed after two days. the second patient showed positive results in two consecutive salivary swabs, while three consecutive respiratory swabs were negative on the same days. real time reverse transcription polymerase chain reaction (rrt-pcr) on nasopharyngeal and respiratory specimens represents the gold standard for the qualitative detection of sars-cov-2 infection. 17 however, the nasopharyngeal swab requires a close contact between healthcare workers and the patients, which poses a risk of transmission of the virus to nurses and physicians. 18 furthermore, the collection of these specimens may be associated with various degrees of discomfort for the patient. these features related to the nasopharyngeal swab collection have led clinicians to test rrt-pcr on other biological specimens, like urine, stools, sputum and posterior oropharyngeal secretions. 19 , 20 sputum is the mucous secretion that is coughed up from the lower airways. several papers have recently pointed out that sputum represents a reliable source for the diagnosis of sars-covtable 2 ). (for interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.) 2 infection. 12 collecting sputum is less invasive than carrying out a nasopharyngeal swab, and, not less important, this procedure can be performed by the patient themselves. 21 however, sputum is not free from drawbacks: it should be provided before toothbrushing and breakfast, since nasopharyngeal secretions move posteriorly, and bronchopulmonary secretions move by ciliary activity to the posterior oropharyngeal area, while the patients are in a supine position during sleep. 22 besides, not all patients can easily provide sputum with respiratory secretions. conversely, saliva is an oral fluid that is produced by the salivary glands and may represent an easily manageable specimen to be easily used for diagnosing covid-19. 23 in the past, saliva has proved to be an ideal organic fluid for the isolation of proteins, peptides, and viral shedding via many molecular assays. 24 several authors demonstrated its reliability in studies regarding the detection of zika and ebola viruses. 25 , 26 in 2004, a study found out a large amount of viral rna in the saliva of a patient affected by sars-cov in taiwan. 27 up to the present time, there are not available studies dealing with the role of salivary and oral fluids in the detection of sars-cov-2, an issue that has been recently claimed. 28 in our research, we collected salivary samples from 25 patients affected by severe covid-19 admitted at our hospital. saliva was collected through the drooling technique or with a pipette, depending on the patient's clinical condition; thus, sputum and oropharyngeal secretions were excluded from the collection. the samples were analysed by rrt-pcr, which showed positive results for all the 25 subjects, with variable threshold cycles (ct), but always under 33 cycles (range 18.12-32.23, mean 27.16 + / − 3.07). these results reinforce the hypothesis that saliva is a reliable tool to be used in qualitative covid-19 diagnosis through the rrt-pcr procedure. surprisingly, in two patients the salivary samples proved positive while their respiratory swabs showed negative results on the same days. this finding, together with the fact that chinese colleagues reported similar results in sputum and feces samples, 19 rises the concern about how to manage recovering patients at the moment of hospital discharge, because some of them could be contagious through their saliva even after two consecutive pharyngeal swabs that converted to negative, a serious danger for their own family and a troublesome issue for the social community. for this reason, last week we decided that the patients who had recovered should be discharged only after two pharyngeal swabs and one salivary swab tested negative. the population analyzed in our study was homogeneous, without any clinical or anamnestic features interfering with the results. their medical history is consistent with that reported in other studies: most of the patients were affected by cardiovascular and/or dysmetabolic disorders. 29 a difference was noted between males and females as regards the haematochemical levels of ldh, with females showing higher levels ( p = 0.025). this finding could be explained by the fact that males are more commonly affected by the severe forms of covid-19 than females 30 ; the latter require intensive care less frequently, but when it happens, they show worse clinical parameters. indeed, ldh is commonly released during tissue damage, it can be associated to the lung damage that takes place in covid-19 patients. 31 within this frame, we reported an inverse association when comparing the ct values in salivary rrt-pcr analysis with the haematochemical ldh levels recorded on the same day of the swab: this means that the higher the salivary viral load is, the higher the ldh levels in the bloodstream are. therefore, our research shows that saliva is not only a biological fluid that could be used for qualitative detection of sars-cov-2, but it may represent a useful tool to follow the course of the illness together with other biological markers. finally, we collected a second salivary specimen after 4 days on 8 of the 25 recruited subjects, and we found that the ct values were consistent with the previous findings, without relevant oscillation. this study suffers several limitations: the use of the ct values highlights a trend in viral load but does not allow a quantification of the viral copies per ml, due the absence of a reliable positive control in our laboratory to be used for the analysis. in addition, the population analyzed in this study is homogeneously composed of individuals affected by the more severe form of covid-19; therefore, more samples should be collected on a less restricted population, especially when mild symptoms occur or when the identified subjects are asymptomatic. asymptomatic patients represent an urgent issue to be addressed by public health policies against covid-19, but to date there are not reliable procedures that can be used for a mass screening. 32 recently, rapid serologic tests have elicited interest in the public opinion, but the scientific community does not agree that they can be used in a mass screening program to detect the asymptomatic carriers. 33 saliva is a reliable biological fluid that could be a candidate for a diagnostic rapid test, because it can be easily performed also by non-healthcare professionals in a screening program. therefore, it is fundamental that the salivary load in asymptomatic carriers be analyzed to establish a sensitivity threshold for a future test. in conclusion, our study highlights that saliva represents a promising tool in covid-19 diagnosis. however, it should be understood why the virus is detectable in the oral cavity. it may appear in the mouth because it migrates from the nasopharynx or the lower respiratory tract to the oral cavity, but it can't be excluded that a role may be played by the secretory activity of the salivary glands. it has been suggested that the oral cavity may play an active role in the pathogenesis of covid-19, and this was highlighted by a chinese study that showed a high expression of ace2 receptors on the epithelial cells of the oral mucosa. 34 none. world health organization 2019-ncov situation report -1 21 outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle evolutionary trajectory for the emergence of a novel coronavirus sars-cov-2 novel coronavirus (2019-ncov) outbreak: a new challenge centers for disease control and prevention (cdc) update: novel influenza a (h1n1) virus infections -worldwide world health organization novel coronavirus (covid-2019) situation reports italian health ministry covid-19 situation reports covid-19 and italy: what next? lancet 2020 combination of rt-qpcr testing and clinical features for diagnosis of covid-19 facilitates management of sars-cov-2 outbreak detection of antibodies against sars-cov-2 in patients with covid-19 antibody response to sars-cov-2 in patients of novel coronavirus disease 2019 yjinf [m5g consistent detection of 2019 novel coronavirus in saliva 2019-ncov: new challenges from coronavirua human saliva as a diagnostic material comparative evaluation of saliva colletion methods for proteome analysis chinese clinical guidance for covid-19 pneumonia diagnosis and treatment guidelines for laboratory diagnostics of coronavirus disease 2019 (covid-19) in korea covid-19 and the risk to health care workers: a case report the presence of sars-cov-2 rna in feces of covid-19 patients evaluation of sars-cov-2 rna shedding in clinical specimens and clinical characteristics of 10 patients with covid-19 in macau virological assessment of hospitalized patients with covid-19 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study saliva and oral diseases human saliva collection devices for proteomics: an update human saliva can be a diagnostic tool for zika virus detection find the right sample: a study on the versatility of saliva and urine samples for the diagnosis of emerging viruses detection of sars-associated coronavirus in throat wash and saliva in early diagnosis human saliva: non-invasive fluid for detecting novel coronavirus (2019-ncov) prevalence of underlying diseases in hospitalized patients with covid-19: a systematic review and meta-analysis clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study analysis of clinical characteristics and laboratory findings of 95 cases of 2019 novel coronavirus pneumonia in wuhan, china: a retrospective analysis alert for non-respiratory symptoms of coronavirus disease 2019 (covid-19) patients in epidemic period: a case report of familial cluster with three asymptomatic covid-19 patients performance of vivadiagtm covid-19 igm/igg rapid test is inadequate for diagnosis of covid-19 in acute patients referring to emergency room department high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa the authors gratefully acknowledge all the colleagues fighting covid-19 at each level. key: cord-270258-9vgpphiu authors: ko, jae-hoon; park, ga eun; lee, ji yeon; lee, ji yong; cho, sun young; ha, young eun; kang, cheol-in; kang, ji-man; kim, yae-jean; huh, hee jae; ki, chang-seok; jeong, byeong-ho; park, jinkyeong; chung, chi ryang; chung, doo ryeon; song, jae-hoon; peck, kyong ran title: predictive factors for pneumonia development and progression to respiratory failure in mers-cov infected patients date: 2016-08-09 journal: j infect doi: 10.1016/j.jinf.2016.08.005 sha: doc_id: 270258 cord_uid: 9vgpphiu background: after the 2015 middle east respiratory syndrome (mers) outbreak in korea, prediction of pneumonia development and progression to respiratory failure was emphasized in control of mers outbreak. methods: mers-cov infected patients who were managed in a tertiary care center during the 2015 korean mers outbreak were reviewed. to analyze predictive factors for pneumonia development and progression to respiratory failure, we evaluated clinical variables measured within three days from symptom onset. results: a total of 45 patients were included in the study: 13 patients (28.9%) did not develop pneumonia, 19 developed pneumonia without respiratory failure (42.2%), and 13 progressed to respiratory failures (28.9%). the identified predictive factors for pneumonia development included age ≥45 years, fever ≥37.5 °c, thrombocytopenia, lymphopenia, crp ≥ 2 mg/dl, and a threshold cycle value of pcr less than 28.5. for respiratory failure, the indicators included male, hypertension, low albumin concentration, thrombocytopenia, lymphopenia, and crp ≥ 4 mg/dl (all p < 0.05). with ≥ two predictive factors for pneumonia development, 100% of patients developed pneumonia. patients lacking the predictive factors did not progress to respiratory failure. conclusion: for successful control of mers outbreak, mers-cov infected patients with ≥ two predictive factors should be intensively managed from the initial presentation. middle east respiratory syndrome (mers) is an emerging lethal respiratory disease caused by a novel betacoronavirus (mers-cov). 1 from may to july 2015, there was a hospital-associated mers outbreak in the republic of korea reporting 186 laboratory-confirmed cases, which is the largest recorded outbreak outside the arabian peninsula. 2e6 the outbreak featured several super-spreading events with unexpectedly high human-to-human transmission rate: 136 of 186 cases (73.1%) were transmitted from only three patients. 5,7e10 as these large transmission clusters were exclusively originated from patients with pneumonia, prediction of pneumonia development has been emphasized in control of mers outbreak. 9 in addition, pneumonia progression to respiratory failure should be anticipated in advance to avoid urgent intubation or cardiopulmonary resuscitation which might break protection of healthcare workers. although several studies analyzed prognostic factors for fatal outcome, 11e16 predictive factors for pneumonia development and progression to respiratory failure have not been reported. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we evaluated mers-cov infected patients managed in a tertiary care center during the 2015 mers outbreak in korea. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we reviewed the electronic medical records of who were diagnosed with mers-cov infection and admitted at samsung medical center, a 1950 tertiary care university hospital which managed the largest number of mers-cov infected patients as a single center during the 2015 korean mers outbreak. as it is still unclear whether initially asymptomatic patients would develop pneumonia or not, 17 we included all the mers-cov infected patients managed at our hospital during the outbreak regardless of symptoms presence. to avoid confusion with the case definition of mers which did not included asymptomatic cases, 18 we used the term of 'mers-cov infected patient' rather than 'mers patients' throughout the present paper. mers-cov infections were confirmed on the basis of rrt-pcr assays targeting upstream of the e gene and the open-reading frame gene 1a. 18, 19 disease status of included patients was assessed at six weeks from their symptom onset and patients were divided into three groups depending on pneumonia development and progression to respiratory failure: patients without the development of pneumonia (group 1), patients who developed pneumonia without respiratory failure (group 2), and pneumonia patients who progressed to respiratory failure (group 3). for practical purposes, respiratory failure was defined as the need for mechanical ventilation (mv). the institutional review board of our hospital approved the present study. we retrospectively collected data from electronic medical records and epidemiologic investigation. to identify factors which can predict pneumonia development and progression to respiratory failure at the early course of the disease, we evaluated clinical variables measured within three days from symptom onset. during the 2015 korean mers outbreak, fever was defined as body temperature !37.5 c to increase sensitivity of screening and the same definition was used in the present analysis. 9 thrombocytopenia was defined as a platelet count lower than 150 â 10 3 cells/mm 3 , lymphopenia as an absolute lymphocyte count lower than 1,000 cells/mm 3 , and hypoalbuminemia as albumin concentration lower than 3.5 g/dl. lower respiratory tract specimens including sputum and endotracheal aspirates were used for mers-cov rrt-pcr. cycle threshold (ct) values of mers-cov rrt-pcr were used as a surrogate of viral load. pneumonia development of mers-cov infected patient was defined as presence of parenchymal infiltration on chest x-ray with respiratory symptoms. test days or events were counted from the day of symptom onset for each patient: day 1 was defined as the day of symptom onset. for asymptomatic patients, the day of diagnosis of mers-cov infection was considered as day 1. to identify predictive factors for pneumonia development and progression to respiratory failure, clinical variables measured within three days from symptom onset were compared. for evaluation of pneumonia development, patients who developed pneumonia (group 2 and 3) were compared to those who did not (group 1). for factors for respiratory failure, patients who progressed to respiratory failure (group 3) were compared to those who did not (group 1 and 2). student's t-tests or mannewhitney u tests were used to compare continuous variables, and chi-square tests or fisher's exact tests were used to compare categorical variables. statistically significant continuous variables were re-categorized into binary factors using threshold values between mean of each group, which showed lowest p value. statistically significant categorical variables and binary factors re-categorized from continuous variables were defined as predictive factors. for significant predictive factors, as a measure of association, odds ratio (or) and 95% confidence interval (ci) for or were calculated using the woolf procedure. 20 multivariate analysis was not performed due to the limited sample size. all pvalues were two-tailed, and those <0.05 were considered to be statistically significant. ibm spss statistics version 20.0 for windows (ibm, armonk, ny, usa) was used for all statistical analyses. time course of pneumonia development and progression to respiratory failure a total of 45 mers-cov infected patients were hospitalized during the outbreak with 13 patients in group 1 (including 3 asymptomatic patients), 19 patients in group 2, and 13 patients in group 3. the clinical course of symptomatic mers patients progressed serially: patients developed initial symptoms after a median 5-day incubation period (iqr 3.5e7.0), pneumonia after a median of 6 days from symptom onset (iqr 5.0e7.0), and respiratory failure after a median of 12 days from symptom onset (iqr 10.0e13.0). in group 3 patients, it took a median of 2 days from desaturation to respiratory failure (iqr 1e3 days). the development and progression of pneumonia by time sequence is depicted in fig. 1 . no one developed pneumonia before day 4 of symptom onset. to evaluate predictive factors for pneumonia development, demographics, underlying diseases, and clinical variables of patients in group 2 and 3 were compared to those of patients in group 1 (tables 1 and 2 ). identified predictive factors are summarized in table 3 with odd ratios (or). increasing age was significantly associated with pneumonia development as a continuous variable (p z 0.015), and age older than 45 years was a predictive factor for the development of pneumonia (or, 8.04; 95% ci, 1.52e42.43; p z 0.007). although proportion of male also increased with progression of pneumonia (38.5%, 57.9%, and 84.6% for group 1, 2, and 3, respectively), statistically significant association between male sex and the pneumonia development was not identified (p z 0.097). fever over 37.5 c by day 3 were more frequently detected in patients with pneumonia (18.2%, 71.4%, and 77.8% in groups 1, 2, and 3, respectively), and was identified as a predictive factor for the development of pneumonia (or, 12.75; 95% ci, 2.12e76.57; p z 0.002). thrombocytopenia (or, not applicable (na); p z 0.007), lymphopenia (or, 17.50; 95% ci, 1.88e163.02; p z 0.003), elevated c-reactive protein (crp ! 2 mg/dl; or, na; p z 0.018), and high viral load (ct value < 28.5; or, 14.00; 95% ci, 1.14e172.65; p z 0.024) were distinctly observed in pneumonia patients from the initial presentation, and identified as predictive factors for pneumonia development. to evaluate predictive factors for progression to respiratory failure, patients in group 3 were compared to those in group 1 and 2 (tables 1e3). although the mean age of patients in each group tended to increase with progression of pneumonia (37.3, 47.7, and 55.2 years in groups 1, 2, and 3, respectively) and increasing age was significantly associated with respiratory failure as a continuous variable (p z 0.036), there was no statistically significant cut-off value for prediction of respiratory failure. proportion of male also increased with progression of pneumonia, and male sex was a predictive factor for respiratory failure (or, 5.50; 95% ci, 1.05e28.88; p z 0.045). among underlying diseases, hypertension was identified as a predictive factor for respiratory failure (or, 6.04; 95% ci, 1.18e30.88; p z 0.021). initial symptoms including fever were not significantly different between patients who progressed to respiratory failure and those who did not. among initial laboratory test results, thrombocytopenia (or, 6.67; 95% ci, 1.18e37.78; p z 0.023), lymphopenia (or, 14.88; 95% ci, 1.56e142.20; p z 0.006), hypoalbuminemia (or, 14.17; 95% ci, 1.83e109.86; p z 0.005), and elevated crp (crp ! 4 mg/ dl; or, 23.00; 95% ci, 2.01e262.57; p z 0.002) were distinctly observed in group 3 patients, and identified as predictive factors for respiratory failure. sensitivity, specificity, positive predictive value (ppv) and negative predictive value (npv) by number of predictive factors were presented in table 4 . when patients presented with ! two of the predictive factors for pneumonia development, 100% of these patients developed pneumonia (sensitivity 56.3%, specificity 100.0%, ppv 100%, and npv 48.1%). patients lacking the predictive factors for respiratory failure did not progress to respiratory failure. when patients presented with ! two of these predictive factors, 50.0% of these patients progressed to respiratory failure (sensitivity 69.2%, specificity 75.0%, ppv 52.9%, and npv 85.7%). initial rapid propagation of mers-cov during the korean mers outbreak was caused by three super-spreading events responsible for 73.1% of all transmissions. 5, 7, 8 in addition to these super-spreaders, transmission of mers-cov despite application of personal protective equipment (ppe) occurred from patients with progressed pneumonia at our hospital. 9 in this regard, identifying the predictive factors for pneumonia development and progression is not only important in patient care, but also in infection control to prevent further in-hospital transmission. the present analysis of predictive factors for pneumonia development and progression to respiratory failure using variables obtained by day 3 of symptom onset could be conducted owing to the observation of entire clinical course of the disease from the exposure to mers-cov. compared to mers outbreaks in the arabian peninsula where community-acquired infections might simultaneously occur from animals, identifying epidemiologic links, exposure date, and symptom onset were relatively clear for each case. 5, 19 in our observation, the clinical course of symptomatic mers patients progressed serially and no one developed pneumonia before day 4 of symptom onset. this is the reason why we used clinical data obtained by day 3 of symptom onset. there is no other comparable data to which presented time interval from the symptom onset to the development of pneumonia. although there were no ideal cut-off scores of predictive factors with good sensitivity and specificity, it should be noted that 100% of patients with ! two predictive factors for data are expressed as the number (%) of patients or mean ae sd. as missing values were also removed from the population parameter, variables with missing values are expressed with modified population parameters. continuous variables with statistical significance were re-categorized into binary factors which are presented in italics. abbreviations: res., respiratory; wbc, white blood cell; alc, absolute lymphocyte count; ast, aspartate transaminase; alt, alanine transaminase; bun, blood urea nitrogen; crp, c-reactive protein; ld, lactate dehydrogenase; ct, threshold cycle; rrt-pcr, real-time reverse transcriptase polymerase chain reaction. a data are presented as mean value of day 1e3 ae sd. pneumonia actually progressed to pneumonia. thus, careful and intensive management should be implemented for such patients including adequate isolation of patient in an airborne infection isolation room (aiir), minimizing chance for exposure, application of ppe with hooded coverall, and consideration of experimental antiviral treatment. 9,21e24 for patients with ! two predictive factors for respiratory failure, aiirs in intensive care units should be prepared for early elective intubation. although the time interval from symptom onset to mv support was much longer than in previous reports (median 12 days versus 7 days), 1 we also experienced rapid progression of pneumonia from the moment of desaturation: 73% of group 3 patients required mv within 2 days from desaturation (median 2, iqr 1e3 days). to avoid urgent situations which might break protection of healthcare workers, elective intubation should be considered when desaturation begins to progress. in addition, sensitivities of predictive values are relatively low with cut-off value of ! two factors, clinical course of patients with any predictive factors also should be carefully monitored. of note, thrombocytopenia, lymphopenia, and increased crp level were shared predictive factor for the pneumonia development and respiratory failure. they were observed in the very early course of the illness, indicating that inflammation had already been enhanced. lymphopenia and thrombocytopenia presenting from the initial presentation of severe mers-cov infected patients were also observed in the recent report by min et al. 25 although time of measurements were not specifically described, these laboratory abnormalities were previously observed in severe mers cases and other respiratory viral illnesses including severe acute respiratory syndrome (sars) and influenza, which are caused by intense inflammatory response to the viruses. 15,26e33 this is the first report that identified these laboratory findings as predictive factors for pneumonia development and progression to respiratory failure in mers. although other predictive factors for pneumonia development and respiratory failure were different due to discordance of statistical significance, they shared the same spectrum of etiology. age increased according to pneumonia progression and was associated with both pneumonia and respiratory failure as a continuous variable (p z 0.015 and p z 0.036, respectively). these findings correlate with previous data suggesting that old age is associated with poor prognosis. 11,13e15,34,35 similarly, the proportion of males increased according to disease severity, though male sex was only significant for predicting respiratory failure. although the mean age of males was older than that of females (49.7 and 42.6 years, respectively), it was not statistically significant (p z 0.169). previous data also reported that overall proportion of male was higher and was associated with severe infection. 15, 34 it could be meaningful observation that the same finding was observed in the republic of korea where the social activity of females is not restricted, especially among healthcare workers. on the other hand, hypoalbuminemia and hypertension were predictive factors only for respiratory failure, while high viral load was predictive factor for the development of pneumonia. these factors were related with severe disease and poor prognosis of mers in previous reports. 1, 12, 13, 15, 16, 34, 35 our study has several strengths and limitations. due to its retrospective nature, there may be a bias regarding collecting medical information in retrospective manner. however, as all electronic medical records were standardized to record symptoms and signs in the same way from the beginning of the outbreak, bias was minimized. secondly, there were missing values when calculating the sensitivity and specificity of predictive factors, which is another limitation of retrospective study. lastly, the present study did not perform multivariate analysis due to limited sample size and need to be validated. prospective studies with sufficient number of patients are required for validation of the predictive factors identified in the present study. despite these limitations, our data would be suitable for identifying predictive factors because we could observe entire course of the disease from exposure and apply homogenous management to patients. in conclusion, based on 45 cases from a single tertiary care hospital during the largest mers outbreak outside of the arabian peninsula, we identified six predictive factors for the development of pneumonia and progression to respiratory failure, respectively. thrombocytopenia, lymphopenia, and high crp level were shared predictive factors. mers-cov infected patients with ! two predictive factors should be intensively managed from the initial presentation for successful control of mers outbreak. there are no potential conflicts of interest relevant to this article to report. this work was supported by the samsung biomedical research institute (sbri) grant [#smx1161321]. middle east respiratory syndrome summary of current situation, literature update and risk assessment middle east respiratory syndrome coronavirus (mers-cov) e republic of korea. world health organization an unexpected outbreak of middle east respiratory syndrome coronavirus infection in the republic of korea the characteristics of middle eastern 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apoptosis by mers coronavirus middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates both the extrinsic and intrinsic apoptosis pathways epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease we would like to express our sincerest condolences to the patients and families who suffered from the mers outbreak. we also greatly appreciate the health care personnel and staff members at samsung medical center and all other hospitals who worked together to overcome the mers outbreak. key: cord-254635-gtgahlqm authors: sun, guanghao; matsui, takemi; hakozaki, yukiya; abe, shigeto title: an infectious disease/fever screening radar system which stratifies higher-risk patients within ten seconds using a neural network and the fuzzy grouping method date: 2014-12-23 journal: j infect doi: 10.1016/j.jinf.2014.12.007 sha: doc_id: 254635 cord_uid: gtgahlqm objectives: to classify higher-risk influenza patients within 10 s, we developed an infectious disease and fever screening radar system. methods: the system screens infected patients based on vital signs, i.e., respiration rate measured by a radar, heart rate by a finger-tip photo-reflector, and facial temperature by a thermography. the system segregates subjects into higher-risk influenza (hr-i) group, lower-risk influenza (lr-i) group, and non-influenza (non-i) group using a neural network and fuzzy clustering method (fcm). we conducted influenza screening for 35 seasonal influenza patients and 48 normal control subjects at the japan self-defense force central hospital. pulse oximetry oxygen saturation (spo(2)) was measured as a reference. results: the system classified 17 subjects into hr-i group, 26 into lr-i group, and 40 into non-i group. ten out of the 17 hr-i subjects indicated spo(2) <96%, whereas only two out of the 26 lr-i subjects showed spo(2) <96%. the chi-squared test revealed a significant difference in the ratio of subjects showed spo(2) <96% between hr-i and lr-i group (p < 0.001). there were zero and nine normal control subjects in hr-i and lr-i groups, respectively, and there was one influenza patient in non-i group. conclusions: the combination of neural network and fcm achieved efficient detection of higher-risk influenza patients who indicated spo(2) 96% within 10 s. the highly pathogenic avian influenza virus subtype h5n1 causes severe respiratory disease in humans, inducing threats of pandemic to increase. 1 such a severe influenza infection elevates the risks of developing influenzarelated complications, which is one of the leading causes of death during the epidemic season. 2, 3 when a pandemic occurs, the rapid screening of infected patients with a severe infection can help clinicians make better medical decisions and provide improved patient care. 4 to conduct mass screenings of people with higher-risk influenza, we developed a radar system based on a neural network and the fuzzy clustering method for screening of influenza. infrared thermography has been applied as a means of fever screening at airports for almost 10 years, having been implemented after the severe acute respiratory syndrome outbreak of 2003. 5e8 however, the taking of an antifebrile drug results in the rapid modification of the body temperature and directly affects the efficacy of the thermography. some recent studies have indicated that fever screening using thermography does not provide a satisfactory method of detecting febrile passengers. 9 considering the defective fever screening method, we previously developed a noncontact screening system for performing medical examinations within 10 s using measured vital signs (i.e. heart rate, respiration rate, and facial temperature). 10 as a result of being infected, not only body temperature but also heart and respiration rates will invariably increase. therefore by adding heart and respiration rates as new screening parameters, the system provided a higher screening sensitivity than using thermography alone. infection screening using multiple vital signs presents a multi-dimensional data classification problem, given that it is complex and exhibits non-linear boundaries. since a neural network provides an efficient method of classifying multi-dimensional data, we have proposed a method that uses a neural network to distinguish influenza patients from normal control subjects. this method was developed in our previous study, which uses kohonen's self-organizing map 11 (som) and the k-means clustering algorithm 12 (a non-linear clustering algorithm). 13 the advantage of using som together with the non-linear clustering algorithm is that it allows the specification of any number of classification groups, not just two. therefore, it is rational to increase the number of groups to three to investigate whether the higher-risk patients can be gathered together into a newly created group. in present study, we enhanced the som by incorporating a fuzzy clustering method (fcm) to cluster the subjects into three groups, i.e. a higher-risk influenza (hr-i) group, a lower-risk influenza (lr-i) group, and a non-influenza (non-i) group. fcm is a non-linear clustering method that is used in a wide range of fields, including biometric recognition, pattern recognition, and medical data mining. 14e16 unlike the k-means clustering method, fcm supports the use of classified data, which may belong to more than one group but with different degrees of membership. the membership represents the probability that the data belongs to a specific group based on fuzzy logic. therefore, fcm is suitable for classifying multi-dimensional data without clearly defined boundaries, such as our multiple vital-signs data. the aim of this study was to evaluate the efficacy of the radar screening system for detecting higher-risk influenza patients in clinical settings. we tested the system at the japan self-defense forces central hospital during the 2012e2013 influenza season. the neural-network-based infectious disease screening radar system we redesigned our previously developed system 10,17 to improve its portability and stability. the main advantage of this portable system is that it can be used in confined spaces such as inside an aircraft. 18 the system consists of three biosensors, namely, a thermograph to monitor facial temperature (nec/avio infrared technologies co., ltd., c-200, japan), a 10-ghz microwave radar for the noncontact determination of the respiration rate 19 (new-jrc, njr-4175, japan), and a finger-tip photo-reflector to measure the heart rate (rohm, rpr-220, japan). all of the figure 1 a pulse oximeter module was used to measure the spo 2 levels. the respiration rate was measured using the 10-ghz respiration radar by monitoring the respiratory motion of the chest, the heart rate was measured by a finger-tip photoreflector, and the facial temperature was measured by means of thermography. the thermograph was placed 45 cm from the subject's face, and the respiration radar was placed 30 cm from the subject's chest. biosensors were integrated into a single instrument body measuring 27 cm long, 28 cm wide, and 10 cm thick. the system is illustrated in fig. 1 . the signals from the biosensors are sent to a laptop computer, where they are analyzed and displayed in real time. the software environment was developed in labview (national instruments, austin, texas, usa) for recording the signals and in matlab (mathworks, natick, ma, usa) for calculating the neural network based discriminant function. within 10 s, the pulse waves, respiratory curves, and facial thermal image are displayed on the laptop screen. the screening results (i.e. '0 z non-influenza', '1 z lower-risk influenza', or '2 z higher-risk influenza') are obtained by the neural network and fuzzy clustering algorithm, based on the derived multiple vital signs (fig. 2) . the present study was carried out at the japan self-defense force central hospital from january to february 2013. a total of 35 patients, admitted with influenza-like illnesses were diagnosed as having seasonal influenza. the patients were all male and were self-defense forces members in japan with an average age of 22 years (18e35 years). all of patients were treated with an anti-viral medication (oseltamivir or zanamivir). their axillary temperatures averaged 37.2 ae 0.8 c (35.7 c body temperature 39.1 c). all of the 48 normal control subjects were male students at tokyo metropolitan university with no symptoms of fever, headache, or sore throat. the average age of the normal control subjects was 21 years (18e30 years). their axillary temperatures averaged 36.6 ae 0.4 c (34.9 c body temperature 37.1 c). measurements using the screening system were performed between 10:00 a.m. and 12:00 noon, with all of the above-mentioned influenza patients and normal control subjects being examined. the heart rate, respiration rate, facial temperature, and spo 2 of each subject were determined using the system. the axillary temperatures of both the influenza patients and normal control subjects were measured using a clinical thermometer (terumo, c220, japan). the study was approved by the ethics committee of the japan self-defense force central hospital and the committee on human research of the faculty of system design, tokyo metropolitan university. all subjects gave their informed written consent. classification of patients with higher-risk of influenza by using a neural network and the fuzzy clustering method to assess the possibility of identifying higher-risk influenza patients, we enhanced our previous version 13 of the neural network by using fcm to increase the number of classification groups. the neural network and fcm were created in matlab by using neural network toolbox 7.0.1 and fuzzy logic toolbox 2.2.18. a two-layer neural network, with an input layer and an output layer, was constructed. the input layer has three inputs which are derived parameters: heart rate, respiration rate, and facial temperature. the proposed clustering algorithm consists of two steps, as follows (fig. 3 ). the pulse wave, respiratory curves, and facial thermal image are displayed on the laptop screen, followed by the screening result ('0 z non-influenza', '1 z lower-risk influenza', or '2 z higher-risk influenza') obtained by the som and fuzzy clustering algorithm. firstly the vital-sign data for all of the 83 subjects, that are, the 35 influenza patients and 48 normal control subjects, were used to create som clusters. the som clustering results were visualized on a color-coded, twodimensional map based on a unified distance matrix. however, it proved the difficulty of detecting a specified number of clusters simply by visually inspecting the twodimensional som map. as a second step, to reduce the number of som clusters to three, fcm was applied to identify the three specified clusters. fcm is based on the minimization of an objective function j, which is defined as follow. 20 where u ij is the degree of membership of each item of data between the cluster center, and the membership represents the probability of the data belonging to a specific group based on fuzzy logic. the k k is the distance between the data x j and cluster center v i , c is the number of clusters, n is the number of items of data, and m is the degree of fuzziness. fcm iteratively updates the membership function u ij and cluster center v i until the maximum difference between cluster center is reached. finally, the screening results ('non-i', 'lr-i', or 'hr-i') can be obtained from the output layer. we evaluated the performance of the screening by using som and fcm to detect influenza patients by calculating the clinical sensitivity, specificity, positive predictive value (ppv), and negative predictive value (npv). moreover, the classified vital-sign values among the non-i group, the lr-i group, and the hr-i group were compared using the nonparametric kruskalewallis one-way analysis. a p-value of less than 0.05 was considered to indicate statistical significance. spo 2 level (<96%) was used as a reference to evaluate whether a patient can be placed in the hr-i group, since one of the most significant features of higher-risk influenza patients is their lower spo 2 level. 21 a comparison of the spo 2 levels of the patients in the hr-i and the lr-i groups was conducted by using the chi-squared test. statistical analysis was performed using statmate iii (atms, tokyo). the screening results of som and fcm are shown as a threedimensional scatter plot in fig. 4(a) . the 83 subjects were divided into three groups: 17 were placed in the hr-i group, indicated by red spheres and cone dots bounded by red ellipses; 26 were placed in the lr-i group indicated by the blue sphere dots bounded by blue ellipses; and 40 were included in the non-i group indicate by the green sphere dots bounded by green ellipses. the screening results are summarized in fig. 4(b) . the 43/83 subjects were classified into the influenza group including 34 influenza patients (hr-i and lr-i) and 9 misclassified normal control subjects; 40/ 83 subjects were classified into the non-i group including 39 normal control subjects and one misclassified influenza patient. the corresponding sensitivity, specificity, ppv, and npv were 97.1%, 81.3%, 79.1%, and 97.5%, respectively. the spo 2 value was used as a reference to determine the stage of influenza infection. with an spo 2 cut-off value of 96%, the hr-i group and the lr-i group were compared. the details are summarized in table 1 . ten out of the 17 hr-i group cases exhibited an spo 2 of less than 96%, whereas only two out of the 26 lr-i group cases exhibited an spo 2 of less than 96%. the chi-squared test revealed significant differences between the hr-i group and the lr-i group (c 2 z 13.36; degree of freedom z 1; p < 0.001). in the non-i group, there were no subjects with an spo 2 of less than 96%, which indicates that no higher-risk influenza patients were misclassified as normal. to investigate the clustering tendency, the vital-signs data and reference data are shown in part in table 2 . the 17 influenza patients classified into the hr-i group all have an spo 2 level of less than 96%, or have notably increased vital signs. moreover, the vital-sign values and spo 2 were compared for the non-i, the lr-i, and the hr-i groups. fig. 5a shows that the average heart rate differed significantly among the three groups (non-i z 68 bpm, lr-i z 84 bpm, hr-i z 98 bpm; p < 0.05). fig. 5b shows that a significant difference was identified between the non-i and the lr-i groups (p < 0.05), but there is no significant difference between the lr-i and the hr-i groups in terms of respiration figure 3 schematic representation of the som combined with fuzzy clustering algorithm to create a non-linear discriminant function for distinguishing the hr-i and lr-i groups from the non-i group. rate (non-i z 16 bpm, lr-i z 19 bpm, hr-i z 20 bpm). fig. 5c shows that a significant difference was found between the non-i and the lr-i groups (p < 0.05), but there is no significant difference between the lr-i and the hr-i groups in terms of facial temperature (non-i z 35.7 c, lr-i z 36.3 c, hr-i z 37.0 c). for the reference spo 2 data (fig. 5d) , there was a significant difference among the three groups (non-i z 97%, lr-i z 96%, hr-i z 95%; p < 0.05). in march 2013, the first human infection by the novel influenza a (h7n9) virus was reported in mainland china. 22 an influenza virus such as h7n9 can trigger severe pneumonia or acute respiratory distress syndrome, which results in significant morbidity and mortality. 23, 24 when a novel influenza virus emerges, enhanced public health surveillance is essential during the epidemic season. to attain this, we set out to develop this screening system for the mass screening of infected individuals, based on multiple vital signs. the infection screening radar system quickly measured the vital signs and comprehensively analyzed the derived data by using a neural network in real time. the most significant advantage of this system is that it can be used to detect influenza patients who have taken medication with normal body temperature. in the present study, although the 35 influenza patients were treated with anti-viral medication and more than half of the patients had normal body temperature, our system attained higher detection sensitivity than that reported in some recent studies using only thermography. 9, 25 this higher sensitivity can be attributed to the fact that, those patients even with normal body temperature under antifebrile medication, exhibited relatively high rates of heart and respiration in comparison with the normal control subjects. the idea of using vital signs stems from the fact that infectious diseases are associated with inflammation when patients become symptomatic. body temperature, heart, and respiration rates figure 4 results of som screening with fuzzy clustering algorithm, shown as a three-dimensional scatter plot. the 83 subjects were divided into three groups: the non-i group (n z 40), lr-i group (n z 26), and hr-i group (n z 17). the 43/83 subjects were classified into an influenza group (red and blue clusters) including 34 influenza patients and 9 misclassified normal control subjects; 40/83 subjects were classified into a non-influenza group (green cluster) including 39 normal control subjects and one misclassified influenza patient. the corresponding sensitivity, specificity, ppv, and npv were 97.1%, 81.3%, 79.1%, and 97.5%, respectively. are also included in the diagnostic criteria for the systemic inflammatory response syndrome (sirs). in addition, the abnormal white blood cell (wbc) count (>12,000/mm 3 , <4000/mm 3 , or >10% bands) are also included in the sirs diagnostic criteria. however, testing for wbc count requires a blood sample, and this would not be compatible with a fast-screening process. therefore, we did not adopt wbc count as a screening parameter in this study. furthermore, it is important to know the extent to which the screening parameters affect the results while performing a multiple-parameter-based screening for infection. therefore, the classified parameters were compared statistically (fig. 5) ; the most informative parameter was heart rate, while facial temperature and respiration rate were the second most informative parameters. in this study, our enhanced neural network (i.e., kohonen's self-organizing map) combined with the fuzzy clustering method showed a sensitivity of 97.1% and a npv of 97.5%. these results are comparable to our previous work, in which we used a k-means clustering algorithm. 13 more importantly, the proposed optimal neural network and fuzzy clustering method were used to classify the multiple-dimensional vital-sign data to detect higher-risk influenza patients. the high level of accuracy of the table 2 the vital signs and reference data (spo 2 and axillary temperatures) of higher-risk influenza (hr-i) group, a lower-risk influenza (lr-i) group, and a non-influenza (non-i) group are shown in part in this figure 5 heart rate, respiration rate, facial temperature, and spo 2 compared within the hr-i group, lr-i group, and non-i group. automatic infection screening system has a number of clinical implications. the system can be used as a first step for screening infectious patients at an emergency outpatient unit or at an airport quarantine station. the proposed system also appears to offer a promising means of identifying and selecting higher-risk groups for further assessment. these features enable the system to be used for preventing secondary exposure of physicians during outbreaks of highly pathogenic infectious diseases such as the ebola virus disease. however, the main limitation of our study was that it was conducted in a specialized hospital. the subjects were inpatients and were from a limited age group (18years) and were all of the same gender. the system should be further tested with a large and completely random sample of influenza patients. in summary, the neural network and fcm could efficiently detect higher-risk influenza patients within 10 s using multiple vital signs. our system has the potential to serve as a helpful tool for rapid screening of infectious diseases in clinical settings at places of mass gathering. avian influenza a (h5n1) infection in humans clinical review: primary influenza viral pneumonia mortality associated with influenza and respiratory syncytial virus in the united states pandemic flu: clinical management of patients with an influenza-like illness during an influenza pandemic analysis of ir thermal imager for mass blind fever screening the use of infrared thermometry for the detection of fever mass screening of suspected febrile patients with remotesensing infrared thermography: alarm temperature and optimal distance field test studies of our infrared-based human temperature screening system embedded with a parallel measurement approach fever screening during the influenza (h1n1-2009) pandemic at narita international airport a novel screening method for influenza patients using a newly developed non-contact screening system the self-organizing map some methods for classification and analysis of multivariate observations a novel infection screening method using a neural network and k-means clustering algorithm which can be applied for screening of unknown or unexpected infectious diseases fuzzy c-means clustering with spatial information for image segmentation robust information gain based fuzzy c-means clustering and classification of carotid artery ultrasound images a robust fuzzy local information c-means clustering algorithm the development of a non-contact screening system for rapid medical inspection at a quarantine depot using a laser doppler blood-flow meter, microwave radar and infrared thermography a portable infection screening system designed for onboard entry screening based on multi-parameter vital signs noninvasive microwave measurement of respiration a possibilistic fuzzy cmeans clustering algorithm what is the role of pulse oximetry in the assessment of patients with community-acquired pneumonia in primary care? human infection with a novel avian-origin influenza a (h7n9) virus zanamivir for the treatment of influenza a and b infection in high-risk patients: a pooled analysis of randomized controlled trials emerging risk of h7n9 influenza in china international travels and fever screening during epidemics: a literature review on the effectiveness and potential use of non-contact infrared thermometers this research was supported by the tokyo metropolitan government asian human resources fund and the japan society for the promotion of science research fellowships for young scientists (13j05344). the authors declare no conflicts of interest. key: cord-257489-ruf4rzxm authors: kee, sae yoon; lee, jin soo; cheong, hee jin; chun, byung chul; song, joon young; choi, won suk; jo, yu mi; seo, yoo bin; kim, woo joo title: influenza vaccine coverage rates and perceptions on vaccination in south korea date: 2007-06-28 journal: j infect doi: 10.1016/j.jinf.2007.04.354 sha: doc_id: 257489 cord_uid: ruf4rzxm objective: this survey was performed to assess the level of influenza vaccine coverage, to understand the driving forces and barriers to vaccination and determine vaccination interventions for the following year in korean population. methods: a national sample of 1720 community dwelling adults of age 18 and older were surveyed by individual visits during april 2005. demographics, state of influenza vaccination, reasons for vaccination or non-vaccination and perceptions on vaccinations were asked by questionnaire. results: influenza vaccination coverage in general population and high risk group was 34.3% and 61.3%, respectively. predictors for vaccination were ≥65 of age, performance of regular exercise, vaccination in the previous season, experience of influenza-like illness, belief that vaccine can prevent common cold and opinion that vaccine must be taken annually. the most common reason for vaccination for both whole population and high risk groups was to prevent both influenza and common cold, while the most common reason for non-vaccination was the thought that he/she was healthy enough not to be in need for vaccination. having more information on influenza and vaccination as well as doctor's recommendation for vaccination appeared to be the most important modus operandi to encourage influenza vaccination among non-vaccinees. conclusions: doctor's recommendation was the most important factor in encouraging people to be vaccinated against influenza. doctors should be geared up with precise information and actively encourage high risk population in order to increase vaccination coverage. summary objective: this survey was performed to assess the level of influenza vaccine coverage, to understand the driving forces and barriers to vaccination and determine vaccination interventions for the following year in korean population. methods: a national sample of 1720 community dwelling adults of age 18 and older were surveyed by individual visits during april 2005. demographics, state of influenza vaccination, reasons for vaccination or non-vaccination and perceptions on vaccinations were asked by questionnaire. results: influenza vaccination coverage in general population and high risk group was 34.3% and 61.3%, respectively. predictors for vaccination were !65 of age, performance of regular exercise, vaccination in the previous season, experience of influenza-like illness, belief that vaccine can prevent common cold and opinion that vaccine must be taken annually. the most common reason for vaccination for both whole population and high risk groups was to prevent both influenza and common cold, while the most common reason for non-vaccination was the thought that he/she was healthy enough not to be in need for vaccination. having more information on influenza and vaccination as well as doctor's recommendation for vaccination appeared to be the most important modus operandi to encourage influenza vaccination among non-vaccinees. influenza causes significant morbidity in both healthy population and patients with high risk conditions. healthy adults may suffer from high fever, headache and myalgia, whereas clinical manifestations are more serious in high risk patients such as elderly or patients with comorbid conditions and may even cause death due to respiratory complications. 1e3 the clinical course of influenza differs by age, immune status, characteristics of circulating influenza strains, comorbidities and pregnancy status. 4 changes at antigenic sites of influenza virus render a new strain that can avoid the immunity induced by previous strains, thus causing influenza epidemics. 5 the most effective way of preventing influenza is to immunize with vaccines made after prediction of antigenic variation. in one study, inactivated vaccine showed efficacy of 86% reduction in influenza-like illness in healthy adults when vaccine strain was well matched with predominant circulating strain. 6 although antibody production rate is lower in people over the age of 65, various studies proved influenza vaccine to be effective in reducing influenza related diseases and complications, hospitalizations and mortality in this group. 7e11 the priority group who are recommended for annual vaccination includes persons aged !65 years, persons with chronic illness such as chronic cardiopulmonary disease, diabetes, chronic liver disease and malignancy, residents of long term care facilities, health-care personnel and pregnant women. 12 the center for disease control and prevention is expanding the priority group for vaccination in recognition of the significance of influenza and importance of vaccination. the priority group for influenza vaccination have been also expanded in korea; pregnant women and persons aged 50e64 years were newly added in 2003 and children of age 6e23 months were added in 2004. 13 people working in organizations dealing with sars (severe acute respiratory syndrome) have been newly added in response to the movement of cdc. influenza vaccine production and import are increasing in korea; while vaccines for 8e10 million people, which can cover about 19% of total population, were supplied in the season 2002e2003, vaccine for 15 million people were distributed in the season 2003e2004. in the season 2004e 2005, vaccines for 17 million people were supplied, and according to the sales statistics, it is estimated that 33% of total population have been vaccinated. 13 these percentages are comparable to other countries: fedson 14 reported in 2000 that influenza vaccine distribution per 1000 population was 183 doses in korea, and this number is relatively high compared to northern america (265 doses), western europe (170 doses), southeast asia (0.04 dose) and worldwide (37 doses). 15 vaccine distribution rate grew even higher to 359 doses per 1000 population in 2004. 13 korea shows relatively high influenza vaccine distribution rate, however, exact vaccination coverage among total population or priority group have not yet been studied. the korea centers for disease control and prevention set a goal to increase vaccination rate in the priority group to reach at least 60%. 13 nevertheless, vaccination coverage rate has been calculated according to the sales record, and nationwide vaccination rate by self-report of the whole population or priority group has never been studied. precise identification of vaccination rate in the whole population as well as high risk groups is urgently needed in order to accomplish objectives of influenza vaccination policy. therefore, the aim of this study was to investigate the level of influenza vaccination coverage in adults and high risk groups, identify factors related to vaccination and opinions about influenza and influenza vaccine, and discover the way to increase vaccination coverage. this is a population based cross-sectional descriptive study. the target study population included non-institutionalized persons aged !18 years living in south korea. the survey was conducted by gallup korea â , a professional research company, and face-to-face interviews were performed by 80 trained professional interviewers from 19 to 29 april 2005. in order to represent the total population, multistratified random sampling according to the principle of proportionate probability sampling was adopted to select the subjects. south korea is divided into eight provinces and seven cities and each province or city is further subdivided and stratified into 4e5 units. the number of households to be interviewed in each administrative district was calculated and decided proportionately according to the location and sizes of the district, age and gender. the statistics of 2005 from the national statistical office was used for the calculation. 16 if the selected household could not be surveyed, an alternative household was chosen in the same manner. before the interview, the interviewer explained the purpose of the study to all the subjects and verbal informed consent was obtained by respondents who agreed to participate. the questionnaire contained 22 questions. data on demographics such as age, gender, level of education, and level of income were obtained. questions about drinking, smoking and exercise habits and comorbid conditions were asked. the interview continued with asking whether or not the respondent was vaccinated in the season 2004e2005 and 2003e2004. if the respondent was vaccinated in the season 2004e2005, further questions on the reason of vaccination was asked. thirteen reasons were presented, and respondents were to choose as many as they wish. for non-vaccinated respondent, the reasons of non-vaccination were asked in a form of multiple choice questions with 15 reasons. six yes-or-no questions on opinions about influenza vaccine were presented to all respondents. further 11 yes-or-no questions about opinions on influenza and influenza vaccination were presented to high risk group. all respondents were asked whether they intended to have vaccinated in the following season. regular exercise was defined as performing exercise more than once a week, smoker as currently smoking, and regular alcohol consumer as drinking alcohol more than twice a week. high risk group was defined as either age ! 65 years or having comorbid conditions. comorbid conditions included cardiovascular diseases such as congestive heart failure and myocardial infarction, diabetes, lung diseases including asthma and chronic obstructive pulmonary disease, chronic liver diseases including chronic hepatitis and liver cirrhosis, and malignancy. univariate analysis of factors associated with vaccination/non-vaccination was performed using c 2 test and fischer's exact test. to describe statistical significance, the 95% confidence interval (ci) was computed. in logistic regression, gender, presence of comorbid conditions, age (!65), level of education, size of dwelling town, monthly income, smoking habit, drinking habit, exercise habit, vaccination in previous season, history of influenza-like illness and six opinions about influenza were included. all statistical analyses were performed using the spss 10.0ko for windows (spss inc.) of the total responses from 1730 subjects, 10 insincere responses were excluded and, therefore, data of 1720 (99.4%) subjects were analyzed. mean age was 43.1 ae 14.59 years and 848 subjects (49.3%) were male. one hundred and seventy-four subjects (10.1%) were !65 years and 224 subjects (13.0%) had one or more comorbid conditions; 328 subjects (19.1%) were classified as high risk group. the coverage rates for influenza vaccination were 34.3%, 61.3%, 79.7%, and 54.9% among total adult population, high risk group, persons aged !65 years and persons with comorbid conditions, respectively (table 1) . influenza vaccination coverage was higher in females, increasing with age (18.3% in age 18e29 years versus 84.8% in age ! 70 years), and in persons with any comorbid condition. as for the socio-demographic variables, the likelihood of receiving the vaccine increased when the education level was lower, the size of town was smaller, and the income level was lower. persons on regular exercise, non-smokers and not so regular alcohol consumers showed higher vaccination rates compared to subjects not doing regular exercise, current smokers and regular alcohol consumers, respectively. persons who had been vaccinated in the preceding season (2003e2004) and those with a history of influenza-like illness also showed higher rates (table 1) . reasons for vaccination among vaccinees are described in table 2 . the most common reason for vaccination in total population was 'to prevent not only flu but also common cold' (79.5%), followed by 'influenza being a serious disease' (29.0%), 'recommendations from friends or family members' (22.2%) and 'received information from mass media' (11.5%). reasons such as 'having seen people get sick/ die from flu' (11.2%), 'not in good health' (10.7%), 'doctor's advice' (7.8%), 'have chronic disease' (5.4%) were among less common reasons. the most common reasons for vaccination were not different in high risk group, however, 'have interest in vaccination because of bad health status' showed higher rank (18.4%) than the total population. reasons for refusal among non-vaccinee are described in table 3 . in total population, 'perception of good health' was the most common cause of non-vaccination (70.5%) followed by 'not enough time' (26.0%), 'troublesomeness of vaccination' (18.4%), 'distrust in the effectiveness of vaccine' (11.3%), and 'missed vaccination time' (10.5%). the rank of reasons for non-vaccination was not different in high risk group but less people (58.3%) chose 'in good health' as the reason. factors influencing future vaccination are summarized in table 4 . 'more information on importance of vaccination' (27.7%) was the most common factor to increase the drive for vaccination, followed by 'recommendation from doctors or nurses' (27.4%). in the high risk group, this rank was reversed, and doctors or nurses' recommendation was the most influential factor for future vaccination (38.1%). other options included 'if vaccine is cheaper' (18.8% in total population and 21.3% in high risk group), 'if more information is provided about influenza' (18.8% and 15.0%, respectively), 'if i have enough time' (18.0% and 18.9%, respectively), 'if i can get vaccinated at workplace' (8.1% and 10.2%, respectively) and 'if there is a way other than shots' (2.9% and 5.5%, respectively). of the total population 17.3% and of the high risk group 16.5% were negative about getting vaccinated and answered 'i would not take it in any situation'. opinion about influenza vaccine is described in table 5 . more than 60% of both vaccinees and non-vaccinees agreed that 'vaccine can prevent influenza' and 'vaccine is safe'. more vaccinees compared to non-vaccinees agreed that 'vaccine can prevent common cold' and 'vaccine should be taken annually'. however, more non-vaccinees thought vaccine was expensive. less than 20% of vaccinees and non-vaccinees thought that 'you never get influenza once you are vaccinated'. in all opinions, the difference in the percentages of vaccinees and non-vaccinees were statistically significant. further questions were presented to persons of the high risk group. most of both vaccinees and nonvaccinees agreed that complications of influenza might be serious (93.5% and 90.6%, respectively) and that they had chance to hear about influenza and influenza vaccination from mass media (77.1% and 70.9%, respectively). however, more vaccinees of the high risk group compared to non-vaccinees agreed on the following opinion; influenza might be dangerous to high risk group, influenza might aggravate underlying diseases, vaccination might reduce chances of hospitalizations, and vaccination might reduce expenses for extra medication. furthermore, more than 60% of vaccinees agreed that they were at high risk of catching influenza, and at bad health, and that acquaintances advised them to get vaccinated, however, less than 50% of non-vaccinees agreed on the same opinion. in comparison, nearly 50% of non-vaccinees thought themselves to be in good health whereas only 12.4% of vaccinees thought the same way. also, although more vaccinees than nonvaccinees were advised to get vaccinated, it was less than 50% in both groups (table 6) . results of multivariate analysis to determine factors associated with vaccination are summarized in intention for vaccination in the next season was as follows: 43.3% of total subjects and 68.9% of the high risk group were willing to get vaccination. self-reported influenza vaccination coverage of 34.3% in this study corresponded well to the percentage estimated from the number of vaccine doses sold (33%). 13 moreover, the coverage in high risk group met the target set by korean cdc (>60%). these coverage rates in korea in the season 2004e2005 is relatively high, compared to the coverage in the united states (8.8% in whole population and 42% in high risk groups) and europe (19e24% in priority group 17 ). nevertheless, the rates are not satisfactory enough, because who set the goal of attaining vaccination coverage of the elderly population to at least 50% by 2006 and 75% by 2010 18 and more efforts are needed to increase the coverage rates. in univariate analysis, people of older age or persons having comorbid condition were more likely to get vaccinated, which is in concordance with studies from other countries. 19e21 since these two groups are the main target for vaccination, it implies that vaccination program in south korea is quite successful. people with healthy lifestyle habits such as regular exercise, non-smoking and no regular alcohol consumption also had higher vaccination rate. people with healthy lifestyle may have more interest in general health, seek for preventive health care and therefore are more willing to get vaccinated. vaccination coverage in females was significantly higher in univariate analysis, and similar result was shown in another study. 17 the fact that more females (56% versus 44% males) were !65 years who had higher vaccination rate might be the explanation in south korea. interestingly, vaccination coverage was higher among people of lower education level, and lower income and living in smaller towns. this may be partially explained by the fact that both persons !65 years and persons with chronic illnesses are more likely to be undereducated and have lower income, as is shown by south korean statistics, 22 and similar results were also presented by jimenez et al. 23 the government policy to administer influenza vaccine free of charge to low income group at public health centers may be another explanation: survey showed that people vaccinated at public heath centers were older, and had lower level of education and were living in a smaller town (data not shown). to prevent common cold as well as flu was the most common reason for vaccination. this is concordant with the high percentage of agreement (62.2%) that vaccine can prevent common cold. also, the perception that 'influenza vaccine can prevent common cold' was a predictor for vaccination. this idea might have been responsible for the increase of vaccination rate, however, wrong attitude due to wrong knowledge must be corrected. self-perception of bad health, interest in vaccination and chronic illness were common reasons for vaccination in high risk group, showing their interest in health. 'confidence in health' was the most common reason for non-vaccination (60%) in both all adults groups and high risk groups, followed by 'being too busy', 'because it is troublesome' and 'miss vaccination time'. among nonvaccinees with non-vaccination reason of 'miss vaccination time', 49% were willing to get vaccination in the following season. 'not believing in the effectiveness of vaccination' accounted for about 11% of the responses. these results led us to suggest some intervention to increase vaccination uptake: more efforts should be paid to convince people in the priority group who are at high risk, and to provide information on influenza and effectiveness of vaccination to increase vaccination motive. also, improvement of accessibility to vaccines such as providing vaccination at workplace may contribute to an increase of vaccination uptake. less than 1% of non-vaccinees reported 'side effects of vaccination' or 'fear of getting influenza by vaccination' as the reason for non-vaccination. the above result is different from other studies 21, 24 and implies that people have correct knowledge on side effects of vaccines. health-care workers' recommendation for vaccination was the most important factor to influence future vaccination habit in high risk group, in agreement with other studies. 20,21,23,25e27 booth et al. 28 reported that 71e82% of general physicians recommend vaccination to priority group, and song et al. 29 showed that reminding persons of age ! 65 to get flu shots by telephone calls or postcards significantly increased vaccination rate. in this present study, recommendations to the high risk groups by doctors and public health centers were 10% and 3%, respectively, inferring that recommendation rate from doctors in clinical practice is very low. perenboom and davidse 30 reported that active recommendation to persons with chronic illness increased the rate of vaccination from 42% to 75.5%. therefore, the role of health-care workers, especially doctors, appears to be very important in increasing vaccination rate, and therefore, they should give active recommendations. in the high risk group most of the persons were aware of the fact that influenza is a serious disease and it may be more dangerous or produce more complications in persons with chronic illness. furthermore, more than half of them believed that influenza vaccination might reduce hospital admission and extra medical expenses, showing that they have correct perception on influenza and influenza vaccine. however, while more than 60% of vaccinees in the high risk group agreed that they were not in good health and at high risk of catching influenza, and they are interested in vaccination because of bad health, only 28.3% and 42.5% of non-vaccinees, respectively, agreed on that. also, 48.3% of vaccinees were advised to get vaccinations while only 28.3% of non-vaccinees did receive the advice. this shows apparent difference in the perception of one's health between vaccinees and non-vaccinees and, therefore, efforts should be made to inform people about the priority group of vaccination in order to increase coverage rate. forty-three and three-tenth of total subjects and 68.9% of the high risk groups were willing to get vaccination in the coming season, and the percentage in high risk groups exceeds the rate in the season 2004e2005 as well as the target of korea cdc (61.3% and 60%, respectively). 13 persons who had been vaccinated previously were more willing to have vaccination in the following season (table 7) , and this correlates with other studies 24,31,32 that previous vaccination is the most significant predictive factor for future vaccination. moreover, belief that 'vaccine must be taken annually' was a predictor for vaccination. efforts to increase vaccination rate in priority group for at least one season may have influence over vaccination for several years. this may be particularly useful in the situation of vaccine shortage, when it is recommended by authorities that supply of vaccines should take precedence to priority group. 33 the strength of this study lies in the fact that survey was conducted on individual interview basis and meanings of questionnaire were explained thoroughly even to the elderly, and thus receiving precise answers. there are some limitations in the study. first, high risk group consisted of only persons !65 or persons with comorbid condition, and therefore, the whole priority group were not included in the analysis. secondly, the survey was conducted in april, when it was past the influenza season and therefore recall bias might have occurred. thirdly, the presence of comorbid condition and vaccination uptake were totally relied on self-reports of the subjects and therefore actual presence of illness or vaccination uptake might have been over-or under-estimated. in summary, the significance of influenza and importance of vaccination were well perceived, especially, among the high risk groups and 43.3% in total population and 68.9% of the high risk group showed intention to have vaccination, which is very encouraging. since giving correct information and health-care personnel's recommendation to vaccination would greatly influence vaccination rate, doctors should be geared up with precise information and actively recommend them to get 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vaccination in persons younger than 65 years with high-risk medical conditions: the prisma study prevention and control of influenza, recommendations of the advisory committee on immunization practices pandemic influenza and the global vaccine supply the macroepidemiology of influenza vaccination (miv) study group. the macroepidemiology of influenza vaccination in 56 countries korea statistics information system influenza vaccination coverage rates in five european countries-a population-based cross-sectional analysis of two consecutive influenza seasons world health organization. prevention and control of influenza pandemics and annual epidemics (agenda item 14 prevalence and predictors of influenza vaccination among frail, community-living elderly patients: an international observational study influenza vaccination coverage rates in germany factors associated with influenza vaccination among elderly spanish women influenza coverages in spain and vaccination-related factors in subgroup aged 50e64 predictors of influenza vaccine acceptance among healthy adults crosssectional study on influenza vaccination factors affecting influenza vaccination among attendees at a senior center factors associated with influenza and pneumococcal vaccination behavior among high-risk adults implementation of influenza immunisation policy in general practice: 1997 to 1998 effectiveness of telephone and postcard reminders for the influenza vaccination: a study in the elderly who have visited a family practice center in a tertiary care hospital increasing the coverage of vaccination against influenza by general practitioners patient acceptance of influenza vaccination influenza vaccination. knowledge, attitudes, and behavior among high-risk outpatients update: influenza vaccine supply and recommendations for prioritization during the 2005e06 influenza season. mmwr morb mortal wkly rep:850 key: cord-265772-diahoew3 authors: zhang, yue; li, jianguo; xiao, yan; zhang, jing; wang, ying; chen, lan; paranhos-baccalà, gláucia; ren, lili; wang, jianwei title: genotype shift in human coronavirus oc43 and emergence of a novel genotype by natural recombination date: 2014-12-18 journal: j infect doi: 10.1016/j.jinf.2014.12.005 sha: doc_id: 265772 cord_uid: diahoew3 background: human coronavirus (hcov) oc43 is the most prevalent hcov in respiratory tract infections. its molecular epidemiological characterization, particularly the genotyping, was poorly addressed. methods: the full-length spike (s), rna-dependent rna polymerase (rdrp), and nucleocapsid (n) genes were amplified from each respiratory sample collected from 65 hcov-oc43-positive patients between 2005 and 2012. genotypes were determined by phylogenetic analysis. recombination was analyzed based on full-length viral genome sequences. clinical manifestations of each hcov genotype infection were compared by reviewing clinical records. results: sixty of these 65 samples belong to genotypes b, c and d. the remaining five strains had incongruent positions in the phylogenetic trees of the s, rdrp and n genes, suggesting a novel genotype emerging, designated as genotype e. whole genome sequencing and bootscan analysis indicated that genotype e is generated by recombination between genotypes b, c and d. temporal analysis revealed a sequential genotype replacement of c, b, d and e over the study period with genotype d being the dominant genotype since 2007. the novel genotype e was only detected in children younger than three years suffering from lower respiratory tract infections. conclusions: our results suggest that hcov-oc43 genotypes are evolving. such genotype shift may be an adapting mechanism for hcov-oc43 maintaining its epidemic. coronaviruses (covs), belonging to the family coronavirinae, are a large group of viruses with a broad infection spectrum in human and animals. covs are related to respiratory tract disorders, gastroenteritis, as well as to systemic and neurological diseases. 1 covs are the largest rna viruses, containing a positive-sense, single-stranded rna genome with a length of 27,000e31,500 nucletides. 1, 2 based on genome phylogeny and serological characterization, covs are divided into four genera, alphacoronavirus (a-cov), betacoronavirus (b-cov), gammacoronavirus (g-cov), and deltacoronavirus(d-cov). 1e3 since the isolation of hcov-229e and -oc43 in 1960s, a total of six hcov species have been identified, including severe acute respiratory syndrome cov (sars-cov) in 2003, nl63 and hku1 in 2004, and middle east respiratory syndrome cov (mers-cov) in 2012. 1, 4 hcovs belong to a-(229e and nl63) and b-genera (oc43, hku1, sars-cov and mers-cov). hcovs were previously not considered to be of great importance with respect to human diseases as most hcovinfections were thought to be associated with mild symptoms and occasional lower respiratory tract infections (lrtis) until an outbreak of sars in 2003. that has led to increased concerns about hcovs, while the identification of mers-cov in 2012 reinforced the public health significance of hcovs. although sars-cov is no longer detected since 2004, mers-cov continued as an epidemic, spreading to more patients and countries. this spread indicates a high adaption capability of mers-cov in humans. 5, 6 insight into the epidemic characteristics of hcovs at the molecular level will allow us to predict viral pathogenesis and transmission activities and inform hcov prevention and control, particularly against newly emerging hcovs. hcov-oc43 has been more prevalent than other common hcovs including hcov-229e, enl63 and ehku1, in pediatric and adult respiratory infections, and can also cause outbreaks in human respiratory tract infections. 1,7e10 however, our understanding of the molecular epidemiology of hcov-oc43 has been very limited. the genetic diversity of hcov-oc43 was first reported in belgium in 2005 and three clusters were identified based on the analysis of the spike (s) gene of the prototype strain atcc vr-759 and seven clinical strains. 11 subsequently, lau et al. gave the first description on the molecular epidemiology of hcov-oc43 using sequences from 29 clinical samples in 2011. 12 four genotypes, a, b, c and d, were identified based on the viral genome and the phylogeny of the main structural genes, s, rna-dependent rna polymerase (rdrp), and nucleocapsid (n) genes, and genotype d was reported to have arisen due to natural recombination. 12 however, these observations were based on only a limited number of hcov-oc43 positive cases. due to the limited availability of virus sequences, the molecular epidemiological characterization of hcov-oc43, particularly its genotyping, was poorly deciphered. in this study, we genotyped hcov-oc43 by analyzing fulllength sequences of s, rdrp, n genes and viral genomes directly from respiratory samples collected from 65 hcov-oc43 positive patients with acute respiratory tract infections (artis) recruited from 2005 to 2012. we observed a genotype shift in hcov-oc43 over the study period and confirmed the emergence of a new genotype e arising through natural recombination. patients suffering from artis were recruited from the beijing children hospital and the peking union medical college hospital in beijing, china from march 2005 to december 2012 when they seek health care at these hospitals. criteria for including patients in our study encompassed acute fever (body temperature !37.5 c) with respiratory symptoms such as cough or wheezing, normal or low leukocyte count, and with or without radiological pulmonary abnormalities. nasopharyngeal aspirates (npas) were collected from pediatric patients. nasal and throat swabs were collected from adult patients. the respiratory samples were stored in viral transport medium (vtm) at à80 c before use. clinical information of each enrolled patient was recorded in standard form and reviewed retrospectively. written informed consent was obtained from all participants or guardians on behalf of the minors/children participants. the study was approved by the medical ethic review board of the institute of pathogen biology, chinese academy of medical sciences. viral nucleic acids were extracted from 200 ml respiratory samples using a nuclisens easymag apparatus (biomérieux, marcy l'etoile, france) according to the manufacturer's instructions and were stored at à80 c until use. hcov-oc43 positive respiratory samples were tested by rt-pcr with hcov-conserved primers and were confirmed by sequencing methods as described elsewhere. 13 the presence of other common respiratory viruses was also determined as described elsewhere, including influenza virus (ifv) a, b and c, human parainfluenza virus (hpiv) 1e4, adenovirus (adv), respiratory syncytial virus (rsv) a and b, human metapneumovirus (hmpv), human bocavirus (hbov), rhinovirus (hrv) and enterovirus (hev). 14 total rna from respiratory specimens was converted to cdna using combined random primers and oligo(dt) primers and the superscript iii reverse transcription system (invitrogen, carlsbad, ca). the full-length s, rdrp, n genes and viral genomes were amplified from each respiratory specimen which was positive for hcov-oc43, using specific primers (table s1 ) with a genome walking method. pcr was performed using the following conditions: 94 c for 5 min, 40 cycles of amplification at 94 c for 30 s, 50 c for 30 s, and 72 c for 90 s, with a terminal elongation step at 72 c for 10 min. pcr products were sequenced directly using an abi 3700 dna sequencer (applied biosystems, usa). sequences were assembled manually through alignment to the reference strain hk04-02 (genbank accession no. jn129835). all hcov-oc43 sequences available in genbank (www.ncbi. nlm.nih.gov) were retrieved on may 30, 2013. the background information of all the sequences used for phylogenetic analysis is summarized in table s2 . the full-length s, rdrp, n genes, and viral genomes of hcov-oc43 were aligned using clustalw program implemented in mega 5.1 with sequences deposited in genbank. 15 pair-wise sequence identities in each region were calculated for the comparison of sequence divergence using bioedit. maximum likelihood (ml) trees were constructed with the best fit model of general time reversible with gammadistributed rate variation across sites and 1000 bootstrap pseudo-replicates implemented in mega 5.1. the bovine coronavirus was used as the outgroup sequence, but is not shown in the presented figures to make the phylogenetic relationships more clear. substitution models were selected using modeltest (version 3.7) according to the akaike information criterion. 16 phylogenetic trees of each gene region of hcov-oc43 were constructed by using the neighbor-joining method with kimura's two-parameter model and 1000 bootstrap pseudo-replicates implemented in mega 5.1. 15 to analyze the recombination events, the genomes of hcov-oc43 were aligned and analyzed using boot scanning method implemented in simplot (v3.5.1, http://sray.med.som.jhmi.edu/scroftware). distribution frequencies of hcov-oc43 genotypes were compared by using pearson's chi square test or fisher's exact test. one-way analysis of variance was used to analyze the continuous variables for population parameters. p values <0.05 were considered statistically significant. the nucleotide sequence data of s, rdrp, n genes and viral genomes of hcov-oc43 used in this study have been lodged in genbank and the accession numbers are shown in table s2 . to genotype the hcov-oc43 samples, we constructed ml trees using the full-length sequences of s, rdrp and n genes amplified from the 65 respiratory samples of hcov-oc43 positive patients in this study and compared them to those retrieved from genbank (fig. 1) . the hcov-oc43 sequences fell into four distinct clusters on the phylogenetic tree of the s gene as reported by lau et al. 12 however, incongruities were observed in ml trees of rdrp and n genes, indicating genetic diversity. briefly, oc43 strains identified in this study (designated cn strains) fell into three clusters in s gene, i.e., b, c and d genotypes, similar to those from hong kong, china (hk) and belgium (be). eleven cn strains fell into genotype b together with five 2004 hk strains and the belgium strain be03. 11, 12 the sequences of this genotype possessed nucleotide (nt) identities of 98.7%e99.6%. three cn strains and 15 hk strains formed genotype c, possessing 99.6%e99.8% nt identities; while 51 cn strains clustered with nine hk strains and a be04 strain to form genotype d, possessing 99.3%e100% nt identities. genotype a contained only the cell culture strain atcc vr-759 as previously reported. 11, 12 the strains that fell into genotype c clustered together in the ml tree of the rdrp gene, as well as in the ml tree of the s gene. strains belonging to genotype d clustered together with strains of genotype b, and these sequences possessed 99.7%e99.8% nt identities. notably, five cn strains (1783a/10, 2058a/10, 2941a/11, 3074a/12 and 3194a/12), which belong to genotype b in the ml tree of the s gene, formed a distinct clade in the tree of rdrp gene. multiple alignment of rdrp results showed that these five cn strains possessed 99.5e99.6% nt identities to b_be03, c_hk04-01 and d_hk11-01, while other b strains possessed 99.7e100% nt identities to b_be03 ( table 1 ). analysis of the n genes showed that the strains that belong to genotype b (other than the five distinct cn stains) in the ml tree of the s gene clustered together, while the strains belonged to genotype c and d in the ml tree of the s gene clustered together. the aforementioned five distinct cn strains were separated from all the known genotypes and formed two clades. multiple alignment results were consistent with our phylogenetic analysis as the five distinct cn strains had lower nt identities with representatives of b, c and d genotypes than other genotype b strains had with the reference strain, including b_be03 (97.6e98.7%), c_hk04-01 (97.6e99.1%) and d_hk11-01 (97.5e99.0%) ( table 1) . taken together, the incongruities in the phylogenetic trees together with the analysis of nt identities showed that a novel genotype, may have arisen, which we designated as genotype e. the incongruent phylogenetic pattern of the s, rdrp and n genes in the five genotype e strains, particularly the dropout of 1783a/10 from the linage formed by other genotype e strains in the phylogenetic tree of n genes, indicate the occurrence of potential recombination events. to further demonstrate the emergence of genotype e strains, we amplified the whole viral genome sequences directly from respiratory samples. we obtained the whole genome sequences of four of the five distinct strains (1783a/10, 2058a/10, 3074a/12 and 3194a/12; 2194a/11 was not available due to the very low viral load in the specimen). we then analyzed the potential recombination by constructing the phylogenetic trees of all known 23 gene regions of these four strains. ten other whole genome sequences of oc43 were used as reference strains, including be03 and 2145a/10 (genotype b), hk04-01 and 3647/06 (genotype c), be04, hk04-02 and 5240/07 (genotype d), and the atcc strain (genotype a) (fig. 2) . bovine cov (accession no. u00735) was used as outgroup sequence, which was not displayed in the figure to save spaces. we found that these four strains form a separate linage (genotype e) in the phylogenetic trees of complete genome, s, rdrp and most of the nonstructual protein (nsp) genes. these findings further confirmed that these distinct cn strains belong to a novel genotype e, despite the incongruent phylogenetic pattern was observed in ns5a, e, m and n genes. notably, in the phylogenetic trees of the nsp2-nsp6 genes, these four genotype e strains were closely related to genotype c; while clustered more closely with the strains of genotype b in the trees of nsp1, nsp8, hemagglutininesterase (he) and the s genes. strains 3074a/12 and 3194a/ 12 were also clustered together with genotype d in envelope (e) and membrane (m) genes. these results support our hypothesis that recombination events occur among oc43 genotypes. to verify these findings, we then carried out boot scanning analysis and the genome sequences of b_2145a/ 10, c_3647/06 and d_5240/07 were used as references. when the genomes of 1783a/10, 2058a/10, 3074a/12 and 3194a/12 were used as query sequences, we identified several potential recombination sites in the viral genomes of genotype e (fig. 3 ). here 3074a/12 was used as an example to show the recombination analysis results. from positions nt 1000 to 14,500, most of the region of 3074a/ 12 were closely related to c_3647/06, except positions upstream of nt 1,000, nt 2500 to 4,500, and nt 11,500 to 12,500, where 3074a/12 was closely related to b_2145a/ 10. from positions of nt 14,500 to nt 28,000, most of the region was closely related to b_2145a/10. from positions nt 28,000 to the 3 0 end of the viral genome, most of the region was closely related to d_5240/07. potential recombination sites were at the junctions of nsp2/nsp3, nsp6/nsp7, nsp9/ nsp10, nsp12/nsp13, ns5a/e and m/n corresponding to the schematic diagram of the whole viral genome (fig. 3) . these findings were consistent with the observations in phylogenetic analysis of s, rdrp and n genes described above. similar boot-scanning results were obtained when 3194/12 was used as query strain. most of the recombination sites were also found when 1783a/10 and 2058a/10 were used as query strains. however, lower similarities were found in ns5a, m and n gene regions between 1783a/10, 2058a/10 sequences and references, which indicates the diversity of parent strains of recombination. taken together, these findings indicate that natural recombination events led to the emergence of novel genotype e and suggest complicated recombination events in the circulation of hcov-oc43 strains in nature. genotype shift plays an important role in virus adaption to hosts. 17e19 to determine whether genotype shift occurred in hcov-oc43, the yearly distribution of genotypes during the study period (2005e2012) were determined. hcov-oc43 positive cases were identified for each year analyzed, and their detection rate ranged from 1.9& to 13.9& with the highest detection rates in 2007 (fig. 4) . we found that the detection rate of hcov-oc43 spiked every other year except in 2010. shifts of hcov-oc43 genotypes over time were observed. after a low level epidemic of genotypes c and b, genotype d became the major epidemic to characterize the clinical manifestations of different hcov-oc43 genotypes, the clinical data of the 65 hcov-oc43 positive cases were analyzed (detailed information of each patient is summarized in table s3 ). of the 65 cases, 28 were children less than 14 years old, one was a 16-year-old teenager, and 36 were adults more than 16 years old. patient age ranged from 0.2 to 90 years old (mean 29.6 years; median 20 years), with 33 males and 32 females ( table 2 ). in 17 (26.2%) of all patients an additional virus was co-detected. each of the genotypes showed codetection except genotype c. the most frequent codetected viruses were rsv and hrv. the age distributions in different genotypes differed significantly (one-way analysis of variance, p z 0.0094). genotype d was detected in patients with a broad age range (0.2e90 year old), although the majority (35 out of 51 cases) occurred in children and adults less than 50 years old. genotype b was detected in one young adult (21 years old) with urti in 2006, and in five children with lrtis after 2010. genotype c was detected in three older adults aged 58, 72 and 88 years with urtis, whereas genotype e was only detected in five children less than 3 years of age (0.8e2.7 years old) with lrtis. as an important human respiratory virus, the epidemic features of hcov-oc43 at molecular level have not been well addressed. in this study, we describe the molecular epidemiological features of hcov-oc43 in detail based on 65 cases. our results showed marked variations of hcov-oc43 genotype prevalence from year to year, similar to that observed in other hcovs. 7,20e22 in line with previous reports, 11, 12 genotypes b and c were detected before 12 in our study, genotype d was not detected in 2011 but in 2012, albeit at lower numbers (four out of seven hcov-oc43 positive samples). it seems that immunity developed in the human population after the wide-spread of genotype d had blocked its epidemic as the overall prevalence of genotype d showed decreased over time. additional analysis of the evolution of antigenic genes, particularly the s gene will help to further our understanding of the adaption of viral genotypes. recombination is a common phenomenon among coronaviruses. a special random template switching mechanisms can be used during rna replication. 23, 24 the high frequency of homologous recombination together with the high mutation rates of the genome may lead to the adaptation of covs and allow the generation of new strains and genotypes. 25e29 for example, recombination has been reported to generate new genotypes and to contribute to the genetic diversity in hcov-hku1 and -nl63, with recombination sites on nsp6/nsp7, nsp16/he, and nsp3, and the s genes, respectively. 17, 26 our work, which included a larger number of samples over a longer surveillance period than previous studies, shows that a novel genotype e emerged in 2010. this highlights again the role of recombination in the evolution of hcov-oc43. based on nucleotide identity comparison, phylogenetic analysis of different genes, and boot scanning analysis, genotype e might be generated from a recombination between genotypes b, c and d. potential recombination sites may be at the junctions of nsp2/nsp3, nsp6/nsp7, nsp9/ nsp10, nsp12/nsp13, ns5a/e and m/n gene. however, these observations need to be clarified based on more whole genome sequences of oc43. in addition, our results together with those of previous reports on the recombination analysis of hcovs, indicate that the amplification of genes including at least nsp2/nsp3, nsp12/nsp13 (corresponding to pol gene) and the s and n genes is needed for genotyping and recombination analysis. 12, 20, 26 the association of hcov-oc43 genotypes with disease severity has not been well defined. a previous study found that among eight genotype d positive patients, seven were diagnosed with pneumonia. 12 however, in our study, genotype d showed no associations with severe symptoms, as most of the patients suffered from urtis. this difference in results may be attributed to the studied cohort and number of positive cases. however, host immune pressure in response to genotype d during a long epidemic period may also affect virulence. all cases of the novel genotype e and those of genotype b identified after 2010 were found in children younger than three years with lrtis, but not detected in adults with lrtis or urtis. however, as the number of positive cases was limited, it is unclear whether the association of genotypes with lrtis and special age groups is significant. this association may require further investigations for a larger number of samples. in addition, it should be further investigated whether the genetic configuration of genotype e allow it to spread rapidly, leading to the replacement of other genotypes such as genotype d. in summary, our results on the evolving genotypes of hcov-oc43 and the emergence of a novel genotype e indicate that genotype shift may be one of the major ways for hcov-oc43 to maintain its epidemic. our findings provide insight into the evolution of hcovs and its epidemicity, and can help inform cov surveillance and control in humans and animals. the authors have declared that no competing interests exist. philadelphia: lippincott williams &wilkins discovery of seven novel mammalian and avian coronaviruses in 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in normandy circulation of genetically distinct contemporary human coronavirus oc43 strains molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination characterization and complete genome sequence of a novel coronavirus, coronavirus hku1, from patients with pneumonia prevalence of human respiratory viruses in adults with acute respiratory tract infections in beijing mega5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods modeltest: testing the model of dna substitution replacement of previously circulating respiratory syncytial virus subtype b strains with the ba genotype in south africa genomewide analysis of reassortment and evolution of human influenza a(h3n2) viruses circulating between 1968 and rapid evolution of pandemic noroviruses of the gii.4 lineage genomic analysis of 16 colorado human nl63 coronaviruses identifies a new genotype, high sequence diversity in the n-terminal domain of the spike gene and evidence of recombination coronavirus hku1 and other coronavirus infections in hong kong epidemiology and clinical presentations of the four human coronaviruses 229e, hku1, nl63, and oc43 detected over 3 years using a novel multiplex real-time pcr method nidovirus transcription: how to make sense the molecular biology of coronaviruses infectious diseases emerging from chinese wet-markets: zoonotic origins of severe respiratory viral infections comparative analysis of 22 coronavirus hku1 genomes reveals a novel genotype and evidence of natural recombination in coronavirus hku1 feline coronavirus type ii strains 79-1683 and 79-1146 originate from a double recombination between feline coronavirus type i and canine coronavirus molecular cloning and sequence determination of the peplomer protein gene of feline infectious peritonitis virus type i intraspecies diversity of sars-like coronaviruses in rhinolophus sinicus and its implications for the origin of sars coronaviruses in humans supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2014.12.005 key: cord-254556-1zthrgy1 authors: taylor, sylvia; lopez, pio; weckx, lily; borja-tabora, charissa; ulloa-gutierrez, rolando; lazcano-ponce, eduardo; kerdpanich, angkool; angel rodriguez weber, miguel; mascareñas de los santos, abiel; tinoco, juan-carlos; safadi, marco aurelio p.; lim, fong seng; hernandez-de mezerville, marcela; faingezicht, idis; cruz-valdez, aurelio; feng, yang; li, ping; durviaux, serge; haars, gerco; roy-ghanta, sumita; vaughn, david w.; nolan, terry title: respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multi-country population sample date: 2016-09-22 journal: j infect doi: 10.1016/j.jinf.2016.09.003 sha: doc_id: 254556 cord_uid: 1zthrgy1 background: better population data on respiratory viruses in children in tropical and southern hemisphere countries is needed. methods: the epidemiology of respiratory viruses among healthy children (6 months to <10 years) with influenza-like illness (ili) was determined in a population sample derived from an influenza vaccine trial (nct01051661) in 17 centers in eight countries (australia, south east asia and latin america). active surveillance for ili was conducted for approximately 1 year (between february 2010 and august 2011), with pcr analysis of nasal and throat swabs. results: 6266 children were included, of whom 2421 experienced 3717 ili episodes. rhinovirus/enterovirus had the highest prevalence (41.5%), followed by influenza (15.8%), adenovirus (9.8%), parainfluenza and respiratory syncytial virus (rsv) (both 9.7%), coronavirus (5.6%), human metapneumovirus (5.5%) and human bocavirus (hbov) (2.0%). corresponding incidence per 100 person-years was 29.78, 11.34, 7.03, 6.96, 6.94, 4.00, 3.98 and 1.41. except for influenza, respiratory virus prevalence declined with age. the incidence of medically-attended ili associated with viral infection ranged from 1.03 (hbov) to 23.69 (rhinovirus/enterovirus). the percentage of children missing school or daycare ranged from 21.4% (hbov) to 52.1% (influenza). conclusions: active surveillance of healthy children provided evidence of respiratory illness burden associated with several viruses, with a substantial burden in older children. respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multi-country population sample introduction acute respiratory tract infections (artis) comprise the most common illnesses worldwide, and children often experience several episodes a year. 1 in children, clinical presentation can range from mild, uncomplicated upper respiratory tract illness to severe lower respiratory tract infection (lrti) including pneumonia, bronchiolitis, croup and exacerbations of asthma or wheezing. 2 the world health organization estimated that 1.9 million children died from arti in 2000, 70% in africa and south east asia. 3 in 2011, pneumonia alone led to 1.3 million deaths worldwide in children less than 5 years of age. 4 most artis are caused by viruses, but until the advent of multiplex polymerase chain reaction (pcr) techniques, it was often not possible to identify accurately specific viral infections in clinical cases. 5, 6 the pathogens considered responsible for most arti are respiratory syncytial virus (rsv), influenza a and b, parainfluenza viruses types 1, 2 and 3, and adenovirus. 2 several new pathogens associated with arti have been identified more recently, including human metapneumovirus (hmpv), rhinovirus, coronavirus, human bocavirus (hbov) and parainfluenza 4. 2, 7 vaccine efficacy trials provide intensive, active followup of a well-defined population and can be used to evaluate viral epidemiology. as part of a trial of pandemic influenza vaccines, which included 1 year of prospective, active, community-based surveillance for influenza-like illness (ili) in 17 centers in eight countries, 8 we evaluated the prevalence and incidence of respiratory viruses in children 6 months to less than 10 years of age at first vaccination. samples were obtained from an efficacy trial of two pandemic influenza h1n1 vaccines (nct01051661 sponsored by gsk vaccines). 8 an analysis estimating the prevalence of rsv in ili has been previously reported. 9 here, we report data on all respiratory viruses evaluated, a secondary analysis objective. healthy children 6 months to <10 years of age were enrolled in a randomized, observer-blind, parallel group, multi-country trial of as03-adjuvanted versus nonadjuvanted monovalent pandemic h1n1 vaccines. 8 the trial was conducted in 17 centers in australia, brazil, colombia, costa rica, mexico, the philippines, singapore, and thailand between 15 february 2010 and 19 august 2011; enrollment took up to 6 months and timing varied by country. the trial was approved by an institutional review board for each center and written informed consent was obtained from parents/guardians. parents were instructed to contact the study center within 24 h if the child became ill. active surveillance via scripted telephone contact was conducted from 2 weeks after first vaccination, and contact made every 1e2 weeks to day 385 for each child, regardless of time of enrollment. ili was defined as temperature !38.0 c by any route and at least one of: new/ worsening cough, sore throat, stuffy nose, or runny nose. study staff visited the child's home to collect one anterior nasal swab and one throat swab, ideally within 24 h of ili onset, and at most within 7 days. collection could also take place at a study center or hospital if necessary. swabs were transported in a single tube of m4rt transport medium, stored at à70 c and maintained on dry ice during transport. a 7-day symptom-free period was required between new ili episodes. sample testing was performed by standard multiplex pcr techniques. 8, 10 analysis of viral epidemiology the viruses evaluated were influenza (subtypes a-h1, a-h3 and b), parainfluenza (subtypes 1, 2, 3 and 4), rsv (subtypes a and b), hmpv, rhinovirus/enterovirus (the assay did not distinguish between them), adenovirus, coronavirus (subtypes 229e, oc43, nl63 and hku1) and hbov. first analyzed separately, influenza, parainfluenza and coronavirus subtypes were grouped in a post-hoc analysis. the main outcome variable was pcr-confirmed infection with the stated viruses in nasal/throat swabs in children with ili. this included infection with the virus under consideration alone (single infection) or with the virus under consideration plus one or more of the other viruses (co-infection). single and co-infections were also recorded separately. clinical characteristics of ili episodes were reported by the parents of the children, and any hospitalization or medical attendance (by a doctor or other healthcare professional, not including sample collection by study staff) were recorded. pneumonia was defined as acute illness (one or more of fever !38 c, new or worsening cough, dyspnea, consistent auscultation findings [rales or diminished breath sounds], pain in the chest or abdomen when breathing, or purulent or blood-stained sputum production) and radiologic findings consistent with pneumonia. the total cohort included all children enrolled in the randomized trial. the total cohort with ili episodes tested by multiplex pcr included all children enrolled who experienced an ili and had an adequate nasal/throat sample tested by multiplex pcr. the analysis of prevalence of respiratory viruses in ili and clinical characteristics associated with ili was performed in the total cohort with ili episodes tested by multiplex pcr. the analysis of overall incidence of ili, medically-attended ili and hospitalized ili in which respiratory viruses were detected was performed in the total cohort. the prevalence of respiratory viruses among ili episodes was calculated as: where x is the number of ili episodes with nasal/throat samples positive for the virus and n is the total number of ili episodes with samples collected within 7 days and tested. as there was at least 7 days between two ili episodes, it was assumed that each episode was independent. exact 95% confidence intervals (ci) were computed. 11 prevalence was stratified according to country, age at the time of the ili episode (6e11, 12e23, 24e35, 36e59, 60þ months) and whether the child was medically attended or hospitalized. the incidence per 100 person-years (py) of virusassociated ili in the study population was calculated as: where n is the total number of children enrolled in the trial, ε i is the total number of virus-positive ili episodes for subject i, and d i is the follow-up period for subject i. incidence rates were stratified according to country and age group at the time of the ili episode. exact 95% poisson cis were calculated. 12 observations with incomplete data for the outcome variable and ili episodes for which no nasal/ throat sample was taken were removed from the analysis. missing data were accounted for by calculating the missing proportion for each country and age group, then multiplying the py by (1 minus missing proportion). the trial included 6266 children (total cohort). after excluding children with no ili or inadequate samples, 2421 children experienced 3717 ili episodes (total cohort with ili episodes tested by multiplex pcr). participant flow is shown in supplement fig. 1 . demographics were similar in both cohorts, except that children in the total cohort were older (median 55 versus 42 months) (supplement table 1 ). a respiratory virus was detected in 2958 of 3717 ili episodes (79.6%). rhinovirus/enterovirus had the highest overall prevalence (41.5%), followed by influenza (15.8%), adenovirus (9.8%), parainfluenza and rsv (both 9.7%), coronavirus (5.6%), hmpv (5.5%) and hbov (2.0%) ( table 3 ]). co-infection was detected more often with adenovirus and hbov (fig. 1 ). single infections with parainfluenza, hmpv and coronavirus were identified at approximately the same frequency as co-infections ( fig. 1 ). however, parainfluenza 4 and coronavirus 229e were identified more often as co-infections, whilst coronavirus nl63 was identified more often as a single infection (supplement table 3 ). in all countries, rhinovirus/enterovirus was the most prevalent (36.9e59.2%), whilst hbov was least prevalent (0.8e4.7%) ( table 1) . influenza prevalence ranged from 6.1% to 18.5%; parainfluenza prevalence was approximately 10% except in brazil (5.8%) and singapore (6.1%) ( table 1) . (table 1) . influenza was most prevalent (21.3%) in the oldest children (60þ months), followed by 36e59 months (15.6%) and the other age groups (9.8e12.3%) ( table 2 ). all other viruses were least prevalent in the oldest group (table 2) . there was a less obvious pattern in the other age groups, but, in general, prevalence declined with age except for influenza ( table 2) . the incidence of detected respiratory viruses associated with ili reflected their prevalence. the overall incidence per 100 py (total cohort, all children randomized) was 29.78 for rhinovirus/enterovirus, 11.34 for influenza, 7.03 for adenovirus, 6.96 for parainfluenza, 6.94 for rsv, 4.00 for coronavirus, 3.98 for hmpv and 1.41 for hbov (table 3) . australia had the highest incidence of hmpv (5.08) and the second highest of rsv (7.03), but low incidence of the other viruses relative to other countries ( table 3 ). the philippines, singapore and thailand also had low incidences of most viruses in ili relative to the latin american countries ( table 3) . detection of the respiratory viruses at different times during the year was highly variable across countries (fig. 2aeh) . the overall incidence of medically-attended ili associated with viral infection per 100 py (total cohort, all children randomized) ranged from 1.03 for hbov to 23.69 for rhinovirus/enterovirus (table 3) . corresponding values for incidence of hospitalized ili associated with viral infection were 0 for hbov and 0.81 for rhinovirus/enterovirus (table 3) . clinical characteristics of ili episodes associated with a single respiratory virus (i.e. no co-infection) are shown in table 4 . median duration of ili episodes ranged from 8.9 to 13.4 days. few children were hospitalized but most were medically attended outside study procedures. the percentage of children missing school or daycare was highest with influenza-associated ili (52.1%), followed by hmpv (41.5%), adenovirus (39.0%), rhinovirus/enterovirus (37.6%), coronavirus (31.1%), rsv (30.2%), parainfluenza (28.1%) and hbov (21.4%) ( table 4 ). sore throat was experienced by 25e52% of children, cough by 62e97%, stuffy nose by 40e62%, and runny nose by 66e84% (table 4 ). fever was part of the ili definition and therefore experienced by all children. cough was reported in almost all children with influenza, parainfluenza, rsv, hmpv and coronavirus infections, but only in 60e70% of children with rhinovirus/ enterovirus, adenovirus and hbov infections. there were no medically important differences in clinical characteristics between children with a single viral infection compared with children with multiple infections (supplement table 4 ). a total of 58 pneumonia cases were identified among the 6266 children enrolled in the overall clinical trial, corresponding to a detection rate of 0.9%. of the 58 cases, 32 met the definition of ili and were therefore eligible for sample collection as per the clinical trial protocol. a sample was collected within 7 days of onset of ili symptoms for 20 of these 32 cases: one case in thailand, three in the philippines, five in brazil, five in mexico and six in colombia (table 5) . no virus was detected in three cases, a single infection was detected in 10 cases (four rhinovirus/enterovirus, two parainfluenza, one influenza, two rsv and one hmpv), and co-infection was detected in seven cases (table 5) . nine children were hospitalized. rhinovirus/enterovirus had the highest prevalence and incidence in ili of all respiratory viruses tested in all countries, followed by influenza, adenovirus, parainfluenza and rsv, coronavirus, hmpv and hbov. the burden of ili associated with respiratory viruses was considerable, with a high proportion of children being seen by a medical professional and many missing school or daycare. our analysis benefited from being part of a clinical trial, as previously described. 9 most importantly, we conducted 1 year of prospective, active community surveillance of healthy children in tropical and southern hemisphere countries where prospective data are lacking. most studies of viral epidemiology use hospital-based surveillance because community-based surveillance is difficult and expensive. however, hospital-based surveillance tends to capture only the most severe illness and many cases are missed in developing countries because of limited hospital access. our analysis avoided these limitations and allowed us to capture the burden of virus-associated ili in communities. understanding community epidemiology is essential to implement effective control measures. other advantages of being part of a clinical trial included a wellcharacterized population, wide age range up to 10 years, samples taken from a high proportion of children, consistent methodology between countries, and use of sensitive and validated pcr assays. the trial was conducted in eight countries encompassing australia, south east asia and latin america. the exact timing of enrollment varied somewhat between countries, but was planned so that data collection was performed during the peak 2010e2011 influenza season for each individual country. as stated in the methods, all children were followed for 385 days, with the complete period of surveillance for the study occurring between 15 february 2010 and 19 august 2011. this allowed us to compare the distribution of viruses across the different countries. there was considerable variation in the incidence and prevalence of the viruses by country, although rhinovirus/enterovirus had by far the highest incidence and prevalence in all countries. hbov had consistently the lowest incidence and prevalence. several other studies have evaluated the prevalence of viruses in children with respiratory illness. the relative prevalence of the different circulating viruses varied by study. however, the main circulating viruses were similar between studies and with our study, and included picornaviruses (including rhinovirus), adenovirus, rsv, bocavirus, pivs, hmpv, influenza and coronavirus. 13e17 rhinoviruses are classified in the picornavirus family, of the enterovirus genus. 7 a high prevalence of this family has been reported in other studies in different settings. 13,14,18e20 as in our study, an australian study with active community-based surveillance of healthy preschool-age children with arti found that picornaviruses (including rhinoviruses) were the most frequently detected (41.3%). 13 however, other viruses were detected less frequently than in our study: rsv (6.6%), parainfluenza (4.1%), influenza a and hmpv (both 3.7%), adenovirus (3.1%) and coronavirus nl63 (1.5%). 13 in another prospective australian study in children aged 6 months to 3 years reporting ili, rhinovirus was again the most commonly detected. 15 however, in contrast to our results, adenovirus was detected at the same frequency as rhinovirus, followed by parainfluenza 3, polyomavirus, hmpv and hbov. 15 influenza (a/h1n1) and rsv were relatively uncommon; approximately 40% of children were fully or partially vaccinated against influenza. rhinovirus is not always the most commonly detected virus in children with respiratory disease. in children under 5 years of age hospitalized for lrti in thailand, the most commonly detected viruses were rsv (19.5%), rhinovirus (18.7%), hbov (12.8%) and influenza (8.2%). 16 a study of children aged <3 years hospitalized for lrti in brazil found that rsv was most prevalent (53.5% of episodes), followed by hmpv (32.3%), rhinovirus (20.8%), influenza (12.7%), hbov (10.4%), parainfluenza and adenovirus (both 6.5%) and coronavirus (1.2%). 17 in our analysis, influenza prevalence increased with age. the other viruses showed the opposite trend, with the lowest prevalence observed in the oldest children (60þ months). there was a less obvious pattern in younger ages, but, in general, prevalence of all viruses except influenza declined with age. despite this, the burden of illness remained considerable in older children. there was a clear seasonal pattern for influenza, rsv and hmpv in most countries, and to a lesser extent for rhinovirus/ enterovirus. a previous study found that, although there was no clear seasonal peak for rhinovirus/enterovirus, onset seemed to correspond with the start of the school year in the usa. 18 a limited one year analysis of human rhinoviruses and enteroviruses in ili in latin america showed a year-round temporal distribution throughout central and south america. 21 however, human rhinovirus c species displayed opposite seasonal trends on either side of the equator, accounting for a higher percentage of ili cases north of the equator between september and january, while south of the equator detection increased between april and july. 21 as part of the study, all children received a monovalent influenza a/h1n1 pandemic vaccine; one or two doses of an as03-adjuvanted vaccine were administered or two doses of an unadjuvanted vaccine. trivalent seasonal influenza vaccination rate in the present study was approximately 18%. influenza a subtype h1 was not isolated in any children; influenza a subtype h3 was isolated in 9.0% of children; and influenza b was isolated in 5.7% of children. no difference between the study vaccine groups was observed. cases associated with influenza were least likely to be co-infected with other respiratory viruses. rhinovirus/ enterovirus was also more common as a single infection. adenovirus and hbov were found more often as a coinfection. bacterial co-infection was not measured as part of this study. in the us-based influenza incidence surveillance project, which evaluated the most commonly detected viruses in outpatients with arti or ili, threequarters of all co-infections involved adenovirus and rhinovirus/enterovirus. 18 a uk-based analysis found negative associations between influenza a and hmpv, and between influenza a and rhinovirus. 22 positive associations were found between parainfluenza and rhinovirus, rsv and rhinovirus, adenovirus and rhinovirus, and parainfluenza and rsv. 22 no correlation was found between co-infection and clinical severity in a study in brazil evaluating children under 5 years who sought medical care for respiratory tract infections. 23 more research is needed to understand the interaction of respiratory viruses, and the host response to infection. there were no clear differences between viruses in the severity of illness. most ili episodes were medically attended. ili associated with influenza resulted in the highest proportion of children missing school or daycare (52%), although 20e40% of children infected with the other viruses also missed school or daycare. there was no difference between viruses in the proportion of children hospitalized. clinical features were variable depending upon the viral infection associated with the ili episode. study limitations have been described previously. 9 only healthy children participated in the trial, limiting generalizability. in addition, our study did not include any children aged <6 months and only a limited number of children aged 6e11 months, so our findings are mainly relevant to older children. fever was part of the ili definition, and therefore we would have missed cases in children with no fever. to put this into perspective, in the influenza incidence surveillance project, 34% and 43% of cases among children aged 1e4 years and 5e17 years, respectively, met the arti definition which did not require fever, but did not meet the ili definition which did require fever. 18 however, our definition of ili was somewhat broader (except in children under 2 years of age) and us data may not be generalizable to the tropical and southern hemisphere countries in our study. the inclusion of only healthy children in the study and the exclusion of cases with no fever would have underestimated the burden. we also could not discriminate between rhinovirus and enterovirus by pcr, therefore the exact prevalence and incidence of each one could not be determined. finally, our study included only a small number of pneumonia cases (n z 20), limiting the conclusions that can be drawn regarding the distribution of viral infection in these cases. the overall pneumonia detection rate in the clinical trial (0.9%) is higher than, but in line with, what has been reported in the us for hospitalized cases. 24 however, our sample collection rate among pneumonia cases was only 62.5% compared with 80.0% for ili overall. in conclusion, our active surveillance of healthy children as part of a vaccine efficacy trial provided evidence of the burden of respiratory illness associated with a range of viruses. a substantial burden of illness occurs in older children. data on the epidemiology of respiratory viruses determined from active surveillance of healthy children are generally lacking, and are particularly sparse in the developing countries included in our study. a considerable amount of the burden would not be identified through hospital-based surveillance. these novel data fill an important gap in our knowledge of the epidemiology of viruses contributing to the substantial burden of respiratory disease in children, and may be useful in informing priorities for implementation of existing vaccine programs and development of new vaccines. this work was supported by glaxosmithkline biologicals s.a. who was the sponsor of the study and was involved in all stages of study conduct, including analysis of the data, and in addition paid the costs related to the development of the publication of this manuscript. epidemiology of viral respiratory infections viral infections of the lower respiratory tract: old viruses, new viruses, and the role of diagnosis estimates of world-wide distribution of child deaths from acute respiratory infections global burden of childhood pneumonia and diarrhoea comparison of multiplex pcr assays and conventional techniques for the diagnostic of respiratory 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illness in latin america respiratory viral infections during the 2009e2010 winter season in central england, uk: incidence and patterns of multiple virus co-infections concurrent detection of other respiratory viruses in children shedding viable human respiratory syncytial virus community-acquired pneumonia requiring hospitalization among us children all authors participated in the design, or implementation, or analysis and interpretation of the study results; as well as in the development of this manuscript. all authors had full access to the data and gave final approval before submission.terry nolan, charissa borja-tabora, pio lopez, lily weckx, rolando ulloa-gutierrez, eduardo lazcano-ponce, angkool kerdpanich, miguel angel rodriguez weber, abiel mascareñas de los santos, marco aurelio p safadi, aurelio cruz-valdez and juan-carlos tinoco were coordinating investigators, and together with sumita roy-ghanta, david w vaughn and ping li were responsible for the conduct of the flu q-pan h1n1-035 pri (nct01051661) trial. fong seng lim, marcela hernandez-de mezerville and idis faingezicht also contributed to study material and data collection.sylvia taylor led the epidemiology team in collaboration with gerco haars.yang feng was responsible for the statistical input; statistical expertise was also provided by gerco haars, sumita roy-ghanta, ping li and terry nolan. serge durviaux led the laboratory analysis.terry nolan, charissa borja-tabora, yang feng, david w vaughn and sylvia taylor were members of the core writing team. terry nolan and sylvia taylor contributed equally to this manuscript and the corresponding author was responsible for the submission of the publication. the the authors moreover thank magali ribot (gsk vaccines) for multiplex pcr testing, jean-yves pirçon (gsk vaccines) for critical review of the statistical analysis and mich ele seil (keyrus biopharma on behalf of gsk vaccines) for writing the study report.mary greenacre (independent medical writer) was paid by gsk vaccines to prepare the manuscript draft and sophie vanwetswinkel (xpe pharma and science on behalf of gsk vaccines) provided editorial assistance and manuscript coordination. lily weckx declares research grants from gsk to federal university of são paulo for conduct of three clinical trials and received payment from gsk, novartis, pfizer, and sanofi for board membership or lectures.rolando ulloa-gutierrez discloses having received honoraria from gsk for the original influenza a h1n1 clinical trial discussed here, as well as from gsk, sanofi pasteur, pfizer/ wyeth and merck as a speaker in the past.marco aurelio p safadi has received grants to support research projects and consultancy fees from novartis, gsk, pfizer and sanofi pasteur.fong seng lim discloses having received travel grants from gsk as well as a grant from gsk to his institution to perform clinical trials.marcela hernandez-de mezerville declares having received honoraria from gsk for the original influenza a/ h1n1 clinical trial discussed here, as well as travel support from gsk, sanofi pasteur and pfizer outside the submitted work in the past.idis faingezicht received payment from gsk as principal investigator in a previous vaccine clinical trial and as co-investigator in the influenza a/h1n1 clinical trial.pio lopez, eduardo lazcano-ponce, angkool kerdpanich, miguel angel rodriguez weber, abiel mascareñas de los santos, juan-carlos tinoco, and aurelio cruz-valdez report having nothing to disclose. supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2016.09.003. key: cord-276328-08ava9ni authors: kunutsor, setor k.; laukkanen, jari a. title: hepatic manifestations and complications of covid-19: a systematic review and meta-analysis date: 2020-06-21 journal: j infect doi: 10.1016/j.jinf.2020.06.043 sha: doc_id: 276328 cord_uid: 08ava9ni nan coronavirus disease 2019 (covid-19), which is caused by severe acute respiratory syndrome coronavirus 2 (sars cov-2), was declared a global public health emergency on 30 january 2020. emerging data suggests that covid-19 has extrapulmonary manifestations and complications, subsequently leading to multiorgan failure and death. common cardiovascular and renal complications reported to be associated with covid-19 include myocardial injury, heart failure, acute kidney injury and electrolyte disturbances. (1, 2) in addition to the observation that older patients, males and those with pre-existing comorbidities such as cardiovascular disease, diabetes, chronic kidney disease and chronic liver disease are at highest risk for severe illness or death, (3, 4) covid-19 complications have been shown to correlate with the disease severity or mortality. (5, 6) emerging data also suggests covid-19 contributes to adverse hepatic manifestations such as acute hepatic injury. wang and colleagues in their recent published study to investigate characteristics and prognostic factors in 339 elderly patients with covid-19, a high proportion of severe and critical cases were observed as well as a high fatality rate. over hospital stays ranging from 2 to 28 days, the pooled incidence was 69.1% (67.3-70.9) for elevated alt (2 studies); 34.8% (16.2-56.0) for elevated ast; 11.2% (6.4-17.1) for acute hepatic injury (15 studies) and 7.9% (5.9-10.2) for hypoproteinaemia (n=2 studies) ( figure 1a) . subgroup analyses suggested that the incidence of acute hepatic injury was higher in chinese populations and groups with a higher prevalence of pre-existing chronic liver disease; however, the incidence of acute hepatic injury was similar in older (≥60 years) or younger age (<60 years) groups ( figure 1b) . patients with pre-existing liver dysfunction appear to have worse outcomes in covid-19, (8) which has been attributed to their immunocompromised status. none. renal complications in covid-19: a systematic review and meta-analysis cardiovascular complications in covid-19: a systematic review and meta-analysis clinical course and mortality risk of severe covid-19 markers of liver injury and clinical outcomes in covid-19 patients: a systematic review and meta-analysis cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) comorbid chronic diseases and acute organ injuries are strongly correlated with disease severity and mortality among covid-19 patients: a systemic review and meta-analysis. research (wash d c) coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on 4-week follow-up liver injury in covid-19: management and challenges longitudinal association between markers of liver injury and mortality in covid-19 in china hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china key: cord-266455-rbblg4pu authors: poole, stephen; clark, tristan w. title: rapid syndromic molecular testing in pneumonia: the current landscape and future potential date: 2019-12-03 journal: j infect doi: 10.1016/j.jinf.2019.11.021 sha: doc_id: 266455 cord_uid: rbblg4pu community acquired pneumonia (cap), hospital-acquired pneumonia (hap) and ventilator associated pneumonia (vap) are all associated with significant mortality and cause huge expense to health care services around the world. early, appropriate antimicrobial therapy is crucial for effective treatment. syndromic diagnostic testing using novel, rapid multiplexed molecular platforms represents a new opportunity for rapidly targeted antimicrobial therapy to improve patient outcomes and facilitate antibiotic stewardship. in this article we review the currently available testing platforms and discuss the potential benefits and pitfalls of rapid testing in pneumonia. lower respiratory tract infections were accountable for an estimated 2.7 million deaths in 2015, making them the third most common cause of death worldwide 1 . community acquired pneumonia (cap) caused nearly 30,0 0 0 deaths in england and wales in 2015 2 and costs europe around €10 million annually 3 . it is estimated that 25 per 10,0 0 0 adults are hospitalised with pneumonia each year 4 . hospital acquired pneumonia (hap) is defined as occurring > 48 h after admission to a healthcare facility. it is caused by a different spectrum of more antibiotic resistant bacterial pathogens than those occurring in the community. ventilator associated pneumonia (vap) is defined as occurring > 48 h after intubation for invasive artificial ventilation. the two entities combined (hap and vap) are the most common nosocomial infection in the developed world 5 with hap complicating around 2% of hospital admissions 6 . the incidence of vap in intubated patients is around 10% 7 and is associated with mortality of around 10% 8 . a retrospective matched cohort study by kollef et al. 9 found patients who developed vap were intubated for longer, spent longer on icu, and were in hospital for a greater period of time. they estimated the additional cost of vap from to be $40,0 0 0 per patient. large amounts of empirical 'broad spectrum' antibiotics are used to treat pneumonia which inadvertently promote antimicrobial resistance (amr): a problem identified by the who as one of the leading threats to global health today. the o'neill report, commissioned by the uk government in 2014, has highlighted the need for developed nations to take a lead in tackling amr. as part of this there is a specific recommendation that all antibiotic prescriptions should be supported by diagnostic tests where available by 2020 10 . the uk government recently published a five-year action plan for tackling amr, which emphasised the need for improved diagnostics to support antibiotic prescribing. this included a target to be able to report the percentage of antimicrobial prescriptions which are supported by a diagnostic test or decision making tool by 2024 11 . timely administration of appropriate antibiotics is a central tenant of care for patients with pneumonia 12 , 13 and yet the goldstandard for microbiological diagnosis remains traditional, slow, culture based methods. these take greater than 24 h to identify an organism and often greater than 72 h to provide phenotypic antibiotic sensitivity data. culture is insensitive, only detecting a pathogen in 23-40% of patients with clinically diagnosed pneumonia 4 , 14-16 and an even smaller proportion after the administration of antibiotics. in recent years several rapid syndromic molecular tests for pneumonia have been developed. these offer the potential to revolutionise treatment by providing information to clinicians in 'real-time' on the pathogens present and their likely antibiotic sensitivity by also detecting genotypic markers of resistance. multiple studies have demonstrated the superior diagnostic accuracy of pcr based platforms for detecting bacterial pathogens in the sputum compared with standard culture [16] [17] [18] [19] . this review will discuss the commercially available syndromic molecular panels for pneumonia, their potential clinical impact and the challenges to implementing them as a 'front line' diagnostic test. the greatest potential clinical benefit of a rapid syndromic test for pneumonia is being able to better utilise antibiotics. the superior diagnostic yield of multiplex pcr means that a pathogen is detected rapidly in a much greater proportion of patients, so therapy can be quickly tailored to the responsible organism. in some situations, this will allow narrowing of antimicrobial therapy: for example, identification of streptococcus pneumoniae facilitating a change of antibiotics to penicillin, in geographical areas with a low prevalence of penicillin resistant s. pneumoniae . in other cases, it may facilitate a change or escalation of antimicrobial therapy: for example, the identification of methicillin resistant staphylococcus aureus (mrsa) which would not be covered by empirical regimens in many areas. the absence of detection is also helpful: the sensitivity when compared to culture of molecular assays is very high so can reassure clinicians that organisms are not present and so support decisions to stop unnecessary antibiotics or to deescalate antibiotics that were used empirically to cover organisms subsequently not detected. the impact of this improved use of antibiotics are twofold: firstly, earlier appropriate antibiotics should improve clinical outcomes including mortality and length of stay. secondly, it prevents unnecessary broad-spectrum antibiotic use, which facilitates antibiotic stewardship and reduces antibiotic related adverse events. the aetiology of cap and hap/vap are highly variable between different regions and times, and this is reflected in studies of causative microbial agents as identified by culture. patients with underlying lung diseases, for example chronic obstructive pulmonary disease, can be colonised with microbial flora which are more typical pathogens of hap. as a result, they may develop community acquired infections caused by these agents. s. pneumoniae, haemophilius influenzae, s. aureus, moraxella catarrhalis and 'atypical' organisms including mycoplasma pneumoniae and legionella pneumophila are all cultured from the sputum of patients with cap. many of these organisms have predictable resistance patterns when interpreted with local epidemiological data. gadsby et al. developed and internally validated their own syndromic molecular assay for pneumonia. they used this to test sputum samples of 323 adults admitted to hospital with cap 16 . their assay detected a pathogen in 87% of patients (as opposed to 39% of patients using only routine culture). as a result, they proposed that 77% of antibiotic prescriptions in cap could have been deescalated based on results from multiplex pcr testing. the majority of these potential interventions involved stopping clarithromycin when atypical organisms were not detected or 'narrowing' antibiotics when a likely sensitive pathogen had been detected. in hap and vap, frequently cultured bacterial pathogens include s. aureus, pseudomonas aeruginosa, klebsiella species, escherichia coli, acinetobacter species and enterobacter species 20 . empirical regimens are therefore broad spectrum and large numbers of antibiotics are consumed. the absence of certain organisms (for example p. aeruginosa ) could facilitate a narrowing of the antimicrobial spectrum with a knock-on effect of reducing antibiotic related adverse effects and improving stewardship. furthermore, common gram negative isolates are increasingly resistant in pneumonia surveillance studies 21 . rapid molecular detection of these resistance genes should facilitate earlier initiation of effective antibiotics and this should lead to better outcomes. in adults, respiratory viruses are found in approximately one third of community acquired pneumonia cases 4 , 22 . one study found that 36% of patients admitted to intensive care with pneumonia were positive for a respiratory virus, with a broad range of viruses detected 23 . detection of certain viruses such as influenza and adenovirus which are known to cause pneumonia, coupled with the absence of detection of bacteria and low levels of serum biomarkers such as procalcitonin (which is elevated in patients with bacterial infection), could support decisions to stop or use an abbreviated course of antibiotics. the respoc trial was a pragmatic randomised controlled trial that tested patients with community acquired acute respiratory illness using the biofire respiratory panel (which tests comprehensively for respiratory viruses and atypical bacteria) at the point-of-care. it found that patients who were tested with the filmarray were significantly more likely to receive a single dose or shorter course of antibiotics 24 than those who were not. it also found a significant reduction in length of hospital stay in the intervention group along with improved use of neuraminidase inhibitors (nai) in patients with influenza. currently there are no licenced antiviral agents for respiratory viruses other than influenza. the benefit from nai treatment is greatest when they are started within 48 h of symptom onset but there is evidence in adults to suggest ongoing benefit when started beyond this time 25 and a recent study suggests that treatment earlier in admission to hospital improves outcome irrespective of overall duration of illness 26 . as such, timely identification and treatment is critical. antiviral treatments for other respiratory viruses, including respiratory syncytial virus (rsv) are in development. since the 1990s infection control methods including patient source isolation and deep cleaning with targeted decolonisation have been highly successful at reducing the spread of mrsa. enhanced infection control practices are recommended for a number of pathogens that may be present in patients with pneumonia. early identification of these should reduce the spread of these organisms, especially in hospitalised patients. some examples of these which are found on commercially available molecular tests are extended spectrum beta lactamases (esbls), carbapenemase producing enterobacteriaceae (cpes), mrsa, influenza and rsv. in the uk there is a mandatory requirement to report certain infectious diseases to public health england, so they can be investigated. l. pneumophilia is associated with outbreaks from devices that aerosolize water. there were 532 cases in the uk in 2018 27 , earlier sensitive detection of these would allow outbreak investigation to occur sooner and potentially stop further cases occurring. at the current time there are 2 fda approved, ce marked syndromic molecular panels for pneumonia which are commercially available: the filmarray (biofire diagnostics llc, salt lake city, utah, us) pneumonia panel and the unyvero (curetis gmbh, holzgerlingen, germany) hospitalised pneumonia (hpn) panel. fast track diagnostics respiratory panel 33 (fast track diagnostics sarl, luxembourg) is another available platform with a large number of targets, but insufficient bacterial targets for it to be considered a true pneumonia panel so this will only be considered in brief. the commercially available platforms are summarised in table 1 . the authors are aware of further panels in development from mobidiag, bruker, accelerate and axo science 28 but published data is only available for the latter. there are also several research groups who have developed their own syndromic molecular pneumonia tests, most notably gadsby et al 16 . there are a multitude of other 'respiratory pathogen' multiplex panels which have targets only for respiratory viruses, atypical bacterial targets or a very small range of typical bacteria. these are beyond the scope of this review article. we have only included assays with targets for a wide range of typical pathogens for pneumonia. this is an fda approved and ce marked platform that uses nested real-time pcr to detect 34 clinically important respiratory targets (15 semi-quantitative bacterial targets, 3 qualitative atypical bacterial targets, 8 [30] [31] [32] furthermore, the pneumonia panel detects pathogens in a much higher proportion of samples than culture. buchan et al 31 reported that the filmarray detected a bacterial target in 71% more specimens than routine culture, equating to over 100% increase in total bacterial detections. the relative abundance of organism for the 15 bacterial targets is estimated based on real-time pcr relative to a material of known quantity and is grouped for reporting into bins. these represent approximately 10 4 , 10 5 , 10 6 and > 10 7 genomic copies of bacterial nucleic acid per millilitre of specimen respectively. concordance with reference molecular testing is very high 33 but as expected the overall concordance between bin and reference sputum culture (cfu/ml) concentration was lower at around 40% 29 and was highly variable between organisms. as such the manufacturer advises clinical correlation in interpretation of semi-quantitative results. to date there have been no published prospective interventional studies evaluating the clinical impacts of using the pneumonia panel in patients with pneumonia. observational data based on lower respiratory tract assays which preceded the final, fda approved pneumonia panel suggested change of antibiotics could be supported in > 50% of cases 31 , 34 . the hpn panel is ce marked and runs on the unyvero platform which includes the unyvero lysator, the unyvero cockpit and the unyvero analyzer. amplicons generated by 8 parallel multiplex pcr reactions are qualitatively detected by hybridisation on arrays in a single use cartridge. it has a wide range of bacterial and resistance gene targets including 29 the assay is validated for use on sputum (including expectorated sputum, bal and et aspirate). like the filmarray, the unyvero is a platform designed as a 'sample-to-answer' solution taking 5 min of low skill hands-on time with a total turnaround time of 4-5 h. an equivalent test, the lower respiratory tract panel (lrt) has fda approval in the us but is only validated for use on tracheal aspirates. manufacturer reported diagnostic sensitivity for bacterial detection (when compared to reference culture and molecular detection in cases of discrepancy) is between 80 and 100% with the majority of targets in the diagnostic performance data presented by the manufacturer, resistance marker detection aligned poorly with organism antibiogram: for example, matching in only 4/11 meca detections or 9/13 quinolone resistance markers in e. coli . this issue was noted by gadbsy et al 39 for the p55 assay where the sensitivity for antibiotic resistance detection was 18%. two predecessors to the hpn cartridge have been developed and ce marked: the earlier p50, and the later p55. the former of these was evaluated most extensively by personne et al. who found the test to be sensitive for bacterial detection but with a run failure rate of 12.6% and extensive discrepancies with regards to sensitivity testing 40 . furthermore, the test was unable to differentiate s. pneumoniae from the s. mitis group. papan et al. reported that the p50 had a low sensitivity for gram positive organisms (when evaluated on paediatric samples) 41 . the resistance panel on the p50 was broad but lacked several key emerging carbapenemase gene targets. the p55 panel rebalanced this by removing less clinically relevant resistance genes. it added targets for s. pneumoniae and m. pneumoniae . again, the sensitivity for bacterial detection remained high when assessed by ozongwu et al. albeit with a high overall run failure rate of 10% 17 . the targets on the panel for the hpn are the same as the p55, but the manufacturer claims it has a higher sensitivity and specificity. to date there are no published randomised controlled trials evaluating the clinical impact of the unyvero hpn system in patients with pneumonia. jamal et al 42 performed a non-randomised interventional study using the p50 assay where antibiotics were adjusted based on the results and pathogens detected were compared to culture. the turnaround time for result was very quick ( ∼4 h) compared to culture (48-96 h) and a large proportion of patients had antibiotics changed based on the p50 results, however the small number of patients studied and the lack of a comparator group make definitive conclusions impossible. gadsby et al. retrospectively tested bal samples with the p55 and reviewed patient notes. they reported that 53.6% of patients who had positive standard of care microbiology could potentially have had a change in antibiotics earlier based on p55 results 39 . conversely, they reported a false negative p55 result in ∼20% of those with a positive culture which could have caused harm if acted upon. the respiratory pathogens 33 panel differs from the first two tests discussed in that it is exclusively a laboratory centred assay. the ce marked respiratory pathogens 33 kit can be used on several standard laboratory cyclers. as such there is no reported standard turnaround time although it is greater than 6 h. positive signals are detected from eight multiplex real-time pcr reactions. it is not an automated process so will have a considerably longer hands-on time requiring skilled extraction and setup. the there is very little published data comparing different syndromic molecular pneumonia tests. enne et al. and the inhale group presented data at eccmid 2019 where they compared the unyvero and the filmarray on 654 surplus intensive care respiratory tract samples 36 . the filmarray had slightly greater sensitivity for common pathogens, fewer major discordances (defined as routine culture finding 1 or more undetected organisms) and fewer machine failures. the unyvero had slightly higher specificity and overall concordance with reference culture. whilst the data presented for syndromic molecular test for pneumonia clearly demonstrates high accuracy and the detection of many more pathogens than culture, no data has yet been published showing that this translates into improved antibiotic use or clinical benefit. other molecular diagnostics studies for blood stream infection 45 have shown improved diagnostic performance, but negligible impact on clinical outcomes when results were not provided to clinicians along with infection specialist advice. it seems likely that such a wealth of information generated will require careful interpretation by an infection specialist in consultation with the clinicians directly caring for the patient, for these benefits to be maximised. rapid syndromic molecular platforms have the potential to significantly improve the use of antibiotics and clinical outcomes in patient with pneumonia, but high quality randomised controlled trials are urgently required to evaluate their clinical impact. we are aware of 5 trials that are currently underway or in set up that may address this evidence gap: the saripoc study is a single centre randomised controlled trial (rct) recruiting critically unwell patients with pneumonia in southampton, uk. the inhale study is a uk multicentre, rct recruiting critically unwell patients with hap and vap. pibcap is a uk multicentre rct recruiting patients with cap. the norcap trial, in norway is a single centre rct in set up, also aiming to recruit patients with cap. a further single centre rct in edinburgh is using molecular testing for broader community acquired lrti microbial diagnosis. the first of these two studies are testing patients at the point-of-care, whereas the others use rapid laboratory-based testing. antibiotic de-escalation based on results is a key component of antibiotic stewardship and is widely accepted as good practice. trials looking at the efficacy and safety of antimicrobial de-isolation based on culture results are sparse. the vast majority of published studies are observational and comparison between studies for so many variables (hap, cap, vap, icu/ non-icu, severe sepsis etc.) are fraught with difficulties. furthermore, due to the geographic variability in causative organisms and prescribing practices, they are often poorly transferrable between regions. to our knowledge no interventional studies have looked at the safety or efficacy of antimicrobial de-escalation based on multiplexed pcr for pathogens of pneumonia. studies to date have made their de-escalation intervention after at least 48 h when the patient has stabilised, and culture results are available. both the idsa and the national institute for clinical excellence (nice) cite an urgent need for well-run rcts on the impact of de-escalating antimicrobial therapy 46 , 47 . the idsa and the american thoracic society advise antibiotic de-escalation in hap/vap according to culture results on the basis of expert opinion, citing a high level of confidence that it 'reduces costs, burdens, and side effects, and that it is very likely that deescalation also reduces antimicrobial resistance' 47 . there a small number of interventional studies looking at antibiotic de-escalation based upon microbiological culture results in hap/vap which have suggested this practice is safe 48 , 49 . high quality data for outcomes, including length of intensive care stay and antibiotic savings, are lacking and conflicting. a meta-analysis by khan et al 50 of observational studies reviewing antibiotic de-escalation in pneumonia in icu (hap and vap only) found no difference in mortality between those who were de-escalated according to culture result and those that weren't. in the context of cap, both the idsa 51 and nice/bts 46 guidelines recommend organism directed therapy when a pathogen has been identified by culture. high quality data is lacking but observational data and limited interventional data suggests this is safe [52] [53] [54] . a systematic review by paul et al 55 included studies with cap, hap, vap and blood stream infection. the reviewers found no association between de-escalation and survival with pneumonia (or 0.97, 95% ci 0.45-2.12). concern has been raised that the high sensitivity of molecular tests will lead to excessive detection of colonising flora which may paradoxically increase unnecessary antibiotic use. this is particularly pertinent in expectorated sputa where small numbers of potentially pathogenic bacteria can be present in the absence of disease. a potential solution to this is the development of semi-quantitative molecular methods such as with the biofire r filmarray r pneumonia panel. this provides a representation of the amount of bacterial dna present which is highly concordant with reference molecular techniques. as highlighted by studies using the unyvero 17 , 39 , molecular detection of resistance genes may correlate poorly with phenotypic sensitivity in its current form. detection of genes from 'off panel' organisms, for example mec a genes in colonising coagulase negative staphylococci, may be incorrectly attributed to those organisms which are on the panel. as such, clinicians will need to be cautious in interpreting these results. as well as having relatively quicker run times, syndromic multiplex molecular tests could potentially be deployed at the pointof-care. the resppoc trial by brendish et al., demonstrated with a respiratory viral panel that this was logistically feasible and associated with a number of clinical benefits compared to routine clinical care 24 . a post hoc analysis 56 of patients who tested positive for respiratory viruses in the trial highlighted an association between rapid turn-around time (defined as < 1.6 h), shorter hospital admission and shorter durations of antibiotic therapy. it is our belief that point-of-care testing represents the ideal strategy for new, rapid diagnostic test platforms allowing clinicians to maximise the benefit from such accurate tests early in the decision-making process. clearly, rigorous quality assurance is essential for any diagnostic test irrespective of the site of testing. it should also be noted that the tests described in this article are not currently clia waivereda requirement for use at the point-of-care in the us. rapid syndromic molecular tests for pneumonia have improved diagnostic accuracy compared to the current gold standard of culture and can provide results in real time. in the era of widespread amr their use has the potential to dramatically improve the rational use of antibiotics and to improve clinical outcomes in patient with pneumonia. high quality data from well conducted randomised controlled trials are now urgently needed to assess the impact of these platforms on antibiotic use and patient outcome. poole, s. -declarations of interest: none. clark, t.w. has received speaker fees, reimbursement for travel and honoraria from biofire llc and biomerieux and has also received equipment and consumables from these companies for the purposes of independent research. no commercial entities had any input into this manuscript. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. global, regional, and national life expectancy, all-cause mortality, and causespecific mortality for 249 causes of death, 1980-2015: a systematic analysis for the global burden of disease study 2015 480 -486): directly standardised rate, all ages, annual trend: nhs digital risk factors for community-acquired pneumonia in adults in europe: a literature review community-acquired pneumonia requiring hospitalization among u.s. adults changes in prevalence of health care-associated infections in u.s. hospitals the 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comparison of unyvero p55 pneumonia cartridge, inhouse pcr and culture for the identification of respiratory pathogens and antibiotic resistance in bronchoalveolar lavage fluids in the critical care setting n sample-in, answer-out'? evaluation and comprehensive analysis of the unyvero p50 pneumonia assay assessment of the multiplex pcr-based assay unyvero pneumonia application for detection of bacterial pathogens and antibiotic resistance genes in children and evaluation of curetis unyvero, a multiplex pcr-based testing system, for rapid detection of bacteria and antibiotic resistance and impact of the assay on management of severe nosocomial pneumonia sample-in, answer-out'? evaluation and comprehensive analysis of the unyvero p50 pneumonia assay a review of novel technologies and techniques associated with identification of bloodstream infection etiologies and rapid antimicrobial genotypic and quantitative phenotypic determination diagnosis and management of community-and hospital-acquired pneumonia in adults. nice clinical guideline 191. nice, national clinical guidelines centre management of adults with hospital-acquired and ventilatorassociated pneumonia: 2016 clinical practice guidelines by the infectious diseases society of america and the american thoracic society early use of imipenem/cilastatin and vancomycin followed by de-escalation versus conventional antimicrobials without de-escalation for patients with hospitalacquired pneumonia in a medical icu: a randomized clinical trial de-escalation versus continuation of empirical antimicrobial treatment in severe sepsis: a multicenter non-blinded randomized noninferiority trial antibiotic de-escalation in patients with pneumonia in the intensive care unit: a systematic review and meta-analysis infectious diseases society of america/american thoracic society consensus guidelines on the management of communityacquired pneumonia in adults prospective, randomised study to compare empirical treatment versus targeted treatment on the basis of the urine antigen results in hospitalised patients with community-acquired pneumonia comparison between pathogen directed antibiotic treatment and empirical broad spectrum antibiotic treatment in patients with community acquired pneumonia: a prospective randomised study antibiotic de-escalation for bloodstream infections and pneumonia: systematic review and meta-analysis impact of turnaround time on outcome with point-of-care testing for respiratory viruses: a post hoc analysis from a randomised controlled trial the authors would like to thank paula sands, research engagement librarian at the southampton general hospital healthcare library for her help and expertise in constructing search terms for literature review. key: cord-277763-ihg3te63 authors: moynan, david; cagney, maura; dhuthaigh, aoife ni; foley, margaret; salter, aisling; reidy, niamh; reidy, paul; de barra, eoghan; fitzpatrick, fidelma title: the role of healthcare staff covid-19 screening in infection prevention & control date: 2020-06-25 journal: j infect doi: 10.1016/j.jinf.2020.06.057 sha: doc_id: 277763 cord_uid: ihg3te63 nan the current covid-19 pandemic presents many unique challenges for occupational health medicine and infection prevention and control (ipc) in hospitals. while many hospital services were postponed during the pandemic's first peak, the resumption of routine services in an environment where patients still present with covid-19 raises concerns over the potential for significant nosocomial covid-19 transmission. we read with interest a recent study in this journal that emphasises the importance of identifying covid-19 cases to prevent onward transmission of sars-cov-2 1 . while the rapid identification and subsequent testing of symptomatic healthcare workers (hcw) is paramount, we believe there is a role for testing irrespective of symptoms. while hcw can acquire infections and contribute to cross-transmission during hospital outbreaks such as influenza or norovirus, asymptomatic staff are usually not routinely screened as part of the outbreak control measures. the role of covid-19 surveillance of asymptomatic hcw has recently been highlighted and may become increasingly pertinent with new infection waves 2,3 . however, the rapid detection of pcr-positive, asymptomatic staff is also crucial in the management of hospital clusters of covid-19. here, we discuss the high rate of sars-cov-2 pcr positivity among hcw during three inpatient ward outbreaks in beaumont hospital, dublin, ireland. in march and april 2020, on three wards with two or more positive covid-19 patients after three days of admission (designated as potential nosocomial infection), we implemented universal staff sars-cov-2 testing on that ward as part of outbreak management. a combined throat/nasopharyngeal swab was taken on hcw who had been working on the designated wards. rt-pcr was performed using altona diagnostics realstar sars-cov-2 rt-pcr according to the manufacturer's instructions, on the roche light cycler 480ii 4 . demographics including age, sex and job title were collected. all results were disclosed to staff by occupational health and those testing positive were advised to remain off work for minimum 14 days, with appropriate follow up. in addition, details of symptoms, contacts and co-habitation were documented. asymptomatic, sars-cov-2 pcr positive hcw were followed up by telephone for symptom development. asymptomatic, sars-cov-2 pcr negative hcw were advised to self-monitor for symptoms and isolate if they were deemed a close contact of a known positive case. interestingly, 11 (61%) of the symptomatic, pcr-positive hcw worked while symptomatic, similar to other reports 7 . this may reflect hcw presenteeism which has been reported previously during influenza seasons 8 . as asymptomatic (or indeed, pre-symptomatic) hcw may have similar viral loads and may be capable of transmission as much as symptomatic individuals 9 , their detection and subsequent exclusion from work is an important aspect of a hospital's covid-19 strategy. in conclusion, as hospitals begin to reopen to routine non-covid-19 services, hcw sars-cov-2 testing irrespective of symptoms should be considered, particularly as part of outbreak management to rapidly prevent onward transmission to patients and other staff. detecting sars-cov-2 positive staff will benefit not only public health measures, but in the case of staff cohabiting with other hcw, will also benefit other healthcare institutions. clinical presentation of covid-19 in health care workers from a french university hospital covid-19: pcr screening of asymptomatic health-care workers at london hospital covid-19 screening of healthcare workers in a london maternity hospital universal masking in hospitals in the covid-19 era temporal dynamics in viral shedding and transmissibility of covid-19 sars-cov-2 infection in 86 healthcare workers in two dutch hospitals in working with influenza-like illness: presenteeism among us health care personnel during the 2014-2015 influenza season sars-cov-2 viral load in upper respiratory specimens of infected patients key: cord-280350-ay4cnzn5 authors: chan, jasper f.w.; chan, kwok-hung; kao, richard y.t.; to, kelvin k.w.; zheng, bo-jian; li, clara p.y.; li, patrick t.w.; dai, jun; mok, florence k.y.; chen, honglin; hayden, frederick g.; yuen, kwok-yung title: broad-spectrum antivirals for the emerging middle east respiratory syndrome coronavirus date: 2013-10-03 journal: j infect doi: 10.1016/j.jinf.2013.09.029 sha: doc_id: 280350 cord_uid: ay4cnzn5 objectives: middle east respiratory syndrome coronavirus (mers-cov) has emerged to cause fatal infections in patients in the middle east and traveler-associated secondary cases in europe and africa. person-to-person transmission is evident in outbreaks involving household and hospital contacts. effective antivirals are urgently needed. methods: we used small compound-based forward chemical genetics to screen a chemical library of 1280 known drugs against influenza a virus in biosafety level-2 laboratory. we then assessed the anti-mers-cov activities of the identified compounds and of interferons, nelfinavir, and lopinavir because of their reported anti-coronavirus activities in terms of cytopathic effect inhibition, viral yield reduction, and plaque reduction assays in biosafety level-3 laboratory. results: ten compounds were identified as primary hits in high-throughput screening. only mycophenolic acid exhibited low ec(50) and high selectivity index. additionally, ribavirin and interferons also exhibited in-vitro anti-mers-cov activity. the serum concentrations achievable at therapeutic doses of mycophenolic acid and interferon-β1b were 60–300 and 3–4 times higher than the concentrations at which in-vitro anti-mers-cov activities were demonstrated, whereas that of ribavirin was ∼2 times lower. combination of mycophenolic acid and interferon-β1b lowered the ec(50) of each drug by 1–3 times. conclusions: interferon-β1b with mycophenolic acid should be considered in treatment trials of mers. a novel lineage c betacoronavirus, previously known as human coronavirus emc/2012 and later renamed as middle east respiratory syndrome coronavirus (mers-cov), has emerged in the arabian peninsula since april 2012 to cause a "severe acute respiratory syndrome (sars)-like" disease in 136 laboratory-confirmed cases with 58 fatalities in 9 countries in the middle east, europe, and north africa as of 4 october 2013. 1e5 animal-to-human transmission has been suspected in view of mers-cov's close phylogenetic relatedness to other lineage c betacoronaviruses found in bats in hong kong, mexico, europe, and africa, 6e13 and its broad species tropism in various animal cell lines including those of bats, primates, pigs, civets, and rabbits. 14, 15 recently, a serological study of major livestock suggested dromedary camels to be a possible host based on the high prevalence of mers-cov neutralizing antibodies in dromedary camels from oman. 16 however, targeted studies are needed to confirm this finding and its possible relevance to human cases of mers-cov infection as most cases did not have contact with camels and the virus has not been isolated in animals yet. the epidemic continues to evolve with recent outbreaks occurring among epidemiologically-linked household contacts in the kingdom of saudi arabia, the united kingdom, italy, and tunisia, and hospital contacts in the kingdom of saudi arabia, jordan, the united kingdom, and france providing evidence for mers-cov's potential for person-to-person transmission. 17e23 unlike most other human coronavirus infections which are generally mild, most patients with mers have suffered from rapidly progressive pneumonia with some also developing acute renal failure, hepatic dysfunction, gastrointestinal upset, pericarditis, disseminated intravascular coagulation, and/or cytopenias. 2, 24 the resulting crude mortality rate of nearly 50% in documented cases far exceeded those seen in all other human coronavirus infections including sars despite aggressive supportive treatment including extracorporeal membrane oxygenation in some of the mers cases. while mild and asymptomatic cases have been recognized, 2,19,24 these recent case clusters signify a global health threat especially in view of the unusual clinical severity of mers, travel of infected persons to other countries and influx of religious pilgrims to the kingdom of saudi arabia, and the lack of proven effective specific antiviral treatment. after our initial success in applying chemical genetics in probing novel targets and compounds for antiviral development, 25 we started looking for broad-spectrum antiviral compounds that may be active against both influenza a viruses and coronaviruses, the two viral pathogens responsible for causing the recent 2009 pandemic and largescale epidemics. 9 while neuraminidase inhibitors such as oseltamivir and zanamivir remain effective against most seasonal and avian influenza a viruses, 26e30 proven antiviral therapeutic options for coronavirus infections is lacking. given the limited time available to develop novel anti-mers-cov agents in this evolving epidemic, we attempted to provide an alternative solution by identifying potential broad-spectrum antiviral agents against mers-cov and influenza a viruses by a small compound-based forward chemical genetics approach using chemical libraries consisting of 1280 drug compounds already marketed or having reached clinical trials in the united states, europe, or asia (microsource discovery systems, usa). 25 we then assessed the anti-mers-cov activities of the identified drug compounds in cell culture by cytopathic effect (cpe) inhibition, viral yield reduction, and plaque reduction assay (pra) assays, as well as drug cytotoxicity. a clinical isolate of mers-cov was kindly provided by r. fouchier, a. zaki, and colleagues. 3 the isolate was amplified by one additional passage in vero cells to make working stocks of the virus (4 â 10 5 tcid 50 /ml). all experimental protocol involving live mers-cov isolate followed the standard operating procedures of the approved biosafety level-3 facility as we previously described. 31 the influenza a/ wsn/1933 (h1n1) virus was expanded in chick embryo as we previously described. 25 chemical reagents and high-throughput screening (hts) a total of 1280 pre-existing drug compounds (microsource discovery systems) were screened against influenza a/ wsn/1933 (h1n1) virus. high-throughput screening (hts) was carried out in a fully automated beckman coulter core system (beckman coulter, usa) integrated with a kendro robotics co 2 incubator (thermo fisher scientific) at chemical genetics unit, department of microbiology, research center of infection and immunology, li kashing faculty of medicine, the university of hong kong as we previously described with modifications. 25 briefly, compounds were added in 96-well microtitre plates (tpp) in duplicate with a final concentration of 10 mm or 100 mm and 20,000 madinedarby canine kidney (mdck) cells per well in 100 ml complete eagle's minimal essential medium (emem) supplemented with 1% heat-inactivated fbs. cells were then inoculated at an moi of 0.01 with influenza a/ wsn/1933 (h1n1) virus for detection of broad-spectrum antivirals. after infection, the plates were incubated at 37 c with 5% co 2 and monitored daily using a leica dm inverted light microscope for virus-induced cpe. drugs that gave full protection of mdck cells (no cpe) were selected for further evaluation with mers-cov in a biosafety level-3 laboratory. the cytotoxicity of selected drug (ribavirin: 1600e0.1 mg/ml; introna 75,000e4.58 iu/ml; avonex: 75,000e4.58 iu/ml; rebif: 250,000e15.26 iu/ml; betaferon: 50,000e3.05 iu/ml; mmf: 32e0.25 mg/ml) was determined by thiazolyl blue tetrazolium bromide (mtt) assay according to manufacturer's instructions. the endpoint was the 50% effective cytotoxic concentration (tc 50 ). the drug compounds identified as primary hits showing a ec 50 of less than or equal to 50 um and a selectivity index of more than 100 were diluted with serum free mem and added to confluent vero cells in 96-well culture plates in triplicate for 2 h at 37 c. after incubation, the drugcontaining media was removed, and mers-cov at 0.0001 moi was added together with fresh drug-compound media to each well containing approximately 60,000 cells. following 1 h adsorption at 37 c, the virus-compound mixture was removed and the cells were washed 2 times with mem to remove unbound virus. subsequently, media with antiviral compounds were added to the cells for further incubation for 72 h at 37 c in a 5% co 2 humidified environment. cpe was examined by inverted light microscopy, and 50 ml of supernatant was collected for virus quantification, as we previously described with modifications. 14 thereafter, 50 ml of serum free mem and 10 ml of 5 mg/ml mtt solution (prepared in 1 â pbs, filtered) were added to the wells. the monolayers were incubated as above for 4 h (away from light). finally, 100 ml of 10% sds with 0.01 m hcl was added and further incubated at 37 c with 5% co 2 overnight. the activity was read at od 570 with reference wavelength at od 640 . the interferon and non-interferon drug compound with the lowest 50% effective inhibitory concentration (ec 50 ) and highest selectivity index were selected for combination studies using the cpe inhibition assay. for the drug compounds with antiviral activity in the mtt assay, further evaluation by quantitative virus yield reduction and plaque reduction assays (pra) was performed. virus yield quantification was performed by quantitative rt-pcr using total nucleic acid extracted from culture supernatants of the vero cells infected by mers-cov on day 3 post-infection as we previously described. 14 pra was performed as we previously described with modifications. 32 briefly, it was performed in duplicate in 24well tissue culture plates (tpp). the vero cells were seeded at 1 â 10 5 cells/well in mem (invitrogen) with 10% fbs on the day before carrying out the assay. after 16e24 h incubation, 70e100 plaque-forming units (pfu) of mers-cov virus were added to the cell monolayer with or without the addition of drug compounds and the plates further incubated for 2 h at 37 c in 5% co 2 atmosphere before removal of unbound viral particles by aspiration of the media and washing once with mem. monolayers were then overlaid with media containing 1% low melting agarose (cambrex) in mem and appropriate concentrations of drug compounds and incubated as above for 72 h. next, the wells were fixed with 10% formaldehyde (bdh) overnight. after removal of the agarose plugs, the monolayers were stained with 0.7% crystal violet (bdh) and the plaques counted. the percentage of plaque inhibition relative to the control (without the addition of compound) plates was determined for each drug compound concentration. the ec 50 and the 50% cellular cytotoxicity concentration (cc 50 ) were calculated using sigma plot (spss) in an excel add-in ed50v10. the pra were carried out in triplicate and repeated twice for confirmation. high-throughput screening (hts) ten drugs compounds, namely mycophenolic acid, flufenamic acid, tolfenamic acid, meclofenamate sodium, mefenamic acid, ribavirin, mercaptopurine, pyrimethamine, emetine, and estradiol were identified as primary hits with protective results in chemical library screening against influenza a/wsn/1933 (h1n1) virus (table 1) . neuraminidase inhibitors were not identified because they were not included in the chemical library. amantadine was not identified because the virus strain had an m2 gene mutation (s31n) conferring drug resistance. using both ec 50 and tc 50 as the hit selection criteria, only mycophenolic acid exhibited a low ec 50 of <10 mm with a high selectivity index of >100. mercaptopurine, which is a competitive, selective, and reversible inhibitor of the sars-cov papain-like protease, 33 in addition to mycophenolic acid (sigmaealdrich, usa), ribavirin (tianxin pharmaceutical, china), intron a (recombinant interferon-a2b, schering-plough, usa), avonex (recombinant interferon-b1a, biogen idec, denmark), rebif (recombinant interferon-b1a, merck serono, italy), betaferon (recombinant interferon-b1b, bayer schering pharma, germany), imukin (recombinant interferon-g1b, boehringer ingelheim, germany), nelfinavir mesylate hydrate (agouron pharmaceuticals, usa), and lopinavir (abbott, usa) were also tested in the mtt assays because of their documented in vitro anti-sars-cov activities in previous reports. 34e37 among them, only mycophenolic acid, ribavirin, intron a, avonex, rebif, and betaferon showed anti-mers-cov activity at the tested concentrations ( table 2) . cpe was completely absent in vero cells infected with mers-cov on day 3 post-infection at concentrations of !0.063 mg/ml for mycophenolic acid and !100 mg/ml for ribavirin, and was decreased but not absent in the tested concentrations of intron a, avonex, rebif, or betaferon (table 3) . combination studies showed that the ec 50 of mycophenolic acid was lowered by 1.7e2.8 times in the presence of 6.25e12.5 iu/ml of betaferon, and that the ec 50 of betaferon was lowered by 1.1e1.8 times in the presence of 0.016e0.063 mg/ml of mycophenolic acid (table 2) . the mean baseline viral load in the cell culture supernatants without drugs was 12.110 ae 0.003 log 10 copies/ml. there was a 50% reduction in viral load as compared to the baseline in cell culture supernatants inoculated with each of the six drugs (fig. 1 ). there was a >2-log reduction in viral load in cell culture supernatants inoculated with mycophenolic acid, ribavirin, rebif, and betaferon. there was >1-log reduction in the viral load in cell culture supernatants at 40 iu/ml of betaferon and >3-log reduction at the highest concentration of 50,000 iu/ml (fig. 1c) . the largest reduction in viral load at clinically relevant drug levels was a nearly 4-log reduction at 16 mg/ml of mycophenolic acid. mycophenolic acid, ribavirin, and rebif achieved 100% plaque reduction at concentrations of 6.4 mg/ml, 400 mg/ml, and 62,500 iu/ml respectively (figs. 2 and 3) . the maximum percentages of plaque reduction achieved by intron a, avonex, and betaferon were 76.2% at 70,000 iu/ml, 70.2% at 5000 iu/ml, and 66.6% at 400 iu/ml respectively (fig. 3) . in pra, betaferon achieved 40e50% plaque reduction at 40 iu/ml (fig. 3c ). novel antiviral targets for sars coronavirus and influenza a virus have been identified previously using small compoundbased forward chemical genetics approaches similar to ours. 25, 38, 39 in this study, we identified ten compounds among approved drugs with as primary hits in chemical library screening that possess antiviral activities. some may offer potential therapies in the evolving mers-cov epidemic. influenza a/wsn/1933 (h1n1) virus, instead of mers-cov, was used for initial screening because its manipulation did not require a biosafetly level iii laboratory. other human betacoronaviruses such as hcov-oc43 and hcov-hku1 were not used because of their slow replication and low viral titres in cell culture. 14 among the 10 identified drug compounds, only mycophenolic acid exhibited an ec 50 of <10 mm, which is a common cut-off value for lead compound detection, and a high selective index of >100. additionally, we tested other agents reported to have in vitro activities against sars-cov and/or mers-cov. 32, 34, 40 imukin (interferon-g1b) and the hiv protease inhibitors, nelfinavir mesylate hydrate and lopinavir, showed suboptimal ec 50 in the initial cpe inhibition assay and were therefore not further evaluated. together with mycophenolic acid, four other drug compounds in five preparations, namely ribavirin, intron a, avonex, rebif, and betaferon, showed in vitro anti-mers-cov activity of varying magnitude across four assays. mycophenolic acid is a selective, non-competitive, and reversible inhibitor of inosine-5 0 -monophosphate dehydrogenase (impdh). it inhibits the proliferation of t and b lymphocytes and production of immunoglobulins by depletion of the lymphocyte guansine and deoxyguanosine nucleotide pools. 41 its major clinical indication is prevention of graft rejection in solid organ and haematopoeitic stem cell transplantations. in addition to potent immunosuppressive activity, mycophenolic acid also has broad activity in vitro and/or in animal models against different viruses including west nile, 42 japanese encephalitis, 43 yellow fever, 44 dengue, 45 chikungunya, 46 and possibly hepatitis b viruses. 47 furthermore, it inhibited the in vitro and in vivo replication of hepatitis c virus by augmentation of interferon-stimulated gene expression and depletion of guanosine. 48, 49 combination treatment with interferon-a showed additive effects on interferon-stimulated gene expression and enhanced interferon-induced luciferase reporter activity. 48 as for coronaviruses, mycophenolic acid test 1 test 2 test 3 12,500.000 t t t 50,000.000 t t t remarks: -, negative; 1þ is defined as 1%e25% involvement; 2þ is defined as >25%e50% involvement; 3þ is defined as >50%e 75% involvement; 4þ is defined as >75% involvement; t, druginduced toxic effects in vero cells. was found to be ineffective against sars-cov in an animal model, although it did not significantly increase the viral load in the lungs of sars-infected balb/c mice as ribavirin did. 50 we are unaware of data on its activity against other human coronaviruses. our study is the first to demonstrate the anti-coronavirus activity of mycophenolic acid against the novel mers-cov. in addition to mycophenolic acid, our in vitro findings indicated that ribavirin, interferon-a, and interferon-b had anti-mers-cov activities in vitro. in the case of sars-cov, their antiviral activities in in vitro susceptibility tests had been conflicting. 34 none of them were tested systemically in large-scale randomized controlled trials and the results from clinical trials involving their use in sars were often confounded with the concomitant use of corticosteroids. 51, 52 although their clinical use in mers-cov infection has not been described, a recent study found that ribavirin had in vitro anti-mers-cov activity at very high concentrations which was potentiated when given together with interferon-a2b. 40 another study showed that mers-cov is 50e100 times more sensitive to pegylated interferon-a than sars-cov in vero cells, which is possibly related to the lineage-specific genetic differences between the two coronaviruses with mers-cov lacking the homolog of the sars-cov orf6 protein responsible for the blockade of interferon-induced nuclear translocation of phosphorylated transcription factor stat1. 53 furthermore, the delayed and aberrant induction of inflammatory cytokines and chemokines by mers-cov might support the use of adjunctive immuno-modulatory treatment combined with antivirals in patients with mers. 54, 55 among the four preparations of interferons tested, betaferon exhibited the lowest ec 50 of 17.64 iu/ml, which was below the mean peak serum concentration of 40 iu/ml after a subcutaneous dose of 16 million iu or an intravenous dose of 0.2 million to 64 million iu. 56 although the other preparations of interferons also demonstrated in vitro anti-mers-cov activities, their ec 50 were generally above the peak serum concentrations achievable with usual therapeutic dosing. combination treatment consisting of mycophenolic acid and betaferon resulted in a 1.7e2.8-fold reduction in the ec 50 of mycophenolic acid in vero cells with 6.25e12.5 iu/ml of betaferon, and 1.1e1.8-fold reduction in the ec 50 of betaferon in vero cells with 0.016e0.063 mg/ml of mycophenolic acid. our finding may provide the basis for combinational mycophenolic acid and betaferon in future clinical trials. compared with ribavirin and interferons, mycophenolic acid exhibits a number of attributes that support its practical use in mers-cov infection. it is commonly available in two forms, the prodrug mycophenolate mofetil and the salt mycophenolate sodium, and could be given orally or parenterally. the serum concentration of mycophenolic acid peaks at around 10e50 mg/ml after a 1000 mg oral dose of mycophenolate mofetil or 26.1 mg/ml after a 720 mg oral dose of mycophenolate sodium. these far exceeds its ec 50 of 0.17 mg/ml and is 60e300 times higher than the concentrations at which the replication of mers-cov is inhibited in cell culture and pra. 57 with average plasma elimination half-lives of 17.9 h and 16.6 h after a 1000 mg oral dose and 1500 mg intravenous dose of mycophenolate mofetil respectively, 41 the usual regimens consisting of 1000 mg twice daily oral or 1500 mg twice daily intravenous mycophenolate mofetil would be sufficient to achieve levels well above the ec 50 throughout the dosing interval. in contrast, the ec 50 of ribavirin for mers-cov between 9.99 and 41.45 mg/ml is just marginally effective in some cell lines and greatly exceeds the drug's serum concentration with usual oral doses. peak concentrations with high intravenous doses may reach approximately 24 mg/ml in humans, but steady-state requires at least 4 weeks to achieve. 40, 58 furthermore, the use of ribavirin, and hence also its combination with interferon-a2b, may be limited in the clinical setting, because a significant proportion of patients with mers-cov infection have developed acute renal failure often requiring renal replacement therapy. 2, 24 it has been suggested that systemic ribavirin should best be avoided in patients with a creatinine clearance of <50 ml/min because of the increased risk of haemolytic anaemia. 59 although mycophenolic acid may also be associated with acute renal impairment, the dosage adjustment in such a setting is generally well established. 41 the potent in vitro anti-mers-cov activity of mycophenolic acid may allow it to be used as a monotherapy if concomitant interferon is not available or tolerated by the patient. finally, drug level monitoring for mycophenolate mofetil is generally available in most tertiary hospitals which are the usual referral centers for cases of severe mers-cov infections requiring intensive care facilities such as extracorporeal membrane oxygenation. however, the risk of immunosuppression at the onset of adaptive immune responses or polarization towards a deleterious th1 response by mycophenolic acid needs to be considered. 60 one possible approach is a short course of mycophenolate mofetil combined with an interferon, particularly interferon-b1b, to provide synergistic antiviral and immune-enhancing effects against mers-cov. these options should be considered for study in randomized control clinical trials for this highly fatal disease. there are a number of limitations in our study. firstly, the cytotoxicity assay likely underestimated more subtle effects of candidate compounds on host cell growth and metabolism. for example, ribavirin inhibits replication of uninfected mdck cells at concentrations of 10 mg/ml and above but does not cause overt cytotoxicity until much higher concentrations are reached. 61, 62 secondly, we used vero cells alone to study the antiviral activity of ribavirin. vero cells have been described as being comparatively resistant to ribavirin due to their inefficient conversion of the drug into its mono-and tri-phosphate forms. 63 however, we decided not to perform the experiment using another cell line as this has been done in another recent report using vero and llc-mk2 cell lines which also demonstrated anti-mers-cov activity of high ribavirin concentrations similar to our findings. 40 it would be important to extend these in vitro studies to human respiratory epithelial cell systems and explants. to optimize treatment options for mers-cov infection, further studies on the anti-mers-cov activities of other potential anti-coronavirus agents which have been previously identified for sars-cov should be undertaken. replication of many coronaviruses including sars-cov and mers-cov requires proteolytic processing of the replicase polyprotein by two viral cysteine proteases, a chymotrypsin-like protease (3clpro) and a papain-like protease (plpro). however, the protease inhibitors such as nelfinavir and lopinavir were not found to be active in our in vitro study. helicase inhibitors are another group of agents with in vitro anti-sars-cov activities but their anti-mers-cov activities remain undetermined. 39, 64 inhalational nitric oxide was used as rescue therapy for sars and might be useful for treating mers-cov infection if organic nitric oxide donors such as s-nitro-n-acetylpenicillamine also show anti-mers-cov activity. 65, 66 antiviral peptides or neutralizing antibodies designed against heptad repeat region 2 of s2 which may inhibit membrane fusion and cell entry of sars-cov could theoretically be harnessed for mers-cov since the s2 region shared significant homology amongst betacoronaviruses. 67e70 other agents with in vitro anti-sars-cov activities such as glycyrrhizin, baicalin, reserpine, aescin, valinomycin, niclosamide, aurintricarboxylic acid, mizoribine, indomethacin, chloroquine, and experimental agents like small interfering rna (sirna) and inhibitors targeting the binding interface between the s1 domian and receptor in vivo, should also be evaluated. 34, 35, 71 we did not test these agents in this study because most of them have the problems of either not being commercially available or having therapeutic levels that are not easily achievable clinically. recently, cyclophilin inhibitors, such as cyclosporine which is available commercially, have also been reported to exhibit anti-mers-cov and anti-coronavirus activity in cell culture and viral load 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regions as potent inhibitors of sars-associated coronavirus suppression of coronavirus replication by cyclophilin inhibitors pneumonia from human coronavirus in a macaque model the authors have no financial or any other conflicts of interest regarding the contents of the investigations. key: cord-277705-6lgt2i7f authors: luan, junwen; lu, yue; gao, shan; zhang, leiliang title: a potential inhibitory role for integrin in the receptor targeting of sars-cov-2 date: 2020-04-10 journal: j infect doi: 10.1016/j.jinf.2020.03.046 sha: doc_id: 277705 cord_uid: 6lgt2i7f nan integrins are heterodimeric proteins comprising α and β subunits in cell surface. many integrins recognize arg-gly-asp (rgd) and lys-gly-asp (kgd) motifs which are displayed on the exposed loops of proteins. rgd could associate broader types of integrins such as αvβ1, αvβ3, αvβ5, αvβ6, αvβ8, αⅱbβ3, αmβ2, αlβ2 and α3β1, while kgd-recognizing integrins are restricted to αⅱbβ3, αvβ5, αvβ6 and αvβ8 (2) . a recent study proposed a proviral role for integrins in sars-cov-2 entry (3). in current study, we found rgd/kgd motif presents not only in s protein but also in its receptor ace2. we suggested inhibitory roles for integrins in the entry of both sars-cov-2 and sars-cov. rgd and kgd are two classical integrin-binding motifs. first, we performed sequence analysis of s proteins from sars-cov-2 (yp_009724390.1) and sars-cov (np_828851.1). an rgd motif (403-405) was identified in sars-cov-2 s protein ( figure 1a ). in sars-cov s protein, there is a kgd motif (390-392) ( figure 1a ). by searching ncbi databases and literatures, we identified similar rgd/kgd motifs in several coronaviruses including pangolin/mp789/2019 (4), pangolin/gx/p5l/2017 (4), bat_sarsr-cov_rs3367 (agz48818.1). these results suggested that rgd/kgd integrin-binding motif is conserved in several coronaviruses including sars-cov-2 and sars-cov. in order to associate with integrin, rgd/kgd motif should be exposed to the surface of the protein. to investigate whether rgd/kgd motif in s proteins from sars-cov-2 and sars-cov is on the surface, we analyzed the structures of sars-cov-2 s trimer (pdb: 6vsb) (5) and sars-cov s trimer (pdb: 5xlr) (6) by chimera software ver 1.14. rgd located at the top of protrusions in sars-cov-2 s, and kgd is displayed on exposed loop in sars-cov s ( figure 1b and 1c) . these results suggested s proteins are accessible to integrins. rgd/kgd motif in s protein is close to rbm of s protein. next, we checked the structure of ace2 with sars-cov-2 s rbd (pdb: 6lzg) and ace2 with sars-cov s rbd (pdb: 2ajf) (7) by chimera software ver 1.14. rgd/kgd in s protein is near the groove of complex of rbd and ace2 ( figure 1d and 1e ). if s protein associates with integrin, there would be no space for ace2 to contact with s. ace2 is known to associate with integrin. an rgd sequence (203-205) in ace2 is partially buried inside the protein and thus not responsible for integrin association (8) . we identified a kgd motif in 353-355 of ace2 (bab40370.1), which is a key region for binding s protein ( figure 1f ). this kgd motif is on the exposed small loop in the structure of open ace2 dimer (pdb: 6m1d) and closed ace2 dimer (pdb: 6m18) (9), which indicated that integrin could interact with ace2 through this motif ( figure 1g ). we proposed the following model for the role of integrin in receptor recognition of s protein ( figure 2 ). upon sars-cov-2 or sars-cov infection, integrin could interact with ace2 and s protein individually. integrin associates with ace2 through its kgd motif including k353, which is one of key aas for s protein recognition. integrin associates with s protein by its rgd/kgd motif, which would shield the space of rbm for contacting with ace2. in those scenarios, both ace2 k353 (inside kgd) and s protein rbm (near rgd/kgd) are shielded by integrin. thus, ace2 could not recognize s protein, leading to a suppressed viral entry. in the condition of ace2 targeting of s protein, integrin no longer blocks ace2-s interaction, resulting in a robust virus entry. several host proteins were predicted to associate with sars-cov-2 s protein (3, 10). recently, people reported that the sars-cov-2 s protein acquired an rgd integrinbinding motif and claimed that this motif was absent from other coronaviruses (3). they further speculated that rgd acquired by sars-cov-2 would promote virus entry by association of integrins, thus enhance the transmission ability. we disagree with that because both rgd and kgd could recognize integrins. the difference is that rgd recognizes more types of integrins than kgd. when integrins bind to s protein, the stereo-hindrance effect will prevent ace2 targeting by s protein. moreover, ace2 contains a kgd integrin-binding motif inside the s-binding region. when integrins bind to s protein, ace2 targeting by s protein will also be inhibited. taken together, our model favors an inhibitory role for integrins in virus entry by associating with both s protein and ace2. potential association of s protein and integrins provide mechanistic insights for the pathogenesis of sars-cov-2 and sars-cov. because rgd recognized a broader spectrum of integrins than kgd, more integrins could block receptor binding of sars-cov-2 s than that of sars-cov s. consequently, sars-cov-2 would infect fewer organs than sars-cov, which might partially explain why sars-cov-2 caused less mortality than sars-cov. in conclusion, we identified an rgd/kgd integrin-binding motif in s proteins from sars-cov-2 and sars-cov. we also discovered a kgd integrin-binding motif in ace2. integrins were predicted to inhibit receptor targeting of s proteins from sars-cov-2 and sars-cov by shielding both s protein and ace2. we proposed a previous unappreciated inhibitory role for integrin in virus entry, which will improve our understanding on the virus entry for both sars-cov-2 and sars-cov. the authors declare that there are no conflicts of interest. in uninfected condition, integrin could associate with ace2 through kgd motif. upon sars-cov-2 or sars-cov infection, integrin could interact with ace2 (through kgd) and s protein (through rgd/kgd) individually, which will mask the interface between s protein and ace2. thus, ace2 could not recognize s protein, leading to a suppressed viral entry. when ace2 associates with s protein, a robust virus entry will occur. public health might be endangered by possible prolonged discharge of sars-cov-2 in stool from discovery of snake venom disintegrins to a safer therapeutic antithrombotic agent a potential role for integrins in host cell entry by sars-cov-2 identification of 2019-ncov related coronaviruses in malayan pangolins in southern china cryo-em structure of the 2019-ncov spike in the prefusion conformation cryo-electron microscopy structures of the sars-cov spike glycoprotein reveal a prerequisite conformational state for receptor binding structure of sars coronavirus spike receptor-binding domain complexed with receptor angiotensin converting enzyme (ace) and ace2 bind integrins and ace2 regulates integrin signalling structural basis for the recognition of the sars-cov-2 by full-length human ace2 covid-19 spikehost cell receptor grp78 binding site prediction key: cord-263671-2b54qfo7 authors: soriano, maría cruz; vaquero, concepción; ortiz-fernández, almudena; caballero, alvaro; blandino-ortiz, aaron; pablo, raúl de title: low incidence of co-infection, but high incidence of icu-acquired infections in critically ill patients with covid-19 date: 2020-09-19 journal: j infect doi: 10.1016/j.jinf.2020.09.010 sha: doc_id: 263671 cord_uid: 2b54qfo7 nan lansbury et al. recently reported in this journal that 7% of hospitalized covid-19 patients had a bacterial co-infection. this proportion increased to 14% in studies that only included patients who required admission to the intensive care unit (icu) 1 . icu admission is a risk factor for hospital-acquired infections and nosocomial infections by multidrug-resistant (mdr) bacteria 2, 3 . here, we report our findings of a retrospective cohort study to asses the incidence of co-infections, icu-acquired infections and their relation to mortality in patients with covid-19. we retrospectively include all consecutive patients who were admitted to the intensive care department at hospital universitario ramón y cajal in madrid (spain), with the primary diagnosis of sars-cov-2 between march 10th and june 19th, 2020. madrid was one of the pandemic epicenter cities in europe. all patients had a diagnosis of covid-19 confirmed by sars-cov-2 viral rna polymerase-chain-reaction (pcr) test from nasopharyngeal swabs or lower respiratory tract aspirates as well. we excluded patients in whom no positive pcr was detected despite the clinical diagnosis of covid-19 and patients with less than 48 hours of admission at the icu admissions of less than 48h. clinical data were collected from institutional healthcare clinical database record and managed using redcap  (research electronic data capture) tool hosted at irycis (instituto ramón y cajal de investigación sanitaria). frequency measurements have been calculated using the incidence rates of each icu-acquired infections expressed in relation to the number of patients at risk or the number of days at risk. data were expressed as mean ± standard deviation (s.d) or percentages as appropriate. since most variables did not always fulfill the normality hypothesis, we compared continuous data by the mann-whitney u test and categorical data by chisquare or fisher's exact test as appropriate. study protocol was approved by the institutional ethics and clinical research committee. a total of 83 patients were enrolled. clinical characteristics of critically ill patients are shown in table 1 . overall mortality in the icu was 24.1 %. community-based bacteria and viruses were screened at hospital admission in 91.5% (76/83) of patients. in our series, the incidence of bacterial coinfection at admission was only 8.4% and no patient was diagnosed at admission with any other virus than sars-cov-2. isolated bacteria were: s. pneumoniae n=1, legionella pneumophila n=2, pseudomonas aeruginosa n=1, klebsiella oxytoca n=1 and methicilin-sensitive s. aureus n=2. a low prevalence of bacterial co-infection might be underestimated having regard to the high proportion of patients who received empiric antibiotic therapy, such as azithromycin because its antiviral properties. these data are in agree lansbury et al. and with others reports 1, 4 these findings support stopping empirical antibiotics in the vast majority of patients when covid-19 infection is confirmed. however, it is important to remark that mortality in the subgroup of patients with co-infection was very high, with a mortality rate of 57.1% versus 21.1% in patient without co-infection (p = 0.033). therefore, it is essential to suspect and look for the presence of bacterial co-infection to establish appropriate antibiotic therapy as soon as possible. conversely, the incidence of icu-acquired infection was as high as 51.2% (43/84). in patients undergoing mechanical ventilation for more than 5 days (93.3%), microbiological surveillance samples were obtained during their icu stay. table 2 shows incidence rate of icu-acquired infection. the respiratory tract was the most common site of infection, accounting for 38.5%, followed by bloodstream (30.7%), urinary tract infection (28.0%), soft-tissue (1.7%) and abdominal focus 0.8%. icu mortality was significantly different for patients with or without icu-acquired infection (15/20, 75.0% versus 28/63, 44.4%; p= 0.017), respectively. there's controversy regarding to nosocomial infection and its relationship with mortality due to several confounding factors that converge in patients admitted to icu. in large european epidemiological studies of critically ill patients such as the epic ii study, among 13,796 patients, 51% were considered infected, the icu mortality rate of infected patients was more than twice than in non-infected patients 2 . there is a lack of evidence related to superinfections acquired during covid-19 in patients who require hospitalization. a study conducted in wuhan, china shows a series of 150 hospitalized covid-19 patients in whom the presence of secondary infection during hospital admission was one of the risk factors for increased mortality 5 . a recent study found that frequency of hospital-acquired superinfections remained low and this finding was mainly related with icu admission 6 . to the best of our knowledge, there are no previous data on the influence of nosocomial infection in the icu and its relationship with mortality. in conclusion, our results reveal that co-infections in patients diagnosed with covid-19 admitted to the icu is uncommon; however, the incidence of icu-acquired infections very high. when one of both types of infections comes out, this is associated with worse outcomes including higher mortality. assessment of necessary diagnostic workup could assist clinicians in decision-making to optimize antibiotic therapy in critically ill patients with covid-19. co-infections in people with covid-19: a systematic review and meta-analysis international study of the prevalence and outcomes of infection in intensive care units risk factors for multidrug-resistant gram-negative bacteria infection in intensive care units: a meta-analysis epidemiological and clinical characteristics of 99 cases of china: a descriptive study clinical predictors of mortality due to covid-19 based on an analysis of data of 150 patients from wuhan, china incidence of co-infections and superinfections in hospitalised patients with covid-19: a retrospective cohort study key: cord-282499-baia5prj authors: lei, pinggui; fan, bing; yuan, yingnan title: the evolution of ct characteristics in the patients with covid-19 pneumonia date: 2020-03-19 journal: j infect doi: 10.1016/j.jinf.2020.03.014 sha: doc_id: 282499 cord_uid: baia5prj nan in that study, significant statistical differences were observed in ct features of peripheral involving central ( p = 0.028), consolidation ( p = 0.009), and thickened interlobular septa ( p = 0.040) between moderate ( n = 28) and sever/critically severe ( n = 13). 1 we would like to share our opinions about the evolution of radiological features in the patients with covid-19 infection. actually, the ct features are various at different stages in the patients with covid-19 infection. currently, the "gold standard" is real-time reverse transcriptase polymerase chain reaction (rt-pcr) amplification of the viral dna for diagnosis of covid-19 infection. however, medical imaging is of great significance in the detection and surveillance of covid-19 infection. recently, the studies demonstrated that the ct findings were typical signs for diagnosis at different stages of covid-19 pneumonia. 3 , 4 particularly, ground glass opacities (ggo) and consolidation were the principal manifestation in the ct images (ct scans before onset of symptoms or ct scans done ≤ 1 week after symptom onset), and ggo was decreased with increasing the stages of covid-19 pneumonia. however, the consolidation or ggo mixed consolidation increased, and reticular was also observed in the later stages (scan > 1 week after symptom onset). pleural effusion or lymphadenopathy was rarely seen. 4 ct features were distributed primarily in the lower lobes and subpleural (especially in the right lower lobe), 4 , 5 with rapid evolution from focal unilateral pulmonary parenchyma to diffuse bilateral ggo or ggo with consolidation within 1-3 weeks. 4 therefore, knowing the corresponding ct feature of covid-19 pneumonia at different stages, which could be helpful to precisely diagnose and understand ct characteristics of the novel coronavirus pneumonia beyond the radiological findings itself. in conclusion, there were some ct characteristics concerning the location of lesion, ggo, consolidation, etc. detected in the patients with covid-19 pneumonia. however, the evaluation of pri-mary ct findings in covid-19 pneumonia is various at different stages. none. clinical and computed tomographic imaging features of novel coronavirus pneumonia caused by sars-cov-2 clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (covid-19):a multi-center study in wenzhou city time course of lung changes on chest ct during recovery from 2019 novel coronavirus (covid-19) pneumonia radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study chest radiographic and ct findings of the 2019 novel coronavirus disease (covid-19): analysis of nine patients treated in korea key: cord-025980-85jbwmfv authors: iwasaki, sumio; fujisawa, shinichi; nakakubo, sho; kamada, keisuke; yamashita, yu; fukumoto, tatsuya; sato, kaori; oguri, satoshi; taki, keisuke; senjo, hajime; sugita, junichi; hayasaka, kasumi; konno, satoshi; nishida, mutsumi; teshima, takanori title: comparison of sars-cov-2 detection in nasopharyngeal swab and saliva date: 2020-06-04 journal: j infect doi: 10.1016/j.jinf.2020.05.071 sha: doc_id: 25980 cord_uid: 85jbwmfv nan in this journal, azzi et al. reported that saliva was a reliable tool to detect sars-cov-2(1). we prospectively compared the efficacy of pcr detection of sars-cov-2 between paired nasopharyngeal and saliva samples in 76 patients including ten coronavirus disease 2019 (covid-19) patients. the overall concordance rate of the virus detection between the two samples reached as high as 97.4% (table 1) ; we confirmed that saliva is a noninvasive and reliable alternative to nasopharyngeal swabs and facilitate widespread pcr testing in the face of shortages of swabs and protective equipment without posing a risk to healthcare workers. nasopharyngeal swab and saliva samples were simultaneously collected from patients suspicious of covid-19 and those with the diagnosis of covid-19. this study was approved by the institutional ethics board and informed consent was obtained from all patients. to collect nasopharyngeal samples, the swab was passed through the nostril until reaching the posterior nasopharynx and removed while rotating. saliva were self-collected by the patients and spit into a sterile pp screw cup 50 (asiakizai co., tokyo, japan). 200 l saliva was added to 600 l pbs, mixed vigorously, then centrifuged at 20,000 x g for 5 minutes at 4 o c, and 140 µl of the supernatant was used as a sample. real-time reverse transcription-quantitative pcr (rt-qpcr) was conducted according to the manual for the figure 1a) . the ct values were also not significantly different at earlier time points but tended to be higher in saliva later ( figure 1b) . figure 1c shows to our knowledge, a few studies compared viral load between nasopharyngeal and saliva samples. the viral loads were 5-times higher in saliva than in nasopharyngeal samples in one study (5) , whereas they were lower in saliva in two studies (6, 8) . in one study, viral loads were equivalent in symptomatic patients, but lower in asymptomatic patients in saliva(9). our results showed that the viral load was equivalent at earlier time points but lower in saliva than in nasopharyngeal samples at convalescent phase. timing of sampling, severity of the disease, different methodologies of saliva collection and processing, different skill of swab sampling may be related to inconsistent results. although our study has several limitations due to the small number of samples, there have been few prospective studies to date comparing the two samples. given the large benefits of saliva collection that does not require health worker specialists and protective equipment, our results together with recent studies support the use of saliva as a noninvasive alternative to nasopharyngeal swabs to greatly facilitate widespread pcr testing in the face of shortages of swabs and protective equipment without posing a risk to healthcare workers. the authors declare that they have no competing interests. saliva is a reliable tool to detect sars-cov-2 functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses consistent detection of 2019 novel coronavirus in saliva temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study saliva is more sensitive for sars-cov-2 detectionin covid-19 patients than nasopharyngeal swabs. medrxiv saliva as a non-invasive specimen for detection of sars-cov-2 sensitivity of nasopharyngeal swabs and saliva for the detection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) saliva is less sensitive than nasopharyngeal swabs for covid-19 detection in the community setting key: cord-281984-en9825p9 authors: wang, shijie; fu, lingli; huang, kejie; han, jianglong; zhang, rui; fu, zhenming title: neutrophil-to-lymphocyte ratio at admission is an independent risk factors for the severity and mortality in patients with coronavirus disease 2019 date: 2020-09-24 journal: j infect doi: 10.1016/j.jinf.2020.09.022 sha: doc_id: 281984 cord_uid: en9825p9 • the level of nlr at admission could be independent risk factors for the severe disease and the mortality of covid-19. • the predictive value of nlr for poor prognosis was more significant in patients without other potential risk factors. • nlr could help physicians rapidly identify high-risk patients and adopt timely intervention.  the level of nlr at admission could be independent risk factors for the severe disease and the mortality of covid-19.  the predictive value of nlr for poor prognosis was more significant in patients without other potential risk factors.  nlr could help physicians rapidly identify high-risk patients and adopt timely intervention. the study by liu et al. have been published in your journal and reported that the neutrophil-to-lymphocyte ratio (nlr) is an independent risk factor for the mortality of the covid-19 patients. 1 based on it, we reported the association between levels of nlr at admission and the disease severity in covid-19, and further explored the predictive role of nlr for mortality of the covid-19 patients in more subgroups. key epidemiological, clinical, laboratory, radiological and outcomes data were obtained through detailed medical chart review from january 1st to february 10th, 2020 at the renmin hospital of wuhan university. all the peripheral venous blood samples were collected on admission and examined at the laboratory following standard procedures. multivariable logistic regression analyses with stepwise procedure were used to estimate odds ratios (or) and 95% confidence intervals (ci 3 previous studies of coronavirus suggested that the lymphocyte loss might be associated with the immune-escape mechanism of the virus. 3 nowadays, it has also been suggested that lymphocyte loss might be associated hypertension. they only found that the male had a more significant association with the risk of mortality than the female. interestingly, we further found that nlr was of greater value in predicting severity and mortality for patients with no other clinical risk factors (i.e., those with a theoretically better prognosis), such as patients with younger age, or without comorbidities. in conclusion, we found that the level of nlr at admission could be independent risk factors for the prognosis of covid-19, not only for the mortality but also for the disease severity. and the predictive value was more significant in patients without other potential risk factors. nlr could help physicians rapidly identify high-risk patients and adopt timely intervention, so as to reduce the rates of severe disease and mortality. the authors state that they have no conflicts of interest to disclosure. there was no funding source for this study. at admission, liver and renal function tests were all found to be within normal range, and thus excluded from the data collection. neutrophil-to-lymphocyte ratio as an independent risk factor for mortality in hospitalized patients with covid-19 longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology snapshot: covid-19 cytokine storm and leukocyte changes in mild versus severe sars-cov-2 infection: review of 3939 covid-19 patients in china and emerging pathogenesis and therapy concepts clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. the lancet respiratory medicine 2020 immune system and chronic diseases abbreviation: covid-19, coronavirus disease derived from multivariate stepwise analysis of logistic regression models. 2 final model 1: for disease severity, retained in this model after stepwise selection was age (as continuous variable), fever (yes, no), dyspnoea (yes final model 2: for disease severity, retained in the final model were fever (yes, no), dyspnoea (yes, no), nlr, and crpr for survival status, retained in the final model were hypertension (yes, no), cancer (yes copd (yes, no), cancer (yes, no), fever (yes, no), dyspnoea (yes, no), and nlr tertile group; for survival, retained in the final model were age group (<60, ≧60), hypertension (yes, no), dyspnoea (yes, no), crpr, nlr tertile group no acknowledgements. key: cord-030594-xhp8kin0 authors: nan title: dear the editor, date: 2020-08-19 journal: j infect doi: 10.1016/j.jinf.2020.08.021 sha: doc_id: 30594 cord_uid: xhp8kin0 nan kunutsor sk et al. have written to this journal regarding elevated admission levels of markers of liver injury (alanine aminotransferase and aspartate aminotransferase, gamma-glutamyltransferase, alkaline phosphatase and total bilirubin) may be associated with progression to severe disease or death in covid-19. [1] on the other hand, serum cholinesterase plays an important role in the inflammatory response and may be associated with prognosis in sepsis. [2] [3] [4] we focused on the similarities between severe covid-19 pneumonia and sepsis. we examined associations between cholinesterase levels on admission and the severity, and mortality of patients with covid-19 pneumonia, as well as the interaction between cholinesterase and the previously reported factors of severity and mortality. we included patients who had tested positive for severe acute respiratory syndrome coronavirus 2 from february to may 2020 at yokohama city university hospital and yokohama city university medical center. ultimately, 26 patients were included in the study. outcomes were aggravation of symptoms and in-hospital death. the clinical characteristics of the patients grouped by severity are shown in table 1 . there was no significant difference in patient characteristics between the groups. supplementary materials 1 shows the time course of cholinesterase and other factors in critically ill patients with good outcome and death. in critically ill patients with favorable outcome, cholinesterase, lymphocytes, albumin, and pao 2 /fio 2 ratio decreased but c-reactive protein increased toward the peak of inflammation. later, c-reactive protein decreased with improvement in inflammation, but there was a tendency for cholinesterase, lymphocytes, albumin, and pao 2 /fio 2 ratio to increase. in contrast, in the severely ill patient who died, c-reactive protein poorly decreased, and cholinesterase, lymphocytes, albumin, and pao 2 /fio 2 ratio were not elevated. our results demonstrate that the potential of cholinesterase levels and their interactions were significantly associated with severity and mortality in covid-19 pneumonia patients. cholinesterase is an enzyme produced in the liver that hydrolyzes cholinesters, and measured as a liver function test. cholinesterase levels are high in patients with nephrotic syndrome, diabetes, hyperthyroidism, fatty liver, dyslipidemia, and obesity and low in patients with liver cirrhosis, hepatitis, malignant tumor, malnutrition, sepsis, and organophosphate poisoning. [5] although the mechanism underlying cholinesterase reduction in sepsis has not yet been determined, it is thought to be affected by acute-phase infections and inflammatory processes. [6] it has been hypothesized that cholinesterase synthesis decreases owing to hepatic dysfunction with disease progression, capillary permeability enhancement, dilution with fluid challenges, cholinesterase catabolism enhancement, and cholinesterase inhibition by inflammatory mediators (cytokines). [7] levels of "positive" acute-phase proteins such as c-reactive protein, amyloid a, and ferritin generally increase in patients with inflammatory diseases. in contrast, levels of "negative" acute-phase proteins such as albumin, prealbumin, and transferrin decrease in response to inflammation and increase during the recovery period. [2, 8] cholinesterase and lymphocytes behave similar to the "negative" acute-phase proteins in response to inflammation. [9, 10] even in patients with covid-19 pneumonia, amyloid a, which is classified as a "positive" acute-phase protein; albumin, which is classified as a "negative" acute-phase protein; and lymphocytes showing similar reactions as those of "negative" acute-phase proteins against inflammation have been suggested to be related to severity. [4] our study suggests that cholinesterase, which responds similar to the "negative" acute-phase proteins in response to inflammation, is reduced even in the acute phase of severe covid-19 pneumonia. following the changes in cholinesterase over time, we found that it decreased with deterioration of the condition and increased with improvement. cholinesterase level on admission is suggested to be an independent predictor of severity and mortality for covid-19 pneumonia. cholinesterase levels on admission were significantly lower in the severe group than in the mild-to-moderate group, and they were also significantly lower in the death group than in the survival group. cholinesterase was comparable to other markers, such as c-reactive protein, pao 2 /fio 2 ratio, albumin, ・funding this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. markers of liver injury and clinical outcomes in covid-19 patients: a systematic review and meta-analysis butyrylcholinesterase as a prognostic marker: a review of the literature a sustained reduction in serum cholinesterase enzyme activity predicts patient outcome following sepsis value of serum cholinesterase in the prognosis of septic shock serum cholinesterase is an excellent biomarker of cirrhosis acetylcholinesterase and butyrylcholinesterase as possible markers of low-grade systemic inflammation value of serum cholinesterase activity in the diagnosis of septic shock due to bacterial infections advances in understanding and assessing malnutrition protein-energy malnutrition: its effects on 4 metabolic parameters plasma esterases and inflammation in ageing and frailty median laboratory values (iqr) che (u/l) *the mild-to-moderate group was defined based on the need for oxygen inhalation or no oxygen inhalation the severe group was defined as having a respiratory condition requiring ventilator management (pao2/fio2 ratio <200 mmhg to respiratory rate >30 /min) key: cord-008676-35dgybwy authors: armero, georgina; launes, cristian; hernández-platero, lluïsa; alejandre, carme; muñoz-almagro, carmen; jordan, iolanda title: severe respiratory disease with rhinovirus detection: role of bacteria in the most severe cases date: 2016-08-05 journal: j infect doi: 10.1016/j.jinf.2016.07.010 sha: doc_id: 8676 cord_uid: 35dgybwy nan severe respiratory disease with rhinovirus detection: role of bacteria in the most severe cases we read with interest a recent paper in this journal about how the nasopharyngeal bacterial burden may influence in the severity in infants with respiratory syncytial virus (rsv) bronchiolitis. 1 we performed a study which aim was to analyze the epidemiologic and clinical characteristics of patients with severe lower-respiratory-tract infection (lrti) with rhinovirus (rv) detection in comparison to the patients without rv detection in a pediatric intensive care unit (picu), and the role of viral and/or bacterial codetections as risk factors of severity. we observed that the severity was related with bacterial detection on tracheal aspirates, independently of fulfilling diagnostic criteria of bacterial pneumonia. these results are similar to those of suarez-arrabal et al. with rsv infection. rv is the most common respiratory virus detected in all groups of age and probably the main agent causing acute respiratory infections in humans. 2 more than 30% of general admissions for acute lrti in children lower than 5 years of age are caused by rv, and the number of admissions to picu is not negligible. 3, 4 the study was conducted in a picu of a pediatric tertiary care hospital (january 2012edecember 2013). epidemiologic and clinical data of admitted patients 6-month to 18 years-old, with severe lrti (bronchospasm or bronchopneumonia) were consecutively and prospectively collected. patients with chronic conditions, previous episodes of wheezing and nosocomial respiratory infection were excluded. severe lrti was considered as that of those patients in need of admission to picu for any of the following treatments: invasive (imv) or non-invasive (niv) mechanical ventilation; or high flow oxygen therapy with fio 2 greater than or equal 0.6. suspected bacterial infection was defined as fever >38 c with laboratory abnormality (reactive c-protein>70 mg/dl or procalcitonin >1 ng/ml), and one or more thoracic radiography infiltrates, and antibiotic/s prescription during the first 24 h of admission. the total virus analyzed with a real-time pcr (anyplex ii rv16 detection (seegene, south korea)) in respiratory samples were: there were recruited 96 patients with lrti. in 66% rv was detected, similar to the literature. 3, 5 no differences in severity (requirements of ventilatory support, length of ventilatory support and picu stay) were found between patients infected with rv and patients with other viral detections. the main viral co-detection was rsv (4/11, 36%). no differences in severity between patients with rv and rv plus other viral co-detections were found (table 1 ). some series have described that rv could cause a more severe disease in comparison to other frequently detected viruses, such as influenza and respiratory syncytial virus (rsv). 5 in contrast, children with bronchiolitis and rv detection had a significantly shorter hospital length of stay as compared with children with rsv bronchiolitis in other series. 6 in our opinion, age, comorbidities 5 and differences in the diagnosis of included patients (bronchiolitis, bronchopneumonia, and bronchospasm) could be an important bias when interpreting these different results with regard to the severity of rv infection in comparison to other viruses. we want to remark that we didn't include children younger than 6 months, so the diagnosis of bronchiolitis was importantly avoided. 7 the distribution of patients who met criteria for suspected bacterial infection was similar between those with or without rv. the rate of patients with positive tracheal aspirates cultures was also similar between groups (table 1) . with regard to variables leading to severity of lrti in patients in whom rv was detected. 16 of 55 (29%) patients with rv infection required a picu stay over the 75th percentile of the total sample. there were not significant differences in the need for respiratory support with invasive imv, non-invasive mv, nor the duration of these techniques between patients with rv and rv plus other viral co-detections (table 1) . considering only the 15 patients in whom cultures of tracheal aspirates were performed within the first 72 h of hospital admission, the 2 more severely ill patients, those who required hfov, had 10 3 colonies/field of bacterial grown (staphylococcus aureus and haemophilus influenzae). all the patients (9) with confirmed bacterial growth required a long picu stay, whereas only 2 patients of 6 without bacterial detection required it; p z 0.01. of them, 2/9 (22%) do not fulfilled the criteria of bacterial infection ( table 2) . these results are in accord to those reported by kloepfer et al., who described that children with both, rv and bacterial detection in nasal samples, experienced greater airway inflammation, 8 similarly to the results of su arez-arrabal et al. 1 we feel that bacterial carriage in children with virus infection influences either in predisposing to bacterial pneumonia more easily (but 2 of 9 patients in our study do not fulfilled this criteria) or to suffer a greater airway inflammation such as yu et al. 9 recently, hofstra et al. performed an experimental study in healthy volunteers infected with rv. they observed changes of upper respiratory-tract microbiota that could help explain why rv infection predisposes to bacterial otitis media, sinusitis and pneumonia. 10 for this reason, bacterial carriage and, moreover, bacterial infection must be considered when analyzing the severity of rhinovirus infection in comparison to other viruses, and it is often missed. the main limitations of this study are the small sample size and the difficulty in distinguishing bacterial growth in the context of low-respiratory-tract colonization or bacterial pneumonia that did not meet the mentioned criteria of bacterial infection. to conclude, this study did not found differences in epidemiologic and clinical variables between children infected with rv and children with other viral infections. the study also highlights the important role of bacterial detection in tracheal aspirates, even without fulfilling criteria of bacterial pneumonia: all the intubated patients with rv infection and bacterial grown on tracheal aspirates required for a long picu stay. differences in the severity of patients with rv, with or without viral co-detection were not found. recent articles in this journal have referred to the problems caused by enteroviruses 1 particularly in china. 2 hand, foot, and mouth disease (hfmd) has been a serious public health problem in the asia-pacific region. 1e4 human enteroviruses a (hev-a) species with enterovirus 71 (ev-a71) and coxsackievirus a16 (cv-a16) have accounted for major hfmd outbreaks worldwide. 3, 4 recently, coxsackievirus a6 (cv-a6), another virus from hev-a, has also been recognized as an important pathogen for hfmd. 2, 4 infants and children are susceptible to cv-a6 infection, 4 but seroepidemiological studies on cv-a6 are lacking. in this study, neutralizing antibodies (ntabs) in serum samples from a prospective cohort study were analyzed to reveal the epidemic characteristics of cv-a6, cv-a16 and ev-a71 in the context of hfmd epidemic in infants and children. a total of 319 participants aged 6e35 months old, who were previously enrolled in a clinical trial to assess the immunogenicity of ev-a71 vaccine in jiangsu province (clinical trial no. nct01508247), were followed for two years (january, 2012ejanuary, 2014). sera were collected at the start of study (january 2012), in march 2012, september 2012 and january 2014. from this cohort, 180 participants whose sera were collected at all four scheduled visits were analyzed in this study. altogether, there were 117 males and 63 females, while 41 participants were in the infant group (aged 6e11 months) and 139 participants were in the child group (aged 12e35 months). titers of ntabs nasopharyngeal bacterial burden and antibiotics: influence on inflammatory markers and disease severity in infants with respiratory syncytial virus bronchiolitis new aspects on human rhinovirus infections rhinovirus associated with severe lower respiratory tract infections in children rhinovirus infection in hospitalized children in hong kong. pediatr clinical severity of rhinovirus/enterovirus compared to other respiratory viruses in children prospective multicenter study of viral etiology and hospital length of stay in children with severe bronchiolitis upper age limit for bronchiolitis: 12 months or 6 months? detection of pathogenic bacteria during rhinovirus infection is associated with increased respiratory symptoms and asthma exacerbations impact of bacterial colonization on the severity, and accompanying airway inflammation, of virus-induced wheezing in children changes in microbiota during experimental human rhinovirus infection the authors declare that there are no conflicts of interest. key: cord-256633-vls23fu5 authors: dimeglio, chloé; loubes, jean-michel; deporte, benjamin; dubois, martine; latour, justine; mansuy, jean-michel; izopet, jacques title: the sars-cov-2 seroprevalence is the key factor for deconfinement in france date: 2020-04-29 journal: j infect doi: 10.1016/j.jinf.2020.04.031 sha: doc_id: 256633 cord_uid: vls23fu5 a new virus, sars-cov-2, has spread world-wide since december 2019, probably affecting millions of people and killing thousands. failure to anticipate the spread of the virus now seriously threatens many health systems. we have designed a model for predicting the evolution of the sars-cov-2 epidemic in france, which is based on seroprevalence and makes it possible to anticipate the deconfinement strategy.  failure to anticipate the spread of sars-cov-2 after the containment phase seriously threatens many health systems.  we have developed a method for measuring how seroprevalence affects the deconfinement strategy in france.  seroprevalence must be at least 50% before confinement constraints can be relaxed.  deconfinement should be progressive in order to avoid rebound of the epidemic. the sars-cov-2 seroprevalence is the key factor for deconfinement in france. chloé dimeglio 1,2* , jean-michel loubes 3 a new virus, sars-cov-2, has spread world-wide since december 2019, probably affecting millions of people and killing thousands. failure to anticipate the spread of the virus now seriously threatens many health systems. we have designed a model for predicting the evolution of the sars-cov-2 epidemic in france, which is based on seroprevalence and makes it possible to anticipate the deconfinement strategy. keywords: sars-cov-2, seroprevalence, statistical model, deconfinement severe acute respiratory syndrome coronavirus 2 (sars-cov-2) emerged in wuhan, china in december 2019 and spread largely by sustained human-to-human transmission (1). the who declared the resulting disease a pandemic (2) . the virus, which causes severe respiratory illness in susceptible individuals, has spread so rapidly that it threatens to saturate health services, particularly their intensive care capacity. while this presently concerns mainly italy (3), spain (4), and france, other countries, such as the united states, may well succumb as it is difficult to predict how the infection will spread in the general population. a calibrated response to the epidemic must take into account the number of cases, including infected asymptomatic individuals, most of whom are not detected due to a lack of testing. the existing models for sars-cov-2 are based on published positive cases and do not take into account either people's age or any evolutive diffusion coefficient (5). our statistical model for predicting the spread of sars-cov-2 in france is based on a diffusion and transmission coefficient that varies with an individual's age, the likelihood of contagion, and two administration parameters (confinement and quarantine). we use models to measure how the dynamics of the sars-cov-2 infection is affected by these different factors and how to adapt the deconfinement strategy. consider the variables . we define as the age class variable: . on day for a given age class is the number of healthy people, is the number of undetected contagious carriers infected for days . similarly, is the number of detected contagious carriers infected for days on day . is the mortality rate per target age group. we assume that there is the same risk of virus-caused death at any stage of the infection. is the number of people who are immunized or have died for a given age class . we define as the number of days a person is contagious and as the percentage of the population tested on day . is the number of healthy people who a contagious person contacts and infects. we assume that the contagion coefficient varies over time and peaks when the virus load is maximal: 7 days after the start of infection (6) . is the total population at the start of the epidemic phase, is the multiplier for the pace of the epidemic throughout the confinement , and is the same multiplier during the quarantine period . and are equal to 1 when there is no confinement or quarantine phase. and ∑ ∑ is given by: on the transition from day to day , we have: 3. the model is a discretized version of a susceptible infectious and recovered (sir)-type model (7) we have chosen (8) . we estimated the initial model settings using data published by johns hopkins university for france (9) and data collected by the toulouse virology laboratory. without containment the prevalence of infection is 8.3% on march 17 and 97.5% on may 11 ( figure 1.a) . containment began on march 17 in france and is scheduled to end on may 11. figures 1.b, 1 .c, 1.d, 1.e showed predictions of new cases per day depending on the sars-cov-2 seroprevalence before and after the containment phase. figure 1 .b indicates that the seroprevalence before march 17 was 8.3%, and 17.5% after containment. there was an infection rebound on may 29. however, if the seroprevalence at the beginning of containment was 15.44% (figure 1.c) , the seroprevalence on may 11 would be 29.1% and there was an infection rebound. if 31.95% of the population contracted sars-cov-2 before march 17 (figure 1.d) , then 49% would be infected, immunized or dead on may 11 and the rebound would be smaller. lastly, when the seroprevalence before containment was 40.05% (figure 1 .e) there was almost no rebound. the seroprevalence on may 11 was 56.7%. this is why we conclude that the seroprevalence after the containment phase should be between 49% and 56% in order to avoid a rebound. in figure 2 .a we assumed that the containment did not end completely on may 11, but was abolished gradually from may 11 to june 30, to be totally relaxed from july, 1. we considered that this progressive phase consisted of a relaxation of 50% of the restrictive containment measures. seroprevalence on june 30 was 31.7% and leaded to a rebound at the end of july. the ideal situation is that shown in figure 2 .b, with a progressive deconfinement phase lasting longer, from may 11 to october 25, at which time seroprevalence reached 51.2%. relaxation of all restrictions after this date did not lead to a rebound. our data indicate that seroprevalence must reach approximately 50% after total deconfinement on may 11 or a gradual exit phase over several months starting on may 11 if an infection rebound is to be avoided (figure 1 .d, 1.e and figure 2 .b). while the seroprevalence in france before the confinement phase was not measured (march 17) it is probably possible to determine the proportion of the population who came into contact with the virus at the end of the confinement phase. this should provide a basis for estimating the size of any infection rebound. it remains highly unlikely that the case shown in figure 1 .e, with about 50% of seroprevalence, will apply. it will therefore be a question of adapting the deconfinement strategy to reach 50% seroprevalence gradually and so avoid a rebound. this may require wearing masks for several months. the predictive power of the model may be hampered by the way in which casualties are measured, as can the strategy used to detect the number of positive cases. postprocessing the data to handle such biases will be the topic of future reports. according to the seroprevalence before and after containment. a: no containment, b: seroprevalence before 8.3% and after 17.5%, c: seroprevalence before 15.4% and after 29.1%, d: seroprevalence before 31.9% and after 49%, e: seroprevalence before 40% and after 56.7%. who virtual press conference on covid-19-11 covid-19 in europe: the italian lesson the resilience of the spanish health system against the covid-19 pandemic nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study time kinetics of viral clearance and resolution of symptoms in novel coronavirus infection a contribution to the mathematical theory of epidemics who report of the who-china joint mission on coronavirus disease.(covid-19). available at the english text was edited by dr owen parkes. key: cord-288366-xe3pxrhv authors: wellbelove, zoe; walsh, chloe; perinpanathan, tanaraj; lillie, patrick; barlow, gavin title: comparing the 4c mortality score for covid-19 to established scores (curb65, crb65, qsofa, news) for respiratory infection patients date: 2020-10-25 journal: j infect doi: 10.1016/j.jinf.2020.10.015 sha: doc_id: 288366 cord_uid: xe3pxrhv nan comparing the 4c mortality score for covid-19 to established scores (curb65, crb65, qsofa, news) for respiratory infection patients we have taken great interest in ying et al's comparison of crb-65 and qsofa for predicting intensive respiratory support in covid-19 patients. 1 the initial assessment of severity is a key part of clinical decision making, guiding management and treatment escalation. this is particularly pertinent with the recent publication of knight et al's 4c mortality score for patients hospitalised with covid-19 and the upcoming winter respiratory infection season. 2 respiratory illnesses often present with symptoms similar to that of covid-19; fever, cough, shortness of breath and fatigue. this presents a challenge in clinically differentiating patients with covid-19 from other viral or bacterial infections. therefore, for clinical assessment and prognostication, a scoring system that can be applied to a wide range of respiratory infections would be beneficial. we compared the newly validated 4c mortality score to the established curb65, crb65 and qsofa scores in the prediction of 30-day mortality in a variety of existing respiratory infection cohorts in an exploratory analysis. data from various previous studies performed in dundee 3 , hull 4 and south yorkshire 5 of community-acquired pneumonia (cap), invasive pneumococcal disease (ipd), and influenza (flu), respectively, plus a covid-19 cohort (local isaric study patients 2 ) were analysed. a total of 606 patients with required data for 4c calculation were analysed from the existing databases described above. baseline characteristics are presented in table 1 . overall, the mean age was 60 years old, 30-day mortality was 12% and the median time to death was 5 days. the area under the receiver operating curve (auroc) with associated 95% confidence intervals was calculated for each scoring system in the respective cohorts (see table 2 ). the 4c mortality score had the greatest auroc in covid 19, cap and ipd patients (0.83, 0.78 and 0.74, respectively) and had a similar auroc, compared to the other scores (except news, which was not calculable), in the influenza cohort (0.88). the 4c score was the only score that performed statistically significantly better than chance across all four cohorts. this supports the findings of knight et al 2 , which showed that the 4c mortality score outperformed existing scores in covid-19 patients. the findings of our analyses also suggest the potential for application of the 4c score in other common, but potentially fatal respiratory infections. a larger prospective validation study of the 4c mortality score versus established scoring systems is needed this winter to confirm its utility in undifferentiated respiratory infection, focusing on the potential for ongoing use of the 4c mortality score, even after the pandemic has ended and the incidence of covid-19 is much lower. in conclusion, the 4c mortality score performed well (auroc of 0.74 to 0.88 across all the cohorts) in predicting 30-day mortality in covid-19 and other common respiratory infection populations. the 4c score has the potential to be applied broadly this winter, guiding initial escalation and management plans in patient's presenting with symptoms of respiratory infection, prior to a formal diagnosis and regardless of whether they are subsequently confirmed to have covid-19. comparison of crb-65 and quick sepsis-related organ failure assessment for predicting the need for intensive respiratory or vasopressor support in patients with covid-19 risk stratification of patients admitted to hospital with covid-19 using the isaric who clinical characterisation protocol: development and validation of the 4c mortality score the curb65 pneumonia severity score outperforms generic sepsis and early warning scores in predicting mortality in community-acquired pneumonia causes of early and late mortality following invasive pneumococcal disease in hull and east yorkshire severity assessment of influenza virus infection in secondary care thank you to the infectious diseases team for their hard work throughout the pandemic and help in compiling the isaric data. key: cord-019982-hyxrgamj authors: brookfield, d.s.k.; cosgrove, b.p.; bell, e.j.; madeley, c.r. title: viruses demonstrated in children in tanzania: studies in diarrhoea and measles date: 2005-04-14 journal: j infect doi: 10.1016/s0163-4453(79)91285-4 sha: doc_id: 19982 cord_uid: hyxrgamj causes of diarrhoea with particular reference to viral agents were investigated in 123 infants and young children in dar es salaam, tanzania. twenty-six of the patients also had measles. viruses were found in 52 of the 123 patients (43 per cent) and rotavirus occurred in 38 children (31 per cent). enteroviruses were found in 10 patients, adenoviruses in nine and ‘small round virus’ in one (six patients had dual infection). four patients died and only one of these children had viral particles in the stools. breast milk formed part or all of the diet in 77 children (63 per cent) and virus isolation showed a similar pattern in breast fed infants and those not receiving breast milk. in 26 patients with measles only five were excreting viruses in their stools. therefore no strong evidence was found to link the diarrhoea associated with measles in tanzanian children to any particular virus. the pattern of virus infection causing infantile diarrhoea was similar in dar es salaam to other parts of the world. diarrhoeal illness is a major cause of morbidity and mortality in developing countries. in the main hospital in dares salaam it was the commonest reason for admission to the paediatric wards (children aged seven years and under) in 1976. in that year 2410 children were admitted with a diganosis of gastroenteritis (kigadye, kimboi and kimati, 1976) and 75 of them died (3.1 per cent). this study was carried out to examine viral aetiological agents in diarrhoeal illness: bacterial diarrhoea is not included in this study. a number of patients with measles who also had diarrhoea were included in the study to see whether diarrhoea in measles was caused by a detectable viral agent. such diarrhoea occurring in children in the tropics indicates severe measles and often these infants require parenteral fluids (morley, 1969) . in order to provide a complete examination the study was a joint one between the university of dar es salaam and the university of glasgow. the patients in this study were selected randomly without any specific bias from the paediatric infectious disease ward in muhimbili hospital, dar es salaam, which serves mainly an urban african population. stool samples were examined for viruses from children with diarrhoea irrespective of whether bacterial pathogens were isolated. the study was performed between march 1976 and september 1976, a period which covers the cooler months of the year. studies in europe have suggested that stool viruses are more common in autumn and winter (flewett, davies, bryden and robertson, 1974) . a total of 123 children were investigated, of whom 26 had measles. the following information was collected from each patient: age, sex, a short feeding history and the degree of dehydration assessed clinically, using the criteria of ironside, tuxford and heyworth, 1970 . measles was diagnosed clinically by the agreement of two doctors who had considerable experience of paediatric infectious diseases in tanzania. a sample of faeces was collected within the first 48 hours after admission and usually within the first 24 hours. the faeces were stored at -20°c for between six and 12 months before being flown to scotland by air freight. on receipt in glasgow an extract of each stool in phosphate buffered saline was made and, after clarification at 3000 rev/min for 30 min in a bench centrifuge, was inoculated into cell cultures and prepared for electron microscopy (em) as described by madeley, cosgrove, bell and fallon (1977) . briefly, the extracts were centrifuged at 100,000 g for one hour, the pellet resuspended in two drops of em diluent (0.1 per cent bacitracin) and mixed with three per cent potassium phosphotungstate ph 7.0 as a negative stain. stool samples were taken from 123 patients and viruses were detected either by electron microscopy or culture in 52 of these patients. the results are listed in table i . three morphological types of virus (rotavirus, adenovirus and 'small round virus') were observed by electron microscopy but no astroviruses, caliciviruses or coronaviruses were detected cosgrove, 1975, 1976; caul, paver and clark, 1975) . adenoviruses and enteroviruses (polioviruses of two types and echoviruses of six types) were isolated in culture. the adenovirus cultured was not detected by electron microscopy, and no virus was cultured from the stool in which the 'small round virus' was observed. four patients (three per cent) died in hospital following admission for diarrhoea and one child died during a subsequent admission with intussusception. three had additional causes contributing to the severity of their illness: measles, measles and pneumonia, and marasmic kwashiorkor respectively. the fourth was severely dehydrated on admission. all of the children except two were under four years of age when admitted to hospital. the remaining two were six and seven years old. the numbers were too small for further analysis of the age distribution to be possible, but they fell into the age group in which most viruses have been observed. seventy-seven of the children (63 per cent) were still receiving breast milk on admission, though 68 of these were being weaned. in this part of africa, weaning, usually on to maize porridge, is a prolonged process, and children may be offered breast milk up to two to three years of age. a comparison of the type of feeding with the viruses identified is shown in table ii . it is apparent that breast feeding did not prevent the acquisition of viruses with rotaviruses being found most frequently (28 out of 77 (36 per cent)). in the three groups shown in table ii five out of nine children (56 per cent) in the 'breast fed' group had virus in their stools, 34 out of 68 children (50 per cent) in the 'breast and other food' group, and 13 out of 44 children (30 per cent) in the 'other food' group. the lowest rate was in the 'other food' group, which was also the oldest group with an average age of 22 months, compared with four months of the 'breast-fed' and the eight months of the 'breast milk with other food' group. though breast feeding did not prevent the acquisition of microorganisms it six of the 52 infected patients had a dual infection (see footnote on table i ). might reduce the severity of the disease. tables iii and iv explore this possibility by comparing firstly, the organism with the severity of the disease and secondly, severity with diet. the numbers are small but there is no suggestion that any of the viruses were associated with a more severe disease. table v . in five stools a virus was detected. there have been many reports of rotavirus-associated diarrhoea from different parts of the world particularly from europe, north america and australasia. reports from africa have been less frequent and have been mostly confined to those from south africa. the present study attempted to investigate the viruses associated with diarrhoea in dares salaam and, since electron microscopy was considered essential, the study was limited to the number of stools that could be sent in one consignment by air to scotland. examination of stools from 123 children resulted in the detection of 58 viruses of which 38 were rotaviruses. viruses were isolated in similar patterns both in breast fed and weaned babies. any antibody or other inhibitory substance in breast milk (matthews, nair, lawrence and tyrell, 1976) should present an immediate barrier to any organism attempting to colonise the gut. that it failed to do so in a number of cases suggests that later breast milk may be more deficient in inhibitors, antibody or otherwise, than colostrum and investigations into the content of breast milk after prolonged lactation have yet to be done; our numbers would not permit detailed analysis by age. it is also possible that in an area where protein malnutrition is common the quality of breast milk may be poorer than elsewhere. chrystie, totterdell and banatvala (1978) have reported that breast-fed babies excrete fewer rotavirus particles than bottle-fed babies in symptom free infections. the amount of virus seen in the stools of our breast-fed infants appeared to vary considerably but we were unable to standardise our methods enough for any conclusions to be drawn. there was no evidence that any one of the viruses detected caused a more severe disease than the others, judged by the amount of dehydration. rotavirus was the commonest organism identified and it was found in all degrees of severity from no dehydration to severe (10 per cent) dehydration. since this virus as well as others found in stools has also been observed in normal healthy babies (totterdell, chrystie and banatvala, 1976; madeley, cosgrove and bell, 1978) any observations must be interpreted with caution. recent reports (zissis and lambert, 1978; thouless, bryden and flewett, 1978) have suggested that there may be more than one serotype of rotavirus and, if so, the apparent variations in pathogenicity of the virus in this study may be due to a multiplicity of serotypes. no serotyping was attempted. examination of stools from 26 cases of measles failed to implicate any particular virus as a likely cause of the associated diarrhoea. since diarrhoea will be due to a disturbance of gut equilibrium this may occur through the considerable systemic upset caused by measles, which in malnourished infants may be sufficient to depress cellular function. there is evidence that measles virus may be found in the cells of the gut (fraser and martin, 1978) but there has been little investigation into its relationship to the associated diarrhoea which occurs in children with measles in the tropics. however the diarrhoea associated with measles in tanzanian children does not appear to be caused by any of the electron microscopically detectable viruses. the absence of suitable electron microscopy in tanzania meant that the stools had to be flown to scotland. the virus identification rate was similar to a local study (madeley, cosgrove, bell and fallon, 1977) but no astrovirus, calicivirus or coronavirus was identified. whether these were present and failed to survive the journey or whether their absence reflects different viral flora in different parts of the world is unknown at present. (we are grateful to the nursing and medical staff of the department of child health, muhimbili hospital; dares salaam, for help and encouragement; to the university of dares salaam for a grant to carryout the clinical studies and to the scottish hospitals endowment research trust for a grant (hert 484) towards the expenses of the study in scotland.) cornavirus particles in faeces from patients with gastroenteritis. lancet, i asymptomatic endemic rotavirus infection in the newborn acute gastroenteritis associated with reovirus like particles measles virus and its biology a survey of infantile gastroenteritis annual and academic report of the department of child health and muhimbili hospital paediatric services, university department of child health and paediatric department of the ministry of health 28 nm particles in faeces in infantile gastroenteritis caliciviruses in man. lancet, i stool virus in babies in glasgow. 1. hospital admissions with diarrhoea stool viruses in babies in glasgow. 2. investigation in normal newborns in hospital antiviral activity in milk of possible clinical importance severe measles in the tropics serotypes of human rotavirus rotavirus infections in a maternity unit different serotypes of human rotavirus key: cord-260247-akujsk0s authors: hamed, ehab title: rates of recurrent positive sars-cov-2 swab results among patients attending primary care in qatar date: 2020-11-02 journal: j infect doi: 10.1016/j.jinf.2020.10.029 sha: doc_id: 260247 cord_uid: akujsk0s nan we read with interest the work of cocorec (collaborative study covid recurrences) study group 1 . recurrent positive rt-pcr results for sars-cov-2 infection were reported from early in the epidemic [1] [2] [3] [4] [5] [6] [7] [8] . viral genomic sequences provided concrete evidence for reinfection by distinct sars-cov-2 infection [9] [10] [11] [12] . the number of days in between both infections in viral genomic proven reports ranged from 48 to 142 days. while viral genomic sequencing provides robust evidence, it does not lend itself well to everyday practice. the cocorec study identified 11 cases of reinfection using well-defined criteria 1 . the group suggested recurrent positive rt-pcr results of more than 21 days following the resolution of symptoms as criteria for reinfection. the criteria though less specific, are more feasible to use in primary health care settings. utilising the criteria set by the cocorec study group, this record-based study reports on the cases with recurrent positive rt-pcr nasopharyngeal swab for sars-cov-2 results in primary health care corporation (phcc) settings in qatar. phcc is the largest primary care provider in qatar with 27 health centers covering all the country. the organisational employs an electronic medical record (emr), which links all public primary health care centers. for this study, all electronic data were extracted from the primary healthcare setting visits, and no sampling was needed. the study population included patients attending with documented sars-cov-2 rt-pcr results during the study period. the study period was from february 10 th , 2020, to july 30 th , 2020, a total of 171 days. the recurrent positive population included all patients with a minimum number of 2 positive swabs and a minimum number of 21 days in between positive swab results. inconclusive and reactive rt-pcr results were considered negative. during the study period, patients were entitled to a repeat swab if they are attending with new symptoms following the resolution of initial symptoms. a maximum number of days in between any positive swab results was calculated for those who met our definition criteria. the study aims to answer the following questions. what is the maximum number of days in between positive swab results? what are the rates of recurrent rt-pcr sars-cov-2 positive results of more than 21 days, and what are the population characteristics? during the study period, we retrieved a total of 63444 patient records with 76742 swab results. only 62 patients met our inclusion criteria (62/63444;0.1%). the population was predominantly young. the mean age is 37. 3 the rates for recurrent positive results are reported for the total recurrent positive (62) and the total study population, (63444). the recurrent positive results of more than 42 days were rare (7/63444,0.01%) (table 2) . recurrent positive findings could occur in all age groups and different population types, including paediatric, elderly, and pregnant patients. current smoking status was highly prevalent among patients with recurrent positive results. no previous studies reported to the rates of recurrent positive rt-pcr for sars-cov-2 infections. given the extensive reporting of the sars-cov-2 infections, the number of case reports of recurrent positive and reinfection to date is extremely low, which agrees with our findings. earlier studies reported that viral shedding is dynamic and continue in most cases 20-22 days but positive results were generally rare beyond 30 days 13 . so, one could theorise that recurrent positive results in symptomatic patients should be considered reinfection, especially if more than 42 days. the rare occurrences of recurrent infections are reassuring to the world given the current surge and in favour of immunity. however, it does not allude to the length of that immunity. given the rarity of recurrent positive results which is supported by our findings, vaccination should be recommended for patients with no earlier sars-cov-2 infection. the study utilised centralised database records that allowed for large sample size, 63444 and long study period of 6 months. however, the record-based study does not report on the severity or the resolution of symptoms or the patients' outcomes. data request and analysis were anonymous, and no patient consent was required. anonymous data request approved by the department of clinical research, primary health care corporation with reference number phcc/dcr/2020/04/031. clinical recurrences of covid-19 symptoms after recovery: viral relapse, reinfection or inflammatory rebound? positive rt-pcr test results in patients recovered from covid-19 clinical characteristics of severe acute respiratory syndrome coronavirus 2 reactivation recurrence or relapse of <scp>covid</scp> -19 in older patients: a description of three cases recurrence of positive sars-cov-2 rna in covid-19: a case report is novel coronavirus 2019 reinfection possible? interpreting dynamic sars-cov-2 test results through a case report recurrence of covid-19 after recovery: a case report from italy covid-19 relapse with prolonged viral shedding up to 60 days or reinfection, in 3 frontline healthcare workers with recurrent symptoms and persistent sars-cov-2 pcr positivity in ireland, a developing diagnostic challenge: a case report. epub ahead of print genomic evidence for reinfection with sars-cov-2: a case study covid-19 reinfection by a phylogenetically distinct sars-cov-2 variant, first confirmed event in south america symptomatic sars-cov-2 reinfection by a phylogenetically distinct strain. clin infect dis. epub ahead of print covid-19) reinfection by a phylogenetically distinct severe acute respiratory syndrome coronavirus 2 strain confirmed by whole genome sequencing dynamic profile of rt-pcr findings from 301 covid-19 patients in wuhan, china: a descriptive study we acknowledge the support we receive from the primary health care corporation (phcc) research department. key: cord-271957-osaycpe8 authors: zuin, marco; rigatelli, gianluca; zuliani, giovanni; rigatelli, alberto; mazza, alberto; roncon, loris title: arterial hypertension and risk of death in patients with covid-19 infection: systematic review and meta-analysis date: 2020-04-11 journal: j infect doi: 10.1016/j.jinf.2020.03.059 sha: doc_id: 271957 cord_uid: osaycpe8 nan we read with great interest the recent manuscript published by zhou et al. describing the clinical characteristics of myocardial injury in severe and very severe patients with coronavirus 2019 disease [1] . also other recent investigations have reported a higher prevalence of cardiovascular disease (cvd) and a direct association between the severity of covid-19 infection [2] . however, to the best of our knowledge, no previous meta-analyses have globally estimated the risk of death in hypertensive patients with covid-19 infection. we therefore perform a systematic review and meta-analysis to evaluate the risk of death in covid-19 infection patients with and without ht. the analysis was conducted following the preferred reporting items for systematic reviews and meta-analyses (prisma) statement (supplementary file 1) [3] . an electronic search, based on medline (pubmed interface), scopus and web of science, was performed to locate articles any time up to march 23, 2020 comparing the survival between ht and no-ht patients with covid-19 infection. three reviewers (g.r., m.z. and l.r.) independently screened and selected the studies according to the inclusion and exclusion criteria. the following mesh terms were used for the search: "arterial hypertension" and " coronavirus 2019 mortality" or "covid-19 mortality" or "coronavirus 2019 survivors" or " covid-19 survivors" and "coronavirus 2019" or "covid-19" and "mortality". case reports, review articles, abstracts, editorials/letters, and case series with less than 10 participants were excluded. extracted data included: number of patients enrolled, mean age, male gender, prevalence of ht, diabetes mellitus and other cardiac comorbidities. the inclusion criteria for studies were as follows: (1) the study selection and quality of the included studies was independently performed by two reviewers (g.r. and m.z.). discrepancies were resolved by consensus with a third independent reviewer (l.r.). specifically, quality assessment was performed using the newcastle-ottawa quality assessment scale (nos) [4] . in this regard, investigations were classified as having low (< 5 stars), moderate (5-7 stars) and high quality (> 7 stars). publication bias was evaluated according to the funnel plot asymmetry [5] . the primary outcome was the overall prevalence of ht obtained by the reviewed cohorts of patients with covid-19. the secondary outcome was the risk of death in hypertensive patients with covid-19 infection. continues variables were expressed as mean while categorical variables, were presented as proportions. data were pooled using the mantel-haenszel random effects models with odds ratio (or) as the effect measure with the related 95% confidence interval (ci). statistical heterogeneity between groups was measured using the higgins i 2 statistic. specifically, a i 2 =0 indicated no heterogeneity while we considered low, moderate, and high degrees of heterogeneity based on the values of i2 as <25%, 25-75% and above 75% respectively. all analyses were carried out using review manager 5.2 (the cochrane collaboration, oxford, england). a total of 213 articles were retrieved after excluding duplicates. after the initial screening, 197 were excluded for not meeting the inclusion criteria, leaving 16 articles to assess for eligibility. after a comprehensive and careful evaluation of the full-text articles, 13 articles including editorial/letter, reviews, case reports, and investigations not in english language were excluded. finally, 3 articles were included into the analysis [6] [7] [8] (figure 1, panel a) . among 419 patients (259 males (61.8%), mean age 55.6 years), ht resulted the most frequent cv comorbidities (24.3%), followed by diabetes mellitus (15.2%) and cardiac disease (6.2%). however, for the last comorbidity, minor variations were used in the definitions in the studies reviewed. according to the nos, two studies resulted of high-quality and one of moderate quality ( table 1) . as showed figure 1 , panel b, hypertensive patients with covid-19 infections had a significant higher mortality risk compared with normotensive patients (or 3.36, 95% ci 1.96-5.74, p<0.0001, i 2 = 21%). our brief meta-analysis demonstrated that patients with covid-19 infection and ht have a significant high mortality risk. the association between the presence of ht and a poor outcome in covid-19 patients was partially demonstrated in single analysis, but never into a meta-analysis [6, 9] . doubtless, considering that the data used for our investigation derive only from chinese cohorts, other analysis based on different cohorts should be performed to validate our observations because the prevalence of ht, as well as for others cv risk, can have different results across different regions and races. however, despite our findings should be considered as prelaminar results, we believe that remains fundamental to preliminary identify the "phenotype" of those patients which could be at higher risk of death during the covid-19 infection [10] . the preliminary knowledge of those comorbidities incrementing the risk of death of covid-19 patients results fundamental both for outpatients and hospitalized subjects to maintain a high degree of surveillance until the resolution of the disease, since covid-19 infection could rapidly turn into an acute respiratory distress syndrome (ards) up to a multiorgan failure (mof). in our analysis we included only those studies which stratified the cohorts into survivors and not survivors because the mortality represents an undeniable result. in fact, many other investigations used more "uncertain" outcome as the severity of the disease which was often defined using different criteria. our study has several limitations related to the observational nature of the studied reviewed with all inherited biases. secondly, very few investigations on the covid-19 infection have stratified the cohort into survivors and non survivors, limiting the number of investigations included into the meta-analysis. thirdly, in the analysis, the degree of increased risk of mortality in ht patients was largely due to two studies having 65.9& and 32.8% of weight. moreover, the high heterogeneity observed, which depends from the participants' inclusion criteria as well as by the studies design, may have resulted in relatively weak conclusions. in conclusion, ht is the most common cv comorbidity which seems to significantly increase the mortality risk in covid-19 patients. further studies are needed to explain the underline pathophysiological mechanisms linking ht and covid-19 infection. none of the authors have conflicts of interest to declare. the clinical characteristics of myocardial injury 1 in severe and very severe patients with 2019 novel coronavirus disease prevalence of comorbidities in the novel wuhan coronavirus (covid-19) infection: a systematic review and metaanalysis preferred reporting items for systematic reviews and meta-analyses: the prisma statement the newcastle-ottawa scale (nos) for assessing the quality if nonrandomized studies in meta-analyses funnel plots for detecting bias in meta-analysis: guidelines on choice of axis risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease association of radiologic findings with mortality of patients infected with 2019 novel coronavirus in wuhan clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical course and mortality risk of severe covid-19 key: cord-008671-k0vda7fy authors: mccormack, j.g. title: clinical features of rotavirus gastroenteritis date: 2005-04-14 journal: j infect doi: 10.1016/s0163-4453(82)93777-x sha: doc_id: 8671 cord_uid: k0vda7fy five hundred and eighteen children under the age of five years admitted to hospital with a diagnosis of gastroenteritis over a twelve-month period were studied prospectively. rotaviruses were demonstrated by stool electron microscopy (em) in 132 of these cases (25·4 per cent), but in none of io8 ageand sex-matched controls. non-specific cases, where no potentially pathogenic organism could be demonstrated in stools submitted for em, viral and bacterial culture accounted for 46 per cent of cases. if em of the stools had not been performed the proportion of non-specific cases would have risen to 85 per cent, thus demonstrating the importance of this technique in diagnosis. rotaviruses were most commonly found in winter and between the ages of six and eighteen months. a history of contact with an adult with diarrheoa, vomiting occuring before diarrhoea, accompanying upper respiratory tract infection (urti), otitis media and pyrexia and the need for administration of intravenous fluids were all significantly more prominent features of the rotavirus than the non-specific cases of gastroenteritis, and are suggested as pointers to such a diagnosis. pneumonia is described in three patients as an accompanying illness with rotavirus gastroenteritis. rotaviruses were first described in the duodenal mocosa of six infants with acute gastroenteritis in r973,1 and they are now considered the commonest agents responsible for such an illness. 2-6 acquired infection with rotaviruses in the pre-school child is usually followed by a gastroenteritic illness with the interesting exception of the neonatal period when such infection is often symptomless, presumably due to maternally-derived immunity. 7 infection in children over the age of five and in adults usually produces mild symptoms or is symptomless, probably due to acquired immunity. 6,8 diagnosis of rotavirus gastroenteritis is usually made by electron microscopy (em) of stools although techniques of immune em 9 and various serological methods are also available. 6,~°,11 culture of the virus has not so far been universally successful, 3,4,9 although more definite progress in this respect seems imminent. 12, ~3 the laboratory facilities for the diagnosis of this condition are, as yet, not widely available, and it would seem desirable for clinicians and epidemiologists to appreciate any clinical features that might distinguish rotaviral from other forms of gastroenteritis. this study was embarked upon in an attempt to identify any such features. the children in the study were those under the age of five years referred to a large infectious diseases unit over a twelve-month period from may i978 to april 1979 inclusive, because of acute diarrhoea regardless of any other factors. they were studied prospectively. a detailed history, which included data on feeding, contact and past history, was taken and examination was performed with special reference to the temperature, ears, nose, and throat, chest, weight and state of hydration. haemoglobin, white-cell count, sedimentation rate, urea, electrolytes and bicarbonate values in the blood were estimated. notes were made of treatment, feeding requirements and progress. three bacterial cultures of the patients' faeces were prepared, each using mcconkey's agar, desoxycholate agar, selenite selective broth, mannitol salt agar and latterly, skirrow's medium. potentially pathogenic strains of escherichia coli were identified for the following serotypes: oi sac, o26, o44, o55, o86, oiii, oii2ac, oii4, oii9, 0124, 0125, 0126, 0127, oi28 and oi42. viral cultures of the faeces using rhesus monkey or baboon kidney, green monkey kidney and hep 2 tissue cultures were instituted. stool specimens for em were prepared by suspension with phosphatebuffered saline, differential centrifugation, deposition on to standard copper grids pre-coated with formvar and carbon and stained with phosphotungstic acid. em scanning was performed for 3o minutes on each specimen, mostly at a magnification of 4oooo. all stool specimens were, if not immediately transported to the laboratories, stored at 4 °c. as an adjunct study, stool specimens were collected from io8 controls with other illnesses but without diarrhoea. em and viral cultures as described above were performed on these. a total of 518 patients were studied and the isolation rates for the various organisms are outlined in table i . the adenoviruses were predominantly found on em rather than culture. the largest single group was made up of non-specific cases whichamountedto 238 patients (46per cent). the contribution of em to the diagnosis can be measured by the fact that if this procedure had not been employed, 44o patients (85 per cent) would have fallen into the nonspecific category. picornaviruses were defined as small round particles of about 28 mm diameter seen on em, but not cultured. of the i32 rotavirus cases, 23 were associated with other organisms: i i type-specific esch. coli, five echo viruses, four adenoviruses, two picornaviruses and one coxsackie b 2. the identification rate for rotaviruses was higher in winter than in summer; a reversal of this pattern was noted for adenoviruses (table ii) . the control group comprised a total of io8 cases, 59 males and 49 females, with a mean age of i i-6 months and a range of four days to five years. the adenovirus was less commonly found compared to the study group and in all five cases was cultured. no rotaviruses were identified in this group (table iii) . in the study group the isolation rate for rotaviruses was relatively higher in the 6-i8 month age group (35"8 per cent) and lower in infants of less than six months (i6"4 per cent). comparison of the rotavirus and non-specific groups yielded many results outlined below (tables iv, v and vi) . pyrexia was significantly commoner in the rotavirus cases (p < o.oo2) and maximum temperatures recorded in hospital tended to be higher in this group (table iv) . minor differences were observed in mean age, sex distribution, hospital stay, duration of diarrhoea, previous history of gastroenteritis and the incidence of vomiting, but these differences were not statistically significant (p > o-o5). the reporting of vomiting occurring before diarrhoea was significantly commoner in the rotavirus group (p < o.0i). although a history of contact with another case of gastroenteritis was found more commonly in the rotavirus group, the difference between the two groups did not reach statistical significance (o. io > p > o'o5); however, when such a history was confined to contact with adult cases, the difference was more pronounced and was statistically significant (p < o'o5). a significantly larger proportion of the rotavirus cases had symptoms or signs of an upper respiratory tract infection (urti) (p < o.oi) and a similar difference was noted for patients with visibly inflamed tympanic membranes (p < o.ooi). three cases with pneumonia, diagnosed on both clinical and radiological criteria, and two of bronchitis, were observed amongst the rotavirus group; no such features were encountered in the non-specific group. rectal bleeding, rashes, conjunctivitis, meningism and febrile convulsions were noted in a small number of cases and, interestingly, in none of the cases with a rash was there evidence of an enteroviral infection. the necessity to use intravenous fluids was significantly higher in the rotavirus group (p < o.ooi). the gastroenteritis patients as a whole tended to be of lower weight than normal on admission although the skewing of these figures may be partly accounted for by dehydration. small differences in haemoglobin, esr, urea, electrolytes and bicarbonate levels in the blood were noted, but none of these reached statistical significance. hypernatraemic dehydration, a condition seen less frequently nowadays in association with gastroenteritis, was not significantly commoner in any group of patients. isolation rates for rotaviruses in acute gastroenteritis in children less than six years old have varied within the range i8 per cent to 60 per cent. 2, ~, 6,14,15 the rate for this study of 25"5 per cent would have been higher if we had excluded cases which did not appear to be infective, e.g. feeding and social problems and drug-induced diarrhoea, but such differentiation can be very arbitrary and indefinite. similar patterns of seasonal variation in rotavirus and adenovirus identification in faeces have been documented, 5, 7 and one report suggests that this variation for rotaviruses may be lost in tropical climates. 17 the failure in this study to find rotaviruses in the stools of control patients without diarrhoea correlates with other similar reports, a, 5, 15,16 a similar age distribution to that shown in this study has been previously described for rotavirus gastroenteritis, the relatively lower incidence in the under six months age group being explained by maternally-derived antibody. 18, 19 the male predominance in gastroenteritis, a well-documented but poorly understood feature, was reproduced in both rotavirus and non-specific groups in this study. pyrexia has been said to occur in 5o-6o per cent of cases of rotavirus gastroenteritis, 9, 20 although one group in romford noted fever as a feature of all ioo of their cases. 14 in this study, pyrexia is seen to be significantly commoner and generally more severe in the rotavirus than in the non-specific cases. the incidence of vomiting in this survey of 93"2 per cent in the rotavirus cases correlates well with other reports,*, 18 and although one of these studies suggested that vomiting was commoner in rotavirus than in other forms of gastroenteritis, 2 this study, based on larger numbers of patients and statistical analysis, cannot confirm this finding. however, this study does show that vomiting occurring prior to the onset of diarrhoea is a significant feature of the rotavirus infection. as the study year went by, an impression was formed that a definite history of contact was obtainable more often in the rotavirus cases than in other groups, and although there was a difference in this respect between the rotavirus and non-specific groups, this difference did not qualify for statistical significance. although an outbreak ofrotavirus gastroenteritis has been described involving adults without the known infection of children, 8 the finding of rotaviruses in adult stools has usually been acompanied by no intestinal symptoms, zl the finding in this study of a significantly higher incidence of a history of contact with an adult with gastroenteritis in the rotavirus than in the non-specific cases would suggest that adults may represent a reservoir for potential rotavirus infection in infants. upper respiratory tract infection (urti) as a preceding or accompanying event in rotavirus gastroenteritis has been previously described in 29-42 per cent of cases ;2.6, la~ in one survey of gastroenteritis cases, patients with urti were excluded. 12 the current study shows that urti is a significantly commoner event in rotavirus than in non-specific gastroenteritis. the frequency of otitis media in rotavirus gastroenteritis in this study was lower than in two other reports, 21,1~ but this series demonstrates it as a significant feature of this illness. the finding of three cases with pneumonia and two with bronchitis amongst the rotavirus group without any such cases in other groups was surprising. these findings suggest that when gastroenteritic symptoms occur in a patient with urti, otitis media, pneumonia or bronchitis, rotaviruses should be sought in the stools rather than accepting the diarrhoea or vomiting as being secondary to the respiratory or ear infection. the failure to find rotaviruses in the stools of any control patients with similar infections, but without diarrhoea, would support the role of the rotavirus as being responsible for the intestinal features of such illnesses. what role rotaviruses play, if any, in the non-intestinal features of such illnesses is not known; there is no record of demonstration of these viruses from throat, bronchial or aural secretions of such cases. although one report showed clinically apparent passage of blood in the stools in six out of sixty patients with rotavirus gastroenteritis, 2° this study suggests that this is an infrequent complication. the requirement for intravenous fluids in rotavirus gastroenteritis has been reported as between 3 and 34 per cent :2,14, 21 such variation probably represents varying indications for this form of therapy in different hospitals. this study, where similar criteria were applied to all cases in this regard, showed that intravenous fluids were considered necessary in a significantly higher proportion of patients with the rotavirus rather than the non-specific form ofgastroenteritis, and under such conditions these former cases would probably be said to be more severely dehydrated than the non-specific cases. (the valuable advice and encouragement ofdrs j. stevenson, h. pullen, m. hambling and g. gibson is gratefully acknowledged. i also wish to thank drs stevenson and pullen for allowing me to study the patients who were under their care, and drs hambling and gibson and all the staff of the public health laboratory in leeds for their assistance, especially mr george bellamy who manned the electron microscope. i also wish to thank mr a. steel for assistance with the statistics and, last but not least, the many nurses who diligently collected stool specimens around the clock.) virus particles in epithelial cells of duodenal mucosa from children with acute non-bacterial gastroenteritis at british paediatric association meetings, york stool viruses in babies in glasgow rotaviruses of man and animals importance ofa new virus in acute sporadic enteritis in children asymptomatic endemic rotavirus infection in the newborn rotavirus infections in adults in association with acute gastroenteritis immunological response to human reovirus-like agent: measurement of anti-human reovirus-like agent igg and igm levels by the method of enzyme-linked immunosorbent assay measurement of rotavirus antibody by an enzyme-linked immunosorbent assay-blocking assay viruses and diarrhoea-a review immunization of infants and young children against rotaviral gastroenteritis -prospects and problems, ff the clinical features of infantile gastroenteritis due to rotaviruseso a survey of rotaviruses associated with gastroenteritis in aboriginal children in western australia comparison of human rotavirus disease in tropical and temperate settings epidemiological aspects of rotavirus infection in hospitalized venezuelan children with gastroenteritis clinical features of acute gastroenteritis associated with human reovirus-like agent in infants and young children, ff pediatr a study of the prevalence of rotavirus infection in children with gastroenteritis admitted to an infectious diseases hospital a clinical study of rotavirus gastroenteritis an investigation into the possible role of the family unit in the transmission of rotavirus infections of children the techniques of la and cie were used to seek cases of pneumococcal, meningococcal and h. influenzae meningites. gram-staining provided 80 per cent of diagnosis, gram-staining and culture together, 85 per cent. cie alone provided 9o per cent of diagnosis, la alone, 82 per cent (but meningococcal cases were not so well detected, being 72"5 per cent by cie and 71"2 per cent by la). the best combination of tests was gram-staining plus cie which provided a 95 per cent successful diagnosis.difficulties were encountered in that h. influenzae and tuberculous meningitis sometimes produced false-positive pneumococcal cie, and some pneumococcal and meningococcal types produced cross reactions with la. some of the undiagnosed cases had epidemiological features of meningococcal meningitis. the authors concluded that serological and gram-stain diagnosis of the three common types of bacterial meningitis might avoid the need for expensive cultural techniques, which require a good standard of laboratory facilities. these simple techniques can be performed on csf samples which have been stored at low temperatures, or transported long distances, and so lend themselves particularly to use in developing countries. key: cord-269389-x8i5x62v authors: gensini, gian franco; yacoub, magdi h.; conti, andrea a. title: the concept of quarantine in history: from plague to sars date: 2004-04-12 journal: j infect doi: 10.1016/j.jinf.2004.03.002 sha: doc_id: 269389 cord_uid: x8i5x62v the concept of ‘quarantine’ is embedded in health practices, attracting heightened interest during episodes of epidemics. the term is strictly related to plague and dates back to 1377, when the rector of the seaport of ragusa (then belonging to the venetian republic) officially issued a 30-day isolation period for ships, that became 40 days for land travellers. during the next 100 years similar laws were introduced in italian and in french ports, and they gradually acquired other connotations with respect to their original implementation. measures analogous to those employed against the plague have been adopted to fight against the disease termed the great white plague, i.e. tuberculosis, and in recent times various countries have set up official entities for the identification and control of infections. even more recently (2003) the proposal of the constitution of a new european monitoring, regulatory and research institution has been made, since the already available system of surveillance has found an enormous challenge in the global emergency of the severe acute respiratory syndrome (sars). in the absence of a targeted vaccine, general preventive interventions have to be relied upon, including high healthcare surveillance and public information. quarantine has, therefore, had a rebound of celebrity and updated evidence strongly suggests that its basic concept is still fully valid. the concept of 'quarantine' is radically embedded in local and global health practices and culture, attracting heightened interest during episodes of perceived or actual epidemics. the term, however, evokes a variety of emotions, such as fear, resentment, acceptance, curiosity and perplexity, reactions often to be associated with a lack of knowledge about the origins, meaning, and relevance of quarantine itself. historically quarantine has been defined as the detention and segregation of subjects suspected to carry a contagious disease. more recently, the term quarantine has come to indicate a period of isolation imposed on persons, animals or things that might spread a contagious pathology. 1 nowadays the word quarantine should be used to refer to compulsory physical separation (including restriction of movement) of groups of healthy individuals who have been potentially exposed to a contagious disease. 2 the term 'isolation' must be kept separate from the term quarantine, since the former denotes the separation and confinement of subjects already known to be infected with a contagious disease to prevent them from transmitting disease to other people; the latter, essentially the same procedures but with suspected transmitters of disease. from ancient times different populations have adopted varying strategies to prevent and contain disease. one of these is exactly what we would now call isolation. the old testament evidences how individuals affected by diseases were separated from others, and people with leprosy, as leviticus informs, had to live isolated all their lives. in the new testament, too, leprosy continues to be considered a reason for social discrimination, and is represented as curable only through the phenomenon of a divine intervention. the isolation, temporary or otherwise, of sick people has thus always been extensively used as one of the approaches to limit the spread of disease. 3 another strategy was the establishment of a time limit to the manifestation of diseases. in the v century b.c. hippocratic teaching had established that an acute illness only manifested itself within forty days. the case of plague was representative with respect to this; since a disease manifesting itself after 40 days could not be acute, but chronic, it could not be plague. in the ancient past the term pestis (plague) was used in a broad way to indicate every epidemic characterised by high mortality, and magical practices were implemented to fight different diseases since the idea of preventive instruments (such as quarantine) was still not present. 4 with regard to the real plague (the disease caused by yersinia pestis), one may remember the first great pandemic wave of the greek -roman period, and the recurrent epidemics throughout europe in the vi and vii centuries a.d. against acute, fatal diseases such as bubonic plague attempts were made by healthy communities to prevent entry of goods and people from infected communities. in the vii century a.d. armed guards were stationed between plague-stricken provence and the diocese of cahors. 5 particularly virulent was the impact of the disease on the whole of europe in the middle of the xiv century, when the plague spread from southern europe to germany and russia, causing the death of more than 30% of the european population. 6 medieval laws, renaissance health achievements and xvi -xviii centuries overview the concept of (modern) preventive quarantine is strictly related to plague and dates back to 1377, when the rector of the seaport of ragusa, today called dubrovnik (croatia), officially issued the socalled 'trentina' (an italian word derived from 'trenta', that is, the number 30), a 30-day isolation period. ships coming from infected or suspected to be infected sites were to stay at anchor for thirty days before docking. this same period of time became 40 days for land travellers, probably because the shorter period was not considered sufficient to prevent the spread of disease, and precisely from the italian number forty ('quaranta') comes the term quarantine. 7 furthermore, the chief physician of ragusa, jacob of padua, also advised establishing a place outside the city walls for the treatment of sick (or suspected to be infected) citizens. 8 the imposition to remain 30 -40 days in an isolated site was determined not only by health reasons, but also by economic necessity, since the quality and safety of the trade network needed to be protected from the black death. the attention dedicated by the ragusan rulers to the plague was, therefore, responsible for the creation of the first 'official' quarantining as a legal system aimed at defending both health and commercial aspects. 9 the following were the main tenets of the 1377 law of ragusa: visitors from areas where plague was endemic would not be admitted into ragusa until they had remained in isolation for a month; whoever did not observe this law would be fined and subjected to a month of isolation; no one from ragusa was allowed to go to the isolation area; people not assigned by the great council to care for quarantined persons were not allowed to bring food to isolated people. in 1423 venice set up one of the first known 'lazaretto' (quarantine station) on an island near the city, and the venetian system became a model for other european countries. 3 during the next 100 years similar laws were introduced in italian ports (pisa) and in french ones (marseilles), and they gradually acquired other connotations with respect to their original implementation in the context of the middle ages. 10 one such connotation was the institution of a social body to provide the necessary isolation structures (dispositions, facilities, implementation of the laws themselves); another, of more intellectual and medical content, was the gradual acquisition of the essential mechanisms of contagion. in effect, even during the early renaissance, physicians did not have a clear idea of infectiousness, though many waves of epidemics had succeeded one another in the course of the previous centuries. 11 it was only during the xvi century that girolamo fracastoro defined and empowered the concept itself, through the hypothesis that small particles were able to transmit disease. 4 this led the medical profession to integrate previously adopted remedies, simple and insufficient, with more precise quarantine interventions (even if not at an international level) that, however, became the remote bases for modern epidemiology and health sciences. 12 in the xvi century the quarantine system was expanded through the introduction of bills of health, a type of certification that the last port visited by travellers was free from disease. a clean bill, with the visa of the consul of the country of arrival, entitled the ship to the use of the port without quarantine. 5 in the course of the xviii century the practice of quarantine had become, on the one hand a notable nuisance, and on the other, a source of abuse. with regard to the former point, the periods of quarantine were variable across different countries, so that there was no certainty concerning the time needed to implement the quarantine itself. as consequence, not only delay, but perplexity was caused to travellers. with regard to the latter question, instances of bureaucratic and restrictive implementation of quarantine regulations were rife, and the disinfection of correspondence was used as an excuse for political espionage. 5 the upshot of this diffused dissatisfaction with quarantine measures was the emergence of the awareness of the need for a shared standardisation, which, in turn, led to the call for xix century international conferences. from a scientific-epidemiological point of view the concept of quarantine had come to be defined quite precisely in the course of the xix century, but the contemporaneous health organisation was not systematic and capillary enough to confront bursts of epidemics across europe in an organic way. the mid-xix century cholera epidemics, for example, evidenced the scantiness of international uniformity in quarantine practices. even if france had proposed, already in 1834, a meeting for the discussion of the international standardisation of quarantine, 13 it was only in 1851 that the first international sanitary conference took place in paris. collaboration at the international level was hard to achieve since quarantine policies mirrored not only health organisation views, but also national trade protection issues that varied from state to state. 14 open negotiation on quarantine was strongly limited by economic and political agendas, as documented by the 1885 rome conference, where a proposal regarding the inspection of quarantine of ships from india, using the suez canal, produced a violent discussion between britain and france based not on health questions, as much as on the extent of british dominance over the canal. with regard to the united states of america, protection against imported pathologies had always been retained a local issue, and so handled by the single states. only sporadic attempts had been performed to impose quarantine requirements until repeated and serious yellow fever epidemics led to the passing of federal quarantine legislation by congress in 1878, a set of laws that paved the way for federal involvement in quarantine activities. in 1892 the arrival of cholera from abroad prompted a reinterpretation of these laws so as to endow the federal government with more authority in the imposition of quarantine requirements. 13 it was only in 1893 that, after a number of conferences held in the second half of the xix century, an agreement was achieved both in europe and in the united states, concerning the notification of disease and other issues. after this achievement conventions and regulations began to be ratified regarding, in particular, relevant principles for the standardisation of quarantine measures. in the united states, as local authorities realised the benefits of federal involvement, local quarantine stations were gradually turned over to the government; in europe established periods of detention were fixed with special reference to cholera, yellow fever and plague. 14 it is interesting to observe how measures analogous to those employed against the plague have been adopted to fight against the disease that, not by chance, has been termed the great white plague: we refer to tuberculosis (tb). 15 before the tubercle bacillus was recognised as the causative agent of the disease, sanatoria had been set up as the only remedy for sufferers from tuberculosis; this may be considered as an application of the broad concept of 'preventive-therapeutic' quarantine. sanatoria constituted a simple and inexpensive tool to break the chain of transmission of the disease, since they guaranteed isolation. they, therefore, had a precise role in controlling tuberculosis, and it is for this reason that, between 1880 and 1930, sanatoria spread across the whole of europe and north america. even during the 1950s, although streptomycin was already on the market (1947), tb hospitals were considered important for tuberculosis therapy as sites dedicated to the isolation of tb patients, as recommended by quarantine practice. 16 in the scenario of contagious diseases of the past, the so-called 'health officers' deriving partly from medieval and renaissance predecessors and partly from figures created by the schools of hygiene, acquired fundamental importance. among their various functions were those of furnishing the single national health systems with appropriate corporate entities and legislative organisms, as well as obviously caring for the health of the whole population. in many european countries, including italy, these 'officers' represented, even in the second half of the xx century, the basis of all public health organisation devoted to the monitoring and control of infectious diseases. in the first 30 years of the xx century, a deep medicalization of quarantine measures occured. in 1903 the term 'lazaretto' (used especially for plague) was substituted by that of 'health station', since in europe, particularly in france and in italy, the distinction among sick, suspectedly sick, and healthy people, began to acquire a real medical value. four years later an international office of public health was established, and more than twenty nations adhered to it in less than 2 years. variola and typhus were added to the three (plague, cholera and yellow fever) historical quarantining diseases in 1926, and 2 years later this same international office imposed a set of quarantine rules targeted to all kinds of travellers (by land, sea and air). when the world health organisation replaced the international office of public health the expression 'quarantining diseases' disappeared, and pathologies controlled by international health laws (such as plague, cholera and yellow fever) or pathologies under surveillance (such as poliomyelitis, recurrent fever and typhus) appeared. 17 in the face of this resurgence of attention towards infectious diseases, tuberculosis was again made the object of specific measures, which, however, served to monitor and control other diseases. consequent to the high transmission and seriousness of tuberculosis in the europe of the nineteenth century, various countries set up official entities for the identification and control of infections. in the united kingdom a government-funded agency, the medical research council (mrc), was created in 1913 in the hope of finding scientific solutions to the illness. its activity was specifically directed to research. with reference to the other side of the atlantic, in the twentieth century (1967) quarantine measures became the task of the national communicable disease centre, at present called the centre for disease control (cdc) and prevention, an organisation, already equipped, in the sixties, with more than 50 quarantine stations located at every port and international airport, and, in the seventies, shifting its field of action from routine inspection to problem management, intervention and regulation. 10 more recently (2003) the proposal of the constitution of a new european monitoring, regulatory and research institution was made, since the already available system of surveillance, set up in europe to control the onset of epidemics, came up against an enormous challenge in the global emergency of the severe acute respiratory syndrome (sars). 18 in the absence of a targeted vaccine, general preventive interventions had to be relied upon, including high healthcare surveillance and public information. quarantine has, therefore, had a rebound of celebrity, as witnessed by the 'fact sheets' prepared and published by the cdc, in which one may read that 'quarantine is medically very effective in protecting the public from disease'. 19 the 'modern' quarantine for sars is a 10-day period (the incubation period of sars is in fact 2 -9 days) and, like the quarantines of the past, has been applied to persons who have been exposed to the disease and who may be infected, while, once again, isolation has been implemented to separate healthy people from sick ones. as mentioned above, the health emergency of sars has constituted a real challenge for health systems. however, it has also put into discussion the real effectiveness of quarantine measures, for, precisely as for every other health intervention, quarantine has limits of application of which the medical and social community should be perfectly aware. 20 a recent paper proposing a compartmental model for the geographical spread of infectious diseases shows how this scheme may be adopted to address the effectiveness of human quarantine. the model itself was applied to data deriving from a canadian historical record regarding the time period of the so-called spanish influenza pandemic (1918 -19) . information on the daily mobility patterns of subjects engaged in the fur trade throughout central canada before, during and after the epidemic were used to establish whether rates of travel were affected by informal quarantine policies, and then the same methodology was adopted to analyse the impact of observed differences in travel on the diffusion of the epidemic. this same model has suggested that quarantine effectiveness varies depending on when the limitation on travel between communities is applied, and on how long it lasts. 20 an operative template of such a type appears particularly interesting from our historical-scientific point of view since it links historical features to current scientific epidemiological evidence. similar to other effective health measures, quarantine is not a panacea, and has its limits. this is highlighted by the recent risk of bioterrorism, where a potentially large and diverse number of agents may be implicated. 21 in addition, other recent epidemics, such as the acquired immuno deficiency syndrome (aids), cannot be considered quarantine-able not only because of medical but also because of ethical and legal issues. 22 however, good quality evidence overall suggests that the basic concept of quarantine is still fully valid, and that the implementation of correct quarantine procedures must be tailored according to single health, social and geographical conditions. 20, 23, 24 large-scale quarantine following biological terrorism in the united states the origin of quarantine quarantine and isolation. 15th ed. the new encyclopaedia britannica the black death past and present. 2. some historical problems a state of deference: ragusa/dubrovnik in the medieval centuries storia della medicina dall'antichità a oggi trade and health policies in ragusa-dubrovnik until the age of george armmenius-baglivi history of the concept of quarantine plague, policy, saints and terrorists: a historical survey a short history of quarantine (victor c. vaughan) politics of quarantine in the 19th century international law and infectious diseases the evolution of the concept of 'fever' in the history of medicine: from pathological picture per se to clinical epiphenomenon (and vice versa) compliance, coercion, and compassion: moral dimensions of the return of tuberculosis international sanitary regulations. 3rd annotated ed. world health organization guideline on management of severe acute respiratory syndrome (sars) severe acute respiratory syndrome. fact sheet: isolation and quarantine simulating the effect of quarantine on the spread of the 1918-19 flu in central canada plague as a biological weapon: medical and public health management. working group on civilian biodefense quarantine and the problem of aids some historical comments on quarantine: part one some historical comments on quarantine: part two the authors would like to thank professor luisa camaiora, b.a., m.phil., for her correction of the english. key: cord-265504-yq9wsugy authors: anim, desmond ofosu; ofori-asenso, richard title: water scarcity and covid-19 in sub-saharan africa date: 2020-05-21 journal: j infect doi: 10.1016/j.jinf.2020.05.032 sha: doc_id: 265504 cord_uid: yq9wsugy nan lv et al recently predicted that many countries could face similar covid-19 situation as experienced in hubei in china [1] . several factors may impact their prediction. current evidence suggest that the covid-19 virus is transmitted via respiratory droplets or contact [2] . contact transmission happens when contaminated hands touch the mouth, nose, or eyes. consequently, hand hygiene (regular hand washing) is extremely recommended to control the spread of covid-19 virus [2] . in this paper, we highlight the issues that characterize water services amid the covid-19 pandemic in sub-saharan africa (ssa) and discuss avenues for improving water management during this pandemic and future infectious disease outbreaks. in response to the promotion of hand hygiene by the world health organization (who) and national public health agencies as a means to curbing the spread of covid-19, water service providers (wsp) in most developed countries have outlined drastic measures with the goal of ensuring continuous provision of essential water and sewerage services to all during this pandemic. for example, in the us and australia, wsp have suspended water shutoff with service to be temporarily restored to thousands of households disconnected [3, 4] . access to water is a key determinant for infectious disease control and prevention; thus, limited access creates a challenge for transmission control [5] . nevertheless, across many ssa countries where inequalities in access to safe water is pervasive [6] , there is a need to be worried in light of covid-19 pandemic. nearly 300 million people in ssa live in water stressed environment (table 1 ) [5] . this present a major challenge towards controlling the spread of the covid-19 virus. indeed, poorly developed water and sanitation systems was reported to be a key determinant of the rapid spread of the 2014 ebola outbreak, as well as an underlying factor in the high number of deaths [7] . so how do the recommended precautionary measures relative to covid-19 fit within the everyday practices in ssa countries characterised by overwhelming water scarcity? as of 5 may 2020 (13:00 gmt), only lesotho remains a covid-19-free country in ssa (supplementary figure s1 ). in response to the increasing threat from covid-19, most ssa cities have instituted a lockdown (partial in most places). however, residents are concerned about a potential increased spread of covid-19 due to water rationing. in ghana (www.youtube.com/watch?v=kl5v8a8toyg) and kenya (www.bbc.com/news/world-51929598) for example, many households struggle to comply with the advice to 'frequently wash hands under running water' because of the water rationing. it is worth noting that social distancing is almost impossible as residents are likely to queue to access or buy water. notably, one of the key public health preventive messages for covid-19 is: 'washing hands with soap and water for 20 seconds, repeatedly throughout the day, is critical to prevent transmission of the virus' [8] . in many ssa settings, this is an unimaginable luxury due to the inequalities that characterise the provision of water services as well as the limited opportunity to wash their hands regularly at home. consequently, in this period, it is important to reflect on the water and sanitation services situation in the ssa region. in particular, the ongoing covid-19 pandemic provides an opportunity to remind water authorities and the respective governments of the with the water demand pressures from rapid population growth and urban expansion in ssa [20], there is the urgent need to increase the efficiency of use by implementing strategies for improving the conservation of available water. green or nature-based solutions can help to improve water storage and supply, thus increasing water availability. this is particularly needed today considering expectations that water shortage will worsen in ssa due to climate change and risk of droughts causing the decline of water levels of dam and freshwater supply sources [9, 10] . water scarcity and security issues will be exacerbated by recent trends of climate variability and consequent rise in droughts. thus, climate resilient water resource management will require an integrated strategy to ensure resilience for water-related policy making to address both short-and long-term impacts of climate change by balancing robustness with flexibility. with future uncertainties and the likelihood of other potential infectious disease outbreaks, there is the need for robust adaptation options that have the primary objective of supporting sustainable water resources use. ensuring affordable access to safe water, sanitation and hygiene (wash) services is important to address the current covid-19 and future pandemics. particularly, improved access to wash facilities could help to minimize transmission, and reduce healthcare and other societal costs. for example, the ghana government is currently spending money to use water tanks to provide water for poor communities severely hit by the pandemic. residents in these communities are unable to socially distance or conform to lockdowns for reasons such as the need to get out to access water and toilets. in response, long-term efforts should focus on addressing wash access issues among the poor communities especially in urban areas. most people living in informal settlements as is the case for many ssa cities rely on communal water stands and toilets. high cost coupled with limited access could stop generous use of water for hand washing. leaving homes to use communal services and queuing for access also makes social distancing difficult to implement. the poor access to water in ssa presents a major barrier to effective containment of the covi-19 outbreak. it is essential that this unfolding moment trigger collaborative efforts between all stakeholders in rethinking and acting for improved water services during this pandemic and future infectious disease outbreaks. notes: population living in water scarcity (i.e. with less than 1000m 3 per capita per year) and stress (i.e. with less than 1,500m 3 per capita per year) in selected ssa countries. water scarcity reflects the lack of sufficient available water resources to meet demand of usage within a region whereas water stress refers to the inability to meet human and ecological demand for water. source: world data lab (https://worldwater.io/about.php; accessed on 25 th april 2020) global covid-19 fatality analysis reveals hubei-like countries potentially with severe outbreaks features, evaluation and treatment coronavirus (covid-19) us cities and states suspend water shutoffs to tackle coronavirus pandemic here's how australian water utilities are responding to covid-19 progress on household drinking water, sanitation and hygiene 2000-2017. special focus on inequalities water scarcity and related environmental problems in parts of sub-saharan africa: the role of the transboundary environmental impact assessment convention the emergence of ebola as a global health security threat: from 'lessons learned' to coordinated multilateral containment efforts covid-19 hand wash timer water crisis in africa: myth or reality? international journal of water resources development the projected timing of climate departure from recent variability key: cord-266033-gbx48scp authors: xu, yu-huan; dong, jing-hui; an, wei-min; lv, xiao-yan; yin, xiao-ping; zhang, jian-zeng; dong, li; ma, xi; zhang, hong-jie; gao, bu-lang title: clinical and computed tomographic imaging features of novel coronavirus pneumonia caused by sars-cov-2 date: 2020-02-25 journal: j infect doi: 10.1016/j.jinf.2020.02.017 sha: doc_id: 266033 cord_uid: gbx48scp purpose: to investigate the clinical and imaging characteristics of computed tomography (ct) in novel coronavirus pneumonia (ncp) caused by sars-cov-2. materials and methods: a retrospective analysis was performed on the imaging findings of patients confirmed with covid-19 pneumonia who had chest ct scanning and treatment after disease onset. the clinical and imaging data were analyzed. results: fifty patients were enrolled, including mild type in nine, common in 28, severe in 10 and critically severe in the rest three. mild patients (29 years) were significantly (p<0.03) younger than either common (44.5 years) or severe (54.7) and critically severe (65.7 years) patients, and common patients were also significantly (p<0.03) younger than severe and critically severe patients. mild patients had low to moderate fever (<39.1 °c), 49 (98%) patients had normal or slightly reduced leukocyte count, 14 (28%) had decreased counts of lymphocytes, and 26 (52%) patients had increased c-reactive protein. nine mild patients were negative in ct imaging. for all the other types of ncp, the lesion was in the right upper lobe in 30 cases, right middle lobe in 22, right lower lobe in 39, left upper lobe in 33 and left lower lobe in 36. the lesion was primarily located in the peripheral area under the pleura with possible extension towards the pulmonary hilum. symmetrical lesions were seen in 26 cases and asymmetrical in 15. the density of lesion was mostly uneven with ground glass opacity as the primary presentation accompanied by partial consolidation and fibrosis. conclusion: ct imaging presentations of ncp are mostly patchy ground glass opacities in the peripheral areas under the pleura with partial consolidation which will be absorbed with formation of fibrotic stripes if improved. ct scanning provides important bases for early diagnosis and treatment of ncp. the outbreak of an epidemical pneumonia with initially unknown reasons in december 2019 in wuhan, china was ultimately found to be caused by a new virus, the "2019 novel coronavirus" (2019-ncov) or sars-cov-2 formally named by the world committee on virus classification, [1] [2] [3] and the disease caused by this virus was named as covid-19 by the world health organization. 4 , 5 this virus has a characteristic crown representing the spike peplomers on the viral envelope, with strong infectivity and general suscepti-bility to people of all ages and across the globe. on the 8th february 2020, the disease was named as "novel coronavirus pneumonia (ncp)" in china. 6 by february 16, 2020, china had reported a cumulative total of 70,548 patients being infected with sars-cov-2, 57,934 cases currently treated (10,644 severe cases), 1770 deaths, 10,844 cases being discharged from hospitals, 7264 suspected cases and 546,016 cases in close contact including 150,539 people being isolated and closely watched. the sars-cov-2 is primarily propagated through respiratory droplets and close contact, with the incubation period usually between 1 and 14 days and the typical symptoms after onset of fever, dry cough, fatigue and gradual appearance of dyspnea. people carrying this virus are the source of infection even in the incubation period, and early diagnosis of this disease or carrier of the virus is crucial to preventing it from further spread. however, confirmation of infection of this virus requires presence of the virus nucleic acid detected in swabs, secretions and sputum from the respiratory tract, blood or stools. 7 nonetheless, detection of the virus nucleic acid may not be convenient, and early diagnosis of the ncp is essential for timely isolation and treatment of the patient. computed tomography (ct) is easily available and can be used to screen patients for rapid confirmation of sars-cov-2 infected ncp because ct, especially high resolution ct, has the advantages of high spatial resolution, freedom of disturbance from other structures outside the scanning plane and ability to display every details of the lesion in multiple planes and directions. this study was performed to analyze the clinical and ct imaging features in patients with sars-cov-2 infected ncp or covid-19 and to familiarize radiologists and doctors with the common clinical and ct imaging findings of this disease for quick recognition, rapid isolation and treatment of the patient. this retrospective study was conducted in january and february 2020, and the ethics committee of our hospital waived written informed consent because of its retrospective and emergent nature and evaluation of only the imaging and clinical data of the patients, involving no potential risk. inclusion criteria were patients infected with 2019-ncov, positive nucleic acid of the virus and ct scanning of the lung. when patients were admitted, ct pulmonary scanning was performed for all patients with the high-resolution lightspeed vct ct64 scanner (ge medical systems, china branch, beijing, china). the patient was in the supine position and scanned with breath holding at the end of inhaling. the scanning range was from the thoracic entrance to the level of posterior costophrenic anglem with the scanning parameters of tube voltage 120 kv, with the automatic milliampere technology (40-250 ma), noise index (ni) 25, pitch 0.984:1, matrix 512 × 512, slice thickness 5 mm, window width/level 20 0 0/-60 0 hu for the lung window, 350/40 hu for mediastinal window, and slice thickness 0.625 mm for reconstruction of the lung window in the axial position. imaging analysis was performed by three experienced radiologists with over 10-year experience, and in case of disagreement, they would consult to reach an agreement. the ct imaging was analyzed according to the following parameters: lesion distribution: left lung (upper or lower lobe), right lung (upp, middle or lower lob) and single or multiple lesions within each lobe; lesion location: peripheral, central or involving both peripheral and central locations; lesion density: ground glass opacity, consolidation and mixed type of ground glass opacity and consolidation; thickness of interlobular and intralobular septa, enlarged lymph nodes within the mediastinum and pleural effusion. based on the fifth edition of the china guidelines for the diagnosis and treatment plan of novel coronavirus (2019-ncov) infection by the national health commission (trial version 5), 6 the ncp was classified into four types: mild with slight clinical symptoms but no imaging presentations of pneumonia; common with fever, respiratory symptoms and imaging presentations of pneumonia; severe type with any of the following: respiratory distress with rr > 30 times/minutes, oxygen saturation at rest < 93%, or pao2/fio2 < 300 mmhg (1 mmhg = 0.133 kpa); critically severe type with any of the following: respiratory failure needing mechanical ventilation, shock, or combination with other organ failure needing icu intensive care. the statistical analysis was performed with the spss software (version 19.0, ibm, chicago, il, usa). continuous data were presented as mean ± sd (standard deviation) and tested with paired t -test. categorical data were presented as number (%) and tested with chi square test or fisher's exact test. the significant p value was set at < 0.05. fifty patients with ncp caused by infection of the sars-cov-2 virus were enrolled and had high-resolution pulmonary ct scanning, including mild type in nine, common in 28, severe in 10 and critically severe in the rest three ( table 1 ). there were 29 (58%) male patients and 21 (42%) female patients, with an age range of 3-85 years (mean 43.9 ± 16.8). five (10%) patients were below the age of 18 years, 30 (60%) between 18 and 50 years and 15 (30%) over 50 years. the mean age and age range were 29 and 3-47 years for mild patients, 44.5 and 17-62 years for common, 54.7 and 34-85 years for severe, and 65.7 and 50-79 years for critically severe, respectively. mild patients were significantly ( p < 0.03) younger than either common or severe and critically severe patients, and common patients were also significantly ( p < 0.03) younger than severe and critically severe patients. in disease exposure history, thirty (60%) patients had been to wuhan or the nearby regions, 18 (36%) in close contact with patients of ncp and 2 (4%) with no definite exposure history. clinical presentations of ncp were fever, cough, expectoration, fatigue, headache, gastrointestinal discomfort, dyspnea and muscle ache. the body temperature was below 37.3 °c in seven patients (14%), between 37.3 °c and 38 °c in 22 (44%), between 38 °c and 39 °c in 16 (32%), and over 39 °c in 5 (10%). cough was in 20 (40%) patients, expectoration in 7 (14%), fatigue in 8 (16%), headache in five (10%), sore throat in four (8%), gastrointestinal discomfort in one (2%), dyspnea in four (8%) and muscle ache in 8 (16%). mild patients had low to moderate fever ( < 39.1 °c), 49 (98%) patients had normal or slightly reduced leukocyte count, 14 (28%) had decreased counts of lymphocytes, and 26 (52%) patients had increased c-reactive protein ( table 1 ) . among 50 patients infected with sars-cov-2, nine mild patients were negative in ct pulmonary imaging ( fig. 1 ) , including five cases below the age of 18 years, suggesting that children, teenagers and younger patients were mostly mild. among 41 common, severe and critically severe patients who had positive pulmonary imaging ( table 2 over two lobes, and single lobe involvement was only in two cases in the right lower lobe, with the lesion within lobes being mostly multiple ( table 3 ) . severe and critically severe ncp involved most commonly 4-5 lobes and most significantly ( p < 0.05) bilateral lower and upper lobes compared with common ncp ( tables 3-4 ). the lesion was primarily located in the peripheral area under the pleura with possible extension towards the pulmonary hilum in big lesions ( table 5 ) . symmetrical lesions were seen in 26 cases and asymmetrical in 15. on ct imaging, common ncp was mostly at the early stage with bilaterally scattered irregular patches of ground glass opacity. some lesions might have a mixed pattern of consolidation in the center and ground glass opacity in the peripheral like a "halo sign". the lesion density was mostly non-uniform with air bronchogram and thickened interlobular or intralobular septa. in severe or critically severe patients, multiple patches or an integrated larger patch of ground glass opacity, consolidation or mixed consolidation and ground glass opacity might present in bilateral lungs, with these three imaging manifestations being possibly presented in one patient at the same time. consolidation and thickened interlobular septa were presented in severe or critically severe patients more than common ones, suggesting progression of the disease. the lesion was mostly located in the peripheral area under the pleura but might extend towards the center in bigger lesions, and peripheral lesions with involvement of the center were mostly seen in severe and critically severe patients. four to ten days after treatment in this series, repeated ct scanning revealed that most lesions were absorbed and improved with reduced extent, decreased density and formation of fibrotic stripes ( fig. 2 -6 ). in one common patient, the lesion in the right lower lobe was improved four days after treatment, however, a new lesion appeared in the left lower lobe which was improved after treatment for three additional days ( fig. 3 ) . this might indicate that the lesion might change rapidly in a short period of time and repeated ct scanning could provide basis for clinical diagnosis and treatment. follow-up ct scanning was performed in 30 cases, with 27 cases having repeated ct scanning within one week and three cases over one week. the interval of repeated ct scanning was 3-13 days, with 1-4 ct scanning in total. nineteen patients showed improved imaging findings at the first ct follow-up, eight patients showed disease progression at the first but improvement at the second ct follow-up, one case had disease progression at the first two ct follow-up but improvement at the fourth follow-up, and the rest two patients had no marked improvement at ct followup. the disease was improved between 3 and 13 days. two mild patients remained negative on ct pulmonary imaging. patients infected with sars-cov-2 may present primarily with low fever, fatigue and dry cough with accompanied symptoms of nasal congestion, runny nose and diarrhea like common cold in some cases. 1-3 , 8-11 mild patients may just have low fever and slight weakness without pneumonia, severe patients will have dyspnea and/or hypoxemia, and those with critically severe illness will quickly progress to acute respiratory distress syndrome, septic shock, uncorrectable metabolic acidosis, coagulation dysfunction and even death. unlike infection with sars-cov and h7n9 avian influenza which usually result in high fever at the beginning of infection, 12 the initial symptoms of sars-cov-2 infection are atypical with only low fever and even a long incubation period, which leads to its strong infectiousness. laboratory tests usually reveal at early stages normal or reduced counts of peripheral blood leukocytes and lymphocytes. 5 . severe novel coronavirus pneumonia in a 48-year-old man with fever for seven days before admission. a&b. computed tomography axial (a) and coronal (b) plane revealed multiple lesions (arrows) of ground glass opacity accompanied with consolidation under or near the pleura in bilateral lower lobes, with air bronchogram and thickened interlobular septa. a large piece of ground glass opacity (square box) could also be seen in the right lower lobe (b). c&d. seven days after treatment, the right lesion was significantly reduced with formation of fibrotic stripes, and the left lesion was also absorbed with decreased density like ground glass opacity (arrow in c). e&f. ten days after treatment, bilateral lesions were mostly absorbed with only some nodules and stripes of fibrosis left (arrows). in some patients, the liver enzyme, lactate dehydrogenase (ldh), muscle enzyme and myoglobin may increase while some severe patients may have increased troponin. most patients have increased c-reactive protein and elevated erythrocyte sedimentation rate. the virus nucleic acid can be detected in swabs, secretions and sputum from the respiratory tract, blood or excrement. in some patients with negative virus nucleic acid, chest ct scanning may detect abnormality. 7 , 13-16 the primary ct imaging findings are summarized here. most lesions occur in the peripheral area or under the pleura along the bronchovascular bundles. multiple locations are involved with occasionally single or double lesions, lower lobes are involved more often than the upper and middle lobes, and the right middle lobe is the least to be infected. the lesion may be patchy, nodular, honeycomb, grid or strips, and the lesion density is mostly uneven with the primary presentation of ground glass opacity accompanied by thickening of interlobular or intralobular septa. the lesion may also present as paving stones with consolidations and formation of fiber stripes. the lesion may be accompanied by air bronchogram but rarely by pleural effusion and enlarged mediastinal nodes. the imaging manifestations of this group are sometimes inconsistent with the clinical manifestations. mild ncp has no abnormality in the pulmonary imaging, but these patients still have infectivity and should be properly isolated and treated. mild patients were significantly younger than the other types of patients, suggesting that children, teenagers and younger patients were mostly mild. patients with severe (mean 54.7 year) and critically severe (mean 65.7 years) ncp were significantly ( p < 0.05) older than those with mild and common (44.5 years) ncp. severe and critically severe ncp involved more commonly 4-5 lobes and most significantly ( p < 0.05) bilateral lower and upper lobes compared with common ncp. the lesion was primarily located in the peripheral area under the pleura with possible extension towards the pulmonary hilum in big lesions or when the disease is deteriorated. in a short period of time, the lesion may change quickly with occurrence of new lesions in other areas of the lung or improvement at three days after treatment, necessitating repeated ct imaging scan for guiding disease progression and implementing proper treatment. ncp should be differentiated from other pneumonia caused by other viruses like influenza virus, parainfluenza virus, adenovirus and sars-cov or by other microorganisms including mycoplasma, chlamydia and bacteria. bacterial pneumonia presents as small pieces of shadow distributing along the bronchus, which can fuse into a large lesion or a large piece of consolidation. laboratory tests can show increased count of leukocytes in bacterial pneumonia for differentiation. other viruses cause pneumonia with large diffused lesions of ground glass opacity in both lungs accompanied with interlobular septa, which may be difficult to differentiate from sars-cov-2 pneumonia, however, definite epidemical history is useful for this disease. virus nucleic acid detection helps determine the diagnosis. in summary, imaging presentations of ncp are mostly patchy ground glass opacities in the peripheral areas under the pleura with partial consolidation which will be absorbed with formation of fibrotic stripes if improved. the lesion may have quick changes with formation of new lesions in other areas and extend from the peripheral to the central area if deteriorated. repeated ct scanning is helpful for monitoring disease progression and implementing timely treatment. clinical features of patients infected with 2019 novel coronavirus in wuhan, china clinical characteristics of novel coronavirus cases in tertiary hospitals in hubei province clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china does sars-cov-2 has a longer incubation period than sars and mers novel coronavirus (sarscov-2) epidemic: a veterinary perspective [interpretation of "guidelines for the diagnosis and treatment of novel coronavirus (2019-ncov) infection by the national health commission (trial version 5 imaging changes in patients with 2019-ncov the progress of 2019 novel coronavirus (2019-ncov) event in china diagnosis, treatment, and prevention of 2019 novel coronavirus infection in children: experts' consensus statement clinical characteristics and therapeutic procedure for four cases with 2019 novel coronavirus pneumonia receiving combined chinese and western medicine treatment overview of the 2019 novel coronavirus (2019-ncov): the pathogen of severe specific contagious pneumonia (sscp) thin-section ct of severe acute respiratory syndrome: evaluation of 73 patients exposed to or with the disease ct imaging features of 2019 novel coronavirus (2019-ncov) ct imaging of the 2019 novel coronavirus (2019-ncov) pneumonia evolution of ct manifestations in a patient recovered from 2019 novel coronavirus (2019-ncov) pneumonia in wuhan chest ct for typical 2019-ncov pneumonia: relationship to negative rt-pcr testing none. key: cord-008686-9ybxuy00 authors: everett, tom; douglas, jenny; may, shoshanna; horne, simon; marquis, peter; cunningham, richard; tang, julian w title: poor transmission of seasonal cold viruses in a british antarctic survey base date: 2019-03-14 journal: j infect doi: 10.1016/j.jinf.2019.03.007 sha: doc_id: 8686 cord_uid: 9ybxuy00 nan recently, it is reported in journal of infection that h5n6 1 and h7n9 2 subtype avian influenza virus may have an increased pathogenicity to humans.the h1n1 subtype influenza virus has emerged in china. not only the h1n1 (95%) subtype but also several h3n2 (5%) subtype influenza viruses have been detected in samples. according to chinese national influenza data ( http: //ivdc.chinacdc.cn/ ), the h1n1 subtype influenza virus was prevalent from the end of 2018 to the beginning of 2019. the h1n1 and h3n2 subtypes of influenza virus are resistant to adamantanes (amantadine and rimantadine), and a small number of h1n1 strains have been found to be less sensitive to na inhibitors (nais; oseltamivir, zanamivir, and peramivir). in this study, we briefly evaluated the evolution patterns of the h1n1 influenza virus and the h3n2 influenza virus. we collected non-repeat 674 h1n1 and h3n2 subtype influenza virus sequences isolated in china over the course of nearly 5 years from the global initiative on sharing avian influenza data (gisaid) database ( www.gisaid.org ) and national center for biotechnology information (ncbi) ( www.ncbi.nlm.nih.gov/genomes/flu ). the haplotype network map shows that the 2014 h1n1 strain has a node in common with the 2016/17 h3n2 strain ( fig. 1 ) , and h1n1 and h3n2 often co-infect the same patients. when multiple strains of influenza infect the same host, they may undergo recombination and reassortment of the gene fragment, which greatly changes the pathogenicity and epidemiological characteristics of the virus. currently, the h1n1 subtype influenza virus is widespread in the population, and the number of children with neurological symptoms increased significantly this year. 3 the influenza virus has shown some variation, and whether this variation occurs along h1n1 and h3n2 lines remains to be seen. when multiple viruses co-infection occurs, it becomes possible for the viruses to undergo genetic communication, which may change the direction of viral evolution and so deserves our attention. we calculated the average gene evolution rate (nucleotide replacement rate) of the h1n1 influenza virus and the h3n2 influenza virus between different years from 2013 to 2019. it can be seen that the genetic evolution rate of the h1n1 influenza virus and the h3n2 influenza virus is 2.91e −5 -4.03e −4 and 2.72e −5 -1.05e −4 ( table 1 ) , respectively, in the same year and there are up and down fluctuations that may be related to the subtypes that were prevalent that year. additionally, we calculated the evolution rate of the h1n1 influenza virus from the end of 2018 to the beginning of 2019 (1.13e −3 ). a large increase in the rate of advancement indicates a rapid change in the virus in the short term, triggering changes in the replication, resistance, and transmission of the virus. the type a influenza virus emerges periodically every year,influenza undergoes continuous evolution, and different lineages have appeared. 4 at the same time, under the action of vaccines and drugs, the antigenicity and antigenic site of the virus are transformed and resistant. 5 however, medical science has also improved. it is necessary to determine the frequency of gene exchange between different subtypes and be alert to possible variations in gene communication between different subtypes. pu et al. 6 and shi et al. 7 reported that duck-derived virus h7nx and recombination of h7n9 can produce new h7n2 that can cause disease death in waterfowl. this recombination event may have occurred in 2013 or earlier, but the recombinant virus has a distinct evolutionary advantage given the use of a vaccine. exchange between h1n1 and h3n2 may allow them to give each other different viral characteristics, hence, ongoing surveillance of h1n1 and h3n2 subtypes of influenza is warranted. the authors declare not conflict of interest. recent studies in this journal revealed that some h7n9 viruses reassorted with duck aivs, and then attained the ability to efficiently infect ducks. 1,2 h7n9 aivs have been endemic in chicken since their emergence in china in february 2013. 3 after its emergence, h7n9 viruses have evolved substantially, and have frequently reassorted, acquiring internal genes from other chicken h9n2 viruses, increasing the genetic diversity of h7n9 viruses. 4 this raises the concern that whether h7n9 can attain internal genes from other aivs. thus, we collected all available h7n9 sequences to detect potential novel reassortments of the h7n9 aivs, and found evidences that three human-isolated h7n9 isolates attained internal genes from duck and human aivs. all available sequences of h7n9 aivs were downloaded from the ncbi ( https://www.ncbi.nlm.nih.gov ), gisaid ( https://www.gisaid.org ) and fludb ( https://www.fludb.org ) public databases. then, phylogenetic trees for ha, mp, np, ns, pa, pb1, pb2 and na genes were reconstructed, respectively, using raxml v.8.0.24 with gtrgamma model, and 10 0 0 bootstrap tests. phylogenetic analyses revealed that five genes (np, ns, pa, pb1, and pb2) of a/fujian/33845/2017(h7n9), mp gene of a/gd-66/2014/h7n9/2014-01-29, and two genes (pb1 and pb2) of a/zhejiang/9/2014(h7n9) did not clustered with chicken h7n9 aivs, respectively ( fig. 1 and supplementary fig. 1 ). further, blastn ( https://blast.ncbi.nlm.nih.gov/blast.cgi ) was used to search the homology sequences of these three abnormal strains ( 5 while domestic ducks act as an interface between the natural gene pool and terrestrial poultry in the influenza virus ecosystem. the 2013 h7n9 viruses cannot replicate efficiently in ducks in the first four waves. however, studies have indicates that the highly pathogenic h7n9 virus has extended its host range by acquiring genes from duck influenza viruses and has now adapted to ducks. 1, 2, 6 the reassortments between 2013 h7n9 and duck aivs would further raise the diversity and spread of the h7n9. 7 in addition to our finding of the reassortments between duck aivs and the human-isolated h7n9 viruses suggest that surveillance and control of duck aivs is critical for the control of h7n9 viruses, which was almost ignored previously. it's surprising that the pb1 and pb2 genes of a/zhejiang/9/2014 (h7n9) showed the most closed relationship with human h3n2 viruses (89% and 88% identity respectively, table 1 ). reassortments between human and avian aivs can make the reassortant viruses replicate efficiently in mammalian hosts. 8 it's very possible that the reassortment between h7n9 and human h3n2, may make the reassortant virus more adaptive to human, even attain the ability to efficiently transmit between humans, and thus raise great thread to the public health. historically, several pandemic human influenza viruses were derived from reassortant virus between avian and human influenza viruses. for example, the h1n1/pdm2009 virus, which was a swine reassortant virus that attained the pb1 from human h3n2, was rapidly transmitted between humans and then, globally circulates as a seasonal virus, posing a substantial risk to human. 9 h7n9 aivs have the genetic makeup associated with human infections, 10 in addition to our finding that the reassortant human-isolated h7n9 virus attained the pb1 and pb2 from human h3n2, possibility like the h1n1/pdm2009 virus, the reassortant virus would become more invasive to humans, and thus pose serious pandemic threat to humans. h7n9 is a novel reassortant aiv subtype, which has surpassed h5n1 in laboratory-confirmed human infections despite its limited dissemination outside of china. thus, whether this subtype could acquire the ability to efficiently human-to-human transmission, and become a new influenza pandemic raise great attention. although a h5/h7 vaccination in chicken has successfully decreased the prevalence of the h7n9 viruses in chicken, our findings that h7n9 viruses attained internal genes from human and duck aivs raise concerns about the potential ability of the viruses to increase their diversity and spread, and especially the possibility to develop better ability to infect human, and eventually attain efficient human-human infections. we note with interest these previous studies into household and hospital influenza outbreaks. 1,2 such community and hospitalbased respiratory virus transmission and outbreak investigations often suffer from the potential confounding arising from possible exposures to undiagnosed index cases outside of the outbreak cohort, leading to an overestimate of virus transmissibility, and potentially unnecessary costly and restrictive infection control interventions. to avoid such confounding, we performed a small pilot study in a closed population of adult research scientists ( n = 43 out of a possible 48). all participants signed informed consent forms, following ethical approval from plymouth university ethics committee. these scientists were confined to a british antarctic survey base for 1 month (march 2017), during which no personnel entered nor left the base. therefore any detectable human respiratory viruses could only have been brought into the base by personnel at the beginning of this 'closed period'. participants were given anonymous codes to maintain confidentiality. each agreed to give nasal swabs (collected in virus transport medium, virocult, medical wire and equipment ltd, corsham, wiltshire, england) upon entry (day 0, 14/3/17), then at days 4 (18/3/17), 10 (24/3/17) and 17 (31/3/17) post-entry. all viral swabs were stored at −80 °c until they could be shipped back to the uk and tested at the leicester royal infirmary. this was performed using a respiratory multiplex pcr assay (16-well, ausdiagnostics uk ltd., chesham, uk) that could detect any of: influenza a, b, respiratory syncytial virus (rsv), parainfluenza (piv) types 1-4, human metapneumo (hmpv)-, entero-/rhino-, corona-(229e, oc43, nl63, hku1) and adeno-viruses. no specific instructions about infection control were given to the participants. they were left to act as they would normally behave throughout the period of the study. any participants who developed any of 9, self-assessed, influenza-like symptoms (fever, cough, stuffy nose and/or sinuses, headache, sore throat, myalgia, fatigue, shortness of breath, nausea or vomiting) would complete a tick-box questionnaire (on a scale of 1-'very mild' to 5-'very severe') to describe the relative severity of their symptoms. this same questionnaire also requested the contact intensity (i.e. number, nature and frequency) of their daily contacts with other participants as a self-assessed, linear graded score (from 1-'sharing just one meal together' to 5-'spending the majority of the day and evening with the other person'), depending on the frequency of contact whilst working, eating meals and socialising together. the daily location of all personnel in any of the four station zones at 0830, 110 0, 140 0, 1630 and 20 0 0 h was also recorded routinely for safety and security, using a 'tagboard' system. out of the 43 participants who consented, 3 later declined to have any viral swabs taken, and of the resulting 160 (i.e. 40 × 4 swabbing time-points) possible swabs, 153 were successfully collected and stored for testing. testing the incubation period of human coronaviruses is around 2-5 days, 3 which can be used to link symptomatic cases together, epidemiologically, 4 with viral shedding being reported for up to 6 days post-symptom onset. 5 so the symptoms and positive nl63 and oc43 results for participants 21 and 74, respectively, could have been acquired from participants 40 and 47, who may have been the original index cases (sources) for these viruses. although no respiratory virus was detected in their samples, participants 40, 47 and 107 all reported similar symptoms to those of 21 and 74 during the study period, which were typical common cold symptoms. note that the participants' self-reported contact intensities were not entirely robust, e.g. both participants 40 and 47 list participant 21 as a contact, so could both have been index cases for him/her. however, 21 did not list either 40 or 47 as a contact (such contacts should be reciprocal). this may have just been a simple oversight, but it makes the contact link less reliable. similarly, participant 74 could have served as the index case for participant 107, but neither lists the other as a contact. regardless of the contact intensities reported in the questionnaires, there were no secondary cases of either nl63 or oc43 coronaviruses detected in any of the other study participants' weekly swabs. one possible explanation for this may have been an insufficient sensitivity of the assay to detect low levels of these respiratory viruses. the limit of detection (lod) for the ausdiagnostics assay varies significantly with each virus (as given in the kit insert, all in copies/ml): influenza a (1900-2375), b (525), rsv (50-2125), piv types 1-4 (50-250 0), hmpv (10 0-625), entero-/rhino-(75-1025), corona-(229e, oc43, nl63, hku1) (1350-4175) and adeno-(1075) viruses. however, in the acute infection stage respiratory viruses are generally present in relatively high copy numbers, with median values of mostly 4-8 log 10 (i.e. 10,0 0 0-10 0,0 0 0,0 0 0 copies/ml) for adeno-, corona-, hmpv, influenza, piv and rsv, as reported in one comprehensive paediatric study. 6 although children generally shed higher viral loads than adults, it is likely that the coronavirus loads in acutely infected adults would still be mostly detectable on this assay, which is approved (i.e. ce-marked) for routine diagnostic testing. yet, it is still possible for viruses that are infecting individuals at the lowest loads within these ranges, to fail to be detected by this assay. given the results that are currently available from this study, one of the key questions is: from where did the nl63 and oc43 coronaviruses arise? in addition, the lack of any secondary nl63 or oc43 coronaviruses cases (symptomatic or asymptomatic) arising from the known positive sources (participants 21 and 74), suggests that the transmissibility of these common cold viruses may be limited. this seems unexpected, given the potential stress on the body immune system whilst living and working in such an extreme environment. however, such relatively poor transmission of respiratory viruses has been previously described in antarctic base personnel for rhinovirus and adenovirus, 7, 8 for reasons that are still unclear. another potential confounding factor is the unknown status of the 5 individuals who were also present at the base (mostly base personnel) but who declined to participate in the study. it is possible that one or more of these non-participants could have been the original sources (i.e. index cases) of the nl63 and oc43 coronaviruses at the start of the study. whilst there are some limitations to this study, there are plans to repeat this on a larger scale, over a longer 'closed' period, with the use of real-time, point-of-care testing (poct) to detect such respiratory viruses. although previous respiratory virus outbreaks have been described in remote research bases, 7,8 these were unable to utilise the greater sensitivity and spectrum of respiratory viral targets provided by modern, molecular, diagnostic tools. 9, 10 thus, some positive cases in these earlier studies may have been missed, leading to an underestimate of the transmissibility of these respiratory viruses in these populations. respiratory infections in research personnel can impact significantly on their productivity, an important consideration when their time at such remote research bases is limited. this and future studies will enable medical teams to enhance the healthcare of research base personnel to optimise their precious research time spent there. none of the authors have any conflicts of interests to declare. we thank the following for their support of this study: uk clinical virology network (cvn), for general funding support; medical wire & equipment ltd., for donating some of the sampling swabs; ausdiagnostics uk ltd., for donating the respiratory multiplex pcr tests. none of these companies were involved in the writing of this article. we read with interest, the article by poller et al. 1 , in this journal, entitled "a unified personal protective equipment….". we understand the importance of proper personal protective equipment(ppe) as an integral component of healthcare workers(hcw) protection in outbreak situations of infections with possible high consequence. but at times, such an outbreak occurs in an unsuspected region, when initial cases present in early course of illness before the development of ominous clinical features. medical staff, particularly in busy rural set ups of resource-poor developing countries may discover that they have been exposed to a high consequence infectious disease after the event of exposure, particularly if these centres are unaware, reluctant or unequipped regarding routine use of ppe. a similar situation occurred in a rural healthcare setting of kerala, india, during the may-2018 outbreak of nipah virus (niv), killing 21 out of 23 reported cases. 2 a 26-year-old male ( index case of the outbreak report 2 ) from kerala's perambra town died undiagnosed with fever, en-cephalitis and respiratory distress in government medical college kozhikode(gmck), after being transferred from taluk hospital, perambra(thp). another 47-year-old male patient ( case-10 ) 2 was admitted in thp for an acute febrile illness and recovered while the unsuspected index case was being treated there in the adjacent bed. two weeks later, case-10 presented to taluk hospital, balussery(thb) (the setting of our intervention), with complaints of fever, headache and vomiting of 4 days duration. he was treated there as inpatient for about 24 h after which he was referred to gmck, owing to clinical deterioration. the next day he developed altered sensorium, respiratory distress and expired. till then, there was no suspicion about the niv outbreak situation, as kerala is at least 2500 km away from the last known outbreak in the indian subcontinent in 2012 3, 4 . in the meanwhile, the brother, father and aunt of the index case , and a nurse, who cared for him at thp, developed similar clinical features of acute encephalitis with respiratory distress and got admitted. all of their samples, along with that of case-10 , were tested positive for niv from the reference laboratory. the state public health authorities swiftly declared the outbreak and ensured containment and protective measures. however, by this time, 17 out of 18 confirmed niv cases were already infected and fell ill, being epidemiologically related as contacts of the index case in family, during transit to healthcare facilities or in hospital 2 . here, we report our experience with eight hcw including two doctors (authors aps and mb of this correspondence) and six nurses working in thb, who had unsuspected, inadvertent, yet significant exposure to case-10 when he was admitted there, without any ppe. both the doctors had closely clinically examined case-10 and the six nursing-staff had repeated bare-hand, unmasked contacts with him ( table 1 ). all of them were extremely panicked once the outbreak notification was out and beseeched aps and mb for an immediate solution. aps and mb contacted the other authors for advice regarding any possible post-exposure prophylaxis(pep). considering the possibility of human-to-human (airborne / contact) transmission of niv, 3, 4 in-vitro 5 and in-vivo 6 effects of ribavirin on niv, evidence of safety and efficacy of short-course high-dose ribavirin pep(rpep) used for lassa fever 7 and unavailability and inexperience of any other alternatives (favipiravir or monoclonal antibody m102.4), were discussed by vkmn, sb, ms, nw and ab, and a consensus opinion of rpep as the only available and reasonably safe option was placed before aps and mb. the importance of psychological factors 8 in the hcws were also considered seriously. the suggested dose was 10 0 0 mg thrice daily for 14 days(cumulative 42,0 0 0 mg) in congruence to the lassa fever recommendation. 7 all the contacts started rpep within 72 h of exposure. the mean cumulative dose of rpep taken by the contacts was 28,750 mg(17,60 0-40,0 0 0 mg) and the mean duration was 12.5 days(11-14 days, table 1 ). their clinical and laboratory parameters were monitored for the next 6 months. mean age of the 8 hcws was 35.4 years(30-43 years). two were males and rest females; none were pregnant ( table 1 ) . most of them experienced minor side effects like fatigue, headache, nausea, dry mouth and palpitations. there was a mean drop of 2.82 g/dl of haemoglobin, predominantly between days 17 and 21 after starting rpep, which started rising in all within a week of stopping rpep. bilirubin levels rose by a mean of 1.65 (range: 0.0 -3.6) mg/dl in 7 of the 8 hcw ( fig. 1 ) . none of them ultimately contracted niv disease. interestingly, one 25-year-old male patient ( case -23 of the outbreak report 2 ), was admitted at an adjacent bed in thb with dysentery, while case-10 was admitted. he was present within a distance of 1 metre for more than 5 h. there was apparently no direct contact between them, except that same hcw served both of them sharing non-critical medical devices. after recovery, case -23 was discharged from thb, to return after a week to gmck with high grade fever, developing encephalopathy and respiratory distress, diagnosed to have niv infection and succumbed to it. in the current outbreak, 3 family members, one staff nurse, one trainee nurse, one radiology assistant who took care of the index case and 13 hospital contacts contracted the infection, proving human-to-human transmission. respiratory aerosols and fomites cause human-to-human spread. 3 the mortality rate, in all recent niv outbreaks in the indian subcontinent is 75-100% with the bangladeshi strain 3 (niv-b) and its close relative in the recent kerala outbreak, 2 has been consistently almost double compared to ebola. further that case -23 getting infected from case-10 in the same premises of thb, makes our case for rpep stronger. the 2 survivors out of 23 infected patients in this outbreak have also received ribavirin. 9 given the recent findings of widespread presence of niv among pteropus bats in india, 10 another outbreak might be just a matter of time. our field notes from emergency, voluntary, off-label rpep among hcw provides evidence, albeit low-quality, of its safety and probable efficacy, strongly suggesting a pre-planned trial for pep to be started immediately once such an explosive outbreak of niv is notified. none. we note the previous report describing a decreasing incidence of eosinophilia in returning travellers by barrett and colleagues. 1 in contrast, another type of hazard reported by returning travellers -monkey bites -appears to be increasing. this makes it necessary for our frontline medical staff to be aware of the potential risks from rabies and simian herpes b virus (shbv or cercopithecine herpesvirus 1 -cehv-1) associated with this type of exposure. 2 although infections are rare as a consequence of bites, 3,4 both viruses can result in very high (80-100%) mortality if the appropriate post-exposure prophylaxis is not initiated promptly. in view of these serious consequences, post-exposure protocols have been developed to reduce likelihood of infection. 2 , 5-7 while this is agreed for rabies, 5, 8 post-exposure prophylaxis is not uniformly recommended for shbv, as cases have only been reported with captive monkeys, 3,9 despite numerous monkey bite exposures in regions where animals are thought to be infected. assessing risks versus benefits obviously needs to be done judiciously in each case, and national public health specialists can be consulted to support decision making. 8, 9 the main risk is primarily from monkey bites from macaque monkeys (genus macaca ), which are now encountered relatively frequently in various tourist areas in southeast asia (e.g. philippines, indonesia, malaysia, cambodia, vietnam, thailand). after a monkey bite, the patient should perform immediate wound cleansing: irrigation with soap and water, or other skincleansing detergent, or sterile water alone, for at least 15 min. later, when the patient presents to the emergency department (ed), all medical staff need to be aware of both the rabies and shbv post-exposure protocols (peps) associated with such bites. whilst most ed teams will likely know of the rabies pep protocol, 5 fewer will be aware of the guidelines for shbv pep. 6 along with the wound cleansing and post-exposure rabies immunoglobulin (rig) and vaccination, any risk of shbv requires that high dose acyclovir (preferably valaciclovir 1 g tds po; or acyclovir 800 mg 5 times daily po, for adults) pep for at least 14 days should be considered. immediate pcr and later serological testing for signs of shbv infection are possible. however, recommendations for such testing are somewhat variable, with some advising testing in symptomatic cases only, whilst others will test all potentially exposed cases, regardless of symptoms. 6, 7 symptoms of possible shbv disease include vesicular lesions, pain and itching near the bite site, local lymphadenopathy, flulike illness (fever, headache, myalgia, fatigue), and any focal or progressive neurological symptoms, including dyspnoea. outcomes are generally fatal (80% mortality without any treatment), once there is central nervous system involvement. 2, 7 however, with antiviral prophylaxis and treatment, such fatal outcomes are rarer. bacterial infections (e.g. staphylococcus and streptococcus spp.) can also arise from the bite itself, especially in children, for which systemic antibiotics can be given, 10 and tetanus vaccination. to highlight this issue, we present three cases of returning travellers with monkey bites. case 1: a 24-year old male was admitted with headache, lethargy and myalgia following a trip to indonesia (monkey forest, ubud, bali), where he sustained a penetrating bite to his right shoulder from a macaque monkey. there were no immediate post-bite complications. however, 16 days later, he developed paresthesia and neuropathic pain in his right thigh. after seeing a local physician, oral aciclovir 800 mg 5 times daily for 14 days was prescribed, as prophylaxis for possible shbv. one day later he developed a vesicular rash on his right thigh, which subsequently resolved on the antiviral therapy. on return to the uk, given this history, he was admitted and started rabies post-exposure immunisation, without rabies immunoglobulin (rig). he was then extensively investigated for possible shbv infection. he had five days of intravenous aciclovir and a further nine days of oral valaciclovir, whilst awaiting investigation results. diagnostic testing performed at the department of viroscience, erasmus medical centre (m/c), rotterdam, the netherlands on cerebrospinal fluid (csf), blood, lesion swab and saliva by shbv pcr showed no evidence of infection at that time. at outpatient review, two months later, there had been no further history of any rash or neurological symptoms, though the patient did mention several recurring episodes of genital herpes, for which he was given a 10-day course of oral valaciclovir 500 mg bd po. by this time, the risk of latent shbv was considered negligible and he was discharged from clinic. case 2: a 28-year old male was seen in clinic who gave a history of receiving an unprovoked, penetrating bite on his right upper arm from a vervet monkey ( chlorocebus pygerythrus , previously classified as cercopithecus aethiops ), one week earlier whilst on holiday in barbados. after immediate wound care, he was seen in a local clinic and received tetanus vaccine and oral antibiotics. no aciclovir shbv pep was commenced at this time. the bite wounds healed without complication. after returning to the uk a week later, an outpatient review revealed that he was still asymptomatic for any clinical features of shbv, rabies or other travel-associated illnesses. however, as a precaution, valaciclovir 1 g tds po, for 21 days was prescribed as shbv prophylaxis, due to the possibility of an incubating shbv infection. 3 blood and saliva samples were sent to viroscience for pcr testing to check for any residual shbv, dengue, chikungunya or zika virus infections. all tests were negative. the patient continued to remain asymptomatic, so was eventually discharged from outpatient follow-up. case 3: a 31-year old female was seen in clinic upon return from southeast asia, with a history of receiving a penetrating bite to her right upper arm from a macaque monkey, whilst visiting monkey island, vietnam, 18 days previously. she received her first dose of rabies vaccination (without rig) at a local clinic within four hours of the bite. a week later, whilst still in vietnam, she received a second dose of rabies vaccine from a different clinic, which also started her on acyclovir post-exposure prophylaxis for shbv. the bite wounds healed without sequelae. at her 18-day clinic review once back in the uk, she was still asymptomatic. baseline saliva and blood samples were taken and stored but not tested for shbv. she continued both the shbv and rabies pep whilst continuing her travels a week later, and remained asymptomatic six weeks post-exposure. this small case series demonstrates a diversity of presentations and follow-up management for these patients, notably: case 1 likely presented with genital herpes; case 2 sustained a bite from a vervet, not a macque, monkey; case 3 did not have any shbv testing. to our knowledge, all of these cases remain well. as there are no consensus guidelines available for managing such monkey bites, we suggest a precautionary approach and that to be safe, assume that both rabies and shbv are potential risks, regardless of monkey species. therefore, in the event of a monkey bite, where, after discussion with the patient (based on their individual clinical assessment), a decision is made to give prophylaxis: (i) immediately cleanse the wound for 15 mins with clean water + / − soap or detergent. consider appropriate antibiotic therapy to prevent skin infection (e.g. co-amoxiclav or amoxicillin), and tetanus vaccination. (ii) seek competent clinical help and obtain the first dose (day 0) of rabies vaccine + / − rig, depending on the risk assessment (in the uk, public health england tel: 0208 327 6204, 9-5 pm mon-fri). 8 complete post-exposure rabies vaccination with further doses on days 3, 7 and 21, post-exposure. 5 (iii) start acyclovir (1 g valacyclovir tds po or 800 mg acyclovir 5 times daily po -depending on local availability, for adults. adjust the dose as appropriate for children) as soon as possible after the bite, for at least 14 days, as post-exposure prophylaxis against shbv. (iv) if symptoms compatible with shbv develop within the next 2-4 weeks (e.g. vesicular lesions, pain, itching around the bite, local lymphadenopathy, flu-like illness, focal or progressive neurological symptoms), continue the acyclovir and seek further expert advice. (v) baseline samples (serum, saliva, wound swabs) can be taken and stored for comparison. if acute illness develops, repeat samples (including cerebrospinal fluid -csf) should be taken for diagnostic testing (by pcr) to check for shbv dna, and serology for shbv antibodies -if such testing is available. recent article in this journal has reported dengue patients were associated with a higher risk of autoimmune diseases than nondengue patients 1 . dengue has been a serious public health prob-lem in the world, the number of dengue epidemics has been on the rise worldwide. before 1970, only nine countries had experienced severe dengue epidemics; however, currently more than 100 countries have been severely affected by dengue 2 . after the first dengue-fever epidemic in china, which occurred in may 1978 in foshan, guangdong province, there have been regional outbreaks of dengue every year and the number of cases has increased. guangdong province is the area most seriously affected by dengue in china. the number of cases in guangdong accounts for more than 90% of the total number of cases in china 3,4 . however, the epidemic characteristics of dengue fever in guangdong province have not been reported since 2010 5 . a dengue epidemic is closely related to various factors, such as environmental conditions, imported cases, and migration; thus, its epidemic characteristics and patterns change rapidly. especially after the outbreak of dengue in guangdong province in 2014, the epidemic patterns have changed greatly. therefore, exploring the changing patterns of dengue outbreaks and epidemics in guangdong is of great significance to the prevention and control of dengue. this study aimed to investigate the changing patterns of the epidemic characteristics of dengue in guangdong province and to propose prevention and control measures. we collected dengue cases from 2008 to 2017 from the web-based disease reporting information system of the chinese national center for disease control and prevention. a total of 52,792 cases were reported for this period. population data were obtained from the statistics bureau of guangdong province ( http://www.gdstats.gov.cn/ ). a chi-square test was used to determine spatial differences in cities. this study was approved by center for disease control and prevention of pla review board. in terms of temporal distribution ( fig. 1 ) , dengue cases showed an increasing trend from 2008 to 2014 in guangdong province, and a decreasing trend from 2014 to 2017 (93 cases in 2008, 45,170 cases in 2014, and 1662 cases in 2017). the dengue outbreak in 2014, with 45,170 cases and 6 deaths, was the largest dengue outbreak in china in the past 20 years. before the outbreak in 2014, an average of 617 people was infected with dengue annually. following the outbreak, an average of 1305 people were infected with dengue annually-nearly twice the number of cases per year compared to that before 2014. dengue fever had typical characteristics in population distribution. adults aged 20-65 years accounted for 76.76% of patients, and children aged 0-10 years only accounted for 4.16% of patients. in terms of distribution across occupations, housekeepers and the unemployed (23.1%), retired individuals (12.82%), those involved in commercial services (11.52%), industrial worker (9.56%), and students (7.49%) formed the majority of patients with dengue fever. these five groups accounted for 64.48% of the total number of cases. the number of male patients was comparable to that of the number of female patients (male: female = 1:1.01), and the mortality rate of dengue was low at only 0.11 ‰ . monitoring data of the past 10 years showed that although the number of cases increased greatly after the outbreak in 2014, the population distribution did not change significantly. the key population for the prevention and control of dengue fever were adults over the age of 20 years. although cases have been reported in various regions, there are significant differences ( p < 0.001) in the distribution of dengue among the different cities in the guangdong province ( fig. 2 ) . incident cases were mainly concentrated in guangzhou city and foshan city. guangzhou city had a total of 40,170 cases during 2008-2017, which accounted for 76.09% of cases among the different cities. foshan city had a total of 4660 cases, which accounted for 8.83% of cases. however, according to annual data trends, 2014 was the turning point in spatial distribution patterns for dengue fever. from 2008-2013, the incidence of dengue was concentrated in foshan city, guangzhou city, jiangmen city, and zhongshan city (93.14% of cases). however, by 2014, except for meizhou city, there were case reports from all 20 cities in the province. among them, there were 13 cities with more than 200 cases each. after 2014, dengue fever was prevalent throughout the province. even in zhanjiang city, zhaoqing city and jieyang city, which are distant from guangzhou city, and the number of cases in remote areas increased significantly. this paper is the first to analyze the characteristics of the change in dengue epidemics in guangdong province in the past 10 years. in general, the incidence of dengue fever has typical spatial differences. the cases were mainly concentrated in urban areas with large populations and developed economies, such as guangzhou city and foshan city. theinflux of many migrants, especially those from southeast asia, could have caused the higher incidence of dengue fever in these areas. studies have shown that dengue epidemics in china were initiated by imported cases and then became prevalent in the local areas 6 . however, attention should be given to the large number of dengue epidemics in remote areas, which appeared after 2014. a possible reason for this could be global climate warming, which has caused the natural environments in some remote areas to become suitable for mosquito breeding. moreover, rapid development in tourism and trade has also led to an increase in local and imported cases. in summary, after 2014, the epidemic characteristics of dengue fever in guangdong province have undergone major changes. the key areas for prevention and control are no longer confined to traditional epidemic areas, such as guangzhou city and foshan city. instead, dengue prevention and control should be conducted throughout the province. in addition, according to the national dengue surveillance in 2018, dengue fever has shown a trend of "moving up north" in the country 7 . therefore, the changing characteristics of this epidemic warrant high attention of relevant departments. the authors declare that they have no competing interests. this work was supported by national key r&d program of china ( 2016yfc120 070 0 ), beijing nova program ( z17110 0 0 01117102 ) and military medical innovation project ( 16cxz046 ) and pla youth training project for medical science ( 16qnp127 ). the funders had no role in the study design, data collection and analysis, decision to publish, or preparation of the manuscript. pathogenicity and transmissibility of three avian influenza a (h 5 n 6 ) viruses isolated from wild birds evolving ha and pb 2 genes of influenza a (h 7 n 9 ) viruses in the fifth wave -increasing threat to both birds and humans? genetic and antigenic characterization of a(h 1 n 1 )pdm09 in yantai, china, during the 2009-2017 influenza season neurologic manifestations of influenza a(h 3 n 2 ) infection in children during the distinct molecular evolution of influenza h 3 n 2 strains in the 2016/17 season and its implications for vaccine effectiveness rapid evolving h 7 n 9 avian influenza a viruses pose new challenge rapid evolution of h 7 n 9 highly pathogenic viruses that emerged in china in 2017 hemagglutinin characteristics, changes in pathogenicity, and antigenic variation of highly pathogenic h7n9 avian influenza viruses in china two novel reassortant high pathogenic h7n9 viruses isolated in southern china in fifth wave shows internal genomic diversity and high virulence in chickens and ducks human infection with a novel avian-origin influenza a (h7n9) virus evolutionary dynamics of avian influenza a h7n9 virus across five waves in mainland china evolution and ecology of influenza a viruses rapid evolution of h7n9 highly pathogenic viruses that emerged in china in 2017 deciphering the sharp decrease in h7n9 human infections h5n1 hybrid viruses bearing 2009/h1n1 virus genes transmit in guinea pigs by respiratory droplet origins and evolutionary genomics of the 2009 swine-origin h1n1 influenza a epidemic potential pandemic of h7n9 avian influenza a virus in human case-ascertained study of household transmission of seasonal influenza -south africa haemagglutinin and neuraminidase sequencing delineate nosocomial influenza outbreaks with accuracy equivalent to whole genome sequencing incubation periods of acute respiratory viral infections: a systematic review identifying the probable timing and setting of respiratory virus infections human coronaviruses and other respiratory infections in young adults on a university campus: prevalence, symptoms, and shedding. influenza other respir viruses is higher viral load in the upper respiratory tract associated with severe pneumonia? findings from the perch study rhinovirus infections in an isolated antarctic station. transmission of the viruses and susceptibility of the population adenovirus 21 infection in an isolated antarctic station: transmission of the virus and susceptibility of the population a unified personal protective equipment ensemble for clinical response to possible high consequence infectious diseases: a consensus document on behalf of the hcid programme outbreak investigation of nipah virus disease in kerala, india nipah virus-associated encephalitis outbreak nipah virus contamination of hospital surfaces during outbreaks characteristics of nipah virus and hendra virus replication in different cell lines and their suitability for antiviral screening treatment of acute nipah encephalitis with ribavirin review of the literature and proposed guidelines for the use of oral ribavirin as postexposure prophylaxis for lassa fever post-exposure prophylaxis in ebola virus disease: don't forget the psychological factors adaptive immune responses in humans during nipah virus acute and convalescent phases of infection a spatial association between a nipah virus outbreak in kozhikode, kerala, india and nipah virus infection in pteropus bats the changing aetiology of eosinophilia in migrants and returning travellers in the hospital for tropical diseases expert review of evidence bases for managing monkey bites in travelers understanding primate herpesviruses rabies in nonhuman primates and potential for transmission to humans: a literature review and examination of selected french national data immunisation against infectious diseases virus working group recommendations for prevention of and therapy for exposure to b virus (cercopithecine herpesvirus 1) monkey bites in travelers: should we think of herpes b virus rabies and immunoglobulins service follow-up of monkey bites. phe publications gateway number keep children away from macaque monkeys! increased risk of autoimmune diseases in dengue patients: a population-based cohort study developing a time series predictive model for dengue in zhongshan, china based on weather and guangzhou dengue surveillance data the changing epidemiology of dengue in china, 1990-kk2014: a descriptive analysis of 25 years of nationwide surveillance data spatial and temporal patterns of dengue in guangdong province of china clinical and epidemiological features of the 2014 large-scale dengue outbreak in guangzhou city center for disease control and prevention of chinese people's liberation army, 20 dongdajie street xinying du center for disease control and prevention of chinese people's liberation army, 20 dongdajie street beijing 10 0 071, china hongbin song * center for disease control and prevention of chinese people's liberation army the authors declare not conflict of interest. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.03.008 . we thank the following for their support of this study: uk clinical virology network (cvn), for general funding support; medical wire & equipment ltd., for donating some of the sampling swabs; ausdiagnostics uk ltd., for donating the respiratory multiplex pcr tests. none of the authors have any conflicts of interest to declare. key: cord-264255-q5izs39f authors: chieochansin, thaweesak; samransamruajkit, rujipat; chutinimitkul, salin; payungporn, sunchai; hiranras, thitikul; theamboonlers, apiradee; poovorawan, yong title: human bocavirus (hbov) in thailand: clinical manifestations in a hospitalized pediatric patient and molecular virus characterization date: 2007-12-31 journal: j infect doi: 10.1016/j.jinf.2007.11.006 sha: doc_id: 264255 cord_uid: q5izs39f objective: human bocavirus (hbov), a novel virus, which based on molecular analysis has been associated with respiratory tract diseases in infants and children have recently been studied worldwide. to determine prevalence, clinical features and perform phylogenetic analysis in hbov infected thai pediatric patients. methods: hbov was detected from 302 nasopharyngeal (np) suctions of pediatric patients with acute lower respiratory tract illness and sequenced applying molecular techniques. results: the incidence of hbov infection in pediatric patients amounted to 6.62% with 40% co-infected with other respiratory viruses. there were no clinical specific manifestations for hbov; however, fever and productive cough were commonly found. generalized rales and wheezing were detected in most of the patients as well as perihilar infiltrates. the alignment and phylogenetic analysis of partial vp1 genes showed minor variations. conclusion: our results indicated that hbov can be detected in nasopharyngeal aspirate specimens from infants and children with acute lower respiratory tract illness. acute respiratory tract infection is a major cause of pediatric morbidity and mortality worldwide. in most cases, viruses including influenza a and b, parainfluenza viruses, adenoviruses, respiratory syncytial virus (rsv), and human metapneumovirus (hmpv) are the causative agents. recently, a new respiratory tract virus of the parvoviridae family, human bocavirus (hbov), has been discovered applying molecular analysis on pooled respiratory tract aspirations taken from children in sweden. this virus is closely related to the bovine parvovirus and canine minute virus, which have been classified as members of the genus bocavirus. the virus comprises two major open reading frames (orfs) encoding a nonstructural protein (ns1) and at least two capsid proteins (vp1 and vp2), respectively. moreover, the hbov genome also contains a third middle orf encoding a nonstructural protein (np1) of unknown function. 1 the most conserved region of this virus is the ns1 and np1 gene whereas the vp1/vp2 gene constitutes the variable region. 2 the induction of respiratory illness by hbov is not clearly defined due to lack of propagation techniques in cell culture or animal models. 1 however, many studies have reported this virus infection to be associated with acute respiratory illness. 1, 3, 4 upon discovery of hbov in respiratory pools, its global prevalence has been reported to range from 1.5% to 19% and co-infection with other viruses was commonly found. 1,3e24 moreover, in few studies were shown negative results for hbov infection in nasopharyngeal (np) swabs from healthy volunteers. 20, 25, 26 additional epidemiological and clinical investigation will be essential in order to elucidate what exactly engenders hbov related illness. therefore, in the present study we applied polymerase chain reaction to detect hbov from np suctions collected from infants or children who had been admitted with respiratory tract illness. nasopharyngeal suction specimens were collected from 302 individual infants or children (age range: 5 days to 14 years) who were admitted and diagnosed as having acute lower respiratory illness during the period february 14, 2006 to february 28, 2007 . all of the clinical samples were provided by the department of pediatrics, king chulalongkorn memorial hospital, thailand. nasopharyngeal suction samples were collected in transport medium with antibiotics (0.5% bsa, penicillin g (2 â 10 6 u/l), streptomycin 200 mg/l) and stored at à70 c until tested. the study was conducted after receiving approval by the ethics committee of the faculty of medicines, chulalongkorn university. prior to enrolment, all participating parent gave their written informed consent. dna and rna were extracted from 150 ml of np suction using tri reagent â ls (molecular research center inc., cincinnati, oh) and solubilized in 20 ml of 8 mm naoh or 12 ml of depc treated water for dna or rna, respectively. for hbov detection we amplified the np1 gene by conventional pcr modified from a previous study. 1 the reaction mixture contained 2 ml dna, 0.5 mm 188f primer and 0.5 mm 542r primer, 10 ml 2.5â eppendorf mastermix (eppendorf, hamburg, germany), and nuclease-free water to a final volume of 25 ml. the amplification reaction was performed in a thermocycler (eppendorf) under the following conditions: initial denaturation at 94 c for 3 min, followed by 35 amplification cycles consisting of 94 c for 30 s (denaturation), 55 c for 30 s (primer annealing), and 72 c for 1 min (extension), and concluded by a final extension step at 72 c for 7 min. another set of primers specific for the vp1 gene, vpf2 forward primer (5 0 -ttcagaatggt cacctctaca-3 0 : nt 3639e3659) and vpr2 reverse primer (5 0 -ctgtgcttccgttttgtctta-3 0 : nt 4286e4266), were used in a separate pcr reaction to exclude false positives. after 2% agarose gel electrophoresis stained with ethidium bromide, the expected products of 354 and 648 bp representing the np1 and vp1 gene, respectively, were visualized on a uv transilluminator. influenza a virus detection was performed by conventional pcr using 1 ml of cdna, 0.5 mm of flua_m_f: 5 0 -rggccccctcaaagccga-3 0 (nt 76e93), 0.5 mm of flua_m_r: 5 0 -actgggcacggtgagygt-3 0 (nt 235e218), 10 ml of 2.5â eppendorf mastermix, and nuclease-free water to a final volume of 25 ml. the housekeeping gene glyceraldehyde-3-phosphate dehydrogenase (gapdh) of each sample was amplified in one additional reaction mixture of identical volume with primers gapdh_f: 5 0 -gtg aaggtcggagtcaacgg-3 0 (nt 112e131) and gapdh_r: 5 0 -gttgtcatggatgaccttggc-3 0 (nt 603e583) at a 0.5 mm concentration, each. the amplification reaction was performed in a thermocycler (eppendorf) under the following conditions: initial denaturation at 94 c for 3 min, followed by 40 amplification cycles consisting of 94 c for 30 s (denaturation), 55 c for 30 s (primer annealing), and 72 c for 1 min (extension), and concluded by a final extension step at 72 c for 7 min. after 2% agarose gel electrophoresis, the expected products were influenza a virus (160 bp) and the housekeeping gene (492 bp). parainfluenza virus was detected by real-time pcr using sybr green i carried out in a rotor-gene 3000 (corbett research, new south wales, australia). the reaction mixture contained 1 ml of dna sample, 10 ml 2.5â eppendorf mastermix, 0.5 mm paraf: 5 0 -gctaaatactgtcttmah tggagat-3 0 (nt 11,254e11,278), 0.5 mm parar: 5 0 -gtaagg atcaccwacatadawtgta-3 0 (nt 11,392e11,370), 2 ml 1â sybr green and nuclease-free water to a final volume of 20 ml. the amplification reaction consisted of a preincubation step at 95 c for 3 min followed by 35 cycles of amplification including 95 c for 15 s, 55 c for 15 s and 72 c for 30 s. the fluorescent signal was detected once per cycle upon completion of the extension step. after amplification, melting curve analysis was performed by heating to 95 c then cooling to 60 c for 15 s, followed by a temperature increase to 95 c, while continuously collecting the fluorescent signal data. adenovirus, influenza b virus, respiratory syncytial virus (rsv) and human metapneumovirus (hmpv) were detected by the method described by krafft et al., 27 chi et al., 28 samransamruajkit et al. 29 and thanasugarn et al., 30 respectively. all hbov positive samples were subjected to vp1 gene sequencing. the partial vp1 gene at the 5 0 end of hbov was amplified into two segments with two primer sets, vpf1 (5 0 -gataactgacgaggaaatgct-3 0 : nt 3009e3029) and vpr1 (5 0 -agtatgtccatggagttgtga-3 0 : nt 3731e3711) for the first segment and vpf2 and vpr2 for the second. the expected product sizes after 2% agarose electrophoresis were 723 and 648 bp, respectively. pcr conditions have been described elsewhere. 2 the pcr products were purified using the perfectprep gel cleanup kit (eppendorf). dna sequencing was performed using the gene amp pcr system 9600 (perkineelmer, boston, ma). the sequencing products were subjected to a perkin elmer 310 sequencer (perkine elmer) for subsequent sequence analysis. the dna sequences were analyzed with the blast (http://www. ncbi.nlm.gov/blast) program and the phylogenetic analyses and genetic comparisons between hbov strains were performed using the molecular evolutionary genetics analysis (mega) version 3.1 program. we applied pcr and rtepcr to detect hbov, other respiratory viruses and gapdh. of 302 specimens, 20 (6.62%) were positive for hbov. all specimens had been collected throughout the year and plotting of seasonal hbov detection was shown in fig. 1 . among the 302 specimens we also detected co-infection with other respiratory viruses such as rsv in 48 (15.89%) samples, influenza a virus in 33 (10.92%) samples, hmpv in 28 (9.27%) samples, adenovirus in 18 (5.9%) samples, parainfluenza in 14 (4.63%) samples, and influenza b virus in 1 (0.33%) sample. hbov-positive samples co-infected with other respiratory viruses comprised altogether 40% (table 1) . the clinical manifestations of hbov infected patients are summarized in table 1 . the majority of infants positive for hbov were male (14/20) , between 4 and 36 months old (median age 12 months). on average, they were hospitalized for 6.15 days (range 2e22 days). the most common clinical symptom in all hbov infected infants was fever with a mean duration of 3.47 days and other common symptoms such as runny nose (55%) and productive cough (50%) were found. generalized rales were the most common (70%) and wheezing the second most common lung signs (35%). acute bronchiolitis was diagnosed in 9 out of 20, and 11 samples had viral pneumonia. perihilar infiltration was ubiquitously present on the chest x-ray of hbov positive patients (except for cu6 and cu15 where no chest x-ray result was available) (data not shown). additional diseases of some patients with hbov infection included congenital heart disease (cu33 and cu74), chronic lung disease (cu 31 and cu171), asthma (cu71) and cow milk protein sensitive enteropathy (cu157). the nucleotide sequences obtained from this study have been submitted to the genbank database under accession numbers: ef690641eef690657 (table 1 ) except for cu6 (ef203920), cu49 (ef203921) and cu74 (ef203922) that were subjected to analysis for complete coding sequences as described elsewhere. 2 the 5 0 terminal 1182 base pairs (bp) of the vp1 gene were sequenced in all hbov positive samples for phylogenetic analysis. other vp1 sequences from several areas were available at the genbank database and used for phylogenetic analysis including sequences from china: dq778300, ef441262, ef584447, dq457413, dq987595, dq987596, dq677523, dq494203, dq457415, dq988933, dq988934, dq987597, dq987598 and dq494201, japan: ef035488, usa: dq340570, and sweden: dq000495 and nc_007455. the constructed phylogenetic trees of the vp gene (nt 2986e4167) are shown in fig. 2 . alignment of these partial vp1 genes showed minor variations in that the percent identity with the st1 swedish prototype strain ranged from 98.6% to 99.5%. a phylogenetic tree of the hbov vp gene was constructed based on neighbor-joining (nj) trees. confidence values for the tree topologies were evaluated by bootstrap analysis of 1000 pseudo-replicate datasets that showed the low genetic diversity. in the past few years, applying molecular techniques has led to the discovery of hbov as a novel virus apparently associated with respiratory tract illness in humans. 1 however, due to lack of suitable culture systems and animal models to propagate the virus previous studies could not meet with koch's postulates in order to clarify that hbov can cause respiratory illness. it can be concluded from epidemiology data that hbov has been distributed throughout every continent with a different incidence rate (1.5% to 19%). in this study, the np suctions had been collected from pediatric patients hospitalized with acute lower respiratory tract illness. hbov has been detected in 20 (6.62%) out of 302 np suction samples, whereas the previous study in the rural area of thailand had the prevalence of 4.5%. 18 co-infection with other respiratory viruses was found in 40% of the samples tested. rsv and parainfluenza virus were frequently detected as co-infecting viruses. however, some of the respiratory viruses were not included in this study, for examples; rhinoviruses and coronaviruses. therefore, the percentage of co-infection may be higher than this report. thailand has a tropical climate country and seasonal detection of hbov can not be deduced from the few (1e3) hbov-positive samples found in each month. rsv and hmpv were shown the seasonal distribution that peaked in july to september which correlated to the number of samples collection (data not shown). moreover, the study of mannig et al. reported a similarity in seasonal appearance between hbov and rsv 12 whereas weissbrich et al. did not. 16 the clinical features commonly found in hbov positive patients were fever and productive cough. bilateral rales and wheezing were among the most common abnormal lung signs observed and almost equally found in these patients (table 1) . these findings might indicate both lung parenchyma and airway involvement by this pathogen. furthermore, the detection of this virus in specimens aspirated from the nasopharynx together with the significant lower respiratory tract illness indicated the lung pathology in these patients. in conclusion, we detected hbov infection in 6.62% and co-infection with other respiratory viruses in 40% of the np suctions obtained from infants and children with acute lower respiratory tract illness with non-specific clinical features and age distribution. seasonal appearance of hbov was not significant. based on phylogenetic analysis of the vp1 gene, the low genetic diversity was defined. the present study has investigated associations of hbov with clinical manifestations and performed genome analysis but in order to completely describe this novel virus, further studies on serology, tissue and animal culture systems or clinical manifestation inductions will be required. cloning of a human parvovirus by molecular screening of respiratory tract samples complete coding sequence and phylogenetic analysis of human bocavirus (hbov) human bocavirus: prevalence and clinical spectrum at a children's hospital detection of human bocavirus in cannadian children in a 1 year study frequent detection of human rhinoviruses, paramyxoviruses, coronaviruses, and bocavirus during acute respiratory tract infection human bocavirus infection the association of newly identified respiratory viruses with lower respiratory tract infections in korean children bocavirus infection in hospitalized children human bocavirus in french children human bocavirus infection among children detection of human bocavirus in japanese children with lower respiratory tract infection epidermiology profile and clinical associations of human bocavirus and other human parvovirus real-time pcr assays for detection of bocavirus in human specimens evidence of human coronavirus hku1 and human bocavirus in australian children human bocavirus in hospitalized children frequent detection of bocavirus dna in german children with respiratory tract infections human bocavirus and acute wheezing in children human bocavirus: a novel parvovirus epidemiologically associated with pneumonia requiring hospitalization in thailand high prevalence of human bocavirus detected in young children with severe acute lower respiratory tract disease by use of a standard pcr protocol and a novel real-time pcr protocol human bocavirus in italian patients with respiratory diseases human bocavirus in febrile children, the netherlands human bocavirus in iranian children with acute respiratory infections human bocavirus infection, people's republic of china human bocavirus dna detected by quantitative real-time pcr in two children hospitalized for lower respiratory tract infection human bocavirus infection in young children in the united states: molecular epidemiological profile and clinical characteristics of a newly emerging respiratory virus detection of bocavirus dna in nasopharyngeal aspirates of a child with bronchiolitis evaluation of pcr testing of ethanol-fixed nasal swab specimens as an augmented surveillance strategy for influenza virus and adenovirus identification detection and characterization of new influenza b virus variants in 2002 plasma endothelin-1 in infants and young children with acute bronchiolitis and viral pneumonia human metapneumovirus infection in thai children this study was supported by the thailand research fund (senior research scholar), royal golden jubilee ph.d. program, the thailand research fund and the center of excellence in clinical virology, chulalongkorn university. we would like to thank the entire staff of the pulmonary unit, department of pediatrics, faculty of medicine, chulalongkrong university for their assistance in collecting the specimens. we also would like to thank ms petra hirsch for reviewing the manuscript. key: cord-264302-8vo5psgm authors: lu, yue; zhang, leiliang title: social media wechat infers the development trend of covid-19 date: 2020-04-10 journal: j infect doi: 10.1016/j.jinf.2020.03.050 sha: doc_id: 264302 cord_uid: 8vo5psgm nan trol, while the number of cases outside china is on the rise. 3 , 4 the official infectious diseases surveillance system in china is run by the chinese center for disease control and prevention. however, its lag makes it difficult to timely catch the outbreak of epidemics. with the popularity of the internet and smartphones, the focus of social media is often a sign of these major epidemic diseases. 5 information and discussions on covid-19 spread rapidly on social media, so the use of big data allows more people to pay attention to these situations earlier. wechat is the largest social media in china, and the number of monthly active accounts has reached to 1.165 billion. the wechat index is an official wechat mobile index based on the analysis of wechat big data. it reflects the popularity of words in the past 7 days, 30 days, and even 90 days. it is often used to capture current hot events and monitor the trends of public opinion. here, through the keyword query in the wechat index, we analyzed the public attention and demand for the covid-19 pandemic. we have classified keywords from late december of 2019 to the present according to their relevance. these word groups have generated a high degree of popularity in social media at some stages. since these hot spots are in a good correlation with the occurrence and progress of some major events in china, we capture these hot events and follow their tracks. first of all, the hottest words in the pandemic are "wuhan", "novel coronavirus" and "pneumonia" ( fig. 1 a) . their rising and downward trends were similar. the heat of these words began to increase sharply on january 19, 2020. among them, the heat of the word "pneumonia" reached 268,350,505 seven days later, about 35 times that of seven days ago, while the heat of the word "wuhan" reached its peak on january 25. about 16 times what it was six days ago. the heat of "novel coronavirus" remained above 150 m from january 25th to february 16th. thus, during the period from the end of january to the beginning of february, the public attention focused on those words. secondly, we looked at some key figures appeared during the epidemic and regarded as national heroes ( fig. 1 b) . from the comparison chart of wechat index, the heat of these heroes increased on january 20, 2020. zhong nanshan for the first time declared a human-to-human transmission phenomenon in covid-19. at the same time, a large number of medical experts rushed to the front line of wuhan. the "whistler" dr. li wenliang died on february 7, and his index reached an astonishing 179,575,130 on that day ( fig. 1 b) . we also looked at the potential sources and hosts of this outbreak: "bushmeat", "bat", "pangolin" and "masked palm civet" ( fig. 1 c) . those words began to increase on january 20, and the heat lasted until mid-february. during this period, scientists worked hard to find clues about the source of sars-cov-2. in the end, bats had the highest heat because they were identified as the original source of the sars-cov-2. 6 covid-19 s symptoms are public concern, and we found that "fever" is the most concerned one ( fig. 1 d) . studies have shown that covid-19 caused a variety of symptoms, and fever is not a necessary factor for diagnosis. however, because of the understanding of conventional pneumonia symptoms and fever symptoms are more obvious, the public are still concerned about fever. in addition, we paid attention to some daily words related to people's livelihood ( fig. 1 e) . the popularity of the word "mask" began to increase on january 19, peaked on february 6 and 7, and has remained at a high level ever since. we suspect that the highest popularity of masks is due to the fact that most people cannot buy masks, so the online booking system for the purchase of masks has been opened in many places throughout the country. relief supplies from various countries and medical materials purchased domestically are in place in large quantities, providing strong support to front-line medical staff. the words "isolation" and "disinfection" are almost inseparable, so their trends are very consistent. "vaccine" reached a small peak on january 26th, february 12th, february 25th and march 18th, respectively, because both researchers have announced the start of vaccine development or clinical trials of vaccines. we also found that the entries for "starting school" and "resuming work" have been very hot since the beginning of february ( fig. 1 f) . this is probably due to the impact of the epidemic. many enterprises have delayed returning to work, and students who were supposed to go to school have also begun to take classes online. in february, when the epidemic is more serious, the policy of reducing crowds and allowing people to be isolated at home has aroused extensive discussion. by analyzing the hot words in wechat, we found that there is a certain pattern in the development of covid-19. at first, people will focus on which kind of pathogen the disease comes from, where the outbreak is located, what symptoms will appear. then the public will begin to focus on the source of pathogens, the actions and self-protection of medical workers, as well as the needs of daily life. on the other hand, people will pay more attention to the epidemic situation in other places and the local control results. through analysis of public concerns, we review the development trend of covid-19, which will set up an example for future outbreaks of epidemic diseases. it is believed that public health authorities will rely more on these social medias in the future for monitoring the development of the epidemic or pandemic. the authors declare that there are no conflicts of interest. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia emergence of sars-like coronavirus poses new challenge in china the outbreak of coronavirus disease 2019 (covid-19)-an emerging global health threat the continuing 2019-ncov epidemic threat of novel coronaviruses to global health -the latest 2019 novel coronavirus outbreak in wuhan, china retrospective analysis of the possibility of predicting the covid-19 outbreak from internet searches and social media data, china a pneumonia outbreak associated with a new coronavirus of probable bat origin key: cord-274558-1k7bi6ng authors: moiseev, sergey; brovko, michail; tao, ekaterina; bulanov, nikolay; fomin, larisa akulkina victor title: sex differences in mortality in the intensive care unit patients with severe covid-19 date: 2020-09-28 journal: j infect doi: 10.1016/j.jinf.2020.09.031 sha: doc_id: 274558 cord_uid: 1k7bi6ng nan the available evidence suggests that mortality from coronavirus disease 2019 in males is higher than in females. 1 in a recent cross-sectional study, de lusignan et al. evaluated the absolute excess risk (aer) of mortality and excess mortality rate (emr) in the uk from a covid-19 sentinel surveillance network in people aged 45 years and above. 2 the aer in mortality was calculated by comparing mortality for weeks 2 to 20 this year with mortality data from the office for national statistics (ons) from 2018 for the same weeks. the absolute excess mortality was approximately 2 deaths per 100 person years in the first wave of covid-19, whereas the emr for male gender, compared with female, was 1.4 (95% confidence interval [ci] 1.35-1.44, p<0.00) . we investigated the sex-related differences in the occurrence of comorbidities and mortality rates in a nationwide study in 1522 consecutive patients with severe sars-cov-2 pneumonia who were admitted to intensive care unit (icu) for respiratory support. medical records of patients were submitted by the covid-19 hospitals located in 70 regions across russia to the federal center at the sechenov university in the total cohort, the requirement for mechanical ventilation and mortality rates were similar in males and females (table 1) . however, female patients were older and had a higher occurrence of various chronic illnesses, that is, arterial hypertension, obesity and type 2 diabetes, that impair prognosis in patients with covid-19. 4, 5 coronary artery disease (cad) and chronic obstructive pulmonary disease (copd) were more frequent in males than in females, although their occurrence was lower compared to that of the other significant comorbidities. the mortality rates increased with age both in males and females. in patients aged 50 years or younger, the mortality rates were similar in males and females (odds ratio [or] p=0.021), whereas the requirement for mechanical ventilation did not differ between sexes. in all age groups, the occurrence of arterial hypertension, type 2 diabetes and obesity was higher in females than in males, although these differences reached statistical significance only in a proportion of cases (table 1) . on the contrary, cad occurred significantly more frequently in males aged 51-60 and 61-70 years than in females of similar age, whereas the frequency of copd was increased in males aged 61-70 and ≥70 years. in summary, the mortality rate in the icu patients with severe sars-cov-2 pneumonia was higher in males aged >50 years than in females of similar age. our findings are in line with de lusignan et al. data, who reported a higher emr in males during the covid-19 pandemic in the english population. the differences between mortality rates in males and females cannot be explained by comorbidities, given the divergent trends in the occurrence of chronic illnesses that may worsen survival in covid-19 patients. al-lami et al. suggested that low levels of testosterone, as can occur in normally aging men, may account for more severe lung damage since testosterone deficiency has been linked with autoimmune disease and increases in inflammatory markers. 6 moreover, anti-inflammatory effects of estrogens may protect females from progression of sars-cov-2 induced lung disease. our data indirectly support that sex steroid hormones underlie sex-related differences in covid-19 mortality. sergey moiseev, michail brovko, ekaterina tao, nikolay bulanov, larisa akulkina sex differences in severity and mortality from covid-19: are males more vulnerable? disparities in the excess risk of mortality in the first wave of covid-19: cross sectional study of the english sentinel network coronavirus disease 2019 (covid-19): a systematic review of imaging findings in 919 patients coronavirus infections and type 2 diabetes -shared pathways with therapeutic implications covid-19 and obesity: dangerous liaisons sex hormones and novel corona virus infectious disease (covid-19) whitney u test for continuous variables; me(iqr) = median ecmo = extracorporeal membrane oxygenation cad = coronary artery disease (a history of myocardial infarction or interventions on the coronary arteries) af = atrial fibrillation; j obesity was defined as body mass index ≥30 kg/m 2 copd = chronic obstructive pulmonary disease key: cord-019977-kj0eaw6v authors: nan title: neonatal bacterial infection: a changing scene? date: 2005-04-14 journal: j infect doi: 10.1016/s0163-4453(82)91569-9 sha: doc_id: 19977 cord_uid: kj0eaw6v nan the foetus and newborn infant have a particular vulnerability to infection of all kinds, yet most escape this hazard. immune mechanisms are developing at each stage of existence which are appropriate to the likely challenges at such times; 1 the unusual challenge however may not be met, for the immune and inflammatory responses produced by healthy older children and adults cannot be mounted as effectively, particularly if the infant is born pre-term. 2 the risk therefore is ever present, and-at least where neonatal bacterial infection is concerned -its nature may be ever changing. the reasons for such change are sometimes obvious, sometimes obscure, and have to be sought in the maternal and infant hosts, in improved laboratory techniques, and in altering clinical practices which may impose change on host and infecting organism alike. records have been kept over a 5o-year period (i928-i978) at yale of the isolates recovered from the bloodstream of infants in the first weeks of lifef1-6 and accord well with those reported intermittently from other centres. in addition to showing changes in the infecting organisms, they also reveal the growing importance of maternally transmitted intrapartum (early) infections over later infections in recent years, many of the latter being environmentally acquired. in the first quarter of the 5o-year period, two-thirds of the isolates were gram-positive, the majority staphylococci (mostly staphylococcus aureus) and beta-haemolytic streptococci. the introduction of lancefield typing in the early i93os showed these to be mainly group a streptococci. over the next 20 years the position was to be gradually reversed and between i958 and i9655 two-thirds of yale's newborn bacterial isolates were gram-negative. this time period coincided with the entry of paediatricians to newborn nurseries on a much larger scale than hitherto, with an increase in the use of antimicrobial drugs, and with the introduction of apparatus such as incubators, resuscitation and suction units, the humidification parts of which often harboured gramnegative organisms, all capable of causing lethal disease in the infant. the last period (i966-i978) 6 has shown a decrease in gram-negative isolates, and a rise in gram-positive ones, the latter largely due to greatly increased isolation of group b streptococcus. during the half century under review, mortality from neonatal bacteraemia fell from 9o per cent in the period i928-i933, ~ to z6 per cent in i966-i978 ;6 and the proportion of isolates recorded as recovered in the first 48 hours of life (early infections) increased from io per cent to 57 per cent of the total respectively. we can only guess whether this last change was due to a growing awareness on the part of paediatricians of the possibility of maternally transmitted illness, or to a genuine increase of such infection. as the number of blood cultures drawn increased considerably over the years, the former may be more likely. on the other hand an increasing vogue for surgical induction of labour, and the use of pressure transducers for foetal monitoringfl if practised there may have introduced the organism into the amniotic fluid more frequently in recent years. a report from hammersmith hospital 8 suggests a further change may be under way in newborn nurseries, as the io6 editorial predominant isolate from cases of neonatal bacteraemia there since 1976 has been staphylococcus epidermidis. this organism has ousted the group b streptococcus from its prominent place in early infections at the hospital in previous yearsfl and has also displaced gram-negative bacilli as the most important cause of later infection. elsewhere in this issue (p. ii7) oto discusses some of the reasons-the increasing preoccupation of neonatal intensive care units with infants of very low birth weight and the increasing complexity of their care, involving much more intravenous therapy -which may be responsible for this change. twenty years ago the great majority of such infants would have died within 24 hours of birth, and it is likely that intrapartum infection, even as a contributory cause of their death, may have gone unsuspected and undiagnosed. today they live, and their immaturity makes them at risk throughout the many weeks of their hospital stay. oto also records the major infections found in such a unit over a six month period, and bacteraemia is far the commonest. much less often seen, but next in importance, is necrotising enterocolitis. this is a condition which is known to have occurred for over one hundred years, but which reached almost epidemic proportions in certain newborn intensive care units in the late 196os and i97os. 1° bacteria are responsible for the progression and complications of the illness, but whether they have a primary role, or merely invade ischaemic bowel wall which has been damaged by a variety of other factors, is still unsure. the condition has certainly accompanied genuine bowel pathogens (bacterial n and viral12), other cases often occur in clusters, tm may be curtailed by infection control measures, lz and clostridium spp. have occasionally been implicated. 14, 15 some believe it to be part of a spectrum of disease which includes pseudomembranous or antibiotic-associated colitis. 16 clostridium difficile and its toxin may be present in the stools of many well newborn babies, but this organism has not been implicated in the genesis of neonatal necrotising enterocolitis as it has been with pseudomembranous colitis. 17 meningitis is now fortunately rare (o.26/iooo live births in a geographically defined population, excluding that associated with neural tube defects18), but there is still controversy about treatment of the most commonly occurring gram-negative enteric infections, despite controlled trials. omphalitis, the bacteriology of which is described by brook in this issue (p. i27), is now very infrequently seen. many newborn nurseries use some form of antiseptic cord care; and a daily antibiotic spray containing polymyxin, bacitracin and neomycin, prevents bacterial colonisation, 19 and hence infection, at this site in the great majority of babies. improved laboratory techniques for the recognition of organisms such as clamydia trachomatis are leading to a gradually increasing awareness of the extent of its involvement in neonatal conjunctivitis and respiratory illness, s° bacterial infections can be permanently damaging to developing and rapidly growing organs; and constant surveillance of the patterns of infection is nowhere more important than in newborn care. preventive measures such as handwashing and disinfection of apparatus have to be carried out to a uniformly high standard in intensive care units. this is especially important if outbreaks of infection are to be prevented among the immature residents, for the inevitable overuse of antimicrobial therapy in such units leads to a disturbance of bacterial flora which may make the hosts even more unusually vulnerable. the development of the immune response. characterization of the response of the human infant and adult to immunization with salmonella vaccines host defences in the human neonate septicemia in the new-born. am`7 dis child septicemia of the newborn septicemia of the newborn a half century of neonatal sepsis at yale. 1928 to i978 nasal colonization of infants with group b streptococcus associated with intrauterine pressure transducers changing blood culture isolates in a referral neonatal intensive care unit early neonatal bacteraemia. comparison of group b streptococcal, other gram-positive and gram-negative infections neonatal necrotizing enterocolitis. monographs in neonatology. new york: grune and stratton gastroenteritis with necrotizing enterocolitis in premature babies association of coronavirus infection with neonatal necrotizing enterocolitis clustering of necrotizing enterocolitis. interruption by infection-control measures outbreak of necrotising enterocolitis caused by clostridium butyricum fulminant necrotising enterocolitis associated with clostridia pseudomembranous enterocolitis (antibiotic-related colitis) clostridium difficile and the aetiology of pseudomembranous colitis acute bacterial meningitis in childhood. incidence and mortality in a defined population an investigation into the aerobic and anaerobic bacterial flora of normal and ill~low birthweight newborn babies prospective study of chlamydial infection in neonates key: cord-289169-3u7qgxud authors: fang, xiaowei; mei, qing; yang, tianjun; li, lei; wang, yinzhong; tong, fei; geng, shike; pan, aijun title: low-dose corticosteroid therapy does not delay viral clearance in patients with covid-1 date: 2020-04-11 journal: j infect doi: 10.1016/j.jinf.2020.03.039 sha: doc_id: 289169 cord_uid: 3u7qgxud nan drugs and symptomatic therapies. furthermore, patients in the severe group received the necessary supportive treatment. corticosteroids were administrated to a subset of patients according to severity and the individual opinion of clinicians. more severe patients were treated with corticosteroids, leading to inconsistent baseline data (i.e., age, comorbidities and laboratory findings) between patients receiving corticosteroids and those who did not. therefore, the 78 patients were divided into a general and a severe group, and separate data analysis was conducted for each group. table 1 therefore, these two studies involved patients with different severities of illness; however, whether corticosteroids exerted greater effects in critically-ill patients requires further investigation. furthermore, the study reporting on patients with mers included rt-pcr data from 14 intensive care units, and the nucleic acid test results were not protocolized and varied among centers (5) . in the present study, all patients were from a single center, and all swab samples were tested using a unified approach at the chinese center for disease control to avoid measurement bias. in fact, a similar study analyzing data from 72 patients with covid-19 was conducted at the first affiliated hospital of zhejiang university, and the conclusions were consistent with the results of the present study (7) . however, these two retrospective studies have unavoidable limitations, such as small sample size, poor controllability of the data and bias in the process of data collection. in conclusion, low-dose corticosteroid therapy may not delay viral clearance in patients with covid-19; however, this still needs to be confirmed by well-designed and large-scale rcts with a longer follow-up duration. ethics approval was obtained from anhui provincial hospital institutional review board (ethical approval no. 2020-p-008). the authors declare that they have no competing interests. all the authors agree to publish. clinical progression of patients with covid-19 in shanghai clinical features of patients infected with 2019 novel coronavirus in wuhan national health commission of the people's republic of china. the 5th trial version of diagnosis and treatment scheme for pneumonitis with 2019-ncov infection (in chinese) effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients corticosteroid therapy for critically ill patients with the middle east respiratory syndrome adjuvant corticosteroid treatment in adults with influenza a (h7n9) viral pneumonia retrospective study of low-to-moderate dose glucocorticoids on viral clearance in patients with novel coronavirus pneumonia creatinine (µmol/l), median (q1, q3) we thank all medical staff working in the infectious diseases branch of anhui provincial hospital for their essential assistance with case collection. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-286014-cc99e24x authors: jang, t.-n; yeh, d.y; shen, s.-h; huang, c.-h; jiang, j.-s; kao, s.-j title: severe acute respiratory syndrome in taiwan: analysis of epidemiological characteristics in 29 cases date: 2003-11-05 journal: j infect doi: 10.1016/j.jinf.2003.09.004 sha: doc_id: 286014 cord_uid: cc99e24x objectives. to describe the clinical characteristics and outcomes of patients with severe acute respiratory syndrome (sars). methods. between march 28 and june 30 '2003, 29 patients with probable sars seen at shin kong wu ho-su memorial hospital, taipei, were analysed. results. presenting symptoms included fever (100%), cough (69.0%), chills or rigor (62.1%), and shortness of breath (41.4%). mean days to defervescence were 6.8±2.9 days, but fever recurred in 15 patients (51.7%) at 10.9±3.4 days. common laboratory features included lymphopenia (72.4%), thrombocytopenia (34.5%) and elevated c-reactive protein (crp), lactate dehydrogenase (ldh), and aspartate aminotransferase (ast) (93.1, 62.1, 44.8%, respectively). all patients except one had initial abnormal chest radiographs and 20 (69.0%) had radiological worsening at 7.5±2.6 days. nine patients (31.0%) subsequently required mechanical ventilation with four deaths (13.8%). most patients with clinical deterioration responded to pulse corticosteroid therapy (14 out of 17) but six complicated with nosocomial infections. the risk factors associated with severe disease were presence of diarrhoea, high peak ldh and crp, high ast and creatine kinase on admission and high peak values. conclusions. prudent corticosteroid use, vigilant microbiological surveillance and appropriate antibiotics coverage are the key to successful treatment. province, people's republic of china, that has continued since november 2002, was reported to have infected 792 people and caused 31 deaths. 1 this outbreak received no international attention until february 2003, when a nephrologist from guangdong province became ill during a one-day stay on the ninth floor of a hong kong hotel. twelve other guests became infected including 10 staying on the same floor. these hotel guests subsequently became the index patients who transported the disease to vietnam, singapore, canada, ireland, and the united states. 2 the primary mode of transmission of sars appears to be by the airborne spread of large droplets. as the illness has spread, the condition has been particularly prevalent among healthcare workers and their household members. many cases progressed rapidly and often resulted in acute respiratory distress syndrome (ards). 3, 4 the syndrome was designated as 'severe acute respiratory syndrome' (sars) in march 2003. 5, 6 as of july 4, 2003, a total of 8439 cases resulting in 812 deaths (a case-fatality proportion of 9.4%) have been reported from more than 30 countries globally. 7 taiwan has a population of over 23 million. the development of sars became a real concern in taiwan because of the extensive business ties and inter-country travel that exist between taiwan, hong kong, and especially mainland china, which appears to be the origin of sars. the first probable sars patient in taiwan returned from china via hong kong early in the global outbreak in february 2003. 8 for more than a month, the daily incidence cases remained in the single digits until a nosocomial outbreak occurred in a municipal hospital (hospital a) in taipei on april 22, 2003. 9 as of july 4, 674 probable cases and 84 deaths have been reported in taiwan. 7 we analyse the clinical, laboratory, and radiological features of patients with probable sars who were seen at the shin kong wu ho-su memorial hospital (skmh) in taipei, taiwan. in addition, we also report the probable index case for the sars outbreak in taiwan. skmh is an 817-bed teaching hospital in the city of taipei, taiwan. between march 28 and june 30 2003, 82 patients who were suspected of having sars were admitted to the isolation rooms. fiftythree of these patients were subsequently excluded after other explanations for their fever and abnormal chest x-rays were found. the remaining 29 patients fulfilled the world health organization (who) criteria of 'probable sars' infection. 10 routine microbiological tests were performed to exclude other causative pathogens, and reversetranscriptase-polymerase-chain-reaction (rt-pcr) of oropharyngeal swabs were done for sarsassociated coronavirus in each case as described elsewhere. 11 initial treatment included amoxicil-lin/clavulanic acid or ceftriaxone and clarithromycin to target common pathogens causing community-acquired pneumonia, according to current recommendations. 12, 13 oral ribavirin (2.0 g followed by 1000 -1200 mg daily) or intravenous ribavirin (400 mg q8h) for severe cases was also administered. corticosteroid therapy included intravenous hydrocortisone (100 mg four times daily) in mild cases, and methylprednisolone (2 -3 mg/kg/day) in moderate cases. daily pulsed administration of 0.5 -1.0 g of methylprednisolone for two to three days was carried out for those patients with persistent high fever, radiological worsening, increasing shortness of breath, or oxygen desaturation. in severe cases, and depending on the attending physician's judgment, human immunoglobulin could be administered intravenously (1 gm/kg/day for 2 days). we compared risk factors associated with complicated and uncomplicated diseases. the wilcoxon test was used to compare distributions of continuous variables. 14 categorical variables were compared using the chi-square or fisher's exact test, as appropriate. data are reported as meansŝ tandard deviation (sd) unless otherwise specified. all p values were 2-tailed; p , 0:05 was considered statistically significant. for the analysis, the spss software package, version 10.0 (spss inc., chicago, usa) was used for the analysis. the demographic profiles and comorbidities of the 29 cases are shown in table 1 . their median and mean ages were 39 and 42.9 years, respectively, with a range of 22 -82 years. the female-to-male ratio was 2.2:1. six of the 29 patients (20.7%) had comorbidities that included cardiovascular diseases ðn ¼ 4þ; diabetes mellitus ðn ¼ 1þ; and chronic pulmonary disease ðn ¼ 1þ: among the patients, 10 (34.5%) had been to hospitals with known sars outbreak, 6 (20.7%) were healthcare workers, 4 (13.8%) were household contacts, 4 (13.8%) were unknown, 3 (10.3%) had recently traveled to mainland china or hong kong, and 2 (6.9%) had social contact with sars patients. one of these patients is the first native sars case without a contact history in taiwan. she was referred from hospital a on april 9 and may have been the index case of the hospital a outbreak. here we briefly describe her history. a previously healthy 47-year-old housewife was transferred to skmh from hospital a on april 9, 2003 on suspicion of sars. she displayed a persistent fever accompanied by chills and a dry cough for 6 days, and had become dyspnoeic 3 days prior to transfer. she denied any previous hospital visits or recent travel outside of taiwan. the patient recollected a 3-hour train journey to taipei 12 days prior, when she was in close proximity to a female who had a persistent cough. the patient went to the emergency room of hospital a in the morning of april 9, 2003. a chest x-ray showed extensive bilateral lower lung infiltrates ( fig. 1 , panel a). soon after arrival in our hospital, she required 100% oxygen through a non-rebreathing mask to maintain her oxygen saturation. a second chest x-ray taken in the afternoon showed progressive changes ( fig. 1, panel b) . by that evening, the patient developed progressive tachypnea and had to be intubated. the sputum gram-stain obtained after intubation showed many inflammatory cells without visible bacteria (fig. 2) . she was treated with empiric antibiotics, ribavirin and corticosteroids. on the eighth hospital day, her fever subsided, and her oropharyngeal swab came back positive for sars-associated coronavirus by rt-pcr. on the same day, her husband and son were admitted to other hospitals in taipei due to fever and pulmonary infiltrates. both subsequently proved positive for coronavirus by rt-pcr. on april 23, the patient was extubated. the relative frequencies of all reported symptoms at the time of admission are shown in table 2 all patients except one had abnormal chest radiographs on presentation; 12 (41.4%) had unilateral focal involvement, and 16 (55.2%) had either unilateral multifocal or bilateral involvement. the initial radiographic changes were indistinguishable from those associated with other causes of bronchopneumonia. radiological worsening was noted in 20 patients (69.0%) at a mean of 7.5^2.6 days. overall, 12 patients (41.4%) progressed to diffuse ground-glass appearance. sars-associated coronavirus rna was detected in oropharyngeal swabs by rt-pcr in 16 (55.2%) of 29 patients at initial presentation. routine microbiological investigation for known viruses and bacteria was negative in most cases except three patients who had evidence of coinfection with mycoplasma, legionella, and streptococcus pneumoniae, respectively. all patients received empirical antibiotic therapy during the course of their hospitalization. ribavirin was begun at a mean of 5.0^2.4 (range 1 -10) days after onset of illness to all patients. the mean duration of treatment with ribavirin was 9.2^4.0 days (median: 11 days). all patients received oral the sars epidemic started in asia, with the majority of cases occurring in china and the asian-pacific region. 3 prior to the nosocomial outbreak at 'hospital a', most cases of sars in taiwan had been restricted to imported cases from sarsaffected regions. only 23 probable cases were detected in the first month. 15 however, when seven cases of sars were reported among healthcare workers at hospital a on april 22, 2003 , the incidence of sars cases in taiwan increased dramatically. the index patient was a laundry worker who lived in the basement of hospital a and was often in the emergency room chatting with the staff. 9 he noted the onset of fever and diarrhoea on april 12. the source of infection for the index patient remained unclear because hospital a admitted no known sars patients prior to the incident except for our reported case, who was treated in the emergency department of hospital a for a few hours prior to transferring. the possibility of transmission via incidental contact at the emergency room or indirect contact through laundry items cannot be excluded. success in controlling sars relies on early identification of suspect cases, proper isolation, and meticulous infection control measures. 4 recognition of a native case without a prior contact history plays an important role in controlling an outbreak, especially when no local transmission is reported. the important clues in our reported case included rapid progressive chest x-ray changes and lack of identifiable bacterial pathogen from the initial sputum gram-stain. even though the health authority initially excluded the case patient, she was kept in a negative-pressure isolation room, and her family was quarantined. these precautions proved to be prudent, since a positive coronavirus rt-pcr result was obtained one week later. because of our aggressive infection control policy, there was no sars outbreak at our hospital. we found the initial clinical features of our sars patients to be similar to those recently reported by lee et al. in a cohort of 138 sars patients in hong kong, with the exceptions of a higher occurrence of cough in our patient population (69.0% vs. 57.3%), less frequent occurrence of myalgia (37.9% vs. 60.9%), and of headache (31% vs. 55.8%). 4 in addition, we also found that 51.7% of our cases had recurrence of fever in the second week of illness. this is similar to that described by peiris et al. where 64 of 75 patients (85%) exhibited recurrent fevers after initial improvement. 16 in the present study, shortness of breath and diarrhoea typically developed at the end of the first week of illness. this finding is also consistent with the report by peiris et al. 16 common laboratory results on presentation included lymphopenia, thrombocytopenia, and elevated crp, ldh and aspartate aminotransferase. with the exception of crp, these findings have been noted elsewhere. 4, 17, 18 since the crp elevation resulting from most common viral infections is not as pronounced as in sars, crp is a reasonable candidate marker to distinguish sars from other viral infections early in the infectious process. in the present study, the vast majority of cases (96.6%) displayed abnormal chest radiographs on presentation, and a majority of cases (69.0%) had radiological worsening during the first week of hospitalisation (mean onset 7.5 days). a similar trend has been described by peiris et al. with 80% of cases exhibiting radiological worsening at a mean of 7.4 days. 16 since the initial presentation of sars is quite non-specific, early diagnosis largely relies on known history of potential exposure to the infection. however, once the disease develops into an epidemic, contact history becomes unreliable. clinicians must maintain a high index of suspicion since several features of the clinical presentation may be the important clues to differentiate sars from other infectious diseases. the discovery that a novel coronavirus is the probable cause of sars 11,19,20 provides a dramatic example of an emerging coronavirus disease in humans. sars-associated coronavirus fulfills koch's postulates for an infectious microbiological disease. 21 rt-pcr assay is the most rapid method for the laboratory diagnosis of sars-associated coronavirus infection. according to a recent cohort study in hong kong, viral rna detection in the nasopharyngeal aspirate has a sensitivity of only 32% at presentation, but testing of multiple nasopharyngeal and faecal samples increased the sensitivity of the rt-pcr assay. 16 in our study, sars-associated coronavirus rna was detected in oropharyngeal swabs by rt-pcr in 16 (55.1%) of 29 patients at initial presentation. the percentage of patients in our cohort who required mechanical ventilation (31%) is much higher than the 13.8% reported by lee et al. in a cohort of 138 sars patients in hong kong. 4 the different clinical courses may be related to different strains of coronavirus infection. 22 recent reports of intubation rates of 25% (19 of 75 patients in hong kong), 16 and 30% (six of 20 patients in singapore) 23 are more similar to our cases. our mortality rate was 13.8% (4/29) overall and 44.4% in complicated cases. these figures are much higher than those reported from canada (5.6%), 24 but comparable to a recent report from singapore with an overall mortality rate of 13.6% (27 of 199 patients) and 52.5% (24 of 46 patients) in complicated cases. 25 the low canadian mortality rate may be partly due to short-term outcomes, which did not reflect the real mortality after long-term follow-up. at this time, no effective treatment is known for this infection. empirical treatment with a combination of high-dose corticosteroid and ribavirin has been advocated in certain areas. 18, 26, 27 the rationale behind this approach is to reduce both the viral load and the inflammatory response generated by the infection. all of our patients received ribavirin without obvious therapeutic response, and 48.3% of the patients became anaemic. this finding is consistent with booth et al. who described that 49% of their patients experienced a decrease in the haemoglobin level of 2 g/dl or greater after ribavirin use. 24 large-dose corticosteroid became the de facto mainstay of our treatment protocol. most of our patients with clinical deterioration responded to pulse corticosteroid therapy (14 out of 17), including four out of five patients with repeated episodes of deterioration who were successfully rescued by two or more courses of pulse steroid. recently, a report from guangzhou by zhao et al. also supported this approach. 28 however, the use of large-dose corticosteroid is associated with the high attendant risk of infection. six patients developed several episodes of nosocomial infections in our cohort. we emphasize that appropriate antibiotics coverage and careful microbiological surveillance with judicious corticosteroid use are the key to successful treatment of these patients. univariate analyses showed that the presence of diarrhoea, high peak ldh and crp values, high aspartate aminotransferase and ck on admission and high peak values were associated with adverse outcomes. rigorous multivariate analysis could not be meaningfully performed, however, given the small sample size in our series. age and comorbidities were also not associated with an adverse outcome in our series as observed elsewhere. 4, 20, 24, 29 this may also reflect our sample size. further reports involving larger patient populations will be necessary to clarify our understanding of the risk factors and optimal treatment of sars. world health organization, severe acute respiratory syndrome (sars) update: outbreak of severe acute respiratory syndrome-worldwide outbreak of severe acute respiratory syndrome-worldwide a major outbreak of severe acute respiratory syndrome in hong kong severe acute respiratory syndrome (sars)-multi-country outbreak outbreak of severe acute respiratory syndrome-worldwide world health organization. cumulative number of reported probable cases of sars control measures for severe acute respiratory syndrome (sars) in taiwan severe acute respiratory syndrome-taiwan world health organization. case definitions for surveillance of severe acute respiratory syndrome (sars) a novel coronavirus associated with severe acute respiratory syndrome practice guidelines for the management of community-acquired pneumonia in adults guidelines for the management of adults with community-acquired pneumonia: diagnosis, assessment of severity, antimicrobial therapy, and prevention selected nonparametric techniques update 96-taiwan, china: sars transmission interrupted in last outbreak area clinical progression and viral load in a community outbreak of coronavirusassociated sars pneumonia: a prospective study identification of severe acute respiratory syndrome in canada a cluster of cases of severe acute respiratory syndrome in hong kong identification of a novel coronavirus in patients with severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome koch's postulates fulfilled for sars virus coronavirus genomic-sequence variations and the epidemiology of the severe acute respiratory syndrome severe acute respiratory syndrome in singapore: clinical features of index patient and initial contacts clinical features and short-term outcomes of 144 patients with sars in the greater toronto area acute respiratory distress syndrome in critically ill patients with severe acute respiratory syndrome development of a standard treatment protocol for severe acute respiratory syndrome the use of corticosteroids in sars description and clinical treatment of an early outbreak of severe acute respiratory syndrome (sars) in guangzhou, pr china short term outcome and risk factors for adverse clinical outcomes in adults with severe acute respiratory syndrome (sars) we thank the entire staff who worked on the sars ward of shin kong wu ho-su memorial hospital. key: cord-280544-1rhu478r authors: korte, wolfgang; buljan, marija; rösslein, matthias; wick, peter; golubov, valentina; jentsch, jana; reut, michael; peier, karen; nohynek, brigitte; fischer, aldo; stolz, raphael; cettuzzi, michele; nolte, oliver title: sars-cov-2 igg and iga antibody response is gender dependent; and igg antibodies rapidly decline early on date: 2020-08-25 journal: j infect doi: 10.1016/j.jinf.2020.08.032 sha: doc_id: 280544 cord_uid: 1rhu478r nan antibodies rapidly decline early on 1, 3 wolfgang korte*, 2,3 marija buljan, 2,3 matthias rösslein, 2,3 peter wick, 1 valentina golubov, 1 jana jentsch, 1 michael reut, 3, 4 karen peier, 3 brigitte nohynek, 3 aldo fischer, 3 raphael stolz, 3 this cohort study included patients with a history of a positive sars-cov-2 pcr test. the study is registered in the covid-19 database (https://swissethics.ch/covid-19/approved-projects) and approved by the regional ethics committee (id2020-00941). potential participants were identified in the public health database and voluntary participation was based on the informed consent and documented positive sars-cov-2 pcr. after inclusion in the study, antibody tests were performed every week in the first month and then after another four weeks in the second month. quantitative (optical index, oi) antibody measurements were performed using commercially available 3,4 elisa assays (anti-sp igg and iga, euroimmun, lübeck, germany; anti-nc igg, epitope diagnostics, san diego, usa) according to the recommendations of the manufacturers. data were evaluated and visualized with the 3 statistical software r using the implemented statistical tests and the packages "tidyverse" and "ggplot2". results of the antibody course in 159 participants (52·2% females, 47·8% males), effectively spanning the time frame of two to ten weeks after a positive sars-cov-2 pcr test, are provided. upon the first blood sampling (corresponding to the median of 5 weeks after the pcr test (95% ci 5-6 weeks)), 4·6%, 4·6% and 6·5% of participants have not developed measurable anti-sp igg, anti-sp iga or anti-nc igg, respectively. this may suggest a delayed or missing primary humoral response in a sizeable proportion of patients (at a time when any igm response is believed to have worn off 2 ). we speculate this to be secondary to a suspected virus' ability to modify or suppress innate immune responses 5 . after a significant increase, we find the antibody response to peak 4-5 weeks after positive pcr, followed by an early decline. the decline is statistically significant for anti-sp and anti-nc igg at weeks 8-10 ( figure 1) ; this is remarkable, as a continued igg response for more than 34 weeks was seen with the sars-cov(-1) outbreak 6 . moreover, significantly higher antibody concentrations are seen in men for all antibodies ( figure 2 ). in addition, anti-sp iga antibody concentrations showed a striking dichotomy in the distribution of their values among the patients (figures 1c and 2c) . a subgroup of individuals with extremely high values had a significantly higher fraction of men than the rest of the cohort (77% of samples with od > 20, p < 0·01). this observation might help to explain the higher mortality risk in men with covid compared to women 7 , e.g. through an 4 increased inflammatory response 8 . we speculate that the overall course of anti-sp iga (with no further decline despite igg declining, figure 1c ) as well as the sex specific differences with an early, pronounced peak in men and a subpopulation of men with significantly higher iga titers than the remainder (figure 2c) may be the result of an ongoing infection, which needs further attention and clarification (previous work has shown that iga has a protective role against influenza a 9 ). whether this represents a spike-"antibody dependent infection enhancement" in covid-19, as suggested for sars-cov(-1) 10 , remains to be elucidated. figure 2c ). samples from predominantly male (77% of samples with oi > 20, p < 0·01, fisher's exact test) patients with very high, dichotomically separated iga antibody values were seen (inset 2c, see also 1c). dotted lines separate increased (reactive) from non-increased antibody concentrations. the role of serology for covid-19 control population, kinetics and test performance do matter profile of igg and igm antibodies against severe acute respiratory syndrome coronavirus 2 (sars-cov-2) evaluation of the euroimmun elisa assay for detection of iga and igg antibodies performance characteristics of four high-throughput immunoassays for detection of igg antibodies against sars-cov-2 innate immune evasion by human respiratory rna viruses iga antibodies against the severe acute respiratory syndrome (sars) coronavirus nucleocapsid protein in patients with pneumonia due to the sars coronavirus risk factors for mortality in patients with coronavirus disease 2019 (covid-19) infection: a systematic review and meta-analysis of observational studies clinicolaboratory study of 25 fatal cases of covid-19 in wuhan nasal iga provides protection against human influenza challenge in volunteers with low serum influenza antibody titre anti-severe acute respiratory syndrome coronavirus spike antibodies trigger infection of human immune cells via a ph-and cysteine protease-independent fcgammar pathway key: cord-254512-dct045kl authors: chen, libin; cai, juncheng; lin, qiuyan; xiang, bin; ren, tao title: imported covid-19 cases pose new challenges for china date: 2020-04-10 journal: j infect doi: 10.1016/j.jinf.2020.03.048 sha: doc_id: 254512 cord_uid: dct045kl nan libin chen a,b,c,d , juncheng cai a,b,c,d , qiuyan lin a,b,c,d , bin xiang a,b,c,d* ,and tao that translates to a mortality rate of 9.51%, which is more than twice as high as that for china-the initial epicenter of the outbreak-two stages of the epidemic have passed ( figure 1a ). the first stage is the outbreak period (december 31, 2019 to february 29, 2020), which entailed the period from the first detection of cases to the peak of the epidemic which saw a rapid increase in the number of confirmed cases, and to the time when the growth rate slowed down to less than 200 new confirmed cases per day. in the second stage, which lasted from march 1, 2020 to march 21, 2020, the number of existing cases in most chinese provinces was reduced to less than 10, respectively, whilst the number of newly confirmed cases in wuhan, hubei province, the worst-hit city, was slowly approaching zero. it was during this stage-more specifically on the march 4-that foreign imported cases to appear. during these two stages, the chinese government, its populous, and its medical professionals had managed to stabilized the deadly epidemic with great deliberation and sacrifices 6 . currently, however, the situation in china has entered its third was not capable of efficient screening for every arrival, resulting in a large number of passenger flow jams in the airport lobby, and subsequently causing high risk of infection. therefore, the conundrum regarding the control over overseas imported cases as well as the prevention of a second epidemic outbreak that is fast approaching is a problem that china needs to pay special attention to, especially after the first second-generation case imported from abroad had appeared. it shows that the current prevention and control measures have yet proven to be consummate. as the global epidemic continues in the outbreak period, more and more overseas chinese citizens or ethnic chinese, even non-chinese, will choose to come to china to escape from the epidemic 7 . the government has shortened the processing time for immigration procedures and required all arrivals from other countries to be quarantined. however, the chinese government needs to ensure that every overseas arrival would have passed a quarantine period of at least 14 days, and tested negative to covid-19 before they can conduct social activities in the country, and such measures are not easy to implement. china has managed to control the outbreak of the domestic epidemic, but there are still new issues waiting for them to deal with. in addition, china's outstanding performance in the first stage of the epidemic has provided the world with valuable insights and earned two months of breathing room for the world to respond the novel virus. however, due to the lack of attention and the spread of misinformation regarding covid-19 in many countries, the disease has seriously threatened the whole world 4 , bringing china's epidemic to the third stage. therefore, based on the one health model 8 , the outbreak of covid-19 should concern all humankind, for no country can survive alone. the real victory over covid-19 will require concerted efforts from around the globe. none. emergence of a novel coronavirus causing respiratory illness from wuhan a novel coronavirus from patients with pneumonia in china trend and forecasting of the covid-19 outbreak in china covid-19 and italy: what next? case-fatality rate and characteristics of patients dying in relation to covid-19 in italy chinese medical personnel against the 2019-ncov mental health care for international chinese students affected by the covid-19 outbreak. the lancet covid-19: epidemiology, evolution, and cross-disciplinary perspectives three stages of china's covid-19 epidemic the first and second stages of china's covid-19 epidemic. (b) the third stage of china's covid-19 epidemic. data for all cases are from world health organization key: cord-261240-osbk041e authors: bermejo-martin, jesús f; almansa, raquel; menéndez, rosario; mendez, raúl; kelvin, david j; torres, antoni title: lymphopenic community acquired pneumonia as signature of severe covid-19 infection date: 2020-03-05 journal: j infect doi: 10.1016/j.jinf.2020.02.029 sha: doc_id: 261240 cord_uid: osbk041e nan as recently commented by tang and colleagues in this journal, the novel coronavirus emerged at the end of 2019 from wuhan has already spread beyond china [1] , which led who to declare a public health emergency of international concern by jan 30, 2020. infection caused by this virus (covid-19) courses with severe community acquired pneumonia (cap) in a minority of the cases, but given its transmission characteristics, this is a further cause of alarm. in two recent articles from huang c et al. and yang x in the lancet [2 , 3] , 85% of critically ill patients with covid-19 showed lymphopenia. the presence of lymphopenia as a signature of severe covid-19 was confirmed by wang d et al., who, in their study published in jama, reported that icu patients suffering this infection had a median lymphocyte count of 800 cells/mm [3] , with non survivors exhibiting persistent lymphopenia [4] . icu patients present also with high levels of plasma cytokines [2] . the existence of hyper-cytokinemia in covid-19 patients with lymphopenia could indicate a poor control of the pathogen, as showed in severe patients infected with the 2009 pandemic influenza virus. interestingly, hypercytokinemia and lymphopenia were also evident in critical patients with severe acute respiratory syndrome due to the coronavirus emerged in 2003 (sars-cov) [5 , 6] . these features (lymphopenia + hypercytokinemia) fit the characteristics of a particular immunological phenotype of community acquired pneumonia (cap), lymphopenic cap (l-cap), which, as we recently demonstrated in an article published in the journal of infection, is associated with increased severity, mortality and a dysregulated immunological response [7] . in their works, yang x et al. and chen n et al. propose a direct cytotoxic action of the virus to explain the low lymphocyte counts observed in the severe cases of covid-19 [3 , 8] . but, in our opinion, host factors could also contribute to induce lymphopenia in these cases. compared with those patients not requiring intensive care, covid-19 patients admitted to the icu are older and are more likely to have hypertension, diabetes, cardiovascular and cerebrovascular disease [4] . aging and chronic diseases induce chronic endothelial dysfunction. as we recently reviewed in j clin med, endothelial dysfunction induces disassembly of intercellular junctions, endothelial cell death and blood-tissue barrier disruption, along with enhanced leukocyte adhesion and extravasation, which could contribute to explain the lymphopenia observed in severe covid-19 patients [9] . recent findings from our group have evidenced the interconnection between lymphopenia and endothelial dysfunction in patients with cap and organ failure [10] . endothelial dysfunction induces also increased oxidative stress and systemic inflammation, glycocalyx degradation and shedding along with a pro-coagulant and anti-fibrinolytic state [9] . in aged individuals with chronic diseases, these features could represent predisposing factors for presenting a severe respiratory failure following covid-19 infection. in the huang et al. report, 32% of the hospitalised patients required admission to the icu [2] , and 26% in the study of wang d et al. [4] co-circulation of the novel coronavirus in china is coincident with the winter season, a period of high demand for icu resources due to influenza and other respiratory infections. finding new biomarkers that can be used at the earliest stages of hospitalization to identify those individuals with covid-19 who will become critically ill will be important for efficient management of icu resources. lymphocyte count can easily be obtained at admission to the emergency room. in areas with sustained circulation of the new coronavirus, evaluation of lymphocyte counts in patients with cap could help to identify and prioritize those individuals who require or will shortly require critical care. in conclusion, there is growing evidence suggesting that severe infection caused by the novel coronavirus emerged in 2019 induces l -cap. the presence of l -cap suggests the existence of immunological dysregulation as an accompanying event of the critical illness caused by this virus. early recognition of this immunological phenotype could be useful to assist prompt recognition of severe patients. should lymphopenia play a role in the pathogenesis of the disease, drugs targeting lymphocyte proliferation or apoptosis (il-7, pd1/pd-l1 inhibitors) could help to prevent lymphopenia or restore lymphocyte counts in severe patients suffering covid-19. the potential role of endothelial dysfunction as predisposing and pathogenic actor in this disease merits investigation. biomarkers / tests assessing endothelial function could also help to early identify severe cases of covid-19. drugs improving endothelial dysfunction such adrecizumab could play a role in its treatment. preclinical works on animal models should contribute to elucidate the true role of lymphopenia and endothelial dysfunction in this disease. emergence of a novel coronavirus causing respiratory illness from wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study. the lancet clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china interferon-mediated immunopathological events are associated with atypical innate and adaptive immune responses in patients with severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong lymphopenic community-acquired pneumonia is associated with a dysregulated immune response and increased severity and mortality epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study shared features of endothelial dysfunction between sepsis and its preceding risk factors (aging and chronic disease) simultaneous depression of immunological synapse and endothelial injury is associated with organ dysfunction in community-acquired pneumonia letter to the editor / journal of infection 80 (2020) e23-e24 not applicable key: cord-283758-0z3jwwby authors: rokadiya, s.; gil, e.; stubbs, c.; bell, d.; herbert, r. title: covid-19: outcomes of patients with confirmed covid-19 re-admitted to hospital. date: 2020-07-08 journal: j infect doi: 10.1016/j.jinf.2020.07.007 sha: doc_id: 283758 cord_uid: 0z3jwwby nan dear editor, ye et al, in this journal, reported the characteristics of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) reactivation 1 . we aimed to investigate clinical outcomes of patients with confirmed covid-19 who were readmitted to hospital, in order to identify risk factors for patients discharged and subsequent management of covid-19 in clinical practice. clinical outcomes from hospitalised patients with covid-19 are poor, with a reported mortality of 21% 2 . age, sex, comorbidities, ethnicity and deprivation have all been shown to correlate with worse outcomes in patients with covid-19, however the outcomes of hospitalised patients once discharged remains unknown [3] [4] [5] . the north middlesex university hospital (nmuh), a 459 bed hospital on the outskirts of london, serving a population of over 350,000, was identified early as the second most covid-pressured trust in the uk 6 . all patients with covid-19, once discharged, were referred to the home referral team (hrt), a team of clinicians specialising in general practice who would follow up patients with a phone call either daily or every three days depending on covid severity, unless discharged to a care home or another treatment centre. demographic, clinical, biochemical and radiological metadata were retrospectively collected and interrogated for all medical patients with confirmed sars-cov-2 infection between 09/03/2020 and 30/04/2020. nmuh performed 2275 sars-cov-2 pcr tests on 1911 patients, 729 of whom (38%) tested positive, 391 of whom were discharged. of these, 39 patients re-presented to the hospital emergency department, with 25 then requiring re-admission. readmitted patients were further stratified into those with dyspnoea as their primary complaint. chest radiographs were re-reviewed and blindly scored by an experienced radiologist using the british society of thoracic imaging criteria 7 . all re-admitted patients had been appropriately referred to the hrt or transferred to a care provider. the median age of the re-admission group was higher at 73 years (iqr 58 -82) compared to discharged patients of (429/729) in all covid-19 patients and ethnicity was predominantly of black, asian or minority ethnic (bame) background in both groups (absolute 62.5% discharged vs 57.1% re-admission) [ table 1 ]. biomarker results were similar in both groups. of those re-admitted; two patients required intensive care management, three patients died, and three further patients were discharged home as part of their end of life care. in the re-admission group, the average time before being re-admitted to hospital was 10 days (iqr 6 -15), with dyspnoea the presenting complaint in 13/25 (52%). median oxygen saturations (spo2) on re-admission were low at 90.5% (iqr 80.75 -95) and even lower in the dyspnoea sub-group at 82% (iqr 77.0 -89.25%) despite having adequate spo2 on discharge (median 94%). biomarker differences between the two groups showed a higher median crp of 76 in the dyspnoeic cohort (iqr 6.65 -197.5) compared to the total readmission group of 45 (iqr 5.5 -92). 50% of chest radiographs were reported as covid on re-admission, rising to 57.1% in the dyspnoeic cohort. 6/13 (46%) patients in the dyspnoeic cohort underwent computed tomography (ct) of the thorax, one of whom was diagnosed with pulmonary emboli. as far as we are aware, this is the first study looking at clinical outcomes for patients with covid-19 who were readmitted to hospital. the emergency re-presentation rate within 30 days appears low in the context of national emergency re-presentation averages (9.9% vs 14.0%) 8 and whilst this should be interpreted with caution within a pandemic, this may reflect the value of a remote follow up team. however, the high mortality rate (24%), and the median and prevalence of low spo2 results in patients re-admitted is concerning and warrants further studies to evaluate reasons for re-admission, ensuring appropriate safety-netting when discharged. our data suggests patients with covid-19 who are re-admitted may be at increased risk of hypoxia and death. based on our data on the average time before re-presentation at 10 days, enhanced, personalised follow up at 7 days in a formalised covid-19 clinic with radiological imaging and oxygen saturation recording using pulse oximetry probes may help early identification of those at risk of deterioration, thus preventing re-admission. our study has a number of limitations including a moderate sample size, no stratification of covid-19 severity and a higher than average proportion of patients from bame backgrounds, reflecting our local demographic. this study was strengthened with the use of local data from a highly pressurised region of the epicentre of the covid-19 epidemic in the united kingdom. further studies, using large datasets with detailed clinical data are urgently needed to investigate both the drivers of and causes for the risk of re-admission, hypoxia and death in re-admissions from covid-19. clinical characteristics of severe acute respiratory syndrome coronavirus 2 reactivation presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area clinical course and risk factors for mortality of adult inpatients with covid-19 in china: a retrospective cohort study comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis asian and minority ethnic groups in england are at increased risk of death from covid-19: indirect standardisation of nhs mortality data covid-19 bsti reporting templates | the british society of thoracic imaging we would like to acknowledge all staff, from healthcare providers to support staff on the frontline at north middlesex hospital for their dedication and perseverance during the covid-19 epidemic. sakib rokadiya: none this study received no external funding. clinical care and manuscript preparation were performed within the scope of routine employment. key: cord-291750-4s93wniq authors: lv, boyan; li, zhongyan; chen, yajuan; long, cheng; fu, xinmiao title: global covid-19 fatality analysis reveals hubei-like countries potentially with severe outbreaks date: 2020-04-14 journal: j infect doi: 10.1016/j.jinf.2020.03.029 sha: doc_id: 291750 cord_uid: 4s93wniq • cfr in iran in the early stage of the outbreak is highest among all the countries. • cfrs in the usa and italy are similar to hubei province in the early stage. • cfrs in south korea are similar to outside hubei, indicating less severity. • our findings highlight the severity of outbreaks globally, particular in the usa. the outbreak of 2019 novel coronavirus diseases (covid-19) is ongoing in china, 1 but appears to reach late stage and also just starts to devastate other countries. 2 as of 13 march 2020, there have been 80991 confirmed covid-19 cases and 3180 deaths in china, much higher than those outside china with 51767 confirmed cases and 1775 deaths. 3 however, the daily increase in covid-19 cases outside china has greatly surpassed that inside china 3 (over 70 0 0 verse 11 on 13 march), and therefore people raise deep concerns about the outbreaks outside china. here we attempted to uncover their characteristics by comparative analysis on crude fatality ratios (cfrs). we collected data of the officially released cumulative numbers of confirmed cases and deaths (from 23 january to 13 march 2020) with respect to mainland china, epicenter of the outbreak (i.e., hubei province and wuhan city), outside hubei (in china) and outside wuhan (in hubei), as well as to typical countries reported with a substantial number of deaths including south korea, japan, iran, italy, usa, france and spain ( fig. 1 ) . cfrs in hubei and wuhan are significantly higher than those outside hubei and outside wuhan, and they are relatively higher in the early stage of outbreaks than in the late stage ( fig. 1 a) , in line with earlier comprehensive reports by china cdc and who. 4 , 5 the outbreaks outside china are overall lagging approximately one month behind china ( fig. 1 b vs fig. 1 a) . cfr in iran in the early stage (from 21 february to late march) is extremely high while cfr in korea is low and stable over time. notably, cfr in iran has significantly decreased since 2 march while cfr in italy increased a lot in the past 10 days. cfrs in a period of 10-day, i.e., from 23 march to 1 february for china and other specific periods for countries outside china (for detail, refer to s1.xls file), were plotted as mean ±sd at 95% confidence intervals (in the black box), with median being shown as short lines. statistics were performed using spss with anova algorithm, and significance levels ( p value) for all the pairs are shown in table s1 . p values larger than 0.05 between wuhan/hubei and other countries are colored in red, indicating no significant difference (i.e., somehow being similar to each other) and the relative severity of the epidemic therein; p value between outside hubei and south korea is 0.55 (colored in blue), indicating relatively mild or controllable epidemic in south korea. next, we performed comparative statistical analysis on cfrs in a period of 10 days in the early stage of outbreaks between outside china and china. in particular, two periods were set for iran and italy in order to fully cover their changing trends (for detail, refer to s1.xls file). results displayed in fig. 1 c revealed i) cfrs in iran, italy and usa in the past ten days are not significantly different from hubei ( p being 0.24, 0.648 and 0.281, respectively); ii) cfr in usa is not significantly different from wuhan to marginal degree ( p being 0.0504); iii) cfr in iran from 22 february to 2 march is significantly different from any regions of china (p < 0.001; table s1 ). in view of the detailed p values among all pairs (table s1 ), we suppose the ranking for the severity of covid-19 outbreaks in different countries/regions in terms of cfrs as follows: iran > wuhan > hubei ≈usa ≈italy > outside wuhan ≈spain ≈japan ≈france > south korea ≈outside hubei. as cfr is defined as the number of deaths (numerator) among the number of confirmed cases (denominator), both increase of numerator and decrease of denominator lead to higher cfr. in hubei/wuhan there were neither sufficient covid-19 test kits for infection identification nor enough beds in hospitals for effective treatments of patients in the early stage of the outbreak. 6 these shortages led to numerous transmissions in households, reduced the apparent number of cumulative confirmed cases and caused mild patients without treatments to become severe/critical ones and even die, as implicated by earlier reports. 4 , 7 as such, cfrs in hubei/wuhan was relatively high in the early stage. 5 , 7 similar cfrs between hubei and usa/italy, suggest that these countries may face similar situations at present as hubei had experienced before. in support of this, recent news reports show that italy is extremely short of medica resources (beds and acute care equipment) while usa has some problems in covid-19 testing capacity. 8 in iran, these problems might be even more severe such that its cfr is extremely high. to fight against the covdi-19 outbreaks in these hubei/wuhan-like countries, governments may need to implement control measures and timely supply medical resources as hubei/wuhan had done in the past month. 2 , 4 emergence of a novel coronavirus causing respiratory illness from wuhan who: coronavirus disease (covid-19) outbreak . available from who: coronavirus disease (covid-2019) situation reports report of the who-china joint mission on coronavirus disease the novel coronavirus pneumonia emergency response epidemiology team. vital surveillances: the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19)-china corona virus disease 2019, a growing threat to children potential association between covid-19 mortality and healthcare resource availability cnn: the us is starting to look like italy on coronavirus lockdown this work is support by the national natural science foundation of china (no. 31972918 and 31770830 to xf). authors declare no conflict of interests. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2020.03.029 . key: cord-291181-u2t20mgi authors: chin, ken lee; ofori-asenso, richard; jordan, kaylee a.; jones, daryl; liew, danny title: early signs that covid-19 is being contained in australia date: 2020-05-01 journal: j infect doi: 10.1016/j.jinf.2020.04.042 sha: doc_id: 291181 cord_uid: u2t20mgi • the covid-19 pandemic is overwhelming many national healthcare networks. • case fatality from covid-19 infection in australia is between 0.4% to 3.0%. • strict public health measures were enforced to control this outbreak in australia. • australia is on its way joining china and south korea in ‘flattening the curve’. the covid-19 pandemic is overwhelming many national healthcare networks and crippling many economies. as of 24 april 2020 (11:08am gmt), a total of 2,721,354 confirmed cases, 191,231 deaths and 745,605 recovered cases have been recorded in over 150 countries. 1 to date, the reported case-fatality varies from 2.3% (in china) to 7.2% (in italy), with substantially higher fatality among older populations, and those with co-morbidities. 2 australia has a population of approximately 26 million, of whom 15.4% are aged 65 years and above. 3 the first case of covid-19 infection was reported on 26 january 2020. as of 24 april 2020, there were 6,675 known cases, and 78 deaths. both the federal and state/territory governments have enforced strict public health measures to control this outbreak. 4 in the present study, we report on the epidemiology of the covid-19 outbreak in australia observed thus far, as well as the predicted future numbers of cases, deaths and icu admissions, and associated icu costs. in 2018-2019, total icu capacity across public and private hospitals in australia comprised 2,229 beds, and the mean cost of each icu bed-day was aud $5040. 5, 6 we forecasted the number of beds required for covid-19 patients over time and its associated costs by applying the following conditions: (i) allocation of 10%, 30% and 50% of icu beds for covid-19; (ii) 3% (as currently observed in australia), 5% (china) and 12% (italy) of confirmed cases requiring intensive care 7 ; (iii) mean icu stay between 7 and 14 days; and (iv) mean hospital stay prior to intensive care between 7 and 14 days. 8 evaluation of temporal changes in trend and dynamic time series forecasting were performed by box-jenkins autoregressive integrated moving average and regression models. predicted models were validated by review of mean absolute percentage errors and r-squared values. we estimated the mortality rate by dividing the number of deaths on a given day by the number of patients with confirmed covid-19 infection (i) on the same day and (ii) seven days before, given that patients who die on any given day were infected much earlier. on this basis, case fatality from covid-19 infection in australia is presently between 0.4% to 3.0%. australia all reporting less new cases in the three to five days prior (supplement figures a and b) . hence the rate of rise in incidence has slowed, mindful of the limitations of applying trends to short periods of time. based on extrapolation of trends prior to 29 march 2020, the australian healthcare system would have been over-run by over 12,000 confirmed cases by 12 april 2020 (supplement figure c) . furthermore, icu capacity would have been exceeded by mid to end april 2020 (supplement figure d) . the associated icu costs would have amounted to between aud $8.82 million and $34.38 million if capped at the current maximum number of icu beds. notably, australia has successfully averted these outcomes through timely implementation and strict enforcement of bans on travel and social gatherings, as well as concerted diagnostic and management strategies. the results of our analysis suggest that australia is on its way joining china and south korea in 'flattening the curve'. klc designed the study and performed the analysis. all authors contributed to manuscript preparation and revision for intellectual content. all authors approved final manuscript version prior to submission. none coronavirus covid 19 global cases case-fatality rate and characteristics of patients dying in relation to covid-19 in italy population by age and sex, australia, states and territories australian department of health. coronavirus (covid-19) health alert national pricing model: technical specifications 2019-20 australian and new zealand intensive care society. centre for outcome and resource evaluation critical care utilization for the covid-19 outbreak in lombardy, italy: early experience and forecast during an emergency response jama clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study figure 1. public health measures undertaken to contain covid-19 in australia none key: cord-290066-umthoftd authors: jia, xingwang; xiao, liehui; liu, yajie title: false negative rt-pcr and false positive antibody tests ——concern and solutions in the diagnosis of covid-19 date: 2020-10-08 journal: j infect doi: 10.1016/j.jinf.2020.10.007 sha: doc_id: 290066 cord_uid: umthoftd nan sir, we read with interest that antibody testing using a rapid immunochromatographic assay is reliable in the diagnosis of severe acute respiratory syndrome coronavirus 2 ( sars-cov-2 ) infection 1 . however, the accuracy of antibody testing and rt-pcr does not meet the need for a large number of screening tests. false negative rt-pcr and false positive antibody tests are a concern. coronavirus disease 2019 (covid-19), which is caused by sars-cov-2, was first detected at the end of 2019, and was named by the world health organization on january 12, 2020. covid-19 is now a pandemic. it took only 25 days for newly confirmed cases to decrease to zero in beijing in june, which revealed that timely discovery, accurate diagnosis, early isolation and treatment of covid-19 are the most effective measures. positive antibody results could be eliminated after five times dilution with normal human serum, when the rf level was lower than 10 iu/ml. it was not eliminated after five times dilution with physiological saline [fig. 1 ]. we also identified five patients with false antibody results, who had nasopharyngeal carcinoma, colon cancer, duodenal carcinoma, diabetes, and diffuse bronchitis, respectively. serum rf level in these patients was lower than 100 iu/ml. the false positive antibody results could also be eliminated after 5 times dilution with normal human serum. thus, further studies are needed to investigate the false results of this test. 4 we believe that no diagnostic technique has 100% sensitivity and specificity. although the rt-pcr test has become the standard method for the diagnosis of sars-cov-2 infection, false-negative rates have been reported. for the serological antibody test, the detection time needs to consider the window period. moreover, several factors should be considered when diagnosing covid-19, including epidemiology, history of exposure and clinical symptoms, such as fever or respiratory disease. therefore, the combination of serum igm/igg antibody detection, the nucleic acid test, ct scan and clinical features improves the accuracy of covid-19 diagnosis. this study was approved by the ethics committee of shenzhen hospital, southern medical university (nyszyyec20200009). the authors declare that they have no conflict of interest. reliability and usefulness of a rapid igm-igg antibody test for the diagnosis of sars-cov-2 infection: a preliminary report false negative tests for sars-cov-2 infection -challenges and implications detection of sars-cov-2 in different types of clinical specimens potential false-negative nucleic acid testing results for severe acute respiratory syndrome coronavirus 2 from thermal inactivation of samples with low viral loads clinical significance of igm and igg test for diagnosis of highly suspected covid-19 infection humoral immune response to sars-cov-2 in iceland cross-reaction of sars-cov antigen with autoantibodies in autoimmune diseases we thank lijun zhang and qing liu of the department of clinical laboratory medicine center, shenzhen hospital, southern medical university who collected the serum samples. key: cord-267621-oc8bw7ft authors: kevorkian, jean-philippe; riveline, jean-pierre; vandiedonck, claire; girard, diane; galland, joris; féron, florine; gautier, jean-françois; mégarbane, bruno title: early short-course corticosteroids and furosemide combination to treat non-critically ill covid-19 patients: an observational cohort study date: 2020-09-01 journal: j infect doi: 10.1016/j.jinf.2020.08.045 sha: doc_id: 267621 cord_uid: oc8bw7ft nan availability of data and materials j.p.k. had full access to all of the data in the study and takes responsibility for the integrity of the data and the accuracy of the data analysis. all the authors agree to publish. none. the authors would like to thank mrs. alison good (scotland, uk) for her helpful review of the manuscript. acute respiratory distress syndrome (ards), a life-threatening complication of coronavirus disease-2019 (covid-19) is associated with elevated risk of intensive care unit (icu) admission and death, predominantly in the elderlies. 1 based on a randomized controlled trial, early dexamethasone was shown effective to reduce mechanical ventilation (mv) duration and overall mortality in moderate-to-severe ards patients independently of the etiology. 2 therefore, since systemic and pulmonary inflammatory cytokine storm and fibrinous and organizing pneumonia are involved in covid-19 ards, early corticosteroid administration has been considered as appropriate to avoid clinical deterioration and need for mv support. 3 however, cautious has been advised due to potential harmful effects of corticosteroids in viral pneumonia such as during covid-19 epidemic, non-critically ill covid-19 patients for whom intubation could be an option if worsening and those not eligible for intubation due to refusal, comorbidities and/or advanced age in the context of limited access to icu beds were referred to our ward. all patients received standard care, i.e. oxygen with adapted flow to oximetry (including high-flow oxygen), antibiotics, anticoagulants, vasopressors and antiviral drugs if needed. usual monitoring was provided including pulse oximetry, electrocardiogram, finger blood sugar and daily routine chemical tests. decision to administer corticosteroids was left to physicians in charge due to uncertainties regarding the benefit/risk balance of their use in non-critically ill covid-19 patients. 5 interestingly, because pulmonary edema could worsen hypoxemia in patients presenting cardiovascular co-morbidities and/or cardiac involvement in covid-19 at risk of fluid retention, we decided to co-administer furosemide systematically to corticosteroid-treated patients, with a rationale similar to that of conservative fluid management in ards patients. 5 therefore, to address the effectiveness of early short-course corticosteroid/furosemide treatment in the non-critically ill covid-19 patient, we designed a retrospective observational cohort study. all successive covid-19 patients with pneumonia requiring oxygen admitted to our non-critical medical ward from 03/11/2020 to 04/27/2020 were included. patients who received intravenous or oral corticosteroids plus furosemide for at least once daily three consecutive days were compared to those who did not (usual care group). the primary composite endpoint was invasive mv requirement (corresponding to care escalation from ward to icu) or 28-day mortality. data are expressed as median [percentiles 25 th -75 th ] or percentages. univariate comparisons were performed using mann-whitney or fisher exact tests, as appropriate. a multivariate logistic regression model to explain the outcome was tested with the corticosteroid/furosemide treatment as explanatory variable and adjustment for independent covariates (gender, age, body-mass index and comorbidities). odds ratios (or) and their 95%-confidence intervals were determined. p-values ≤0.05 were considered significant. analyses were preformed using the r3.6 environment. whereas ninety-three patients did not. noteworthy, 14/24 control patients (58%) at risk of cardiogenic pulmonary edema (serum brain natriuretic peptide (bnp) ≥100ng/ml) received furosemide without corticosteroids. in the corticosteroid/furosemide treatment group, incidence of invasive mv or death given once daily for up to ten days reduced 28-day mortality by one-third among mechanically ventilated covid-19 patients and by one-fifth among patients treated with oxygen, while no benefit was observed in patients not receiving respiratory support at randomization. 7 in covid-19 patients, viral shedding is elevated early then declines. interestingly, low-dose corticosteroids were shown not to delay viral clearance, thus encouraging their safe prescription aiming to limit the excessive systemic and pulmonary inflammation involved in ventilation worsening. 8 however, the best dose regimen and timing of corticosteroids in covid-19 remain undetermined. various anti-inflammatory therapies including interleukin-1-receptor and interleukin-6 receptor antagonists were proposed to treat non-critically ill covid-19 patients. 9 however, availability and cost-effectiveness of corticosteroids/furosemide (~60-fold less expensive than monoclonal antibodies) remain unbeatable. in aged covid-19 patients with high proportion of cardiac comorbidities, mild-to-moderate pneumonia may be accompanied by some degree of acute heart failure and ischemia, 10 as evidenced in our series by elevations in cardiac biomarkers (bnp, 43 ng/l [16-135] and troponin, 12 ng/ml , respectively). thus, furosemide administration when prescribing corticosteroids is pertinent, possibly beneficial to limit corticosteroid-induced retention and at least safe if adequately monitored. we observed increase in length of hospital stay, undoubtedly corresponding to increased survival resulting in prolonged medical care and rehabilitation. our study limitations include the non-randomized single-center design and relatively small number of patients. the brief study duration determined by covid-19 epidemic duration precluded a more elaborate design. future trials should determine the most appropriate strategy offering the best risk/benefit ratio. to conclude, our data provides evidence that early short-course of corticosteroids combined to furosemide reduces the risk of invasive mv requirement or 28-day mortality in the non-critically ill covid-19 patients. in comparison to the recovery trial results, our findings highly suggest the benefits and safety of adding furosemide to corticosteroids, aiming to improve fluid management especially in the aged patients with comorbidities at risk of pulmonary edema (bnp >100ng/ml on admission). the authors declare that they have no competing interests. this study was part of the french covid-19 cohort registry conducted by the reacting consortium (research and action targeting emerging infectious diseases) and directed by inserm (institut national de la santé et de la recherche médicale) and isaric (international severe acute respiratory and emerging infection consortium). our institutional ethics committee approved the study (n°, idrcb, 2020-a00256-33; cpp, 11-20 20.02.04.68737). figure 1 . impact of the corticosteroid/furosemide treatment in the different patient subgroups defined according to age (using the median value as threshold), gender, presence of diabetes mellitus, serum brain natriuretic peptide (bnp; threshold at 100 ng/ml) and troponin levels (threshold at 16 ng/ml). odds ratio (or) and their 95%-confidence intervals were determined. risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease dexamethasone treatment for the acute respiratory distress syndrome: a multicentre, randomised controlled trial covid-19: consider cytokine storm syndromes and immunosuppression clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and meta-analysis fluid management in ards dexamethasone in hospitalized patients with covid-19-preliminary report low-dose corticosteroid therapy does not delay viral clearance in patients with covid-19 interleukin-1 blockade with high-dose anakinra in patients with covid-19, acute respiratory distress syndrome, and hyperinflammation: a retrospective cohort study covid-19 in patients with heart failure: the new and the old epidemic troponin ic high-sensitivity (ng/ml) 18 11 key: cord-292201-e7k7gn9q authors: fodjo, joseph nelson siewe; pengpid, supa; villela, edlaine faria de moura; van, thang vo; ahmed, mohammed; ditekemena, john; crespo, bernardo vega; wanyenze, rhoda k; dula, janeth; watanabe, takashi; delgado-ratto, christopher; driessche, koen vanden; van den bergh, rafael; colebunders, robert title: mass masking as a way to contain covid-19 and exit lockdown in lowand middle-income countries date: 2020-07-17 journal: j infect doi: 10.1016/j.jinf.2020.07.015 sha: doc_id: 292201 cord_uid: e7k7gn9q in new guidelines published on june 5(th) 2020, the world health organization (who) recommends that in areas with ongoing covid-19 community transmission, governments should encourage the general public to wear face masks in specific situations and settings as part of a comprehensive approach to suppress covid-19 transmission. recent online surveys in 206,729 persons residing in nine lowand middle-income countries showed that 32.7%-99.7% of respondents used face masks with significantly differences across age groups and sexes. targeted health promotion strategies and government support are required to increase mask use by the general population. in new guidelines published on june 5 th 2020, the world health organization (who) recommends that in areas with ongoing covid-19 community transmission, governments should encourage the general public to wear face masks in specific situations and settings as part of a comprehensive approach to suppress covid-19 transmission. recent online surveys in 206,729 persons residing in nine low-and middle-income countries showed that 32.7%-99.7% of respondents used face masks with significantly differences across age groups and sexes. targeted health promotion strategies and government support are required to increase mask use by the general population. we read with interest the research work of cheng and collaborators on community-wide mask use for coronavirus disease 2019 (covid-19) control. 1 indeed, face masks are now recommended by the world health organization (who) to prevent covid-19 transmission, according to new guidelines published on june 5 th 2020. 2 the new recommendations state that in areas with ongoing covid-19 community transmission, governments should encourage the general public to wear masks in specific situations and settings where physical distancing cannot be achieved, as part of a comprehensive approach to suppress covid-19 transmission. 2 long before the issuance of these guidelines, many asian countries were already using face masks and this potentially contributed to the rapid containment of covid-19 in these countries. 1, 3 outside of asia, routine use of masks by the general population is rare. most european countries were applying previous who recommendations whereby face masks were reserved for covid-19 patients, carers or healthcare workers. moreover, there were fears that promoting mass masking could aggravate the shortage of face masks among healthcare workers, especially as cloth (fabric) masks were not initially considered useful for covid-19 prevention in europe. 3 the director-general of the chinese center for disease control and prevention went as far as warning europe and the united states of america (usa) regarding the risks of not enforcing routine wearing of face masks by the general public. 4 most low-and middle-income countries (lmic) outside of asia also initially deprioritised masks and focused on lockdown strategies in an attempt to "flatten the curve". however, lockdowns are associated with major socio-economic losses, which may further exacerbate the precarious conditions in resource-limited settings, and thus compliance to such strategies is implausible (particularly among populations who depend on daily labour for their income). furthermore, in highly congested settings such as urban slums or refugee camp settings, lockdowns and/or measures of physical distancing are not feasible. the benefits of isolation-based strategies are also limited, given that pre-and asymptomatic individuals are potentially contagious for covid-19. 5 we thus welcome the who recommendations to use face masks in the general population, as an important component of strategies to stop the epidemic and/or exit the lockdowns, particularly in lmic. recent evidence supports a predominantly airborne transmission route for covid-19, and strongly encourages face mask use in public to prevent inter-human transmission. 6 modelling studies estimate that the covid-19 pandemic can be brought to an end if 80% of the population would wear a surgical mask. 7 moreover, mass masking could also alleviate fears that prevent people from seeking medical care for non-covid-19 conditions, limiting the collateral damage of the covid-19 pandemic. on the downside, improper mask use may inadvertently increase covid-19 transmission via indirect contact routes with the mask serving as a fomite. mass making may also produce a false sense of security leading to reduced adherence to other preventive measures such as hand hygiene. 3 finally, surgical masks pose an environmental threat if discarded inappropriately due to their plastic content. 8 it is therefore paramount to monitor both compliance and user practices in ensuring the effectiveness of masks in covid-19 control. between march and june 2020, an international consortium (www.icpcovid.com) organised online surveys in lmic to monitor adherence to covid-19 preventive measures, including face mask use. only data of consenting respondents who were at least 18 years old and who self-identified as either male or female were analysed 8 in countries where masking was mandatory or highly encouraged by the government during the early phases of the covid-19 outbreak, adherence rates were >90%. in brazil, the initial low adherence to face mask use together in combination with little or no confinement measures may have contributed to the high covid-19 mortality in this country. where data were available on the type of mask used, reusable cloth masks (more cost-beneficial and environmentally friendly than surgical masks) were the most frequent accounting for 4,413/8,636 (51.1%) of all mask types. our study shows that even in countries where no pre-existing culture of mask use existed, high uptake of mass masking was feasible. the differential rate of uptake between sexes and age groups, as shown in table 2 , suggests that targeted health promotion strategies to (further) stimulate mask use may need to be developed, and that covid-19 prevention strategies need to be contextualized to each setting/population. as there is currently no effective vaccine or treatment against covid-19, the mass masking policy of the who is a prudent move for covid-19 prevention. we therefore urge the public health and scientific communities to invest in strategies to promote mask use among all tiers of the population, and to further build the evidence-base for optimal covid-19 prevention strategies. the authors declare no conflicts of interest. rc receives funding from the european research council (grant number 671055). all participants provided an informed e-consent (checkbox) before submitting their data anonymously. rc conceived the surveys and drafted the initial manuscript; jnsf cleaned and analysed the data, and edited the initial draft; all authors participated in data collection, critical review and approval of the final manuscript. the role of community-wide wearing of face mask for control of coronavirus disease 2019 (covid-19) epidemic due to sars-cov-2 advice on the use of masks in the context of covid-19: interim guidance rational use of face masks in the covid-19 pandemic. the lancet respiratory medicine not wearing masks to protect against coronavirus is a 'big mistake,' top chinese scientist says temporal dynamics in viral shedding and transmissibility of covid-19 identifying airborne transmission as the dominant route for the spread of covid-19 mathematical assessment of the impact of non-pharmaceutical interventions on curtailing the 2019 novel coronavirus discarded covid 19 gear: a looming threat hand hygiene, and influenza among young adults: a randomized intervention physical distancing, face masks, and eye protection to prevent person-to-person transmission of sars-cov-2 and covid-19: a systematic review and meta-analysis. the lancet we thank the following icpcovid research team members in the different countries who were involved in the local organisation of the surveys: key: cord-019968-o5bdb37q authors: goldwater, paul n. title: gastroenteritis in auckland: an aetiological and clinical study date: 2005-04-14 journal: j infect doi: 10.1016/s0163-4453(79)90677-7 sha: doc_id: 19968 cord_uid: o5bdb37q faecal specimens from 60 patients (under six years old), most of whom were maoris and pacific islanders admitted to auckland hospital with gastroenteritis during the months of june and july 1977, were examined for the presence of faecal viruses, bacterial pathogens and parasites. faecal specimens from 18 non-diarrhoeal control patients were also examined, of which three contained rotavirus. forty-three (72 per cent) gastroenteritis patients had rotavirus detectable in stools by electron microscopy or immune electron microscopy. of the remainder, 17 patients were regarded as having non-rotavirus diarrhoea. enterotoxigenic esch. coli. was isolated from seven patients of whom six yielded stable toxin producers (st+), four labile toxin producers (lt+) and two dual toxigenic strains (st+/lt+). all st+ isolates appeared to be of low enterotoxigenicity as indicated by low gut weight/carcass weight ratios in the infant mouse assay. rotavirus was the commonest aetiological agent (72 per cent), bacterial pathogens (alone) accounted for only five per cent and no enteric pathogens were found in 15 per cent of cases. non-agglutinable rotavirus, presumably a different serotype, was seen in both gastroenteritis and control patients. rotavirus ‘satellite’ particles previously undescribed were demonstrated in a number of stool samples. there have been no studies of the relative incidence of agents of gastroenteritis in new zealand. prior to this survey, in the majority of cases admitted to auckland hospital, no pathogen has been found using traditional bacteriological and virological techniques. this study was designed to give some indicaton of the relative importance of aetiological agents and clinical features in cases of gastroenteritis requiring hospital admission. rotavirus has been shown to be a major cause of gastroenteritis in other parts of the world (bishop, davidson, holmes and ruck, 1974; flewett, bryden, davies, woode, bridger and derrick, 1974; middleton, szymanski, abbott, bortolussi and hamilton, 1974; kapikian, kim, wyatt, rodriguez, ross, cline, parrott and chanock, 1974; echeverria, blacklow and smith, 1975; ryder, wachsmuth, buxton, evans, du pont, mason and barrett, 1976; lancet 1975; who 1975/76; british medical journal, 1977) --but with the advance of knowledge in this area and the finding of the virus in large *current address: senior registrar in virology, department of virology, st. thomas' hospital, london sei 7eh. numbers of non-diarrhoeal neonates (t0tterdell , chrystie and banatvala, 1976; murphy, albrey and crewe, 1977) , uncertainty of its relationship with its host is evolving. like rotavirus, exterotoxigenic esch. coli has been implicated as a cause of gastroenteritis in young children in various parts of the world (gorbach and khurana, 1972; nalin, mclaughlin, rahaman, yunus and curlin, 1975; sack, hirschhorn, brownlee, cash, woodward and sack, 1975; ryder, wachsmuth, buxton, evans, du pont, mason and barrett, 1976; evans, olarte, du pont, evans, galindo, portnoy and conklin, 1977; echeverria, ho, blacklow, quinnan, portnoy, olson, conklin, du pont and cross, 1977) . the significance of rotavirus and enterotoxigenic esch. coli as causal agents of gastroenteritis was studied. in june and july, 1977 , patients admitted to auckland hospital with gastroenteritis were studied to determine the relative isolation rates of (1) rotavirus, (2) other viruses identifiable by electronmicroscopy of stools, (3) enterotoxigenic esch. coli producting heat stable (st) and/or heat labile (lt) enterotoxins, (4) salmonella species, (5) shigella species and (6) parasites as aetiological agents. in addition, the presence of red blood cells, leucocytes and mucus strands in stools was recorded. sixty patients (30 males and 30 females) admitted to the infectious disease unit of auckland hospital with gastroenteritis were studied. gastroenteritis was defined as acute development of unusually frequent and loose stools with or without vomiting. the first stool passed after admission was collected in a sterile plastic container and processed on the day of collection or the day following overnight storage at 4°c. eighteen control patients (11 males and seven females) with non-diarrhoeal diseases were studied. clinical features were obtained on review of the patients' notes. viral pellets of stool specimens from patients with diarrhoea and from control patients were prepared for electronmicroscopy on a philips em 300 by the method of totterdell, chrystie and banatvala (1976) . the pellet was resuspended in a few drops of distilled water and stored at -20 ° until examined. specimens were negatively stained with three per cent potassium phosphotungstate (ph6). at least five suitable grid squares were scanned at approximately 41,000 magnification. electronmicrographs were prepared for measurements of virus size and identification of morphologically distinct viruses. all faecal viral pellets were examined after reacting with specific antirotavirus guinea pig serum kindly supplied by dr m. d. holdaway, dunedin hospital. using kayline 96 u-welled microtitre plates, 25/~1 anti-rotavirus serum (complement fixation titre 1:512) was diluted in phosphate buffered salines ph 7.01 to 1:64 and incubated with 25/xl faecal pellet at 37°c for three hours. after negatively staining and coding, grids were stored in lkb 4828b specimen grid boxes at room temperature until examined 'blind'. a wet preparation of stool stained with one per cent loeffler's methylene blue (harris and coleman, 1963; harris, du pont and hornick, 1972) was examined for the presence of red blood cells, leucocytes, mucus strands and parasites. stool speciments were examined for the presence of salmonella sp. and shigella sp. using macconkey and xld agar plates and selenite broth. these species were identified by standard methods (edwards and ewing, 1972) . ten lactose-fermenting colonies with the typical appearance of esch. coli were discriminately selected from the last two streaks on macconkey plates and were stored on nutrient (columbia) agar slopes and inoculated into 3 ml syncase medium (glucose substituted for sucrose) to make a pool suspensions of the 10 colonies for enterotoxin screening. pure and predominant non-lactose fermenting growths were treated in a similar manner. each pool was incubated stationary at 37°c for 48 hours, a 1 ml aliquot of broth suspension was removed and stored at -20°c for lt assay screening. the remainder of the suspension was centrifuged at 6000 rev/min for 30 minutes and the supernatant withdrawn for st assay screening. all specimens were coded and tested 'blind'. the assay for st was carried out as described by dean, ching, williams and harden (1972) . pools of 10 isolates from each patient were tested for st production. each individual isolate from st positive pools was then tested. four mice were used per assay. a mean ratio of > 0.0700 was regarded as weakly positive, > 0.0800 was regarded as positive. positive and negative control cultures were included in each assay. st +/lt + strains b7a (serotype 0148 k? h28), h10407 (serotype 078 hll) and st-/lt-u5/41 (serotype 01k1h7) were kindly supplied by dr b. rowe, salmonella and shigella reference laboratory, central public health laboratory, colindale avenue, london nw9 5ht and st +/lt + strain 408-3 and st -/ lt -strain 408-4 were kindly supplied by dr r. b. sack, baltimore city hospital, 4940 eastern avenue, baltimore, md 21224. a miniculture assay was carried out using whole bacterial pool culture as described by . individual strains of lt+ pools were tested later. all enterotoxigenic isolates were identified by standard biochemical tests. the male:female ratio in diarrhoeic patients with stools positive for rotavirus was 0-75 and for non-rotavirus gastroenteritis was 2.4. for the purpose of comparing clinical features, gastroenteritis patients were placed into two diagnostic categories according to the presence or absence of rotavirus in their stools: (1) rotavirus gastroenteritis group and (2) non-rotavirus gastroenteritis group. further subdivisions into age groups provided comparisions. table i shows the main clinical features associated with rotavirus diarrhoea and gastroenteritis due to other causes. a high incidence of upper respiratory symptoms and/or signs was seen in both gastroenteritis groups (71-100 per cent). otitis media was exclusive to rotavirus gastroenteritis. diarrhoeic patients (in all age groups) with rotavirus in their stools had a propensity to present with marked dehydration and circulatory shock which was not encountered in other forms of diarrhoea. on the whole, rotavirus gastroenteritis patients required intravenous fluids more often than the non-rotavirus group, but the numbers of patients in each group are small, control patients had mainly respiratory infections and other non-diarrhoeal conditions. the relative distribution of enteric pathogens detected according to patient age is illustrated in fig. 1 . rotavirus was seen in non-diarrhoeal control stools in all age groups. three of 18 (17 per cent) control stools contained rotavirus (one of which had non-agglutinable virus detectable on iem). there was no evidence of gastroenteritis in these three patients, however, a three month old female infant had had transient loose bowel motions one week prior to admission to hospital with whooping cough syndrome. the proportion of patients receiving antibiotics prior to and during admission to hospital is shown in table i . complications table ii shows the complications arising in the two gastroenteritis groups of patients. of the 78 pools tested, eight were weakly st+ (ratios > 0.0700 but < 0.0800) and six were lt+. assay of the individual ten strains of each pool identified enterotoxigenic strains. isolates. dual enterotoxigenicity of a strain (st +/lt +) was encountered only in two patients' stools. mixtures of 'toxitypes' within one pool were encountered. strain u5/41 (serotype 01k1h7) was used as the non-toxigenic control and gave a mean ratio of 0.061.2 (range 0.0487-0-0700, standard deviation _+ 0.0062). strains b7a (serotype 0148 k?h28), h10407 (serotype 078hll) and 408-3 (?serotype) were used as positive enterotoxigenic (st +/lt +) strains and gave mean ratios of 0" 1077, 0-0843 and 0.1203 respectively with ranges of 0.0769-0.1466, 0.0700-0.1066 and 0-0707-0.2877 respectively; with standard deviations of __ 0.022, + 0.011 and -0-0516 respectively. assuming that greater ratios are associated with a higher degree of toxigenicity; a comparison of the mean ratios for each control strain was carried out. the mean ratios of strains h10407 and b7a were significantly different (p < 0.001). this suggests that these differences are true and that the idea of degrees of toxigenicity thus can be entertained (klipstein, engert and short, 1977) . the mean ratio of test strains regarded as being st+ was 0.0736 (range 0.0701 to 0" 0793, standard deviation _+ 0.0025) being significantly greater than that of the control st negative strain u5/41 (p < 0.001). from table iii it is seen that enterotoxigenic isolates were found in both groups of gastroenteritis patients and also in non-diarrhoeal controls. table iv shows combinations of enteric pathogens isolated from gastroenteritis patients and control patients. mean rotavirus diameters for smooth and rough particles (flewett, 1977) , were 64.5 nm and 53.8 nm respectively (standard deviation -+ 2.4 nm and --4.3 nm respectively). rotavirus-like particles that failed to agglutinate on iem were seen in two gastroenteritis patients' stools and in one control patient's stool. solitary particles indicated non-agglutination. small round particles (which i shali call 'satellite particles') were seen in close proximity to rotaviruses. these satellite particles were between 13 and 26 nm in diameter (mean 16-9nm, standard deviation -+ 3.8nm) and appeared to have a surface substructure and were seen with increasing frequency with patient age. (plates 1 and 2) . on four occasions direct em of faecal viral pellets failed to show rotavirus that was later easily seen in large clumps on iem. cent incidence of viruses seen by em and iem according to age and patient group. tubular capsid protein structures as described flewett (1977) were common to all groups of stool whether or not rotavirus was present and failed to agglutinate on iem. adenovirus (54-78 rim) were seen in stools from all patient groups. their link with diarrhoea could not be established. coronavirus-like particles (mean size 93 × 161 rim) were commonly seen in all three patient groups. reovirus (80 rim) was seen in one control patient's stool. it was distinguished from rotavirus by its size, capsid structure and failure to react with immune rotavirus serum on iem. small round viruses (srv) 13-29 nm not identifiable as astroviruses or calciviruses were an almost invariable inhabitant of all stool types. bacteriophages of various shapes and sizes were common inhabitants of stools regardless of clinical condition. [fucing page 3471 this article points out the high incidence of upper respiratory symptoms and signs associated with rotavirus gastroenteritis and other causes of diarrhoea. this was alluded to (without reference) in a leading article (1977) but a respiratory mode of transmission of rotavirus was not suggested. from this study there is clinical evidence for this, but the hypothesis would also support a similar mode of transmission of non-rotavirus gastroenteritis and its largely unknown cause or causes. it should be noted that this study took place during winter when respiratory admissions are most common. however, respiratory symptoms and signs were also a common finding in washington children with rotavirus gastroenteritis reporte d by rodriguez, kim, arrobio, brandt, chanock kapikian, wyatt and parrott (1977) and lewis, parry, davies, parry, mott, dourmashkin, sanderson, tyrrell and valman (1979) showed a significant excess of rotavirus infected children with respiratory illness. a female predominance in the younger age groups affected by rotavirus was noted. the finding of rotavirus or non-agglutinable rotavirus in nondiarrhoeal stools in all age groups suggests asymptomatic infection. this is contrary to current evidence elsewhere except that newborn infants in neonatal units appear to be colonised by rotavirus with quite variable consequences (chrystie totterdell and banatvala, 1975; totterdell, chrystie and banatvala 1976; murphy, albrey and crewe, 1977) . the predominance of maoris and pacific islanders in the younger age groups with or without gastroenteritis may be a reflection of socio-economic, nutritional or other factors. evidence of spread within families was seen with equal frequency in maoris and caucasians. rotavirus more often spread to siblings than other agents of gastroenteritis. enterotoxigenic strains were isolated from six patients with diarrhoea and three control patients. no strain producing st had mouse mean gut weight/ remaining body weight ratios exceeding 0. 0800 indicating a low level of st production. from the work of klipstein, engert and short, 1977 , relative degrees of enterotoxigenicity seem to occur amongst toxigenic enterobacteriaceae. whether or not the weakly st+ strains found in this study produced the symptoms in those patients with gastroenteritis remains to be seen. to date there is no information concerning the amount of toxigenic activity required to classify a strain as toxigenic except arbitrary infant mouse mean gut weight body ratios which vary from one published study to another (dean, ching, williams and harden, 1972; morris, merson, sack, wells, martin, de witt, feeley, sack, bessudo, 1976; donta, wallace, whipp and olarte, 1977) . the finding of enterotoxigenic isolates in control patients indicates that lt and st plasmids are probably circulating freely in the community, and as pointed out by pickering, du pont, evans, evans and olarte (1977) , asymptomatic subjects are probably important in transmission of infection. combinations of enteric pathogens isolated from gastroenteritis patients and control patients were a common finding. the isolation of multiple enteric pathogens supports the findings of evans, olarte, du pont, evans, galindo, portnoy and conklin (1977) ; schoub, greef, lecatsas, prozesky, hay, prinsloo and ballard (1977) ; echeverria, ho, blacklow, quinnan, portnoy, olson, conklin, du pont and cross (1977) ; and madeley, cosgrove, bell and fallon (1977) . rotavirus was responsible for a large proportion of non-bacterial gastroenteritis in most age groups accounting for 74 per cent of diarrhoeal illness in the six months to less than two year age group, and 77 per cent in the two to six year group. in the youngest age group (less than six months) its prevalence was 65 per cent. rotavirus' high overall prevalence (72 per cent) may be explained by the winter season during which the study took place. from overseas reports (middleton, szymanski, abbott, bortolussi and hamilton, 1974; bryden, davies, hadley, flewett, morris and oliver, 1975; davidson, bishop, townley, holmes and riuck, 1975; kapikian, kim, wyatt, cline, arrobio, brandt, rodriguez, sack, chanock and parrott, 1976) , rotavirus diarrhoea appears to occur at the cooler times of the year in temperate climates but neonates in sydney showed no seasonal variation of rotavirus excretion (murphy, albrey and crewe, 1977) . it remains uncertain whether seasonal variation of rotavirus infection occurs in new zealand. however, this high detection rate may be reflected by the use of iem. the increased sensitivity of iem over em was shown by the detection of rotavirus in four patients which would otherwise have been missed by the latter method. rotaviruses of two gastroenteritis patients and one control patient failed to agglutinate on iem. the particles were identical in structure to agglutinable rotavirus and the severity of attributable disease appeared to be similar. zissis and lambert (1978) have shown that iem is a valid means of serotyping rotavirus. at least two serotypes of rotavirus (one predominant) were responsible for a large proportion of paediatric gastroenteritis admissions in auckland at the time of the study. adenoviruses, small round viruses and coronavirus-like particles appeared (on em) to a similar extent in gastroenteritis and control patients' stools. without immune serum, identification of parvoviruses and their distinction from other small round viruses by iem was not possible. astroviruses and caliciviruses cosgrove, 1975 and were not seen. 'satellite particles' were a common accompaniment of rotavirus. the proximity of these small isometric particles to rotavirus may only be an accidental encounter in the overcrowded tube journey to the outside world. but that this was seen with a frequency of 54 per cent suggests that some design was involved in their meeting. it is possible that these particles represent small aggregates of coat proteins. they have not been described previously. the author has not seen these particles in rotavfl'us positive specimens from patients at st. thomas' hospital. the capsid protein described by flewett (1977) as an association with rotaviruses of various animal species was seen in all types of stool whether or not rotavirus was detectable. this material failed to agglutinate on iem suggesting that it is not of rotavirus origin. in 15 per cent of gastroenteritis cases no pathogen could be found. it is possible that these infections were due to rotavirus excreted in numbers not great enough for detection even by iem of faecal viral pellets. the features of most cases of rotavirus gastroenteritis and non-rotavirus gastroenteritis were so similar that a separate aetiology for each group could not be distinguished on clinical evidence. that only one stool specimen from each patient was examined may explain negative results, but the overall yield of potential pathogens is higher or similar to 6ther published studies already cited where multiple specimens had been collected. the role of enteropathogenic serotypes of esch. coli (epec) and their relationship with enterotoxin production may cast some light on the area of aetiologically unexplained gastroenteritis. data on epec serotypes is being prepared for publication. (my thanks to dr r. b. ellis-pegler and dr t. e. miller for their helpful criticism. additional thanks to dr w. e. lang, dr c. howden and dr keitha farmer and other clinicians for giving me access to their patients and clinical notes. my gratitude to the nursing staff who collected the specimens and without whose help this work would not have been possible. dr sharon hannan's help with ultracentrifugation is gratefully appreciated. my thanks to merck, sharp and dohme who supported this work by means of a fellowship.) detection of a new virus by electron microscopy of faecal extracts from children with acute gastroenteritis rotavirus infection in a maternity unit importance of a new virus in sporadic enteritis in children test for escherichia coli enterotoxin using infant mice: application in a study of diarrhoea in children in honolulu enterotoxigenicescherichia coli and diarrheal disease in mexican children role of heat-labile toxigenic escherichia coli and reovirus-like agent in diarrhoea in boston children relative importance of viruses and bacteria in the etiology of pediatric diarrhoea in taiwan identification of enterobacteriaceae enteropathogens associated with pediatric diarrhoea in mexico city acute non-bacterial infectious gastroenteritis. an essay in comparative virology relation between viruses from acute gastroenteritis of children and newborn calves toxigenic escherichia coli. a cause of infantile diarrhoea in chicago diagnostic procedures and reagents fecal leucocytes in diarrheal illness human reovirus-like agent as the major pathogen associated with 'winter' gastroenteritis in hospitalized infants and young children reovirus-like agent in stools: association with infantile diarrhoea and development of serologic tests relative enterotoxigenicity of coliform bacteria a year's experience of the rotavirus syndrome and its association with respiratory illness 28 nm particles in faeces in infantile gastroenteritis stool viruses in babies in glasgow. 1. hospital admissions with diarrhoea orbivirus acute gastroenteritis of infancy laboratory investigation of diarrhoea in travellers to mexico: evaluation of methods of detecting enterotoxigenic escherichia coli rotavirus infections of neonates enterotoxigenic escherichia coli and idiopathic diarrhoea in bangladesh isolation of enteric pathogens from asymptomatic students from the united states and latin america clinical features of acture gastroenteritis associated with human reovirus-like agent in infants and young children infantile diarrhoea produced by heat-stable enterotoxigenic enterotoxigenic escherichia coil-associated diarrheal disease in apache children test for enterotoxigenicescherichia coli using yt adrenal cells in miniculture a microbiological investigation of acute summer gastroenteritis in black south african infants rotavirus infections in a maternity unit the new program of the world health organization in medical virology different serotypes of human rotaviruses key: cord-280829-juu8d60q authors: anathallee, mohammad; curphey, andrew; beeching, nick; carley, simon; crawford, ian; mackway-jones, kevin title: emergency departments (eds) in the united kingdom (uk) are not prepared for emerging biological threats and bioterrorism date: 2006-06-09 journal: j infect doi: 10.1016/j.jinf.2006.03.034 sha: doc_id: 280829 cord_uid: juu8d60q objective: to assess the preparedness of emergency departments (eds) in the united kingdom (uk) for the management of potential biological incidents. methods: we telephoned all hospitals in the uk listed as having a major ed. we surveyed their ed facilities and procedures for managing patients with infectious diseases. we determined how many of the eds had an isolation room available and, if present, whether this had an independent ventilation system and separate access from outside the ed. in addition, we determined how many of the eds would isolate patients with suspected cases of chickenpox, tuberculosis (tb), severe acute respiratory syndrome (sars) and other suspicious infections. results: we obtained complete data from 203 (79%) of the 257 hospitals approached. only 49 (24%) of these hospitals had isolation facilities available in the ed. of these 49 eds, 30 (61%) reported an independent ventilation system and 18 (37%) reported a separate access from outside the ed. the majority of eds would isolate patients with potential infectious diseases, however, 47 (23%) would not isolate patients with suspected chickenpox, 37 (18%) eds would not isolate patients with suspected tb, 12 (6%) eds would not isolate patients with suspected sars and 55 (27%) eds would not isolate patients with other suspicious infections. conclusion: eds in the uk are not prepared for emerging biological threats and bioterrorism. with current facilities and procedures it is highly likely that an infectious agent will spread to staff and other patients in any future biological incident. concern is growing about the re-emergence of infectious diseases as a significant health threat in the developed world. periodic natural outbreaks of new and emerging infectious diseases, such as the recent severe acute respiratory syndrome (sars) outbreak in south east asia, 1 the possibility of an influenza pandemic, 2e4 and the threat of the terrorist use of biological weapons against civilian populations, 5, 6 have all led to increasing concern among emergency planners and first responders who may not be prepared to respond safely to such incidents. 7e10 unlike most health services' major incidents, the onset of a biological incident may be insidious, geographically widespread and may demonstrate features unfamiliar to the clinician. it is therefore essential that the emergency departments (eds) have facilities and procedures in place to manage patients who may present with features associated with a biological agent. it is highly likely that in any future biological incident patients will present with clinical features suggestive of infectious disease prior to the nature of the biological agent being known. the objective of this study was to assess current facilities and procedures in eds in the united kingdom (uk) for the management of potential biological incidents. we telephoned all 261 hospitals in the uk listed as having a major ed in the 2004 directory of the british association for emergency medicine. in the uk a major ed is defined as one that accepts patients 24 h per day, 365 days per year and is staffed by accredited emergency physicians. major eds range in size from small district general hospital eds which see less than 25 000 patients per year to large inner-city eds which see greater than 100 000 patients per year. as these are the only health care facilities that provide care 24 h per day, 365 days per year they are therefore at greatest risk of receiving patients with potential infectious diseases. telephone calls were made to the duty sister/ charge nurse or duty shift leader in the ed of each hospital. an initial approach was made in july 2004. follow-up telephone calls to initial non-responders were made in october 2004 and december 2004. data were collected using a standardised data collection sheet (appendix 1). data were collected on hospital characteristics, facilities for isolation of patients presenting to the ed with potential infectious diseases, and procedures for the management of patients with known infectious diseases. the questions used were derived from national guidance current in the uk at the time the survey was undertaken and from experts in infectious diseases, public health medicine and emergency medicine. four hospitals no longer had major eds. we obtained complete data from 203 (79%) of the remaining 257 hospitals approached. only 49 (24%) of these hospitals had isolation facilities available in the ed. of these 49 eds 37 (76%) had one isolation room available, seven (14%) had two isolation rooms available, four (8%) had three isolation rooms available and one (2%) had six isolation rooms available. thirty (61%) reported an independent ventilation system and 18 (37%) reported a separate access from outside the ed. the majority of eds would isolate patients with potential infectious diseases, however, 47 (23%) would not isolate patients with suspected chickenpox, 37 (18%) eds would not isolate patients with suspected pulmonary tb, 12 (6%) eds would not isolate patients with suspected sars and 55 (27%) eds would not isolate patients with other suspicious infections. results for approximately how many cases of suspected chickenpox, pulmonary tuberculosis (tb) and sars were seen in eds in the past year were generally not available and consequently are not reported. table 1 shows the facilities and procedures in uk eds for the management of infectious diseases patients. this survey has shown that the majority of eds in the uk do not have isolation facilities available for the management of patients with potential infectious diseases; even when isolation facilities are available they may be of an inadequate standard. in addition, significant numbers of eds do not have adequate infection control procedures for the management of patients presenting with known infectious diseases that may mimic a more serious biological incident. we chose to telephone a senior member of operational nursing staff rather than to formally write to each ed as we felt that this was more likely to reveal the true departmental response. it is possible that some of the eds reporting no infection control procedures for the management of patients presenting with known infectious diseases do in fact have such procedures; however, if these are not known to the senior nursing staff then they are clearly ineffective. as with all studies of this type we have only been able to assess what people say that they will do rather than what they will do in practice. however, we see no reason why those contacted would deliberately under report their eds response. while up to three approaches were made to collect data there is no reason to suspect 'survey fatigue' since the answers were sought from the duty sister/charge nurse or duty shift leader in the ed of each hospital rather than from a specific individual. we chose chickenpox (varicella) as a marker of preparedness in light of its infectivity and its potential for confusion with smallpox (variola) early in the course of the disease. 11 in the ed, isolation is the single most important intervention in patients in whom variola infection is suspected. pulmonary tb was chosen as a marker of preparedness in light of its increasing prevalence and the emergence of multiple drug-resistant tb. national guidelines have been available for some time regarding the management of multiple drug-resistant tb 12 and are currently being reviewed by the national institute for health and clinical excellence (nice), 13 although specific guidance about the isolation of patients in the ed has not been included. specific questions regarding sars were asked as we sought to determine how recent guidance from the health protection agency for the management of sars 14 had been implemented in practice. the sars guidance at the time stated that patients should be managed by appropriately protected staff in an isolation setting with an independent ventilation system and with a separate access from the main ed or medical assessment unit (mau). any future influenza pandemic is likely to present in a similar way to sars and similar precautions are advocated for cases of avian influenza. 15 strengths and weaknesses in relation to other studies, discussing particularly any differences in results we are unaware of any other assessments of uk preparedness for biological incidents. our findings are in keeping with assessments of uk preparedness for other types of major incident. 16,8e10 meaning of the study: possible mechanisms and implications for clinicians or policymakers the current availability of appropriate isolation facilities in eds is inadequate. such facilities must be provided to improve health services capability to manage patients with potential infectious diseases. the provision of new-build isolation facilities in all eds is an expensive solution and would take a considerable time period to achieve. the designation (rather than dedication) of existing areas within eds as potential isolation facilities is a less ideal but more easily achieved solution and all hospitals should be encouraged to undertake this exercise. in prolonged biological incidents mobile isolation facilities could be delivered to affected hospitals. however, this can only be achieved after the initial outbreak and does not address the underlying ed problem. at times of heightened risk, such as the possibility of an influenza pandemic or the threat of bioterrorism, the first step to ensure an appropriate response is to raise awareness. the true first responders in a biological incident are the health care workers in eds and other primary health care facilities. 17 they must be made aware of the relevant signs and symptoms and taught to react appropriately. the health protection agency has prepared educational material and training courses to improve the ability of health care workers from a variety of backgrounds to deal with biological incidents. 18 however, these must be delivered to all front line staff, especially triage nurses in eds, so that they can identify the effects of at least some of the most likely biological agents. a nationally funded training standard, the structured approach to chemical casualties course, has previously been successfully cascaded down to all eds to improve preparedness for chemical incidents. 19, 20 once a biological incident is identified, be it natural, accidental or deliberate, a wider range of issues must be managed. 21 our study can only demonstrate what persons say that they will do, not what they will do in practice. such questions can only be answered by observational research to see how and where patients with potential infectious diseases are assessed in the emergency setting and if infection control guidelines are adhered to. world health organisation. who guidelines for the global surveillance of severe acute respiratory syndrome (sars) department of health. uk health departments' influenza pandemic contingency plan. london: department of health world health organisation. who global influenza preparedness plan avian influenza: assessing the pandemic threat biological warfare and bioterrorism getting ahead of the curve: a strategy for combating infectious diseases (including other aspects of health protection) making the uk safer: detecting and decontaminating chemical and biological agents facing the challenge: nhs emergency planning in england. london: the stationery office sixth report of session 2001e2002. defence and security in the uk. london: the stationery office are british hospitals ready for the next major incident? analysis of hospital major incident plans bioterrorism e variola (smallpox) and its mimics the prevention and control of tuberculosis in the united kingdom: uk guidance on the prevention and control of transmission of 1. hiv-related tuberculosis 2. drug-resistant, including multiple drug-resistant, tuberculosis. london: department of health tuberculosis: national clinical guideline for diagnosis, management, prevention and control. draft for second consultation. london: national institute for health and clinical excellence sars e hospital infection control guidance h5n1): who interim infection control guidelines for health care facilities making the uk safer: detecting and decontaminating chemical and biological agents emergency department response to the deliberate release of biological agents emergency preparedness and response training programme preparedness of london hospitals for a chemical weapons attack the structured approach to chemical casualties an emergency department response to severe acute respiratory syndrome: a prototype response to bioterrorism we wish to thank dr jennifer hill for discussions that led to this study being undertaken. key: cord-284853-6efhdogi authors: xie, yun; cao, song; li, qingyun; chen, erzhen; dong, hui; zhang, wenkai; yang, luyu; fu, shouzhi; wang, ruilan title: effect of regular intravenous immunoglobulin therapy on prognosis of severe pneumonia in patients with covid-19 date: 2020-04-10 journal: j infect doi: 10.1016/j.jinf.2020.03.044 sha: doc_id: 284853 cord_uid: 6efhdogi • initiation of ivig as adjuvant treatment for covid-19 pneumonia within 48 hours of admission to the icu can reduce the use of mechanical ventilation . • initiation of ivig as adjuvant treatment for covid-19 pneumonia within 48 hours of admission to the icu can reduce hospital length of stay and length of stay in icu. • initiation of ivig as adjuvant treatment for covid-19 pneumonia within 48 hours of admission to the icu can reduce 28-day mortality of patients with severe covid-19 pneumonia. dear editor, we read with interest the recent review by han et al. [1] that addressed the nature of the virus and its clinical characteristics to response to the 2019-ncov outbreak.at present, there is no vaccine or specific drugs for the human coronavirus . the most effective measures to 2019-ncov are still early detection and quarantine of new sources of infection, and early diagnosis and supportive treatments for comfirmed patients. as of march 18, 2020, china had a total of 81,151 confirmed cases of covid-19, including those in health care workers. italy, japan, south korea, the united states and other countries also reported new coronavirus cases, and the total global case load outside of china was 115,682 confirmed cases. the mortality rate of patients critically ill with the covid-19 pneumonia is as high as 61.5% [2] . intravenous immunoglobulin(ivig) has been clinically used as an adjunctive drug in the treatment of severe pneumonia caused by influenza [3] , but there is controversy about its therapeutic effect on covid-19 pneumonia, despite inclusion in the seventh edition of the guidelines stating that it can be considered for use in severe and critically ill patients. for this reason, this study retrospectively observed the relationship between the prognosis of patients with severe and critical covid-19 pneumonia and the adjuvant therapy of ivig and explored whether ivig could improve the clinical symptoms, laboratory examination and prognosis of these patients. in this retrospective study, we reviewed 58 cases of severe or critical illness due to covid-19 diagnosed in the intensive care unit of wuhan third hospital from january to february 2020. the study was approved by the hospital's ethics committee and exempted from written informed consent. inclusion criteria: all patients were diagnosed with covid-19 and confirmed by real-time rt-pcr. exclusion criteria: patients with incomplete data. primary outcome: 28-day mortality. secondary outcomes: 14-day mortality, hospital length of stay, length of stay in the icu, and use of mechanical ventilation. grouping: > 48 h group and ≤48 h group were divided according to the use of intravenous immunoglobulin within 48 h after admission. our treatment plan was as follows: all patients received oxygen therapy and abidor antiviral treatment and were initially administered the antibiotic moxifloxacin, according to the patient's clinical symptoms and signs and laboratory results, which were used to determine whether to adjust the antibiotics. in addition, according to the patient's condition, they were subjected to low molecular heparin anticoagulation, and when the absolute lymphocyte count fell to < 0.5× 10 9 /l at 20 g/day, they received intravenous immunoglobulin and correction for hypoalbuminemia. if the absolute number of lymphocytes was still low five days later, we used thymosin to boost immune function. patients in critical condition received intravenous administration of small doses of glucocorticoids (1-2 mg/kg) for 5-7 days depending on their condition. all other treatments were administered according to the who guidelines. we obtained epidemiological, demographic, clinical, laboratory, management, and outcomes data from patient records. final clinical results were followed up through february 29, 2020. the study included 58 patients diagnosed with covid-19 pneumonia . among them, 36 (62.1%) were males, with an average age of 62. the youngest age was 29 years old, the oldest age was 86 years old, and the median age was 63 (54-72) years old. the cumulative dose of intravenous immunoglobulin over 28 days was significantly increased in the >48 h group (88.57±71.14 vs 64.35±54.74 g, p=0.006) compared to that in the ≤48 h group. after admission, patients in the >48 h group had an average delay of 1 day in using ivig for the first time than patients in the ≤48 h group (2.707±1.427 vs 1.567±0.504 days, p=0.000) .of all enrolled patients, 11 (18.96%) required mechanical ventilation, 5 (8.62%) noninvasive mechanical ventilation, 6 (10.3%) invasive mechanical ventilation, and 2 (3.45%) high-flow oxygen aspiration. a total of 23 of the 58 patients died within 28 days of admission, 7 in the ≤48 h group and 16 in the > 48 h group. there was a statistically significant difference in 28 day mortality between the two groups (p=0.009). the length of stay in the hospital of the ≤48 h group was significantly shorter than in the > 48 h group (11.50 ±1.030 vs 16.96 ±1.620 days, p=0.0055), and the length of stay in the icu of the ≤48 h group was also significantly shorter than that of the > 48 h group (9.533±1.089 vs 13.50 ±1.632 days, p=0.0453) (figure1) . the proportion of patients requiring mechanical ventilation in the ≤48 h group was also significantly lower than in the > 48 h group (6.67% vs 32.14%, p=0.016) (figure 2 ). our study included 58 patients diagnosed with severe covid-19 . twenty-three (39.6%) critically ill patients died within 28 days. all patients were treated with ivig. this is the first clinical study to evaluate the efficacy of ivig in the treatment of severely ill covid-19 patients. ivig is applied in the adjuvant treatment of critical patients. as a blood product purified from the mixed plasma of healthy people, protein is the main component, and it is rich in bacterial antibodies and viral igg, etc. continuous infusion can improve the igg level in the serum, effectively neutralizing the pathogens in the respiratory tract of patients, and thereby promoting the recovery from diseases and shortening the course of disease [4] . ivig can improve the body's defense, block the receptors associated with the target cell, and prevent the pathogen from further damaging the target cell [4] . in addition, the use of ivig can also influence the process of lymphocyte differentiation and maturation, hinder the normal immune response of white blood cells, inhibit the production of inflammatory factors, and thus decrease the inflammatory injury experienced by patients [3, 5, 6] . a previous meta-analysis using ivig in sars infection concluded that whether intravenous infusion of ivig could improve the prognosis is still unclear [7] . there are also literature reports on the use of ivig in mers infection [8] , but there is no evidence that ivig has anti-mers activity, as specific efficacy has not been reported. studies on influenza virus infection, such as h1n1, have shown that ivig can prevent severe pandemic influenza infection [9] . a multicenter, double-blind, randomized, controlled trial using hyperimmune globulin in the treatment of patients with severe 2009 h1n1 infection found that the use of h-ivig in the treatment of severe h1n1 infection within 5 days of symptom onset was associated with reduced viral load and reduced mortality [3] . it is therefore worth examining when to use ivig to assist the treatment of covid-19. in our first analysis, we found that the use of ivig within 24 hours after admission had no significant statistical difference in either the 28-day mortality or the 14-day mortality rates, but the use of ivig within 48 hours could significantly reduce the 28-day mortality rate, indicating that the initiation time of ivig was related to the reduction of the covid-19 mortality rate. in our study, treatment with ivig within 48 hours of admission not only reduced ventilator use, but also reduced hospital and icu length of stay, ultimately improving 28-day mortality. our study demonstrated that ivig treatment in covid-19 patients with severe pneumonia can improve the patients' indicators within a short time and improve the treatment efficiency of the patients with high effectiveness. to the best of our knowledge, this is the first study to evaluate the efficacy of ivig therapy in critically ill patients infected with covid-19. in four previously published studies of critically ill patients, the use of ivig was not mentioned in detail, and it was impossible to summarize the effect of ivig use on prognosis of these patients with covid-19 pneumonia [2, 10] . our research also has several limitations. our study only included 58 patients with severe illness. the 28-day mortality rate of this population cannot represent all patients with severe covid-19, and the mortality rate is also different at based on different times of illness onset within the same center. we included all critical patients in the intensive care unit of wuhan third hospital who met the inclusion criteria. because of the exploratory nature of the study, the calculation of sample size is exempted. meanwhile, the next step is to confirm this conclusion with a larger sample size. this is a retrospective study. the data in this study allowed for a preliminary evaluation of the efficacy of ivig therapy in critically ill patients with covid-19 pneumonia. however, additional prospective randomized controlled studies are needed for further verification. in summary, initiation of ivig as adjuvant treatment for covid-19 pneumonia within 48 hours of admission to the icu can reduce the use of mechanical ventilation, shorten the hospital length of stay, promote the early recovery of patients, and improve the effective treatment of patients to achieve significant clinical efficacy. coronavirus 2019-ncov: a brief perspective from the front line clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered,retrospective, observational study hyperimmune iv immunoglobulin treatment: a multicenter double-blind randomized controlled trial for patients with severe intravenous immunoglobulin and mortality in pneumonia patients with septic shock: an observational nationwide study immunoglobulin a modulates inflammatory responses in an in vitro model of pneumonia efficacy of intravenous immunoglobulin in the prevention of pneumonia in patients with common variable immunodeficiency systematic review of treatment effects current treatment options and the role of peptides as potential therapeutic components for middle east respiratory syndrome (mers): a review intravenous immunoglobulin protects against severe pandemic influenza infection clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china dose of ivig b.time of ivig c. hospital length of stay d. icu length of stay we thank the hospital staff for their efforts in recruiting patients. ethics approval from wuhan third hospital institutional review board: reference number (ky2020-007) .written informed consent was waived due to the rapid emergence of this infectious disease. all the authors have approved the manuscript and agree with publication. after publication, the data will be made available to others on reasonable requests to the corresponding author. we declare no competing interests. key: cord-285179-26ey3fm8 authors: chan, kwok-hung; chan, jasper fuk-woo; tse, herman; chen, honglin; lau, candy choi-yi; cai, jian-piao; tsang, alan ka-lun; xiao, xincai; to, kelvin kai-wang; lau, susanna kar-pui; woo, patrick chiu-yat; zheng, bo-jiang; wang, ming; yuen, kwok-yung title: cross-reactive antibodies in convalescent sars patients' sera against the emerging novel human coronavirus emc (2012) by both immunofluorescent and neutralizing antibody tests date: 2013-04-10 journal: j infect doi: 10.1016/j.jinf.2013.03.015 sha: doc_id: 285179 cord_uid: 26ey3fm8 objectives: a severe acute respiratory syndrome (sars)-like disease due to a novel betacoronavirus, human coronavirus emc (hcov-emc), has emerged recently. hcov-emc is phylogenetically closely related to tylonycteris-bat-coronavirus-hku4 and pipistrellus-bat-coronavirus-hku5 in hong kong. we conducted a seroprevalence study on archived sera from 94 game-food animal handlers at a wild life market, 28 sars patients, and 152 healthy blood donors in southern china to assess the zoonotic potential and evidence for intrusion of hcov-emc and related viruses into humans. methods: anti-hcov-emc and anti-sars-cov antibodies were detected using screening indirect immunofluorescence (if) and confirmatory neutralizing antibody tests. results: two (2.1%) animal handlers had if antibody titer of ≥1:20 against both hcov-emc and sars-cov with neutralizing antibody titer of <1:10. no blood donor had antibody against either virus. surprisingly, 17/28 (60.7%) of sars patients had significant if antibody titers with 7/28 (25%) having anti-hcov-emc neutralizing antibodies at low titers which significantly correlated with that of hcov-oc43. bioinformatics analysis demonstrated a significant b-cell epitope overlapping the heptad repeat-2 region of spike protein. virulence of sars-cov over other betacoronaviruses may boost cross-reactive neutralizing antibodies against other betacoronaviruses. conclusions: convalescent sars sera may contain cross-reactive antibodies against other betacoronaviruses and confound seroprevalence study for hcov-emc. the emergence of the novel human coronavirus emc (hcov-emc) in the middle east since april 2012 has so far led to 17 cases of human infection with 11 being fatal as of 26 march 2013. 1e3 the first 2 laboratory-confirmed cases were reported by the world health organization (who) on 23 september 2012. 1 the index case was a 60-year-old man from jeddah, the kingdom of saudi arabia, who presented with severe acute community-acquired pneumonia and acute renal failure on 6 june 2012 and later succumbed on 24 june 2012 despite maximal supportive treatment. 1, 4 a sputum sample obtained on admission showed cytopathic changes suggestive of virus replication in llc-mk2 and vero cells, and was positive for coronavirus by pan-coronavirus rt-pcr. subsequent phylogenetic analysis of the viral genome sequences showed that the virus was a novel coronavirus with close genetic relatedness to tylonycteris-bat-coronavirus-hku4 (ty-batcov-hku4) and pipistrellus-bat-coronavirus-hku5 (pi-batcov-hku5) discovered in the lesser bamboo bat (tylonycteris pachypus) and japanese pipistrelle bat (pipistrellus abramus) of hong kong, china respectively. 4e7 closely related coronaviruses have also been found in other bat species in europe and ghana. 8, 9 the second case was a 49-year-old man from qatar who kept camels and sheep in his farm and had a travel history to the kingdom of saudi arabia before symptom onset. 1, 10 he developed severe acute communityacquired pneumonia and acute renal failure requiring extracorporeal membrane oxygenation in an intensive care unit of london. the lower respiratory tract samples were positive for coronavirus using pan-coronavirus rt-pcr. the 250 bp pcr fragments of the viral isolates in the first 2 cases showed 99.5% sequence homology with only 1 nucleotide mismatch over the regions compared. 10 subsequently, 15 more laboratory-confirmed cases of hcov-emc infection were reported in the middle east and the united kingdom with a total of 9 in the kingdom of saudi arabia, 2 in qatar, 2 in jordan, 1 in united arab emirates and 3 in the united kingdom. 2, 3 most of the cases developed severe pneumonia, at least 6 cases had concomitant acute renal failure, and 11 cases died. this unusually high crude fatality rate of over 50% and the severe clinical manifestations of acute respiratory and renal failure are unique among human coronavirus infections. 11e18 the source, transmissibility and seroprevalence of hcov-emc are not well established at present. as with other highly pathogenic viruses which are capable of causing epidemics such as sars coronavirus (sars-cov) and avian h5n1 influenza a virus, an animal source of the virus leading to interspecies jumping to humans is possible. 7,11,19e22 this hypothesis is supported by the epidemiological link to animal exposure in some of these patients with laboratory-confirmed hcov-emc infection, 1,3 the close phylogenetic relatedness between hcov-emc and ty-batcov hku4 and pi-batcov hku5, 5,6 and the broad species tropism of hcov-emc in different animal cells including bat, primate, swine, civet, and rabbit. 23, 24 human-to-human transmission appears to be limited at this stage with only 4 epidemiologically-linked clusters being identified so far. the jordanian cluster was retrospectively traced back to april 2012 with no further evidence of spread. moreover, none of 2400 residents in the kingdom of saudi arabia had serum antibody against hcov-emc. 4 thus, hcov-emc is likely different from other human coronaviruses associated with mild respiratory tract infections, namely hcov-oc43, hcov-229e, hcov-nl63 and hcov-hku1 which account for 5e30% of all respiratory infections with up to 21.6% of the general population having serum antibodies. 25, 26 rather, it may be similar to sars-cov which crossed species barriers from its natural bat reservoir to intermediate amplification animal hosts and humans and caused severe infection or subclinical non-pneumonic infection in about 0.5% of the general population. 12 in order to further substantiate the hypothesis of hcov-emc being a zoonotic agent and elicit evidence for intrusion of hcov-emc and its related viruses into humans, we studied the antibody titers using immunofluorescence (if) as screening and neutralization as confirmatory tests in at-risk groups working in a wild life market in guangzhou of southern china who were constantly exposed to a wide range of game food animals, sars patients who might have acquired their infection directly from wild animals, and healthy blood donors. the study was approved by the institutional review board of the hospital authority in hong kong. archived sera obtained from 94 subjects belonging to at-risk groups working in a wild life market in guangzhou, 28 patients with laboratory-confirmed sars by rt-pcr, and 152 healthy blood donors in hong kong special administrative region, southern china were retrieved from à70 c refrigerator. the at-risk groups consisted of game food animal market retailers, animal slaughterers and animal transporting personnel. all subjects were aged 18 years or above. the 94 animal handlers had a mean age of 35.4 years (range, 19e76 years), and the male-to-female ratio was 60:34. all of them had exposure to live and/or dead chickens, ducks, geese, pigeons, sparrows, seagulls, turtledoves, cranes, foxes, wild boars, sika deers, rabbits, and/or cats. their average exposure time was 3.91 years (range, 1 month to 16 years). a clinical isolate of hcov-emc was kindly provided by fouchier and zaki et al. 4 the isolate was amplified by one additional passage in vero cell lines to make working stocks of the virus. all experimental protocol involving live hcov-emc coronavirus isolate followed the standard operating procedures of the approved biosafety level-3 facility as we previously described. 27 hcov-emc and sars-cov-infected vero, hcov-oc43infected bsc-1, hcov-229e-infected mrc-5 and hcov-nl63-infected llc-mk2 cell smears were used for the study. smears were prepared as we previously described. 28 briefly, when 60%e70% of cells had early evidence of cytopathic effect (cpe) as shown by rounding up of cells under inverted microscopy, the cells were harvested by trypsinization and air dried on tefllon slides (immuno-cell int, mechelen, belgium), and fixed with chilled acetone for 10 min at à20 c and were stored at à80 c until use. indirect immunofluorescent antibody test ( fig. 1 ) anti-hcov-emc and anti-sars-cov if antibody detection was performed using indirect if as we previously described with slight modifications. 28 sera were screened at a dilution of 1 in 20 on infected and non-infected control cells at 37 c for 45 min. the cells were washed twice in pbs for 5 min each time. anti-human igg (inova diagnostic, san diego) were then added and the cell smears further incubated for 45 min at 37 c. sera positive at a screening dilution of 1 in 20 were further titrated with serial 2-fold dilutions. a positive result was scored when fluorescent intensity equaled or was higher than that of a positive control used in our previous studies. 28e32 for hcov-emc antibody testing, vero cells were infected with 0.01 moi for 36e40 h before harvesting. the infected cells were then coated on teflon slides 8-well, air dried and fixed with chilled acetone at 20 c for 10 min, and kept at à80 c until use. guinea pig anti-n hyper-immune sera were prepared as positive controls for testing with each new batch of infected and non-infected cells together with non-immune guinea pig sera as a negative control. 23 positive and negative guinea control sera were included in each run of antibody testing. the if antibody titer was taken to be the highest serum dilution giving a positive result. anti-hcov-oc43 if antibody titers were further determined for sera with positive anti-hcov-emc if antibody titers. all sera were inactivated at 56 c for 30 min before neutralizing antibody test. starting with a serum dilution of 1 in 10, serial 2-fold dilutions of sera were prepared in 96-well microtiter plates as we have previously described. 28 each serum dilution of 0.05 ml was mixed with 0.05 ml of 200 50% tissue culture infectious doses (tcid 50 ) of hcov-emc or sars-cov (hk39849), and incubated at 37 c for 1.5 h in a co 2 incubator. then 0.1 ml of the virus-serum mixture was inoculated in duplicate wells of 96-well microtiter plates with preformed monolayers of vero cells and further incubated at 37 c for 3e4 days. a virus backtitration was performed to assess the actual virus titer used in each experiment. cpe was observed using an inverted microscope on day 3 and 4 post-inoculation. the neutralizing antibody titer was determined as the highest dilution of serum which completely suppresses the cpe in at least half of the infected wells. the experiment was read when the virus back-titration showed the virus dose to be 100 tcid 50 as expected. mouse anti-whole hcov-emc hyper-immune sera were used as positive controls. all sera with positive neutralizing antibody titers were repeated for confirmation. anti-hcov-oc43 neutralizing antibody titers were further determined for sera with positive hcov-emc if antibody titers. amino acid sequences of the s proteins of hcov-emc, sars-cov, hcov-oc43 and hcov-hku1 were downloaded from ncbi genbank. structure-based sequence alignment of the s1 and s2 domains of hcov-emc, sars-cov, hcov-oc43 and hcov-hku1 were performed by promals3d server. 33 immunogenic regions containing potential human b-cell epitopes were predicted using epitopia. 34 the transmembrane domain preceding the cytoplasmic tail was predicted using tmhmm version 2.0. 35 heptad repeat regions within the s2 domains were predicted using marcoil. 36 fisher exact test was used to determine the differences in proportion of the 3 groups with positive antibody titers by if and nt between animal handlers and healthy blood donors, sars patients and healthy blood donors, and animal handlers and sars patients. computation was performed using the predictive analytics soft ware (pasw) version 18.0 for windows. correlation between the if and neutralizing antibody titers against hcov-emc, sars-cov and hcov-oc43 was performed using ibm spss statistics 19, with titers of <1:20 and <1:10 regarded as 1:10 and 1:5 respectively. a p-value of <0.05 was considered as statistically significant. two of 94 (2.1%) animal handlers working at a wild game food animal market in south china had positive anti-hcov-emc igg detected by indirect if with titer of 1:20 and 1:40 (table 1) . case 1 was a 38-year-old man with exposure to pigeons for more than 2 years. case 2 was a 39-year-old man with exposure to chickens, ducks, and geese for more than 3 years. both of them also had positive anti-sars-cov igg by indirect if with a titer of 1:40 and anti-hcov-oc43 igg with titers >z1:320 (table 2 ). case 2 who had adequate archived serum for testing of anti-hcov-oc43 neutralizing antibody had a titer of 1:80. another 11 animal handlers had positive anti-sars-cov igg by indirect if and 4 of them had anti-sars-cov neutralizing antibodies ( table 1) . none of the animal handlers had anti-hcov-emc neutralizing antibody. among the 28 sars patients, 17 (60.7%) had positive anti-hcov-emc igg detected by indirect if with titers ranging from 1:20 to 1:320 (table 1) . most had a titer between 1:80 to 1:160 (6/28 or 21.4% each). all 17 patients had anti-hcov-oc43 igg detected by indirect if (table 2) . surprisingly, 7 (25%) of the sars patients also had low titers of anti-hcov-emc neutralizing antibody of 1:20 or less, and all 17 of them had anti-hcov-oc43 neutralizing antibodies. anti-sars-cov if and neutralizing antibodies were found in the majority (96.4%) of the sars patients as expected. most of them had high titers of 1:80 or above. four of the 28 sars patients had paired acute and convalescent sera available for comparison ( table 3 ). the anti-hcov-emc if igg titer rose from <1:20 in the acute sera to 1:40 and 1:320 in the convalescent sera in 2 of these patients, while there was no significant rise in the other two. these patients also had 4-fold rise in if antibody titer against another human betacoronavirus hcov-oc43. none of 152 (0%) healthy blood donors had anti-hcov-emc or anti-sars-cov antibodies by indirect if and neutralization (table 1 ). there was an overall significant correlation between the indirect if igg titers against hcov-emc and sars-cov (pearson correlation 0.587, p < 0.01), and between the neutralizing antibody titers against hcov-emc and sars-cov (pearson correlation 0.422, p < 0.01). for subgroup analysis of sars patients with positive anti-hcov-emc if and/or neutralizing antibodies, the correlation was strongest between antibodies against sars-cov and hcov-oc43 (pearson correlation 0.593 and 0.605 for if and neutralizing antibodies respectively; p < 0.01 in both cases). while there was little amino acid sequence identity (16.6%) between the receptor-binding domain in the s1 proteins of hcov-emc and sars-cov, their s2 proteins showed an amino acid sequence identity of 40.3%. epitopia was used to predict immunogenic regions that might be b-cell epitopes in the s1 and s2 domains. 34 while epitopes were predicted in aligned regions of s1 from hcov-emc and sars-cov, it is unlikely that cross-neutralization by antibodies would occur in these regions as the sequence identity of the predicted epitopes between the two viruses is low (fig. 2) . three and two immunogenic regions were predicted in the s2 domains of hcov-emc and sars-cov respectively (fig. 3) . the immunogenic regions identified in s2 of hcov-emc overlapped the predicted regions in s2 of sars-cov. notably, the identified immunogenic regions sars-i and emc-ii overlapped the heptad repeat 2 region of the s2 domain of both hcov-emc and sars-cov, which is known to harbor an epitope for broadly neutralizing antibody in the case of sars-cov. 37 while looking for evidence of intrusion by the novel betacoronavirus hcov-emc into at-risk groups and the general population, convalescent sars patients' sera were found to contain significant titers of antibodies against other betacoronaviruses. there was a positive correlation between the antibody titers against the sars-cov and hcov-emc using both the indirect if and neutralization antibody tests. the finding of cross-reactive if antibodies was not that unexpected because these could be induced by crossreactive epitopes against structural proteins such as the nucleoprotein which is the most abundant structural protein in the coronaviruses as we had previously reported. 38 indeed, cross-reactive antibodies among human betacoronaviruses by if are well known, and have made large scale surveillance studies and epidemiologic surveys of human coronavirus infections difficult. 39 on the other hand, crossreactive neutralizing antibodies among betacoronaviruses have rarely been reported except between the closely related human and palm civet sars-covs. 40 the significant neutralizing antibody titers against hcov-emc in sars patients' sera in this study were surprising because neutralization is generally considered as the most specific serological test. our previous surveillance study showed that anti-sars-cov neutralizing antibody in our population was extremely low despite a high seroprevalence of anti-hcov-oc43 and anti-hcov-hku1 antibodies. 12 zaki and colleagues also failed to detect cross-reactive anti-hcov-emc antibodies among 2400 patients in the kingdom of saudi arabia who likely also had serum anti-hcov-oc43 and/or anti-hcov-hku1 antibodies. furthermore, none of the 152 healthy blood donors in the present study had serum anti-hcov-emc antibodies detected by indirect if and neutralization. therefore we assessed the structural homologies between these betacoronaviruses for possible explanations of the observed cross-reactive neutralizing antibodies. of all the surface proteins, only the ectodomains of s (spike) and orf3a can induce significant neutralizing antibody with some augmentation from the m (matrix) and e (envelope) proteins. 41, 42 though orf3a is absent in hcov-emc, we cannot completely exclude the possibility that similar orf3a-like proteins are being coded by the accessory protein gene but homology search does not reveal the presence of similar protein. all betacoronaviruses use the s protein for attachment and fusion of the virion with the host cell membrane. trimers of the s protein form the peplomers that radiate from the lipid envelope and give the virus a characteristic corona solis-like appearance under the electron microscope. the spike protein ectodomain consists of the s1 and s2 domains. the s1 domain contains the receptor binding domain and is responsible for recognition and binding to the host cell receptor. the s1 fragment between amino acids 318 and 510 is the receptor binding domain for ace2 in the case of sars-cov. however, the homology of s1 between sars-cov and hcov-emc is low with only 16.6% amino acid identity. indeed, this region is generally more divergent relative to the s2 region for coronaviruses. hence, while the s1 region induces the majority of the neutralizing antibody in convalescent sera of sars patients, 43, 44 it would be unlikely to result in antibodies with significant cross-neutralizing activity. the s2 domain, responsible for fusion, contains the putative fusion peptide and the heptad repeat hr1 and hr2. the binding of s1 to the cellular receptor will trigger conformational changes which collocates the fusion peptide upstream of the two heptad repeats of s2 to the transmembrane domain, and, finally, fusion of the viral and cellular lipid envelopes. an epitope situated between amino acids 1055 to 1192 and around heptad repeat 2 of the s2 subunit is likely to have induced the cross-reactivity of neutralizing antibody against hcov-emc and sars-cov. 63 our phylogenetic and antigenic epitope analysis suggested that this area is highly conserved among these 4 table 3 titers of anti-human-coronaviruses antibodies by immunofluorescence and/or neutralization in sars patients with available paired acute and convalescent serum samples. table 3 and case 3 in table 2 were the same specimens. b case d (convalescent) in table 3 and case 17 in table 2 were the same specimens. table 2 and case c (convalescent) in table 3 were the same specimens. b case 17 in table 2 and case d (convalescent) in table 3 were the same specimens. c test was not performed due to insufficient quantity of archived sera. betacoronaviruses and therefore could not completely explain the presence of cross-reactive anti-hcov-emc neutralizing antibodies among sars patients but not the general population. we postulate that in addition to the structural homologies between hcov-emc, sars-cov, hcov-oc43 and hcov-hku1, the different clinical manifestations and subsequent host immunological response of these infections may account for this pattern of neutralizing antibody cross-reactivity. while sars-cov causes severe infection with viremia, 45 hcov-oc43 and hcov-hku1 predominantly cause superficial mucosal infections of the upper respiratory tract which is self-limiting. therefore unlike the highly virulent sars-cov or hcov-emc which can induce a solid humoral immune response, an insufficient b cell maturation process with failure to induce high avidity antibodies is more likely to occur with figure 2 structure-based protein sequence alignment of the s1 region of hcov-emc, sars-cov, hcov-oc43 and hcov-hku1, constructed using promals3d (http://prodata.swmed.edu/promals3d/). the receptor binding domain is highlighted. identical and similar residues are shaded in black and grey respectively. immunogenic regions predicted by epitopia of at least 10 residues in length are highlighted by a black line. only 1 representative sequence from each virus is used to improve clarity of presentation. other betacoronavirus infections in the general population but their neutralizing antibody titer against these less virulent betacoronaviruses such as hcov-oc43 can be boosted with superimposed sars-cov or hcov-emc infections ( table 2) . these viral, clinical and immunological differences may explain the absence of cross-reactive neutralizing antibody against both sars-cov and hcov-emc in normal blood donors despite that most of them should have been exposed to hcov-oc43 and hcov-hku1 in the past. our finding has important implications in the serodiagnostic testing, treatment and development of vaccine for the prevention of human infection caused by betacoronaviruses. the possibility of cross-reactive antibodies giving rise to false-positive results concurs with the suggestion of a recent report to use anti-hcov-emc if antibody test only in patients with very clear epidemiological linkage. 46 besides the possibility of wrong serodiagnosis due to crossreactivity, this observation would support the use of antiviral peptides in the treatment of this emerging hcov-emc infection as antiviral peptides targeting the heptad repeat 2 has been successfully used in neutralizing sars-cov in cell culture. 47 furthermore, this antigenic epitope could be an important vaccine target though the danger of immunopathology must also be considered. the possibility of low level neutralizing antibody leading to immune enhancement should also be considered if sars convalescent plasma or normal intravenous immunoglobulin are used for the treatment of hcov-emc infection. 48 no definitive evidence of intrusion of hcov-emc into atrisk groups was found in the present study. two out of 94 sera from animal handlers had indirect if antibody against both hcov-emc and sars-cov but no specific neutralizing activity toward these 2 viruses. though this can be due to cross-reactivity with any betacoronaviruses such as hcov-oc43, the possibility of cross-reactivity to ty-batcov hku4 and pi-batcov hku5 remains a distinct possibility which may represent sporadic interspecies jumping in this high risk group. indeed, coronaviruses are found in many mammalian and avian species, 49e53 and have repeatedly crossed species barriers to cause interspecies transmission throughout history and occasionally caused major zoonotic outbreaks with disastrous consequences. 11,54e56 phylogenetic analysis showed that the lineage a betacoronavirus hcov-oc43 might have jumped from a bovine source into figure 3 structure-based protein sequence alignment of the s2 region of hcov-emc, sars-cov, hcov-oc43 and hcov-hku1 constructed using promals3d (http://prodata.swmed.edu/promals3d/). identical and similar residues are shaded in black and grey respectively. immunogenic regions predicted by epitopia of at least 20 residues in length are highlighted by a black line. the heptad repeat regions are highlighted. only 1 representative sequence from each virus is used to improve clarity of presentation. human in the 1890s. 57 the more recent example of interspecies transmission was the jumping of the lineage b betacoronavirus sars-cov from bats to civets and then to humans which caused the sars epidemic in 2003. 11,19,58e62 though the seroprevalence of anti-hcov-emc antibody found no indication of positivity among residents in the kingdom of saudi arabia, their demographic details, particularly the history of animal exposure, were not described. 4 further studies including seroprevalence studies with more refined serological test should be conducted among at-risk groups in the middle east to confirm the zoonotic nature of this emerging human coronavirus. there were a number of limitations in this study. first, only a relatively small number of sars patients were tested because of the lack of archived sera. however, most of the positive anti-hcov-emc igg titers in this group were of high values between 1:80 to 1:160 which made the results less ambiguous. it would be interesting to test a larger group of laboratory-confirmed sars patients with different viral strains to substantiate our observation. second, the low seroprevalence of anti-sars-cov in the general population make the possibility of wrong serodiagnostics due to crossreactivity less important for routine diagnostics. however, the finding is essential for confirmation of serological surveillance studies especially in some southeast asian countries including china where the seroprevalence for anti-sars-cov may not be well established, as hcov-emc may continue to spread and cause an epidemic in this densely populated area in the future. is the discovery of the novel human betacoronavirus 2c emc/2012 (hcov-emc) the beginning of another sars-like pandemic global alert and response: novel coronavirus infection e update latest outbreak news from promed-mail: novel coronavirus e middle east isolation of a novel coronavirus from a man with pneumonia in saudi arabia genetic relatedness of the novel human lineage c betacoronavirus to tylonycteris bat 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interspecies transmission of coronavirus between bats of different suborders molecular epidemiology of human coronavirus oc43 reveals evolution of different genotypes over time and recent emergence of a novel genotype due to natural recombination the severe acute respiratory syndrome coronavirus as a possible cause of severe acute respiratory syndrome aetiology: koch's postulates fulfilled for sars virus a novel coronavirus associated with severe acute respiratory syndrome identification of a novel coronavirus in patients with severe acute respiratory syndrome amino acids 1055 to 1192 in the s2 region of severe acute respiratory syndrome coronavirus s protein induce neutralizing antibodies: implications for the development of vaccines and antiviral agents none. key: cord-282821-qvtvpnrr authors: thijsen, steven; heron, michiel; gremmels, hendrik; van der kieft, robert; reusken, chantal; kremer, kristin; limonard, gijs; bossink, ailko title: elevated nucleoprotein-induced interferon-γ release in covid-19 patients detected in a sars-cov-2 enzyme-linked immunosorbent spot assay date: 2020-06-12 journal: j infect doi: 10.1016/j.jinf.2020.06.015 sha: doc_id: 282821 cord_uid: qvtvpnrr nan various studies suggest a suppressed t-cell immunity in patients with severe covid-19 based on decreased t-cell numbers or abnormal interferon gamma (ifnγ) expression by t-lymphocytes detected by flowcytometry. (2) (3) (4) the objective of the present study was to determine the functional t-cell responses to sars-cov-2 antigens (mosaic surface protein and nucleoprotein), by using an enzyme-linked immunosorbent spot (elispot) interferon-γ release assay, in patients with rt-pcr confirmed covid-19 (n=27) and healthy controls (n=16). of the 27 covid-19 patients, 9 were included from the icu and 18 from the pulmonary ward. the moment of sampling varied from 6 to 32 days post onset of symptoms (dps). in addition, the concomitant humoral immune response was assessed by detection of sars-cov-2 specific iga and igg antibodies directed against the structural protein (s1 domain) of sars-cov-2 with a commercial elisa (euroimmun medizinische labordiagnostika ag, lϋbeck, germany). our results show that the sars-cov-2-specific t-cell response measured in the elispot versus the dps induced by the mosaic surface protein and the nucleoprotein showed different patterns. in all but one of the 27 covid-19 cases the t-cell response against the mosaic surface protein was absent or weak, as shown by the elispot results which were lower than 20 spot forming cells (sfc). the outlier was recruited from the pulmonary ward and exhibited 45 sfc (fig. 1a) . in contrast, the tcell response against the nucleoprotein measured by the elispot assay was elevated (10-150 sfc) in 12 of 19 patients (63%) that were sampled at ≥14 dps ( fig. 1b) . a subgroup of 9 (fig. 1b, red oval) showed a delayed or reduced t-cell response against the nucleoprotein, compared to the other patients. five of these showed practically no response, and four showed a weak response (10-20 sfc) at 18-32 dps. absolute lymphocyte numbers loaded in the sars-cov-2 elispot were not significantly different between the normal and reduced responders (data not shown). however, the number of spot forming cells by using the mitogen control stimulant was also significant lower in the delayed or reduced responders (p<0.001) (fig. 3) . moreover, sars-cov-2 iga and igg antibody levels did not differ between the normal and delayed or reduced responders (data not shown). serology showed a sigmoidal pattern, with a sharp increase in specific iga (fig. s1) and igg antibodies (fig. 1c) against the structural protein (s1 domain) of sars-cov-2 around 14 and 15 dps, respectively. most of the healthy controls showed antibody levels below the cut-off. except 4 controls, who showed detectable anti-sars-cov-2 iga antibody levels, while anti-sars-cov-2 igg antibodies were negative (fig. s2) . as none of the healthy controls had more than nine sfc specific for the sars-cov-2 nucleoprotein, 10 or more spots was determined to be indicative for covid-19 disease. prolonged illness, when sampled more days post onset of symptoms, increased the chance of finding higher number of spots. roc analysis was performed for the nucleoprotein elispot results and >7, >14 and >21 dps. all roc analyses showed significant areas under the roc curve, respectively 0.77 (p=0.004), 0.82 (p=0.001) and 1 (p=0.002) for detection of covid-19 disease (fig. 1f) . interestingly, in a recent published study by grifoni et al.(5) sars-cov-2 epitope pools were used to probe cd4+ t cell responses. they found that m, spike and n proteins were co-dominant each recognized by 100% of covid-19 cases studied. with respect to sars-cov-2 cd8+ t cell responses, the spike protein was even less dominant, significant reactivity was noted for m, n and other antigens. in agreement, in our study t-cell reactivity was detected with sars-cov-2 elispot against the n protein. in contrast however, the mosaic surface protein, consisting of exposed healthy controls (green symbols). the broken line represents the cut-off for the sars-cov-2 antibody elisa. figure 1f depicts the roc analyses of the sars-covdynamic profile for the detection of anti-sars-cov-2 antibodies using four immunochromatographic assays clinical and immunological features of severe and moderate coronavirus disease 2019 suppressed t cellmediated immunity in patients with covid-19: a clinical retrospective study in wuhan dysregulation of immune response in patients with covid-19 in wuhan, china targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals key: cord-301744-rx7ywew5 authors: kelleni, mina t. title: sars cov-2 viral load might not be the right predictor of covid-19 mortality date: 2020-08-15 journal: j infect doi: 10.1016/j.jinf.2020.08.018 sha: doc_id: 301744 cord_uid: rx7ywew5 nan in their recent correspondence, pujadas et al. have reported sars-cov-2 viral load at diagnosis as an independent predictor of mortality. they've reported the mean log10 viral load significantly differed between patients who were alive (n=807; mean log10 viral load 5·2 copies per ml [sd 3]) versus those who had died (n=338; 6·4 copies per ml [2·7]) by the end of the study period 1 . the author would like to argue the clinical significance of their findings as it's apparent from the standard deviation of their reported results that some patients have survived though their initial detected viral load has been like or above the mean viral load in the deceased group and vice versa. noteworthy, a systematic review of literature has previously demonstrated that seven studies observed increases in sars cov-2 viral loads prior to clinical deterioration and vice versa, yet it's also reported other seven studies that found little to no difference in viral load between pre-symptomatic, asymptomatic and symptomatic patients 2 . in a trial to explain the apparent contradictory results found in different studies as well as the lack of a distinct boundary between the viral loads that might be associated with a higher mortality rate or a higher recover rate; the author would like to suggest that sars cov-2 viral load should be only considered as a personalized reflection to the immune response to covid-19 as well as to the genetic polymorphisms in sars cov-2 receptors 3 . ace2 polymorphisms might be a better field of study than sars cov-2 viral load wishing to develop a genetic test that might predict and exempt, if possible, from covid-19 related duty those who are more vulnerable to complications and mortality 4 sars-cov-2 viral load predicts covid-19 mortality sars-cov-2 detection, viral load and infectivity over the course of an infection ace2 receptor polymorphism: susceptibility to sars-cov-2, hypertension, multi-organ failure, and covid-19 disease outcome hypothesis article: covid-19 unexplained mortality in the young adults: could it be due to ace2 polymorphisms? key: cord-292451-2tpef19n authors: komiya, kosaku; yamasue, mari; takahashi, osamu; hiramatsu, kazufumi; kadota, jun-ichi; kato, seiya title: the covid-19 pandemic and the true incidence of tuberculosis in japan date: 2020-07-07 journal: j infect doi: 10.1016/j.jinf.2020.07.004 sha: doc_id: 292451 cord_uid: 2tpef19n nan a recent report in the present journal focused on the decreased incidence of tuberculosis during the covid-19 pandemic in taiwan. 1 a declining trend in influenza following the covid-19 outbreak has already been indicated in brazil, singapore and japan. [2] [3] [4] according to the infectious diseases weekly report japan, 5 besides influenza, the incidences of mycoplasma pneumoniae pneumonia, respiratory syncytial virus infection, infectious gastroenteritis, epidemic keratoconjunctivitis and other droplet or contact transmission diseases are also markedly lower than those in the same periods in previous years. the covid-19 pandemic has brought about lifestyle changes, including the encouragement of wearing masks, handwashing, maintaining social distance and suspension of mass gatherings. these prevention measures for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) transmission may have contributed to a decline in many types of infectious diseases. lai et al. suggested that these interventions for infection control may also positively influence pulmonary tuberculosis. 1 however, whether or not the decline in tuberculosis incidence is actually due to these prevention measures, as with other respiratory infectious diseases, is unclear. tuberculosis can be pathologically divided into primary and secondary (reactivation) tuberculosis. an increase in the numbers of elderly patients with newly diagnosed tuberculosis has been observed worldwide, and this disease typically develops as a result of reactivation of a latent tuberculosis infection a long time, usually several dozen years, after the initial tuberculosis infection. 6 as such, the infection control measures enacted to prevent sars-cov-2 transmission a short time after the covid-19 outbreak are not expected to influence the trend in tuberculosis incidence. in japan, as seen in taiwan none. the covid-19 pandemic and tuberculosis in taiwan. the journal of infection different patterns of influenza a and b detected during early stages of covid-19 in a university hospital in são paulo, brazil. the journal of infection unintended consequence: influenza plunges with public health response to covid-19 in singapore. the journal of infection seasonal influenza activity during the sars-cov-2 outbreak in japan ministry of health law, diseases nioi tuberculosis in the global aging population the authors thank mr. shinya takano (srl, tokyo, japan) for providing data. key: cord-266036-qhlo99l7 authors: axell-house, dierdre b.; lavingia, richa; rafferty, megan; clark, eva; amirian, e. susan; chiao, elizabeth y. title: the estimation of diagnostic accuracy of tests for covid-19: a scoping review date: 2020-08-31 journal: j infect doi: 10.1016/j.jinf.2020.08.043 sha: doc_id: 266036 cord_uid: qhlo99l7 objectives: to assess the methodologies used in the estimation of diagnostic accuracy of sars-cov-2 real-time reverse transcription polymerase chain reaction (rrt-pcr) and other nucleic acid amplification tests (naats) and to evaluate the quality and reliability of the studies employing those methods. methods: we conducted a systematic search of english-language articles published december 31, 2019-june 19, 2020. studies of any design that performed tests on ≥10 patients and reported or inferred correlative statistics were included. studies were evaluated using elements of the quality assessment of diagnostic accuracy studies (quadas-2) guidelines. results: we conducted a narrative and tabular synthesis of studies organized by their reference standard strategy or comparative agreement method, resulting in six categorizations. critical study details were frequently unreported, including the mechanism for patient/sample selection and researcher blinding to results, which lead to concern for bias. conclusions: current studies estimating test performance characteristics have imperfect study design and statistical methods for the estimation of test performance characteristics of sars-cov-2 tests. the included studies employ heterogeneous methods and overall have an increased risk of bias. employing standardized guidelines for study designs and statistical methods will improve the process for developing and validating rrt-pcr and naat for the diagnosis of covid-19. after its emergence in december 2019, the virus now known as sars-cov-2 was identified and sequenced in early january 2020, 1 allowing for the rapid development of diagnostic testing based on the detection of viral nucleic acid (i.e., real-time reverse transcription polymerase chain reaction [rrt-pcr]). 2 because infected patients can present with non-specific symptoms or be asymptomatic, the development of accurate diagnostic tests for both clinical and epidemiological purposes was a crucial step in the response to the covid-19 pandemic. 3 in the united states, the spread of sars-cov-2 rapidly outpaced the capacity to test for it, resulting in the food and drug administration (fda) relaxing regulatory requirements to increase testing availability. the fda granted the first emergency use authorization (eua) for a sars-cov-2 rrt-pcr diagnostic test on february 4, 2020. consequently, hundreds of tests for sars-cov-2, among them rrt-pcrs, other types of nucleic acid amplification tests (naats), and automated and/or multiplex methods based on proprietary platforms, obtained fda emergency use authorization (eua). as of august 4 th , 2020, the fda has granted euas to 203 diagnostic tests, including 166 molecular tests, 35 antibody assays, and 2 antigen tests. although the fda began requiring the submission of validation methods and results as part of eua application for sars-cov-2 diagnostic tests, these tests were not initially required to undergo the rigorous assessment that would normally be part of the fda approval process. researchers also began developing alternative nucleic-acid based methodologies to detect sars-cov-2, including reverse-transcription loop-mediated isothermal amplification (rt-lamp), and others. concurrently with rapid test production, publications emerged reporting clinical diagnostic test performance characteristics, such as "sensitivity" and "specificity", though some lacked the rigorous methodologies usually required to formally estimate diagnostic accuracy. here we present a scoping review of the literature with two main objectives: 1) to assess the methodologies used in the estimation of diagnostic accuracy of sars-cov-2 tests and 2) to evaluate the quality and reliability of the studies employing those methods. searches were performed through medline (ovid), embase (elsevier), scopus, web of science, cinahl, and pubmed following the preferred reporting items for systematic reviews and meta-analyses (prisma) guidelines 4 between december 1, 2019 and june 19, 2020. the following search string was used: (2019-ncov or sars-cov-2 or sars-cov2 or covid-19 or covid19 or covid) and ("positive agreement" or "negative agreement" or "overall agreement" or "diagnostic accuracy" or "positive rate" or "positivity rate" or "test performance" or "reference standard" or "gold standard" or sensitivity or specificity or "percent agreement" or "concordance" or "test agreement" or "predictive value" or "false negative" or "false positive") and ("polymerase chain reaction" or pcr or "reverse transcriptase" or "nucleic acid amplification test" or naat or isothermal or "rt-lamp" or "rt-pcr" or "molecular test"). the literature hub litcovid's "diagnosis" section was screened in its entirety once and then daily for relevant titles. we liberally screened articles by title and abstract for further evaluation. articles were included if they met the following criteria on screening: 1) peer-reviewed publication, 2) study evaluated diagnostic test accuracy of naat, 3) diagnostic test performed on ≥10 patients, 4) diagnostic/clinical sensitivity, specificity, other correlative statistics, or test positive rate were either identified by name or were included in the publication as a numerical value and we could reproduce the calculations. exclusion criteria included: 1) pre-print status, 2) guidelines, consensus, review, opinion, and other summary articles 3) entirely pregnant or pediatric populations, 4) overlap of study population with another included publication. four authors independently extracted data and two authors reviewed data for accuracy. for study characteristics, we extracted: first author name, country, study design, patient population, total number of patients or samples included in test performance calculations, and number of cases according to rrt-pcr (tables 1-5) or total number of cases based on positive result of any platform tested (table 6) . for patient characteristics, we extracted age and sex. for index test and reference standard characteristics, we extracted: test type (naat) or definition (clinical diagnosis, composite reference standards), specimen (naat), specimen dry/collection liquid status (for studies evaluating abbott id now), proprietary automated and/or multiplex systems -henceforth called "platforms" (naat), and target genes of primers (naat). for outcomes, we extracted the values of test performance characteristics with their designation according to the original authors, without our interpretation. for this reason, we indicate these outcomes as "reported" (r): reported sensitivity (rsn), specificity (rsp), positive predictive value (rppv), negative predictive value (rnpv), accuracy (racc), positive percent agreement (rppa), negative percent agreement (rnpa), overall agreement (roa), and kappa coefficient. additionally, we extracted "positive rate," a non-standard term used by the included studies to refer to the number of positive naats in a population of patients suspected to have covid-19 (table 1) , or to the number of positive samples in a total population of positive samples after repeat testing (table 2) . we constructed 2 x 2 contingency tables and reproduced test performance characteristic calculations to demonstrate the methods of how the original authors obtained the values (supplementary table 1) . we report additional pertinent study data in supplementary table 2 : enrollment dates, number of sites of enrollment, symptomatic status, and chest radiology status. no articles were excluded on the basis of quality in order to present the most comprehensive summary of the currently available evidence. we presented the extracted data in tabular form mirrored by a descriptive synthesis 5 in two broad categories: diagnostic accuracy studies for rrt-pcr (tables 1-3) , and diagnostic accuracy or comparative agreement studies of two naats (tables 4-6 ). tables are thematically divided based on the reference standard strategy, or approach to obtaining comparative agreement measures. diagnostic accuracy studies for rrt-pcr were arranged alphabetically in tables by first author last name (tables 1-3) . diagnostic accuracy and comparative agreement studies for two naats were arranged by decreasing order of studies per methodology, then alphabetically by methodology or platform (tables 4-6) for easy comparison. due to significant diversity in methods and reporting of results, we reported grouped summary data for study characteristics, patient characteristics, and outcomes. we used the framework of the quality assessment of diagnostic accuracy studies (quadas-2) 6 to evaluate our selected articles (supplementary table 3 ). we collected data, or noted their absence, for a narrative description of risk of bias and concerns of applicability based on the quadas domains. for assessment of bias in patient selection, we evaluated author conflicts of interest, study design type, inclusion/exclusion criteria, method of patient enrollment, and reporting of patient demographics and characteristics (i.e. symptomatic status). for assessment of bias in reference standard and index test, we evaluated the accuracy of the reference standard, the description of duration of symptoms at time of testing, whether the threshold to determine a positive test was prespecified, and researcher blinding to reference standard and index test results. for assessment of bias in flow and timing, we evaluated whether the reference standard was the same for all patients, the sequence and timing of the performance of the reference standard and index test, whether test performance characteristics were calculated based on sample numbers or patient numbers, and whether indeterminate or invalid results were included in test performance calculations. our search yielded 1537 articles, with 816 unique articles after deduplication. after screening title and abstract, 130 articles underwent full text evaluation. ultimately, 49 articles were included in our review ( figure 1 ). three studies, with 19 to 1014 patients, report a "positive rate" as the number of positive rrt-pcr out of the number of suspected cases of covid-19, with a range of 38.42% to 59% (table 1) . [7] [8] [9] the studies do not report these values as "sensitivity" directly, however these concern that their low calculated positive rates (38.42% and 47.4%, respectively) were indicative of a failure of rrt-pcr to diagnose covid-19. 8, 9 in terms of quality assessment, the studies lack specific details as to how patients were classified as having suspected covid-19 infection. the accuracy of clinical diagnosis based on case definitions is unclear but is likely not ideal for diagnosis. additionally, duration of symptoms at the time of clinical diagnosis or rrt-pcr testing was not provided (supplementary table 3 ). eight studies, with a range of 36 to 22,061 patients per study, attempted to determine the accuracy of rrt-pcr by comparing the initial rrt-pcr result to the result after multiple repeated samples from the patient submitted for rrt-pcr testing, which was called the reference standard (table 2) . [10] [11] [12] [13] [14] [15] [16] [17] three studies reported this value as a "positive rate," ranging from 51.25% to 88%, 10, 14, 17 and five reported sensitivity, with a range of 57.9% to 94.6%. [11] [12] [13] 15, 16 of these studies, only he et al included an rsp of 100%, calculated from patients who remained negative for sars-cov-2 after repeated sample testing (supplementary table 1 ). 13 green et al. included patients in their study regardless of whether they were tested once or multiple times, using data from these subsets of patients to make assumptions for estimating clinical test characteristics. in addition, this study also conducted multiple different naats and rrt-pcrs on patients, whereas other studies employing this strategy used only one type of naat. additionally, the authors do not clarify whether patients who had repeat sars-cov-2 test were consistently tested with the same naat/rrt-pcr test or a different one. they also calculated test performance characteristics differently from other studies: two estimates of sensitivity were calculated, one in which the rate of false negatives for single-tested patients was 0%, and one in which the "false negative" rate was the same as in repeat-tested patients in their study of approximately 16.8%. 12 however, the details of how they calculated test characteristics were not presented. to clarify the two assumptions made in the calculations, we in terms of quality assessment, most of the studies were performed with non-cohort design, and six consisted of only patients who were determined to have covid-19 by rrt-pcr, i.e. cases only (table 2) . 10, 11, [14] [15] [16] five of the studies had inclusion criteria which caused pertinent patients to be excluded by necessitating patients to have had a well-performed ct chest or x-ray (supplementary table 3 ). this excluded several patients who would otherwise have been pertinent to the study of test diagnostic accuracy. 10, 11, 13, 16, 17 the studies involved repeating rrt-pcr several times for a reference standard, but each patient received a different number of repeat tests over a different time period, resulting in each patient receiving a different reference standard. one study tracked negative-to-positive conversion over 1 to 49 days, 12 and another tracked over 1 to 14 days, 13 leading to concern that potentially a patient could have been infected in the time between the initial test and the final test and confounding results. one study counted invalid results as negative results and indeterminate results as positive results when calculating test performance characteristics, 12 otherwise the rationale and ways invalid and indeterminate results were handled were not reported in these studies. three studies determined the accuracy or agreement of rrt-pcr or automated rrt-pcr platforms/instruments compared to a reference standard based on the results of several tests as a "composite reference standard" (table 3) . [18] [19] [20] there were between 58 and 184 patients per study. suo et al considered a positive result of either repeated measurements of rrt-pcr or serology to indicate a positive test according to the reference standard; reported sensitivity of initial rrt-pcr result was 40%, rsp 100%, rppv 100%, and rnpv 16%. 19 zhen et al compared rrt-pcr performed according to the us cdc protocol to a composite reference standard in which the consensus result of 3 or more out of 4 molecular assays was considered the correct result. the rrt-pcr had an rppa of 100%, an rnpa of 98%, and cohen's kappa coefficient of 0.98. 20 cradic et al did not study rrt-pcr but studied three automated molecular assays and used a composite reference standard of the consensus result of two or more of the three assays. while abbott id now had a rppa of 91%, the roche cobas 6800 and diasorin simplexa assays had a rppa of 100%. 18 these studies either did not report how samples were selected for evaluation (supplementary table 3 ), 19, 20 or reported that only samples which had sufficient residual volume and had been properly stored were selected. 18 days after the initial test, after the patient had been discharged from the hospital, leading to potential exposure for initial infection or reinfection. 19 the performance other nucleic acid amplification test methods compared to standard rrt-pcr fourteen studies compared other nucleic acid amplification test methods to detect sars-cov-2 to rrt-pcr (table 4) 30 suo et al evaluated digital droplet polymerase chain reaction (ddpcr), with rsn 94%, rsp 100%, rppv 100%, rnpv of 63%, and racc 95%. 19 bulterys et al study evaluated an isothermal amplification method with rsn 82.8% and cohen's kappa 0.86. 31 wang, cai, and zhang et al evaluated one-step single-tube nested quantitative polymerase chain reaction (osn-qrt-pcr) with cohen's kappa of 0.737. 32 regarding evaluation of quality (supplementary table 3) , the majority of studies did not report how patient samples were selected for evaluation. 21, 22, 24, 25, [27] [28] [29] [30] 32 in the study conducted by bulterys et al, sample selection was a convenience selection of samples with residual volume that had been stored correctly. 31 most studies did not report symptomatic status of the patient 21-28,30-32 or patient demographics. [21] [22] [23] [24] [25] [27] [28] [29] [30] [31] [32] problematically, many of the studies did not report when the reference standard was conducted on the patient samples compared to the index test, or whether actions that could potentially alter test results (such as freeze/thaw cycles) occurred between reference standard or index test. [21] [22] [23] [24] 27, 28, 31 four studies calculated test performance characteristics based on number of samples rather than number of patients. 23, 27, 30, 32 the management of indeterminate and invalid test results went largely unreported. [21] [22] [23] [24] [25] 27, 30 the performance of naat platforms compared to rrt-pcr as the reference standard fifteen studies compared automated naat platforms to various rrt-pcr assays to determine test performance characteristics ( other studies evaluated ausdiagnostics (rsn 100%, rsp 92.16%), 43 hologic panther fusion (rppa 98.7%, rnpa 98.1%), 44 luminex nxtag (rsn 97.8%, rsp 100%), 45 mesa biotech accula (rppa 68.0%, rnpa 100%), 46 or qiastat-dx (rsn 100%, rsp 93%) 47 compared to rrt-pcr. with regards to quality evaluation (supplementary table 3 ), most studies did not report method of sample collection/patient recruitment, 33, 35, 37, [41] [42] [43] [44] [45] [46] [47] and four studies conducted a convenience selection of samples, including enrichment for positive samples. [34] [35] [36] 39 eight studies conducted test performance calculations on sample numbers instead of patient numbers. 33, 35, 36, 38, 39, [42] [43] [44] four studies conducted calculation of test performance characteristics with indeterminate or inconclusive results as "positive," 35, 38, 39, 42 and the management of indeterminate/inconclusive as well as invalid results went unreported in an additional three studies. 33, 37, 47 no study reported the blinding of researchers to the reference standard or index test results. ten studies, containing between 15 and 524 patients per study, evaluated the agreement between two different types of naat platforms ( in the studies, some platforms were identified as the "comparator" or "reference" platforms, including cepheid xpert xpress, 49 abbott realtime, 34, 48 hologic panther fusion, 50, 51 and roche cobas 6800, 52,55 and these were listed as "platform #1" in table 6 . three studies did not identify any studied platform as the "comparator" or "reference standard," and instead only reported general, non-directional measures of agreement such as overall agreement, cohen's kappa, or alternatively, the calculations of ppa and npa were identical no matter their method of calculation (supplementary table 1) . 39, 53, 54 regarding quality evaluation (supplementary table 3 ), the samples used for calculating test performance characteristics were reported to be selected for enrichment of positive samples, 34, 39 for diversity of viral load, 52,54 otherwise curated, 50 or the method of selecting samples was unreported. 49, 51, 53, 55 symptomatic status of the patients was largely unreported. 39, [49] [50] [51] [52] [53] [54] [55] five studies included samples where one test was conducted, then interim freezing, cooling, or other storage, before performance of the second test. 39, [50] [51] [52] 54 two studies did not report the sequence of testing of the two platforms or interim handling or storage of the samples. 49, 53 the status of researcher blinding to either platform result was not reported in any study. in our scoping review of 49 articles concerning test performance characteristics of rrt-pcr and other naat used for the diagnosis of covid-19, we were able to observe several overarching themes. clinical diagnosis by the case definition for covid-19 used in the early period of the pandemic does not correlate well with positive rates of covid-19 rrt-pcr ( table 1 ). the result of the initial rrt-pcr performed on a patient, if negative, may not be reflective of the result after multiple repeated rrt-pcrs for that patient ( table 2) . several alternative naat methods, many of which are easier or faster to perform, may be comparable to standard rrt-pcr (table 4 ). proprietary multiplex, automated, and/or point-of-care methods are comparable to in accuracy to rrt-pcr (table 5 ) and to each other (table 6 ), although the abbott id now sars-cov-2 test appears to have lower comparative agreement to other platforms. 34, [48] [49] [50] [51] [52] these findings should be viewed cautiously as the sars-cov-2 tests in these studies have not undergone rigorous evaluation necessary for fda approval due to the emergency state generated by the covid-19 pandemic. in addition, during our scoping review, we found substantial heterogeneity among available studies in terms of test types, reference standards, metrics, and details of study design and methodology. we categorized the included studies by four different reference standard strategies: clinical diagnosis/case definitions (table 1) , repeated index testing (table 2) , composite reference standard (table 3) , and rrt-pcr (table 4 and 5) . additionally, we identified a fifth category, where instead of using a reference standard, comparative agreement between two naat platforms was calculated (table 5 and 6 ). the main limitation of the first group of studies (table 1 ) was the use of a "case definition" as the reference standard to report a "positive rate" of rrt-pcr. during novel disease outbreaks, standard case definitions are often developed to assist clinicians in case identification before a diagnostic test is available. unfortunately, the studies included in this group were unable to use a clear case definition; instead they refer to a population of "suspected cases," for which the definition is not reported. [7] [8] [9] because this group enrolled patients prior to february 15, 2020 in china, during the time in which the chinese national guideline for diagnosis and treatment of covid-19 (ngdtc) published five different versions of the covid-19 case definition, the case definitions in use at the time of these studies varied. 56 a recent study estimated that if a single guideline (specifically, version 5 of the ngdtc) had been used to identify cases from the beginning of the outbreak to february 20, 2020, there would have been more than three times as many identified cases in hubei province. 56 this is relevant to our review because the two largest studies that evaluated the rrt-pcr positive rate of patients with a clinical diagnosis of covid-19 took place in wuhan, hubei province, and included patients evaluated before february 14, 2020 7, 8 (supplementary table 2 ). this increased case estimate due to diagnosis of covid-19 based on case definition complicates the legitimacy and reported accuracy of the "positive rate" of rrt-pcr referred to in these studies. the second group assessed rrt-pcr test performance characteristics via repeated index rrt-pcr testing ( table 2 ). most studies in this group reported "sensitivity" by dividing the number of participants with positive baseline rrt-pcrs by the total number of participants who eventually had a positive rrt-pcr after repeated measurements. while such an approach may have some advantages over the use of a case definition alone as a reference standard, this strategy is, nonetheless, an imperfect solution with its own set of inherent limitations. sars-cov-2 infection is transient and the associated viral loads are time-varying because of the natural pathophysiology of the infection. therefore, the time interval between each repeated test becomes crucially important, and even relatively small time differences (and/or lack of uniformly used intervals) could complicate the interpretation of re-test results and their quality as reference standards. furthermore, repeated use of the same test as a reference standard for itself does not eliminate the inaccuracies or limitations of the test. such comparisons ultimately reflect the reliability of the test (assuming a short, uniform time interval between tests), rather than providing a true view of test accuracy. the third group of three studies calculated test performance characteristics of rrt-pcr according to a composite reference standard (table 3 ). using arbitrary rules to combine multiple different and imperfect tests inevitably creates a reference standard with some degree of bias. 57 furthermore, all three studies in this group included the test under evaluation as part of the composite reference standard, which leads to additional bias, described below. 58 use of a biased composite standard is likely to lead to reduced sensitivity, among other errors affecting true test performance characteristics. 59 the fourth group of studies evaluated sars-cov-2 diagnostic tests that are under development as well as proprietary testing platforms (most of which are based on standard rrt-pcr methods). these studies used traditional rrt-pcr as a reference standard; results are summarized in tables 4 and 5 , respectively. importantly, while these studies were not designed to estimate the accuracy of rrt-pcr, their results indicate that the index tests did not identify significantly more positive samples than rrt-pcr. finally, the last group of studies compared sars-cov-2 naat platforms (table 6) . these comparative accuracy studies examined the agreement between two non-reference standard tests. although most of the testing platforms evaluated in these studies were based on standard rrt-pcr, the agreement between two non-reference standard tests is not equivalent to test accuracy, as mentioned previously. this scoping review is limited by the lack of reporting of several key study features in the majority of the articles evaluated, which is an important indicator of quality and potential bias. based on the quadas-2 criteria, most of the included studies had concern for bias (supplementary table 3 ). the most prominent concerns were unclear inclusion/exclusion criteria, unclear method of enrollment/selection of patients and samples, and unclear handling of indeterminate/inconclusive and invalid results. additionally, many of the studies were conducted in a so-called "two gate" (case-control) design, in which cases and controls were known and selected ahead of time, rather than performing the test on a group of patients or samples with suspected covid-19. these factors likely incorporate bias that significantly confounds the results of the studies, thus, the accuracy of the tests in other settings with different prevalences (such as asymptomatic screening, other age groups) may not be truly generalizable. furthermore, few studies were able to evaluate both the index and reference tests simultaneously or within a short period of time, which is key to avoiding biases caused by changes in the patient's true disease status; this bias can also affect the diagnostic accuracy of the index test. the best approach to determining diagnostic test performance characteristics in the absence of a "gold" standard is an open question in diagnostic accuracy methodology. while many methods have been described, there are only a few well-defined statistical approaches that use a reference standard in lieu of a gold standard, reviewed elsewhere. 60 latent class analysis is one commonly used approach in situations in which neither the true error rates of the reference standard nor the true prevalence of the disease are known. this approach uses the results of a set of imperfect tests to estimate parameters related to sensitivity, specificity, and prevalence often using maximum likelihood methods. however, this is not the only method available and every method has its own strengths and limitations. 57 therefore, careful interpretation by studies that attempt to estimate test characteristics is warranted to account for and clarify the inherent limitations of assessing accuracy-related metrics when a gold standard is unavailable. evaluation of the performance characteristics of sars-cov-2 diagnostic tests is vital to control of the ongoing covid-19 pandemic. while more than 200 sars-cov-2 molecular diagnostic tests have received fda euas, we have described in this scoping review that the performance of few of these tests has been assessed appropriately. the lack of robust test performance that we noted in many studies published to date is undoubtably due in part to the critical need for tests, which resulted in accelerated test development. however, our scoping review also uncovered imperfect methods for estimating diagnostic test performance in the absence of a gold standard and demonstrate that the accuracy of these tests should be interpreted with caution. future studies would benefit from employing statistical methods such as latent class analysis and other methods referenced above to accurately analyze their data. indeed, instituting national requirements for test performance analysis and reporting, perhaps based on the existing fda guidelines on diagnostic tests, 61 would advance the goal of standardizing the evaluation sars-cov-2 diagnostic test performance. such an initiative would lead to statistically robust conclusions regarding the accuracy of the index test, which will in turn support hospitals and clinicians as they determine the optimal test to use for covid-19 diagnosis. table 2 . abbreviations-aigs: automatic integrated gene detection system, bal: bronchoalveolar lavage, ci: confidence interval, ddpcr: digital droplet polymerase chain reaction, e: envelope, iamp: isothermal amplification, iqr: interquartile range, κ: kappa statistic, n/a: not applicable, n: nucleocapsid, no.: number, nps: nasopharyngeal swab, nr: not reported, ops: oropharyngeal swab, orf1ab: open reading frame 1ab, osn-qrt-pcr: one-step single-tube nested quantitative real-time polymerase chain reaction, racc: reported accuracy, rdrp: rna-dependent rna polymerase, ref stnd: reference standard, rnpa: reported negative percent agreement, rnpv: reported negative predictive value, roa: reported overall agreement, rppa: reported positive percent agreement, rppv: reported positive predictive value, rrt-pcr: real-time reverse transcription polymerase chain reaction, rsn: reported sensitivity, rsp: reported specificity, rt-lamp: reverse transcription loop-mediated isothermal amplification, rt-raa: reverse-transcription recombinase-aided amplification, s: spike, y: years. a novel coronavirus from patients with pneumonia in china genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding presymptomatic transmission of sars-cov-2 -singapore preferred reporting items for systematic reviews and meta-analyses: the prisma statement synthesis without meta-analysis (swim) in systematic reviews: reporting guideline quadas-2: a revised tool for the quality assessment of diagnostic accuracy studies correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases positive rate of rt-pcr detection of sars-cov-2 infection in 4880 cases from one hospital in comparison of different samples for 2019 novel coronavirus detection by nucleic acid amplification tests chest ct findings in coronavirus disease-19 (covid-19): relationship to duration of infection sensitivity of chest ct for covid-19: comparison to rt-pcr clinical performance of sars-cov-2 molecular testing diagnostic performance between ct and initial real-time rt-pcr for clinically suspected 2019 coronavirus disease (covid-19) patients outside wuhan, china testing for sars-cov-2: can we stop at two? clin infect dis diagnosis of the coronavirus disease (covid-19): rrt-pcr or ct? frequency and distribution of chest radiographic findings in covid-19 positive patients detection and analysis of nucleic acid in various biological samples of covid-19 patients clinical evaluation and utilization of multiple molecular in vitro diagnostic assays for the detection of sars-cov-2 ddpcr: a more accurate tool for sars-cov-2 detection in low viral load specimens comparison of four molecular in vitro diagnostic assays for the detection of sars-cov-2 in nasopharyngeal specimens development of a reverse transcription-loop-mediated isothermal amplification as a rapid early-detection method for novel sars-cov-2 evaluation of rapid diagnosis of novel coronavirus disease (covid-19) using loop-mediated isothermal amplification real-time reverse transcription loop-mediated isothermal amplification for rapid detection of sars-cov-2 a novel reverse transcription loop-mediated isothermal amplification method for rapid detection of sars-cov-2 rapid and visual detection of 2019 novel coronavirus (sars-cov-2) by a reverse transcription loop-mediated isothermal amplification assay multiple-centre clinical evaluation of an ultrafast single-tube assay for sars-cov-2 rna a reverse-transcription recombinase-aided amplification assay for rapid detection of the 2019 novel coronavirus (sars-cov-2) multiplexing primer/probe sets for detection of sars-cov-2 by qrt-pcr triplex real-time rt-pcr for severe acute respiratory syndrome coronavirus 2 development of an automatic integrated gene detection system for novel severe acute respiratory syndrome-related coronavirus (sars-cov 2) comparison of a laboratory-developed test targeting the envelope gene with three nucleic acid amplification tests for detection of sars-cov-2 novel one-step single-tube nested quantitative real-time pcr assay for highly sensitive detection of sars-cov-2 evaluation of the covid19 id now eua assay comparison of two commercial molecular tests and a laboratory-developed modification of the cdc 2019-ncov rt-pcr assay for the detection of sars-cov-2 comparison of abbott id now, diasorin simplexa, and cdc fda eua methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from individuals diagnosed with covid-19 validation and verification of the abbott realtime sars-cov-2 assay analytical and clinical performance multi-center evaluation of the cepheid xpert xpress sars-cov-2 assay for the detection of sars-cov-2 in oropharyngeal swab specimens comparison of commercially available and laboratory developed assays for in vitro detection of sars-cov-2 in clinical laboratories multicenter evaluation of the cepheid xpert xpress sars-cov-2 test rapid and sensitive detection of sars-cov-2 rna using the simplexa tm covid-19 direct assay clinical evaluation of the cobas sars-cov-2 test and a diagnostic platform switch during 48 hours in the midst of the covid-19 pandemic comparison of sars-cov-2 detection from nasopharyngeal swab samples by the roche cobas 6800 sars-cov-2 test and a laboratory-developed real-time rt-pcr test interpret with caution: an evaluation of the commercial ausdiagnostics versus in-house developed assays for the detection of sars-cov-2 virus comparison of the panther fusion and a laboratory-developed test targeting the envelope gene for detection of sars-cov-2 clinical performance of the luminex nxtag cov extended panel for sars-cov-2 detection in nasopharyngeal specimens of covid-19 patients in hong kong comparison of the accula sars-cov-2 test with a laboratory-developed assay for detection of sars-cov-2 rna in clinical nasopharyngeal specimens evaluation of the qiastat-dx respiratory sars-cov-2 panel, the first rapid multiplex pcr commercial assay for sars-cov-2 detection comparison of abbott id now and abbott m2000 methods for the detection of sars-cov-2 from nasopharyngeal and nasal swabs from symptomatic patients performance of abbott id now covid-19 rapid nucleic acid amplification test in nasopharyngeal swabs transported in viral media and dry nasal swabs, in a new york city academic institution five-minute point-of-care testing for sars-cov-2: not there yet clinical evaluation of three sample-to-answer platforms for the detection of sars-cov-2 comparison of cepheid xpert xpress and abbott id now to roche cobas for the rapid detection of sars-cov-2 the detection of sars-cov-2 using the cepheid xpert xpress sars-cov-2 and roche cobas sars-cov-2 assays comparison of two high-throughput reverse transcription-polymerase chain reaction systems for the detection of severe acute respiratory syndrome coronavirus 2 clinical evaluation of a sars-cov-2 rt-pcr assay on a fully automated system for rapid ondemand testing in the hospital setting effect of changing case definitions for covid-19 on the epidemic curve and transmission parameters in mainland china: a modelling study evaluation of diagnostic tests when there is no gold standard. a review of methods using a combination of reference tests to assess the accuracy of a new diagnostic test value of composite reference standards in diagnostic research diagnostic test evaluation methodology: a systematic review of methods employed to evaluate diagnostic tests in the absence of gold standard -an update food and drug administration cfdarh. statistical guidance on reporting results from studies evaluating diagnostic tests key: cord-305330-mklkugj5 authors: moiseev, sergey; avdeev, sergey; brovko, michail; akulkina, larisa; fomin, victor title: cancer in intensive care unit patients with covid-19 date: 2020-05-28 journal: j infect doi: 10.1016/j.jinf.2020.05.053 sha: doc_id: 305330 cord_uid: mklkugj5 nan susceptible to severe acute respiratory syndrome corona virus 2 (sars-cov-2) infection and complications, although data on covid-19 and malignancies remain limited. 1 in a small study, liang et al. noted that patients with cancer were more likely to experience severe sequelae of sars-cov-2 infection, such as intensive care admission, invasive ventilation or death. 2 however, wang and zhang argued that the most important morbidity factor is exposure to an infection source, whereas worse outcomes from sars-cov-2 infection could be associated (at least partly) with older age of patients with cancer 3 . xia and colleagues also concluded that current evidence is insufficient to confirm an association between cancer and covid-19. 4 these issues have important implications for management of patients with malignancies during pandemics of covid-19 that continues to evolve in many countries including russia. in a nationwide study, we evaluated the prevalence of malignancies among 1307 intensive care unit (icu) patients with sars-cov-2 pneumonia who required respiratory support. medical records were submitted via internet by the covid-19 hospitals located in 60 regions across russia to the federal center at the sechenov university (moscow) that provided advice on management of patients. diagnosis of sars-cov-2 pneumonia was confirmed both by polymerase chain reaction (pcr) and ct. in patients with inconclusive or pending results of pcr, sars-cov-2 pneumonia was defined as severe acute respiratory infection with typical ct findings (bilateral multilobar ground-glass opacification with a peripheral or posterior distribution, or multifocal consolidative opacities superimposed on ground-glass opacification) 5 and no other obvious aetiology. various tumors were reported by the local physicians in 31 patients (2.4%). however, only 19 patients (1.5%) had active tumors or underwent chemotherapy or surgery in the past 3 months ( table 1) . as expected, lung and breast cancers were the most common malignancies. median age of patients was 66 years. most patients were older than 60 and/or had comorbidities, such as cardiovascular diseases and type 2 diabetes. in summary, the prevalence of malignancies was low among icu patients with sars-cov-2 infection and did not exceed that in the general population. therefore, patients with malignant tumors were not overrepresented in this cohort of patients with severe infection. we realize that our reassuring findings may be misleading, since we do not know the total number of cancer patients who contracted sars-cov-2 in russia and cannot definitely conclude that malignancy did not worsen outcomes of covid-19. however, our data suggest that other factors, such as older age and comorbidities, contribute significantly to the more severe course of sars-cov-2 infection in cancer patients. we declare no competing interests. clinical characteristics and prognosis in cancer patients with covid-19: a single center's retrospective study key: cord-019964-9leljj8j authors: nan title: recent research in infectious disease date: 2005-01-22 journal: j infect doi: 10.1016/j.jinf.2004.11.005 sha: doc_id: 19964 cord_uid: 9leljj8j nan value was 83.8%, and negative predictive value was 98.6%. the sensitivity of the assay was significantly higher than that of antepartum culture (54%). furthermore, many clinicians use a risk-based approach to gbs colonization by treating high-risk women with empiric antibiotics without waiting for culture results. the assay was also significantly more sensitive than the risk-factor approach (94% versus 42%) with regard to predicting intrapartum status. the quick and accurate results obtained with the idi-strep b assay during labor suggest that its use would lead to reduced rates of neonatal gbs colonization and the more judicious use of antibiotics. 2004;39:1129-35. how positive blood culture results are reported has an impact on outcomes of bsi st. louis (md consult)-many patients with bloodstream infection (bsi) do not receive adequate therapy, even after the final microbiologic report is available. the method of reporting microbiologic information to the physician has a significant impact on whether and how this information is used, according to the october 15 clinical infectious diseases. dr. emilio bouza and colleagues of hospital general universitario 'gregorio marañón' in madrid evaluated data from 197 bsis. microbiologic results were reported in 3 different ways. in 109 cases (group a), the physician was informed by telephone of the results and given a written report after the definitive results were known. in 99 cases (group b), physicians also received a written report at the bedside, along with written therapeutic recommendations. in 89 cases (group c), physicians also received clinical advice orally. during empiric treatment, 27.7% of patients received inappropriate treatment, and 13.7% received no treatment. these patients had significantly longer hospital stays (mean, 27.2 versus 19.4 days) and a higher risk of having clostridium difficile-associated diarrhoea (8.3% versus 1.9%), infection-related death (23.3% versus 13.6%), and all-cause mortality (30.8% versus 19.4%) than patients whose empiric therapy was appropriate. after the reports were received, therapy was changed for 52.3% of patients in group b and 53.1% in group c but for none of the patients in group a. consequently, the proportion of days during which patients received adequate treatment was significantly smaller in group a than in groups b and c (66.3% versus 92.1% and 91.2%, respectively). group a also received significantly fewer defined daily doses of appropriate antibiotics (16.4 versus 22.2 and 20.7 days) . the duration of hospitalization, mortality rates after reports were received, and costs also tended to be lower in groups b and c. these results suggest that written and/or oralalert reports providing clinical advice in addition to standard reports will improve the management of patients with bsi. dr. lynda anne szczech and colleagues of the women's interagency hiv study recruited 2,038 hiv-positive from october 1994 through november 1995. differences in the development of a new acquired immunodeficiency virus-defining illness (adi) or death were compared according to renal status and haart status. at baseline, 287 patients (14.1%) had proteinuria. these patients had significantly lower cd4c lymphocyte counts and higher viral loads than patients without proteinuria, both at baseline and before starting haart. before haart was widespread, proteinuria was associated with a significantly increased risk of developing a new adi (hazard ratio [hr], 1.37) and death (hr, 1.35). an elevated creatinine level was also a significant independent risk factor for mortality (hr, 1.72 per decrease in the inverse creatinine level). however, even after haart was initiated, proteinuria was a significant independent predictor of mortality (hr, 2.07). an elevated creatinine level was also a significant predictor of new-onset adi and death (hrs, 1.54 and 1.96 per decrease in the inverse creatinine level, respectively. whether the kidney modulates hiv disease or whether these findings merely reflect the impact of greater comorbidity is not known. what is clear, however, is that hiv-positive women with these findings need improved monitoring and more aggressive treatment. genetic and transmission analysis of helicobacter pylori strains within a family raymond j, thiberge j-m, chevalier c, kalach n, bergeret m, labigne a, et al. to look for evidence of intrafamilial infection, we isolated 107 helicobacter pylori clones from biopsied specimens taken from both parents and four children. we compared the sequences of two housekeeping genes (hspa and glmm) from these clones with those of 131 unrelated strains from patients living in different geographic regions. strain relationships within the family were determined by analyzing allelic variation at both loci and building phylogenetic trees and by using multilocus sequence typing. both hspa-and glmm-based phylogenetic trees showed east asian and african branches. all samples from family members showed natural mixed infection. identical alleles found in some strains isolated from the children and parents, but not in the strains isolated from unrelated patients, demonstrated that strains have circulated within the family. several mechanisms, such as point mutations, intragenic recombination, and introduction of foreign (african) alleles, were shown to enhance strain diversity within the family. increasing reports of the appearance of novel nonmultiresistant methicillin-resistant staphylococcus aureus mrsa (mrsa) strains in the community and of the spread of hospital mrsa strains into the community are cause for public health concern. we conducted two national surveys of unique isolates of s. aureus from clinical specimens collected from nonhospitalized patients commencing in 2000 and 2002, respectively. a total of 11.7% of 2,498 isolates from 2000 and 15.4% of 2,486 isolates from 2002 were mrsa. approximately 54% of the mrsa isolates were nonmultiresistant (resistant to less than three of nine antibiotics) in both surveys. the majority of multiresistant mrsa isolates in both surveys belonged to two strains (strains aus-2 and aus-3), as determined by pulsed-field gel electrophoresis (pfge) and resistogram typing. the 3 aus-2 isolates and 10 of the 11 aus-3 isolates selected for multilocus sequence typing (mlst) and staphylococcal chromosomal cassette mec (sccmec) analysis were st239-mrsa-iii (where st is the sequence type) and thus belonged to the same clone as the eastern australian mrsa strain of the 1980s, which spread internationally. four predominant clones of novel nonmultiresistant mrsa were identified by pfge, mlst, and sccmec analysis: st22-mrsa-iv (strain emrsa-15), st1-mrsa-iv (strain wa-1), st30-mrsa-iv (strain swp), and st93-mrsa-iv (strain queensland). the last three clones are associated with community acquisition. a total of 14 sts were identified in the surveys, including six unique clones of novel nonmultiresistant mrsa, namely, sts 73, 93, 129, 75, and 80slv and a new st. sccmec types iv and v were present in diverse genetic backgrounds. these findings provide support for the acquisition of sccmec by multiple lineages of s. aureus. they also confirm that both hospital and community strains of mrsa are now common in nonhospitalized patients throughout australia. use of multiple nucleic acid amplification tests to define the infected-patient 'gold standard' in clinical trials of new diagnostic tests for chlamydia trachomatis infections martin dh, nsuami m, schachter j, hook ew 3rd, ferrero d, quinn tc, gaydos c nucleic acid amplification tests (naats) can be used to define the infected-patient 'gold standard' for the purpose of designing studies of the performance of chlamydia trachomatis diagnostic tests. it is unclear how many test results run by different naats and what combinations of specimens comprise the best infected-patient gold standard. we approached this question with data from a large study of the performance of a new naat. data were available from three endocervical swabs and a urine specimen collected from each of 1,412 women and tested by three different naats. results from all three assays were used equally in a rotating fashion to define the infected-patient gold standard. multiple different infectedpatient gold standards for estimating swab and urine specimen sensitivity and specificity for one naat method were created by varying the number and combinations of swab and urine comparator results with two different naats, the effect of changing the infected-patient gold standard definition was determined by constructing receiver-operator-like curves with calculated sensitivities and specificities for each test. the one-positive-of-two-results or two-positive-oftwo-results (same or two different assays) infected-patient gold standard definitions produced low sensitivity and low specificity estimates, respectively. if four comparator naat results were used, the any-three-positive-offour-results definition or the at-least-one-specimen-positive-by-each-of-two-comparator-assays definition appeared to provide better combinations of sensitivity and specificity estimates. the any-two-positive-out-of-three-results definition resulted in estimates that were as good as produced with the former two definitions. this analytic approach provides a means of clearly visualizing the effects of changing naat-based infected-patient gold standards and should be helpful in designing future studies of new c. trachomatis diagnostic tests. prospective study of human metapneumovirus infection in children less than 3 years of age konig b, konig w, arnold r, werchau h, ihorst g, forster j most lower respiratory tract infections (lrtis) in children under the age of 3 years are due to respiratory syncytial virus (rsv). epidemiological, host, and viral factors eventually account for the severity of lrtis, but they do not completely explain it. human metapneumovirus (hmpv) was recently identified in children with lrtis. in a population-based prospective multicenter study (the pri.de study, conducted in germany over 2 years), we tested 3,369 nasopharyngeal secretions from children younger than 3 years of age with lrtis for rsv a and b, influenza viruses (ivs) a and b, and parainfluenza viruses (pivs) 1 to 3. of the children requiring intensive care (nz85), 18% had hmpv infections, and 60% of these children were infected with hmpv in combination with rsv. we did not detect hmpv in a randomly selected subset of rsv-positive nasopharyngeal secretions (nz120) from children not requiring intensive care support. hmpv was detected in !1% of virus-negative samples from patients without intensive care support (nz620). our data support the hypothesis that coinfections with rsv and hmpv are more severe than infections with either rsv or hmpv alone, at least in children younger than 3 years of age. candida parapsilosis is an important cause of bloodstream infections in the health care setting. we investigated a large c. parapsilosis outbreak occurring in a community hospital and conducted a case-control study to determine the risk factors for infection. we identified 22 cases of bloodstream infection with c. parapsilosis: 15 confirmed and 7 possible. the factors associated with an increased risk of infection included hospitalization in the intensive care unit (adjusted odds ratio, 16.4; 95% confidence interval, 1.8 to 148.1) and receipt of total parenteral nutrition (adjusted odds ratio, 9.2; 95% confidence interval, 0.9 to 98.1). samples for surveillance cultures were obtained from health care worker hands, central venous catheter insertion sites, and medical devices. twenty-six percent of the health care workers surveyed demonstrated hand colonization with c. parapsilosis, and one hand isolate was highly related to all case-patient isolates by tests with the dna probe cp3-13. outbreak strain isolates also demonstrated reduced susceptibilities to fluconazole and voriconazole. this largest known reported outbreak of c. parapsilosis bloodstream infections in adults resulted from an interplay of host, environment, and pathogen factors. recommendations for control measures focused on improving hand hygiene compliance. heikkinen t, silvennoinen h, peltola v, ziegler t, vainionpaa r, vuorinen t, kainulainen l, puhakka t, jartti t, toikka p, lehtinen p, routi t, juven t background: influenza vaccination of healthy children is encouraged because children are frequently hospitalized for influenza-attributable illnesses. however, most children with influenza are treated as outpatients, and scarce data are available on the burden of influenza in these children. methods: we performed a prospective study of respiratory infections in preenrolled cohorts of children %13 years old during 2 consecutive respiratory seasons (2231 child-seasons of follow-up). at any sign of respiratory infection, we examined the children and obtained a nasal swab for the detection of influenza. the parents filled out daily symptom diaries. of all the enrollees, 94% remained active participants in the study. results: the average annual rate of influenza was highest (179 cases/1000 children) among children !3 years old. acute otitis media developed as a complication of influenza in 39.7% of children !3 years old. for every 100 influenza-infected children !3 years old, there were 195 days of parental work loss (mean duration, 3.2 days). influenza causes a substantial burden of illness on outpatient children and their families. vaccination of children !3 years old might be beneficial for reducing the direct and indirect costs of influenza in children. moscicki ab, ellenberg jh, crowley-nowick p, darragh tm, xu j, fahrat s background: the risk of developing the human papillomavirus (hpv)-associated precancer high-grade squamous intraepithelial lesion (hsil) in human immunodeficiency virus (hiv)-infected adolescents is unknown. we examined the risk of developing hsil among adolescents with and without hiv infection. methods: hiv-infected (nz172) and-uninfected (nz84) girls aged 13-18 years who were participating in a multicenter study of primarily horizontally acquired hiv infections in adolescents (reaching for excellence in adolescent health care) and who did not have hsil on cytologic examination at study entry or at the first follow-up visit were followed at 6-month intervals. hiv-uninfected girls were recruited for comparison in a 2:1 ratio (hiv infected: hiv uninfected). the primary outcome was cytologic diagnosis of hsil confirmed by expert review. results: incidence of hsil by the end of follow-up was higher for hiv-infected girls than for hiv-uninfected girls (21.5% vs. 4.8%, respectively). in multivariate analysis, use of hormonal (either estrogen/progesterone oral combination or medroxyprogesterone acetate intramuscular) contraceptives, high cervical mucous concentrations of interleukin (il)-12, a positive hpv test, and persistent low-grade squamous intraepithelial lesion (lsil) were significantly associated with the development of hsil. conclusions: the incidence of hsil was alarmingly high in hivinfected adolescent girls. however, when other predictors were considered in multivariate analysis, hiv status was not retained in the model. the heightened risk for hsil associated with persistent lsil underscores the need to closely monitor hiv-infected adolescents with lsil. the risk for hsil associated with high concentrations of il-12 may be suggestive of a local immune dysregulation. the role of hormonal contraception as a risk factor deserves further investigation. little is known about the epidemiologic profile of trichomoniasis in men and its relationship to human immunodeficiency virus (hiv) infection. among men presenting for care for symptomatic sexually transmitted infections (stis) in malawi, trichomoniasis is not considered for first-line treatment. methods: we conducted a cross-sectional survey of 1187 men attending either a dermatology or sti outpatient clinic in the capital of malawi. men were interviewed, and the etiologies of the stis were determined. results: at the sti clinic (nz756 men), we identified 150 men (20%) with trichomonas vaginalis infection, 358 men (47%) with hiv infection, and 335 men (44%) with neisseria gonorrhoeae infection. at the dermatology clinic (nz431 men), we identified 54 (13%), 118 (27%), and 2 (0.5%) men, respectively. at both clinics, a lower education level and reporting never having used a condom were predictive of t. vaginalis infection. only at the dermatology clinic was older age associated with infection, and only at the sti clinic were marital, genital ulcer disease, and hiv-infection status associated with t. vaginalis infection. at the sti clinic, urethral symptoms attributable to trichomoniasis were more severe among hiv-positive men than among hiv-negative men. conclusions: given its high prevalence and the increased risk for hiv transmission, t. vaginalis infection should be reconsidered for inclusion in the malawi stitreatment regimen for men. 2004; 190(8):1448-55. pmid: 15378437 [pubmed-in process] role of human neutrophil peptide-1 as a possible adjunct to antituberculosis chemotherapy kalita a, verma i, khuller gk we report the role of human neutrophil peptide (hnp)-1 as an adjunct to antituberculosis (anti-tb) drugs. the combination of hnp-1, isoniazid, and rifampicin was evaluated against mycobacterium tuberculosis h(37)rv in vitro, ex vivo, and in vivo, and synergism was observed on the basis of reductions in minimum inhibitory concentrations (mics) of these agents. in vitro results revealed o1log unit reductions even when hnp-1 and anti-tb drugs were used at 1/16 mics. this combination was also found to be bactericidal against intracellular mycobacteria even at 1/8 mics of hnp-1 and drugs. hnp-1 used in conjunction with anti-tb drugs resulted in significant clearance of bacterial load from lungs, liver, and spleen of infected, compared with control animals. the effective therapeutic dosage of drugs could be reduced to half by supplementing hnp-1 in the therapeutic schedule. these results clearly suggest that hnp-1 can be used as adjunct chemotherapy with conventional drugs against tb. in this phase iii trial, dr. jo-anne h. van burik and the national institute of allergy and infectious diseases mycoses study group studied 882 children and adults undergoing hsct. about half were randomized to receive micafungin (50 mg; 1 mg/kg for patients !50 kg), whereas the others received fluconazole (400 mg; 8 mg/kg for patients !50 kg). drugs were administered once a day via infusion during the neutropenic phase of hsct (i.e., before engraftment), and they were continued until neutropenia resolved or day 42 after hsct, whichever came earlier, or until fungal infection developed. time to treatment success was also significantly shorter with micafungin. no excess toxicity occurred with micafungin as compared with fluconazole, and fewer patients receiving micafungin dropped out of the study due to drug-related adverse events (4.2% versus 7.2%, pz0.058). these results suggest that micafungin is an effective alternative to fluconazole for antifungal prophylaxis in neutropenic patients during hsct. the authors obtained baseline blood samples from 174 girls 12 to 15 years old (76% african-american; 24% white). seropositivity for hsv-1, hsv-2, and cmv was also assessed 3 years later. at baseline, 41% of subjects (71 girls) had a history of sexual activity; this proportion increased to 73% (127 girls) by the 3-year follow-up. overall, seropositivity for cmv increased from 71% to 81%, seropositivity for hsv-1 increased from 44% to 49%, and seropositivity for hsv-2 doubled, from 7% to 14%. among sexually experienced girls, hsv-2 seropositivity increased from 15% to 19%. these increases correspond with attack rates of 13.8 cases of cmv per 100 person-years and 3.2 cases of hsv-1 per 100 person-years in the entire cohort. among sexually experienced girls, hsv-2 attack rates were 4.4 cases per 100 person-years of sexual activity. at follow-up, generalized estimating equation modeling identified 4 factors significantly associated with hiv-2 seropositivity: it also saves money, according to a new study. dr. kent k. hu and colleagues of veterans affairs puget sound health care system in seattle, wash, and other institutions discuss their findings in the november 15 clinical infectious diseases. the authors developed a decision analysis model based on hospitalized patients most likely to require a cvc for 2 to 10 days (i.e., those in intensive care units, with immunosuppression, or receiving total parenteral nutrition). cvcs were inserted according to either msb techniques (person inserting the catheter must wear a head cap, face mask, sterile body gown, and sterile gloves and use a full-size sterile drape around the site) or in accordance with lessstringent measures (only sterile gloves and a small regional sterile drape). in the base-case analysis, the use of msbs actually lowered costs by $252 as compared with less-stringent infection control measures ($369 versus $621 per catheter inserted). the incidences of catheter-related bsi (2.8% versus 5.3%), catheter colonization (2.9% versus 5.5%), and death (0.4% versus 0.8%) were also lower with msbs. during sensitivity analyses-even over a wide range of clinical and economic assumptions-the use of msbs resulted in improved patient safety. these data suggest that the improved management of infection with the use of msbs also pays off by lowering medical costs. thus, even though msbs are sometime cumbersome and time consuming, the authors suggest that they should be routinely used during cvc insertion. 2004;39:1441-5. hypogonadism is a risk factor for gynecomastia in hiv-positive men st. louis (md consult)-some studies suggest antiretroviral therapy is a risk factor for gynecomastia in men with human immunodeficiency virus (hiv) infection. however, no association has ever been found between the duration of exposure to any antiretroviral drug and the development of gynecomastia. instead, it appears hypogonadism is a predisposing factor to gynecomastia in hiv-infected men, according to the november 15 clinical infectious diseases. dr. alejandra biglia and colleagues of the university of barcelona in spain and other institutions studied 2,275 men with hiv infection. clinical examination with confirmatory sonography showed 40 men, or 1.8%, had gynecomastia. this is similar to the prevalence of gynecomastia in the general population. these 40 cases were matched with 44 men with hiv but not gynecomastia, and clinical, demographic, and laboratory features were extensively compared between the 2 groups. the mean free testosterone index was significantly lower in the cases than in the controls (42.6% vs. 58.0%). hypogonadism was the strongest independent predictor of gynecomastia (adjusted odds ratio [or] 7.6). other factors increasing the risk of gynecomastia included hepatitis c virus infection (adjusted or 6.1) and lipoatrophy (adjusted or 5.6). furthermore, gynecomastia resolved in many patients with persistent gynecomastia after transdermal or intramuscular testosterone administration, and in fact improved without any intervention in a substantial proportion of men. the authors also compared antiretroviral drug use between the cases and controls. significantly more cases were taking stavudine (73% vs. 41%) or efavirenz (33% vs. 14%), but significantly more controls were taking zidovudine (34% vs. 10%). however, the duration of exposure to each drug did not differ significantly between cases and controls, which further argues against antiretroviral therapy as the cause of gynecomastia in hivpositive men. the authors conclude gynecomastia in hivinfected men appears to be related more to hypogonadism than to adverse effects of antiretroviral therapy. if gynecomastia proves to be related to hypogonadism, then hormone treatments may be able to reverse this lipodystrophy, and further study is warranted. clin infect dis 2004;39:1514-9. taira av, neukermans cp, sanders gd human papillomavirus (hpv) has been implicated as the primary etiologic agent of cervical cancer. potential vaccines against high-risk hpv types are in clinical trials. we evaluated vaccination programs with a vaccine against hpv-16 and hpv-18. we developed disease transmission models that estimated hpv prevalence and infection rates for the population overall, by age group, by level of sexual activity within each age group, and by sex. data were based on clinical trials and published and unpublished sources. an hpv-16/18 vaccine for 12year-old girls would reduce cohort cervical cancer cases by 61.8%, with a cost-effectiveness ratio of $14,583 per quality-adjusted life year (qaly). including male participants in a vaccine rollout would further reduce cervical cancer cases by 2.2% at an incremental cost-effectiveness ratio of $442,039/qaly compared to female-only vaccination. vaccination against hpv-16 and hpv-18 can be cost-effective, although including male participants in a vaccination program is generally not costeffective, compared to female-only vaccination. yokoe ds, noskin ga, cunningham sm, zuccotti g, plaskett t, fraser vj, et al. we evaluated antimicrobial exposure, discharge diagnoses, or both to identify surgical site infections (ssi). this retrospective cohort study in 13 hospitals involved weighted, random samples of records from 8,739 coronary artery bypass graft (cabg) procedures, 7,399 cesarean deliveries, and 6,175 breast procedures. we compared routine surveillance to detection through inpatient antimicrobial exposure (s9 days for cabg, s2 days for cesareans, and s6 days for breast procedures), discharge diagnoses, or both. together, all methods identified ssi after 7.4% of cabg, 5.0% of cesareans, and 2.0% of breast procedures. antimicrobial exposure had the highest sensitivity, 88% to 91%, compared with routine surveillance, 38% to 64%. diagnosis codes improved sensitivity of detection of antimicrobial exposure after cesareans. record review confirmed ssi after 31% to 38% of procedures that met antimicrobial surveillance criteria. sufficient antimicrobial exposure days, together with diagnosis codes for cesareans, identified more postoperative ssi than routine surveillance methods. this screening method was efficient, readily standardized, and suitable for most hospitals. miner al, sands ke, yokoe ds, freedman j, thompson k, livingston jm, et al. we investigated using administrative claims data to identify surgical site infections (ssi) after breast surgery and cesarean section. postoperative diagnosis codes, procedure codes, and pharmacy information were automatically scanned and used to identify claims suggestive of ssi ('indicators') among 426 (22%) of 1,943 breast procedures and 474 (10%) of 4,859 cesarean sections. for 104 breast procedures with indicators explained in available medical records, ssi were confirmed for 37%, and some infection criteria were present for another 27%. among 204 cesarean sections, ssi were confirmed for 40%, and some criteria were met for 27%. the extrapolated infection rates of 2.8% for breast procedures and 3.1% for cesarean section were similar to those reported by the national nosocomial infection surveillance program but differ in representing predominantly outpatient infections. claims data may complement other data sources for identification of surgical site infections following breast surgery and cesarean section. several studies have shown that nasopharyngeal sampling is more sensitive than oropharyngeal sampling for the detection of pneumococcal carriage in children. the data for adults are limited and conflicting. this study was part of a larger study of pneumococcal carriage on the navajo and white mountain apache reservation following a clinical trial of a seven-valent pneumococcal conjugate vaccine. persons aged 18 years and older living in households with children enrolled in the vaccine trial were eligible. we collected both nasopharyngeal and oropharyngeal specimens by passing a flexible calcium alginate wire swab either nasally to the posterior nasopharynx or orally to the posterior oropharynx. swabs were placed in skim milktryptone-glucose-glycerin medium and frozen at k708c. pneumococcal isolation was performed by standard techniques. analyses were based on specimens collected from 1,994 adults living in 1,054 households. nasopharyngeal specimens (11.1%; 95% confidence interval [ci], 9.8 and 12.6%) were significantly more likely to grow pneumococci than were oropharyngeal specimens (5.8%; 95% ci, 4.8 to 6.9%) (p!0.0001). few persons had pneumococcal growth from both specimens (1.7%). therefore, both tests together were more likely to identify pneumococcal carriage (15.2%; 95% ci, 13.7 to 16.9%) than either test alone. although we found that nasopharyngeal sampling was more sensitive than oropharyngeal sampling, nasopharyngeal sampling alone would have underestimated the prevalence of pneumococcal carriage in this adult population. sampling both sites may give more accurate results than sampling either site alone in studies of pneumococcal carriage in adults. microbiol 2004; 42(11):4974-6. pmid: 15528682 [pubmed-in process] effects of intrapartum penicillin prophylaxis on intestinal bacterial colonization in infants jaureguy f, carton m, panel p, foucaud p, butel mj, doucet-populaire f early-onset group b streptococcal (gbs) infections remain a leading cause of morbidity and mortality in infants. to prevent the vertical transmission of gbs and neonatal gbs infection, guidelines recommend intrapartum penicillin or amoxicillin prophylaxis. this intrapartum antibiotic prophylaxis (iap) is suspected to favor colonization by antibiotic-resistant bacteria. however, the effects of this prophylaxis on the patterns of acquisition of gastrointestinal bacterial flora in infants have never been studied. we collected stool samples from 3-day-old infants born to mothers who received intrapartum amoxicillin (antibiotic-exposed group; nz25) and to untreated mothers (non-antibiotic-exposed group; nz25). the groups were matched for factors known to affect intestinal microbial colonization: gestational age, type of delivery, and type of feeding. qualitative and quantitative differential analyses of the bacterial flora in stool samples were performed. similar numbers of infants in the nonantibiotic-exposed and antibiotic-exposed groups were colonized by aerobic bacteria and amoxicillinresistant enterobacteria (75 and 77%, respectively) (pz0.79). in contrast, significantly fewer infants in the antibiotic-exposed group than in the nonantibiotic-exposed group were colonized by anaerobic bacteria, especially clostridium (12 and 40%, respectively) (p!0.05). regarding intestinal bacterial colonization, the differences between antibiotic-exposed and non-antibiotic-exposed infants were remarkably few. the only statistically significant effect was the reduced initial bacterial colonization by clostridium in the antibioticexposed group. in our study, the use of iap did not favor colonization by beta-lactam-resistant bacteria. however, further evaluations are required to highlight the potential risks of the widespread use of antibiotics to prevent early-onset gbs infection. microbiol 2004; 42(11):5184-8. pmid: 15528713 [pubmed-in process] usefulness of routine epicardial pacing wire culture for early prediction of poststernotomy mediastinitis mekontso-dessap a, honore s, kirsch m, houel r, loisance d, brun-buisson c poststernotomy mediastinitis (psm) is one of the most serious complications of cardiac surgery, and its associated morbidity and mortality demand early recognition for emergency therapy. in this study, we investigated the usefulness of epicardial pacing wire (epw) cultures for the prediction of psm. among 2,200 patients who underwent a cardiac surgical procedure at our hospital between 1 january 1999 and 31 december 2001, 82 (3.7%) had psm; staphylococcus aureus was the organism (45.1%) most frequently isolated at the time of surgical debridement. epws from 1,607 (73.0%) patients, 73 (4.5%) of whom developed psm, were cultured. epw cultures from 466 (29.0%) were positive, most often (74.9%) for coagulase-negative staphylococci. epw cultures were truly positive in 26 cases, truly negative in 1,106 cases, falsely positive in 428 cases, and falsely negative in 47 cases (with sterile cultures in 35 cases and a culture positive for an organism different from that isolated at the time of debridement in 12 cases). epw culture had a positive predictive value of only 5.7% and a high negative predictive value (95.9%) for the diagnosis of psm, with an accuracy of 70.4%. however, the likelihood ratio of positive (1.27) and negative (0.89) tests indicated only small changes in pretest-to-posttest probability. therefore, a strategy of routine culture of epws to predict psm seems questionable. dual-probe assay for rapid detection of drug-resistant mycobacterium tuberculosis by real-time pcr wada t, maeda s, tamaru a, imai s, hase a, kobayashi k mutations in particular nucleotides of genes coding for drug targets or drug-converting enzymes lead to drug resistance in mycobacterium tuberculosis. for rapid detection of drug-resistant m. tuberculosis in clinical specimens, a simple and applicable method is needed. eight taqman minor groove binder (mgb) probes, which discriminate one-base mismatches, were designed (dual-probe assay with four reaction tubes). the target of six mgb probes was the rpob gene, which is involved in rifampin resistance; five probes were designed to detect for mutation sites within an 81-bp hot spot of the rpob gene, and one probe was designed as a tuberculosis (tb) control outside the rpob gene hot-spot. we also designed probes to examine codon 315 of katg and codon 306 of embb for mutations associated with resistance to isoniazid and ethambutol, respectively. our system was m. tuberculosis complex specific, because neither nontuberculous mycobacteria nor bacteria other than mycobacteria reacted with the system. detection limits in direct and preamplified analyses were 250 and 10 fg of genomic dna, respectively. the system could detect mutations of the rpob, katg, and embb genes in dnas extracted from 45 laboratory strains and from sputum samples of 27 patients with pulmonary tb. this system was much faster (3 h from dna preparation) than conventional drug susceptibility testing (3 weeks). results from the dual-mgbprobe assay were consistent with dna sequencing. because the dual-probe assay system is simple, rapid, and accurate, it can be applied to detect drugresistant m. tuberculosis in clinical laboratories. to define methicillin-resistant staphylococcus aureus (mrsa) reservoirs in the community and their population dynamics, we studied the molecular epidemiology of a random sample (nz490) from a collection of 2154 inpatient and outpatient mrsa isolates during a 7-year period in san francisco. we noted a progressive replacement of type ii staphylococcal chromosomal cassette (scc)mec-bearing isolates with type iv sccmec-bearing isolates, which coincided with o4-fold increase in methicillin resistance between 1998 and 2002. type iv sccmec-bearing isolates involved in the increase in methicillin resistance belonged to 4 molecular genotypes. these 4 genotypes were associated predominantly with community-onset disease, rather than hospital-or long-term-care facilityonset disease (76.9% vs. 19.4% vs. 3.7%; pz0.0005), suggesting that they are not feral descendants of hospital isolates. the longitudinal results linked the dramatic increase in mrsa infections to an expanding community reservoir of mrsa genotypes with intrinsic community survival advantage. the outbreak of monkeypox in the midwestern united states during june 2003 marks the first documented human infection in the western hemisphere. consistent with those in outbreaks in africa, most cases in this outbreak were associated with febrile rash illness. we describe a cluster of monkeypox in a family with a spectrum of clinical illness, including encephalitis, and outline the laboratory confirmation of monkeypox. methods: standardized patient information was collected by questionnaire and medical chart review; all cases described were laboratory confirmed. laboratory methods included nucleic acid detection, viral culture, serologic testing, histopathologic evaluation, and immunohistochemical testing. results: of 3 family members with monkeypox, 2 had rash illness only, and 1 required hospitalization for severe encephalitis. the family member with the mildest clinical course had previously received smallpox vaccination. diagnostic testing by both polymerase chain reaction and culture revealed infectious monkeypox virus in skin lesions of all 3 patients; 2 patients had orthopoxvirus detected by immunohistochemistry in skin lesions. the patient with encephalitis had orthopoxvirus-reactive immunoglobulin m (igm) in cerebrospinal fluid. all patients had detectable igm responses to orthopoxvirus antigens. conclusions: these 3 patients illustrate a spectrum of clinical illness with monkeypox despite a common source of exposure; manifestation and severity of illness may be affected by age and prior smallpox vaccination. we report that monkeypox, in addition to causing febrile rash illness, causes severe neurologic infection, and we discuss the use of novel laboratory tests for its diagnosis. the proportion of infected cells to total cells was measured in serial breast milk samples collected from 291 hiv-1-infected women in nairobi, kenya, by use of real-time dna polymerase chain reaction amplification of bmcs. the number of infected bmcs per million cells was associated with levels of cell-free viral rna in breast milk previous studies demonstrated that the concentration of bmcs varies throughout lactation, and we used these data to transform infected bmcs per million cells to infected bmcs per milliliter. the estimated concentration of infected bmcs per milliliter was higher in colostrum or early milk than in mature milk (p!0.001) factors influencing increases in cd4 cell counts of hiv-positive persons receiving long-term highly active antiretroviral therapy smith cj, sabin ca, youle ms, kinloch-de loes s, lampe fc, madge s, cropley i, johnson ma, phillips an background: highly active antiretroviral therapy (haart) results in an improvement in immunologic function. we sought to investigate the factors associated with increases in cd4 cell count among human immunodeficiency virus (hiv)-positive antiretroviral-naive patients starting haart.methods: five hundred ninety-six subjects were followed for a median of 2.5 years (interquartile range, 1.0-4.0 years). factors associated with changes in cd4 cell counts in the first 3 months of haart and from 3 months onwards were analyzed.results: after 6, 12, and 24 months of haart, the median increases in cd4 cell counts were 114, 181, and 248 cells/mm(3), respectively; 84%, 84%, and 80% of subjects had a virus load of !400 copies/ml during the same periods. white ethnicity, higher pre-haart virus load, and lower pre-haart cd4 and cd8 cell counts were associated with greater increases in cd4 cell counts during the first 3 months of haart. from 3 months onward, a greater cumulative proportion of time spent with virus load !400 copies/ml was associated with a more favorable change in cd4 cell count (an average increase of 5.2 cells/mm(3)/year [95% confidence interval [ci], 3.8-6.7 cells/mm (3)/year] for each extra 10% cumulative time spent with a virus load !400 copies/ml) (p!0.0001). for every 100 cells/mm(3) higher in baseline cd4 cell count, the increase was 6 cells/mm(3)/year less (95% ci, 2-11 cells/mm(3)/year) (pz0.02). sex, risk group, age, and haart regimen were not associated with increases in cd4 cell counts.conclusions: these findings emphasize the importance of maintaining virological suppression and suggest other factors that influence long-term cd4 cell response. 2004; 190(10):1860-8. pmid: 15499544 [pubmed-in process] association of levels of hiv-1-infected breast milk cells and risk of mother-tochild transmission rousseau cm, nduati rw, richardson ba, john-stewart gc, mbori-ngacha da, kreiss jk, overbaugh j understanding how the level of human immunodeficiency virus type 1 (hiv-1)-infected breast milk cells (bmcs) affects hiv transmission via breastfeeding can shed light on the mechanism of key: cord-288814-1zayb21f authors: lu, qing-bin; jiang, wan-li; zhang, xin; li, hui-jun; zhang, xiao-ai; zeng, hao-long; du, juan; yang, guo-liang; zhang, lei-ke; li, rui; fang, li-qun; li, hao; liu, wei title: comorbidities for fatal outcome among the covid-19 patients: a hospital-based case-control study date: 2020-07-27 journal: j infect doi: 10.1016/j.jinf.2020.07.026 sha: doc_id: 288814 cord_uid: 1zayb21f nan since the discovery of coronavirus disease 2019 (covid-19), there have been numerous evidences supporting the pathogenic role of chronic comorbidities in the prognosis of infections, including the study by galloway et al. (1) , however, with the extent of the risk remained controversial (2) (3) (4) (5) (6) . the existing univariate models do not adequately distinguish between risk due to age and that due to increased presence of co-morbidities in older patients, thus these assessments of real effect from comorbidities are inevitably confounded. here by performing a retrospective multi-center study, we try to evaluate the adjusted effect of the common preexisting comorbidities on covid-19 related death, based on which, the therapy effect of three widely used anti-hypertension drugs were assessed. from 18 january to 24 february 2020, 1138 confirmed covid-19 patients consisting of 920 survivals and 218 deaths from three designated hospitals for covid-19 treatment in hubei province were included for analysis (table s1 ). the presence of comorbidities was reported in 49.12% (559/1138) of the total patients, 2 with significantly higher frequency in the deceased than in the survivals (77.06% vs. 42.50%, p < 0.001; table s1 ). as a whole, hypertension was the most prevalent comorbidity (32.95%), followed by diabetes mellitus (dm, 15.64%) and chronic heart diseases (chd, 9.31%). the chronic obstructive pulmonary diseases (copd), malignancy, cerebrovascular diseases (cvd), chronic kidney diseases (ckd), and chronic viral hepatitis (cvh) were less frequent, with prevalence ranging from 2.11% to 6.41%. by multivariate logistic regression model adjusting age, sex, and delay from symptom onset to hospital admission, six comorbidities showed significant association with the disease outcome, with malignancy exhibiting the highest risk of death, followed by ckd, cvd, hypertension, chd, and dm ( figure 1a and table s2 ). an age-stratified analysis revealed the effect of comorbidities on death was reduced as the age increased. among the patients ≤60 years, cvd had the highest effect on death, followed by hypertension. among the patients aged 60-70 years, only malignancy and dm were related to fatal outcome. among the patients aged >70 years, none of the eight comorbidities demonstrated significant association with fatal outcome. the sex-stratified analysis disclosed that male patients presenting with any of the three comorbidities (hypertension, dm, or cvd) had increased risk of developing fatal outcome, in contrast, female patients presenting any of the four comorbidities (chd, copd, malignancy, or ckd) had increased risk of fatal outcome (table s3) . for patients with isolated dm, four parameters displayed significantly higher 3 abnormal levels than those without any comorbidity, i.e., fibrinogen, activated partial thromboplastin time, prothrombin time and il-6 ( figure 2a-d) . for patients with isolated hypertension than those without, five laboratory indicators were deviated from normal value with greater extent, i.e., higher levels of d-dimer, fibrinogen degradation products, lactic dehydrogenase (ldh) and neutrophil percentage, and lower lymphocyte percentage ( figure 2d -i). among the 1138 patients, 149 (13.09%) had two coexisting comorbidities (table s1 ), with hypertension-dm most frequently observed (table s4) . fifty-seven (5.01%) had three coexisting comorbidities, and 27 (2.37%) had four or more. the coexisting of multiple comorbidities had significantly increased the risk of death ( figure 1b and table s5 ). in the multivariate analysis, over 4-fold risk of death was observed in the patients with ≥3 comorbidities. age-stratified analysis again revealed the effect of comorbidities on death was reduced as the age increased. forty-one (50.00%) of 82 fatal patients had taken calcium channel blocker (ccb) drugs, significantly lower than those among the survived (68.15%, 92/135; table s6 ). the effect of ccb drugs on reducing fatal outcome was shown to be significant. the use of angiotensin receptor blockers (arbs) or angiotensin converting enzyme inhibitors (aceis) was comparable between the fatal patients (24.39%, 20/82) and the survivals (24.44%, 33/135), showing no effect in reducing risk of death. decreased levels of fibrin degradation product, d-dimer, c-reactive protein, il-6 and ldh, less incidence of leukocytosis, and more rapid recovery of lymphocytes and neutrophils percentages were observed in the patients with ccb drugs treatment ( figure 2j -q). 4 the patients who regularly received oral hypoglycemic agents or insulin treatment had over 70% reduced risk of death without significance ( table 3) . as is known, mechanisms that lead to hypertension, dm, and cvd were increasingly recognized to overlap with pathways that regulate immune function. most importantly, older age is an important risk factor for these conditions and the effect of aging on immune function was equally important for covid-19 severity. therefore, the effect of age was mixed with those from the comorbidities, resulting in heterogenicity of effects among age groups. for those aged >70 years, none of the underlying condition played role in affecting the outcome any more. it's suggested that old age had exerted the strongest effect on death, that all effects from the comorbidities could be compromised when the patients are old enough. dm was found to be a strong risk factor for adverse outcome, with its risky effect also observed in those aged 60~70 years, which was reported for the two earlier cov infections, severe acute respiratory syndrome (7) and the middle east respiratory syndrome (8) . in this study, inflammation-related biomarker such as il-6, was elevated to a significantly higher level among the dm patients, indicating more intense induction of inflammatory storm. it's suggested accordingly that the anti-inflammatory drugs in treating diabetes-covid-19 should be proposed. moreover, the higher risk of diabetes-covid-19 death could also be reduced by good glycaemic control, as displayed by the therapy effect of insulin. raas blockade might decrease proinflammatory activity of ang ii, decreasing the risk of ards, myocarditis, or mortality in covid-19 (9, 10). we provided evidence 5 that ccb drugs offered beneficial effect of reducing risk for fatal outcome in hypertension-covid-19 patients, mostly mediated through enhancing the recovery of abnormal parameters and reducing host inflammatory response that had been proven to aggravate the disease severity. hence the current study provided further pharmacoepidemiologic data supporting the effect of ccbs in treating sars-cov-2 infection combined with hypertension. in conclusion, the comorbidities significantly affected the outcome of ocvid-19 but are age-dependent. the anti-hypertensive treatment, especially ccbs can offer beneficial effect in reducing the mortality of covid-19. the study was conducted in accordance with guidelines approved by the ethics role of the funder/sponsor: 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 access to data: after publication, the data will be made available to others on reasonable requests to the corresponding author. a proposal with detailed description of study objectives and statistical analysis plan will be needed for evaluation of the reasonability of requests. additional materials might also be required during the process of evaluation. deidentified participant data will be provided after approval from the corresponding author and wuhan tongji hospital. a clinical risk score to identify patients with covid-19 at high risk of critical care admission or death: an observational cohort study who. report of the who-china joint mission on coronavirus disease characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72314 cases from the chinese center for disease control and prevention risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study clinical course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective clinical outcomes of current medical approaches for middle east respiratory syndrome: a systematic review and meta-analysis aldosterone system inhibitors in patients with covid-19 is there an association between covid-19 mortality and the renin-angiotensin system-a call for epidemiologic investigations key: cord-289364-p31gt533 authors: alfehaidi, alanoud; ahmed, syed ali; hamed, ehab title: a case of sars-cov-2 re-infection date: 2020-10-25 journal: j infect doi: 10.1016/j.jinf.2020.10.019 sha: doc_id: 289364 cord_uid: p31gt533 nan we read with interest the study of gousseff et al 1 . the study reported a second acute covid-19 episode in 11 patients. a novel coronavirus (sars-cov-2) caused a pandemic end of 2019. common signs of sars-cov-2 include fever, cough and shortness of breath with no definitive treatment to date 2 . reverse transcriptase polymerase chain reaction (rt-pcr)-based assays are the current reference diagnostic test 3 . positive result does not necessitate the presence of infection and viral rna shedding declines following the resolution of symptoms. viral nucleic acid could be detectable in throat swabs up to 6 weeks after symptom onset 2 . different reports have proposed the reactivation of sars-cov-2 infection, with 2 rt-pcr positive results following resolving symptoms and interim rt-pcr negative results [4] [5] [6] [7] [8] [9] [10] [11] . early studies reported patients with negative results having positive results within one week of discharge. later, reports used 3 weeks (21 days) as the cut-off point, following which another positive rt-pcr result with symptoms would support the possibility of reinfection 4 . a recent report confirmed the possibility of reinfection with genetically distinct sars-cov-2 infection in 2 different cases 12 . here we describe a case with 2 positive rt-pcr results in a symptomatic female with 84 days apart, patient had a complete resolution of symptoms and interim negative swab results on day 13. a 46-year-old female attended primary healthcare settings in qatar with a history of sars-cov-2 contact and sore throat on the 23 rd of may, 2020. the patient sars-cov-2 rt-pcr results swab result was positive. her vital signs, blood investigations and chest x-ray were normal. the patient had a past medical history of mild asthma, which was controlled with only occasional use of salbutamol inhaler. the patient was admitted to a quarantine facility for observation. the patient pcr swab result was negative on the 5 th of june and she was discharged on the 6 th june. on the 11 th of august she presented with fever, sore throat and body pain following a second contact with sars-cov-2 positive case. sars-cov-2 rt-pcr results swab result was inconclusive on 13 th august. the patient also reported chest pain, cough, and mild dyspnea. her vital signs were normal, but blood investigations showed leucocytes of 1.7 ×10 9 cells per l and lymphocytes 0.9 ×10 9 cells per l. a repeat covid-19 pcr swab was positive on the 15 th of august with ct value of 25.49. chest x-ray was normal and patient progression to recovery was unremarkable. she was discharged home on the 29 th of august, 2020. during both events, the patient received only symptomatic treatment. early studies reported that re-detectable positive virus nucleic acid among patients with sars-cov-2 with an average duration of 15 days from discharge to a re-positive results 13 . patients in those early reports did not show signs of infection with the second positive results and had negative swab results within one week later. researchers have suggested the persistence of viral rna with no recurrence of infection. the cocorec (collaborative study covid recurrences) study suggested that recurrence of infection is likely if the patient has two confirmed sars-cov-2 rt-pcr positive results over 15 days apart with one major clinical sign and no other cause to explain the symptoms. the study identified 11 patients similar to our case. the longest time to second positive test results in this cohort of patients was 49 days 4 . our case presented with symptoms, positive contact history and positive swab results with a timeline significantly longer than any reported case. the persistence of positive rt-pcr for sars-cov-2 is reported only up to 6 weeks 4 . a re-infection or to the least a re-activation following long-lasting carriage seems more likely in this patient report. reports of such occurrences are rare to date in view of the number of worldwide reporting of the infection rates which is reassuring. this case report adds to current evidence of possibility of reinfection and provides a basis for future cohort studies. detection of viral rna in symptom 1 atic patients following complete remission of symptoms and full recovery should be considered as reinfection or recurrence at the least. the patient gave consent for the material to appear in bmj publication the case report received ethical approval from primary health care corporation (phcc) research committee. the patient consented to publication and reporting is fully anonymized. clinical recurrences of covid-19 symptoms after recovery: viral relapse, reinfection or inflammatory rebound? transmission, diagnosis, and treatment of coronavirus disease diagnostic testing for severe acute respiratory syndrome-related coronavirus 2: a narrative review clinical recurrences of covid-19 symptoms after recovery: viral relapse, reinfection or inflammatory rebound? cause analysis and treatment strategies of "recurrence" with novel coronavirus pneumonia (covid-19) patients after discharge from hospital zhonghua jie he he hu xi za zhi clinical characteristics of severe acute respiratory syndrome coronavirus 2 reactivation recurrence of covid-19 after recovery: a case report from italy reactivation of sars-cov-2 after recovery recurrence of positive sars-cov-2 rna in covid-19: a case report recurrent pcr positivity after hospital discharge of people with coronavirus disease seven discharged patients turning positive again for sars-cov-2 on quantitative rt-pcr asymptomatic reinfection in two healthcare workers from india with genetically distinct sars-cov-2 a systematic review of re-detectable positive virus nucleic acid among covid-19 patients in recovery phase we acknowledge the support we receive from primary health care corporation (phcc) research committee. key: cord-316501-fl2wvhia authors: noh, ji yun; yoon, jin gu; seong, hye; choi, won suk; sohn, jang wook; cheong, hee jin; kim, woo joo; song, joon young title: asymptomatic infection and atypical manifestations of covid-19: comparison of viral shedding duration date: 2020-05-21 journal: j infect doi: 10.1016/j.jinf.2020.05.035 sha: doc_id: 316501 cord_uid: fl2wvhia • more than 25% of patients with covid-19 were asymptomatic. • among patients with covid-19, 26.1% presented anosmia, and 22.6% complained of ageusia with median duration of 7 days. • mean duration of sars-cov-2 viral shedding was 24.5 days. • irrespective of clinical manifestations, all patients with covid-19 showed prolonged viral shedding. to evaluate the prevalence of asymptomatic infection, anosmia (smell loss) and ageusia (taste 8 loss) among patients with mild covid-19 in a residential treatment center (rtc). we also 9 compared the duration of sars-cov-2 viral shedding between groups with different clinical 10 manifestations. an observational cohort study was conducted for 199 patients with covid-19 in a rtc at among 199 patients with covid-19, male was 34.7% and mean age of the patients was 26 38.0 years (table 1) . most patients (187, 94.0%) were healthy without chronic medical 27 conditions. among 199 patients, 26.6% were asymptomatic. in the early study, asymptomatic this study has some limitations. anosmia and ageusia were subjective symptoms. olfactory test was not performed, and quantitative scale of olfactory dysfunction was not 58 measured. in addition, viability of sars-cov-2 detected by pcr was not proven using viral covid-19 in south korea -challenges of 80 subclinical manifestations rapid asymptomatic transmission of covid-19 during 82 the incubation period demonstrating strong infectivity in a cluster of youngsters aged 16-83 23 years outside wuhan and characteristics of young patients with covid-19: a 84 prospective contact-tracing study self-reported olfactory and taste disorders in sars-86 clinical infectious diseases : an official publication 87 of the infectious diseases society of america estimating the asymptomatic proportion of 89 coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship the covid-19 pandemic and otolaryngology: 93 what it comes down to? smell and taste disorders, a study of 750 patients 96 from the university of pennsylvania smell and taste center identification of viruses in patients with postviral 100 olfactory dysfunction severe acute respiratory 103 syndrome coronavirus infection causes neuronal death in the absence of encephalitis in 104 mice transgenic for human ace2 high expression of ace2 receptor of 2019-ncov on the 107 epithelial cells of oral mucosa epithelial cells lining salivary gland ducts are early target 110 cells of severe acute respiratory syndrome coronavirus infection in the upper respiratory 111 tracts of rhesus macaques duration, mean ± sd 7.5 ± 5.6anorexia 1 (0.5) key: cord-276345-xsjh3766 authors: arshad, yasir; mahmood, nayab; zaidi, syed sohail zahoor; sharif, salmaan; ikram, aamer; ali, muhammad qaisar; usman, muhammad; akhtar, ribqa; hassan, muhammad; salman, muhammad; adil, naveed; rana, muhammad suleman title: detection of sars-cov-2 in ophthalmic secretions in pakistan: a preliminary report date: 2020-08-25 journal: j infect doi: 10.1016/j.jinf.2020.08.035 sha: doc_id: 276345 cord_uid: xsjh3766 nan out of total 35 patients, 25(71.4%) were male and 10(28.5%) were female. age of the patients enrolled for this study was ranging from 20-65 years. symptoms associated with suspected covid-19 patients, 35(100%) had fever, 21(60%) had dry cough, and 17(48.6%) had difficulty in breathing. low lymphocytes and platelet count was noted in both group. there was no any significant difference was observed in the hematological and biochemical markers between the both groups. all demographic, clinical and laboratory characteristics of patients given in the table-1. nucleic acid (rna) was extracted from all samples including oropharyngeal and conjunctival swabs and were tested by using one-step real-time rt-pcr. all 35 oropharyngeal swab samples were detected positive for sars cov-2, however out of total 35 conjunctival swab samples, 3(8.5%) were detected positive by using real-time rt-pcr. 2(66%) out of 3 conjunctival sars-cov-2 positive patients were having dry cough whereas 3(100%) patients were suffering from fever and difficulties in breathing. there was no ocular manifestation observed among patients with positive conjunctival specimens and similar information has already been reported by the previous study [4] . results of the present study support the evidence that ophthalmic secretions may not be the main source of transmission for the novel sars-cov-2, but the role of eye in the transmission of this highly contagious virus must not be ignored. our results are consistent with other reports although the percentage of positivity is low from ophthalmic secretions as compared to respiratory secretions; this route of transmission cannot be ruled out and needs for further detail investigation. previously, coronaviruses are reported as less infectious, causing common cold like symptoms by infecting upper respiratory tract. because of the previous investigations on coronaviruses it is known that its spread occurs through infectious respiratory droplets and contaminated fomites. therefore, primary source of sampling is the throat/nasal swab, and samples from the lower respiratory tract [5] . in the current pandemic situation, additional knowledge is required to understand the transmission patterns of sars-cov-2 in order to overcome the spread of this newly emerged virus. tears can be a potential body fluid to harbor coronaviruses, as human eye conjunctiva usually remains exposed to respiratory droplets in air and if rubbed with contaminated hands. in 2004, sarscoronavirus was detected from tear samples in 37.5% positive cases and in another study, positivity of sars-cov-2 from conjunctival swabs was 16.6% which contributed to the evidence of eye as a carrier [6, 7] . it is already reported that many health care workers have been infected by covid-19 and three ophthalmologists died of covid-19 in wuhan china [8] . presence of sars-cov-2 up to 27 days after the onset of infection reported previously [9] is a matter of great concern. high infection rate and rapid spread of sars-cov-2 intrigued the need to investigate the possible role of eyes as shedding route or portal of entry of this sars cov-2. however, our study is limited by its small ): p. e122. 2. world health organization declares covid-19 a 'pandemic covid-19 coronavirus pandemic covid-19: limiting the risks for eye care professionals emerging threats from zoonotic coronaviruses-from sars and mers to 2019-ncov the severe acute respiratory syndrome coronavirus in tears sars-cov-2 in the ocular surface of covid-19 patients. eye and vision new evidence of sars-cov-2 transmission through the ocular surface. graefe's archive for evaluation of ocular symptoms and tropism of sars-cov-2 in patients confirmed with covid-19 the authors extend their gratitude to the patients and medical staff of pakistan institute of medical sciences (pims). we declared that there is no conflict of interest no funding source key: cord-309577-438fotfd authors: xing, yuhan; ni, wei; wu, qin; li, wenjie; li, guoju; wang, wendi; tong, jianning; song, xiufeng; wong, gary wing kin; xing, quansheng title: dynamics of faecal sars-cov-2 in infected children during the convalescent phase date: 2020-04-10 journal: j infect doi: 10.1016/j.jinf.2020.03.049 sha: doc_id: 309577 cord_uid: 438fotfd nan we read with interest the recent paper in this journal by he et al. who reviewed the current evidence on covid-19 and concluded that faecal shedding of sars-cov-2 should not be ignored as one of the possible transmission routes of the virus. 1 we would like to share findings from our paediatric patients who were positive for nucleic acid testing for sars-cov-2 in stools up to 8-20 days after clearance of viral rna in respiratory specimens. with the world health organization declaring a pandemic, the outbreak of coronavirus disease 2019 (covid-19) poses a global threat with the highest risk impact. 2 the aetiological agent is a newly identified pathogen which was later renamed as severe acute respiratory syndrome coronavirus 2 (sars-cov-2). there has been accumulating evidence suggests that the virus plagues people ubiquitously, although paediatric patients seem to present with distinct characteristics from infected adults. 3 -6 between january 17, 2020 and february 23, 2020, a total of 3 children with laboratory confirmed covid-19 was reported in qingdao, shandong province, china. baseline characteristics and clinical, laboratory, and radiological data were collected. dynamic profiles of real-time reverse transcription polymerase chain reaction (rt-pcr) results in throat swabs and faecal specimens were closely monitored till march 10, 2020, the final date of follow-up. all the three paediatric patients were diagnosed with mild pneumonia and fever was the most consistent and predominant symptom at any time during the illness. only one child had gastrointestinal symptoms. all children were in stable condition during the course of hospitalisation and none of them required respiratory support or intensive care. their laboratory and radiological features were not typical for covid-19. the three infected children showed good response to supportive and anti-viral treatment with a relatively short time to resolution. surprisingly, we found sars-cov-2 remained detectable in faeces of paediatric patients for approximately 4 weeks, whereas negative conversion of viral rna in respiratory specimens occurred within 2 weeks after disease onset. two children showed negative results for faecal detection of sars-cov-2 20 days after clear-ance of viral rna in the respiratory tract, while another child persistently tested positive on faecal samples even 8 days after respiratory samples turning negative. chronological changes in rt-pcr results of respiratory and faecal specimens are shown in fig. 1 . in general, children appear to be less severely affected by sars-cov-2 in contrast with adult patients. 3 -6 the mechanism underlying this youthful resilience to covid-19 is yet to be systematically determined. notably, asymptomatic infection is not uncommon amongst children. a recent study on the prevalence of covid-19 in children demonstrated that 39 of 171 (27.7%) laboratory confirmed cases did not have any symptoms of infection. 3 an asymptomatic child had sars-cov-2 rna detectable in faeces at least 9 days after viral clearance in the respiratory tract. 7 it might be possible that asymptomatic children infected with sars-cov-2 go undetected and represent as important contributors to virus transmission in the community, causing the pandemic to propagate. aggressive efforts should be made to prevent spreading of the infection in schoolyard environments. faecal shedding of viral rna has been constantly reported in patients infected with sars-cov-2. 7 -10 one study reported over half of the 17 patients had faecal samples positive for sars-cov-2 detection, although virus copies in stools were less than those in respiratory specimens. 8 however, the researchers did not mention age distribution of these patients, neither did they investigate the duration of faecal samples positive for nucleic acid testing. another study on paediatric cases indicated that viral loads in the gastrointestinal tract might be greater than that in the respiratory system. eight out of ten paediatric patients were positive for nucleic acid testing in rectal swabs after respiratory specimens showing negative. 9 hence, sars-cov-2 may exist in the digestive system for a longer duration and convalescent patients with prolonged faecal shedding of sars-cov-2 may be an infectious source if fitness for discharge is based on respiratory testing. in the face of a novel disease, scientists and clinicians still have much to learn about the potential routes of virus transmission. evidence so far raises the possibility of faecal-oral transmission, reinforcing the need for nucleic acid testing of stool samples from covid-19 patients during the convalescent phase. close surveillance of convalescent patients would be crucial to curb the covid-19 pandemic, and maybe outbreaks yet to come. days since negative conversion of viral rna in throat swabs are shown in numbers with grey boxes. boxes with red plus sign denote the days when faecal specimens were positive for reverse transcription pcr testing. boxes with minus sign represent6 the days when viral rna was not detectable in faecal samples. na means faecal specimen was not collected from the patients on that day and laboratory result was not available. public health might be endangered by possible prolonged discharge of sars-cov-2 in stool sars-cov-2 infection in children a case series of children with 2019 novel coronavirus infection: clinical and epidemiological features epidemiological characteristics of 2143 pediatric patients with 2019 coronavirus disease in china novel coronavirus infection in hospitalized infants under 1 year of age in china detection of novel coronavirus by rt-pcr in stool specimen from asymptomatic child viral load of sars-cov-2 in clinical samples characteristics of pediatric sars-cov-2 infection and potential evidence for persistent fecal viral shedding evidence for gastrointestinal infection of sars-cov-2 key: cord-309294-ax6sr3zr authors: garrigues, eve; janvier, paul; kherabi, yousra; bot, audrey le; hamon, antoine; gouze, hélène; doucet, lucile; berkani, sabryne; oliosi, emma; mallard, elise; corre, félix; zarrouk, virginie; moyer, jean-denis; galy, adrien; honsel, vasco; fantin, bruno; nguyen, yann title: post-discharge persistent symptoms and health-related quality of life after hospitalization for covid-19 date: 2020-08-25 journal: j infect doi: 10.1016/j.jinf.2020.08.029 sha: doc_id: 309294 cord_uid: ax6sr3zr nan in this journal, we recently reported a series of 279 hospitalized patients with novel coronavirus 2019 disease and their short-term outcome. 1 however, only a few studies have assessed post-discharge persistent symptoms and health-related quality of life (hrqol) after hospitalization for covid-19. 2, 3 here, we describe a single-centre study assessing post-discharge persistent symptoms and hrqol of patients hospitalized in our covid-19 ward unit more than 100 days after their admission. covid-19 diagnosis was based on positive sars-cov-2 real-time reverse transcriptase-polymerase chain reaction on nasal swabs, and/or typical abnormalities on chest computed tomography. patients who were directly admitted to the icu without being hospitalized in our covid-19 unit were excluded. demographic and clinical data at admission were extracted from electronic medical records. we designed a short phone questionnaire to collect post-discharge clinical symptoms, modified medical research council (mmrc) dyspnoea scale scores, professional and physical activities, and attention, memory and/or sleep disorders. hrqol was assessed using the eq-5d-5l questionnaire, a widely used, validated european questionnaire 4 . patients are asked to rate their health state from 1 to 5 in five domains (mobility, self-care, usual activities, pain/discomfort, and anxiety/depression) and on a scale ranging from 0 ("the worst possible health") to 100 ("the best possible health") on a visual analogue scale (eq-vas). based on the answers, an eq-5d-index can be calculated, ranging from states worse than dead (<0) to 1 (full health). 5 all eligible patients were contacted by phone by trained physicians and were asked to answer to the questionnaire. deceased, unreachable, demented, bedridden and non-french speaking patients were excluded. we compared patients managed in hospital ward without needing intensive care ("ward group") with those who were transferred in intensive care units (icu) for artificial ventilation, including non-invasive ventilation, high flow nasal cannula and/or mechanical ventilation (icu group), with t-tests for quantitative variables and chisquare tests for qualitative variables. all tests were two-sided, and a p-value <0.05 was considered statistically significant. all analyses were performed with r version 3. after a mean of 110.9 days, the most frequently reported persistent symptoms were fatigue (55%), dyspnoea (42%), loss of memory (34%), concentration and sleep disorders (28% and 30.8%, respectively) ( table 1) . loss of hair was reported by 24 (20%) patients, including 20 women and 4 men. comparisons between ward-and icu patients led to no statistically significant differences regarding those symptoms. thirty-five (29%) patients had a mmrc grade ≥2 ("walks slower than people of the same age because of dyspnoea or has to stop for breath when walking at own pace"). before covid-19 infection, 56 (46.7%) were active workers. among them, 38 (69.1%) had gone back to work at the time of the phone interview. among the 39 patients who had regular sports activity before their hospitalizations for covid-19, 28 (71.8%) have been able to resume physical activity, but at a lower level for 18 (46%). there was no statistically significant difference between ward and icu groups, but there was a nonsignificant trend towards a reduced proportion of patients returning to work among icu patients (46.7% versus 77.5%, p=0.061). in both group, dimensions of the eq-5d (mobility, self-care, pain, anxiety or depression, usual activity) were altered with a slight difference in pain in the icu group, but no statistically significant difference in the other groups (figure 1 ). mean eq-vas was 70.3% and mean eq-5d index 0.86, with no difference between icu and ward patients ( table 1 ). the present study shows that most patients requiring hospitalization for covid-19 still have persistent symptoms, even 110 days after being discharged, especially fatigue and dyspnoea. these results highlight the need for a long-term follow-up of those patients and rehabilitation programs. surprisingly, many patients (mainly women) spontaneously reported significant hair loss, which may correspond to a telogen effluvium, secondary to viral infection and/or a stress generated by the hospitalization and the disease. 6 nevertheless, hrqol was quite satisfactory, as most patients who had a professional activity before the infection went back to work. except pain or discomfort, we found no significant difference regarding persistent symptoms and hrqol between ward patients versus icu patients. this clearly supports the interest of a full resuscitation for covid patients despite heaviness of cares. however, patients from our "icu group" were relatively non-severe, as those who were directly admitted to icu (thus corresponding to the most severe forms) were not included in our study. other limitations of our study include the limited number of patients, the single-centre nature of our series, and the high rate of unreachable patients, which could lead to differential bias. in conclusion, many symptoms persist several months after hospitalization for covid-19. while there were few differences between hrqol between ward and icu patients, our findings must be confirmed in larger cohorts, including more severe icu patients. applicability of the curb-65 pneumonia severity score for outpatient treatment of covid-19 gemelli against covid-19 post-acute care study group. persistent symptoms in patients after acute post-discharge symptoms and rehabilitation needs in survivors of covid-19 infection: a cross-sectional evaluation development and preliminary testing of the new five-level version of eq-5d (eq-5d-5l) a french value set for the eq-5d-5l sener serpil, colak cemil. evaluation of the effects of covid-19 pandemic on hair diseases through a web-based questionnaire the authors are indebted to all persons (physicians, surgeons, radiologists, biologists, medical students, and paramedical staff) who were involved in the beaujon covid-19 unit. the writing review and editing: all authors. none of the authors declared any competing interest in link with the present study. key: cord-315300-v3pxb997 authors: zhang, haipeng; wu, ti title: cd4+t, cd8+t counts and severe covid-19: a meta-analysis date: 2020-06-20 journal: j infect doi: 10.1016/j.jinf.2020.06.036 sha: doc_id: 315300 cord_uid: v3pxb997 nan key words: cd4+t, cd8+t, cd4/cd8 ratio, covid-19, meta-analysis to the editor: we read an interesting study in your journal. a retrospective study by liu et al was conducted to investigate the associated between lymphocyte subset counts and severe covid-19 [1] . they found low counts of cd4+t and cd8+t were more common in patients with severe covid-19. and cd4/cd8 ratio showed no significant difference between non-severe and severe covid-19 groups. cd4+t and cd8+t play a vital role in maintaining immune function and viral clearance in the body. it has been reported that cd4+t and cd8+t counts significantly decreased in covid-19 patients [2] . however, whether their status were correlated with the clinical type of covid-19 patients has not reached consistent conclusions. therefore, we conducted this meta-analysis to investigate the relationship between cd4+t counts, cd8+t counts, cd4/cd8 ratio and the severity of covid-19 patients. we searched pubmed, embase and web of sciences, using the keywords as "cd4", "cd8", "covid-19", "severe 2019-ncov", and "sars-cov-2" without date (until jun 2, 2020) or language restrictions. trials providing data of counts of cd4+t, cd8+t or cd4/cd8 ratio in patients with non-severe or severe covid-19 were included. according to guidelines for the diagnosis and treatment of covid-19 [3] , covid-19 is classified as mild, moderate, severe, and critical pneumonia. we categorized severe and critical pneumonia into the severe group, mild and moderate pneumonia into the non-severe group. we independently screened every article and extracted the data. any disagreement were resolved by discussion and consensus. mean difference (md) with 95% confidence intervals (95% ci) was calculated in this meta-analysis using review manager 5.3 software. study heterogeneity was assessed using i 2 statistic, when i 2 <50%, a fixed-effects model was used, otherwise a random-effects model was chosen. sensitivity analysis were performed by sequential removal of each trial. 13 studies included a total number of 1647 patients were considered in our meta-analysis.(supplementary material) all the studies, except for 1 in spain [4] , were conducted in china. 10 studies distinguished non-severe and severe groups, 2 studies only reported icu and non-icu groups [4 5] , and 1 study only reported decease and survivor groups [6] . data of cd4+t, cd8+t counts and cd4/cd8 ratio were provided in 13, 12 and 8 studies, respectively. both cd4+t and cd8+t counts significantly reduced in severe covid-19 group compared with non-severe group [cd4+t (md: -0.22×10 9 /l, 95%ci: -0.27 to -0.17×10 9 /l, i 2 =89%); cd8+t (md: difference between two groups in cd4/cd8 ratio (md: 0.17, 95%ci: -0.12 to 0.46, ).the details of our meta-analysis are presented in figure 1 . covid-19 is an acute inflammatory infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). sars-cov-2 has a genome sequence 79.6% identical to the sars-cov [7] . similar clinical features, such as fever, dry cough, dyspnoea, and bilateral ground-glass opacities on chest ct scans, were found between covid-19 and severe acute respiratory syndrome (sars) [8] . it has been reported that low counts of cd4+t and cd8+t were associated with adverse outcome in patients with sars, and the counts would rise dramatically when clinical symptoms improved [9] . as wang et al. reported, after 1 week of covid-19 treatment, cd8+t counts increased only in patients with attenuated symptoms or improved radiological abnormalities, while no similar change of cd4+t counts was found [10] . it appears that, unlike sars, cd8+t may be a more sensitive predictor of clinical outcome than cd4+t in covid-19 patients. however, both cd4+t and lymphocyte subset (cd4+, cd8+) counts reflect the severity of infection and predict the clinical outcomes in patients with covid-19 t cell subset counts in peripheral blood can be used as discriminatory biomarkers for diagnosis and severity prediction of covid-19 national health commission of the people's republic of china. guidelines for the diagnosis and treatment of covid-19 989/files/ce3e6945832a438eaae415350a8ce964 selective cd8 cell reduction by sars-cov-2 is associated with a worse prognosis and systemic inflammation in covid-19 patients reduction and functional exhaustion of t cells in patients with coronavirus disease 2019 (covid-19) predictors of mortality for patients with covid-19 pneumonia caused by sars-cov-2: a prospective cohort study a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical features of patients infected with 2019 novel coronavirus in wuhan haematological manifestations in patients with severe acute respiratory syndrome: retrospective analysis characteristics of peripheral lymphocyte subset alteration in covid-19 pneumonia key: cord-317315-yyssvbbl authors: mao, ming-hui; guo, jing-jing; qin, li-zheng; han, zheng-xue; wang, ya-jie; yang, di title: serial semiquantitative detection of sars-cov-2 in saliva samples date: 2020-10-06 journal: j infect doi: 10.1016/j.jinf.2020.10.002 sha: doc_id: 317315 cord_uid: yyssvbbl nan we read with interest the paper by azzi and colleagues who report on the reliability of saliva testing for sars-cov2 infection. 1 we have carried out a study to analyze the efficiency of saliva testing in monitoring the viral load of confirmed patients and get a similar conclusion. saliva testing has been widely used in diagnosing and screening suspected covid-19 patients due to it being easy to collect and noninvasiveness and having a high positive rate. 2, 3 for inpatients, the current standard for discharge is a negative rt-qpcr result from two sets of nasopharyngeal and throat swab specimens. multiple throat swab specimens from each patient are needed to monitor the viral load, which will not only inevitably increase the risk of cross-infection but also increase the discomfort of the patient and cause possible complications such as bleeding. 4 there is no doubt that saliva testing can greatly improve patient comfort and reduce the risk of medical staff contracting the virus. in this study, inpatients with a diagnosis of covid-19 provided by real-time reverse-transcriptase polymerase chain reaction (rrt-pcr) on oropharyngeal swabs in beijing ditan hospital, capital medical university from july 10, 2020, to july 20, 2020, were included. saliva was collected and one-step rrt-pcr was performed using the da'an gene 2019-ncov detection kit (fluorescent pcr method, batch number: 2020032). ct values of the orf1a gene and n gene were also tested simultaneously. the results were considered 'positive' when the cycle threshold (ct) values of fam and vic channels were less than 40, and there were obvious amplification curves. spss 24.0 and prism 8.0 were used for statistical analyses, the difference between groups was analyzed by anova and student's t-test, p < 0.05 was considered to be statistically significant. a total of 34 patients were included (table 1) table 1 ). the ct value of most patients increased with time. however, in some patients, the ct value first decreased with increasing time and finally increased and became negative ( fig. 1c, d) . univariate analysis found that the reduction in red blood cells significantly affected the peak value of the orf1a gene (p=0.027), while for the n gene, there was no significant difference (p=0.059). in multivariate analysis, no related factors that significantly affected the ct peak were found (see appendix table 2 ). the total positive rate of nucleic acid detection from sputum was the highest (67.2%), followed by oropharyngeal swabs (53.1%) and saliva (36%). according to the number of weeks after hospitalization, the positive rate of nucleic acid detection from the three sample types gradually decreased, the positive rate of nucleic acid detection from saliva was 83.33% in the second week, 48% in the third week, and 0% in the seventh week (see appendix fig. 1 ). while the positive rates of nucleic acid detection from saliva, sputum, and oropharyngeal swab samples were significantly different at 3 and 6 weeks (see appendix table 3 ). the average time for nucleic acid detection results to become negative was 27.29±7.73 days for sputum samples, 27.82±12.09 days for oropharyngeal swab samples, and 24.53±13.59 days for saliva samples (see appendix table 4 ). univariate analysis revealed that the clinical classification had a significant impact on both the time of the positive to negative conversion of sputum, oropharyngeal swab and saliva samples (p=0.001, p=0.001, p=0.012), while only red blood cell reduction had a significant effect on the positive to negative conversion time of saliva samples (p=0.032). multivariate analysis found that clinical classification had a significant impact on the time of sputum and oropharyngeal swab samples to become negative (p=0.007, p=0.002) (see appendix table 5 ). taking sputum specimens as an example, the average time for test results to become negative in asymptomatic patients was 14 days, while the average times for patients with mild and moderate disease were 25 days and 32 days, respectively. using the sputum-oropharyngeal swab test results as a reference, that is, a negative result was when the nucleic acid results of both specimen types were negative, and if one of the samples had a positive test result, it is considered a positive result. the efficiency of saliva single detection method and saliva-sputum combined detection method was tested. the results showed that the total sensitivity, efficiency and specificity of saliva single detection method were 74.10%, 83.90% and 94.40%, respectively. the overall sensitivity, efficiency and specificity of saliva-sputum combined detection method were 93.40%, 94.00% and 95.20%, respectively (see appendix table 6 ). studies have conducted research on the effectiveness of saliva to diagnosis covid-19, and the overall efficiency rate differs, ranging from 30.7% to 100%. 1,5-9 total efficiency and specificity of the saliva detection method in this study were higher than those of the sputum and oropharyngeal swab detection methods (83.90% and 94.40%, respectively). the saliva-sputum combined diagnosis is more effective, with a total efficiency and specificity of 94.00% and 95.20%, respectively. in addition, to verify the specificity of saliva testing, the saliva and oropharyngeal swab samples of 50 patients were tested, and the results of all of these patients were negative. however, only 34 patients were included and it was not possible to collect all three sample types from every patient at the same time. we also fails to obtain the true copy of the virus, that is, the viral copies per ml of sample. nonetheless, our results show that combined sputum-saliva detection is a reliable method for monitoring the viral load of patients recovering from covid-19. this research received no specific grant from any funding agency in the public, commercial, or not-for-profit sectors. data directly supporting the study results can be found in beijing ditan hospital in paper form. saliva is a reliable tool to detect sars-cov-2 additional molecular testing of saliva specimens improves the detection of respiratory viruses saliva as a diagnostic specimen for testing respiratory virus by a point-of-care molecular assay: a diagnostic validity study a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster consistent detection of 2019 novel coronavirus in saliva two cases of covid-19 with positive salivary and negative pharyngeal or respiratory swabs at hospital discharge: a rising concern key: cord-318126-gg68o52z authors: zhou, juan; tan, yingzheng; li, dan; he, xiaojin; yuan, ting; long, yunzhu title: observation and analysis of 26 cases of asymptomatic sars-cov2 infection date: 2020-04-03 journal: j infect doi: 10.1016/j.jinf.2020.03.028 sha: doc_id: 318126 cord_uid: gg68o52z nan we read with interest the article in this journal which revealed that ct scanning provides important bases for early diagnosis and treatment of covid-19 (corona virus infection disease 2019) which is caused by sars-cov2 (severe acute respiratory syndrome coronavirus 2). 1 as a new infectious disease, the early identification of the source of the infection and the determination of the isolation time are the primary issues. 2 recent studies have found that the emergence of asymptomatic sars-cov2 infections. 3 unexpectedly, the asymptomatic proportion was nearly about 18% in light of a statistical modeling analysis on the diamond princess cruise where an outbreak of sars-cov2 infections occurred. 4 therefore, it is necessary to further clarify the infectivity and outcome of these asymptomatic infections. we observed and analyzed the phenotypic characteristics of asymptomatic individuals originating from the active detection of high-risk individuals who had close contacts with covid-19 patients during isolated observation with viral nucleic acid positive. the epidemiological history, laboratory, radiologic data and outcomes were collected according to the medical records. a total of 26 cases of asymptomatic infection were detected as sars-cov2 positive through swab specimen between january 20 to february 30. among these, there were 10 males (38.5%), which was in line with another similar report, 5 and the average age was 37 years old, with the youngest being 3 years old and the oldest beings 90 years old. five cases were under 18 (19.2%) and 4 cases were over 60 (15.4%). these data showed that people of different ages are generally susceptible to sars-cov2, but the average age of asymptomatic patients is lower than the reported age of covid-19 patients which was 40-70 years old (propinquity 73%). 6 except for one case from wuhan, the others were local infection cases. fig. 1 . ct check of lung in one case of asymptomatic infection. a. on february 13, a first lung imaging examination was performed after a positive nucleic acid test was found. mild local lesions were observed. b. rechecking on february 18, it was found that the lesion had obvious absorption. at this time, the viral nucleic acid test was still positive. c on february 24th, the re-examination showed that lung lesions were almost completely absorbed, and the viral nucleic acid test was also negative. it was found that the period between exposure to the source of infection to the discovery of nucleic acid positive was 5-22 days, with an average of 8 days. they had no obvious discomfort or clinical manifestations related to covid-19 when were found to be positive for sars-cov2. during the process of the quarantine, body temperature was daily monitored, and chest computed tomography (ct) regularly checked. peripheral blood test results showed that the total number of white blood cells and lymphocytes in these patients were basically normal. the screening for pneumonia by chest ct found five cases with small area of glass exudative lesions on the edge of lung ( fig. 1 a-c) , implying that these individuals would be difficult to detect except through pathogen tests and chest ct scanning. finally, 9 patients subsequently had respiratory infections symptoms such as cough and fever, lately they were diagnosed as covid-19. in order to observe the outcome of the virus carriers, these asymptomatic infected persons were managed in isolation wards, and took lopinavir-ritonavir as well as chinese medicine under the guidance of physicians to avoid possible progression to covid-19. eventually, these asymptomatic patients were discharged from hospital after two consecutive negative of viral nucleic acid test. most importantly, a 37-year-old woman was confirmed to be significantly contagious. her father was diagnosed as a covid-19 patient on february 12 after her return home on january 22 from wuhan; thereafter, an isolated observation was conducted. during this period, the virus was not detected to be positive until the fifth nucleic acid sampling test on february 15th which suggested an incubation period up to 22 days, and then turned negative on march 2. meanwhile, no clinical symptoms and pulmonary imaging changes were found throughout the process. in addition, other cases who had close contact with a confirmed patient from wuhan also caught our attention, and members of her family were in isolation after she was found to be an asymptomatic carrier. subsequently, her father-in-law and mother-in-law were both detected as sars-cov2 positive. these cases proved that asymptomatic sars-cov2 carriers can also spread the virus before the https://doi.org/10.1016/j.jinf.2020.03.028 0163-4453/© 2020 the british infection association. published by elsevier ltd. all rights reserved. nucleic acid test was positive, fully illustrating the importance of home isolation during the epidemic. the outbreak of the new coronavirus infection in 2019 was unexpected and poses a great threat to international public health security. 6 note that the sars-cov2 spreads extremely quickly, and it probably resulted from the unrecognized asymptomatic carriers in the population which accounted for approximately 18% of total. 4 , 5 early detection and isolation of asymptomatic virus carriers is necessary in the current situation of epidemic control. none. clinical and computed tomographic imaging features of novel coronavirus pneumonia caused by sars-cov-2 early identification and prevention of the spread of ebola -united states substantial undocumented infection facilitates the rapid dissemination of novel coronavirus (covid-19) estimating the asymptomatic proportion of 2019 novel coronavirus onboard the princess cruises ship clinical characteristics of 24 asymptomatic infections with covid-19 screened among close contacts in nanjing epidemiology working group for ncip epidemic response. the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china key: cord-284862-nhihxog0 authors: kroemer, marie; spehner, laurie; vettoretti, lucie; bouard, adeline; eberst, guillaume; floury, sebastien pili; capellier, gilles; lepiller, quentin; orillard, emeline; mansi, laura; clairet, anne-laure; westeel, virginie; limat, samuel; dubois, maxime; malinowski, léa; bohard, louis; borg, christophe; chirouze, catherine; bouiller, kevin title: covid-19 patients display distinct sars-cov-2 specific t-cell responses according to disease severity date: 2020-08-25 journal: j infect doi: 10.1016/j.jinf.2020.08.036 sha: doc_id: 284862 cord_uid: nhihxog0 adaptive immune responses generated by sars-cov-2 virus in convalescent patients according to disease severity remain poorly characterized. to this end, we designed a prospective study (nct04365322) that included 60 covid-19 convalescent patients (1-month post infection) in two cohorts respectively entitled mild illness and severe pneumonia. the monitoring of peripheral immune responses was performed using ifnᵧ elispot assay. the serology index of each patient was investigated at the same time. patients with severe pneumonia were older and had more comorbidities than patients with mild illness. t-cell responses in term of frequency and intensity were clearly distinct between mild illness and severe pneumonia patients. furthermore, our results demonstrated that recent history of covid-19 did not hamper viral memory t-cell pool against common viruses (cytomegalovirus, epstein-barr-virus and flu-virus). the presence of potent adaptive immunity even in patients who underwent severe pneumonia sustain the rationale for the development of protective therapeutics against sars-cov-2. adaptive immune responses generated by sars-cov-2 virus in convalescent patients according to disease severity remain poorly characterized. to this end, we designed a prospective study (nct04365322) that included 60 covid-19 convalescent patients (1month post infection) in two cohorts respectively entitled mild illness and severe pneumonia. the monitoring of peripheral immune responses was performed using ifnᵧ elispot assay. the serology index of each patient was investigated at the same time. patients with severe pneumonia were older and had more comorbidities than patients with mild illness. t-cell responses in term of frequency and intensity were clearly distinct between mild illness and severe pneumonia patients. furthermore, our results demonstrated that recent history of covid-19 did not hamper viral memory t-cell pool against common viruses (cytomegalovirus, epstein-barr-virus and flu-virus) . the presence of potent adaptive immunity even in patients who underwent severe pneumonia sustain the rationale for the development of protective therapeutics against sars-cov-2. sars-cov-2 virus induces symptoms of variable severity; some patients only have mild illness whereas other rapidly become critically ill progressing to an acute respiratory distress syndrome (ards). this critical state of the disease supports the immediate relevance for the development of protective therapeutics against sars-cov-2 but requires fundamental knowledge concerning adaptive immune responses induced by the virus. therefore we have read with interest the recent findings published by xu bo and colleagues describing in the early stage of the disease a positive correlation between t-cells decrease and covid-19 severity 1 . however, thijsen s and colleagues demonstrated the presence of specific t-cell responses against s and n proteins few days post onset symptoms in patients with severe pneumonia hospitalized in intensive care unit (icu) 2 . although the existence of sars-cov-2 specific t-cells has been described 2,3 , the frequency and the intensity of sars-cov-2 specific t-cell responses among mild illness and severe pneumonia convalescent covid-19 patients remains to be investigated. in this prospective study, 60 patients who had covid-19 were enrolled in a two cohorts study that were entitled mild illness (n=30) and severe pneumonia (n=30) at least 21 days after the first symptoms of ; table 1 for cov-n) might be explained by the sequence homology between structural proteins from various coronavirus suggesting the existence of cross reactive memory t-cells 5 . indeed, high degrees of similarities between coronaviruses and sars-cov-2 concerning s, m and n structural proteins have been recently described 6, 7 . beyond specific cellular responses, typical humoral responses to acute viral infection are wildly induced in covid-19 patients 8 . zhao et al. showed that the seroconversion rate and antibody levels increased rapidly during the first two weeks with a cumulative seropositive rate of 50.0% on the 11th-day and 100% on the 39th-day. high titers of igg antibodies detected by enzyme immunoassays have been shown to positively correlate with neutralizing antibodies 8, 9 . in this study, the median serology index of severe pneumonia patients was equal to 7.19 s/co [iqr: 6. fig. 1c) . we observed that all patients with severe pneumonia had a positive serology index and most of them had at least one specific cellular response for sars-cov-2 proteins (28 out of 30). in contrast, patients with mild illness had less specific cellular responses (20 out of 29) than severe pneumonia patients (p=0.0211) (fig. 1d) . among mild illness patients, three had a negative serology index (fig. 1c) . specific t-cell responses for s, m and n proteins were simultaneously shown for 70.0% of severe pneumonia patients while only for 37.9% of mild illness patients (p=0.0191) (fig. 1e) . of note, levels of t-cell responses were not influenced by previous exposition to a specific covid-19 treatment (lopinavir/ritonavir; interferon-beta-1a; hydroxychloroquine and remdesivir) (data not shown). we notice that despite a lower intensity of response in terms of infᵧ secretion ( fig. 1 b) , patients with severe pneumonia had frequencies of responses clearly distinct from the one of mild illness patients. suppressed t cell-mediated immunity in patients with covid-19: a clinical retrospective study in wuhan elevated nucleoprotein-induced interferon-γ release in covid-19 patients detected in a sars-cov-2 enzyme-linked immunosorbent spot assay targets of t cell responses to sars-cov-2 coronavirus in humans with covid-19 disease and unexposed individuals clinical and immunological features of severe and moderate coronavirus disease 2019 immunology of covid-19: current state of the science preliminary identification of potential vaccine targets for the covid-19 coronavirus (sars-cov-2) based on sars-cov immunological studies *elispot was not performed for one patient, ards = acute respiratory distress syndrome we thank olivier adotévi for scientific support and proof reading. we thank sylvie cour (nurse) of the clinical investigation center (inserm cic 1431) for her help to collect blood sample. we also thank the biomonitoring platform (eléonore gravelin, adeline renaudin, harmonie simonin, caroline laheurte) for technical support. authors declare no competing financial interests. key: cord-281130-9tawihti authors: schirinzi, annalisa; pesce, francesco; laterza, riccardo; d'alise, maria gabriella; lovero, roberto; fontana, antonietta; contino, renato; serio, francesca di title: pentraxin 3: potential prognostic role in sars-cov-2 patients admitted to the emergency department date: 2020-11-02 journal: j infect doi: 10.1016/j.jinf.2020.10.027 sha: doc_id: 281130 cord_uid: 9tawihti nan recent manuscript by hansen c et al. (4) that evaluated the role of complement related pattern recognition molecules, including c-reactive protein (crp) and pentraxin 3 (ptx3), as markers of short-term mortality in intensive care patients. ptx3 and crp are the well-known, prototypic short and long pentraxin, respectively, differing for gene organization, protein oligomerization and expression pattern. crp is produced by the liver, whereas ptx3 is an inflammatory mediator produced by various cells in peripheral tissues (5) . crp is a typical acute phase biomarker since it is produced as a result of systemic inflammatory responses. conversely, ptx3 defines early and local acute phases, being rapidly produced and released by mononuclear phagocytes, neutrophils, fibroblasts, and epithelial and endothelial cells in response to primary inflammatory signals (e.g. il-1 and tnf-) (6) . in patients with community-acquired pneumonia (cap), the plasma concentration of ptx3, but not crp, was correlated with the severity of cap based on the pneumonia severity index (psi), curb-65, acute physiology and chronic health evaluation (apache) ii scores, and the length of hospital stay (7) . in order to evaluate the potential prognostic value of ptx3 and its correlation with the severity of sars-cov-2, measurement of ptx3 in serum samples of patients (n= 75, male/female 47/28, age 69 years (median) 59-75 years (iqr)) with covid-19 microbiology proven infection (from march to may 2020) was carried out using an enzyme-linked immunosorbent assay (elisa) (dsx, technogenetics srl, milano, italy), in addition to routine laboratory tests performed at admission. forty patients were admitted in the intensive care unit (icu), 35 patients in infectious disease division or in pneumology division (nicu). according to the severity and the evolution of the disease, 37 icu patients died and 3 were moved to nicu divisions for improved clinical conditions. in our cohort, routine laboratory tests showed an increase of crp (dimension vista, siemens healthcare diagnostics inc, tarrytown usa), ddimer (cs 5100, siemens healthcare diagnostics inc), psp (pathfast, chemical medience corporation, tokyo, japan), procalcitonin (pct) and interleukin-6 (il-6) (cobas 8000 system, roche diagnostics, gmbh, mannheim, germany), in line with other studies (8, 9) . in particular 85% of patients showed crp >10 mg/l, 73% d-dimer >500 ug/l, 66% ldh >241 u/l, 88% psp >250 pg/ml, 26% pct >0.5ng/ml, and 82% il-6 >7 pg/ml. ptx3 was measured at the admission of patients in emergency covid room and values higher than the cut-off suggested by the manufacturer (2000 pg/ml) were observed in patients who died (median, iqr =13589,11734-15000) as well as in patients who survived (median, iqr =5729, 3362-9470). according to roc curve analysis of all biomarkers considered in our study, the auc of ptx3 values in predicting the mortality was 0.93 (95% ci: 0.86-0.99) reaching a sensitivity of 89% and a specificity of 92% at the threshold level of 10792 ( figure 1 ). the auc resulting from the combination of ptx3, il-6 and pct was significantly higher than that of ptx3 alone (0.95, 95% ci: 0.90-1.0). figure 2 shows the median values of ptx3 between patients who died and those who survived (p<0.001). moreover, ptx3 correlated (spearman test) with some inflammation biochemical parameters commonly evaluated in sars-cov-2 patients, in particular with il-6 (r =0.69, p<0.001), pct (r =0.52, p<0.001), psp (r =0.52, p<0.001), ldh (r =0.62, p<0.001), crp (r =0.59, p<0.001), and d-dimer (r =0.43, p<0.001). furthermore, a multivariate logistic regression analysis, using all considered variables, confirmed the independent prognostic role of ptx3 with an or = 1.001 (95% ci: 1.000-1.001, p =0.005). taken together, data obtained from our preliminary study suggest a potential prognostic role of ptx3 in sars-cov-2 patients, with higher levels associated with poor outcome. moreover, we observed that the combination of ptx3 with il-6 and pct associated with covid-19 disease progression improves the accuracy of prognosis prediction. as such, ptx3, peaking within 6 to 8 hours of the inflammatory stimulus, might have important implications for the clinical management of patients with covid-19 allowing to identify, at admission, the patients headed for adverse outcomes. our study presents some limitations, namely the limited number of patients. then, ptx3 concentrations should be assessed during the hospitalization period to better estimate the prognostic role of this biomarker. if further studies will confirm our preliminary findings, the manufacturer could be encouraged to improve the current diagnostic method in order to reduce the analytic turnaround time (tat) according to clinical needs. interestingly, ptx3 is not only present in blood samples but can also be found in other biofluids, including pleural fluid (10) . it is hence possible to hypothesize that ptx3 concentration measurement in bronchoalveolar lavage fluid correlates with disease severity in sars-cov-2 patients presenting frequent pulmonary complications such as acute lung injury. ethical approval: the study has been cleared by the local ethical committee (aou policlinico consorziale of bari; no. 6388 covid19 dom -protocol number 0034687/12-05-2020). clinical features of patients infected with 2019 novel coronavirus in wuhan, china biomarker associated with covid-19 disease progression presepsin in risk stratification of sars-cov-2 patients complement related pattern recognition molecules as markers of short-term mortality in intensive care patients multimer formation and ligand recognition by the long pentraxin ptx3: similarities and differences with the short pentraxins c-reactive protein and serum amyloid p component cellspecific regulation of ptx3 by glucocorticoid hormones in hematopoietic and nonhematopoietic cells plasma long pentraxin 3 (ptx3) concentration is a novel marker of disease activity in patients with communityacquired pneumonia laboratory abnormalities in patients with covid-2019 infection taste and smell disorders in covid-19 patients: role of interleukin-6 pentraxin 3 in acute respiratory distress syndrome: an early marker of severity competing interests: authors state no conflict of interest.author contributions: all authors have accepted responsibility for the entire content of this manuscript and approved its submission. key: cord-317846-str9i01o authors: chen, dr. xian; shan, yuheng title: mesenchymal stem cell therapy in severe covid-19: a retrospective study of short-term treatment efficacy and side effects date: 2020-05-15 journal: j infect doi: 10.1016/j.jinf.2020.05.020 sha: doc_id: 317846 cord_uid: str9i01o nan we read with interest the recent paper by cantini et al., describing the safety and clinical impact of baricitinib therapy in coronavirus disease 2019 (covid-19) [1] . covid-19, caused by 2019 novel coronavirus (2019-ncov), is increasing rapidly in an epidemic scale and has spread in over 200 countries, causing more than three million confirmed cases and two hundred thousand deaths as of may 5 th , 2020. but currently, there is no vaccine against 2019-ncov or effective treatment for covid-19 [2] . the typical symptoms of covid-19 are fever, cough and dyspnea, and the leading cause of mortality is acute respiratory distress syndrome (ards). as an immunopathologic event, ards is characterized by cytokine storm, which is an excessive systemic inflammatory response triggered by the release of proinflammatory cytokines [3] . therefore, diminishing the cytokine storm may be an important part of treatment in patients with severe covid-19 [4] . mesenchymal stem cells (mscs) have been shown to possess powerful immunomodulatory properties and beneficial effects for preventing or reducing the cytokine storm [5] . hence, mscs therapy may be a promising option for the treatment of severe covid-19. on this basis, we conducted a retrospective study to evaluate the treatment efficacy and side effects of mscs therapy on severe covid-19. all hospitalized patients met the following criteria were consecutively recruited from clinical grade mscs were given at a dose of 1×10 6 mononuclear cells per kilogram of weight. promethazine hydrochloride (intramuscular injection, 25 mg) was used before the injection of mscs to prevent allergies. for patients received two or three times mscs therapy, the interval of injection was 5 days. laboratory tests were conducted 2 to 3 hours before the injection and 48 to 72 hours after the injection. data were presented as mean±sd for continues variables with normal distribution, and median and interquartile range (iqrs) otherwise. independent continuous variables were compared using the student t test or the mann-whitney test. paired continuous variables were compared using the paired t test or the wilcoxon signed-rank test. categorical variables were compared using the chi-square test or the fisher exact test (if any expected value <5). all of the analyses were conducted as 2-sided tests and p<0.05 was considered statistically significant. totally, 25 patients were enrolled according to the criteria. among them, 20 cases (80%) were male and 5 cases (20%) were female. the median age was 70 (iqr: 59,71) years. seven cases received mscs therapy for one time, 7 cases received for two times and 11 cases received for three times. after mscs therapy, 16 cases (64%) gained apparently ct scan improvement and all cases gained clinical improvement (figure 1 ). no fatalities occurred during hospitalization. however, 3 cases experienced treatment related side effects, specifically liver dysfunction, heart failure and allergic rash. the laboratory findings before and after mscs therapy were shown in table 1 . there are two main mechanisms of mscs therapy for covid-19. firstly, mscs could lodge in the pulmonary vascular bed after injection, release anti-inflammatory mediators and reduce the cytokine storm caused by viral infection [7] . secondly, mscs could secrete angiopoietin-1 and keratinocyte growth factor, which are pivotal in the restoration of alveolar capillary barriers disrupted by covid-19 [8] . in our series, all the patients with severe covid-19 survived and entered recovery after mscs therapy, and only 3 patients experienced treatment side effects. this result indicated that mscs therapy might be an effective therapeutic for severe covid-19. however, none of the inflammation indexes changed significantly after mscs therapy. the reason is unclear, may be related to three factors. firstly, inflammation indexes, such as wbc counts and crp were totally normal before mscs therapy in most cases, which means that cytokine storm was mild to moderate and not serious in these cases. secondly, relative studies have shown that mscs will be cleared within 24 to 48 hours after injection [9] . nevertheless, in our study, laboratory tests were conducted 48 to 72 hours after injection. as a result, we might miss the optimal time to track the changes of inflammation indexes. thirdly, the inflammation indexes tested and analyzed in this study were limited, and whether other cytokines like il-2 and il-7 would decrease after mscs therapy is unknown. additionally, we found that the serum levels of lac, ctnt and ck-mb were elevated significantly after mscs therapy. the reason is unclear, but remind us that the use of mscs therapy should be extremely cautious in patients with metabolic acidosis or coronary heart disease. moreover, the infusion speed of mscs must be slow enough. in this study, we injected mscs saline solution at a speed of ~20 drops per minute, but there was still a patient experiencing heart failure while on treatment. the major limitations of this study were small series, retrospective and no placebo. therefore, additional prospective studies involving large cohort of patients are needed in order to confirm and supplement the present findings. in conclusion, we suggested that mscs therapy might be a promising option for the treatment of severe covid-19, but should be used cautiously, especially in patients with metabolic acidosis or coronary heart disease. values are presented as mean ± sd or median (p25, p75). wbc, white blood cells; crp, c-reaction protein; pct, procalcitonin; il-6, interleukin-6; lac, lactate; alt, alanine aminotransferase; cr, creatinine; ctnt, cardiac troponin t; ck-mb, creatine kinase-mb. baricitinib therapy in covid-19: a pilot study on safety and clinical impact the epidemiology, diagnosis and treatment of covid-19 pathogenic human coronavirus infections: causes and consequences of cytokine storm and immunopathology the pathogenesis and treatment of the `cytokine storm' in covid-19 cytokine storm intervention in the early stages of covid-19 pneumonia interpretation of "guidelines for the diagnosis and treatment of novel coronavirus (2019-ncov) infection by the national health commission (trial version 5 current status of cell-based therapies for respiratory virus infections: applicability to covid-19. the european respiratory journal expanded umbilical cord mesenchymal stem cells (uc-mscs) as a therapeutic strategy in managing critically ill covid-19 patients: the case for compassionate use the authors wish to thank all the clinicians for their hard work and sacrifices. as a retrospective study and data analysis was conducted anonymously, written informed consent was not required in this study. the authors declare that there are no conflicts of interest. fig.1 chest ct scans of severe covid-19 cases before and after mscs therapy. a, cases with apparently ct scan improvement; b, cases without apparently ct scan improvement key: cord-281753-neur9nmc authors: ji, jingjing; wu, ming; zhong, li; liu, zheying; wang, conglin; shao, ziyun; xie, qifeng; liu, zhifeng title: early, low-dose, short-term methylprednisolone decreased the mortality in critical covid-19 patients: a multicenter retrospective cohort study date: 2020-11-08 journal: j infect doi: 10.1016/j.jinf.2020.11.001 sha: doc_id: 281753 cord_uid: neur9nmc nan we read with interest the recent paper by yang et al, concluding that corticosteroids overall have a negative impact on covid-19 outcomes from a meta-analysis 1 . critical covid-19, characterized by refractory hypoxemia caused by acute respiratory distress syndrome (ards), is a life-threatening multi-organ dysfunction syndrome resulted from host response to severe acute respiratory syndrome coronavirus 2 (sars-cov-2). glucocorticoid (gc) was one of the antiinflammatory medications used in critical patients 2 . efficacy of glucocorticoids has been reported in numerous clinical studies in the treatment of coronavirus pneumonia 3 . yang and colleagues demonstrated that patients treated with gc had a higher mortality 1 , suggesting that not all patients could benefit from gc treatment. present study aimed to evaluate the effect of gc on different patient population. since critical patients were more likely to receive gc therapy, only severe type and critical type patients, according to clinical classification of the chinese recommendations for diagnosis and treatment of novel coronavirus (sarscov2) infection (trial 7th version) 4 , were enrolled in present study. we retrospective collected the clinical and outcome data of critical covid-19 patients, and taking methylprednisolone ( few studies have discussed the application time, dosage and duration of mp, which were mostly based on the physician experience. to further clarify when and how to employ mp application on the critical type patients, the hazards ratios were analyzed in each group according to the starting time, dosage, and treatment duration ( figure 2 ). in all 107 critical type patients, 33 of them were not received mp treatment, 59 of them received mp treatment in 7 days after admission to hospital and 12 of them were received after 7 days. 3 patients received mp treatment, but the starting time were missed, and they were not enrolled in analysis. our results showed mp treatment in 7 days after admission could decrease the 60-day fatality (hr: 0.294, 95 % ci: 0.159-0.543, p-value < 0.001), while mp treatment after 7 days has no effect on the fatality. subgroup with different doses of mp (=< 80 mg/d or > 80 mg/d) were also analyzed. we found that small dose mp showed significant effect on the fatality (hr: 0.329, 95 % ci: 0.178-0.605, p value < 0.001). in addition, most patients benefited from mp were received treatment no more than 7 days. mp long-term treatment might increase the death risk. present multicenter retrospective cohort study showed that methylprednisolone therapy could decrease the 60-fatality for the covid-19 patients diagnosed as critical type, that is, those occurred respiratory failure and needs mechanical ventilation, or shock, or multiple organ failure and needs icu monitoring. early (starting in 7 days after admission), low-dose (no more than 80 mg/d), and short-term (no more than 7 days) methylprednisolone therapy could significant decrease the 60-day fatality. the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. the corresponding author had full access to all the data in the study and had final responsibility for the decision to submit for publication. the authors declare that they have no competing interests. the study was approved by the research ethics commission of general hospital of southern theater command of pla. the requirement for informed consent was waived by the ethics commission. the effect of corticosteroid treatment on patients with coronavirus infection: a systematic review and metaanalysis glucocorticoid attenuates acute lung injury through induction of type 2 macrophage the use of anti-inflammatory drugs in the treatment of people with severe coronavirus disease 2019 (covid-19): the perspectives of clinical immunologists from china national health commission of the people`s republic of china. chinese recommendations for diagnosis and treatment of novel coronavirus (sarscov2) infection (trial 7th version) independent roles of macrophage migration inhibitory factor and endogenous, but not exogenous glucocorticoids in regulating leukocyte trafficking guidelines for the diagnosis and management of critical illnessrelated corticosteroid insufficiency (circi) in critically ill patients (part i): society of critical care medicine (sccm) and european society of intensive care medicine (esicm) 2017 short-term glucocorticoid treatment normalizes the microcirculatory response to remote ischemic conditioning in early complex regional pain syndrome the data sets used and/or analyzed during the current study are available from the corresponding author on reasonable request. not applicable. all authors 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. zhifeng liu was responsible for study concept and key: cord-278225-d0gxb6bx authors: meng, yifan; guo, ensong; liu, jia; huang, xiaoyuan; sun, chaoyang; wu, peng; chen, gang title: value and challenges: nucleic acid amplification tests for sars–cov-2 in hospitalized covid-19 patients date: 2020-04-30 journal: j infect doi: 10.1016/j.jinf.2020.04.036 sha: doc_id: 278225 cord_uid: d0gxb6bx nan to date, most countries have confirmed covid-19 cases of person-to-person spread, and the number of confirmed cases worldwide is expected to continue to increase. it has been emphasized that diagnostic testing for sars-cov-2 was an especially important tool in the diagnosis and management of patients with covid-19. 2 we included 3232 consecutive patients with covid-19 who were hospitalized between january 18 th and march 27 th , 2020 (data cutoff date) at tongji hospital, a designated hospital for severe covid-19 patients in wuhan, china. all patients included in the present study were verified as positive for sars-cov-2 infection by reverse transcriptase polymerase chain reaction (rt-pcr). the specific operation methods were 3 followed according to the instructions and were consistent with other literature. 3, 4 according to the covid-19 diagnosis and treatment plan issued by the national health commission, all patients included were diagnosed as moderate to severe cases. clinical data were collected from medical records. the ethical committee of tongji hospital of tongji medical college at huazhong university of science and technology approved this study (tj-irb20200311). written informed consent was not obtained because the data were analyzed retrospectively and anonymously. as of march 27th 2020, 3075 of these patients had at least one rt-pcr test during hospitalization, contributing 12 110 results. in total, 10 309 oropharyngeal swabs (op) from 3003 patients and 1141 nasopharyngeal swabs (np) from 567 patients were tested. in addition, there were 660 specimens by other sampling methods (e.g., bronchoalveolar lavage fluid, anal swabs) being collected and tested. the overall positive rate of np was 18.1% (207/1141), which was higher than that of op (16.7%, 1718/10 314). the positive rates also differed between patients who were died and discharged (37.0% vs. 16.0%). it should be noted that only 42.5% of death cases (62/146) were tested positive in the last rt-pcr test before death. the average intervals between two viral tests during hospital stay were 6.2 days for death cases, with 6.0 days for survivors. currently, the us cdc recommended collecting only np, 5 while current public health england guidance advises samples from the upper respiratory tract should be sought as np, op, or both in combination. 6 in the present study, the overall positive rate of np was higher than that of op. we also evaluated the proportion of false-negative results (negatives between two positive results during hospitalization) among all negative results. 7 the false-negative rate of op was 10.0% (863/8596), while np was 8.4% (78/934). however, three patients 4 have contributed 33 false-negative oropharyngeal swabs (33/78), indicating significant individual bias. here we suggested that the nasopharyngeal specimen is the preferred choice for swab-based sars-cov-2 testing with higher sensibility and specificity. moreover, the negative predictive value of viral tests should be carefully evaluated. at present clinical practice, patients with improved respiratory symptoms, improved pulmonary imaging, and nucleic acid tests negative twice consecutively (sampling interval ≥ 24 hours) can be discharged. however, the data showed that people can test positive for the virus even after two consecutive negative results. pan et al. reported that potential false-negative nucleic acid testing results for sars-cov-2 could be caused by thermal inactivation of samples with low viral loads. 8 according to our study, repeated viral rt-pcr testing separated by prolonged duration is needed for viral clearance evaluation. other immunological parameters or antibody test should also be used in combined with rt-pcr negative test. negative results must be interpreted with clinical observations, patient history, and epidemiological information. for 2876 survivors, sars-cov-2 infection persistence curves were generated based on kaplan-meier analysis (figure) . the median duration from onset of symptoms to pathogens clearance was 24 days (iqr 17-33). the median duration from hospital admission to pathogens clearance was 8 days (iqr 3-14) . for patients with reliable preadmission pathogens-identified records, the median duration from pathogens identified to pathogens clearance was 16 days (iqr 11-24). generally, it takes a person several days to weeks to show symptoms after being exposed to the virus. our analysis indicated that the median duration from onset of symptoms to hospital admission was 16 days in wuhan, china. the clinical sampling frequency for inpatients with covid-19 should be viral dynamics of sars-cov-2 across a spectrum of disease severity in covid-19 report from the american society for value of diagnostic testing for sars-cov-2/covid-19 epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical features of patients infected with 2019 novel coronavirus in wuhan, china covid-19) stability issues of rt-pcr testing of sars-cov-2 for hospitalized patients clinically diagnosed with covid-19 potential false-negative nucleic acid testing results for severe acute respiratory syndrome coronavirus 2 from thermal inactivation of samples with low viral loads we would like to show our great respect to all the workers and volunteers in the fight against covid-19, especially to the medical workers who work with the authors on the frontline. none. all authors declare that they have no financial or other conflicts of interest. 7 key: cord-320826-o6ih2f23 authors: blairon, laurent; mokrane, saphia; wilmet, alain; dessilly, géraldine; kabamba-mukadi, benoît; beukinga, ingrid; tré-hardy, marie title: large-scale, molecular and serological sars-cov-2 screening of healthcare workers in a 4-site public hospital in belgium after covid-19 outbreak date: 2020-07-31 journal: j infect doi: 10.1016/j.jinf.2020.07.033 sha: doc_id: 320826 cord_uid: o6ih2f23 nan we read with great interest the study of chen y et al., who analyzed, during the chinese epidemic peak, the seroprevalence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) among 105 healthcare workers (hcws) exposed to covid-19 patients [1] . they found 17.14% of seropositive asymptomatic or paucisymptomatic hcws although their nasopharyngeal swab samples were sars-cov-2 rna negative. our purpose was to document at the end of the belgium epidemic the seroprevalence of sars-cov-2 in hcws exposed to covid-19 at varying degrees and to compare these rates with those observed by other teams worldwide. another objective was to highlight sars-cov-2 carriage in a priori healthy staff members to sensitize them to the need to respect individual protection measures and distancing to avoid patient contamination. in belgium, the covid-19-outbreak peak was reached on 10 april 2020 [2] . at the end of may, when the epidemic spread was greatly slowed down, our management decided to offer screening tests to all staff members (n=3145), regardless of their status and function. the campaign took place from may 25 to june 19, 2020 in the network of iris hospitals south (his-izz, brussels, belgium), a 550-bed public hospital spread over 4 sites. participation was voluntary and regardless of whether the hcw had already contracted the disease or not. a questionnaire was prepared focusing on the type of service the participant works in, the practice of medical procedures potentially at risk for sars-cov-2 infection, its status, function and perception of being infected or not. people with covid-19 symptoms [3] were excluded from routine screening. on the same day, all asymptomatic hcws who agreed to participate benefited from both serological and rt-qpcr sars-cov-2 tests. the quantitative analysis of igg antibodies directed against the s1 and s2 subunits of the virus spike protein was carried out using the liaison®sars-cov-2 igg kit (diasorin, saluggia, italy). this clia method was extensively evaluated in our laboratory and showed 100% sensitivity two weeks after positive qrt-pcr diagnosis using an adapted cut-off [4] . equivocal results were confirmed by a semi-quantitative elisa method directed against the s1 subunit spike protein (euroimmun medizinische labordiagnostika, lübeck, germany). hcws with a previous covid-19 documented history and a persistent positive rt-qpcr benefited from a viral culture. statistical analyses were carried out using medcalc version 10.4.0.0 (medcalc software, ostend, belgium). a pvalue <0.05 is considered statistically significant. during the study period, 1499 staff members participated (47.7%). table 1 [5] . our seroprevalence result of 14.6% is closer to that reported by chen et al [1] . to the best of our knowledge, seroprevalence in hcws after the epidemic peak was never studied in as many participants. at the end of may, the belgian public health institute, sciensano, assessed the seroprevalence of hcws at 8.4% among 785 samples [6] . the difference between our results and those of sciensano can be explained by the outbreak evolution which led to seroprevalence increase. unexpectedly, our screening campaign failed to identify a single new case of covid-19 among the participants. people positive to rt-qpcr were not living-virus carriers. this confirm that molecular methods can give positive results at a distance from a documented infection with an up to 67-day delay. seroprevalence is higher than that documented by sciensano during the epidemic peak and higher among hcws who worked in covid units. this shows that it is important to re-evaluate national seroprevalence in both the general population and hcws at the end of the outbreak, especially as sars-cov-2 infection may be paucisymptomatic or asymptomatic and therefore infected people might ignore their status. high sars-cov-2 antibody prevalence among healthcare workers exposed to covid-19 patients covid-19 bulletin épidémiologique du 10 avril covid-19 diagnosis and management: a comprehensive review first experience of covid-19 screening of health-care workers in england sars-cov-2-specific antibody detection in healthcare workers in germany with direct contact to covid-19 patients covid-19 study: 8,4% of belgian health workers have antibodies to sars-cov-2 n the authors thank the general and medical managements of the iris hospital south for taking the lead on this massive screening; the blood sampling centre, the technologists and administrative staff who contributed to the analytical, pre-analytical and post-analytical steps of the laboratory tests and all those who participated in this investigation. ethical statement: the study design, the procedure of results communication, the information circular and the questionnaire have been submitted to and approved by our hospital's ethics committee (ethical agreement number: cehis/2020-19). an informed consent form has been requested from each participant, guaranteeing anonymity of the data and requesting permission to use them for statistical analysis. out of respect for everyone's privacy, the participant was free to not answer to certain questions.funding: all molecular tests were supported by the federal covid-19 platform. key: cord-288010-i9zrojoo authors: jia, yuanyuan; yang, cuixian; zhang, mi; yang, xianyao; li, jianjian; liu, jiafa; liu, ying; yang, xinping; feng, yue; dong, xingqi; xia, xueshan title: characterization of eight novel full-length genomes of sars-cov-2 among imported covid-19 cases from abroad in yunnan, china date: 2020-05-15 journal: j infect doi: 10.1016/j.jinf.2020.05.016 sha: doc_id: 288010 cord_uid: i9zrojoo nan recent correspondence in this journal has highlighted the current threat posed by recently-emerging corona virus disease 2019 in the world. 1 the covid-19 is infection caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and is characterized by fever, dry cough, weak, and so on. 2,3 sars-cov-2 has already caused a global pandemic. by 26 apr, 2020, the spread of sars-cov-2 has led to more than 3.0 million infections and above 200,000 deaths; 4 thus, its outbreak has become a global public health problem. recently, covid-19 epidemic in china has been well controlled, whereas the risk of imported covid-19 cases has increased dramatically. 5 as of april 26, 2020, a total of 1,636 abroad imported patients were reported in china. 6 however, limited studies on full-length genome characterization of sars-cov-2 from covid-19 cases imported from abroad. here, we characterized the genotype and mutation characteristics of sars-cov-2 isolated from eight imported cases from abroad in yunnan, china. eight covid-19 patients imported from overseas were admitted to yunnan provincial infectious disease hospital from march 15, 2020 to march 26, 2020. the epidemiological history and respiratory symptoms of the eight patients were summarized in figure 1a and 1b. the 8 patients include 4 males and 4 females, with ages ranging from 6 years to 70 years old. no patient has ever been to wuhan city in china. two cases yn_im01 and yn_im03 were from spain to yunnan, yn_im02 from france, yn_im04 from cambodia, yn_im05 from sri lanka, and yn_im06-08, a family cluster of covid-19 patients from the united states (fig.1a) . six cases showed different degrees of respiratory symptoms before hospitalization. yn_im06 was severe, yn_im01, yn_im05, yn_im07, and yn_im08 were moderate, yn_im02 was mild, and yn_im03 and yn_im04 were asymptomatic according to the latest covid-19 diagnostic criteria (5th edition) published by the national health commission of china (fig.1b) . so far, three main clades involving g, v, and s have been identified based on marker mutations in the complete sars-cov-2 genome according to the latest genotyping rules recommended by the gisaid databas. 7 g clade containing d614g variant in s protein is predominant in europe, v clade possessing g251v mutation in orf3 is more common in asia and europe, and s clade having l84s substitution in orf8 is move prevalent in north america. 8 in this study, eight complete genome sequences of sars-cov-2 isolated from sputum samples were successfully amplified and sequenced with 38 overlapping fragments. dataset comprise sars-cov-2 full-length sequences of representative clade g, v, and s as previously reported, and reference sequences with the highest similarity (12 sequences) based on blast in genbank using the eight sequences obtained in this study as the query set. further, phylogenetic trees for sars-cov-2 full-length nucleotide sequences were constructed based on the obtained datasets with the maximum-likelihood method using iq-tree. phylogenetic analyses revealed that the six isolates, including one from france (yn_im02), two from spain (yn_im01 and yn_im03), and three from the united states (yn_im06-08) were clustered as g clade with a high bootstrap value of 99%, one strain from cambodia (yn_im04) was grouped into s clade with a bootstrap value of 80%, and the remaining one from sri lanka was classified within other clade, a large unclassified sequences because lack the signature variants (fig.1c) . of note, the three sequences yn_im06-08 isolated from a family cluster of sars-cov-2 infection formed a close monophyletic subclade supported by a bootstrap value of 100% and had 99.99% nucleotide identity, indicating the three sequences originate from the same strain. to further characterize the characteristics of virus variation, the sequence analyses based on sars-cov-2 full-length nucleotide and amino acid sequences was performed using the strain wuhan-hu-1 (genbank no. nc_045512) identified earliest in wuhan seafood market, in hubei, china as the reference strain for nucleotide and amino acid positions. 9 the results revealed that 15, 12 and 10 nucleotide mutations to clades g, s, and other, respectively, were mapped across the sars-cov-2 full-length genome. corresponding to these nucleotide substitutions, 8, 6, and 5 non-synonymous amino acid substitutions were detected in clades g, s, and other, respectively. of note, all clade g strains possessed another p4715l marker substitution in nsp12 besides the signature mutation d614g variant in s protein. yn_im05 strain belonging to other clade have a unique 3-nucleotide deletion between 518 and 520 nt that was not found in g and s clades, leading to a methionine deletion at position 58 in leader protein. moreover, three novel mutations, including d1962v in nsp3 from the strain yn_im03, l1375f in nsp3 and a829t in s protein from the isolate yn_im04 were first identified in this study according to the comparison with 11,231 genomic sequences available at gisaid on 4/26/2020. 10 interestingly, s23t and r203w mutations located in n protein were identified in the three isolates from a family cluster of sars-cov-2 infection. given that the three covid-19 patients were diagnosed on the same day and the viruses originated from the same strain, indicated that the strain is replicating and mutating rapidly in different individuals. in summary, we characterized the full-length genomes of sars-cov-2 strains from eight covid-19 cases imported from abroad in yunnan, china. our results showed that the predominant sars-cov-2 clade was g (6 cases), followed by s clade (one case) and unclassified clade (one case). further, comparative genomic analyses revealed that a novel signature amino acid substitution p4715l in nsp12 was found in the g clade strains. moreover, three novel mutations, including d1962v in nsp3, l1375f in nsp3, and a829t in s protein were first identified in this study. the present study highlights the urgent need for continuous molecular screening and epidemic surveillance for sars-cov-2 among covid-19 individuals imported from abroad to prevent future outbreaks of sars-cov-2 infection in china. the authors declare no competing financial interests. global covid-19 fatality analysis reveals hubei-like countries potentially with severe outbreaks a novel coronavirus outbreak of global health concern clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study imported covid-19 cases pose new challenges for china phylodynamics of sars-cov-2 transmission in spain a new coronavirus associated with human respiratory disease in china we thank the members of the yunnan infectious disease hospital for the data and sample collection. key: cord-300038-1fjb6b8e authors: cantini, fabrizio; niccoli, laura; matarrese, daniela; nicastri, emanuele; stobbione, paolo; goletti, delia title: baricitinib therapy in covid-19: a pilot study on safety and clinical impact date: 2020-04-23 journal: j infect doi: 10.1016/j.jinf.2020.04.017 sha: doc_id: 300038 cord_uid: 1fjb6b8e • baricitinib at 4 mg/day/orally was given to 12 patients with moderate covid-19. • in baricitinib-treated patients no adverse events were recorded, after 2 weeks. • clinical and respiratory parameters significantly improved at 2 weeks. • none of the baricitinib-treated patients required admission to icu. • proper control group was missing; this is required to demonstrate the efficacy. hospital of prato, italy; phone: +39 0574 807578; fax: +39 0574 802939; mobile: +39 340 8075607 e-mail: fbrzcantini@gmail.com as discussed in the journal recently 1 the sars-cov-2, a new β-coronavirus, uses the angiotensin converting enzyme-2 receptor to enter airway cells. viral endocytosis is mediated by several factors, including clathrin, the adaptor protein-2 complex (ap2) and the adaptor-associated kinase-1 (aak1) 2 . according to a recent report 3 , covid-19, the disease caused by sars-cov-2, is characterized by three clinical patterns: no symptoms, mild to moderate disease, severe pneumonia requiring admission to intensive care unit (icu) in up to 31% of the patients 3 . thus far, there is no specific therapy for covid-19 infection. no benefit of lopinavir-ritonavir treatment resulted in a recent trial 4 . hydroxychloroquine, currently used in view of its "in vitro" observed effect of reduction of viral replication, seems unsatisfactory 5 . elevated proinflammatory cytokine/chemokine responses seem associated with respiratory failure 3 . recently, tocilizumab, an interleukin-6 inhibitor, was reported as effective in patients with severe covid-19 pneumonia 6 . baricitinib, another inhibitor of cytokine-release, seems an interesting anti-inflammatory drug. it is a janus kinase inhibitor (anti-jak) licensed for the treatment of rheumatoid arthritis (ra) with good efficacy and safety records 7 . moreover it seems to have anti-viral effects by its affinity for ap2-associated protein aak1, reducing sars-cov-2 endocytosis 8 . on this basis, we assessed the safety of baricitinib therapy combined with lopinavir-ritonavir in moderate covid-19 pneumonia patients and we evaluated its clinical impact. all consecutive hospitalized patients (march 16 th -30 th ) with moderate covid-19 pneumonia, older than 18 years, were treated for 2 weeks with baricitinib tablets 4 mg/day added to ritonavirlopinavir therapy. the last consecutive patients with moderate covid-19 pneumonia receiving standard of care therapy (lopinavir/ritonavir tablets 250 mg/bid and hydroxychloroquine 400 mg/day/orally for 2 weeks) admitted before the date of the first baricitinib-treated patient served as controls. antibiotics were scheduled only in the case of suspected bacterial infection. inclusion criteria were: a. sars-co-v2 positivity in the nasal/oral swabs; b. presence of at least 3 of the following symptoms: fever, cough, myalgia, fatigue; c. evidence of radiological pneumonia. after discharge, patients treated with baricitinib were planned to be followed for additional 6 weeks. exclusion criteria: history of thrombophlebitis (tp), latent tuberculosis infection (quantiferon plus-test positivity, qiagen, germany 9 ), pregnancy and lactation. mild to moderate covid-19 disease definition: presence of bilateral pneumonia with or without ground glass opacity and in absence of consolidation, not requiring intubation at enrollment; arterial oxygen saturation (spo2) >92% at room-air, and ratio arterial oxygen partial pressure/fractional inspired oxygen (pao2/fio2) 100-300 mmhg. parameters daily accessed were: fever, pulmonary function, modified early warning score (mews) 10 , pulse rate, blood pressure. after the initial execution, radiology imaging was performed on demand. laboratory investigations included blood cell counts with differential counts, tests for liver and kidney functions, erythrocyte sedimentation rate (esr), c-reactive protein (crp), and procalcitonin. the trial was approved by the azienda-usl toscana centro committee for off-label use of drugs. all patients signed a written informed consent to participate to the study. descriptive statistics, presented as median and interquartile range (iqr), were calculated using comorbidities were similar in the two groups. baricitinib-treatment was well tolerated with no serious adverse events (aes). therapy was withdrawn in 1 patient after 10 days of treatment due to consistent transaminases elevation (ast: 267 u/l; alt: 298 u/l), probably due to the antiviral therapy rather than to the baricitinib treatment, which is mainly renal-metabolized. in addition, no bacterial or opportunistic infections, trombo-flebitis or hematologic toxicity were observed. results are summarized in table 2. overall, in the baricitinib-treated group, all clinical characteristics and respiratory function parameters significantly improved both at week 1 and week 2 compared to baseline. crp values significantly decreased at the same timeframes. in the controlgroup, no significant changes were recorded at week 2 compared to baseline. fever, spo2, pao2/fio2, crp, and mews significantly improved in the baricitinib-treated group compared with controls (p: 0.000; 0.000; 0.017; 0.023; 0.016, respectively). icu transfer was requested in 33% (4/12) of controls and in none of the baricitinib-treated patients (p=0.093). discharge at week 2 occurred in 58% (7/12) of the baricitinib-treated patients vs 8% (1/12) of controls (p=0.027). at discharge, 57% (4/7) had negative viral nasal/oral swabs. these preliminary results on 12 patients with moderate covid-19 pneumonia confirmed the safety of baricitinib therapy in a clinical context different from ra 7 . no infections, cardiovascular and hematologic aes occurred after 2 weeks treatment. the short-term drug exposure may probably explain the absence of the supposed aes. to confirm the long-term safety, the patients will be followed-up for further 6 weeks, but the restricted time of treatment and the short half-life of the drug (12.5 hours) suggest as unlikely the later aes occurrence. remarkably, both at week 1 and week 2, baricitinib therapy significantly improved the clinical and laboratory parameters, none of the patients required icu support, and the majority of the patients were discharged. these results were likely due to the rapid action of the drug and the short median interval of 6 days from symptoms-onset and therapy starting, the major limitations of this pilot study were its open-label design with no randomization and the low number of treated patients. a proper control group was missing and this is indeed required to formally demonstrate the efficacy of the therapy. the use of baritinicib therapy may limit the cytokine-release syndrome associated with covid-19 and it may be useful because it acts against a wide-range of cytokines. although our results cannot be generalized to all covid-19 patients, we believe that these data are encouraging in terms of safety, improvement of clinical impact and reduction of severity progression, and it may be the first-step for future controlled, larger studies. this work was partly supported by the italian ministry of health "ricerca corrente" linea 1. , laboratory and respiratory parameters of covid-19 patients after 1-or 2week treatment in the baricitinib-treated group and in the standard-treated group: comparison within the same treatment group and between the 2 different treatment groups. baricitinib analysis of angiotensin-converting enzyme 2 (ace2) from different species sheds some light on cross-species receptor usage of a novel coronavirus 2019-ncov identification of an adaptor-associated kinase, aak1, as a regulator of clathrin-mediated endocytosis clinical characteristics of patients infected with 2019 novel coronavirus in wuhan, china a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 chloroquine and hydroxychloroquine in covid-19 effective treatment of severe covid-19 patients with tocilizumab a systematic review and meta-analysis of infection risk with small molecule jak inhibitors in rheumatoid arthritis the authors are grateful to all the patients, nurses and physicians who helped to perform this study. key: cord-008672-luoxomif authors: mwachari, c.; batchelor, b.i.f.; paul, j.; waiyaki, p.g.; gilks, c.f. title: chronic diarrhoea among hiv-infected adult patients in nairobi, kenya date: 2004-10-29 journal: j infect doi: 10.1016/s0163-4453(98)90561-8 sha: doc_id: 8672 cord_uid: luoxomif objectives: chronic diarrhoea and wasting are well recognized features of aids in africa. however, because of resource constraints few ocmprehensive aetiological studies have conducted in sub-saharan africa which have included a broad range of microbiological investigations. we undertook a prospective cross-sectional study of adult patients admitted to a government hospital in nairobi, kenya, to determine possible bacterial, mycobacterial, parasitic and viral causes of diarrhoca; to consider which may be treatable; and to relate microbiological findings to clinical outcome. methods: stool specimens from 75 consecutive hiv-seropositive patients with chronic diarrhoca admitted to a nairobi hospital were subjected to microbiological investigation and results were compared with clinical findings and outcome. stool samples were cultured for bacteria and mycobacteria and underwent light and electron microscopy; lawns of escherichica coli were probed for pathogenic types and aliquots were tested for the presence of clostridium difficile cytotoxin. blood cultures for mycobacteria and other bacterial pathogens were performed as clinically indicated. results: thirty-nine (52%) patients yielded putative pathogens, the most common being cryptosporidium sp. (17%), salmonella typhimurium (13%), and mycobacterium tuberculosis (13%). of 41 patients investigated for pathogenic escherichia coli, enteroaggregative e. coli and diffusely adherent e. coli were each found in four patients. thirty-one (41%) patients died. detection of cryptosporidium cysts was the single most significant predictor of death (x(2) = 5.2, p<0.05). many patients did not improve (21; 285) or self-discharged whilst still sick (5; 7%) but five (7%) were diagnosed ante mortem with tuberculosis and treated and a further 13 (17%) showed improvement by time of discharge. conclusions: hiv-infected patients with chronic diarrhoea in nairobi have a poor outcome overall, and even with extensive investigation a putative pathogen was identified in only just over half the patients. the most important step is to exclude tuberculosis: and the most useful investigation appears to be ziehl-neelsen staining. other potentially treatable gram-negative bacterial pathogens, s. typhimurium, shigella sp. and adherent e. coli were, however, common but require culture facilities which are not widely accessible for definitive identification. further studies focussing on simple ways to identify sub-groups of patients with treatable infections are warranted. chronic diarrhoea and wasting, slim disease is a well recognized feature of african aids. 1'2 in nairobi it is a common cause of hospital admission in hiv-infected individuals, ranking fourth in cause of admission (after tuberculosis, acute pneumonia and enteric fever-like illness) and third as cause of death. 3 community studies also suggest that it is also a common cause of death, particularly when associated with significant body wasting. in europe the aetiology of hiv-associated chronic diarrhoea has been comprehensively investigated 4 because * please address all correspondence to: c. f. gilks, liverpool school of tropical medicine, pembroke place, liverpool l3 5qa, u.k. accepted for publication 20 april 1998. of the routine use of intensive diagnostic investigations, access to well equipped microbiology laboratories and frequent post-mortem studies. such services are not often routinely available in most government hospitals in sub-saharan africa; and despite the frequency of hiv-related chronic diarrhoea, the aetiology is far less well defined. most investigations into the cause of chronic diarrhoea have limited themselves to small patient numbers, 5' 6 to an intensive search for an individual pathogen 7-9 or have been narrowly based due to limited culture facilities, either ante-mortem or post-mortem. 6' ~o in particular, it is not clear whether any treatable causes of chronic diarrhoea are frequently missed because of the lack of appropriate diagnostic facilities. we undertook a 4-month study in nairobi, kenya, to determine possible bacterial, viral and parasitological causes of chronic diarrhoea and wasting using a broad range of investigations and related findings to patient outcome. from april to july 1992, consecutive patients reporting chronic diarrhoea (loose or watery stool for at least 4 weeks) were enrolled into a prospective clinical and microbiological study. all were adults (>16 years) admitted directly to the acute medical wards of the kenyatta national hospital (knh), the main government hospital serving nairobi. patients referred from other hospitals were excluded. recruitment occurred within 5 days of admission. all patients were examined, a brief history was taken and details were recorded on a standard clinical entry and follow-up form by a single observer (cm). patients received hiv counselling and informed consent was obtained before recruitment and testing. samples of stool and blood for serology were collected from all study patients. blood culture for mycobacteria and other bacterial pathogens was performed on patients according to clinical assessment. because of resource constraints there was limited access to the general diagnostic microbiology service when the study was conducted, and few stool or blood samples were routinely cultured in the service laboratory. all culture work was therefore carried out in the kenya medical research institute (kemri) microbiology research laboratory. relevant results were given by the study team to the clinicians caring for the patients in knh as soon as they were available. patients were otherwise investigated and treated as was appropriate and in line with local policy, and followed daily or until death. the majority of patients (75%) were given cotrimoxazole with or without metronidazole on admission. cd4 counts are not routinely performed as a service investigation in knh, and few families or patients were prepared to pay for them. the study had insufficient funds to carry out cd4 counts on all patients recruited. at the time of the study, sigmoidoscopy was not routinely carried out because of the limited capacity of the service, and no biopsies were taken. post-mortems were occasionally requested, but usually only a few relatives consented. it was concluded that it would not be possible in this study to obtain biopsy or autopsy material in a consistent fashion, so this was not attempted. the study was approved by the kenya hospitals national ethical committee and the kemri research committee. unless otherwise stated, the investigations described were applied to all patients. all specimens were processed for culture on the day of collection using oxoid (unipath, basingstoke, u.k.) media and incubated in air at 37 °c for 18 h, unless specifically stated. identification of isolates was by standard bacteriological techniques and confirmation of identification, along with phage typing and serotyping, where appropriate, were carried out by the public health laboratory service (phls), u.k. culture of stool samples for salmonella spp. and shigella spp. was carried out by direct inoculation onto xylose lysine desoxycholate agar (xld), brilliant green agar (bg) and selenite p broth. after incubation the broth was subcultured onto both xld and bg agars. campylobacter blood-free medium, with cefoperazone selective supplement, was directly inoculated and incubated microaerophilically at 37 °c for 48 h before examination. cefsulodin-irgasan-novobiocin (cin) medium was used to culture yersinia spp. after direct inoculation and after cold enrichment in phosphate buffered saline at 4 °c for 14 days. cin plates were incubated at 30 °c for 24h before examination. direct inoculation onto thiosulphate citrate bile salt sucrose (tcbs) medium and enrichment in alkaline peptone water were used to culture vibrio spp. aeromonas spp. were cultured on ryan's selective medium with 5 mg/1 ampicillin added. blood culture was carried out, for 18 patients, in brain-heart infusion broth incubated in coa for up to 7 days. patients were investigated for mycobacterial infection not as a putative cause of diarrhoea per se, but because a role for tuberculosis has been implicated in slim disease. 9 mycobacterial culture from stool was in selective kirchner medium (loml), following decontamination with 5% sodium hydroxide for 15 min. blood samples (2 ml) from 17 patients for mycobacterial culture were inoculated directly into kirchner medium (loml) at collection. all samples were incubated at 37 °c for up to 6 weeks. phenol-auramine stained smears were examined directly by fluorescent microscopy for mycobacterium spp. and cl~ptosporidium sp. modified ziehl-neelsen (zn) staining was used to confirm equivocal results. specimens were examined by light microscopy for ova, cysts and parasites directly and following formalin-ether concentration as wet preparations. the uvitex 2b fluorescent method was applied to examine for microsporidia in faecal smears from 36 patients. in addition, aliquots of faeces were preserved in 50% (vol/vol) formalin solution and transported to oxford phl, u.k. grids were prepared and examined for enteric viruses under a phillips 301 electron microscope following negative staining with 1% methylamine tungstate. of clinical features and outcome of hospitalization are shown in table i . one observer (cm) recruited all patients and 'marked weight loss' reflected the subjective impression of whether the patient was clinically wasted or not. most patients were unable to state their pre-morbid weight. thirty-seven (49%) patients reported fever and 18 (24%) were febrile on recruitment or during hospitalization. from stool samples from a random selection of 41 patients, lawns of presumptive e. coli were harvested and shipped to the laboratory of enteric pathogens, central public health laboratories, u.k. on columbia agar slopes for detection of pathogenic e. coli using dna probes directed at the following groups: enteropathogenic e. coli aliquots of faeces were stored at -70 °c and then transferred to oxford phl, u.k. for detection of c. difficile cytotoxin using mrc 5 human fibroblast cell lines. hiv antibody testing was performed using wellcozyme hiv-1 (wellcome diagnostics, dartford, u.k.) and confirmed using enzynergost hiv 1 + 2, (behringwerk ag, marburg, germany). one hundred and sixteen adults fulfilled the entry criteria for the study. all patients gave their consent and were entered into the study. of these, 21 (18%) were found to be hiv-seronegative, three (3%) had equivocal hiv serology and 17 (15%) either had inadequate clinical information recorded or inadequate samples collected to enable any useful analysis to be performed; all were excluded. results are presented for the remaining 75 patients. the wide range of potential pathogens detected can be seen in table ii . clostridium difficile toxin, vibrio spp. and yersinia spp. were not detected. light microscopy failed to detect ova, cysts or parasites normally associated with diarrhoea, although the following were detected: chilomastix mesnili (one), ascaris iumbricoides (two), entamoeba coli (two) and hookwork (two). no cases of isospora belli were seen. no patient yielded etec, epec, eiec or vtec. one patient had both eaggec and daec. the only salmonella sp. isolated was s. typhimurium, of which the commonest phage type (pt) was pt 56 (40%). four of 18 standard blood cultures (22%) were positive. one of 17 mycobacterial blood cultures was positive, yielding mycobacterium avium-intracellulare. fifteen patients had multiple pathogens isolated: most had just two, the commonest combinations being s. typhimurium with cryptosporidium sp. (n=5). potential pathogens (apart from hiv) were not found during the investigation of 36 (48%) patients. there was no signifcant difference in mortality rate between overall groups of patients yielding pathogens (17/39, 44%) and not yielding pathogens (14/36, 39%) (table iii) . however, mortality was significantly associated with detection of cryptosporidium cysts. the mortality rate in patients with cryptosporidium cysts was 9/13 (69%) compared with 22/62 (36%) for cryptosporidium-negative patients (x2=5.2; p<o.05). of the 44 patients who survived, 13 (17%) improved with bed rest, broad spectrum antibiotics and symptomatic therapy: five patients (7%) were diagnosed ante-mortem with tuberculosis and started on treatment; 21 patients (28%) failed to show any significant benefit from the hospital admission and were discharged home, and five patients (7%), all still sick, took their own discharge. all patients were considered to have clinical ads. cd4 counts were not available for the study patients. details in africa, chronic diarrhoea is common in adult patients presenting to hospital and is often associated with hiv the spectrum of pathogens recovered was largely in accordance with previous reports from africa, but was in some respects at variance with the experience of hivrelated illness in europe and america. cryptosporidium was the most commonly detected pathogen, being found in 17% of patients, compared with reported rates of 13% in burundi, 13 30% in zaire s and 32% in zambia. 14 other protozoa associated with diarrhoeal illness were not detected in our study. we saw no cases of isospora belli, a pathogen with marked variation in geographical distribution. 15 nor, like other workers in zambia, did we identify any cases of giardia lamblia or entamoeba. 16 a single patient yielded enterocytozoon, the only microsporidian seen, and our low detection rate differed from other studies in zimbabwe and zambia. 9' 17 although simple staining methods on unconcentrated stool samples have been shown to be effective, 9'15'17 small numbers may be revealed only after prolonged examination of preparations from at least two stool samples, and it is possible that more meticulous analysis would have yielded greater numbers. when profound weight loss is a feature of hiv-related illness in africa it is often referred to as slim disease. 1 in our study chronic diarrhoea was the primary entry criterion, but as this is inextricably linked with wasting in many patients it was considered important to seek evidence of mycobacterial infection as a cause of wasting and m. tuberculosis was isolated from the faeces of 10 (13%) patients. although these patients presented complaining of chronic diarrhoea, subsequent clinical evaluation identified five of these patients as likely to be suffering from pulmonary tuberculosis and appropriate therapy was started. these five patients survived, providing further evidence that initially unrecognized tuberculosis may be a significant and treatable contributing factor to the wasting seen in slim disease. 1° nevertheless, in a further five patients who were stool culture positive for m. tuberculosis, tuberculosis was not diagnosed as a result of routine clinical procedures. wasting may be the only noticable feature in a significant number of patients with tuberculosis; or it may be an agonal infection. only one patient yielded mycobacterium avium-intracellulare, which was grown from a blood culture. unlike europe and america, atypical mycobacterial infection is rarely seen in nairobi. 12 salmonella typhimurium was the leading bacterial pathogen in this study and was isolated from 12 patients, two of them from blood culture but not from stool. it is unclear how such acute disseminated salmonellosis, and the single case of e. coli bacteraemia, relate to chronic diarrhoea. these may be secondary infections in debilitated patients with marked immunosuppression. in a previous study, non-typhi salmonellae were found to be the most common cause of hiv-related bacteraemia in hospital admissions in nairobi. tm detection of eaggec or daec from 9/41 (22%) patients was an interesting finding. one patient yielded both of these forms of pathogenic e. coli. a significant association between colonization with adherent e. coli and chronic diarrhoea and wasting in aids patients has been demonstrated by kotler et al., 19 who found such strains in 17% of study patients in the usa. adherent e. coli have also been described from hiv-positive patients with chronic diarrhoea in zambia. 2° despite use of a broad range of microbiological investigations, there was failure to detect a pathogen in almost half the patients. it is likely that analysis of multiple stool samples taken at separate times might have increased yields, as might invasive procedures such as duodenal aspiration and rectal biopsy. ~ it is possible that novel agents remain to be characterized and associated with chronic diarrhoea and wasting in hiv patients, and hiv itself may be the cause of chronic diarrhoea in some patients. 21 whether any such additional investigations would identify more treatable infections remains to be seen. how then may these results contribute to the care of hiv-infected patients with chronic diarrhoea in areas with limited diagnostic and microbiological facilities? the study results clearly show that seropositive patients presenting with chronic diarrhoea have a poor outcome irrespective of whether a potential pathogen is isolated or not. careful diagnostic evaluation, in particular looking for pulmonary tuberculosis, should nevertheless be undertaken. sputum (or stool) zn microscopy for mycobacteria should be feasible almost everywhere and some patients with active tuberculosis will respond well to therapy. stool microscopy using modified zn staining can also identify cryptosporidium cysts, the presence of which is associated with a very limited prognosis. for such patients in the absence of effective therapy, it may be better to aim for symptomatic therapy and early discharge home as soon as the cysts are identified. what of the potentially treatable gram-negative enterobacterial pathogens identified, which were cultured from over 20% of study participants.) furthermore, as many as 20% of patients may be colonized with adherent e. coil, which may also play a role in pathogenesis and may respond to antibiotic treatment. to identify such infections requires access to a microbiology laboratory routinely performing culture and sensitivity testing. unfortunately, few hospitals in sub-saharan africa can at present afford to provide such a service routinely because of severe resource constraints. clinical trials focussing on these patients may be able to define more clearly signs and symptoms which predict bacterial infection without necessarily needing access to culture facilities, and thus more effectively identify a sub-group of patients with chronic diarrhoea for whom hospital admission and intensive antimicrobial therapy may be justified. slim disease: a new disease in uganda and its association with htlv-iii infection persistent diarrhoea, strongly associated with hiv infection in kinshasa, zaire the presentation and outcome of hiv-related disease in nairobi gastrointestinal infections in aids persistent diarrhoea in zairian aids patients: an endoscopic and histological study enteropathic aids in uganda. an endoscopic, histological and microbiological study prevalence of enteric viruses among hospital patients with aids in kinshasa faecal mycobacteria and their relationship to hiv-related enteritis in lusaka, zambia high prevalence of enteroeytozoon bieneusf infections among hivpositive individuals with persistent diarrhoea in harare contribution of tuberculosis to slim disease in africa parasitological observations of chronic diarrhoea in suspected aids adult patients in kinshasa disseminated mycobacterium avinm infection among hiv-infected patients in kenya infectious diarrhoea in african acquired immunodepression syndrome (aids). a prospective survey of 100 patients studied in bujumbura (burundi) hiv-related enteropathy in zambia: a clinical, microbiological and histological study cryptosporidial and mi-crosporidiai infections in hiv-infected patients in northeastern brazil intestinal parasites in zambian patients with human microsporidiosis in african aids patients with chronic diarrhoea life-threatening bacteraemia in hiv seropositive adults admitted to hospital in chronic bacterial enteropathy in patients with aids hep-2 cell-adherent e. coli in patients with human immunodefiency virus-associated diarrhoea small intestinal structure and function in patients infected with human immunodefieiency virus (hiv): evidence for hiv-induced enteropathy we would like to thank the medical and nursing staff of the kenyatta national hospital who participated in these studies and the director, kemri for permission to publish these findings. we are indebted to dr b. rowe for help with pathogenic e. coli detection. this study was funded by the wellcome trust (uk). key: cord-301592-n5ns3m34 authors: ivaska, lauri; niemelä, jussi; lempainen, johanna; österback, riikka; waris, matti; vuorinen, tytti; hytönen, jukka; rantakokko-jalava, kaisu; peltola, ville title: aetiology of febrile pharyngitis in children: potential of myxovirus resistance protein a (mxa) as a biomarker of viral infection date: 2017-01-07 journal: j infect doi: 10.1016/j.jinf.2017.01.002 sha: doc_id: 301592 cord_uid: n5ns3m34 objectives: besides group a streptococcus (gas), microbial causes of pharyngitis in children are not well known. we aimed to document the viral and bacterial aetiology of pharyngitis and to assess the pathogenic role of viruses by determining the myxovirus resistance protein a (mxa) in the blood as a marker of interferon response. methods: in this prospective observational study, throat swabs and blood samples were collected from children (age 1–16 years) presenting to the emergency department with febrile pharyngitis. microbial cause was sought by bacterial culture, polymerase chain reaction, and serology. blood mxa level was determined. results: a potential pathogen was detected in 88% of 83 patients: gas alone in 10%, gas and viruses in 13%, group c or g streptococci alone in 2% and together with viruses in 3%, and viruses alone in 59% of cases. enteroviruses, rhinoviruses, and adenoviruses were the most frequently detected viruses. blood mxa levels were higher in children with viral (880 [245–1250] μg/l; median [iqr]) or concomitant gas-viral (340 [150–710] μg/l) than in those with sole gas (105 [80–160] μg/l) infections. conclusions: detection of respiratory viruses simultaneously with elevated blood mxa levels supports the causative role of viruses in the majority of children with pharyngitis. keywords viral aetiology; pharyngitis; myxovirus resistance protein a; mxa; group a streptococcus; gas summary objectives: besides group a streptococcus (gas), microbial causes of pharyngitis in children are not well known. we aimed to document the viral and bacterial aetiology of pharyngitis and to assess the pathogenic role of viruses by determining the myxovirus resistance protein a (mxa) in the blood as a marker of interferon response. methods: in this prospective observational study, throat swabs and blood samples were collected from children (age 1e16 years) presenting to the emergency department with febrile pharyngitis. microbial cause was sought by bacterial culture, polymerase chain reaction, and serology. blood mxa level was determined. results: a potential pathogen was detected in 88% of 83 patients: gas alone in 10%, gas and viruses in 13%, group c or g streptococci alone in 2% and together with viruses in 3%, and viruses alone in 59% of cases. enteroviruses, rhinoviruses, and adenoviruses were the most introduction acute pharyngitis accounts for a substantial portion of visits to physicians in outpatient setting. 1e4 group a streptococcus (gas) is the only common bacterial cause of pharyngitis justifying antimicrobial treatment according to current guidelines. 5, 6 however, overuse of antibiotics for pharyngitis occurs both in children and adults. 7e10 non-gas pharyngitis is suggested to be mainly a viral infection, 11e13 but only a few comprehensive studies have evaluated the microbiological aetiology of the disease in the paediatric population. 14e18 there is a lack of studies addressing the viral aetiology of pharyngitis in children and adolescents by molecular diagnostic methods. the prevalence of asymptomatic streptococcal carriage in the oropharynx complicates the causal interpretation of gas findings in children with symptomatic pharyngitis. 19, 20 in addition, the significance of respiratory virus detection by polymerase chain reaction (pcr) has been questioned because of frequent virus findings in asymptomatic subjects. 21, 22 among patients with positive viral pcr results, low cycle threshold (ct) and serological response can indicate true infections. an alternative approach distinguishes between viral and bacterial infections by differences in the biomarker blood concentrations or host gene expression patterns. 23e25 the aim of this study was to document the microbial causes of acute pharyngitis in children and adolescents in an outpatient setting and to evaluate the causative role of viruses by determining myxovirus resistance protein a (mxa) and other biomarker levels. this prospective observational study was conducted at the department of emergency services, turku university hospital, turku, finland from november 25, 2013 through january 31, 2015. the department of emergency services (ed) serves as a walk-in clinic with approximately 25,000 yearly visits by patients under the age of 17. children or adolescents aged 1e16 years with acute febrile pharyngitis were eligible for the study. there were no exclusion criteria in the study. the study protocol and inclusion criteria were introduced to ed physicians who diagnosed acute pharyngitis, defined as exudates or intensive redness in the tonsils/oropharynx and fever (body temperature !38 c measured at the ed or reported by the parents during the current illness episode). the patients were treated according to the judgement of the attending ed physician. local guidelines recommend oral phenoxymethylpenicillin as the first line and cephalexin as the second line antimicrobial therapy for microbiologically proven gas pharyngitis. at enrolment, the patient's symptoms and their duration, clinical examination findings, preceding illnesses and vaccinations, and underlying conditions were recorded, and oropharyngeal swab samples and blood sample were obtained. symptom diary cards were given to the parents of the participating children. each family was contacted by telephone after the enrolment visit to record the duration of the patient's symptoms. follow-up calls were continued every second day until the fever and soreness of the throat had resolved. all patients were invited to a follow-up visit approximately 2e4 weeks after the enrolment. at the follow-up visit, possible symptoms and clinical findings were recorded, and an oropharyngeal swab sample and blood sample for paired serology were obtained. the ethics committee of the hospital district of southwest finland approved the study protocol. the legal guardians of all participating children and adolescents and the adolescent patients themselves gave their written, informed consent. at enrolment, oropharyngeal samples were collected in standardized order by rubbing the swabs against both tonsils. throat swabs were handled as follows: 1) the sample for bacterial culture was collected by a flocked swab that was put immediately after the collection in a tube with liquid transport media (eswab, copan, brescia, italy) and transferred to the department of clinical microbiology, turku university hospital. 2) the sample for virus pcr was collected by a flocked swab (copan, brescia, italy) and transferred in a dry, clean tube to the department of clinical virology, turku university hospital. at the followup visit, a study physician collected a flocked swab sample for virus pcr. fifty microlitres from the vortexed eswab was inoculated on streptococcal-selective blood agar, on standard blood, mcleod, and fastidious anaerobe agars as well as in a streptococcal-selective broth, followed by subculture on streptococcal-selective blood agar. beta haemolytic streptococci and other colonies of interest were identified by standard methods, including maldi-tof (bruker daltonics, bremen, germany). anti-streptolysin o (aso) antibody levels were determined using the rapitex ò asl kit (siemens healthcare diagnostics products gmbh, marburg, germany) according to the manufacturer's instructions. the pharyngeal swab was suspended into phosphatebuffered saline, and nucleic acids were extracted using the nuclisens easymag (biomerieux, boxtel, the netherlands) automated extractor. the anyplex ii rv16 multiplex pcr system (seegene, seoul, korea) was used for the detection of influenza a and b viruses, respiratory syncytial virus (rsv) groups a and b, adenovirus, metapneumovirus, coronaviruses 229e, nl63, and oc43, parainfluenza virus types 1 to 4, rhinoviruses, enteroviruses, and bocavirus. in addition, a laboratory-developed pcr assay was used for the detection of rhinoviruses and enteroviruses. 26 all amplifications were performed using rotor-gene 6000 or qiagen q (qiagen, hilden, germany) instruments. all enterovirus-and adenovirus-positive specimens were subjected to genotyping by pcr amplification of the partial gene region of the enterovirus vp1 protein region 27 or the adenovirus hexon protein. 28 the amplicons were purified with the exosap protocol and sequenced at gatc biotech (constance, germany), and the obtained sequences were analysed with the ncbi basic local alignment tool. paired serum samples were stored at à70 c until analysed. commercial test kits were used for the detection of igg and igm antibodies to mycoplasma pneumoniae (labsystems diagnostics, vantaa, finland) and epsteinebarr virus (ebv) (vidas, biomerieux, marcy l' etoile, france) according to the manufacturers' instructions. igg antibodies against adenovirus, enteroviruses, influenza a and b viruses, parainfluenza virus types 1, 2, and 3, and rsv were analysed by in-house enzyme immunoassays routinely used in the virus diagnostic laboratory. 29, 30 a m-capture enzyme immunoassay was used to measure enterovirusspecific igm antibodies. a mixture of coxsackievirus a16, coxsackievirus b3 and echovirus 11 antigens was used in igg and igm tests for enteroviruses. 31 biomarkers whole blood, plasma, and serum samples for biomarker level determinations were collected during the enrolment visit by antecubital venepuncture. the white blood cell count and plasma levels of c-reactive protein (crp) and procalcitonin (pct) were determined in the hospital central laboratory. whole blood samples for mxa measurement were transported to the laboratory where samples were diluted 1:20 in hypotonic buffer and stored at à70 c until the eia analysis was performed as described earlier. 32 serum samples for tumour necrosis factor (tnf)-related apoptosis-inducing ligand (trail) level determination were stored at à70 c until the analysis by elisa (human trail, quantikine, r&d systems inc., minneapolis, usa) according to the manufacturer's protocol. optical density was determined using a microplate reader set to 450 nm with wavelength correction at 570 nm. for the statistical comparison, all patients enrolled in the study were classified in two etiological groups: patients with gas (diagnosis based on bacterial culture) or gas-viral pharyngitis were classified as gas, and patients with all other aetiologies, or with undetermined aetiology, were classified as non-gas. the groups were compared by the c 2 or mannewhitney u tests. the areas under the receiver operating characteristic (roc) curve were determined for biomarkers. furthermore, patients were classified according to their microbial findings in six separate groups for the descriptive analysis. all analyses were performed using ibm spss statistics, version 22 (ibm corp., armonk, ny, usa). the study population comprised 83 children and adolescents with febrile pharyngitis ( table 1 ). the median age of the children was 5.5 years (interquartile range, 3.2e12.2), 37 (44.6%) were girls, and none of them were admitted to a includes cardiological (n z 1) and neurological (n z 1) conditions, ankylosing spondylitis (n z 1), and vitiligo (n z 1). b number of applicable patients n z 74. hospital. five children (6.0%) had received antibiotics within 5 days before enrolment. a potential causative agent was detected in 73 (88.0%) patients: gas alone in 8 (9.6%), gas together with virus in 11 (13.3%), group c or g b-haemolytic streptococci (gcs, ggs) alone in two (2.4%), gcs or ggs together with virus in three (3.6%), and one or more viruses alone in 49 (59.0%) cases (fig. 1) . one of the gas cases was diagnosed by enrichment culture only, and the remaining 18 were detected by standard culture on a streptococcal-selective blood agar plate. other bacteria found by throat culture were considered to be non-pathogenic (supplemental table 1 ). there was no significant difference in the mean initial serum aso levels between the gas and non-gas patients. five patients showed a 2-fold aso increase in paired serum samples: three of them were gas positive, one of them was gcs positive, and one was negative for streptococci by throat culture. overall, paired serum samples were available in 14/19 patients with gas. the most frequently detected viruses were enteroviruses (in 25.3% of the children), rhinoviruses (21.6%), and adenoviruses (15.7%). the most common types of enteroviruses were coxsackievirus types a6 and a16. adenoviruses were typed as c2 in seven patients and as c1 in two patients. ebv igm antibodies were detected in five patients. however, when the duration of symptoms, the presence of igg antibodies, and the results from paired serum samples were considered, acute infection caused by ebv was confirmed only in two (2.4%) patients. m. pneumoniae igm antibodies were detected together with igg antibodies in the serum of 10 patients, seven of whom were 14e16 years old. paired serum samples were available in four of these patients. none of them showed !2-fold increase in their m. pneumoniae igg antibody levels. a b-haemolytic streptococci or virus was detected in 9 of these 10 patients. figure 1 aetiology of febrile pharyngitis in different age groups (n z 83). the vertical axis (y) displays the percentage of microorganisms detected: group a streptococcus, gas (light blue); gas þ virus (orange); group c streptococcus, gcs or group g streptococcus, ggs (grey); gcs/ggs þ virus (yellow); virus (dark blue); microbiological aetiology unknown (green). the horizontal axis (x) represents different age groups included in the study (all, 1e4, 5e8, 9e12 and 13e16 years old). with serological results, we were able to plausibly estimate the most important virus in most patients with multiple virus findings (supplemental table 1 ). these results suggest that despite their frequent co-detection with other viruses, enteroviruses and adenovirus were often also the most probable pharyngitis pathogens. viruses caused the majority of cases in all age groups, but a viral aetiology was particularly common in the age group of one to four years (fig. 1) . the occurrence of selected symptoms and clinical findings in patients with gas and non-gas pharyngitis are presented in table 2 . no clinical scoring system was used in the clinical management of the patients, but the mcisaac scores were determined retrospectively for all eligible patients (n z 66). none of the symptoms, clinical findings, or the mcisaac score were specific for a gas or non-gas aetiology of febrile pharyngitis. the white blood cell count and crp, pct, mxa, and trail levels were measured in the blood, plasma or serum of all pharyngitis patients. the mxa/crp ratio was calculated. the levels of all biomarkers, except that of pct, were significantly different between gas and non-gas patients ( table 2 ). however, none of the biomarkers was accurate in discriminating gas from non-gas aetiology (see supplemental figs. 1e6). based on our previous study, we used 175 mg/l as a cut-off level for increased blood mxa concentration. 32 blood mxa levels were elevated in 79.4% of patients with virus findings, and remained low in 90.0% of patients with streptococcal pharyngitis without virus detection. blood mxa levels were also elevated in most of the patients (90.0%) without a confirmed microbiological diagnosis (fig. 2) . viruses, but no bacteria, were identified in all five patients with recent antibiotic exposure. these patients had also markedly elevated blood mxa levels indicating an acute virus infection. in total, 57 patients (69%) returned for a scheduled follow-up visit after 10e40 days (median 18 days, interquartile range 17e21). at the follow-up visit, a throat swab was taken from 56 and blood sample from 52 children. fever and throat soreness of the initial pharyngitis episode had resolved in all patients. two patients presented to the follow-up visit with a relapsed gas pharyngitis and another of them was febrile. none of the other patients were febrile on the follow-up visit but 29/57 (51%) of them reported milder respiratory symptoms. overall, viruses were detected in 37/56 (66.1%) patients. virus detection was more common in the symptomatic 24/29 (82.8%) than in the asymptomatic 13/27 (48.1%) patients. blood mxa levels were lower in virus positive patients at the follow-up visit (110 [70e218] mg/l; median [iqr]) than they were during the febrile pharyngitis episode (780 [180e1190] mg/l). in this study, viruses had a dominant role in the aetiology of febrile pharyngitis. a possible causative agent could be detected in 88.0% and virus in 75.9% of patients, whereas in earlier studies from the era before pcr, viruses were detected in 11e42% of children or adolescents with pharyngitis or tonsillitis. 16e18 furthermore, an elevated blood mxa level, as a marker of type i or type iii interferon production, demonstrated an active innate immune response against acute virus infection in the majority of patients with a detected virus. we detected group a b-haemolytic streptococci less frequently than earlier reported. 19 this is partly explained by the fact that half of the children in this study were table 2 comparison of the clinical parameters and biomarkers in gas and non-gas pharyngitis. non-gas (n z 64) p a younger than five years old. group c and g b-haemolytic streptococci were detected only infrequently and often together with viruses. it has been suggested that m. pneumoniae is a major pharyngitis pathogen in children. 15 in our study, the detection rate of m. pneumoniae igm antibodies was 12%. however, considering the duration of the patients' symptoms, the presence of other potential pathogens, the presence of igg antibodies, and the missing titre changes in the paired sera, the clinical significance of m. pneumoniae igm antibody findings remain unclear. respiratory viruses can be detected by pcr in many asymptomatic individuals as well, and therefore, their role as etiologic agents can be argued. 21, 22, 33 we and others have shown earlier that a mxa response is strongly associated with a symptomatic viral infection. 32, 34 indeed, in the present study, most patients with virus findings had elevated levels of mxa protein in their blood. in contrast, at the follow-up visit when the patients were asymptomatic or had only minor, non-febrile respiratory symptoms, their blood mxa levels remained low. our finding suggests that at the enrolment they had a true, symptomatic virus infection rather than coincidental virus detection. still, all but one patient with febrile pharyngitis positive for group a, c, or g b-haemolytic streptococci without virus detection demonstrated no mxa response. interestingly, nine of ten patients without a confirmed microbiological diagnosis had increased mxa levels. this finding suggests that the majority of these patients might have had an infection caused by an undetected virus. enteroviruses were the most often detected pathogens in this study. this observation is probably a result of two facts: first, the use of a pcr assay instead of viral culture in diagnostics increases the detection rate of enteroviruses. second, epidemiological factors influence the results. during autumn 2014, there were substantially more reported enterovirus infections in finland than in the two previous years, especially in children younger than 10 years old. 35 viruses were detected together with b-haemolytic streptococci in 14 patients. rhinoviruses and adenoviruses were the most frequent virus findings in these patients. because detection of rhinovirus in asymptomatic subjects is not uncommon, and because adenovirus can persist in tonsillar tissue, the pathogenic role of these viruses is not always clear. 33,36,37 however, 11 out of 14 of the patients had an mxa response, which suggests that these patients either had a viral-bacterial co-infection or they had a virus infection and an incidental gas carriage. biomarkers of bacterial infection have limited clinical utility in distinguishing between gas and non-gas pharyngitis. 38e40 we found a significant difference in the blood levels of wbc, crp, mxa, trail, and the mxa/crp ratio between patients with gas and non-gas illness, but their analytical performance measured by the auc was poor. mxa could not discriminate between a gas and non-gas aetiology of pharyngitis, mainly because streptococcal and viral co-infection induced an mxa response comparable to that of virus infection. because gas can be detected relatively easily in throat swabs, there is probably no clinical need for a biomarker in the routine diagnostics of gas pharyngitis. here, we studied the biomarkers primarily to assess the pathogenic role of detected viruses or bacteria, not to evaluate their performance in differential diagnostics in clinical practice. however, the mxa level and mxa/crp ratio remained low in patients with sole streptococcal infection and increased in the majority of patients with a virus infection. therefore, they could be helpful represents the blood myxovirus resistance protein a (mxa) levels in the different aetiological groups of patients with febrile pharyngitis (n z 83). the vertical axis (y) displays the mxa protein concentration (mg/l) in whole blood. scale of the y axis is logarithmic. the horizontal axis (x) represents different aetiological groups: group a streptococcus, gas; gas þ virus; group c streptococcus, gcs or group g streptococcus, ggs; gcs/ggs þ virus; virus; unknown. tools in the differential diagnosis of viral and bacterial infections in other clinical settings. in our study, blood level of trail, another virus specific biomarker, was less accurate in differentiating between viral and bacterial aetiology. this study had several limitations. first, this was a single-centre study and, therefore, the results might not be fully generalizable to other centres. moreover, patient recruitment continued only for 14 months, and epidemiological variation might have influenced the etiological outcomes. additionally, the sample size was rather small. several different ed physicians carried out the preliminary recruitment of the patients, which could have influenced the diagnostic accuracy of pharyngitis. however, our setting pragmatically reflects how the clinicians identify febrile pharyngitis. furthermore, we could possibly have increased the detection rate of viruses by collecting also nasopharyngeal swab samples. 41, 42 our rationale for sole oropharyngeal sampling was that we were investigating pharyngeal infection and that nasopharyngeal sampling might have increased the detection of viruses with a questionable role in causing pharyngeal symptoms. viruses are the most common cause of febrile pharyngitis in children and adolescents. this study reinforces the current practice of sparing patients with non-gas illness from antibiotic treatment. biomarker measurements seem clinically irrelevant in the diagnostics of pharyngitis. nevertheless, blood mxa has potential as a biomarker of acute virus infection. there are no conflicts of interest. antibiotic prescribing by primary care physicians for children with upper respiratory tract infections trends in childhood illness and treatment in australian general practice national hospital ambulatory medical care survey: 2006 outpatient department summary burden of acute sore throat and group a streptococcal pharyngitis in school-aged children and their families in australia idsa clinical practice guideline for the diagnosis and management of group a streptococcal pharyngitis: 2012 update by the infectious diseases society of america guideline for the management of acute sore throat overprescribing and inappropriate antibiotic selection for children with pharyngitis in the united states antibiotic prescribing to adults with sore throat in the united states bacterial prevalence and antimicrobial prescribing trends for acute respiratory tract infections prevalence of inappropriate antibiotic prescriptions among us ambulatory care visits acute pharyngitis acute pharyngitis: etiology and diagnosis sore throat: is it such a big deal anymore? are empiric antibiotics for acute exudative tonsillitis needed in children? aetiology of acute pharyngitis: the role of atypical bacteria febrile exudative tonsillitis: viral or streptococcal? viral and bacterial organisms associated with acute pharyngitis in a school-aged population group a streptococci, mycoplasmas, and viruses associated with acute pharyngitis prevalence of streptococcal pharyngitis and streptococcal carriage in children: a metaanalysis the group a streptococcal carrier state reviewed: still an enigma identification of respiratory viruses in asymptomatic subjects: asymptomatic respiratory viral infections frequent detection of respiratory viruses without symptoms: toward defining clinically relevant cutoff values a novel host-proteome signature for distinguishing between acute bacterial and viral infections detecting specific infections in children through host responses: a paradigm shift host gene expression classifiers diagnose acute respiratory illness etiology simultaneous detection and differentiation of human rhinoand enteroviruses in clinical specimens by real-time pcr with locked nucleic acid probes sensitive, seminested pcr amplification of vp1 sequences for direct identification of all enterovirus serotypes from original clinical specimens genotyping of clinically relevant human adenoviruses by array-in-well hybridization assay influenza a and b virus igg and igm serology by enzyme immunoassays enzyme immunoassays for detection of igg and igm antibodies to parainfluenza types 1, 2 and 3 enterovirus infection as a risk factor for beta-cell autoimmunity in a prospectively observed birth cohort: the finnish diabetes prediction and prevention study rhinovirus detection in symptomatic and asymptomatic children: value of host transcriptome analysis diagnosis of viral infections using myxovirus resistance protein a (mxa) the institution of health and welfare, finland use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms latent species c adenoviruses in human tonsil tissues are procalcitonin or other infection markers useful in the detection of group a streptococcal acute tonsillitis? diagnostic value of serum procalcitonin and c-reactive protein in egyptian children with streptococcal tonsillopharyngitis. pediatr c-reactive protein in the differentiation of adenoviral, epstein-barr viral and streptococcal tonsillitis in children comparison of nasal swabs with throat swabs for the detection of respiratory viruses by real-time reverse transcriptase pcr in adult hajj pilgrims what is the added benefit of oropharyngeal swabs compared to nasal swabs alone for respiratory virus detection in hospitalized children aged <10 years? we thank all the study subjects, their families, staff at the department of emergency services and research nurse ulla torkko for her contribution in the data collection. supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2017.01.002. key: cord-304176-yloqrblw authors: tunesi, s.; bourgarit, a. title: prescribing covid-19 treatments: what we should never forget date: 2020-05-13 journal: j infect doi: 10.1016/j.jinf.2020.05.018 sha: doc_id: 304176 cord_uid: yloqrblw nan prescribing covid-19 treatments: what we should never forget s. tunesi, a. bourgarit sorbonne paris-nord university (paris 13), bobigny, france; inserm, umr 1135 cimi, paris, france; aphp, hupssd, internal medicine department, bondy, france authors equally contributed to write and edit this paper. both authors have no conflicts of interest to declare. no financial sources were needed to redact this paper and any ethical committee was required. sars-cov-2 pandemics is challenging health care systems worldwide. governments are investing a huge amount of financial resources both to support health facilities and to prevent economic collapse. half of the humanity is currently confined at home in order to stop covid-19 transmission. health care workers are multiplying their efforts while they are struggling to cope with the shortage of personal protective equipment (ppe) globally. respiratory support appears to be the gold standard of treatment in severe forms of sars-cov-2 (1). however, multiple efforts were done to find a possible medical treatment. since the beginning of the epidemics, many drugs were used despite the lack of strong scientific evidence. covid-19-induced proinflammatory status looks to trigger most severe sars-cov-2 forms (2). based on this assessment, many antinflammatory drugs have been proposed and used off randomized trials due to the epidemics spreading. despite the hope and the first evidences, a real clinical utility of these molecules which mostly act on il-6 and il-1 (such as tocilizumab and anakinra) is still to be demonstrated, while their potential side effects are well known (3). similarly, corticosteroids (cs) have been proposed and largely used worldwide to tackle the proinflammatory status. despite some encouraging preliminary data, there is still no evidence of a reduction of mortality in patients receiving cs, and standard side effects, including septic shock, have been reported (4). many drugs have been hypothesized to be directly active against covid-19 only because of a supposed antiviral activity: remdesivir, a molecule originally tested against ebola virus, shows in vivo activity against mers-cov (5) but there is actually no real evidence of in vivo activity against covid-19; lopinavir/ritonavir, a well-known protease inhibitor used in hiv treatment, has been widely used before randomized clinical trials showed his inefficacy in mortality reduction (6); chloroquine and hydroxychloroquine, which are largely used in systemic lupus erythematosus (sle) and rheumatoid arthritis (ra) treatment, show modest antiviral effects, but mortality due to qt elongation-related cardiac events is a matter of concern (7). shifting drugs with proven activity against other diseases to sars-cov-2 empiric treatment is leading to a drug global shortage, that can significantly impact on the quality of life of those patients, such as those affected by sle and ar, who could face hydroxychloroquine stock exhaustion (8) . conversely, preliminary data about a potential role of ace inhibitors in favouring the onset of severe forms of sars-cov-2 infection induced a massive change in antihypertensive drugs prescription that caused the onset of severe cardiovascular events (9). in conclusion, sars-cov-2 spreading requires strong and emergency medical actions to face the first global pandemics since ebm approach was established. nevertheless, this is not the time to encourage clinical practices that can lead to severe adverse events in both covid-19 and non covid-19 patients. the basic motto that must drive clinical decision remains "primum non nocere", even during this global emergency. intubation and ventilation amid the covid-19 outbreak: wuhan's experience covid-19: consider cytokine storm syndromes and immunosuppression. the lancet drug tolerability and reasons for discontinuation of seven biologics in elderly patients with rheumatoid arthritis -the answer cohort study-. plos one clinical evidence does not support corticosteroid treatment for 2019-ncov lung injury. the lancet comparative therapeutic efficacy of remdesivir and combination lopinavir, ritonavir, and interferon beta against mers-cov a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 covid-19: us gives emergency approval to hydroxychloroquine despite lack of evidence use of hydroxychloroquine and chloroquine during the covid-19 pandemic: what every clinician should know position statement of the esc council on hypertension on ace-inhibitors and angiotensin receptor blockers key: cord-304787-fohgekp4 authors: prazuck, thierry; giaché, susanna; gubavu, camelia; colin, mathilda; rzepecki, vincent; sève, aymeric; pialoux, gilles; hocqueloux, laurent title: investigation of a family outbreak of covid-19 using systematic rapid diagnostic tests raises new questions about transmission date: 2020-06-29 journal: j infect doi: 10.1016/j.jinf.2020.06.066 sha: doc_id: 304787 cord_uid: fohgekp4 nan we read with interest the letter by jiang and colleagues who described the spread of sars-cov-2 in a family cluster in china. 1 herein, we describe the epidemiological, clinical and biological characteristics of 30 households in close contact with sars-cov-2-infected members, living in a confined environment during the french national lockdown. this report highlights important issues about transmission. two families (26 and 4 members, respectively) were included in the study. during the 5 days before the french national lockdown, both families moved from their usual parisian residence to a closed property in the countryside, composed by 3 neighboring houses (a, b and c) in a park. house a was inhabited by 8 persons from a single family, including 3 couples who shared their bed for at least 4 days after the onset of symptoms. in house b, there were 2 families composed of 9 persons. there were 2 married couples who shared their bed during all the time, even in presence of symptoms. in house c, there were 2 families composed of 13 persons. there were 4 married couples who shared the bed until the hospitalization of the 84-year-old subject for severe sars-cov-2 infection. with the exception of the elderly couple who already lived in house c, all the remaining people arrived there between march 12 and 16, before the beginning of the national lockdown (march 17, 2020). thereafter, all residents were not allowed to quit until the end of lockdown (may 11, 2020). during the first week of cohabitation there were regular and close contacts among households from the 3 houses. since the occurrence of the first 3 symptomatic cases, contacts among the 3 houses were reduced although they indirectly continued through children displacements. within their stay, all residents were clinically examined at least once. rt-pcr testing of sars-cov-2 was performed for symptomatic cases. 2 serologic testing using the approved covid-presto® rapid diagnostic tests 3 (aaz, boulogne-billancourt, france), detecting both igm and igg, was performed on whole blood finger-stick more than 45 days after the onset of symptomatic cases on all the 30 subjects. population characteristics are detailed in table 1 . the first diagnosed case was a 67-year-old man (resident #1) living in the house a and referring to the infectious diseases outpatient clinic on march 17 for cough, fever and asthenia for 4 days. investigation of the cluster began soon after resident #1 was formally diagnosed with covid-19. this first recognized case was acquired before the arrival of resident #1, who probably transmitted the infection to resident #3 (his son-in-law) after arrival at home a. no transmission occurred between husbands and wives in house a although couples moved to separated rooms only 4 days after the onset of symptoms of the index case. as resident #6 was asymptomatic with igg+/igm-, it is not sure that she was infected prior or after her arrival at home a. in house b, a single resident (#9) was found to be infected with covid-19. he probably acquired infection by the end of february after a close contact with a confirmed case in paris. symptoms occurred on march 12, four days before arriving at home b, with cough, asthenia, anosmia, cutaneous lesions lasting one week. as the covid-19 cluster was unrecognized at this time, he was not separated from the rest of the residents, he continued to share the bedroom with his wife and he had remarkably close contacts with his children. although he had close contacts with all his family and friends during all the symptomatic phase, no secondary cases occurred. in house c, resident #22 was the first (retrospectively) identified with covid-19. she developed typical symptoms (cough, fever, headache and asthenia) on march 10 when she was in paris. at her arrival in house c on march 13, she still had mild symptoms. she probably infected her husband, father and mother (residents #23, #18 and #19, respectively). resident #18 developed a severe lower respiratory tract infection and was hospitalized for 2 weeks while. resident #23 developed a moderate cough for two days. resident #20 may have been infected by resident #23, during a round trip by car on march 17, when resident #23 was already symptomatic. resident #20 presented with diarrhea, cough and fever lasting four days. figure 1 shows distribution of residents in each house, kinship, confirmed covid-19 cases and the temporal occurrence of each case. overall, 9 out of 30 residents (30%) were diagnosed with covid-19. we identified 3 independent index cases (residents #1, #9, #22) that infected 6 secondary cases: 2 in house a, none in house b and present were infected, although they had very closed contacts with their infected parents. among the 9 married couples who were present, all shared the same bed at least some days after the onset of symptoms or during all the time of disease. nevertheless, partners were infected in only 3 couples, partners remained discordant in 5, and none of the partners were infected in 1. after careful questioning, discordant couples confirmed they had continued sexual activity during the period at risk of contagiousness of their partner. this cluster investigation illustrates important facts concerning sars-cov-2 transmission. first, as no case occurred in children we are confident they did not participate in the viral transmission network, either within or between houses. this supports the hypothesis that, usually, children are not asymptomatic carriers who transmit the sars-cov-2. 4-6 second, there was a limited transmission of sars-cov-2 among couples, even though discordant couples continued to be sexually active during the contagious period of the infected partner. to our knowledge, there are no similar reports published to date. if the determinants of such a "resistance" to sars-cov-2 remain uncertain, some of our results (data not shown) allow us to speculate about a potential role of a genetic factor, currently under investigation. characteristics of a family cluster of severe acute respiratory syndrome coronavirus 2 in henan verrill kelly a. how to obtain a nasopharyngeal swab specimen evaluation of performance of two sars-cov-2 rapid whole blood finger-stick igm-igg combined antibody tests spread of sars-cov-2 in the icelandic population covid-19 in children: the link in the transmission chain nasal gene expression of angiotensin-converting enzyme 2 in children and adults this study was approved by the local institutional review board. a written informed consent was obtained from all patients. key: cord-299018-eo73yl4v authors: gandini, o.; criniti, a.; ballesio, l.; giglio, s.; galardo, g.; gianni, w.; santoro, l.; angeloni, a.; lubrano, c. title: serum ferritin as an independent risk factor for acute respiratory distress syndrome in covid-19 patients in rome italy date: 2020-09-15 journal: j infect doi: 10.1016/j.jinf.2020.09.006 sha: doc_id: 299018 cord_uid: eo73yl4v nan identifying the significant parameters for early progression toward worse prognosis is fundamental for the management of covid-19 patients. in this journal, zhi lin and colleagues (1) recently reported that chinese patients with severe sars-cov2 disease showed higher levels of serum ferritin than patients with not severe one, confirming data from other authors on chinese (2) (3) and caucasian populations (4) (5) . here we aimed to establish the most suitable panel for routine prognostic serum laboratory testing in covid-19 patients upon first admission to the emergency department. we thus enrolled 141 patients (59 females and 82 males, aging 64,48±16,58 years) diagnosed as covid-19 by means of real-time polymerase chain reaction testing and admitted to the isolation ward of emergency department at policlinico umberto i hospital in rome, italy, between march 2020 and june 2020. serum samples were collected from patients upon admission before starting any treatment and tested by laboratory department of all patients included, 81 patients (57%) showed mild disease (control group) and 60 (43%) showed acute respiratory distress syndrome (ards) and systemic inflammation (severe group). our data strongly confirm that increased levels of ferritin were directly related with the disease severity (fig 2a) . particularly, not only severe group showed 2.6 times higher ferritin levels respect to mild group, but patients who needed admission to the icu showed 5.8 times higher ferritin compared to patients with mild covid-19. among all parameters considered, we also noted that the neutrophil to lymphocyte ratio (nlr) was statistically correlated with the severity of disease. (fig 2b) . conversely, d dimer, ldh and crp increased only in the group of critical patients (group 3), being substantially stable in the other groups characterized by mild, moderate and severe disease ( fig. 2 , panel c, d, e) multivariate logistic regression model adjusted for several disease-related risk factors at admission, including age, sex, nlr, dd, ldh, ferritin and crp, demonstrated that serum ferritin resulted as an independent predictor of disease severity in covid-19 patients (or = 1,0048, 95% ci, 1,0029 to 1,0083, p < 0,001.). if patients were grouped according to the serum ferritin level with a cut off of 500 µg/ml derived from the hlh-2004 (7) criterion, hyperferritinemia accounted for 48,22% (68/141) of patients and the hyperferritinemia group had a higher proportion of severe cases (77,94% vs 10,30%, p <0,001) than patients without hyperferritinemia. this is the first italian report about the prognostic value of laboratory biomarkers considering 4 groups of mild, moderate, severe and critical patients with covid-19. we clearly demonstrated that serum levels of ferritin progressively increased with the severity of disease and correlate with poor prognosis in covid-19 patients. increased ferritin levels could be indicative of a strong inflammatory reaction in covid-19 and recent studies suggest that increased levels of circulating ferritin levels play a critical role by contributing to the development of a cytokine storm (8-9) resembling macrophage activating syndrome (10) . timely control of the cytokine storm in its early stage through immunomodulators and cytokine antagonists, as well as the reduction of lung inflammatory cell infiltration, is the key to improving the treatment success rate and reducing the mortality rate of patients with covid-19. in this regard, ferritin evaluation could be an early, available and easy to use screening tool to assess the disease severity at the first admission in the emergency department. this test might be of crucial importance for the timely identification of patients at higher risk of an adverse outcome. serum ferritin as an independent risk factor for severity in covid-19 patients risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19): a metaanalysis covid-19 in critically ill patients in the seattle region -case series clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series clinical management of covid-19 -interim guidance hlh-2004: diagnostic and therapeutic guidelines for hemophagocytic lymphohistiocytosis. pediatr blood cancer hyperferritinemia and inflammation crossing the iron gate: why and how transferrin receptors mediate viral entry covid-19) time musings: our involvement in covid-19 pathogenesis, diagnosis, treatment and vaccine planning legends to figure fig key: cord-318013-5om35tu8 authors: marie, tré-hardy; laurent, blairon; alain, wilmet; ingrid, beukinga; hugues, malonne; jean-michel, dogné; jonathan, douxfils title: the role of serology for covid-19 control: population, kinetics and test performance do matter date: 2020-05-15 journal: j infect doi: 10.1016/j.jinf.2020.05.019 sha: doc_id: 318013 cord_uid: 5om35tu8 nan to the editors, accumulating evidence in the literature exemplifies the failings of real-time polymerase chain reaction (rt-pcr) as a sole diagnostic method in covid-19 surveillance, because of its inability to detect past infection. (1-4) the authors of these reports or correspondence highlighted the added value of serological testing, which, if captured within the correct timeframe after disease onset, can detect both active and past infections.(1) by providing estimates of who is and is not immune to sars-cov-2, serological data can be used to estimate epidemiological variables, such as the attack rate or case-fatality rate, which are necessary to assess how much community transmission has occurred and its burden.(5) they can also be used to strategically deploy immune health-care workers to reduce exposure of the virus to susceptible individuals or to assess the effect of non-pharmaceutical interventions at the population level and inform policy changes to release such measures. (6) in the near future, serological testing will be required to assess the effectiveness of vaccine candidates and finally, they are also useful to identify individuals who developed a strong immunological response to the virus and whose antibody isolates can be used to treat patients via plasma therapy. (7) however, several challenges still remain to correctly address the appropriate implementation, validation and interpretation of serological testing. among them, understanding the kinetics of the antibodies matters as divergent opinion are reported in the literature. (8, 9) our group recently reported the validation of a chemiluminescence immunoassay (clia) for igg determination (liaison®sars-cov-2, diasorin®, saluggia, italy) and reported the excellent analytical and clinical performance of the assay. however, data on antibody kinetics and assessment of iga were not conveyed yet on this cohort. statistical analyses show they are representative of the full cohort. figure 1 reports the change from the baseline and sensitivity at weeks 0, 1, 2 and 3 for iga and igg determinations. both immunoglobulin levels increase over time and tend to stabilize after two weeks. using our adapted cut-off, iga determination shows a sensitivity of 100% after one week while it reaches 87% for igg testing. the cut-off provided by the manufacturer still shows a sensitivity of 100% for iga but it diminishes to 80% and 67% for igg elisa and igg clia, respectively. after two weeks, all tests demonstrate a sensitivity of 100%, as reported by other groups, (8, 10) except when the cut-off provided by the manufacturer were used for igg detection (i.e. one of our 15 patients was never considered as positive). these observations are of upmost importance for at least two analytical and clinical matters: first, the selection of the appropriate timeframe is essential for the detection of immunity. namely, these results show that iga immunity can be accurately detected one week after the rt-pcr positivity while igg immunity has to be assessed after two weeks to avoid false negative results. secondly, adapted cut-offs have to be established by each laboratory in order to improve the sensitivity of the commercial assays. however, this implies that the sera selected to define the adapted cut-off is crucial. in our case, the cut-off was determined on samples from symptomatic patients collected 14 days after the positive rt-pcr. of note, there is to date no consensus on how to define the disease onset, i.e. date of first covid-19 symptoms or date of rt-pcr. despite being both validated and approved by competent authorities, these results show that the two igg assays are not similar for determining positivity if measurement is performed at the time of rt-pcr determination (i.e. sensitivity of 7 and 20% for the clia and the elisa method, respectively using the manufacturer cut-off). this further complexifies the interpretation of the results and highlights the need for competent national authorities and learned societies to establish guidance and procedures for serological testing to avoid misinterpretation of too early determination, leading to a high rate of false negative results. this study was not funded by governmental or industrial grant. authorship: the important role of serology for covid-19 control connecting clusters of covid-19: an epidemiological and serological investigation significance of serology testing to assist timely diagnosis of sars-cov-2 infections: implication from a family cluster role of serology in the covid-19 pandemic characteristics of a family cluster of severe acute respiratory syndrome coronavirus 2 in henan the sars-cov-2 seroprevalence is the key factor for deconfinement in france convalescent plasma as a potential therapy for covid-19. the lancet infectious diseases analytical performances of a chemiluminescence immunoassay for sars-cov-2 igm/igg and antibody kinetics. clinical chemistry and laboratory medicine : cclm / fescc reliability and usefulness of a rapid igm-igg antibody test for the diagnosis of sars-cov-2 infection: a preliminary report figure 1: antibody kinetics expressed as change from baseline or sensitivity at week 0, 1, 2 and 3 post rt-pcr covid-19 positivity. week 0 corresponds to the day of rt-pcr determination and confirmation of sars-cov-2 infection. true positivity rates (sensitivity) have been assessed with the cut tré-hardy marie, blairon laurent, wilmet alain, beukinga ingrid and douxfils jonathan were responsible for data collection, analysis and interpretation. douxfils jonathan was responsible for writing the first draft.tré-hardy marie, dogné jean-michel, malonne hugues, and douxfils jonathan were responsible for the final version. key: cord-321033-fusyx0t9 authors: nan title: follow-up study on pulmonary function and radiological changes in critically ill patients with covid-19 date: 2020-05-27 journal: j infect doi: 10.1016/j.jinf.2020.05.040 sha: doc_id: 321033 cord_uid: fusyx0t9 nan dear editor: the clinical manifestations of coronavirus disease 2019 , caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), 1 range from asymptomatic, mild pneumonia to acute respiratory distress syndrome (ards). 2 an epidemiological study with 72,314 patients has reported that around 4.7% of patients with covid-19 developed ards, and the mortality rate in these patients was up to 49%. 3 most patients surviving from ards caused by other coronaviruses like sars-cov and middle east respiratory syndrome coronavirus suffered from impaired pulmonary function and radiological abnormalities. 4, 5 up till now, follow-up data regarding critically ill covid-19 survivors is rare. to facilitate the understanding of the prognosis of these patients, we here present a follow-up study of two covid-19 patients with severe ards up to 3 months after the illness onset. two patients with covid-19 who developed severe ards in the second people's hospital of fuyang (anhui, china) were enrolled. institutional review board approval and written informed consent were obtained. patient 1 was a 20-year old female working in wuhan and returned to fuyang to celebrate the spring festival with her family. she presented in the hospital with fever, dry cough, and deteriorated dyspnea for 10 days on 29 january 2020. on admission, her vital signs were unstable (temperature 39℃, heart rate 112 beats per minute, respiratory rate 32 breaths per minute, blood pressure 104/65 mmhg) with obvious dyspnea (spo2 89% under conventional oxygen therapy of 40% fio2). laboratory results indicated leukopenia (2.65×10 9 /l), lymphopenia (0.45×10 9 /l), elevated c-reactive protein (crp) (102 mg/l), il-6 (62 pg/ml) and d-dimer (1.06 mg/l). arterial blood gas analysis (abga) reported impaired oxygenation of pao2/fio2 at 213 mmhg. the diagnostic rt-pcr on a nasopharyngeal swab specimen was positive for sars-cov-2 and a chest ct scan illustrated bilateral pneumonia ( figure 1a ). lopinavir-ritonavir (500 mg, twice per day) was administrated immediately. her condition progressed rapidly on illness day 12 and she required intubation and mechanic ventilation because of severe ards (pao2/fio2 92 mmhg). on illness day 17, both oxygenation (pao2/fio2 348 mmhg) and chest ct significantly improved (figure 1b) , and she was then weaned from mechanical ventilation. she was discharged on illness day 21 with mild lung abnormalities on chest ct images ( figure 1c ). one month later after discharge, both chest ct scan ( figure 1d ) and pulmonary function test (forced vital capacity (fvc) of predicted 103.7%, fev1/fvc 84.64%, carbon monoxide diffusing capacity (dlco) of predicted 94%) indicated no-abnormalities. patient 2 was a 68-year old man who permanently resides in fuyang. he visited the hospital because of fever (37.5 ℃) and cough for one week on 4 february 2020. the nasopharyngeal swab was positive for sars-cov-2 by rt-pcr and a chest ct revealed bilateral ground-glass opacity (figure 2a ). laboratory results indicated leukopenia (3.33×109/l), lymphopenia (0.55×109/l), elevated crp (116 mg/l), and il-6 (357 pg/ml). his condition was stable on admission, with oxygen saturation of 97% on ambient air. lopinavir-ritonavir (500 mg, twice per day) was administrated. he reported the history of hypertension and diabetes for around 10 years, blood pressure and glucose were controlled well, no history of chronic respiratory diseases. on illness day 12, he was transferred to the intensive care unit and converted to invasive mechanical ventilation because of worsening oxygenation (pao2/fio2 84 mmhg) and progressing abnormalities on chest ct scan with extensive ground-glass opacities and partial consolidation on bilateral lungs (figure 2b ). his oxygenation improved slowly, but could not be weaned from mechanic ventilation, hence he received a tracheostomy on illness day 22. during this period, bronchoalveolar lavage culture reported escherichia coli that was sensitive to carbapenems but resistant against third-generation cephalosporins. therefore, imipenem was administrated. on illness day 30, oxygenation of the patient suddenly deteriorated, and a chest ct scan revealed right-sided pneumothorax (figure 2c ). the patient then received thoracic drainage with a closed system which was removed 2 days later. on illness day 30, the oxygenation of the patient improved and he was successfully weaned from mechanic ventilation. he was discharged from hospital on illness day 45 with obvious abnormalities on the chest ct scan (figure 2d) . because of the impaired pulmonary function on discharge, he was transferred to a rehabilitation center. two months after the onset of illness, he went to the hospital for the first follow-up visit. the pulmonary function test indicated restrictive lung function defect, with decreased fvc of predicted (62.3%) and dlco of predicted (49.6%), but fev1/fvc was at the normal range of 80.1%, which was consistent with the manifestations on chest ct images (figure 2e ). on the second follow-up visit (3 months after illness onset), almost all ground-glass opacities were dissolved, but with obvious architectural distortion, bronchial dilatation and volume loss in bilateral lungs suggestive of fibrotic changes on chest ct (figure 2f ). lung ventilation was worse than that of the previous month, featured as the restrictive pulmonary disease with decreased fvc of predicted (54%), but diffusion capacity improved significantly, albeit it was still lower than that of the normal range (dlco of predicted increased from 49.6% to 64.3%). the patient complained of shortness of breath and general weakness (6-minute walking distance was 200 meters), and abga indicated low pao2 (61.6 mmhg) on ambient air. little is known about the sequelae of covid-19. the two patients reported in this study showed distinct consequences. the young patient completely recovered with non-abnormality on both chest radiology and function tests, while the older patient manifested with obviously radiological changes and functional defects during the follow-up period. the results of the older patient in this study, suggest that a proportion of severe covid-19 patients developed fibrosis. similar fibrotic changes had been reported for sars, which seem to have the ability of self-rehabilitation as gradual improvements were observed over time. 6, 7 nevertheless, the restrictive ventilatory defect and impaired diffusion capacity still affect the patient's physical abilities significantly in the early recovering stage, which suggests the importance of early rehabilitation. 8 the limitation of this study was the short-term follow-up of only two cases; therefore, to understand any long-term effects of covid-19 long-term follow-up studies with large cohorts of patients are warranted. a novel coronavirus from patients with pneumonia in china characteristics of covid-19 infection in beijing the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china. zhonghua liu xing bing xue za zhi = correlation between pneumonia severity and pulmonary complications in middle east respiratory syndrome follow-up study on pulmonary function and lung radiographic changes in rehabilitating severe acute respiratory syndrome patients after discharge severe acute respiratory syndrome: temporal lung changes at thin-section ct in 30 patients sars: prognosis, outcome and sequelae early rehabilitation for critically ill patients with covid-2019: more benefits than risks we thank dr siu kuanglok (union hospital, hongkong, china) and dr zhechun xu (conch hospital, anhui, china) for helping with the interpretation of the chest ct scans. the work was supported by the special fund for coronavirus disease 2019 of wuhu (grant number 2020dx2-1). key: cord-308852-qdn04pun authors: lei, hao; xu, xiaolin; xiao, shenglan; wu, xifeng; shu, yuelong title: household transmission of covid-19-a systematic review and meta-analysis date: 2020-08-25 journal: j infect doi: 10.1016/j.jinf.2020.08.033 sha: doc_id: 308852 cord_uid: qdn04pun • infection risk of household contacts is 10 times higher than other contacts. • risk of household transmission in adults is about 3-times higher than that in children. • sars-cov-2 is much more transmissible than sars-cov and middle east respiratory syndrome coronavirus in households, which challenges the home isolation of covid-19 patients. thus timely household studies can be highly informative for covid-19 prevention. here, we report a systematic review of household transmission studies of and try to assess the secondary attack rate of household covid-19 transmission. studies containing data on household transmission of covid-19 were retrieved from the electronic databases: pubmed, embase, and a chinese database, china national knowledge infrastructure (cnki) on 1 july, 2020. all titles identified by the search strategy were independently screened by 2 authors (h.l. and x.x.). eligible articles reported a household transmission for covid-19 or sufficient data to determine a secondary attack rate, and must have reported data based on >=10 households. case reports with only one family involved were also excluded for analysis, because the household transmissions event collection was biased towards infection caused more serious illness. the bias could be reduced with more families involved. when multiple reports of the same dataset were identified, only the most comprehensive report of the study was included. household secondary attack rate (sar) was calculated as the number of identified cases divided by the number of household contacts. further, the sar was calculated separately for adults and children, using the age criterion of the study, further, the sar by other contacts was are summarized, when data were available. sars were calculated as the number of cases divided by the number of contacts, using the fisher's exact test for the 95% confidence interval (ci). we assessed statistical heterogeneity among studies using the the i 2 index. all analyses were performed by the software spss. the meta-analysis was conducted in review manage 2020. a total of 463, 402 and 212 titles were identified from pubmed, embase and cnki, respectively; and 24 articles were included in the mata-analysis. the characteristics of 24 included articles are summarized in supplementary table s1. 10 studies are retrospective cohort studies, one is a prospective study, and the other 13 are case ascertainment studies. most studies (19/24) were conducted in china, with two in south korea, two in the usa and one in germany. all the studies were conducted between jan 1 and march 31, 2020. reported sars were substantially heterogeneous, ranging from 4.6% to 90.0% (i 2 =96%), with pooled rate of 27% (95% ci: 21%-32%) (figure 1 ). in the 10 retrospective cohort studies, the sars ranged from 11.2% to 68.2%, with mean value 29.7%. in the 13 case ascertainment studies, the sars ranged from 4.6% to 90.0%, with mean value 28.3%. there is no significantly difference between the sars in the retrospective cohort and case ascertainment studies (p=0.93). in household sar with h1n1 virus were also widely varying, from 3% to 38%. 3 in these studies, with 10 or more families involved, the highest sar were observed in wuhan, china, which also had the greatest number of covid-19 confirmed cases in china. generally, the data-based sar estimated from literatures would be higher than the real sar since data-based estimates might have included some untraced exposures from outside. household sar were greater than sar by other contacts (or=10.72, 95% ci: 5.70-20.17, p<0.001), suggesting much higher rates of intra-family transmission of covid-19. several studies have reported that adults were more vulnerable to sars-cov-2. 4 in this study, we also found that within households, adults were about 4 times as susceptible to covid-19 from a household member as children (or=3.67, 95% ci: 2.76-4.87, p<0.001). in the study with the highest sar (62.1%, 95% ci: 52.4%-71.9%) among the 10 retrospective cohort studies, the mean age of the households (58.7 16.0) 5 was also much higher than these in other studies with detailed age of the households. the age of the households might partly explain the varying sars. however, because the age of the households was not available in most studies, the quantitative analysis of the sars and age of the households were not done. for sars-cov, the sar was estimated to be 6.2-10.2% in beijing, hong kong, toronto and singapore. [6] [7] [8] information about the household transmission of mers-cov is relatively rare. in a study in saudi arabia, the household sar of mers-cov was estimated to be 4% (95% ci: 2-7). 9 we conclude that sars-cov-2 is more transmissible than sars-cov and mers-cov in households. in addition, pre-symptomatic and asymptomatic cases were estimated to contribute 53% of covid-19 transmission. 10 all these challenge the value of home isolation for covid-19 patients, as it may put household members at high risk of infection, propagating the disease. when the hospital isolation of all cases becomes unfeasible, other sheltering facilities, such as the fangcang shelter hospital used in wuhan, china, might be a better option. error! reference source not found. household transmission of sars-cov-2 fangcang shelter hospitals: a novel concept for responding to public health emergencies household transmission of 2009 pandemic influenza a(h1n1): a systematic review and meta-analysis changes in contact patterns shape the dynamics of the covid-19 outbreak in china clinical features of familial clustering in patients infected with 2019 novel coronavirus in wuhan, china efficiency of quarantine during an epidemic of severe acute respiratory syndrome household transmission of sars secondary household transmission of sars transmission of mers-coronavirus in household contacts quantifying sars-cov-2 transmission suggests epidemic control with digital contact tracing we thank prof. racheal mary jones (school of medicine, university of utah) for helpful discussion and comments. this project was supported by natural science foundation of zhejiang province (grant no. lq20h260009). all authors declare no competing interests. hl and xx contributed equally. hl conceived the study, hl and xx designed the study. ys and xw supervised the study. hl, xx and sx searched the references, collected and cleaned the data. hl wrote the drafts of the manuscript. ys, xw, xx and sx commented on and revised drafts of the manuscript. all authors read and approved the final report. key: cord-327246-idmo5ds7 authors: m, montero-baladía; l, buzón; i, astigarraga; p, delgado; e, iglesias; f, callejo; m, lópez-veloso; j, minguito; m, fernández-regueras; ubeira m, pharmacy; hermida g, hematologist title: etoposide treatment adjunctive to immunosuppressants for critically ill covid-19 patients: etoposide for severe covid-19 patients date: 2020-06-21 journal: j infect doi: 10.1016/j.jinf.2020.06.006 sha: doc_id: 327246 cord_uid: idmo5ds7 nan . ubeira m, pharmacy (7) , hospital universitario de burgos, burgos, spain. mubeira@saludcastillayleon.es postal address: avenida islas baleares s/n, 09006, burgos 11. hermida g, hematologist (8) , hospital universitario de burgos, burgos, spain. ghermida@saludcastillayleon.es postal address: avenida islas baleares s/n, 09006, burgos title page 1 montero-baladía m, callejo f. critical care department. 2 buzón l , fernández-regueras m . internal medicine. infection disease unit. 3 astigarraga i, pediatrics, biocruces bizkaia health research institut 4 delgado p. neurosurgery department. 5 lópez-veloso m , iglesias e. internal medicine. autoinmune systemic disorders unit 6 in a recent article in the journal, cantini and colleagues 1 present favorable outcomes in a small cohort of moderate covid-19 pneumonia patients treated with lopinavir-ritonavir in addition to baricitinib, a janus kinase inhibitor (anti-jak). baricitinib is a cytokine release inhibitor and is active against sars-cov-2 endocytosis. current evidence suggests that systemic hyperinflammation and immune dysregulation play a key role in the development of severe lung and multiorgan damage found in critically ill covid-19 patients 2, 3, 4 . this massive cytokine release closely resembles that of macrophage activation syndrome or secondary hemophagocytic lymphohistiocytosis , hematological conditions in which acute respiratory distress syndrome (ards) is also common 2, 5 . changes in lymphocyte sub-populations, cytokines dysregulation, presence of highly cytotoxic cd8+ t cells, and the accumulation of pro-inflammatory monocytes/macrophages in the lungs, seem to participate in the immune-mediated tissue damage 3,4,6 . etoposide is a who essential medicine and powerful selective suppressor of activated t-cells and monocytes that reduces the production of inflammatory cytokines. given its effectiveness against hyperinflammation 5, 7 , essentially by targeting monocytes and activated t cells and by moderating the cytokine storm, we propose a rationale for its use in critically ill covid-19 patients. in this report we review the clinical course and outcome of 11 severe covid-19 patients treated with etoposide as salvage therapy following prior immunosuppressants. patients eligible for etoposide treatment were older than 18 years, presented biochemical alterations suggestive of severe hyperinflammation (ferritin levels >1000 ng/ml and/or il-6 values >50 pg/ml), ards (defined by pao 2 /fio 2 < 300) and were not under mechanical ventilation. prior treatment consisted on combinations of oxygen support, lopinavir-ritonavir, antimicrobials, methylprednisolone, and interleukin inhibitors. patients not responding to a 3day course of methylprednisolone plus tocilizumab (il-6 inhibitor) or anakinra (il-1 inhibitor) were offered etoposide. orotracheal intubation, mechanical ventilation and prone positioning were applied when necessary according to the course of respiratory function. prophylactic enoxaparin (40mg per day) gas given regularly, and therapeutic anticoagulation was prescribed if thrombotic complications appeared. the study was conducted at the university hospital of burgos, spain, and approved by the local institutional ethics committee (ceim reference number, 2307) for off-label use of the drug. informed consent from every participant (or relative) was obtained. thirteen patients received etoposide (50-150 mg/m 2 ) out of 709 covid-19 patients admitted to our center during the study period (march 2 to april 10, 2020). overall, 412/709 developed ards (58.1%), of which 169 received methylprednisolone plus tocilizumab (23.8%). two out of 13 patients receiving etoposide were excluded because they were already intubated. a total of 11 patients (1.8%), 9 males and 2 females, with a median age of 58 (range, 41 to 79) were included. clinical characteristics are shown in table 1 . median pao 2 /fio 2 at admission was 98 (range, 52 to 174). following etoposide treatment, the pao 2 /fio 2 ratio improved an average of 195% (fig.1) . three patients needed mechanical ventilation. nine patients fully recovered and were finally discharged home. two patients died as a consequence of thrombotic complications. patient #4 markedly improved her respiratory function allowing extubation but developed massive cerebral ischemic stroke (cardiac ultrasound detected a large thrombus in the right atrium), 2 days after ventilation withdrawal, and died 16 days after admission. patient #5 recovered from severe ards with profound leukopenia, was discharged from the icu, and died suddenly at day 24, presumably due to massive pulmonary thromboembolism, although autopsy was not performed. apart from hematological toxicity and infection in patient #5, no other adverse effects attributable to etoposide were observed and the tolerance was good. noticeably, in our experience, only 1-2 doses of etoposide were enough to observe clinical improvement among severely ill covid-19 patients, in terms of inflammatory serum markers (ferritin, crp, d-dimer), vasopressor therapy requirement and respiratory support. in fact, only 3 patients ultimately required intubation, yet 2 of which died. these preliminary results on 11 patients confirmed the safety and efficacy of etoposide as adjunctive salvage treatment for critically ill covid-19 ards patients, exhibiting systemic hyperinflammation and previously treated with corticosteroids and interleukin inhibitors. a growing evidence suggests that covid-19 disease is a biphasic disease 3, 4, 8 . the initial stage, at which pre-symptomatic or pauci-symptomatic patients exhibit a preliminary and reversible state of immune-suppression associated to the viral load, ideally benefits from antivirals. later on, patients may develop more severe leucopenia (mainly lymphopenia) along with increased inflammatory markers (crp, ferritin, il-6) that may end in a systemic hyperinflammatory state with accompanying cytokine discharge, accumulation of activated cells responsible for the lung damage, need for mechanical ventilation, thrombotic complications, and eventual death 2, 3, 4, 8 . although corticosteroid therapy in covid-19 remains controversial, recent studies suggest a clinical benefit for severely ill covid-19 ards patients in terms of mortality rate, need for mechanical ventilation, and hospital stay 9, 10 . regarding oxygenation parameters, we observed that many severe covid-19 patients presented with alarming pao 2 /fio 2 ratios (commonly under 150, see table1) that, according to berlin ards criteria, were immediate candidates for orotracheal intubation and mechanical ventilation. however, many of them exhibited a relatively preserved pulmonary function (mild dyspnea with or without tachypnea), showed preserved oxygen extraction and adequate organ perfusion without lactic acidosis, and ultimately avoided intubation. we hypothesize that sars-cov-2 related ards distinct pathophysiologic features permit management of many critically ill patients with non-invasive ventilatory support, waiting for the anti-inflammatory reversal effect of etoposide plus adjunctive immunomodulators. the lack of comparison group and the low number of participants are obvious limitations of this study. due to the severity of patients included in the study, with remarkable hyperinflammation data, all patients had received methylprednisolone and tocilizumab or anakinra prior to etoposide, so both drugs can be potential confounders in the interpretation of the results. however, etoposide was administered whenever patients did not respond to prior anti-inflammatory treatment, and at the stage of progressive organ dysfunction. in this preliminary experience, salvage treatment with etoposide in adjunction to immunosuppressants resulted in overall favorable outcome of a small cohort of severely ill covid-19 ards patients presenting with systemic hyperinflammation. the currently ongoing clinical trial nct04356690, started on may 8, 2020, will likely contribute to evaluate the safety and efficacy of etoposide in covid-19 patients. mimonbal@msn.com postal address: avenida islas baleares s/n luisbuzonmartin78@gmail.com postal address: avenida islas baleares s/n itziar.astigarraga@gmail.com postal address: avenida islas baleares s/n com postal address: avenida islas baleares s/n eiglesiasjulian@gmail.com postal address: avenida islas baleares s/n fcaltor@hotmail.com postal address: avenida islas baleares s/n mlopezvel@saludcastillayleon.es postal address: avenida islas baleares s/n avenida islas baleares s/n, 09006, burgos 9. fernández-regueras m (2), internal medicine and infectious diseases baricitinib therapy in covid-19: a pilot study on safety and clinical impact covid-19: consider cytokine storm syndromes and immunosupression the pathogenesis and treatment of the 'cytokine storm' in covid-19 the many faces of the anti-covid immune response recommendations for the management of hemophagocytic lymphohistiocytosis in adults pathogenic t cells and inflammatory monocytes incite inflammatory storm in severe covid-19 patients recommendations for the use of etoposide-based therapy and bone marrow transplantation for the treatment of hlh: consensus statements by the hlh steering committee of the histiocyte society should we stimulate or suppress immune responses in covid-19? cytokine and anti-cytokine interventions rationale for prolonged corticosteroid treatment in the acute respiratory distress syndrome caused by coronavirus disease early short course corticosteroids in hospitalized patients with covid-19 key: cord-310001-qng7h5cj authors: tomlins, jennifer; hamilton, fergus; gunning, samuel; sheehy, caitlin; moran, ed; macgowan, alastair title: clinical features of 95 sequential hospitalised patients with novel coronavirus 2019 disease (covid-19), the first uk cohort date: 2020-04-27 journal: j infect doi: 10.1016/j.jinf.2020.04.020 sha: doc_id: 310001 cord_uid: qng7h5cj nan clinical features of 95 sequential hospitalised patients with novel coronavirus 2019 disease (covid-19), the first uk cohort. sir, wang and colleagues recently reported in this journal the characteristics and prognostic factors of novel coronavirus 2019 (covid-19) disease in 339 patients over 60 years of age presenting to renmin hospital of wuhan university in wuhan, china 1 . this highlighted the higher case fatality rate in this patient group with 19.2% dying within 30 days and frequent comorbidities including hypertension, diabetes and cardiovascular disease. numerous other case series of hospitalised patients in china have provided valuable insight into the clinical features of disease, risk factors for severity and case fatality rate. these have informed diagnostic criteria, treatment strategies and public health policy worldwide. in the largest of these, patients over 65 years of age represented 27% of patients with severe disease and 49.2% of patients admitted to the intensive care unit 2 . to date there has been limited clinical data published outside of china and none from the epidemic in the uk which is estimated to be now nearing its peak (14 th april, 2020). it is anticipated that age and the frequency of coexisting comorbidities in the uk population are likely to be strong drivers of outcome of and mortality in patients hospitalised with covid-19 disease. here, we describe a retrospective single-centre study of all patients hospitalised with sars-cov-2 infection from march 10 th to march 30 th within north bristol nhs trust, a large, regional teaching hospital in the uk. during this period, 95 cases were admitted to the trust and by the final day of follow up on april 6th, 21 patients (21%) had died, 44 patients (43%) had been discharged, and 30 (29%) were still inpatients. of the 21 patients that died, 20 died within 14 days suggesting that most mortality occurs within two weeks. 7 patients were admitted to the intensive care unit, of whom 4 had died by the 6th of april, and 3 remained in intensive care. length of stay for patients who were discharged from hospital was a median of 4 days (iqr 1-16), for those that died 8 days (iqr 6-9) and for those that remained inpatients 9 days (iqr 3-23). longer length of stay was influenced by the timing of a positive test, which for 23 patients (24%) was more than 7 days after admission. fifteen patients (16%) had a negative test preceding the positive result indicating some delay in diagnosis due to false negative results. the demographics, symptoms, radiology, laboratory findings and comorbidities of our patient group are presented in tables 1 and 2. the median age of patients was similar in both patients alive at 14 days and those that had died, at 74 and 77 respectively. no differences by gender were observed, but there were more men in the study overall (63%). cardiovascular and cerebrovascular disease was significantly more common in those that had died by 14 days (90% vs 48%) and of these; congestive cardiac failure was the most notably associated with non-survival (35% vs 11%). diabetes was also significantly more common in those that had died at 14 days (65% vs 32%) whilst respiratory disease was equally distributed between the two groups (30% vs 33%).the most common symptoms were fever (72%) cough (74%) and shortness of breath (43%), followed by confusion (20%). two patients presented with anosmia. this has recently been recognised as an early clinical feature in european patients 4 and may be underrepresented in our cohort due to the frequency of advanced disease and confusion. shortness of breath was the only symptom that was significantly more common in patients that died within 14 days (p= 0.013). we found significantly higher crp and creatinine in those that died in keeping with progressive inflammation and end organ damage. median lymphocyte count was low in both groups, alt was raised in 5 patients and ferritin was > 2000 in 6 patients but was performed infrequently and showed no significant difference between survivors and nonsurvivors. we found little evidence of viral or bacterial co-infection with rhinovirus and human metapneumovirus in 2 of the 88 patients tested and one significant respiratory isolate (k. oxytoca). however, sputum culture and testing for legionella and pneumococcal antigens was performed infrequently. there were 3 positive blood cultures (d. hominis, s. aureus and e. faecium) none of which were felt to be respiratory in origin. 55 patients received antibiotic therapy, including 20 of the 21 patients that died and 2 patients received antivirals (aciclovir for suspected meningoencephalitis). consistent with evidence supporting the use of curb65 as a predictor of mortality secondary to community acquired pneumonia 5 we found a significantly higher median score in non-survivors versus survivors (2.5 versus 1 respectively). patients who did not survive were more likely to have chest x-ray findings, and in particular, were more likely to have bilateral consolidation than unilateral. 40% of survivors did not have any radiological evidence of consolidation. only 6 patients had a ct chest performed which may be useful in detecting early disease in patients that test negative by rtrt-pcr 6 . to our knowledge, this is the first description of a uk cohort of patients with sars-cov-2 infection and the largest descriptive study of the infection outside of china. we found a much higher median age and case fatality rate than that reported by other studies of all hospitalised patients with covid-19. all the patients that died were over the age of 60 and only 4 were admitted to intensive care. given the current availability of beds and ventilatory equipment in the hospital during this study this does not represent deficiencies of medical care. rather it suggests that there was an anticipated deterioration in these patients in the context of poor premorbid state, and planned decision making around intensive care unit admission. nice guidance published during this period endorsed the use of a clinical frailty scale (cfs) score in the assessment for critical care admission which has been shown to perform better than evaluation of cognitive function or comorbidity in estimating risk of death and has been validated in intensive care outcomes 78 . further assessment of its application to the covid-19 pandemic is required and may be instrumental in guiding further public health policy, particularly in areas with a low prevalence where the suspension of health care services such as cancer services may be detrimental to other preventable health outcomes. in summary, despite limited stress on our health care service, around 20% of our hospitalised population died, with the majority dying outside intensive care with significant comorbidities. further work is needed to characterise other uk cohorts. coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on 4-week follow-up clinical characteristics of coronavirus disease 2019 in china olfactory and gustatory dysfunctions as a clinical presentation of mild-tomoderate forms of the coronavirus disease (covid-19): a multicenter european study severity assessment tools for predicting mortality in hospitalised patients with community-acquired pneumonia. systematic review and meta-analysis the role of ct in case ascertainment and management of covid-19 pneumonia in the uk: insights from high-incidence regions a global clinical measure of fitness and frailty in elderly people the impact of frailty on intensive care unit outcomes: a systematic review and metaanalysis this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-311853-k6efd9vg authors: lim, rachel hf; chow, angela; ho, hanley j title: decline in pneumococcal disease incidence in the time of covid-19 in singapore date: 2020-08-15 journal: j infect doi: 10.1016/j.jinf.2020.08.020 sha: doc_id: 311853 cord_uid: k6efd9vg nan chow et al reported a marked decline in influenza incidence in singapore during the first four months of 2020, likely attributable to public health measures aimed at controlling the coronavirus disease 2019 (covid-19) pandemic (1) . measures, such as social distancing, mask-wearing, and limiting the size of group gatherings, might also have reduced the transmission of other respiratory infections. we report on the decreased incidence of pneumococcal disease in our institution during the first five months of the covid-19 pandemic. singapore confirmed its first case of covid-19 on 23 january 2020 (2), following which enhanced surveillance, contact tracing and infection control measures were implemented to contain the disease. when the dorscon (disease outbreak response system condition) risk assessment level was raised to orange on 7 february 2020, further measures including air travel restrictions, stay-home notices for newly returning travellers, and limiting large gatherings were introduced. these culminated in a partial lockdown period from 7 april 2020 (epidemiological week, e-week 15) to 1 june 2020 (e-week 23), termed the 'circuit breaker'. during this period, closure of schools and non-essential workplaces, prohibition of dining in at eateries, and mandatory social distancing and mask-wearing were enacted. these resulted in a significant reduction in the community transmission of covid-19, from a high of 58 reported community cases in a day at the start, to 0 community cases on the last day of the circuit breaker (3). subsequently, phased reduction of restrictions was implemented, although mask-wearing, social distancing and limitations to group gathering sizes continued to be enforced. as of 19 july 2020 (e-week 30), the number of reported covid-19 cases in singapore was more than 47,000, with the majority (n=45,150) of cases being dormitory-based migrant workers on work permits (4). streptococcus pneumoniae is transmitted via respiratory droplets and contributes to significant morbidity and mortality in singapore, especially at the extremes of age (5) . pneumococcal vaccination is recommended for adults aged 65 years and above or who have specific medical conditions; however, it is currently non-mandatory and vaccination rates have been low (6) . tan tock seng hospital (ttsh) is a 1,600-bed adult tertiary care hospital, co-located with the national centre of infectious diseases (ncid), a 330-bed purpose-built facility for centralised management of emerging infectious diseases. as part of routine infectious disease surveillance for these institutions, we reviewed the results of all urinary streptococcal antigen tests performed, as well as the number of notifications submitted to the ministry of health for invasive pneumococcal disease (ipd) (a notifiable disease for which reporting is mandatory for all clinicians and clinical laboratories), from the years 2010 to 2020. contrasting 2020's data against the preceding 10 years by e-week, the mean number of positive urinary streptococcal antigen results in 2020 fell to 1.81/week, from a mean of 3.81/week for the years 2010 to 2019 (standard deviation [sd] 2.38, p<0.0001), from e-weeks 1-27. the mean weekly positivity rate was 1.76% in 2020, a decrease from a mean rate of 4.07% (sd 2.44%, p<0.0001) over the previous 10 years (e-weeks 1-27). examining the weekly trend, the number of positive urinary streptococcal antigen results started to fall in 2020 from e-week 9 onwards to 0-3 positive results per week, a decrease from the usual trend of 3-5 positive results per week from 2010-2019 (fig. 2 ). this corresponded with the time period when public health measures to combat covid-19 were implemented. comparing the yearly trends from 2010 to 2020 (for e-weeks 1-27 inclusive), the positivity rate of urinary streptococcal antigen testing was 1.84% in 2020, compared to an overall positivity of 3.98% for years 2010 to 2019 (fig. 1 ). in addition, the number of notifications to the ministry of health for ipd fell to its lowest point of 8 notifications during e-weeks 1 to 27 of 2020, compared to a median of 14 (iqr 11-18, p=0.18) over the previous 10 years during the same period. this is on a background of similar numbers of urinary streptococcus antigen tests carried out in e-weeks 1-27 of 2020 (2660 tests) compared to a median of 2702 tests (p=0.91) in 2010-2019 for the same period. we postulate that the public health covid-19 prevention measures introduced in singapore resulted in an inadvertent decrease in pneumococcal disease transmission. a marked decline in influenza incidence in the first few months of the covid-19 pandemic has been reported (1, 7) . this was also observed in studies from china and other countries in east asia (8, 9) . similarly, researchers in taiwan reported record lows of incidence rates of severe complicated influenza and ipd (10). there has not been any widespread national campaigns over the past one year to increase pneumococcal vaccination in the population, no practice changes affecting the ordering of urinary streptococcal antigen tests, and no changes in reporting requirements for ipd, to account for our findings. historically, public health messaging for pneumococcal disease prevention has centred around vaccination. while this remains paramount, our study highlights the key role of behavioural and public health measures in reducing the transmission of s. pneumoniae. accordingly, older adults and high-risk individuals should consider adopting more meticulous infection control practices such as frequent hand hygiene and mask-wearing during seasonal increases in respiratory illnesses, even during non-outbreak periods. at a national level, there is scope for sustained public health education and infection control policies beyond the covid-19 period to reduce the burden of respiratory infections. our study has some limitations. there may have been some altered health-seeking behaviour due to the covid-19 pandemic, which might have reduced overall presentation to medical facilities. our data was limited to that from ttsh and ncid, of which the latter is the dedicated national centre for handling covid-19 cases. patients may have opted to present elsewhere due to this status. however, we believe this is less likely given that the number of urinary streptococcus antigen tests for e-weeks 1-27 of 2020 (ordered by clinicians based on clinical suspicion for pneumococcal disease) was similar to that for the same period from 2010 to 2019. in conclusion, we observed a decreased incidence of pneumococcal disease at our institution that corresponded with the time period when public health measures were implemented to control covid-19, suggesting that these measures had also had an inadvertent effect on the transmission of s. pneumoniae. behavioural and infection control measures should be considered as part of long-term prevention efforts. unintended consequence: influenza plunges with public health response to covid-19 in singapore covid-19 in singapore-current experience: critical global issues that require attention and action a national study of the epidemiology of pneumococcal disease among hospitalised patients in singapore: 1995 to 2004 epidemiological characteristics associated with uptake of pneumococcal vaccine among older adults living in the community in singapore: results from the national health surveillance survey positive effects of covid-19 control measures on influenza prevention does covid-19 infection impact on the trend of seasonal influenza infection? 11 countries and regions, from 2014 to 2020 covid-19 preventive measures showing an unintended decline in infectious diseases in taiwan this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-293714-s6ezxi5r authors: principi, nicola; rigante, donato; esposito, susanna title: the role of infection in kawasaki syndrome date: 2013-04-18 journal: j infect doi: 10.1016/j.jinf.2013.04.004 sha: doc_id: 293714 cord_uid: s6ezxi5r objectives: to analyse the evidence suggesting a possible infectious origin of kawasaki syndrome (ks). methods: pubmed was searched for all of the studies published over the last 15 years using the key words “kawasaki syndrome” or “mucocutaneous lymph node syndrome” and “infectious disease” or “genetics” or “vasculitis” or “pathogenesis”. results: various levels of evidence support the hypothesis that ks is a complex disease triggered by an infection due to one or more pathogens. viruses or bacteria may be the primum movens, although no specific infectious agent can be considered definitely etiological. a number of genetic polymorphisms have been identified in subjects with ks, but none of them can currently be considered a real marker of susceptibility. conclusions: various data suggest that ks is intimately related to infectious diseases and that its clinical expression is influenced by predisposing genetic backgrounds, but our knowledge of the infectious agent(s) involved and the genetic characteristics of susceptible children remains only partial. further studies are needed to address the many still open questions concerning the disease. kawasaki syndrome (ks), originally called "mucocutaneous lymph node syndrome" in 1967, 1 is an acute multisystem vasculitis that causes generalised inflammatory cell tissue injury starting in the vascular endothelium and is mainly encountered in children aged less than five years regardless of their ethnicity. 1 the injuries are particularly severe in the coronary arteries, which are frequently affected by dilatations, aneurysms or fistulae, especially in patients who do not receive prompt treatment with intravenous immunoglobulins and anti-inflammatory doses of acetylsalicylic acid. 2 now that rheumatic fever is largely controlled, ks has become the leading cause of acquired heart disease among children in industrialised countries. 3 as no specific diagnostic test is available, ks is identified on the basis of a constellation of non-specific clinical signs. according to the american heart association guidelines, 4 which are shared by most scientific communities throughout the world, a diagnosis requires prolonged fever lasting more than five days and at least four of the following signs: 1) diffuse oral cavity inflammation (including pharyngeal infection), dry fissured lips, and a strawberry tongue); 2) bilateral non-purulent conjunctivitis; 3) heterogeneous skin rashes; 4) angioedema of the extremities (including peripheral erythema, oedema, or induration of the hands or feet); and 5) non-purulent cervical lymphadenopathy exceeding !1.5 cm in diameter. in what are known as "incomplete" cases, one or more of these clinical signs may be absent but a diagnosis can still be made if there is echocardiographic evidence of coronary artery abnormalities. furthermore, a broad range of unusual clinical finding have been reported as defining the "atypical" variant of ks, including aseptic meningitis, peripheral facial nerve palsy, uveitis, gastrointestinal complaints, acalculous gallbladder hydrops, urethritis, testicular swelling, pulmonary nodules, liver impairment with jaundice, and even hemophagocytic syndrome. 5 laboratory investigations (listed in table 1 ) can only support the diagnosis of ks, but they still need to be validated before they can be used in everyday clinical practice. however, although the clinical features of ks are usually recognisable, its underlying immune mechanisms are still being investigated. most experts consider it to be the consequence of an abnormal immunological response evoked by one or more widely distributed infectious agents in genetically susceptible individuals, but it still remains a medical enigma. the main aim of this review is to analyse the available evidence suggesting that ks may have an infectious origin. pubmed was used to search for all of the studies published over the last 15 years using the key words: "kawasaki syndrome" or "mucocutaneous lymph node syndrome" and "infectious disease" or "genetics" or "vasculitis" or "pathogenesis". more than 1300 articles were found, but only those published in english or providing evidence-based data were included in the evaluation. various levels of evidence support the hypothesis that ks is a complex disease initiated by an infection due to one or more pathogens (table 2) . however, no strict and unmistakable correlation between specific infectious agents and the development of the disease has ever been identified. a number of bacterial and viral infectious agents have been sporadically isolated from ks patients. the most frequently implicated bacteria are staphylococcus aureus, 6 streptococcus pyogenes, 7 and atypical pathogens, 8e10 and the viruses associated with ks over recent years are epsteinebarr virus, 11 adenovirus, 12 parvovirus b19, 13 herpesvirus 6, 14 parainfluenza type 3, 15 measles, 16 rotavirus, 17 dengue virus, 18 and human immunodeficiency virus. 13 varicella, 19 2009 h1n1 pandemic influenza 20 and coxsackie b3 virus 21 have also been described in patients with ks, but they were equally found in the blood or body fluids of both patients and healthy subjects. moreover, no relationship was reported between ks and the circulation of the commonest respiratory viruses. 22 the most recent and numerous studies of ks-related viruses have postulated the etiological role of human coronavirus (hcov) nl63 and bocavirus, 23e25 but this has not been confirmed by subsequent studies. in order to evaluate the importance of hcov-nl63 in ks, shimizu et al. established a multi-institutional collaborative research project to test respiratory samples using realtime polymerase chain reaction (rt-pcr), and found that only one out of 48 ks patients (2%) was positive 26 ; dominguez et al. found exactly the same prevalence in nasopharyngeal wash samples from ks patients and healthy controls over a period of seven months 27 ; and lehmann et al. measured the concentrations of igg, igm and iga antibodies against hcov nl63 and oc43 in the blood of children showing the signs and symptoms of ks for 3e10 days and healthy controls, but did not find any difference between the two groups. 28 the data regarding bocavirus (a virus that has recently emerged as a possible cause of respiratory infection) are also unconvincing 29 : although it was identified in the serum, stool and cerebrospinal fluid of some children with ks, no definitive conclusion could be drawn concerning its etiological role. the limited etiological importance of the pathogens so far identified seems to be supported by the studies of benseler et al. 30 and jordan-villegas et al., 31 who found that that concomitant infections in children with ks did not alter the response to treatment with intravenous immunoglobulins, and did not influence the risk of coronary artery involvement or affect overall cardiovascular outcomes. however, the lack of any clear relationship between one or more pathogens and the development of ks does not exclude the possibility that a real infectious disease may be involved, and other factors support this hypothesis. on the other hand, the unsuccessful identification of a specific pathogen to which ks could be ascribed has led some authors to postulate that variants of normal flora in the gut, oral cavity or skin of young children with a genetic defect of proper immune maturation do not induce immune tolerance as self commensals, but rather induce an imbalance of the immune system, leading to a hyperimmune reaction and the manifesting ks. 32 table 1 laboratory findings supporting a diagnosis of kawasaki syndrome. 1. c-reactive protein !3.0 mg/dl 2. erythrocyte sedimentation rate !40 mm/h 3. albumin 3.0 g/dl 4. age-relative anaemia 5. high alanine aminotransferase levels 6. platelet count !450,000/mm 3 in the subacute phase of the disease 7. white blood cell count !15,000/mm 3 8. white blood cells/high-power field !10 in urinalysis histopathological data some histological findings strongly suggest that ks has an infectious origin, although they do not identify the responsible pathogen. the most important is that persistent intracytoplasmic inclusion bodies (iibs) showing amphophilic staining by haematoxylin-eosin and stain for rna have been found in most of the tissues of patients who have died because of ks. 33 as transmission electron microscopy (tem) of bronchial epithelia has revealed virus-like particles associated with the iibs, it was thought that these may be a "footprint" of a viral infection that may persist indefinitely: rowley et al. speculated that the infection associated with the development of ks could be due to a new and ubiquitous rna virus that caused only asymptomatic infection or very mild disease in the general population, but ks in genetically selected subjects. 34 it was thought that the first site of infection was the respiratory tract, with further dissemination through macrophages to all body sites, including the medium-sized arteries (mainly the coronary arteries) that are the most crucial targets in ks. further findings that strongly support an infectious origin of ks are those of orenstein et al., who used light microscopy and tem to study tissue specimens from 32 autopsies, eight heart transplants and an excised coronary aneurysm of patients with ks and identified three different vasculopathic processes: acute self-limited necrotising arteritis (na), subacute/chronic vasculitis, and luminal myofibroplastic proliferation. on the basis of the changes in coronary and non-coronary arteries during the different phases of ks, they considered na the only self-limiting process of the three, and that it was consistent with an acute viral infection. 35 many epidemiological findings regarding ks are consistent with an infectious origin, as they are quite similar to those of various infectious diseases. first of all, there is the occurrence of epidemics because, although cases of ks are diagnosed, there are also sometimes widespread epidemics. in japan, where nationwide epidemiological surveys of ks have been carried out almost every two years since 1970, 36 three large-scale epidemics were recorded in 1979, 1982, and 1986, with incidence rates that were several times higher than those reported in the previous and following year. secondly, as in the case of a number of infectious diseases, 37, 38 ks is more frequent in boys, and the male to female ratio is about 1.6. 39 thirdly (once again as in the case of many infectious diseases), the incidence of ks seems to be closely related to weather conditions, although the predominant season varies from country to country: winter and spring in the united kingdom, australia and the usa, and spring and summer in china. 40e43 pitzer et al., examined seasonal changes in the age and incidence of ks hospitalisations in the usa, and found that periods of high incidence corresponded to a low average age, and vice versa. 40 comparison of the observed pattern with those predicted by a suite of models based on different etiological hypotheses, the ageincidence pattern of ks suggested the involvement of an imperfectly immunising infection and/or a persistently infectious agent. the possible relationship between seasonal variations in the incidence of ks and an infectious aetiology is also supported by data showing that its incidence in the usa inversely correlates with average monthly temperature (r z à0.47; p < 0.001) and positively correlates with the role of infection in ks average monthly precipitation (r z à0.52; p < 0.001). 41 moreover, analyses of the three major ks epidemics in japan, major non-epidemic inter-annual fluctuations of ks cases in japan and san diego, and seasonal variations in the incidence of ks in japan, hawaii and san diego have revealed a consistent pattern linking ks cases to large-scale wind currents originating in central asia and crossing the north pacific. 42 this seems to indicate that the environmental trigger of ks may be wind-borne, and this has led some experts to suggest that efforts to isolate the causative agent should concentrate on the microbiology of aerosols. 43 finally, children in the first months of life only exceptionally develop ks, which supports the hypothesis that most infants are protected by passively acquired specific maternal antibodies against the possible causative agent(s). on the other hand, the very low incidence of ks beyond the fifth year suggests that most children are infected uneventfully by one or more of the infectious agents associated with ks in early life and can then mount a long-lasting, strong and protective immune response. recurrences of ks have been reported in only 1e3% of children, and are best documented in japan. 44 the clinical findings of fever, an erythematous pharynx, conjunctival injection, rash, oedema of the extremities and cervical adenitis in patients with ks, and the clear tendency of these signs to resolve spontaneously even without treatment also support an infectious aetiology. a number of viral diseases have a similar clinical picture. moreover, some of the clinical features of ks, such as mucous membrane erythema and desquamation of the fingers and toes (usually beginning within 2e3 weeks of the onset of fever), overlap those associated with some bacterial diseases that are considered to be a consequence of the superantigen (sa)-mediated activation of t cells leading to the overproduction of cytokines, systemic inflammation and shock. 45e47 the best examples in this regard are toxic shock syndrome (tss) and scarlet fever due to s. aureus, and streptococcal toxic shock syndrome (stss) due to streptococcus pneumoniae. the similarity between these conditions and ks has led to the conclusion that, at least in some cases, ks may follow an infection due to an saproducing pathogen. a number of sas have been identified: the most widespread bacterial pathogens are s. aureus and s. pneumoniae, but yersinia pseudotuberculosis, mycoplasma pneumoniae and mycobacterium tuberculosis have also been associated with sa formation 45 ; among viruses, sas have been found in epstein barr virus, rabies virus and mouse leukaemia virus. 46 sas are a family of potent immunostimulatory proteins whose particular structures and sequences lead to a shared ability to by-pass the mechanism of conventional major histocompatibility complex (mhc)-restricted antigen processing. when an sa is involved, t cell responses are quantitatively and qualitatively different from conventional t cell activation by normal antigens. in particular, sas activate t cells in a manner that depends on the t cell receptor variable domain (vb), and so a large number of t cells can be simultaneously activated. 85 the activation is extremely potent and a number of studies have found that ks is characterised by the marked activation of t cells and monocytes/macrophages, and increased production of il-1b, tnf-a and il-6, which are the same immunological findings as those of tss. 47 although some attempts to demonstrate the presence of sa-producing pathogens in children with ks have led to negative results, 48e51 others have provided data suggesting the direct involvement of sas. leung et al. blindly studied bacterial sas potentially involved in the pathogenesis of ks in cultures of patients in the acute phase and controls, 52 and found sa-producing bacteria in 13 of the 16 patients but in only one of the 15 controls (p < 0.0001). eleven of the 13 toxin-positive cultures from the patients with ks contained toxic shock syndrome toxin (tsst)-1 secreting s. aureus, and the remaining two contained streptococci producing streptococcal pyrogenic exotoxin b (speb) and streptococcal pyrogenic exotoxin c (spec). twelve of the culture-positive patients had toxin-producing s. aureus isolated from pharyngeal or rectal cultures, thus suggesting the gastrointestinal tract as the primary entry site. similar findings of tsst-1-producing s. aureus and spec-producing streptococci in children with acute ks have been recently published. 47 the sa theory may be supported by anecdotal reports of ks patients with guttate psoriasis because it has been suggested that this form of psoriasis is due to toxinmediated t cell activation. 53 furthermore, a number of studies analysing the t lymphocyte receptor repertoire and the titres of antibodies against selected sas have indirectly demonstrated that these proteins may be related to the development of ks. 52,54e56 in addition, suenaga et al. examined five sa genes in the stools of ks patients, febrile controls and healthy children, 57 and found at least one of the genes in 42 specimens from the patients with ks (70%), in 14 from the febrile group (38.9%), and in seven from the healthy group (26.9%). the detection rate between subjects with and without ks was of at least one of the 5 sa genes (p < 0.001), and more than two sa genes were significantly different (p z 0.002), thus suggesting the direct involvement of sas in the development of ks. despite the direct and indirect evidence supporting the hypothesis that ks is an infectious disease, only "susceptible" subjects seem to develop it and genetics seem to play a role in selecting the patients. ks occurs throughout the world, but is significantly more common in some asian countries, such as japan, korea and taiwan. it has been reported that the annual incidence of ks in japan in 2009 and 2010 was respectively 206.2 and 239.6 per 100,000 children aged 0e4 years 58 ; on the contrary, a recent analysis found that the 2009 ks-related hospitalisation rate in the usa was 19 per 100,000 children aged <5 years, 59 and even lower incidence rates have been calculated for a number of european countries. 60e63 theoretically, various factors could explain this large difference. the incidence of ks is rapidly increasing throughout the world, and so surveys carried out at different times may lead to different results. moreover, although ks is significantly more frequent in younger children than in older children, adolescents or adults, its frequency in younger patients also varies. a comparison of the incidence of ks in northern european and japan found that 86.4% of the japanese patients were aged <5 years, but 67.8% of the cases diagnosed in norway, finland, sweden and denmark (p < 0.001). 63 the incidence of ks in different regions may be affected by differences in surveillance methods, clinical diagnostic and treatment practices, physician awareness of ks, and the occurrences of ks clusters or outbreaks. however, a global evaluation of all these factors suggests that they are unlikely to be the only reason for such a striking difference. furthermore, american studies have clearly shown ethnicity-related variations in the incidence of ks: in comparison with white subjects, the incidence is twice as high among asians and pacific islanders, and about 1.5 times higher among black subjects. 64 similarly, its incidence in hawaii (the state with the highest proportion of asians and pacific islanders) is 2.5 times higher than that reported for the continental usa. 65 it has also been reported that the incidence of ks is 6e10 times higher among the siblings of ks patients than in the general population, 66 and that children with ks are more likely to have parents who have had the disease. 67 it has been calculated that the inheritability of ks (i.e. the ratio of the incidence of ks between siblings and the general population) is only slightly less than that of type 1 diabetes and about four times more than that of allergic asthma. 68 all of the above findings strongly suggest that genetic factors play a substantial role in the occurrence of ks, but studies of the genetic characteristics of ks patients have not definitively identified which genetic marker(s) may favour or protect humans from developing of ks, or regulate its clinical severity. several candidate genes, mainly chosen among those related to innate and acquired immunity, cardiovascular function, and vascular remodelling, have been tested in patients with ks (table 3 ). 69e117 results were frequently negative or conflicting, particularly when the studies enrolled a limited number of patients and were carried out in populations with significant racial differences that impact the allele frequency of some of the single nucleotide polymorphisms (snps) analysed in the studies. good example at this regard are the data collected on the role of matrix metalloproteinases (mmps), fc gamma receptors (fcgr) and of cd40 ligand snps in conditioning susceptibility, evolution and outcome of ks. mmps play an important role in processes that degrade extracellular matrices. their activity is controlled by specific inhibitors (timps) and imbalances between mmps and timps are associated with several pathological conditions, including vascular aneurysm. association of increased mmp9/timp2 and mmp3/ timp1 ratios with risk of coronary artery lesions was found in japanese children. 95 on the contrary, no association was found between snp of mmp-3 in the korean population. 96 debated is also the role of cd40l, a transmembrane protein that engages with cd40 and transduces signals related to cell activation and development because a strict association between snps and development and severity of ks was demonstrated in the japanese patients 77 but not in the taiwanese population. 78 however, in some studies more convincing results were found particularly when they could evidence that the same polymorphisms were present in populations with different racial characteristics. onouchi et al. reported that multiple variants in the caspase-3 gene (casp3) that were in linkage disequilibrium conferred susceptibility to ks in both japanese and us subjects of european ancestry. 73 these authors found that a g to a substitution of one commonly associated snp located in the 5 0 untranslated region of casp3 (rs72689236; p z 4.2 â 10 à8 in the japanese and p z 3.7 â 10 à3 in the european americans) abolished binding of nuclear factor of activated t cells to the dna sequence surrounding the snp suggesting that altered casp3 expression in immune effecter cells influences susceptibility to ks. interesting results were also reported by shimizu et al. that investigated genetic variation in 15 genes belonging to the tgf-b pathway in a total of 771 kd subjects of mainly european descent from the united states, the united kingdom, australia, and the netherlands. 101 genetic variants in tgfb2, tgfbr2, and smad3 and their haplotypes were consistently and reproducibly associated with ks susceptibility, coronary artery aneurysm formation, aortic root dilatation, and intravenous immunoglobulin treatment response in different cohorts. a smad3 haplotype associated with ks susceptibility replicated in 2 independent cohorts and an intronic single nucleotide polymorphism in a separate haplotype block was also strongly associated (a/g, rs4776338; p z 0.000022; or, 1.50; 95% ci, 1.25e1.81). pathway analysis using all 15 genes further confirmed the importance of the tgf-b pathway in ks pathogenesis. because similar data regarding susceptibility were reported by kuo et al. 102 it was concluded that genetic polymorphisms in tgfb signalling pathway are strictly associated with the risk of development of ks. however, more reliable results have been obtained using genome-wide association studies (gwas) because genome scanning without a defined hypothesis has the advantage of identifying disease genes even if the functions of these genes are not associated with previous knowledge about the disease's pathophysiology. even in this case, the most important results were those repeatedly reported in different populations. by linkage disequilibrium mapping performed on the region of chromosome 19q13.2, it was found that an snp within the inositol 1, 4, 5-trisphosphonate 3-kinase c (itpkc) gene, a gene that regulates the signal transduction in t lymphocytes and the degree of inflammatory response, was associated with increased susceptibility to ks and with the development of coronary artery in both a japanese and a usa population. 111 further data confirming the relationships between genetics and ks were collected by burgner et al.. 112 these authors conducted a gwas with dutch ks cases and controls and followed up associations signals with a family-based association study of ks families from australia, usa, and uk. they reported that snps in intron 3 of n-acetylated a-acidic dipeptidase-like 2 (naaladl2), a protein possibly involved in immune homeostasis, were associated with ks (p z 1.13 â 10 à6 ) and that mrna expression of the same protein in erythrocytes was significantly lower in the acute phase of ks than in the convalescence period. 107 the presence of a predisposing genetic system favouring the development of ks and regulating its severity was confirmed by the study of kim et al., 113 korean ks patients and 600 healthy controls, and found that 18 genomic regions with one or more sequence variants were associated with ks, and 26 were associated with coronary artery lesions (cals) (p < 1 â 10 à5 ). an snp within the disabled homologue 1 (dab1) gene locus in chromosome 1 (rs527409) was replicated in 266 children with ks and 600 114 the most strongly associated snps detected in the joint analysis corresponded to three novel loci. among kd-associated snps, three were close to the copb2 (coatomer protein complex beta-2 subunit) gene: rs1873668 (p z 9.52 â 10 à5 ), rs4243399 (p z 9.93 â 10 à5 ), and rs16849083 (p z 9.93 â 10 à5 ). moreover, an snp in the intronic region of the erap1 (endoplasmic reticulum amino peptidase 1) gene (rs149481, p z 4.61 â 10 à5 ) and six snps (rs17113284, rs8005468, rs10129255, rs2007467, rs10150241, and rs12590667) clustered in an area containing immunoglobulin heavy chain variable regions genes, with p-values between 2.08 â 10 à5 and 8.93 â 10 à6 , were also identified. because these kd candidates have been implicated in t cell receptor signalling, regulation of proinflammatory cytokines, as well as antibody-mediated immune responses, these findings strongly supported the relationships among genetics, alterations in immune response and development of ks. association of snps within the fcgr gene cluster on chromosome 1 with ks was identified by khor et al. in european and asian populations. 115 in a large study sample (2173 individuals with ks and 9383 controls) including european and asian populations, these authors found that two loci exceeded the formal threshold for genome-wide significance. the first was a functional polymorphism in the igg receptor gene fcgr2a encoding an h131r substitution (rs1801274; p z 7.35 â 10 à11 , or z 1.32), with the a allele (coding for histidine) leading to a high risk of disease. the second was at 19q13 (p z 2.51 â 10 à9 , or z 1.42 for the rs2233152 snp near mia and rab4b; p z 1.68 â 10 à12 , or z 1.52 for rs28493229 in itpkc ), thus confirming the data previously with previous studies. 79 the involvement of the fcgr2a locus may have implications for understanding immune activation in ks pathogenesis and the mechanism of response to intravenous immunoglobulin, the only proven therapy for this disease. more recently, japanese and taiwanese groups independently reported a significant association between ks and polymorphisms in the intergenic region on chromosome 8p23-p22 between b lymphoid kinase (blk ), a tyrosine kinase involved in b-cell receptor signal transduction and fam167a, a functionally uncharacterized gene. 116, 117 onouchi et al. undertook a gwas involving 428 japanese individuals with ks and 3379 japanese controls genotyped at 473,803 snps. 116 they validated the results in two independent replication panels of 754 cases and 947 controls, and observed significant associations in the fam167a-blk region (rs2254546, p z 8.2 â 10 à21 ). similar results were obtained by lee et al. in 622 individuals with ks and 1107 controls in a han chinese population residing in taiwan, with replication in an independent han chinese sample of 261 cases and 550 controls. 117 they found that polymorphisms at blk gene together with genetic abnormalities at cd40, were associated with ks at genomewide significance (p < 5.5 â 10 à8 ) confirming the role of immune activation and inflammation in the pathogenesis of the syndrome. however, despite these findings, the correlations between genetic markers the risk of developing and severity of ks are far from clear. at the moment the most convincing evidences of a strict correlation between genetic abnormalities and ks regards polymorphisms of itpkc, fcgr, casp3 and tgfb genes. although various data suggest that ks is an infectionrelated clinical syndrome that can only develop in children with predisposing genetic backgrounds, our knowledge of the infectious agent(s) involved and the genetic characteristics of susceptible children is still unsatisfactory. either viruses or bacteria could act as disease, but no specific infectious agent can be considered the definite cause of ks, and so no specific anti-infective 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kawasaki disease serologic evidence that streptococcal superantigens are not involved in the pathogenesis of kawasaki disease prevalence of superantigen-secreting bacteria in patients with kawasaki disease association of psoriasis-like eruption and kawasaki disease analysis of t-cell receptor v-beta 2 in peripheral blood lymphocytes as a diagnostic marker for kawasaki disease selective increase of v beta 2 þ t cells in the small intestinal mucosa in kawasaki disease t cell activation profiles in kawasaki syndrome detection of multiple superantigen genes in stools of patients with kawasaki disease epidemiologic features of kawasaki disease in japan: results of the 2009e2010 nationwide survey epidemiology of kawasaki disease in asia, europe, and the united states rising incidence of kawasaki disease in england: analysis of hospital admission data kawasaki syndrome hospitalizations in ireland increased detection rate of kawasaki disease using new diagnostic algorithm, including early use 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ccr5 and ccl3l1 are important determinants of susceptibility to kawasaki disease the ccr5 (-2135c/t) polymorphism may be associated with the development of kawasaki disease in korean children polymorphisms in chemokine receptor genes and susceptibility to kawasaki disease genetic polymorphisms in the cd40 ligand gene and kawasaki disease inflammatory gene polymorphisms and susceptibility to kawasaki disease and its arterial sequelae polymorphism of fc gamma riia may affect the efficacy of gamma-globulin therapy in kawasaki disease the involvement of fc gamma receptor gene polymorphisms in kawasaki disease association of il-1ra gene polymorphism, but no association of il-1beta and il-4 gene polymorphisms, with kawasaki disease the -590 c/t and 8375 a/g interleukin-4 polymorphisms are not associated with kawasaki disease in taiwanese children interleukin 6 gene promoter polymorphism is not associated with kawasaki disease the il-10 (-627 a/c) promoter polymorphism may be associated with coronary aneurysms and low serum albumin in korean children with kawasaki disease influence of interleukin 18 promoter polymorphisms in susceptibility to kawasaki disease in taiwan inducible and endothelial constitutive nitric oxide synthase gene polymorphisms in kawasaki disease increased frequency of alleles associated with elevated tumor necrosis factor-alpha levels in children with kawasaki disease association of mannose-binding lectin genotype with cardiovascular abnormalities in kawasaki disease polymorphisms in the mannose-binding lectin gene as determinants of age-defined risk of coronary artery lesions in kawasaki disease modulating effects of mannose binding lectin genotype on arterial stiffness in children after kawasaki disease monocyte chemoattractant protein 1 gene regulatory region polymorphism and serum levels of monocyte chemoattractant protein 1 in japanese patients with kawasaki disease polymorphism of transmembrane region of mica gene and kawasaki disease circulating matrix metalloproteinases and their inhibitors in patients with kawasaki disease polymorphism of matrix metalloproteinase-3 promoter gene as a risk factor for coronary artery lesions in kawasaki disease genetic analysis of mmp gene polymorphisms in patients with kawasaki disease association of the matrix metalloproteinase-3 (-439c/g) promoter polymorphism with kawasaki disease in korean children methylenetetrahydrofolate reductase polymorphism in kawasaki disease a polymorphism in plasma platelet-activating factor acetylhydrolase is involved in resistance to immunoglobulin treatment in kawasaki disease polymorphism of slc11a1 (formerly nramp1) gene confers susceptibility to kawasaki disease transforming growth factor-beta signaling pathway in patients with kawasaki disease polymorphisms of transforming growth factor-b signaling pathway and kawasaki disease in the taiwanese population tissue inhibitor of metalloproteinase 2 and coronary artery lesions in kawasaki disease analysis of tumor necrosis factor-alpha production and polymorphisms of the tumor necrosis factor-alpha gene in individuals with a history of kawasaki disease association between levels of tnf-alpha and tnf-alpha promoter -308 a/a polymorphism in children with kawasaki disease tumor necrosis factor-alpha levels and promoter polymorphism in patients with kawasaki disease in korea association of vascular endothelial growth factor (vegf) and vegf receptor gene polymorphisms with coronary artery lesions of kawasaki disease vascular endothelial growth factor gene haplotypes in kawasaki disease association of vascular endothelial growth factor c-634 g polymorphism in taiwanese children with kawasaki disease lack of association of the vascular endothelial growth factor gene polymorphisms with kawasaki disease in taiwanese children itpkc functional polymorphism associated with kawasaki disease susceptibility and formation of coronary artery aneurysms a genome-wide association study identifies novel and functionally related susceptibility loci for kawasaki disease a genome-wide association analysis reveals 1p31 and 2p13.3 as susceptibility loci for kawasaki disease identification of novel susceptibility loci for kawasaki disease in a han chinese population by a genome-wide association study genome-wide association study identifies fcgr2a as a susceptibility locus for kawasaki disease a genome-wide association study identifies three new risk loci for kawasaki disease two new susceptibility loci for kawasaki disease identified through genome-wide association analysis this review was supported by a grant from the italian ministry of health (bando giovani ricercatori 2007). the authors have no potential conflict of interest to declare. key: cord-330387-7lci44w5 authors: bird, paul; badhwar, vinay; fallon, karlie; kwok, kin on; tang, julian w title: high sars-cov-2 infection rates in respiratory staff nurses and correlation of covid-19 symptom patterns with pcr positivity and relative viral loads date: 2020-06-18 journal: j infect doi: 10.1016/j.jinf.2020.06.035 sha: doc_id: 330387 cord_uid: 7lci44w5 nan we read with interest the study in the journal by chen and colleagues from nanjing, china that demonstrated a high positivity rate (17%) in healthcare workers (hcws), 1 but did not attempt to breakdown which type of hcws working in which specialties had the highest infection rates. similarly, another study from leicester, uk compared hospitalised and community patient sars-cov-2 pcr (polymerase chain reaction) positivity rates with that of staff, 2 but again, did not assess which staff groups or clinical specialties were at most risk of acquiring covid-19. finally, one other study from wuhan, china described the clinical features of hcws infected with covid-19, but again did not analyse the staff most infected by role or specialty. 3 here we present an analysis by role and specialty of symptomatic hcws and their household contacts (total n=207) that presented for sars-cov-2 pcr testing, during the early part of the uk covid-19 epidemic, between 17 march and 4 may 2020. during this period, the recommendations from public health england (phe) were for any symptomatic hcw to self-isolate for at least 7 days, or for any individual (including hcws) with symptomatic household contacts to self-quarantine for 14 days. 4 to give some additional context, the uk went into lockdown on 23 march 2020, 5 and all hcws were required to wear some form of surgical mask or better in clinical areas on 26 march 2020. 6 healthcare workers (mean age: 38.2 years, s.d. 9.2, range 17-60 years) also presented with symptomatic household family members (mean age: 23.8 years, s.d. 16.5, range 2-45 years) for swabbing. the rationale for this at the time was that if neither the family contacts, nor the hcw were sars-cov-2 pcr positive, then the hcw could return to work earlier without being a sars-cov-2 risk to other hcws or patients. during this 7 week period, a total of 152 symptomatic and/or self-isolating hcws (54 male: 98 female) with 55 home contacts (including spouses and children) presented for swabbing (a single combined nasal/throat swab). of the 152 hcws, 6 were black, 99 were asian, and 47 were of white ethnicity. the ausdiagnostics sars-cov-2 pcr assay was used for this testing. this kit has a manufacturer's reported sensitivity of 97-98% and a specificity of 99-100%, which has been confirmed elsewhere. 7 the results (fig. 1a) showed that the highest sars-cov-2 pcr positive rate (34.9%) was found in staff nurses compared to those (15-16%) for junior doctors, consultants and other support staff (24%, including healthcare assistants and those based in operating theatres, administration and estates). of note, in this cohort, the home contact sars-cov-2 pcr positivity rate (16.4%) was very similar to those in the non-staff nurse hcws, at this stage of the covid-19 epidemic in this cohort. illustrates the sars-cov-2 positivity rate in hcws working in different clinical specialties, with those working in respiratory wards showing the highest positive rate (38.1%), followed by other medical specialties (28.9%), particularly the renal dialysis wards (23.5%), the adult intensive care unit (icu) and anaesthetics (20.9%). the latter two specialties had a lot of overlap, with many anaesthetists also covering icu, so these hcw totals were combined and plotted together. these findings may not be entirely surprising as most suspected covid-19 patients would be referred initially to the respiratory teams for assessment, and staff nurses are likely to spend more time with the patients on a more frequent basis, taking and recording bedside observations, administering drugs, and being the first hcws onsite for any patient complications. in addition, we compared the sars-cov-2 pcr positivity rate against ethnicity for both the hcw and household contacts combined (fig. 1c) . this showed a high 62.5% (n=8) positivity rate for black individuals, though there were very few cases; and a similar 21.2% (n=137) and 21.0% (n=62) sars-cov-2 pcr positivity rate for asian and white individuals, respectively. these numbers are small and were obtained from the early part of the covid-19 epidemic in the uk so it is not possible to recognise any specifically higher incidence of sars-cov-2 infection in any of these bame (black, asian, minority ethnic) groups. 8 we then compared the sars-cov-2 pcr positivity rates against various demographic parameters, ethnicity and symptom patterns in both the hcws and household contacts (n=47, table 1 ). this showed that most of these positive cases were female (n=29, 61.7%), asian (n=29, 61.7%), and aged 35 years or above (n=31, 66.0%), with at least 1 systemic symptom (i.e. any of: fever, headache, myalgia or fatigue; n=37, 78.7%); and at least 1 respiratory symptom (i.e. any of: cough, sore throat, shortness of breath or chest tightness/pain, n=41, 87.2%). the mean duration from illness onset to specimen collection in these 47 sars-cov-2 pcr positive cases was 6.13 (s.d. 5.28) days. an additional analysis was performed on the pcr positive cases where their sample ct (cycle threshold) values were available (n=39), using the same parameter categories as table 1 . this compared the ct value (a relative indicator of viral load) against presenting symptom patterns. after adjusting for age, ethnicity, sex and time from symptom onset to specimen collection, it was found that the number of respiratory symptoms were positively associated with greater ct values (i.e. lower viral loads, p< 0.01), while an increasing number of systemic symptoms was associated significantly with smaller ct values (i.e. higher viral loads, p< 0.01) ( table s1 ). the mean duration from illness onset to specimen collection in these 39 sars-cov-2 pcr positive cases was 5.67 (s.d. 4 .58) days. this suggests that in this cohort, systemic symptoms may be more closely associated with the presence of higher viral loads, whereas respiratory symptoms may be more immune-mediated and can continue to persist during viral clearance. other studies have found varying associations between symptom patterns, illness severity and viral loads, 9, 10 so additional studies are needed to understand better these host-virus interactions. in summary, we have compared the sars-cov-2 pcr positivity rates in this hcw and household contact cohort, across different clinical roles and specialties, ethnic groups, and explored the correlation between their symptom patterns and swab viral loads. additional work is required to clarify further the relationships between these various parameters, such that dose-response monitoring can be applied in a rational manner when proven antiviral therapies for covid-19 eventually become available. figure 1a . comparing positive and negative sars-cov-2 pcr results in frontline staff by role. figure 1b . comparing positive and negative sars-cov-2 pcr results in frontline staff by clinical specialty. figure 1c . comparing positive and negative sars-cov-2 pcr results in hcws and household contacts by ethnicity. high sars-cov-2 antibody prevalence among healthcare workers exposed to covid-19 patients comparing hospitalised, community and staff covid-19 infection rates during the early phase of the evolving covid-19 epidemic clinical characteristics of 54 medical staff with covid-19: a retrospective study in a single center in wuhan stay at home guidance for households: current guidelines illustrated recommended ppe for healthcare workers by secondary care inpatient clinical setting, nhs and independent sector clinical evaluation of ausdiagnostics sars-cov-2 multiplex tandem pcr assay is ethnicity linked to incidence or outcomes of covid-19? sars-cov-2 viral load in upper respiratory specimens of infected patients quantitative detection and viral load analysis of sars-cov-2 in infected patients figure 1 a, b, c components: key: cord-303763-2784hze3 authors: choi, seong-ho; chung, jin-won; jeon, min-hyok; lee, mi suk title: risk factors for pandemic h1n1 2009 infection in healthcare personnel of four general hospitals date: 2011-05-01 journal: j infect doi: 10.1016/j.jinf.2011.04.009 sha: doc_id: 303763 cord_uid: 2784hze3 to characterize an outbreak of pandemic h1n1 2009 among healthcare personnel (hcp), we conducted a cross-sectional survey of hcp who had worked in four general hospitals during the outbreak. more than one-quarter of responding hcp (27.6%) had influenza-like illness (ili) during the outbreak. the estimated infection rate of pandemic h1n1 2009 was 9.1% in the study of hcp. independent risk factors for ili were female gender, <40 years of age, the presence of chronic diseases associated with influenza complications, having family members with ili or pandemic h1n1 2009, and working in influenza outpatient, influenza inpatient, non-influenza outpatient or emergency departments. during the outbreak of pandemic h1n1 2009, hcp frequently had ili or the influenza infection. the development of the influenza infection in hcp was associated with some of their baseline characteristics, occupational risk factors, and non-occupational ones during the outbreak. during an outbreak of pandemic h1n1 2009, healthcare personnel (hcp) were thought to be at substantial risk for acquiring influenza due to their frequent and close interactions with infected patients. 1e7 influenza infection in hcp may cause an hcp shortage and the spread of influenza to their patients and colleagues. it may also disrupt appropriate healthcare services during the outbreak and result in significant morbidity and mortality among patients with chronic cardiopulmonary disease, immunosuppression or those belonging to either age extreme. 8e10 to engineer effective means of controlling influenza transmission in a healthcare setting, we need more information regarding the characteristics of influenza outbreaks among hcp. recently, there have been several investigations describing clinical characteristics of hcp with pandemic h1n1 2009. 1e7 however, the majority of these studies were conducted in single centers or included only hcp who developed pandemic h1n1 2009. consequently, they may have a limited ability to explain the general features of the outbreaks or to investigate risk factors for the influenza infection among hcp. therefore, we performed a multicenter survey of all hcp who had been on duty during the outbreak. a multicenter survey was performed in four general hospitals in the republic of korea between july and august 2010. the study hospitals included three >500-bed tertiary care centers and a single 350-bed secondary care center. all centers were assigned as local influenza centers by the korean government during the outbreak. during the outbreak, all of these centers established isolated influenza facilities using tents or separate warehouses outside of the main hospital buildings for daytime outpatient visits. emergency departments undertook some outpatient care during holidays or weekday nights. several inpatient rooms were designated for admitted patients with pandemic h1n1 2009. however, these rooms were located in the general ward due to the absence of specialized isolation wards among the study hospitals. hcp who had been on duty during the outbreak were invited to participate in the survey. the anonymous, selfadministered questionnaires were distributed and collected by infection control personnel of the study hospitals. the questionnaire was developed to assess the following characteristics of hcp: age, gender, job type, facility type in which they principally worked during the outbreak, presence of underlying diseases associated with influenza complications, adherence to isolation precautions, presence of influenza-like illness (ili), the receipt of diagnostic tests for pandemic h1n1 2009, the results of the tests, the number of household members, the presence of ili or pandemic h1n1 2009 among household members, the associations of the family member illnesses with those of hcp within one week, and the order of illness onset in hcp families. job types were classified as physician, nurse, nursing assistant, technician, or jobs not directly related to patient care. facility types in which hcp had principally worked were classified as influenza outpatient department, influenza inpatient department, non-influenza outpatient department, non-influenza inpatient department, emergency department, or facilities not directly related to patient care. the levels of adherence to isolation precautions were rated according to a four-point scale (always-often-rarely-never). optimal adherence was determined for responses of "always" ili was considered to be present if hcp experienced an acute episode including at least one respiratory symptom, such as cough, sputum production, rhinorrhea or nasal obstruction, with documented or subjective fever. the presence of pandemic h1n1 2009 was noted if hcp had a positive result in either the rapid influenza antigen detection test (ridt) for type a influenza or in the reverse transcriptase polymerase chain reaction (rt-pcr). statistical analyses were performed using spss software (version 12.0; spss, chicago, il). for univariate analysis, a c 2 test or fisher's exact test was used. logistic regression analysis was performed to investigate risk factors for the development of ili among hcp. all p-values were twotailed, and p < 0.05 was considered statistically significant. the questionnaires were distributed to 4555 hcp, 3431 (75.3%; range, 72.5e85.5%) of whom responded during the study period. after excluding the questionnaires of 66 who did not work in the study hospitals during the outbreak, the results of 3365 (73.9%) were analyzed. the presence or absence of ili was reported in 2681 (79.7% of 3365 the majority of the 141 infected hcp were female (74.4%) and <40 years of age (76.9%). their job types were as follows: nurse (45.4%), physician (17.0%), nursing assistant (13.5%), technician (5.0%), and jobs not directly related to patient care (19.1%). they worked principally at the following facilities: non-influenza inpatient (23.0%), non-influenza outpatient (13.5%), influenza outpatient (8.5%), emergency (7.8%) and influenza inpatient departments (5.0%), and facilities not directly related to patient care (33.6%). the possible source of transmission was as follows: patients of the outpatient (45.7%) and inpatient departments (19.7%), those of the community (11.0%), hcp's colleagues (3.1%), and unknown sources (20.5%). among all responding hcp, the rates of adherence to each part of isolation precautions were as follows, according to a four-point scale: wearing a mask always (32.2%), often (38.8%), rarely (24.4%) and never (4.6%); wearing a gown always (6.9%), often (9.2%), rarely (14.1%) and never (69.8%); wearing gloves always (15.4%), often (22.4%), rarely (25.6%) and never (36.6%); wearing goggles always (0.5%), often (2.2%), rarely (4.3%) and never (93.0%); washing hands always (54.2%), often (35.5%), rarely (7.9%) and never (2.4%). during the outbreak, 82.1% of 3183 hcp were vaccinated for seasonal influenza and 86.6% of 3235 for pandemic h1n1 2009. among 2180 hcp who answered the major parts of the questionnaires and had data for ili, the crude and adjusted odds of clinical factors for the development of ili were calculated (tables 1 and 2 ). female gender, <40 years of age, the presence of chronic diseases associated with influenza complications, the presence of ili or pandemic h1n1 2009 among household members of hcp, and the history of contact with infected patients were more commonly observed in hcp with ili than in those without. physicians, nurses and nursing assistants were at a higher risk for ili than were hcp who had jobs not directly related to patient care. regarding facility types, hcp who worked in influenza outpatient, influenza inpatient, non-influenza outpatient and emergency departments were at a higher risk for ili than were those in facilities not directly related to patient care. the risk of developing ili was lower in hcp who always wore a gown and gloves than in those who did not. in the multivariate analysis, independent risk factors for ili were female gender, age <40 years, the presence of chronic diseases associated with influenza complications, and the presence of ili or pandemic h1n1 2009 among household members of hcp. hcp who worked in influenza outpatient, influenza inpatient, non-influenza outpatient and emergency departments had higher adjusted odds for ili than did those in facilities not directly related to patient care. of 3135 hcp who answered the questions of the presence of ili or pandemic h1n1 2009 in their households, 641 (20.4%) reported that at least one of their household members had ili or pandemic h1n1 2009. among those hcp whose household members exhibited ili or pandemic h1n1 2009, 77 (12.0% of 641) cases occurred within one week prior to or after their household members becoming ill. among these, 57 (74.0%) reported that ili or pandemic h1n1 2009 occurred first in the hcp and then in their household members. physicians and nurses were more common in these 57 than in the remaining 20 (12.3% vs. 5.3%; 45.6% vs. 36.8%). the remaining 20 subjects had jobs not directly related to patient care or worked in facilities not directly related to patient care more frequently than the 57 (31.6% vs. 19.3%; 60.0% vs. 34.8%). also, the former more frequently had affected household members 19 years of age compared to that of the latter (84.2% vs. 57.4%, p z 0.04) ( table 3 ). during the outbreak, 27.6% of the study hcp reported ili. the estimated infection rate of pandemic h1n1 2009 was 9.1%. among the clinical variables, baseline characteristics such as female gender, age <40 years, and chronic underlying diseases were associated with pandemic h1n1 2009 infection. the type of facilities in which hcp had principally worked was the most important occupational risk factor for the infection. non-occupational risk factor, such as having infected household members, was also associated with the development of the influenza infection among hcp. ilis have been reported to develop in 23e36% of hcp during the peak of the influenza season. 11, 12 our data showed that the frequency of ili was 27.6% among the study hcp, in agreement with the results of the previous studies. regarding the incidence of pandemic h1n1 2009 among hcp, there have been no reports except a few seroepidemiological studies. 2,3 one study from taiwan reported a seropositive rate of h1n1 antibodies to be 20% among hcp. 3 in one large singapore center designated for outbreak management, seroconversion was observed in 7% of hcp. 2 the direct comparison of the infection rates between the hospitals of the previous studies and our institutions may not be plausible due to the different study designs and case definitions. however, the estimated rate of this study (9.1%) simply suggest that influenza infection may be considerable among hcp during an influenza pandemic, with the data from several large centers rather than those of single centers. in addition, it suggests that a large number of infected hcp might be unrecognized or untested during the pandemic, because only 4.2% (141/3365) was reported to have the infection in the study hospitals. therefore, these data show that there needs to be a higher level of suspicion for influenza infection and the easy availability of diagnostic tests during an influenza pandemic among hcp. hcp of a younger age or who have underlying chronic diseases were more frequently associated with ili than those without. this association may be due to the relative lack of preexisting immunity to this novel influenza virus among younger age groups, 13 and the lack of local or general immunity associated with chronic diseases. however, the higher susceptibility in female hcp in this study may not be easily understood. one possible explanation is that this is due to women being caregivers in the household setting and, therefore, more likely to be infected from the household. unfortunately, we did not make inquiries about the presence or absence of children for all female table 3 . a recent review has reported a higher incidence of pandemic h1n1 2009 infection in young women than young men of compatible age, although the exact causes have not been suggested. 14 more data may be needed for higher susceptibility to influenza infection in female hcp. hcp who worked at influenza outpatient and emergency departments had the highest risk for ili. it has been previously suggested that much of the risk for infection to hcp during an outbreak likely exists in an outpatient setting. 1 additionally, in korean general hospitals, a huge number of outpatient visits took place only for the confirmatory diagnostic tests for pandemic h1n1 2009, maybe from the pervasive anxiety over fatality potential of the infection. at least in part, it may increase the risk for pandemic h1n1 2009 among hcp working in outpatient settings rather than those in inpatient ones. hcp working in the non-influenza outpatient department were also at risk for ili. this may be due to the variable presentation of influenza infection. 15 a substantial number of patients with atypical or mild symptoms, especially pediatric patients, frequently visited the non-influenza outpatient department. the isolation precautions were not shown to markedly reduce the transmission of pandemic h1n1 2009. although a recall bias might be the cause of this finding, overall low levels of compliance were likely to be another problem. whereas the compliance rates for hand washing and frequent mask wearing (often-always) were 89.7% and 71.0%, compliance rates for frequently wearing a gown, goggles and gloves were 16.1%, 2.7%, and 37.8% of hcp, respectively. the center for disease control and prevention recommended the use of standard and contact precautions with eye protection during the pandemic. 1 besides hand washing and mask wearing, use of other personal protective equipment should be taught and encouraged. this study showed that household members may transmit pandemic h1n1 2009 to hcp or vice versa. hcp whose household members had ili or pandemic h1n1 2009 were more frequently infected than were those whose household members were ili or pandemic h1n1 2009 negative. a seroepidemiological study from singapore reported that having a child with ili was a non-occupational risk factor for hcp seroconversion. however, whether transmission of the infection occurred from the child to hcp or vice versa was not clear in the study. 2 our data suggest that hcp who are involved directly in patient care may transmit pandemic h1n1 2009 to their household members, whereas those who are not involved in patient care are more likely to transmit the infection from their homes to their hospitals. these findings may indicate that variable infection control measures should be implemented for hcp who are actively involved in influenza patient care in order to prevent transmission from hospital to household. additionally, control of influenza among children or adolescents may lead to the reduction of influenza infection in hospitals as well as in the community. this study has several important limitations. first, the proportion of hcp not reporting the receipt of diagnostic tests was substantial. therefore, the total number of pandemic h1n1 2009 among hcp may be underreported. this, in turn, might limit the power of the data to identify meaningful risk factors. instead, we used ili to identify risk factors for influenza infection in hcp. however, the ili case definition of self-reported fever with respiratory illness was not highly specific for the diagnosis of pandemic h1n1 2009. any non-pandemic h1n1 illness classified as ili might result in a bias in this study. the laboratory surveillance data from the korean government reported that rhinovirus, adenovirus, coronavirus and respiratory syncytial virus cocirculated in the community during the influenza pandemic. 16 however, even with this bias prone to nullify any remarkable differences in infected hcp, the identified risk factors for ili of this study were similar to those for pandemic h1n1 2009 identified in other studies. 2, 3, 5 second, the impact of recall bias may be considerable due to the study design, especially on the source of infection, the compliance to the isolation precautions, and the direction of transmission between hcp and their family members. third, working at pediatric wards or clinics may be an important risk factor for the infection, but the data could not be collected mainly due to unseparated wards or clinics in the study hospitals. fourth, we presented the crude odds of seasonal or pandemic vaccination for the development of ili in table 1 . however, influenza vaccination only started in late october 2009 (just before the peak of the pandemic) in korea and we could not collect information on the onset of ili and the date of vaccination among hcp from the study design. consequently, we could not determine the effectiveness of influenza vaccination in this study. the receipt of vaccination was excluded in the multivariate analysis. fifth, we did not present the variation in the characteristics of the outbeak by the study hospital. we calculated the infection rate of each hospital as described in fig. 1 . the infection rate was as follows: hospital a, 14.2%; hospital b, 9.9%; hospital c, 6.9%; hospital d, 3.4%. we compared the characteristics of hcp in the study hospitals with lower infection rates (hospital a and b), with those in the study hospitals with higher infection rates (hospital c and d). however, the comparison did not show any differences in the preventive measures or systems unique to each study hospital, except some differences in demographic data of participating hcp between the study hospitals. in addition, the estimated rate may be less reliable due to the quartered number of the study hcp. therefore, these data were not presented here. in conclusion, ili or pandemic h1n1 2009 was frequently observed among hcp. the risk of the influenza infection was associated with variable characteristics of hcp, such as baseline demographics, occupational or non-occupational risk factors. no authors have any conflicts of interest to report. center for disease control and prevention. novel influenza a (h1n1) virus infections among health-care personnel e united states, aprilemay risk factors for pandemic (h1n1) 2009 virus seroconversion among hospital staff seroprevalence of antibodies to pandemic (h1n1) 2009 influenza virus among hospital staff in a medical center in taiwan prevention of nosocomial transmission of swine-origin pandemic influenza virus a/h1n1 by infection control bundle which health care workers were most affected during the spring 2009 h1n1 pandemic? h1n1 2009 influenza among healthcare workers in a tertiary care hospital in thailand transmission of pandemic (h1n1) 2009 influenza to healthcare personnel in the united states influenza in the acute hospital setting novel (pandemic) influenza a h1n1 in healthcare facilities: implications for prevention and control healthcare personnel and nosocomial transmission of pandemic 2009 influenza preventing healthcare workers from acquiring influenza use of, effectiveness of, and attitudes regarding influenza vaccine among house staff center for disease control and prevention. serum crossreactive antibody response to a novel influenza a (h1n1) virus after vaccination with seasonal influenza vaccination the impact of sex, gender, and pregnancy on 2009 h1n1 disease seasonal influenza in adults and childrendiagnosis, treatment, chemoprophylaxis, and institutional outbreak management: clinical practice guidelines of the infectious diseases society of america korean center for disease control and prevention. novel influenza a (h1n1) homepage we thank our colleagues in each study hospital who actively participated in the clinical and infection control practices during the outbreak and also in this study during the postoubreak period, especially mi ae choi, r.n., department of infection control, chung-ang university yongsan hospital; ji youn choi, r.n., and in soon choi, r.n., department of infection control, chung-ang university hospital; hee kyung chun, r.n., and mee la kim, r.n., department of infection control, kyung hee university medical center; eun ju lee, r.n., and sun mi park, r.n., department of infection control, soonchunhyang university cheonan hospital. key: cord-291960-1is0rv6c authors: piñana, josé luis; giménez, estela; gómez, maría dolores; pérez, ariadna; gonzález, eva maría; vinuesa, víctor; hernández-boluda, juan carlos; montoro, juan; salavert, miguel; tormo, mar; amat, paula; moles, paula; carretero, carlos; balaguer-roselló, aitana; sanz, jaime; sanz, guillermo; solano, carlos; navarro, david title: pulmonary cytomegalovirus (cmv) dna shedding in allogeneic hematopoietic stem cell transplant recipients: implications for the diagnosis of cmv pneumonia date: 2019-02-21 journal: j infect doi: 10.1016/j.jinf.2019.02.009 sha: doc_id: 291960 cord_uid: 1is0rv6c objectives: to date no definitive cut-off value for cytomegalovirus (cmv) dna load in bronchoalveolar lavage (bal) fluid specimens has been established to discriminate between cmv pneumonia and pulmonary cmv dna shedding in allogeneic hematopoietic stem cell transplant (allo-hsct) recipients. methods: the current retrospective study is aimed at assessing the range of cmv dna loads quantified in bal fluid specimens from allo-hsct patients with pneumonia in which different microorganisms were causally involved. results: a total of 144 bal specimens from 123 patients were included. cmv dna was detected in 56 out of 144 bal fluid specimens and the median cmv dna load from patients in whom cmv pneumonia was unlikely or could be tentatively ruled out was 1210 (31–68, 920) iu/ml. the frequency of cmv dna detection and median cmv dna loads were comparable, irrespective of the attributable cause of pneumonia. detection of cmv dna loads in bal fluid specimens >500 iu/ml was independently associated with pneumonia-attributable mortality. conclusions: the current study highlights the difficulty in establishing universal cmv dna load thresholds in bal fluid specimens for distinguishing between cmv pneumonia and pulmonary cmv dna shedding, and suggests that the presence of cmv dna in bal fluid specimens beyond a certain level may have a deleterious impact on patient outcome. the implementation of effective prevention strategies has significantly reduced the incidence of cytomegalovirus (cmv) pneumonia after allogeneic hematopoietic stem transplantation (allo-hsct) 1 ; nevertheless, this clinical entity persists as a major clinical problem owing to poor survival, despite timely initiation of targeted antiviral therapy. 1, 2 currently, diagnosis of cmv pneumonia still remains a challenge. proven cmv pneumonia can only be diagnosed using virological, histopathological or immunochemistry methods on biopsied lung tissue 1 ; this specimen type, however, is rarely obtained due to potential life-threatening complications. traditionally, culture-based bronchoalveolar lavage (bal) testing has been the mainstay for suggesting cmv involvement ex vivo , 3 although detection of viable cmv in bal fluids only points to a probable causality. 4 may be present in the absence of probable or proven disease (pulmonary cmv shedding). [5] [6] [7] [8] recent studies suggested that quantitation of cmv dna in bal fluids may permit discrimination between these two conditions 9 -11 ; specifically, a cut-off cmv dna level > 500 iu/ml was proposed to serve that purpose, this displaying a positive predictive value of roughly 50% for probable cmv pneumonia using current prevalence rates. 10 nevertheless, validation of diagnostic viral load threshold across centers using different real-time pcr assays for cmv dna quantitation and procedures for bal obtention seems of paramount relevance. this task is hampered by the very low incidence of cmv pneumonia nowadays ( < 2%) 3, 12 and the difficulty in establishing an incontrovertible diagnosis, even when lung tissue specimens are available for testing. 13 -16 exploratory studies gathering information on the range of cmv dna loads measured in bal specimens from allo-hsct patients with pneumonia in whom cmv causality is unlikely or can be reasonably ruled out may provide useful information and are thus warranted. here, we report on our experience on this matter. the study cohort consisted of 123 consecutive patients who received an allo-hsct at hospital clínico universitario-hcu-( n = 61) or at hospital universitario politécnico "la fe" -hlf-( n = 62) and underwent diagnostic bronchoscopy. clinical and microbiological data of patients and bal fluid specimens ( n = 144; hcu, n = 80; hlf, n = 64) submitted to the respective microbiology service between may 2012 and may 2017, respectively, were retrospectively reviewed. as per protocol, quantitative cmv pcr testing has been routinely performed on bal specimens at both centers since 2012. all patients had clinical and radiography signs of pneumonia at the time of sampling. bronchoscopy was performed using standard procedures according to international consensus guidelines. 17 the first bal fluid specimen per pneumonia event was taken into consideration for analysis purposes. this study was approved by the hospital clínico, fundación incliva ethics committee and informed consent was obtained from all patients. a preemptive antiviral therapy approach was used at hcu to prevent cmv end-organ disease. 18 cmv dna in plasma was quantified using the realti me cmv pcr assay (abbott molecular, des plaines, il, usa), which exhibits a limit of detection of approximately 31 iu/ml. 19 patients were preemptively treated with oral valganciclovir, i.v. ganciclovir or i.v. foscarnet upon detection of cmv dna levels exceeding 1500 iu/ml or a cmv dna doubling time ≤ 2 days, as previously reported. 18, 20 surveillance for cmv dna detection and quantitation was conducted at least once a week within the first 100 days after allo-hsct and this was extended beyond day 100 in patients at risk for recurrent episodes of cmv dnaemia. 21 in turn, a universal prophylaxis strategy was deployed at hlf. 22 briefly, hla-matched related allo-hsct recipients were given oral valganciclovir (900 mg/day, three times a week) through day 90 after transplantation. unrelated allo-hsct recipients were treated with oral valganciclovir (900 mg/day) through day 180 after transplantation. at this center, plasma cmv dna load was quantified using the cmv r-gene® (biomerieux, l'etoile, paris, france), 23 which displays a limit of detection of 150 iu/ml, prior dna extraction using the virus/pathogens mini kit (qiagen, valencia, ca, usa) on the qiasymphony dsp platform (qiagen). plasma cmv dna monitoring was performed once a week within the first 100 days, fortnightly from day 100 to day 180, and every 2-4 weeks thereafter through day 365 after transplantation. detection of any level of cmv dna in plasma prompted the administration of antiviral therapy with (val)ganciclovir or foscarnet at the doses specified above. anti-cmv therapy was given at the physician discretion when patients with cmv dna detected in bal specimens had concurrent cmv dnaemia with cmv dna levels below the threshold for preemptive antiviral therapy. plasma specimens obtained within 72 h of bal sampling were available from all patients for cmv dna quantitation. all bal fluid specimens underwent cmv pcr testing within 24-48 h. upon reception. samples were kept at 4 °c until processed. bal fluid specimens obtained from different locations (when available) were collected in sterile containers, pooled and vortexed for 30 s after the addition of sterile glass beads. two-hundred μl of each undiluted bal fluid samples were subjected to nucleic acid extraction using the m20 0 0 sp system (abbott diagnostics) or the qiaamp dna blood mini kit (qiagen) on either the qia symphony or the ez-1 platforms (qiagen). cmv dna quantitation was performed using the realti me cmv pcr assay (abbott molecular) at hcu or the cmv r-gene® assay (biomerieux) at hlf. commutability of the cmv who international standard 24 for cmv dna quantitation in bal specimens was assessed. the cmv who standard was reconstituted in 1 ml of deionized water as recommended by the manufacturer. 24 a pool of bal specimens free of cmv dna, as determined by the abbott pcr assay was made and spiked with the who international standard with a predefined cmv dna concentration to achieve nominal values of 10 2 10 3 , 10 4 and 10 5 iu/ml. the reference materials were assayed in triplicate in a single run. cmv dna levels measured in bal fluid specimens closely matched those expected (the mean standard deviation for both pcr assays was < 0.2 log 10 iu/ml for each cmv who standard concentration tested). gram stain, fungal stain and acid-fast bacilli stain were routinely performed. cytology examination (bal fluid cytospins) was performed systematically in patients attended at hcu, and nonroutinely at hlf. in all, bal fluid cytospin data were available from 83 patients. in addition, bal fluid specimens were examined for the presence of respiratory viruses (rvs) using either the luminex xtag rvp fast assay (luminex molecular diagnostics, austin, tx,usa) at hcu, which allows detection of 19 rvs, or the clart® pneumovir assay (genomica, coslada, spain)-at both centers-that makes it possible to detect simultaneously 17 rvs, as previously reported. 25 quantitative cultures of bal specimens for bacterial isolation were performed on conventional media as recommended 26 ; bacterial loads > 10 4 cfu/ml were deemed to be clinically relevant. 26 bal specimens were cultured on bcye-alpha agar, bd (becston dickinson) mgit® (mycobacteria growth indicator tube)/lowenstein-jensen agar slants and sabouraud agar for recovery of legionella pneumophila, mycobacterium spp., and fungal organisms, respectively. the platelia tm aspergillus ag kit (bio-rad, hercules, ca, usa) was used for quantitation of aspergillus spp. galactomannan in bal fluid and serum specimens. calcofluor white, blue toluidine or direct immunofluorescence staining procedures were used for detection of pneumocystis jiroveci. 26 lung biopsy tissue specimens were not obtained. likewise, neither shell vial and conventional viral cultures for cmv detection or recovery nor direct fluorescent antibody testing for cmv detection were performed. [39] . acyclovir/valacyclovir prophylaxis against herpes simplex and varicella zoster viruses was given to all patients as previously described. 18,20 -22 all patients received standard antibacterial and antifungal prophylaxis. 18,20 -22 definitions cmv dnaemia and pulmonary cmv dna shedding were defined as the detection of cmv dna (at any level) in one or more plasma or bal fluid specimens, respectively. proven cmv pneumonia categorization required histopathological evidence (i.e., viral inclusions and immunohistochemical staining) in biopsy (autopsy) lung tissue. cmv pneumonia diagnosis was reasonably excluded if cmv-induced cytopathogenetic effect was not observed in post-mortem (autopsy) lung tissue (data were available from 23 patients) or bal fluid cytospins (data available from 83 patients), there was a lack of chest x ray and computed tomography (ct) evidence consistent with cmv pneumonia (ie. reticulonodular infiltrates, the presence of bilateral ground-glass opacities, air-space opacities, small centrilobular nodules < 1 cm, and absence of larger nodules -see 27 , for review -) and alternative diagnoses that could account for the signs and symptoms, particularly when the presence of other significant bacterial, virus or fungal pathogens was demonstrated. the clinical and radiographic response to targeted antimicrobial therapy (for bacterial, fungal and non-cmv viral infections) was also considered to be against the involvement of cmv. the probable cmv pneumonia category, according to recent criteria 4 was not considered herein, since culture-based bal fluid testing was not performed and no diagnostic cmv dna cut-off level has been definitely established. diagnosis of proven, probable, and possible invasive fungal infection and pneumocystis jiroveci pneumonia was achieved following consensus criteria. 28, 29 acute graft versus host disease (agvhd) was diagnosed and graded according to standard criteria. 30 frequencies were compared using the χ 2 test (fisher exact test) for categorical variables. differences between medians were compared using the mann-whitney u test (for two independent variables) or the kruskal-wallis test (for more than two independent variables). cumulative incidence plots of mortality from pneumonia were generated with the graphpad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test. cox proportional hazards models were used to evaluate unadjusted and adjusted hazard ratios (ahrs) for mortality attributable to pneumonia, as previously reported. 21 for multivariate analyses, only variables with parameter estimates showing a p value ≤ 0.10 in the univariate analyses were included. two-sided exact p values are reported and p values ≤ 0.05 were considered statistically significant. the data were analyzed with the spss (version 20.0) statistical package. table 1 shows relevant demographic and clinical characteristics of the 123 patients in the cohort. bal fluid samples were obtained at a median of 172.5 days after allo-hsct (range 3 days to five years). specific details on the microbiological yield of bal fluid specimens are shown in table 2 . proven cmv pneumonia was diagnosed in 2 patients (1.6%) on the basis of histopathological and immunohistochemistry findings in lung autopsy tissue. in the remaining 142 episodes, pneumonia was attributable to bacteria in 18 cases (12.5%), and to one or more viruses in 37 (25.7%). there were 22 invasive aspergillosis infection cases (13.3%), these being categorized as possible ( n = 11), probable ( n = 7), or proven ( n = 4). mixed infections were documented in 42 patients (29.2%). the individual pathogenetic contribution of each detected or cultured microbial agent to pneumonia was not attempted to be settled. twenty-three pneumonia cases (15.9%) were deemed not to have an infectious cause on the basis of clinical and radiographic data and the lack of detection of any potential pathogenic microorganism (other than cmv) in bal fluid specimens. cmv dna was detected in 56 out of 144 bal fluid specimens (38.9%): 49 from patients with pneumonia episodes in which potential respiratory pathogens (one or more) were either cultured or detected, and 7 from cases deemed not to have an infectious origin. in these latter cases, the involvement of cmv as the causative agent was reasonably ruled out on clinical and radiographic grounds. cmv dna was present in bal fluid specimens from the two patients with proven cmv pneumonia; no other microorganisms were concurrently detected/cultured in these two specimens. the frequency of cmv dna detection was comparable ( p = 0.40), regardless of the established (or presumed) etiology of pneumonia ( table 3 ). in 41 episodes, cmv dna bal detection occurred in the face of an ongoing episode of cmv dnaemia (including two episodes that occurred in the setting of autopsy-proven cmv pneumonitis), and isolately in the remaining 15 episodes. clinical and laboratory characteristics of patients ( n = 33) in whom cmv dna was co-detected in bal and plasma specimens in the absence (presumed) of cmv pneumonitis ( n = 39 episodes) merit special attention, as cmv lung disease usually occurs concomitantly with cmv dnaemia. 3, 4 the data are shown in supplementary table 1 . cmv pneumonitis was deemed to be unlikely in these patients owing to one or more of the following: (i) lack of typical findings in cts (in all episodes); (ii) negative bal cytospin results (in 25 episodes); (iii) negative lung histopathology at autopsy (in table 2 pneumonia attributable etiology and microbiological findings in bronchoalveolar lavage fluid specimens. no. (%) c according to [28] . d according to [4] . e including idiopathic pneumonia syndrome, bronchiolitis obliterans and cryptogenic organizing pneumonia among other causes. 7 out of 16 patients who died); (iv) survival of patients who did not undergo specific anti-cmv treatment courses with appropriate doses for cmv pneumonitis (23 episodes); of note, no patient in this series was treated with anti-cmv drugs for cmv pneumonitis as recommended 3 ; (v) documentation of the presence of bacteria, viruses (other than cmv) or fungal pathogens known to cause pneumonia (in 25 episodes); (vi) clinical response (survival) to targeted anti-bacterial, anti-viral (influenza virus) or anti-fungal therapy (in 17 episodes). nevertheless, in particular, there were four episodes in which the causative involvement of cmv raised doubts (second episode in patient 31, and episodes in patients 35, 37 and 51-supplementary table 1. despite the fact that ct scans were not suggestive of cmv pneumonitis, no alternative microbial etiology was documented and all these patients died (pneumonia was the attributable cause of death). in addition, cmv dna was detected at high levels in both bal (ranging from 1382 to 40,048 iu/ml) and plasma (ranging from 3510 to 54,540 iu/ml) specimens. one of these patients (patient 51) had negative bal cytospin results. in turn, cmv dnaemia was documented in 33 cases in the absence of cmv dna detection in bal fluid samples. recipient cmv seropositivity and treatment with corticosteroids were associated with detection of cmv dna in bal fluid specimens in univariate analyses (supplementary table 2 ). overall, the median cmv dna load in bal fluid specimens from patients categorized as not having cmv pneumonia was 1210 iu/ml (range, 31-68,920 iu/ml). this magnitude was greater ( p = 0.001) in specimens analyzed at hlf (median, 1938 iu/ml; range, 180-68,920 iu/ml) than in those being tested at hcu (median, 345 iu/ml; range, 21-11,263 iu/ml) (supplementary fig. 1) . the cmv dna load was > 500 iu/ml in 32 pneumonia episodes and < 500 iu/ml in the remaining 22 ( table 4 ). the cmv dna load in bal fluid in the two proven cmv pneumonia cases was 1453 and 12,998 iu/ml. a trend towards higher cmv dna loads in bal fluid specimens was observed ( p = 0.09) in episodes in which cmv dnaemia was detected concurrently (1382 iu/ml; range, 31-68,920 iu/ml vs. 289 iu/ml; range, 66-12,839 iu/ml in its absence). cmv dna loads in bal fluid specimens were comparable ( p = 0.62), irrespective of the etiology (attributable) of pneumonia ( fig. 1 , and supplementary table 3) . overall, there was a trend towards an inverse correlation between the level of immunosuppression, as inferred by the immunodeficiency index (isi) -see footnotes in table 1 -, and the cmv dna load quantified in bal specimens (rho, −0.15; p = 0.08). overall, 72 patients (50%) were under anti-cmv therapy at the time of bal sampling (prophylaxis, n = 14; preemptive therapy, n = 58). among those with cmv dna detectable in bal fluids, 37 (66%) were receiving anti-cmv therapy (preemptive therapy, n = 35, and antiviral prophylaxis, n = 2). the cmv dna load in bal was significantly higher in patients who were under antiviral therapy (median, 1585 iu/ml; range, 31-68,920 iu/ml vs. median, 345 iu/ml; range, 39-7159 iu/ml; p = 0.04 in non-treated patients). two out of the 19 patients with cmv dna detected in bal fluids and no concurrent cmv dnaemia were treated with i.v. ganciclovir. forty-six patients (37%) died within 60 days after bal sampling, including one patient with proven cmv pneumonia. the cause of death was deemed to be related to the pneumonia episode in 40 patients (87%). the cumulative incidence of pneumoniaattributable mortality was comparable across the different etiological categories ( fig. 2 ) . we investigated whether the presence of cmv dna in bal fluid specimens was associated with increased mortality, and found this not to be the case. nevertheless, roc curve analysis enabled to determine a cut-off cmv dna level in bal fluid identifying those patients with increased mortality; this threshold was 500 iu/ml (sensitivity: 84.2%; 95% ci 62.4-94.5%; specificity, 53.1%; 95% ci, 36.4 −69.1%) (supplementary fig. 2 ). adjusted cox models including in addition to this variable a number of others that could have had an impact on pneumoniaattributable mortality identified the detection of a cmv dna load in bal fluid specimens at levels > 500 iu/ml, treatment with corticosteroids (at any dose) and lymphocyte counts < 0.7 × 10 9 /l, the [30] . b the cut-off was established by roc analysis (not shown). cumulative incidence of mortality attributable to pneumonia complications by day 60 after bronchoalveolar lavage fluid testing according to the etiology. cumulative incidence plots were generated with the graphpad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test (the p value is shown). latter two at the time of bal sampling, as the parameters that were independently associated with pneumonia-attributable mortality ( table 5 ) . moreover, as shown in fig. 3 , these factors appeared to exert a synergistic impact on mortality. the definitive abandonment of traditional culture-based procedures for cmv testing in most laboratories and the non-availability of lung biopsy specimens for histopathological and immunohistochemistry examination make the diagnosis of proven or probable cmv pneumonia 4 elusive nowadays. moreover, no cut-off value for cmv dna load in bal fluid specimens discriminating between cmv pneumonia and pulmonary cmv dna shedding in allo-hsct recipients has been established at the present time. 4 the assessment of the performance of quantitative cmv dna pcr testing for the diagnosis of cmv pneumonia faces several difficulties including: (i) the low incidence of this clinical event, (ii) the lack of a normalized procedure for cmv dna pcr testing on bal fluids, (iii) the non-negligible possibility of miscategorization of pneumonia cases as either being causally linked or unrelated to cmv when using bal fluid specimens, or even lung tissue material, for cmv diagnosis, 8 (iv) the persistence of a large variability of cmv dna loads provided by different real-time pcr assays, 31 -33 despite their calibration to the who international standard for cmv dna 24 and, as already mentioned, (v) the relegation of virological procedures for cmv detection in clinical specimens. inevitably, all these drawbacks were encountered in this study, so that we aimed not to establish a diagnostic cut-off, but rather to assess the range of cmv dna loads quantifiable in bal fluid specimens from patients in whom the causal involvement of cmv was highly unlikely or could be reasonably discarded; this, having in mind that we surely incurred cmv pneumonia infradiagnosis. nevertheless, we perceived this limitation as not being an insoslayable obstacle to our purpose fig. 3 . cumulative incidence of mortality attributable to pneumonia complications by day 60 after bronchoalveolar lavage fluid testing according to the presence of one or more factors associated with mortality in multivariate cox models (cmv dna load in bal fluid 500 iu/ml, treatment with corticosteroids and total lymphocyte counts < 0.7 × 10 9 /l). cumulative incidence plots were generated with the graph-pad prism software (la jolla, ca, usa) and the curves were compared by using the gehan-wilcoxon test (the p values for comparisons are shown. as: (i) cmv is unlikely to be the etiological agent of more than 10% of pneumonia cases among allo-hsct recipients who undergo routine bronchoscopy, 10 and (ii) survival of patients with cmv pneumonia who do not undergo specific anti-cmv treatment with appropriate doses of (val)ganciclovir or foscarnet is highly unlikely, provided the high rate of cmv pneumonia-associated mortality. 4 we retrospectively reviewed clinical and microbiological data from patients who underwent routine quantitative cmv pcr testing on bal fluid specimens at two transplant centers in our city. we deliberately chose not to include archived bal specimens in our series because of the lack of data on the impact of cryopreservation on cmv dna load quantitation in this sample type. several findings arose from the present study. first, we confirmed previous observations 8 -10 indicating that detection of cmv dna in bal fluid specimens using highly-sensitive pcr assays is a very common finding in allo-hsct patients with pneumonia, irrespective of the definitive etiological diagnosis. in our series, cmv dna was detected in more than one third of bal fluid samples, the frequency of detection varying between 30% in patients seemingly with not having an infectious pneumonia and 45% in patients with mixed infections. in our series, recipient cmv seropositivity and treatment with corticosteroids were associated with detection of cmv in bal fluid specimens. of interest, the two patients with proven cmv pneumonia had cmv dna detectable in bal fluid. second, we found a wide range of cmv dna loads measured in bal fluid specimens from patients with pneumonia in whom cmv causality was unlikely or reasonably ruled out attending to clinical (including therapeutic response to nonanti-cmv drugs), radiographic, lung autopsy histopathology or bal fluid cytology (in some patients) and microbiological criteria. interestingly, median cmv dna loads were comparable irrespective of the nature and the number of co-detected microorganisms (at both participating centers), and were overall higher in the presence of concurrent dnaemia. as for the latter observation, in agreement with boeckh et al., 10 we found this not to be due to pulmonary hemorrhage (not shown). overall, cmv dna loads in bal fluid specimens processed at hlf were of greater magnitude than those analyzed at hcu. since cmv dna loads produced by the argene pcr assay are slightly lower than those measured by the abbott pcr assay (not shown), differences in the net state of immunosuppression of patients at the time of bal sampling across centers, as reflected by the immunodeficiency score index (higher for hlf patients), may account for this observation. in fact, a trend towards an inverse correlation was found between the isi score and the cmv dna load quantified in bal specimens. in a recent study, 10 a cmv dna load cut-off of 500 iu/ml in bal fluid was found to have a positive predictive value of ∼50% for the presence of probable cmv pneumonia (considering a prevalence of this event of 10% among patients at risk and undergoing bal testing). tan et al., 8 in contrast, found cmv dna levels in bal fluid samples to have a limited value to distinguish between cmv and non-cmv pneumonia cases. it is pertinent to mention here that the above threshold is between one and two log 10 lower than those tentatively proposed for diagnosing cmv pneumonia in lung transplant recipients. 34 -38 interestingly, control patients with non-cmv pneumonia in the boeckh study 10 showed a median cmv dna load of 0 log 10 iu/ml (iqr, 0-1.6 iu/ml), with cmv dna levels between 100 and 500 iu/ml in roughly 64% of cases and > 500 iu/ml in 36% of them. here, the opposite was true, with nearly 60% of bal fluid samples from patients with non-proven cmv pneumonia having cmv viral loads > 500 iu/ml, of which 75% had > 1000 iu/ml. it is worth highlighting that these figures were comparable at both participating centers, despite the above-referred differences in cmv dna loads provided by pcr assays used at each center. to gauge the potential relevance of these data, it must be taken into consideration that in nearly 70% of pneumonia episodes in our cohort, bal sampling was performed while patients were under anti-cmv therapy ( > 3 days), whereas in the aforementioned study, 10 only 24% of patients with cmv pneumonia and 35% of patients with non-cmv pneumonia had been treated with antivirals for at least two days. despite this fact, higher cmv dna loads in bal fluid specimens were quantified in the current study, likely reflecting major differences regarding the dnaemia cut-off triggering the inception of antiviral therapy between these studies (much higher in the current cohort). in fact, in this series, the median cmv dna load in bal was significantly higher in patients who were under antiviral therapy than in non-treated patients. as stated above, the limited number of proven cmv pneumonia cases in our series precluded any attempt to establish a diagnostic cmv dna load cutoff; nevertheless, in light of the data presented herein, a threshold value of 500 iu/ml is unlikely to be discriminative between cmv pneumonia and pulmonary cmv dna shedding in our setting. in this sense, we fully support the idea that the magnitude of such a diagnostic cut-off is likely to vary depending upon the patient's characteristics, the bal procedure and processing, the assay used for cmv dna quantitation and the severity of cmv pneumonia at the time of sampling. 4 our data should be interpreted with caution as the exclusion of cmv as the probable causative agent can be judged as dubious in some cases provided that no virological methods were used to investigate the presence of cmv in bal fluid specimens. in particular, there were 4 episodes occurring in 4 patients who died, in whom cmv dna was detected at high levels in both bal and plasma specimens ( > 10 0 0 iu/ml) and no alternative microbial etiology was documented. nevertheless, the conclusions drawn on the basis of the overall dataset stood when cases in which little or no doubt existed on the lack of involvement of cmv (i.e. bacterial pneumonia, tuberculosis.) were analyzed separately (representative examples are shown in supplementary table 4) . cmv is a highly pro-inflammatory and immunosuppressive virus; as such it may act as a synergistic co-pathogen in the absence of documented cmv-induced cytopathogenicity, and may have a relevant impact on patient outcome. 3 in this sense, we found that the presence of cmv dna in bal fluid specimens at levels > 500 iu/ml, in addition to receipt of corticosteroids and low lymphocyte counts at the time of bal sampling, was associated with increased pneumonia-attributable mortality in cox multivariate models. the relative scarce number of pneumonia cases in which bal specimens had cmv dna loads > 500 iu/ml did not allow to investigate whether this apparent effect exhibited a dose-response pattern. again, this finding must be interpreted cautiously, as in order to avoid overfitting, cox models were not adjusted to a number of factors that may have had an impact on mortality (i.e., adequacy of antimicrobial treatment, severity of pneumonia, among others). the limited size of the current cohort also precluded any meaningful statistical analysis evaluating the impact of cmv dna load in bal at each center, separately. further studies are urgently needed to validate this observation, since this subset of patients may benefit from short courses of anti-cmv therapy. 10 limitations of the current study, in addition to the ones highlighted above, are its retrospective nature, the potential biased selection of patients requiring bronchoscopy for the etiological diagnosis of pneumonia, the lack of normalization of cmv dna loads in bal fluids to cellular dna content (although this was reported to be expendable in a previous study 10 ) and the use of different dna extraction platforms and real-time pcr assays for cmv dna quantitation. nevertheless, this study has several strengths, including the inclusion of consecutive specimens, the use of fresh bal fluid specimens and highly-sensitive real-time pcr assays for cmv dna load quantitation, and the performance of a comprehensive and systematic evaluation of specimens for the presence of rvs pathogens using molecular assays. in summary, our study highlights the difficulty in establishing universal 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quality control study group would kinetic analyses of plasma cytomegalovirus dna load help to reach consensus criteria for triggering the initiation of preemptive antiviral therapy in transplant recipients progress in quantitative viral load testing: variability and impact of the who quantitative international standards human cytomegalovirus load in plasma and bronchoalveolar lavage fluid: a longitudinal study of lung transplant recipients clinical utility of cytomegalovirus viral load in bronchoalveolar lavage in lung transplant recipients relationship between cytomegalovirus dna load in epithelial lining fluid and plasma of lung transplant recipients and analysis of coinfection with epstein-barr virus and human herpesvirus 6 in the lung compartment preemptive therapy for systemic and pulmonary human cytomegalovirus infection in lung transplant recipients cytomegalovirus viral load in bronchoalveolar lavage to diagnose lung transplant associated cmv pneumonia immunodeficiency scoring index to predict poor outcomes in hematopoietic cell transplant recipients with rsv infections this research was supported by a grant ( 12/1992 ) from fis ( fondo de investigaciones sanitarias, ministerio de sanidad y consumo, spain).estela giménez holds a río hortega research contract from the carlos iii health institute (ref. cm16/0 020 0 ). none. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.02.009 . key: cord-298639-v9yg80jw authors: chen, yuxin; tong, xin; wang, jian; huang, weijin; yin, shengxia; huang, rui; yang, hailong; chen, yong; huang, aijun; liu, yong; chen, yan; yuan, ling; yan, xiaomin; shen, han; wu, chao title: high sars-cov-2 antibody prevalence among healthcare workers exposed to covid-19 patients date: 2020-06-04 journal: j infect doi: 10.1016/j.jinf.2020.05.067 sha: doc_id: 298639 cord_uid: v9yg80jw the seroprevalence of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) was examined among 105 healthcare workers (hcws) exposed to four patients who were laboratory confirmed with coronavirus disease 2019 (covid-19), the disease caused by sars-cov-2 infection. these hcws were immediately under quarantine for 14 days as soon as they were identified as close contacts. the nasopharyngeal swab samples were collected on the first and 14(th) day of the quarantine, while the serum samples were obtained on the 14(th) day of the quarantine. with the assay of enzyme immunoassay (eia) and microneutralization assay, 17.14% (18/105) of hcws were seropositive, while their swab samples were found to be sars-cov-2 rna negative. risk analysis revealed that wearing face mask could reduce the infection risk (odds ratio [or], 0.127, 95% confidence interval [ci] 0.017, 0.968), while when exposed to covid-19 patients, doctors might have higher risk of seroconversion (or, 346.837, 95% ci 8.924, 13479.434), compared with hcws exposed to colleagues as well as nurses and general service assistants who exposed to patients. our study revealed that the serological testing is useful for the identification of asymptomatic or subclinical infection of sars-cov-2 among close contacts with covid-19 patients. the ongoing pandemic of 2019 novel coronavirus, known as severe acute respiratory syndrome coronavirus 2 (sars-cov-2), was first reported in wuhan, china in late dec 2019 (1) . due to its escalating spread globally, as of may 17, 2020, more than 532,224 patients were confirmed with coronavirus disease 2019 (covid-19), the disease caused by sars-cov-2, across 215 countries and regions, causing a total of 24,087 deaths. a wide spectrum of disease severity of laboratory confirmed covid-19 has been depicted (2, 3) , including asymptomatic or minimally symptomatic cases (4, 5) . the proportion of asymptomatic sars-cov-2 infected patients is unknown, which remains a critical epidemiological puzzle (6) . whether one may seroconvert to sars-cov-2 with minimal or without symptoms still needs to be answered. meanwhile, an efficient human-to-human transmission of sars-cov-2 mostly occurs among close contacts (1) . serological testing to close contacts with covid-19 patients will help define the local transmission rate and the risk factors of infection, especially identify asymptomatic or subclinical infections. healthcare workers (hcws) have been on the frontline for fighting this covid-19 pandemic worldwide, which placed themselves at high risk of catching covid-19. understanding risk factors of sars-cov-2 infection during clinical setting is urgently needed, which not only provides the hcws with essential guidance of self-protection, but also helps policymakers to formulate appropriate measures to control infection in hospital setting. this present study aimed to evaluate the seroprevalence of sars-cov-2 in a cohort of 105 hcws exposed to covid-19 patients using both enzyme immunoassay (eia) and microneutralization assay. furthermore, risk factors of sars-cov-2 seroconversion among these hcws were identified with epidemiological investigation. our study shed lights on the subclinical infection of covid-19 and provided information for pandemic mitigation efforts. from jan 28 th to feb 5 th , 2020, four patients were diagnosed with covid-19 in nanjing drum tower hospital, china. patient 1 (a 30-year-old male surgeon) was occupationally exposed to sars-cov-2, and patient 2 (a 31-year-old female nurse) is his wife. both of them were diagnosed with covid-19 on jan 29th. patient 3 (a 54-year-old male) was diagnosed in the 10th day of his hospitalization, while patient 4 (a 35-year-old male)was in the emergency room (er) for 2 days before diagnosis. the exposure history, the symptom onset timeline of the 4 covid-19 patients and the cycle threshold [ct] values of nucleic acid results were retrieved from their electronic medical records (figure 1 ). because of direct contact with the four aforementioned covid-19 patients during the past 2 weeks, the 105 hcws were immediately quarantined for 14-day observation. at the first day of their quarantine, each hcw was asked to complete a questionnaire designed by local center for disease control and prevention (cdc) during the past two weeks. the questionnaire included clinical symptom, relationship with the patients and exposure history. the relationship between hcws and coivd-19 individuals was categorized as colleague, doctor, nurse, or general service assistant. the exposure history included the time of exposures, date for each exposure, activity, location, distance, duration, with or without face mask (disposable non-surgical face mask, surgical mask or n95 respiratory if wearing face mask) during the past two weeks. disposable non-surgical face mask refers to the face mask made of non-woven textile in two or three layers, which generally lacks the capability of filtering particles, viruses and bacteria. additionally, during their quarantine, their body temperature and clinical symptoms were also recorded twice a day. nasopharyngeal swab specimens were collected on the first and 14 th day of their quarantine by professional certified nurses who have received the training of nasopharyngeal swab collection. blood samples were only obtained on the 14 th day of the quarantine. this study was approved by ethics committee of nanjing drum tower hospital. information consent was waived as part of a public health outbreak investigation. viral rna was extracted from nasopharyngeal swab samples using the qiaamp rna viral kit (qiagen), and quantitative reverse transcriptionpolymerase chain reaction (qrt-pcr) assay was performed (bioperfectus technologies, china). two sets of primers and probes targeting the open reading frame1ab (orf1ab) and nucleocapsid protein (np) genes of sars-cov-2 were used as recommended by the chinese cdc (1) following who guidelines (7) . the primers and probe set for orf1ab are: forward primer (5'-ccctgtgggttttacacttaa-3'); reverse primer (5'-acgattgtgcatcagctga-3'); probe: (5ʹ-vic-ccgtctgcggtatgtggaaaggttatgg-bhq1-3ʹ). the primers and probe set for np are: forward primer (5'-ggggaacttctcctgctagaat-3'); reverse primer (5'-cagacattttgctctcaagctg-3'); probe: (5ʹ-fam-ttgctgctgcttgacagatt-tamra-3ʹ). the thermal cycling condition was 50°c for 30 min, 95°c for 5 min, followed by 45 cycles of 95°c for 10s and 55°c for 40s. the ct value of the amplification curve was defined as positive if less than 40 and negative if greater than 40. to determine the seroprevalence among close contacts with covid-19 patients, an in-house enzyme immunoassay (eia) was conducted as previously described (8) . two sars-cov-2 proteins, recombinant spike protein receptor binding domain (rbd) protein and recombinant nucleocapsid protein (np) were used as detecting antigens, respectively. the genes encoding spike rbd (amino acid residues 319 to 541 of spike protein) and full-length np were codon-optimized and synthesized (genewiz, china). the gene encoding spike rbd was cloned into mammalian expression vector pcdna3.4 in frame respective and upstream of a series of six histidine residues, and np gene was cloned into prokaryotic expression vector pet-28(b). rbd protein was expressed in 293f cells while np protein was expressed in escherichia coli, followed by affinity purification. the purity of np and rbd protein was determined with 10% sodium dodecyl sulphate (sds) polyacrylamide gel electrophoresis. briefly, 96-well plates were coated with 500 ng/ml of recombinant rbd or np protein overnight, incubating with diluted were also collected and the nasopharyngeal swab samples from these patients have been repeatedly tested as negative for sars-cov-2 rna at least twice at a two-day apart. furthermore, 60 serum samples from 20 covid-19 patients were also collected at different time points for assay validation. pseudovirus expressing the sars-cov-2 spike protein was obtained as a general gift from the institute of biological product control from national institute for food and drug control, china. sars-cov-2 pseudovirus was prepared by using vsv g pseudotyped virus (g*δg-vsv) that packages the expression cassette for firefly luciferase instead of vsv-g in the vsv genome, and the serum neutralization capability was determined as described recently (9) . briefly, the sars-cov-2 pseudovirus was preincubated with serum samples at 1:20 dilution at 37°c for one hour, together with the pseudovirus control and cell control wells. serum samples from healthy controls were served as negative control in hexaplicate. then, the 96-well plates were seeded with 100 μg of freshly trypsinized huh7 cells (2 x 10 4 cells/well). after 24 hours of incubation in a 5% co 2 environment under 37°c,the luminescence was measured using luciferase substrate (one-glo tm luciferase assay system, promega, e6120) and the percentage of neutralization was calculated with the following formula as: [(relative light units (rlus) of virus control wells -rlus in cell control wells)-(rlus of serum incubated with virus wells-rlus of the cell control wells)]/ (rlus of virus control wells -rlus of cell control wells) x100%. the percentage of neutralization over 50% was considered to have neutralization activity. all statistical analyses were performed using spss 22.0. the medians (interquartile range (iqr)) were used to present the continuous variables, and the categorical variables were described as the counts and the percentages. the man-whitney u test (non-normal distribution) was used to compared the continuous variables between groups. chi-square test or fisher exact test was used to compare categorical variables. variables with p values < 0.1 in the univariate analysis were further used for a multivariate logistic regression analysis. pearson's correlation coefficients between different assays were calculated. the threshold for statistical significance was established at a p value <0.05. the demographic and epidemiological characteristics of the 105 hcws were summarized in table 1 . the median age of these hcws was 30 years old (iqr 26-62) and 83 (79.05%) of them were female. during the quarantine, 13 (12.38%) hcws were reported having one or more general symptoms, including fever (2/105, 1.90%), headache (2/105, 1.90%), sore throat (3/105, 2.86%), cough (6/105, 5.71%), myalgia (1/105, 0.95%), diarrhea (3/105, 2.86%) and rhinorrhea (2/105, 1.90%). all swab specimens collected on the first and 14 th day of the quarantine showed negative results for sars-cov-2, and none of these close contacts developed covid-19 later. an eia-based sars-cov-2 antibody assay was developed to detect igm and igg antibodies against rbd and np protein, respectively ( figure 2a ). different groups of serum samples were used to determine the specificity and sensitivity of our assay. specifically, as negative control, all the exposure history of covid-19 patients between seropositive and seronegative hcws was summarized ( table 1 ). the exposure events occurred in the early period of the outbreak of covid-19 in china, so the understanding to the sars-cov-2 was still limited at that time. all hcws did not wear personal protective equipment (ppe), including n95 respirators, surgical masks, face shield or googles. first, the proportion of doctors who were exposed to patients was higher in seropositive group than in seronegative group (38.89% vs. 10.34%, p=0.004). it was identified that there was higher percentage of hcws exposed to patient 2 in seropositive group (27.78% vs. 10.34%, p=0.048) than in seronegative group, which might be caused by the relatively high level of viral shedding from patient 2, revealed by a low ct value for her respiratory swab samples at the hospital admission. besides, more seropositive hcws had a previous experience of exposure for over 30 minutes within a distance of 1 meter (55.56% vs. 26.44%, p=0.015), and less seropositive hcws contacted covid-19 patients with disposable non-surgical face mask wearing (55.56% vs. 78.16%, p=0.046), compared to that in seronegative group. neither swab sample collection nor multiple times of exposure with covid-19 patients showed any differences in seroconversion. risk factors associated with sars-cov-2 seroconversion were assessed ( table 2 ). the univariate analysis showed that the exposure for more than 30 minutes at a distance of less than 1 meter (odds ratio [or],3.478，95% confidence interval [ci] 1.224, 9.887), close contact with patient 2 (or, 7.125, 95% ci 1.627, 31.210) and doctors exposed to their patient (or, 3.850, 95% ci 1.131, 13.105) led to higher risk of seropositivity, while contact with covid-19 patient wearing mask (or, 0.349, 95% ci 0.121, 1.008) was associated with a reduced risk of seroconversion. in multivariate analysis, there existed higher risk of seroconversion for close contacts with patient 2 (or, 6.605, 95% ci, 1.123, 38.830) and doctors exposed to their patient (or, 346.837, 95% ci 8.924, 13479.434), while the lower risk of seroconversion was closely related to direct contact with covid-19 patients wearing face mask (or, 0.127, 95% ci 0.017, 0.968). similar to sars-cov, sars-cov-2 has been reported to be highly communicable in hospital setting (10, 11). high attack rate of sars-cov-2 among healthcare workers with direct patient care has been observed worldwide, including china, italy, and united states, etc. our study examined the seroprevalence of the 105 hcws exposed to covid-19 patients without wearing ppe at early period of outbreak in a tertiary hospital of china. of note, 56 hcws were exposed to covid-19 patients as colleagues, while 49 hcws were exposed during performing direct care for patient or general service. despite the swab samples from all hcws collected at twice were negative for sars-cov-2 rna, our serological analysis indicated 17.1% of asymptomatic or subclinical infection of sars-cov-2 in hospital setting. consistent with the efficient transmissibility of sars-cov-2, our serological analysis in the hospital setting highlighted a higher percentage of asymptomatic or subclinical sars-cov-2 infection than that of sars-cov (12) (13) (14) (15) and mers-cov (16) . our data suggested that those with asymptomatic or subclinical infection of sars-cov-2 were able to be seroconverted. all the nasopharyngeal swab samples collected twice were found to be negative for sars-cov-2. this could because that the modest level of viral load from nasopharyngeal swabs was beyond detection. also, the nasopharyngeal swab samples were not longitudinally collected and the positive samples might be missed. therefore, serological testing is an ideal approach to assess the proportion of people who might experience the asymptomatic or subclinical infection of sars-cov-2, which is critical to understand the viral transmissibility and disease burden during covid-19 pandemic. previous studies on human coronaviruses have shown that np protein and rbd region are highly immunogenic, which have been successfully applied in eia-based approach for serological analysis (14, 17, 18) . in order to eliminate the possibility of cross-reactivity with other human coronaviruses, rbd and np protein were used as two antigens in our assay, similar to a recent published study (19) . we found that all the healthy controls in 2019 and the non-covid-19 pneumonia patients were 100% found to be negative, while 93.3% of serum from covid-19 patients were proved to be positive, suggesting our elisa assay has high sensitivity and fine specificity. by using eia assay, 19 samples were discovered positive for detectable igm or igg responses to both rbd and np protein, while the antibody responses among these hcws were relatively lower compared with covid-19 patients. meanwhile, 18 out of 19 sera samples displayed neutralization activities. consistent with recent findings (19), the level of neutralization activity was associated with anti-np igg response and anti-rbd igg response. whether those seropositive hcws acquired protective immunity from sars-cov-2 infection remains unknown. active surveillance and longitudinal followup to close contacts should be attached more importance. firstly, our analysis showed that the secondary attack rate to each covid-19 patients was distinct. in our study, higher percentage of close contacts with patient 2 were seroconverted. such efficient transmission might be caused by higher level of viral load of patient 2, which was determined by the low ct value of nucleic acid assay. it is also possible that sars-cov-2 was evolved in patient 2 after infection, which might further modulate the viral transmissibility and virulence (20) . besides, our data support an essential need to wear face mask, including the disposable non-surgical face mask, because it might provide effective protection against sars-cov-2. of note, since these exposure events occurred at the very first of the outbreak in china, all the hcws in our study only worn the disposable non-surgical face mask, rather than n95 respirator or surgical masks. therefore, our data suggested that the disposable non-surgical face mask might be also beneficial to the reduction of the potential nosocomial infection. consistently, a recent study in hong kong revealed that the community-wide wearing of the face mask plays a critical role in the control of covid-19, not only by preventing the dispersal of droplets from subclinical or mild individuals with covid-19, but also by reducing the environmental contamination of sars-cov-2 (21) . moreover, among four relationships with the covid-19 patients, there is higher risk for doctors who were exposed to covid-19 patients to have seroconversion. it is possible that doctors might share more conversations with covid-19 patients within relatively close distance, which could generate large amount of infected saliva or respiratory droplets. our findings might shed light on the implementation of appropriate infection prevention and control measures, especially at hospital setting. this study has several limitations. firstly, our cohort is limited to only inclusion of 105 hcws exposed to 4 covid-19 patients. however, our cohort is representative since they are either colleagues or health professionals in terms of the relation with the covid-19 patient. and different extents of exposure history in our cohort allow us to identify the potential exposure risk factors. secondly, the serum samples were only collected on the 14 th day of the quarantine, while the serum samples on the first day of the quarantine were not obtained. therefore, the dynamic antibody responses were not determined in our study. thirdly, the nasopharyngeal swab specimens were only collected twice on first and 14 th day of the quarantine, which were not serially collected especially during the early period of the quarantine, and thus the positive nasopharyngeal swab samples might be missed. in summary, the serological testing among 105 hcws exposed to covid-19 patients illustrated that 17.14% (18/105) might have experienced asymptomatic or subclinical infection of sars-cov-2. our study proved that the serological testing is useful for the identification of asymptomatic or subclinical infection of sars-cov-2 among close contacts with covid-19 patients. whether these asymptomatic or subclinical infections play a role in transmission dynamics still remains to be determined. our findings have important implications for the implementation of pandemic mitigation strategies. early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china: retrospective case series clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china epidemiologic and clinical characteristics of novel coronavirus infections involving 13 patients outside wuhan, china a family cluster of sars-cov-2 infection involving 11 patients in nanjing, china association of public health interventions with the epidemiology of the covid-19 outbreak in wuhan, china. medrxiv. 2020 coronavirus disease (covid-19) technical guidance: laboratory testing for 2019-ncov in humans a novel rabbit monoclonal antibody platform to dissect the diverse repertoire of antibody epitopes for hiv-1 env immunogen design establishment and validation of a pseudovirus neutralization assay for sars-cov-2 supporting the health care workforce during the covid-19 global epidemic surface environmental, and personal protective equipment contamination by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) from a symptomatic patient healthcare worker seroconversion in sars outbreak severe acute respiratory syndrome-associated coronavirus infection relative rates of non-pneumonic sars coronavirus infection and sars coronavirus pneumonia sars-cov antibody prevalence in all hong kong patient contacts surveillance of the middle east respiratory syndrome (mers) coronavirus (cov) infection in healthcare workers after contact with confirmed mers patients: incidence and risk factors of mers-cov seropositivity characterization of anti-mers-cov antibodies against various recombinant structural antigens of mers-cov in an imported case in china temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study genomic diversity of sars-cov-2 in coronavirus disease 2019 patients. clin infect dis. 2020. mar 4; ciaa203 the role of community-wide wearing of face mask for control of covid-19) epidemic due to sars-cov-2 the authors have declared that no conflicts of interest. key: cord-312984-rzryn3on authors: pan, daniel; sze, shirley; rogers, benedict; bron, jan; bird, paul w.; holmes, christopher w.; tang, julian w. title: serial simultaneously self-swabbed samples from multiple sites show similarly decreasing sars-cov-2 loads in covid-19 cases of differing clinical severity date: 2020-09-19 journal: j infect doi: 10.1016/j.jinf.2020.09.016 sha: doc_id: 312984 cord_uid: rzryn3on nan detection of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is essential for diagnosis of coronavirus disease . nasopharyngeal swabs (nps) are recommended by the world health organisation (who) as the current standard method to detect sars-cov-2 in suspected patients. 1 however, collection of an nps or oropharyngeal swab (ops) can be technically difficult, uncomfortable for patients, may induce sneezing or coughing, and expose those nearby to aerosolised sars-cov-2). 2 we postulate that alternative easier-to-sample swabs such as those from the nose (ns) or cheek (cs) may be equally sensitive in acute covid-19 cases, and can be effectively taken by patients themselves. in addition, longitudinal analysis of serial samples collected contemporaneously from multiple upper respiratory tract (urt) sites, from the same patient, has not been well described. here, we performed a prospective longitudinal analysis of three healthcare worker volunteers with differing clinical severities of acute covid-19. shortly after a confirmed diagnosis of covid-19 using the local diagnostic ausdiagnostics (ausdiagnostics uk ltd., chesham, england) sars-cov-2 pcr test, 3 each participant volunteered to provide serial self-collected swabs from the nasopharynx (nps, 1 swab), just inside the soft part of the nose (ns, 1 swab), the oropharynx (ops, 1 swab), inside the cheek (cs, 1 swab). in addition, we also decided to check for the presence of sars-cov-2 in the conjunctiva (cjs, 1 swab for both eyes). all of these swabs were taken at the same time-point, on a daily basis -or as frequently as was practical and tolerable -until all the sars-cov-2 viral loads became undetectable. thus, each patient collected up to 5 separate swabs on a daily basis for this study. all three participants (hereafter referred to individually as 'patient' 1, 2, or 3) became symptomatic with confirmed covid-19 during the week of 12th april 2020. patient 1 had mild covid-19, complaining of a 7-day history of anosmia only; patient 2 had moderate covid-19, with a 5-day history of fevers, shivers, dry cough and myalgia; patient 3 had severe covid-19, presenting with a 2-week history of fevers, shivers and a productive cough that required supplemental oxygen therapy, and eventual admission to the intensive care unit (icu) during the second week, after which no further swabs were taken. final follow-up swabs were performed by the participants on 5th may 2020, two weeks after symptom onset for all participants. from 17th april to 5th may 2020, a total of 105 swabs were collected from three participants ( our small longitudinal study cohort demonstrated several findings. firstly, the most symptomatic case, patient 3 was most likely to be viremic at multiple sites in the urt, as reported elsewhere. 4 secondly, self-swabbing from these various urt sites is an effective and sensitive way to collect diagnostic samples, as found elsewhere. 5 note that only one out of these three cases, patient 3 who did not exhibit overt conjunctivitis, exhibited detectable virus from the conjunctiva within the first 5 days of illness. patient 1 s first swab was only taken on day 8 post-illness onset, so it is possible that any virus present earlier in conjunctival fluids may have been missed. however, this lower detection rate for conjunctival swabs (with or without overt conjunctivitis) is consistent with previous reports. 6 thirdly, the relative sars-cov-2 viral loads from the urt decreased with time in the 1-2 weeks post-covid-19 symptom onset, regardless of disease severity. this has been shown elsewhere, 7 though this is not always the case. 8 from this small longitudinal cohort study on serially collected samples in acute covid-19 cases of differing severity, we conclude that for symptomatic patients, it is difficult to obtain a 'false negative' result on nps, ops, ns or cs samples, if sampled early (within 5 days) post-symptom onset, even if the swab was 'poorly' taken. despite a previous meta-analysis showing that sputum testing is possibly more sensitive for sars-cov-2 pcr testing, 9 other studies have shown that self-sampling from various urt sites performed satisfactorily for the diagnosis of acute covid-19. 5 sputum testing is not standard in many virology labs due to long-recognised problems related to its viscosity and risks of pcr inhibition, 10 and not all covid-19 patients will have a productive cough. therefore, we further confirm that early (within 5 days of symptom onset), self-swabbed nps, ops, ns or cs samples for sars-cov-2 diagnostic testing in acute covid-19 cases is a sensitive, practical approach, which reduces patient discomfort (as self-swabbing can be controlled) and minimises virus exposure to healthcare workers. who. laboratory testing of 2019 novel coronavirus (2019-ncov) in suspected human cases 2020 how to obtain a nasopharyngeal swab specimen high sars-cov-2 infection rates in respiratory staff nurses and correlation of covid-19 symptom patterns with pcr positivity and relative viral loads virological assessment of hospitalized patients with covid-2019 swabs collected by patients or health care workers for sars-cov-2 testing detecting sars-cov-2 rna in conjunctival secretions: is it a valuable diagnostic method of covid-19? sars-cov-2 viral load in upper respiratory specimens of infected patients yjinf [m5g presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility sars-cov-2 detection in different respiratory sites: a systematic review and meta-analysis comparison of sputum and nasopharyngeal swabs for detection of respiratory viruses key: cord-337854-5ogip9tz authors: huang, wanqiu; lin, dachuan; wang, cuini; bao, chaohui; zhang, zhaoqi; chen, xinchun; zhang, zheng; huang, jian title: the determination of release from isolation of covid-19 patients requires ultra-high sensitivity nucleic acid test technology date: 2020-07-02 journal: j infect doi: 10.1016/j.jinf.2020.06.075 sha: doc_id: 337854 cord_uid: 5ogip9tz nan the prevention and control of sars-cov-2 has entered a critical period. recent one paper in this journal also discussed weather qualitative rt-pcr be used to determine release from isolation of covid-19 patients [1] . this issue is really important. since the outbreak of covid-19 worldwide, discontinuation of isolation has been presenting a dilemma of covid-19, despite of the test-based strategy or the symptom-based strategy [1] . the reason for the confusion is that nucleic acid testing presents false negative based on qpcr technology, because of its low sensitivity [2] [3] [4] . there are several factors for false negative, including sample collection, preservation, transportation, virus inactivation, nucleic acid extraction and technical sensitivity, among which technical sensitivity and precise sampling are the most important quality control measures to eliminate false negative. it is well known that sars-cov-2 nucleic acid test is the main diagnostic method of covid-19. recombinase polymerase amplification (rpa) is a new technology for testing nucleic acid with some advantages of simple operation, fast speed and low cost based on isothermal amplification. in our study, we developed an improved strategy, termed as nestrpa (nest recombinase polymerase amplification), which could greatly improve the sensitivity of nucleic acid detection for sars-cov-2 than rpa or qpcr. 4 firstly, we designed eight sets of primers and probes for rpa on the conservation regions of sars-cov-2 genes, in which some fragments were designed to span multiple gene regions ( figure 1a ) which is one of the important technical tips. through the two rounds of primer screening, we found that the limit of detection (lod) of 16 pairs of primers and 8 probes is quite different ( figure 1b ), in which fragment 1 against orf1 gene had the worst amplification efficiency. and fragment 5 and 7 had the smallest lod value, 300 and 500 copies/ul ( figure 1c to 1f), respectively. as far as we know, we firstly proposed the concept of nestrpa. the basic principles of nestrpa are as follows: in nestrpa, the first amplification fragment of target gene is amplified by outer primers, then a second fragment of target gene completely within the first amplification fragment is amplified by inner primers. in order to eliminate the influence of the fluorescence signal of enzymes, fluorescent probe is not included in first rpa reaction which is another important technical tips. and in the second rpa reaction, fluorescent probe will be added into reaction system. using nestrpa technology, we found that positive plasmid containing sars-cov-2 with the concentration of 1 copy/ul could also be stably detected by fragment 5 and nucleic acid detection results were negative using qpcr. however, we found 12.50% (4/32) of the samples were positive using fragment 5 and/or fragment 7 by nestrpa ( figure 1i) , which was consistent with those reported by other researchers [5] . our results suggested that the ultra-sensitive nucleic acid detection technique has important implications for early identification of those asymptomatic carriers infected with sars-cov-2. of course, in order to avoid false positive results, the target sequence of positive amplification products was 100% detected by high-throughput sequencing. in summary, 36.18% (55/152) of the samples with qpcr negative results were identified as positive by nestrpa technology in 172 clinical samples from 127 patients, which indicated the analytical sensitivity of nestrpa assay is much better than that of qpcr ( figure 1j ). in addition, many experts of covid-19 prevention and treatment clearly pointed out that the inaccurate sample collection were also one of the important reasons for the false negative result of sars-cov-2 nucleic acid [6] [7] [8] . the most commonly sites used as sampling are oropharynx and nasopharynx. the sample collectors should fix the tongue with a spatula, and the sampling swab is used to scrape the cells from tonsil recess and lateral wall when sampling from the oropharynx [9] . however, the sample collectors were often fear of contagion with sars-cov-2. under great infection pressure, inaccurate sampling sites and inadequate sample volume will lead to false 7 negative test results. therefore, it is helpful to reducing the false negative through strict and normative operation of precise sampling with well protection for sample collectors (figure 2) . except for the technical sensitivity and precise sampling, we also need to pay more attention for the quality control of sample preservation and transportation, virus inactivation, nucleic acid extraction [10] . if all the links in the detection of sars-cov-2 nucleic acid could be strictly administrated, false negative could be completely eliminated, and the discontinuation of isolation will no longer be a dilemma for us. all authors 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. jian huang was responsible for study concept and design. zheng zhang and xinchun chen were responsible for specimens sampling. wanqiu huang and dachuan lin were responsible for the experiment and statistical analysis. wanqiu huang, dachuan lin, cuini wang, chaohui bao and zhaoqi zhang were responsible for the analysis of data. wanqiu huang and jian huang were responsible for drafting the manuscript. should qualitative rt-pcr be used to determine release from isolation of covid-19 patients covid-19 testing: the threat of false-negative results negative nasopharyngeal and oropharyngeal swabs do not rule out covid-19 false-negative of rt-pcr and prolonged nucleic acid conversion in covid-19:rather than recurrence estimating the extent of asymptomatic covid-19 and its potential for community transmission: systematic review and meta-analysis suboptimal biological sampling as a probable cause of false-negative covid-19 diagnostic test results understanding the influence factors in viral nucleic acid test of 2019 novel coronavirus (2019-ncov) expert consensus on specimen sampling technique of sars-cov-2 infected patients expert consensus on nucleic acid detection of covid-19 antibody detection and dynamic characteristics in patients with covid-19 key: cord-335355-lcrbs2op authors: kunutsor, setor k.; laukkanen, jari a. title: markers of liver injury and clinical outcomes in covid-19 patients: a systematic review and meta-analysis date: 2020-05-28 journal: j infect doi: 10.1016/j.jinf.2020.05.045 sha: doc_id: 335355 cord_uid: lcrbs2op nan since january 2020 when it was first isolated in china, coronavirus disease 2019 (covid-19) has spread throughout the world and caused substantial morbidity and mortality. ( abnormalities. in their analyses to explore prognostic factors for fatal outcomes, alanine aminotransferase (alt) and aspartate aminotransferase (ast) were not found to be independently associated with the risk of mortality. though it has been reported liver injury is more prevalent in severe cases of covid-19, (3, 4) whether circulating levels of markers of liver injury could predict clinical outcomes in covid-19 patients is uncertain. in this context, we aimed to determine the nature of the relationships of admission levels of five main markers of liver injury (alt, ast, gamma-glutamyltransferase (ggt), alkaline phosphatase (alp) and total bilirubin) with the risk of clinical outcomes in patients with covid-19 using a systematic meta-analysis. we conducted this review using prisma and moose guidelines (supplementary materials 1-2) and in accordance with a registered protocol in the prospero international prospective register of systematic reviews (crd42020183672). medline, embase, and the cochrane library were searched from 2019 to 17 may 2020 for published studies reporting on relationships between admission levels of markers of liver injury (ggt, alt, ast, alp and total bilirubin) and clinical outcomes in patients with covid-19. the detailed search strategy has been reported in supplementary material 3. outcomes were categorised into severe illness and mortality. mean differences (95% cis) for comparing mean levels of circulating markers across outcomes and relative risks (rrs) (95% confidence intervals, cis) for associations between markers and outcomes were used as summary measures across studies. (5) the inverse variance-weighted method was used to effect estimates using random-effects models to minimize the effect of heterogeneity. stata release mp 16 (statacorp lp, college station, tx, usa) was used for all statistical analyses. sixteen retrospective cohort studies comprising 10,540 covid-19 patients were eligible ( table 1 in pooled results of two studies each, the rrs (95% cis) of mortality associated with elevated alt and ast were 3.35 (2.37-4.75) and 10.42 (7.05-15.40) respectively. in results from single studies, increased levels of alp and total bilirubin were each associated with an increased risk of mortality (supplementary material 6). admission levels of ast and total bilirubin were higher in those who died; whereas alt levels were not significantly different in both groups: mean differences (95% cis) of 17.13 u/l (11.25, 23.01; p<0.001); 4.21 µmol/l (3.97, 4.46; p<0.001) and 5.82 u/l (-2.57, 14.21; p=0.17) respectively. in single reports, levels of alp and ggt were higher in those who died compared with survivors (fig. 1b) . taking the overall evidence together, the data supports a higher prevalence of elevated admission levels of markers of liver injury in severe or mortality due to covid-19 disease, which suggests that patients with elevated levels of liver markers at baseline (during admission) had higher risks of developing worse outcomes in covid-19. the likely explanation for the worse outcomes observed in patients with baseline elevated markers of liver injury (as seen in chronic liver disease) could be attributed to compromised immune status. (3, 4) irrespective of the fact that about 2-11% of patients with covid-19 have liver comorbidities,(3) covid-19 also causes liver injury. however, there is controversy regarding the causes of liver injury in covid-19. (3, 4) proposed explanations include (i) drug-induced liver injury; (ii) direct injury to the liver due to covid-19 hepatitis(4); (iii) covid-19 induced myositis(4); (iv) binding of sars cov-2 directly to angiotensin-converting enzyme 2 (ace2) positive rich cholangiocytes and causing liver damage;(6) (v) hepatic congestion due to high levels of positive end expiratory pressure during mechanical ventilation;(4) and (vi) aggravation of liver injury by sars cov-2 in patients with pre-existing viral hepatitis. (7, 8) in the absence robust association studies and formal risk prediction analyses, the overall evidence suggests that increased baseline levels of markers of liver injury could predict poor outcomes. the global prevalence of chronic liver disease remains high and continues to increase. treatment options for covid-19 are currently supportive; hence, there should be more intensive monitoring of levels of markers of liver injury during admission so that therapeutic approaches can be individually tailored. there are several limitations which deserve mention. first, the heterogeneous reporting of severe illness outcomes prompted the use of composite measures. second, the possibility of patient overlap as all 16 studies were reported from china; there have been concerns with duplicate reporting of study participants in articles.(9) third, due to the limited sample sizes and low events, some studies were unable to assess risk ratios to quantify the relationships. finally, though we extracted data on baseline (admission) levels of these markers, studies were not very specific regarding the exact time of blood sampling in relation to the disease status; hence, these results may have some biases. in conclusion, elevated admission levels of markers of liver injury particularly the aminotransferases, may be associated with progression to severe disease or death in covid-19. monitoring levels of these markers could assist in the optimum management of patients. none. clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study coronavirus disease 2019 in elderly patients: characteristics and prognostic factors based on 4-week follow-up liver injury in covid-19: management and challenges covid-19 and the liver: little cause for concern association of serum total osteocalcin with type 2 diabetes and intermediate metabolic phenotypes: systematic review and meta-analysis of observational evidence specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection liver injury during highly pathogenic human coronavirus infections clinical best practice advice for hepatology and liver transplant providers during the covid-19 pandemic: aasld expert panel consensus statement concern-possible reporting of the same patients with covid-19 in different reports ruan, 2020chen, 2020subtotal yang, 2020ruan, 2020 key: cord-332786-7b6wz6i7 authors: ha, dat p.; van krieken, richard; carlos, anthony; lee, amy s. title: the stress-inducible molecular chaperone grp78 as potential therapeutic target for coronavirus infection date: 2020-06-12 journal: j infect doi: 10.1016/j.jinf.2020.06.017 sha: doc_id: 332786 cord_uid: 7b6wz6i7 nan the current coronavirus pandemic has become the greatest threat to global public health, thus there is an urgent need for identifying therapeutic targets. a recent report in this journal by ibrahim and colleagues describing the potential binding interaction between sars-cov-2 spike protein and the host 78-kda glucose regulated protein (grp78) raised the possibility that grp78 could be a facilitator for viral entry 1 and disruption of such interaction may be used to develop novel therapeutics specific against this virus 2 . our laboratory has a long-standing interest in the regulation and function of grp78, which is a stress-inducible, multi-faceted chaperone protein serving critical functions in the endoplasmic reticulum (er) and other cellular compartments, impacting both health and disease 3, 4 . the er is the major site of synthesis, folding, and maturation for membrane and secretory proteins. when the folding capacity of the er is overwhelmed due to increased protein synthesis, the cell undergoes er-stress which activates the unfolded protein response (upr), a complex network of signaling pathways aiming to restore er homeostasis or trigger apoptosis depending on context, duration, and intensity of the stress 3 . grp78, also referred to as bip/hspa5, is a master regulator of the upr, and is upregulated upon er stress to alleviate proteotoxic stress. as such, grp78 has emerged as a key target to combat diseases, like cancer, where uncontrolled cellular proliferation causes er overload leading to upr activation 3 . interestingly, viral infection also creates er stress and triggers the upr 5 . as outlined below, grp78 is an important host factor for viral infection and targeting grp78 has the potential to disrupt multiple stages of the viral life cycle including entry, production and subsequent cellular infection ( figure 1 ). grp78 has been reported to facilitate viral entry for a wide variety of viruses, including human and bat coronaviruses 6 ( table 1 ). the role of grp78 in these studies was investigated through the use of sirna targeting grp78, antibody against grp78, proteolytic cleavage of grp78 by subab, as well as small molecule ar12 and natural product egcg both of which inhibit the atpase activity of grp78 3,6,7 . how might grp78, normally residing in the er, facilitate viral attachment onto host cells? upon er stress, including coronavirus infection, a fraction of grp78, an abundant er luminal protein, is actively translocated from the er to the cell surface and assume new functions, including viral entry 3,4,6,8 ( figure 1 ). in the case of mers-cov and bcov-hku9 coronaviruses, their spike proteins bind to cell surface grp78 (csgrp78) in addition to their cognate receptors 6 . thus, csgrp78 may enhance viral entry by stabilizing the interaction between host and viral factors required for viral entry, which is consistent with our recent observations that csgrp78 can interact with and stabilize cell surface receptors such as cd44 and cd109 8, 9 . furthermore, in cell types where the primary viral receptor expression is low, csgrp78 may serve as an alternative host factor for viral entry. future studies are required to test out these concepts, as well as to establish whether grp78 is a critical host factor for sars-cov-2 entry. the notion that upregulation of grp78 on the surface of virally infected cells can be exploited to direct antiviral and immunomodulatory drugs to cell populations infected by sars-cov-2 is also worthy of investigation. beyond viral entry, grp78 can play a major role in viral protein synthesis and maturation (table 1) . viruses are obligate intracellular parasites which depend primarily on the cellular machinery to manufacture their proteins required for virion production, assembly, and budding. additionally, many viruses including sars-cov-2 are enveloped by a lipid bilayer containing viral glycoproteins on its surface to bind host cell receptors to facilitate their entry. since these viral envelope proteins are membrane-embedded, they are synthesized and processed in the er. unlike cellular protein synthesis, which is tightly regulated to maintain homeostasis, viruses, such as coronavirus, can selectively shut down host protein production and usurp the host er translational machinery to synthesize the viral proteins in massive quantities. this results in er overload, leading to er stress and upr activation. consequently, er stress and grp78 upregulation have been reported during infection by a wide variety of viruses [5] [6] [7] . in addition to its role in viral protein folding, grp78 upregulation during viral replication could protect the virus-infected host cells from undergoing apoptosis since grp78 is known to bind and maintain the er-associated apoptotic machineries in their inactive forms and exert pro-survival effects especially under er stress 3 . these features make the er a particularly important cellular compartment for viral production and viruses have evolved complex mechanisms to exploit and manipulate the er to enhance their replication. conversely, the dependence of viruses on the er and its key resident chaperone grp78 for viral protein production and host cell survival could be the virus' achilles heel and offers a unique opportunity for combating sars-cov-2 and other virus infections. the last step in a successful viral life cycle is the release of progeny virions to infect new cells. here, grp78 may also be critical for viral infectivity. firstly, grp78 depletion during viral replication could lead to reduced synthesis or improper folding of viral proteins resulting in impaired budding or immature virions with diminished infectivity. secondly, grp78 could facilitate the assembly of various viral components by maintaining er homeostasis and thus provide a conducive environment for virus maturation. lastly, grp78 could be captured into the viral particles and enhances subsequent cellular infection. indeed, it has been reported that grp78 was found in japanese encephalitis virus particles and mature virions that lacked grp78 displayed significant decrease in viral infectivity 10 . it will be interesting to determine the topology of grp78 in these virions and the generality of this interesting and surprising observation. in conclusion, we hope that the current scientific evidence presented here and our perspectives will stimulate further interest in grp78 as a promising target and expand the emerging development of anti-grp78 agents in the fight against sars-cov-2 and viral infection in general. the authors declare no conflict of interest covid-19 spike-host cell receptor grp78 binding site prediction natural products may interfere with sars-cov-2 attachment to the host cell glucose-regulated proteins in cancer: molecular mechanisms and therapeutic potential beyond the endoplasmic reticulum: atypical grp78 in cell viability, signalling and therapeutic targeting modulation of the unfolded protein response by the severe acute respiratory syndrome coronavirus spike protein middle east respiratory syndrome coronavirus and bat coronavirus hku9 both can utilize grp78 for attachment onto host cells ar-12 inhibits multiple chaperones concomitant with stimulating autophagosome formation collectively preventing virus replication endoplasmic reticulum stress activates src, relocating chaperones to the cell surface where grp78/cd109 blocks tgf-β signaling grp78 regulates cd44v membrane homeostasis and cell spreading in tamoxifen-resistant breast cancer. life science alliance japanese encephalitis virus co-opts the er-stress response protein grp78 for viral infectivity we thank vicky yamamoto, frank attenello and paul lee for the helpful discussions. this work is supported by nih grants (r01 ca027607, r01 ca027607-37s1 and r01 ca 238029) and the judy and larry freeman chair in basic cancer research to a.s.l. key: cord-282433-p6jl9gxf authors: tu, xinyi; chong, wai po; zhai, yun; zhang, hongxing; zhang, fang; wang, shixin; liu, wei; wei, maoti; siu, nora ho on; yang, hao; yang, wanling; cao, wuchun; lau, yu lung; he, fuchu; zhou, gangqiao title: functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to severe acute respiratory syndrome coronavirus infection date: 2015-03-27 journal: j infect doi: 10.1016/j.jinf.2015.03.006 sha: doc_id: 282433 cord_uid: p6jl9gxf objectives: to assess associations between the functional polymorphisms g-2518a at the chemokine (c–c motif) ligand 2 gene (ccl2) and mannose binding lectin (mbl) codon 54 variant (a/b) and susceptibility to sars. methods: we genotyped the ccl2 g-2518a and mbl codon 54 variant (a/b) in 4 case–control populations of chinese descent, totally consisting of 932 patients with sars and 982 control subjects. results: both the high-ccl2-producing gg genotype and the low-mbl-producing b allele were consistently associated with increased risks of sars-cov infection in all 4 case–control populations (joint p = 1.6 × 10(−4) and 4.9 × 10(−8), for ccl2 and mbl respectively), with no interaction between polymorphisms could be detected. furthermore, all the 4 case–control studies demonstrated a cumulative effect on risk of sars-cov infection for the combination of polymorphisms (joint p = 1.3 × 10(−10)). however, tests using the area under the curve (auc) indicated that at this stage, the polymorphisms were unlikely to be appropriate for risk prediction testing because of low auc values (all <66%). additionally, no association was observed between the polymorphisms and severity of sars. conclusions: the ccl2 g-2518a and mbl codon 54 variant have a significantly cumulative effect on increased risk of sars-cov infection. functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to severe acute respiratory syndrome coronavirus infection introduction severe acute respiratory syndrome (sars) is a newly emerged infectious disease of humans caused by a novel coronavirus (cov), the sars-cov. 1 pathogenesis of sars is complex and host genetic background is considered to be one of the factors in determining susceptibility to, and outcome of sars. 2 previous reports have shown that polymorphisms of several putatively important genes affect an individual's susceptibility to sars-cov infection or disease severity of sars. 3à10 in particular, our previous two independent association studies have implicated that a functional polymorphism at codon 54 in exon 1 (rs1800450, g230a, denoted as a/b variant) of mannose binding lectin (mbl), which encodes a protein belonging to the family of collectin and plays a critical role in the innate immune response, conferred a significantly increased susceptibility to sars-cov infection. 3, 9 however, on the basis of the fact that the susceptibility to infectious disease is determined at different functional levels of innate and adaptive immunity, 11 we hypothesize that an unknown number of other unidentified genes are likely to mediate the susceptibility to sars, including sars-cov infection and disease severity. chemokines play important role in cells trafficking during immune responses. among the chemokine family, the chemokine (cec motif) ligand 2 (ccl2), also designated as monocyte chemoattractant protein-1 (mcp-1), is known as a potent chemoattractant for monocytes and macrophages, and is considered to be involved in several diseases characterized by intense macrophage infiltration. 12 ccl2 influences both innate immunity, through effects on monocytes and macrophages, and adaptive immunity, through control of t helper cell polarization. 13 in the case of sars, our and other studies have shown that ccl2 was one of the earliest and most prominent chemokines upregulated in either lung epithelial cells or monocyte derived dendritic cells infected with sars-cov. 14, 15 the upregulation of ccl2 mediate the migration of monocytes and macrophages, which were the infiltrating cells indeed observed in the lung tissues of patients with sars. 16 notably, the overexpression of ccl2 was consistently detected in plasma of patients with sars in several independent studies. 17à19 furthermore, after treatment with corticosteroid, which is an effective cytokine modulator and has a beneficial effect on sars patients, the level of plasma ccl2 was reduced significantly from 5 to 8 days. 17 additionally, it has also been reported that the higher level of serum ccl2 in patients is correlated with more advanced disease severity of sars. 17 in the lungs of balb/c mice, the pdzbinding motif of recombinant sars-cov envelope protein is a determinant of viral pathogenesis and induces the deleterious exacerbated immune response including increased expression of ccl2. 20 on the basis of the above relevance of the ccl2 in the pathogenesis of sars, we hypothesize that the ccl2 may be the excellent biologic candidate susceptibility gene for sars. it is expected that the genetic variation within ccl2 could contribute to inter-individual differences in susceptibility to, and outcome of sars. recently, a functional single nucleotide polymorphism (snp) (rs1024611, g-2518a) in the distal regulatory region of the ccl2 at position à2518 relative to the transcription start site has been well characterized. 21 compared with the ccl2 -2518a allele, the à2518g allele conferred a greater ccl2 transcriptional activity mediated by differential protein-dna interactions, an increased ccl2 protein production in vitro and in vivo, and an enhanced leukocyte trafficking to tissues. 21à23 furthermore, prevalence of the high-ccl2-producing à2518g allele has been shown to be associated with increased susceptibility or severity of infectious diseases, including hiv-1 infection and aids dementia, 21,24 hcv infection, 22 hbv clearance, 25 hcmv reactivation, 26 and pulmonary tuberculosis. 27 the role of this functional polymorphism in sars, however, has never been specifically evaluated. in this study, we therefore investigated whether the functional polymorphism g-2518a in the ccl2 gene have any bearing on the sars. to this end, we genotyped the ccl2 g-2518a polymorphism in 4 independent caseecontrol populations of chinese descent, totally including 932 patients with sars and 982 control subjects. we also reassessed the mbl codon 54 variant (a/b) in susceptibility to sars-cov infection in the present study. furthermore, we investigated whether a combination of these two functional polymorphisms of ccl2 and mbl would have a cumulative effect on risk of sars-cov infection. four independent caseecontrol populations of chinese ancestry were included in the present study. the details about enrollment criterion and demographic characteristics of these populations had been described previously, 3, 4, 9 and were also provided in the supplementary materials and methods and supplementary the polymorphism ccl2 g-2518a (rs1024611) was genotyped by polymerase chain reaction-restriction fragment length polymorphism (pcr-rflp) analysis. 28 the sequences of primers and conditions of pcr and enzyme digestion were provided in the supplementary materials and methods. the mbl codon 54 a/b variant (rs1800450, g230a) was genotyped by using pcr direct sequencing as described previously. 9 this polymorphism had been investigated in beijing community population and hong kong population in our previous studies 3, 9 ; therefore, the present study genotyped this polymorphism only in beijing health care worker (hcw) and tianjin population. the results of snp genotyping were tested for deviations from hardyeweinberg equilibrium (hwe) by using the markov chain method implemented in the genepop software (available at: http://wbiomed.curtin.edu.au/ genepop/). associations between snps and risk of sars were assessed by use of logistic regression analyses in spss software (version 9.0; spss inc., chicago, il). for ccl2 g-2518a, the reference category is the combination of the heterozygous genotype ag and the homozygous minor genotype aa, while for mbl codon 54 variant (a/b) the reference category is the homozygous major genotype aa. the odds ratios (ors) and 95% confidential intervals (cis) were calculated and adjusted for potential confounders including age (continuous; years), sex (categorical; female or male) and populations (categorical; beijing community, beijing hcw, tianjin, or hong kong), where it was appropriate. the meta-analysis of data generated from multiple stages was conducted to estimate pooled genetic effects using fix-effect model based on mantelehaenszel method. we calculated cochran's q statistic to test for betweengroup heterogeneity and the i 2 statistic to quantify the proportion of the total variation due to heterogeneity. the heterogeneity was considered significant for p < .05. for there was no significant heterogeneity for multiple populations, we further analyzed the pooled data by logistic regression with adjustment for potential confounders. the ccl2 gene resides on chromosome 17q11.2-q12, while the mbl gene resides on chromosome 10q11.2, meaning that ccl2 g-2518a and mbl codon 54 variant (a/b) are not in linkage equilibrium with each other. to further test whether or not a snp is dependent on the other, independence test was performed for a snp with adjustment for the other and potential confounders. logistic regression with the use of an interaction term was also performed to investigate potential geneegene interaction between two snps. for there was no significant interaction detected, we then tested the cumulative effects of snps on sars by counting the number of at-risk genotypes associated with sars for these two snps in each subject. the odds ratio for patients carrying any combination of one or two atrisk genotypes was estimated by comparing them with those carrying none of the at-risk genotypes by logisticregression analysis. the cochranearmitage trend test was used to assess the disease risk upon the increasing number of at-risk genotypes. the population attributable fractions (pafs) were estimated for the at-risk genotypes of ccl2 g-2518a and mbl codon 54 variant (a/b) with the use of the formula paf z f(or e 1)/[1 þ f(or e 1)], where f is the prevalence of at-risk genotype associated with sars and or is used in the place of relative risk. 29 the paf value indicates percentage of the increase in the risk of developing sars attributed to at-risk genotypes. the specificity and sensitivity of the regression model were calculated by constructing receiver-operatingcharacteristic (roc) curves, and then statistics for the area under the curve (auc) were calculated to estimate the ability of models to distinguish case subjects from control subjects. the values for auc range from 50% (no caseecontrol discrimination) to 100% (perfect discrimination of cases and controls). an association was considered significant at a p value of less than .05 in individual populations, and with more stringent threshold in the pooled samples based on the bonferroni correction for multiple testing of two snps. all statistical tests were two-sided. all analyses were performed using the spss (version 9.0, spss inc., chicago, il, usa) and stata 9.2 software (statacorp lp, college station, tx, usa). initially, we estimated the effect of ccl2 g-2518a on susceptibility to sars-cov infection in beijing community population consisted of 352 patients with sars and 392 controls. the observed genotype frequencies for this polymorphism conformed to the hwe in both patients and controls (p > .05). on the basis of logistic regression analysis with adjustment for age and sex, the subjects carrying the gg genotype had a significantly increased susceptibility to sars-cov infection, compared with ones carrying the à2518a allele (i.e., ag plus aa genotype) (or 1.41, 95% ci, 1.03 to 1.92, p z .031; table 1 ). to test for replication of the association, we then genotyped the g-2518a in three additional caseecontrol sample sets. the genotype distributions of all groups did not deviate from the hwe. again, there was an excess of gg genotype in patients than in controls in beijing hcw (p z .025), tianjin (p z .038) and hong kong population (p z .010), respectively. meta-analysis on pooled data from all the four chinese populations provided unequivocal evidence for a relationship between ccl2 g-2518a and susceptibility to sars-cov infection (fig. 1 ). or associated with gg genotype was 1.58 (95% ci, 1.28 to 1.96, p z 2.3 â 10 à5 , p heterogeneity z .43, i 2 z 0%; fig. 1a ). given there was no significant heterogeneity for the four chinese populations, we further analyzed the pooled data by logistic regression, and a strong support for an association between the g-2518a and susceptibility to sars-cov infection was observed (p z 1.6 â 10 à4 ), with the or being 1.48 (95% ci, 1.21e1.82) for the at-risk gg genotype, compared with the ag plus aa genotype. on the basis of the ors combined with the frequencies of gg genotype, pafs were estimated to account for 12.8e53.1% of sars cases in the 4 individual populations, and 14.6% in the overall population (table 1) . we also reassessed the association between sars and the codon 54 variant (a/b) in mbl, which were previously reported to be associated with sars in both beijing community and hong kong population (table 1 ). 3, 9 again, we replicated that the subjects bearing the variant b allele (bb plus ab genotype), which is associated with low serum mbl, 30 have a significantly increased risk of sars-cov infection in both beijing hcw (p z .019) and tianjin population (p z .012), compared with those homozygous for the wild-type a allele. combined analysis of the 4 caseecontrol populations by meta-analysis yielded a stronger support for the association (or 1.82, 95% ci, 1.47 to 2.25, p z 5.3 â 10 à8 , p heterogeneity z .50, i 2 z 0%; fig. 1b ). logistic regression gave a similar result (or 1.79, 95% ci, 1.45 to 2.21, p z 4.9 â 10 à8 ; table 1 ). the pafs were 18.8e53.3% for the 4 individual populations, and the pooled paf was 21.9% for the overall population (table 1) . we further evaluated whether the effects for the ccl2 g-2518a and mbl codon 54 variant (a/b) on risk of sarsfig. 1c) . the joint pafs for the combination of at-risk genotypes of ccl2 and mbl were 29.1e41.0% in the 4 individual populations, and the joint paf was 28.5% in the pooled population (table 2) . we next calculated statistics for auc to estimate the ability of each of three models to distinguish case subjects from control subjects. in the four individual populations, the auc was 53.2e61.0 for model 1 (sex, age, and the number of at-risk genotypes at ccl2), 53.9 to 63.6 for model 2 (sex, age, and the number of at-risk genotypes at mbl), and 55.7 to 65.8 for model 3 (sex, age, region, and the number of at-risk genotypes at both ccl2 and mbl) with all p values equal or less than .10 ( supplementary fig. 1 ). in the pooled population, the auc was 53.7 (95% ci, 51.1 to 56.3; p z 4.8 â 10 à3 ) for model 1, 55.5 (95% ci, 52.9 to 58.1; p z 2.9 â 10 à5 ) for model 2, and 57.0 (95% ci, 54.5 to 59.6; p z 1.0 â 10 à7 ) for model 3 ( supplementary fig. 1) . we also assessed whether there was an association between the ccl2 g-2518a and mbl codon 54 variant (a/ b) and severity of sars, but found that the two snps were associated with the severity of sars neither individually nor jointly (supplementary table 3 ). this study includes 932 patients with sars totally, which accounts for about 12% of the sars cases worldwide during the sars outbreak, being so far the largest cohort in the field of genetic association studies of sars. additionally, given whether the patients were infected in the hospital or community is a potential confounding factor, we matched the cases and controls for this factor in each caseecontrol series. furthermore, we performed statistical adjustment for age and sex to minimize other potential biases. taken together, the large size of the investigation, the consistency of the observations in 4 independent caseecontrol series and the low p values distinguish our study from previous studies investigating the influence of different other polymorphisms on the development of sars, and strengthen the association between the ccl2 g-2518a and mbl codon 54 variant (a/b) and susceptibility to sars-cov infection. to our best knowledge, this is the first report that functional polymorphisms of the ccl2 and mbl genes cumulatively increase susceptibility to sars-cov infection, confirming the initial hypothesis that these genes may play a role in the pathogenesis of this disorder. several previous studies have consistently demonstrated that the carriers of the à2518g allele confer up-regulated transcriptional activity, higher ccl2 mrna and protein levels in vitro and in vivo, and more infiltration of leukocytes into tissues in comparison with a carriers. 21à23 therefore, one might expect that the individuals who carry the at-risk gg genotype, and thus have higher expression of ccl2 and successively more attraction of monocytes and macrophages, may have an increased susceptibility to sars-cov infection. this hypothesis is biologically plausible. previous studies have consistently reported an overexpression of ccl2 in the lung tissues and sera of sars patients. 17e19 the pulmonary tissues of sars patients were also well characterized by pronounced infiltration of monocytes and macrophages. 17, 31, 32 furthermore, sars-cov was shown to infect the monocyte-derived macrophages in vitro 15, 33 and, to be readily detectable in macrophages in lungs and other target organs of sars patients. 31, 32 based on these observations, gu et al. have argued that the sars virus infects resident, infiltrating, and circulating immune cells, such as macrophages and monocytes; and then, these infected circulating cells may carry sars-cov to various target organs as manifested by widespread dissemination. 31, 32 notably, this kind of spread of infection by infected phagocytes (so-called "trojan horses effect") has been implicated before in the infection of hiv, measles virus and other intracellular microorganisms. 34, 35 considering the known pathophysiologic role of the ccl2 in these disorders, these examples further suggest the high-ccl2-producing gg genotype may be the at-risk genotype conferring increased susceptibility to sars-cov infection. consistent with our previous findings, 3, 9 this study further confirmed the significant association between the low-mbl-producing b allele and increased risk of sars-cov infection in two additional independent sample sets, suggesting that mbl deficiency may be a susceptibility factor for the acquisition of sars. the independent confirmation of associations of these two snps with sars-cov infection supports the validity of genetic association studies in complex diseases. this finding promoted us to investigate the role of geneegene interaction in the genotype-to-phenotype relationship of sars-cov infection. however, no consistent significant genetic interaction between these two snps on risk of sars-cov infection was observed across all caseecontrol populations and in the pooled samples. furthermore, no functional studies reported for the interactive effect of mbl and ccl2 on the susceptibility to sars-cov infection. several previous studies have implicated that mbl could increase the secretion of ccl2 from human monocytic u937 cell lines, peripheral blood mononuclear cells and umbilical vein endothelial cells mediated by a variety of bacterial microbes. 36, 37 whether or not there exit biological interactions between mbl and ccl2 under the condition of sars-cov infection remains to be investigated in future studies. it has to be noted that many chemokines not only take part in the process of viral infection, but are also involved in cell damage and organ dysfunction. indeed, several lines of evidence have indicated that ccl2 secretion is upregulated during the development of sars and correlates with intensifying immuno-mediated damage to the lungs and other target organs, resulting in acute lung injury and, subsequently, multi-organ dysfunction. 38 therefore, we speculate that the high-ccl2-producing gg genotype may enhance the inflammation and associate with more severe clinical outcomes of sars. however, we did not find a genetic association between ccl2 polymorphism and admission to intensive care units or deaths due to sars. this result may be due to the limited number of patients with severe sars or died from sars in the present study. for example, as for disease severity this study has merely powers of 14.6% (two-sided test of significance, a z 0.05) to detect ors of >1.66 in beijing community, and 9.5% to detect ors of <0.73 in hong kong population, for carriers of the gg genotype relative to the carriers of the aa plus ag genotypes at the position à2518 of the ccl2 gene (supplementary table 3 ). in contrast, as for sars-cov infection this study has powers of 56.2% to detect ors of >1.41, and 82.7% to detect ors of >1.57, in beijing community and hong kong populations, respectively, for the same snp (table 1) . it certainly warrants confirmation in additional studies with larger collections of sars patients. the allele and genotype frequencies of the ccl2 g-2518a polymorphism vary with ethnicity (according to the hapmap project database). indeed, in this study with 982 control subjects, we found that the frequency of the à2518g allele and gg genotype was 52.0% and 27.0%, compared with around 34.7% and 12.0%, respectively, among caucasians from the iceland. 39 therefore, although the highly significant associations between ccl2 g-2518a and susceptibility to sars-cov infection were biologically plausible, and strengthened by our 4 independent caseecontrol studies, it remains interesting to investigate that whether there exist population-specific differences for this polymorphism to sars susceptibility between chinese and europeans. interestingly, a significant cumulative effect on the susceptibility to sars-cov infection was observed for the combination of two functional snps in ccl2 and mbl across the four independent caseecontrol series (p trend z 1.3 â 10 à10 in the pooled samples). we estimated that individuals who have both of the at-risk genotypes have an odds ratio of 2.88 for sars-cov infection (table 2) . therefore, pafs can be calculated to be 14.6% for ccl2, 21.9% for mbl, and 28.5% for both in the pooled samples, indicating that, for example, 28.5% of the increase in the risk of sars-cov infection can be attributed to carrying 1 or 2 at-risk genotypes of the two polymorphisms. if the associations are real, then at-risk genotypes of the two polymorphisms are associated with a low to moderate fraction of sars-cov infection among chinese, suggesting that there exist unrevealed genes conferring susceptibility to such an infection. in fact, previous reports have shown that polymorphisms of several genes affect an individual's susceptibility to sars-cov infection or disease severity of sars, 3à10 although most of these susceptibility genes need to be confirmed in independent sample sets. in addition, it is notable that at this stage, ccl2 g-2518a and mbl codon 54 variant (a/b) are unlikely to be appropriate for risk prediction testing individually or in combination, because of low aucs (53.7% for ccl2, 55.5% for mbl, and 57.0% for both) in pooled population. however, as further susceptibility polymorphisms are identified and confirmed, and interaction effects among such polymorphisms together with other risk factors are taken into account, prediction of the susceptibility of sars-cov infection may become more accurate and clinically usable. this study has several potential limitations. first, the small sample size of subjects homozygous for the minor allele b of mbl codon 54 variant (a/b) did not allow calculation of separate ors for the bb genotype (the genotype with the greatest risk). second, the small sample sizes likely meant that there was low power to detect genetic interaction between polymorphisms. third, the small sample sizes of the two new caseecontrol populations (beijing hcw and tianjin) meant that the effect estimates for these groups were larger in magnitude and had wider cis (that is, they were less stable as shown in table 1 and fig. 1 ) than the effect estimates generated from the other two larger caseecontrol populations. last, there was no adjustment for co-morbid conditions (such as chronic diseases) that could affect susceptibility to sars. in summary, our findings indicate that individuals who are genetically predisposed to produce greater amounts of ccl2 protein seem to be more susceptible to sars-cov infection. we also confirmed our previous findings that low-mbl-producing allele is a susceptibility factor for the acquisition of sars. furthermore, a combination of ccl2 and mbl polymorphisms has a stronger genetic association with the susceptibility to sars-cov infection. however, 1) no interaction between polymorphisms could be detected; 2) no association was observed between the polymorphisms and severity of sars; and 3) although there were statistically significant associations between the two polymorphisms and sars-cov infection, tests using the auc curve do not indicate that the at-risk genotypes have clinical usefulness (that is, the at-risk genotypes cannot discriminate cases from controls). since both the ccl2 and mbl are important components in innate immunity system, our findings further support the hypothesis that variability in the innate immune response can be involved in susceptibility to sars-cov infection during the vulnerable period before the production of specific antibodies. 9 sars-associated coronavirus pathogenesis of severe acute respiratory syndrome mannose-binding lectin in severe acute respiratory syndrome coronavirus infection lack of support for an association between clec4m homozygosity and protection against sars coronavirus infection lack of support for an association between clec4m homozygosity and protection against sars coronavirus infection il-12 rb1 genetic variants contribute to human susceptibility to severe acute respiratory syndrome infection among chinese homozygous l-sign (clec4m) plays a protective role in sars coronavirus infection the association of rantes polymorphism with severe acute respiratory syndrome in hong kong and beijing chinese association between mannose-binding lectin gene polymorphisms and susceptibility to severe acute respiratory syndrome coronavirus infection genetic variation of the human alpha-2-heremans-schmid glycoprotein (ahsg) gene associated with the risk of sars-cov infection genetic susceptibility to infectious disease: lessons from mouse models of leishmaniasis monocyte chemoattractant protein-1 control of th2 polarization by the chemokine monocyte chemoattractant protein-1 chemokine up-regulation in sars-coronavirus-infected, monocytederived human dendritic cells modeling the early events of severe acute respiratory syndrome coronavirus infection in vitro lung pathology of fatal severe acute respiratory syndrome plasma inflammatory cytokines and chemokines in severe acute respiratory syndrome an interferon-gamma-related cytokine storm in sars patients characterization of cytokine/chemokine profiles of severe acute respiratory syndrome the pdz-binding motif of severe acute respiratory syndrome coronavirus envelope protein is a determinant of viral pathogenesis hiv-1 infection and aids dementia are influenced by a mutant mcp-1 allele linked to increased monocyte infiltration of tissues and mcp-1 levels a novel mcp-1 gene polymorphism is associated with hepatic mcp-1 expression and severity of hcv-related liver disease ccl2 polymorphisms are associated with serum monocyte chemoattractant protein-1 levels and myocardial infarction in the framingham heart study effect of genetic variants of ccr2 and ccl2 on the natural history of hiv-1 infection: ccl2-2518gg is overrepresented in a cohort of spanish hiv-1-infected subjects association of common promoter polymorphisms of mcp1 with hepatitis b virus clearance polymorphisms in the genes encoding chemokine receptor 5, interleukin-10, and monocyte chemoattractant protein 1 contribute to cytomegalovirus reactivation and disease after allogeneic stem cell transplantation a functional promoter polymorphism in monocyte chemoattractant protein-1 is associated with increased susceptibility to pulmonary tuberculosis association of cytokine and chemokine gene polymorphisms with severe acute respiratory syndrome the use of attributable fraction in the design and interpretation of epidemiologic studies mannose-binding lectin deficiencyerevisited multiple organ infection and the pathogenesis of sars molecular pathology in the lungs of severe acute respiratory syndrome patients sars-coronavirus replication in human peripheral monocytes/macrophages mechanisms of microbial traversal of the blood-brain barrier hiv infection and dementia mannose-binding lectin without the aid of its associated serine proteases alters lipopolysaccharide-mediated cytokine/chemokine secretion from human endothelial cells c1q and mbl, components of the innate immune system, influence monocyte cytokine expression the immunobiology of sars* examination of genetic effects of polymorphisms in the mcp-1 and ccr2 genes on mi in the icelandic population no potential conflict of interest relevant to this article was reported. supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.jinf.2015.03.006. key: cord-341754-rr7ei8o2 authors: aitken, tess; chin, ken lee; liew, danny; ofori-asenso, richard title: rethinking pandemic preparation: global health security index (ghsi) is predictive of covid-19 burden, but in the opposite direction date: 2020-05-08 journal: j infect doi: 10.1016/j.jinf.2020.05.001 sha: doc_id: 341754 cord_uid: rr7ei8o2 nan in the wake of the ebola outbreak in 2014, the global health security index (ghsi) was developed with the aim of gauging countries' capacity to deal with infectious disease outbreaks. 2 the ghsi highlights the shortcomings of existing pandemic policies and procedures, with the aim of spurring improvement of future practices. the index ranges from 0 to 100, and assesses six core elements: prevention, detection and reporting, response, health system, compliance with norms and risk of infectious disease outbreaks. 2 a higher ghsi indicates better preparedness. in the present study, we examined the correlation between ghsi and various measures of covid-19 burden across different countries. we hypothesised that higher ghsi was inversely associated with measures of covid-19 burden. country-level data on covid-19 as at 11 april 2020 were sourced from the 'worldometer'. 3 countries without testing data, or those with no assigned ghsi score were excluded. furthermore, we included only countries with at least 100 confirmed cases of covd-19. data on countries' median age and proportion of females in 2019 were sourced from the united nations population database. 4 we analysed the association between ghsi and covid-19 burden, represented by numbers of tests confirmed cases and deaths per million people per day since the first confirmed case in each country. first, we plotted ghsi against natural log transformed values of these outcomes (to provide more symmetrical distributions). secondly, we used a generalised linear model (glm) to determine the association between ghsi and confirmed cases and deaths per million people per day, with adjustment for testing rate, population median age and proportion of females. we considered ghsi both as a continuous variable and as a categorical variable comprising four quartiles. in the latter analyses, the first (lowest) quarter of ghsi was considered as the reference category. a total of 100 countries with complete data were included in the analysis (supplementary table s1 covid-19 metrics (log transformed) plotted against ghsi are presented in figure 1 . these suggest a positive correlation between ghsi and testing rate, as well as cases and deaths per million people per day since the first recorded case. of note, the us was the highest ranked country in terms of ghsi of all 100 countries analysed yet had the largest number of covid-19 cases worldwide at the time of analyses. 2,3 secondranked uk was also bearing a large burden of disease. the results from the glm model are presented in table 1 . there was no statistically significant association observed between ghsi and testing rate. after adjusting for testing rate, median age and the proportion of females, a positive association was also observed between ghsi and covid-19 cases and deaths, with the biggest burden borne by countries at the highest quartile of ghsi. the findings of our study were unexpected. first, no association was noted between ghsi and testing rate, despite that ghsi should serve as a surrogate for healthcare capacity, including covid-19 testing. effective pandemic response requires significant investment in testing, with adequate training of healthcare workers in testing, as well as sufficient supply of ppe and testing kits. 5 in addition, effective and widespread dissemination of information to the general population regarding testing criteria assists case detection. 5 secondly, the associations between ghsi and covid-19 cases and deaths were positive, meaning that the ghsi can reflect a country's capacity to deal with epidemics or pandemics, but in the opposite manner than intended. no doubt there was confounding by increased globalisation among more developed countries (with higher ghsi). increased exposure to foreigners travelling for the purposes of tourism, business and use of healthcare is likely to increase the risk of new infectious pathogens being introduced. similarly, mass migration contributes to disruption of local bacterial and viral environments. 2 furthermore, the rarity of pandemics in conjunction with false reassurance from a high ghsi may have contributed to more lenient adherence to infection control mechanisms in recent years. 6 the intent of the ghsi is noble, and the findings of our study should not discourage future endeavours to gauge capacity to respond to pandemics. however, as the world becomes increasingly interconnected, the value of assessing the capacity of countries to manage infectious outbreaks individually is redundant. this interconnectedness extends beyond social, political, and business interactions to pathogenic environments. consequently, identifying and controlling spread of newly arising infectious agents is only as effective as the practices within the poorest performing countries. the covid-19 pandemic has revealed insufficiencies in existing knowledge of pandemic preparedness and response. a more integrated global approach is necessary, as is further research into alternative factors related to infection control that have not yet been considered. development of international response protocols and effective communication channels will permit coordinated global action. furthermore, establishment of dynamic models and tools will ensure the world is better prepared for future outbreaks. global covid-19 fatality analysis reveals hubei-like countries potentially with severe outbreaks the economist intelligence unit. global health security index worldometer covid-19 coronavirus pandemic united nations, department of economic and social affairs evaluation of national pandemic management policies-a hazard analysis of critical control points approach world health organisation. the world health report 2007 -a safer future: global public health security in the 21st century key: cord-311766-m9yv4qkm authors: demey, baptiste; daher, nagib; françois, catherine; lanoix, jean-philippe; duverlie, gilles; castelain, sandrine; brochot, etienne title: dynamic profile for the detection of anti-sars-cov-2 antibodies using four immunochromatographic assays date: 2020-05-07 journal: j infect doi: 10.1016/j.jinf.2020.04.033 sha: doc_id: 311766 cord_uid: m9yv4qkm in order to fight the sars-cov-2 pandemic infection, there is a growing need and demand for diagnostic tools that are complementary and different from the rt-pcr currently in use. multiple serological tests are or will be very soon available but need to be evaluated and validated. we have thus tested 4 immunochromatographic tests for the detection of antibodies to sars-cov-2. in addition, we assessed the kinetics of antibody appearance using these assays in 22 patients after they were tested positive by rt-pcr. we observed great heterogeneity in antiboy detection post-symptom onset. the median antibody detection time was between 8 and 10 days according to the manufacturers. all the tests showed a sensitivity of 60 to 80% on day 10 and 100% on day 15. in addition, a single cross-reaction was observed with other human coronavirus infections. thus, immunochromatographic tests for the detection of anti-sars-cov-2 antibodies may have their place for the diagnostic panel of covid-19. in order to fight the sars-cov-2 pandemic infection, there is a growing need and demand for diagnostic tools that are complementary and different from the rt-pcr currently in use. multiple serological tests are or will be very soon available but need to be evaluated and validated. we have thus tested 4 immunochromatographic tests for the detection of antibodies to sars-cov-2. in addition, we assessed the kinetics of antibody appearance using these assays in 22 patients after they were tested positive by rt-pcr. we observed great heterogeneity in antiboy detection postsymptom onset. the median antibody detection time was between 8 and 10 days according to the manufacturers. all the tests showed a sensitivity of 60 to 80% on day 10 and 100% on day 15. in addition, a single cross-reaction was observed with other human coronavirus infections. thus, immunochromatographic tests for the detection of anti-sars-cov-2 antibodies may have their place for the diagnostic panel of covid-19. keywords: sars-cov-2; covid-19, antibody, lateral flow assay since december 2019, the world has been facing a pandemic of covid-19, an infectious disease caused by sars-cov-2, a virus that emerged in china (wu and mcgoogan, 2020) . although rt-pcr testing of sars-cov-2 has become the standard method for direct diagnosis, these real-time pcr tests have some limitations, primarily dedicated infrastructure to avoid any biorisk, limited capacity and a long turnaround time (y. . there is increasing pressure from the medical community and society to screen the population on a large scale. serological tests in elisa format or as immunochromatographic lateral flow assay (lfa) have recently become available from many manufacturers (z. (haveri et al., 2020) . these serological tests will be complementary to pcr tests both for screening and diagnosis of the population, for the purpose of population exits from containment in different countries and finally for future epidemiological studies. however, it is necessary to evaluate the analytical performance of these assays and also their place in clinical practice. thus, the objective of our study was to evaluate four immunochromatographic assays for the detection of igm and igg antibodies to sars-cov-2 and to evaluate the kinetics of their detection by these lfa. twenty two patients diagnosed positive in amiens university hospital for sars-cov-2 on a nasopharyngeal swab using a rt-pcr technique (national reference center in pasteur institute, paris, france) were included in our study. the date of reporting of the first symptoms was retrieved from the medical records. the samples were tested regularly during the hospitalization until the tests were positive, with an evaluation at most on day 24 post-symptoms. in order to evaluate a possible crossreaction with the other human coronaviruses described to date (nl63, hku1, 229e and oc43), sera following such viral respiratory infection diagnosed in our lab were tested. this project was conducted in accordance with the reference methodology (mr-004 france) in accordance with article 30 of the gdpr. we evaluated 4 immunochromatographic tests for the detection of igm and igg directed against sars-cov-2 ( figure 1 ). these tests were kindely provided by asian manufacturers, namely biotime biotechnology co, autobio diagnostics co, isia bio-technology co and biolidics. for the biotime, autobio, and biolidics tests the detection of igm and igg is performed on the same diagnostic cassette. for isia 2 different cassettes are available. each test requires between 10 and 20 µl of serum, plasma or whole blood and is read 10 to 15 minutes after the sample and diluent have been deposited. for the biotime and biolidics assays, respectively 15 and 17 of the 22 patients could be tested for lack of immunochromatographic tests. longitudinal immunochromatographic testing in all patients shows heterogeneity in the time to detection of antibodies after symptom reporting (figure 2 ). the median antibody detection time was 8 days since onset of symptoms for autobio and biotime (igm or igg), 9 days for biolidics (igm or igg) and 9 and 10 days for isia igm and igg respectively (figure 2 and supplementary data). igg was detected in all patients on day 15 since onset of symptoms, while igm was not detected in 3 patients with autobio and isia. igm was detected before igg in 1, 1, 7 and 0 patients with the biotime, autobio, isia and biolidics assays respectively. in the other cases, igm was detected at the same time as igg. thus, the diagnostic interest of detecting igm directed against cov-2-sars appears limited. the clinical sensitivity of the different tests could be assessed longitudinally during follow-up and we observed an increasing sensitivity in the post-symptom period ( figure 3 and table 1 ). as described above, the clinical sensitivity of igm does not appear to be superior to igg for these immunochromatographic tests. with either igm or igg detection for a patient on days 5, 10 and 15 since onset of symptom, we calculated a clinical sensitivity between 9 and 24%, 67 and 82% and 100% respectively ( figure 3b and table 1 ). the autobio test appears to have better sensitivity at day 10 (81.82%) versus 70.59%, 68.18% and 66.67% for biotime, isia and biolidics respectively (not significant). in order to evaluate the specificity of these different immunochromatographic tests, particularly regarding previous infections to other viruses of the human coronavirus family, we evaluated 12 sera from patients who had a rt-pcr diagnosis of respiratory infection by different coronaviruses in 2019 (table 2) . of the 41 tests performed, only one (autobio) was positive from the serum of a patient with a respiratory diagnosis 39 days previously of hcov-229e. the same test for the other three samples with hcov-229e was negative each time. cross-reactions with these different coronaviruses therefore appear to be limited but may require further investigation. in this study we demonstrated the kinetics of detection of antibodies to sars-cov-2 using immunochromatographic lfa. these simple rapid unit tests are also easy to read (figure 1 ). profiling early humoral response were already observed with elisa assay (guo et al., 2020) (zhao et al., 2020) . we calculated increasing clinical sensitivities over time from the onset of symptoms in patients. moreover, we did not observe any real added value in igm staining from these immunochromatographic tests. with this kind of test, it is very difficult to distinguish the very recent infection from the older one because some patients present early with igg without igm and for some a detectable igm threshold appears later. serological elisa tests from research laboratories or portfolios of in vitro diagnostic manufacturers may allow a clearer distinction between igm and igg kinetics and their respective interest. nevertheless, the value of these point of care tests seems obvious in countries with limited resources but perhaps also as the epidemic progresses in individuals in the form of self-homemade assay from a drop of blood on the fingertip. this type of test could be easily delivered to individuals and thus limit contact. in addition, in countries with strong health systems, these rapid detection tests may also find their way as doctor tests in emergency departments. during this covid-19 epidemic there is a rebound in symptoms on days 7 to 10 leading to a visit to a doctor or an emergency department. in figure 3 and table 1, we have calculated a sensitivity of these tests between 22% and 81% during this post-symptom reporting period. even if symptom reporting remains very subjective and time-varying but if we add an average incubation period of 5 days (linton et al., 2020) , we can say that at 14-15 days post-infection these tests seem reliable. finally, regarding specificity that we evaluated with respect to sera of other common coronavirus infections, we observed a single cross-reaction. however, we were unable to test serum from people formerly infected with sars-cov (tian et al., 2020) . we would probably have many more cross reactions between these very close viruses as already report. also, due to the lack of available tests, we could not test for specificity with samples containing antibodies to other viruses (hiv, hcv, hbv and others pathogens). in conclusion, we described the kinetics of detection of post-symptom antibodies in 22 patients using immunochromatographic rapid tests and demonstrated the good performance of these tests for the detection of antibodies after sars-cov-2 infection. our results suggest that these rapid and simple tests should be seriously considered in this time of health and political crisis to monitor both symptomatic and non-symptomatic patients. profiling early humoral response to diagnose novel coronavirus disease (covid-19) serological and molecular findings during sars-cov-2 infection: the first case study in finland stability issues of rt-pcr testing of sars-cov-2 for hospitalized patients clinically diagnosed with covid-19 development and clinical application of a rapid igm-igg combined antibody test for sars-cov-2 infection diagnosis incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data potent binding of 2019 novel coronavirus spike protein by a sars coronavirusspecific human monoclonal antibody characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention antibody responses to sars-cov-2 in patients of novel coronavirus disease key: cord-305462-2wz1f6k6 authors: beckham, j. david; cadena, ana; lin, jiejian; piedra, pedro a.; glezen, w. paul; greenberg, stephen b.; atmar, robert l. title: respiratory viral infections in patients with chronic, obstructive pulmonary disease date: 2004-09-22 journal: j infect doi: 10.1016/j.jinf.2004.07.011 sha: doc_id: 305462 cord_uid: 2wz1f6k6 objectives: the purpose of the present study was to apply reverse transcription-pcr (rt-pcr) assays to clinical specimens collected from patients with acute respiratory illness and chronic obstructive pulmonary disease (copd). methods: one hundred and ninety-four samples from two different study cohorts were analysed using rt-pcr assays for picornaviruses, coronaviruses 229e and oc43, influenza a and b viruses, respiratory syncytial virus, parainfluenza types 1–3 viruses, and human metapneumovirus and a pcr assay for adenoviruses. the results were added to results obtained previously using cell culture and serologic methods. results: rt-pcr assays identified an additional 35 respiratory virus-associated illnesses not identified previously by cell culture or serology (n=46). picornaviruses and coronaviruses were the most common viral infections identified only by rt-pcr. overall, 41.8% of the acute respiratory illnesses evaluated were associated with a respiratory virus infection, with picornaviruses, coronaviruses and influenza viruses being the most common infections recognized. no human metapneumovirus infections were identified by rt-pcr assay. conclusions: respiratory viral infections are commonly associated with acute respiratory illness in copd patients, and the use of rt-pcr assays significantly increases the ability to diagnose these infections. the fourth most common cause of death in the united states is chronic lower respiratory disease. 1, 2 journal of infection (2005) patients with chronic obstructive pulmonary disease (copd) also suffer recurrent exacerbations that increase the morbidity of the disease and contribute to its mortality. 3 acute exacerbations of copd are associated with acute infections of the respiratory tract caused by bacteria, viruses, or both bacteria and viruses. 4 the frequency in which a respiratory virus infection has been identified in association with acute worsening of respiratory function in patients with copd has varied in different studies. [3] [4] [5] [6] [7] [8] [9] we previously performed two studies that evaluated the prevalence of acute respiratory viral infections in patients, including those with copd. 8, 10 both studies utilized cell culture and serologic methods to identify acute respiratory virus infections. study 1 examined the frequency of specific respiratory virus infections in persons with acute respiratory tract condition that lead to hospitalization. 10 in study 2, a longitudinal cohort of copd patients were tested for rtvis in an ambulatory setting, although some patients were also hospitalized. 8 respiratory viral infections were identified in 19% of copd subjects with acute respiratory illness. reverse-transcription polymerase chain reaction (rt-pcr) assays can identify respiratory virus infection missed by cell culture. 11 the number of respiratory viral infections identified in asthmatic patients with acute exacerbations of disease increase significantly when rt-pcr assays are used in addition to other diagnostic methods. 12, 13 a similar observation has been made for acute exacerbations of copd. 3 in order to extend our understanding of the prevalence of respiratory viral infection in acute respiratory illnesses in patients with copd, we used rt-pcr assays to evaluate samples from the previous two prospective studies for evidence of respiratory virus infection. 8, 10 materials and methods patients with copd were enrolled in two prospective clinical studies designed to identify the respiratory viruses associated with exacerbations of their lung disease. 8,10 study 1 evaluated the prevalence of rtvis among patients hospitalized with an admitting diagnosis of acute respiratory illness or congestive heart failure between july 1991 and june 1995. 10 patients were identified by review of the admission log. persons with exacerbations of copd enrolled in this study were identified using discharge diagnosis codes (icd9 codes 491. 2, 491.21, 492, 493.2, 496) . study 2 was a longitudinal study conducted between september 1991 and may 1995 to evaluate the incidence of rtvis in a cohort of 62 patients with copd. 8 the criteria for diagnosis of copd were as defined by the american thoracic society. 14 an exacerbation of copd was defined as meeting one or more of the criteria from anthonisen et al. 15 increased dyspnoea, sputum production or sputum purulence. rtvis in both studies were identified by virus culture of combined nasal wash/throat swab specimens and by serologic assays as previously described. 8, 10 for this evaluation, available samples collected between september 1991 and june 1995 were used for analysis. combined nasal wash/throat swab samples were transported to the laboratory on wet ice, inoculated onto cell culture (usually within 12 h), and then stored at k80 8c prior to the performance of viral rt-pcr assays. microneutralization assays were used to measure antibody levels to influenza a and b viruses, respiratory syncytial virus, parainfluenza types 1, 2 and 3 viruses, and coronavirus 229e. hemagglutination inhibition assays also were used to measure influenza a and b virus antibody, and an enzymelinked immunosorbent assay (elisa) was used to measure antibody to coronavirus oc43. 8, 10 rt-pcr studies the following rt-pcr or pcr assays (limits of detection) were used to assay the clinical specimens for the following: influenza a and b viruses (1-10 tissue culture 50% infectious dose [tcid 50 ]), respiratory syncytial virus (10-50 tcid 50 ), parainfluenza virus types 1, 2 and 3 (10-50 tcid 50 ), coronavirus oc43 and 229e (1-10 tcid 50 ), picornaviruses (!1 tcid 50 ), adenoviruses (10-50 tcid 50 ), and human metapneumovirus (10-50 tcid 50 ) ( table 2 ). these assays consist of three major steps: (1) nucleic acid extraction; (2) complementary dna (cdna) synthesis and amplification (rt-pcr); and (3) identification of virus-specific amplicons by southern blot hybridization. nucleic acids were extracted from nasal wash/throat swab samples using the q1amp viral rna mini kit (qiagen inc.; valencia, ca) according to the manufacturer's instructions. rt-pcr/pcr assays were performed as previously described using primers listed in table 1 . 13, [16] [17] [18] [19] [20] for detection of human metapneumovirus infection, cdna was synthesized for 1 h at 43 8c. after an initial 5-minute heat denaturation at 94 8c, 40 cycles of heat denaturation at 92 8c for 15 s, annealing at 55 8c for 30 s, and primer extension at -iia aat tgc tii tct tgt tct . virus-specific pcr products were detected by southern hybridization using digoxigenin-labeled probes (see table 1 ). human metapneumovirus-specific amplimers were identified following hybridization with a virus-specific digoxigenin-labeled oligoprobe at 50 8c. real-time rt-pcr assays also were performed for influenza a and b viruses and coronavirus oc43 and 229e viruses. the influenza virus-specific assays were performed as previously described. 21 for the coronavirus-specific assays, cdna was generated in a single reaction in the thermal cycler at 43 8c for 1 h. specimens were assayed in duplicate in a 50 ml reaction containing 25 ml of 2! taqman universal pcr master mix (applied biosystems, foster city, ca), 5 ml of cdna, 900 nm forward and reverse primers, and 100 nm fluorogenic probes. amplification and detection were performed using the geneamp 5700 sequence detection system (applied biosystems) at the following conditions: 10 min at 95 8c for denaturation followed by 40 cycles of 15 s at 95 8c and 1 min at 60 8c. extraction and reagent positive and negative controls were used in each experiment to assure the validity of the experimental results. steps to prevent carryover contamination were used routinely such as separation of sample preparation, separate pcr set-up and post-pcr analysis areas, the use of plugged pipette tips, traffic control from 'clean' (pre-pcr) to 'dirty' (post-pcr) areas, and the use of extraction and reagent positive and negative controls. for an assay to be considered valid, negative controls yielded negative results and positive controls yielded positive results. there were 96 study subjects with 194 separate illnesses for which samples were available for analysis ( table 2 ). participants in study 1 were younger (61.3 vs. 66.7 years, pz0.005), more likely to be non-white (71% vs. 21%, p!0.05), and less likely to have received influenza vaccination (49% vs. 89%, p!0.001) than those in study 2. the majority of subjects in both studies had a smoking history, but a larger percentage of persons in study 1 were still smoking at study enrollment (46% vs. 15%, p!0.01). paired sera were available for analysis for fewer illnesses in study 1 than in study 2 (38% vs. 91%, p!0.001). eighty-eight respiratory viral infections were identified in 81 illnesses in the two studies; there were seven illnesses in which dual respiratory infections were identified (table 3) . fifty (57%) of the 88 infections were identified by culture or serologic methods while the rt-pcr assays identified an additional 38 infections. overall, picornaviruses (rhinoviruses and enteroviruses) were the most common respiratory virus infections identified and the most frequent virus group identified only by rt-pcr. twenty-eight of the isolated picornaviruses were further classified to the genus level: 24 were rhinoviruses and 4 were enteroviruses. influenza viruses and coronaviruses were the next most commonly identified viruses. fourteen of the 16 influenza virus infections were caused by influenza a viruses. influenza virus infection was more common in study 1 where none of the influenza virus-infected subjects were documented to have received influenza vaccine in the prior year. twelve of the 16 coronavirus infections identified were caused by oc43-like strains. only one of the four infections with 229e-like strains occurred in hospitalized subjects, and all four of these infections were identified by rt-pcr. parainfluenza virus infections occurred in less than 5% of the illnesses. two, two and five parainfluenza virus infections were seen with types 1, 2 and 3 strains, respectively. rt-pcr identified two additional type 3 infections and one additional type 1 infection. rsv infections were this study this study this study a for real-time pcr assay. seen in 3.6% of the subjects. no additional rsv infections were identified by rt-pcr. rt-pcr was the only assay used to identify human metapneumovirus infections, and no infections with this virus were identified. fifteen of the respiratory illnesses in study 2 were upper respiratory illnesses only, and all were managed in the outpatient setting. eighty percent (12/15) of these illnesses were associated with a respiratory viral infection compared to 38% (39/102) of the acute exacerbations of copd in this study. there were six picornavirus, four coronavirus, two parainfluenza virus, one influenza virus, and one rsv infections (two dual) associated with these common cold illnesses. the application of rt-pcr assays to clinical specimens collected from patients with copd and acute respiratory illness significantly increased the number of illnesses in which an associated respiratory viral infection was identified. when culture and serologic studies were used for virus identification, 23.4% of the sampled illnesses were associated with a respiratory virus infection. the addition of rt-pcr assays increased the overall percentage of respiratory virus-associated illness to 41.8%. this is similar to the w40% of patients seemungal et al. 3 noted to have a respiratory virus infection in association with an acute exacerbation of copd and somewhat lower than the 56% prevalence rate rohde et al. 22 noted in copd patients hospitalized with an acute exacerbation and the 64% reported by seemungal et al. 9 in a later study. the percentage of dual respiratory infections (7/81, 8.6%) is also similar to rates reported in previous studies. 23 picornaviruses and coronaviruses were the most likely viral pathogens to be detected using rt-pcr assays. picornavirus infections, especially those caused by rhinoviruses, are the most prevalent of respiratory virus infections identified in copd patients with acute respiratory illnesses in this (44%) and other recent studies (36-58%). 3, 22 many rhinoviruses grow poorly in cell culture and are only detected when rt-pcr assays are used. 3, 12, 13, 24, 25 similarly, coronaviruses are either difficult to grow (229e) or do not grow at all (oc43) using current cell culture methods, so it is not surprising that the number of infections identified increases with the application of rt-pcr techniques. coronaviruses are associated with 11-20% of viral illnesses in copd patients when rt-pcr assays are used. 3, 25 influenza virus remains an important cause of virus infection in copd patients, especially in the hospitalized patient. fifteen of the 77 (19%) illnesses in study 1 were associated with an influenza virus infection compared to only four of 117 (3.4%) in study 2. less than half of the patients in study 1 had received influenza vaccine in the previous year while almost 90% of the patients in study 2 had accepted influenza vaccine in the same time frame. the use of influenza vaccine is safe, effective and recommended for this population. [26] [27] [28] nevertheless, lack of use of influenza vaccine persists as a modifiable risk factor in patients hospitalized with copd. 29 respiratory syncytial virus is increasingly being recognized as an important viral pathogen in older adults. [30] [31] [32] just under 4% of the illnesses evaluated in the two studies were associated with rsv infection, a number similar to that observed in some studies but lower than that reported in others. 3, 22, 33 differences in methods used to identify rsv infection and differences in the sensitivities in the rt-pcr assays likely contribute to the differences observed between studies. 34 human metapneumovirus is a newly recognized paramyxovirus that causes lower respiratory disease in children. 35, 36 the importance of this pathogen in adults is less well defined. falsey et al. 37 found up to 10% of subjects admitted with acute cardiopulmonary conditions had serologic evidence of acute human metapneumovirus infection. prevalence rates were somewhat lower in an ambulatory geriatric population, and there was considerable variability between the two study years. cases of human metapneumovirus infection also have been observed in elderly patients hospitalized with chronic lower respiratory diseases or immunosuppression. 38, 39 in contrast, we failed to identify any illnesses associated with human metapneumovirus infection. our rt-pcr assay for human metapneumovirus was designed to detect all known groups of human metapneumovirus and has detected these viruses in another study involving hospitalized children (unpublished data), differences in assay performance could explain the results observed. alternatively, virus circulation during the seasons studied may have been low enough to prevent detection. 37 a potential weakness of this study is that we did not evaluate the impact of respiratory viruses in our two populations during times of clinical stability. seemungal et al. 9 reported detection of respiratory viruses other than rsv in 16% of copd patients during clinical stability and detection of rsv in 23.5%. we have recently completed an evaluation of another cohort of chronic bronchitic patients during periods of clinical stability (unpublished data). the prevalence of rt-pcr positivity (excluding coronavirus 229e and human metapneumovirus) ranged from 0 to 2% for individual agents, with an overall prevalence of 6.4% in 143 encounters. the overall frequency of identification of a respiratory viral infection during illness was similar to that seen in this study. thus, our experience with the application of rt-pcr assays to cohorts with copd differs from that described by seemungal et al. 9 in summary, the application of rt-pcr assays to clinical specimens collected from patients with copd during clinical respiratory illness significantly increased the number of respiratory viruses identified. rhinoviruses, coronaviruses and influenza viruses were the most common respiratory virus infections in this patient population, and no human metapneumovirus infections were found. respiratory virus infections are an important cause of respiratory illness in copd patients. health and by a research grant from glaxosmithkline. deaths: final data for 1999 deaths: final data for effect of exacerbation on quality of life in patients with chronic obstructive pulmonary disease severe exacerbations of copd patients: the role of pulmonary infections interactions between viruses and bacteria in patients with chronic bronchitis impact of respiratory virus infection in patients with chronic chest disease chronic airway disease: the infection connection respiratory viral infections in adults with and without chronic obstructive pulmonary disease respiratory viruses, symptoms, and inflammatory markers in acute exacerbations and stable chronic obstructive pulmonary disease impact of respiratory virus infections on persons with chronic underlying conditions role of viruses in exacerbations of chronic obstructive pulmonary disease community study of role of viral infections in exacerbations of asthma in 9-11 year old children respiratory tract viral infections in inner-city asthmatic adults standards for the diagnosis and care of patients with chronic obstructive pulmonary disease (copd) and asthma. this official statement of the american thoracic society was adopted by the ats board of directors antibiotic therapy in exacerbations of chronic obstructive pulmonary disease typing and subtyping clinical isolates of influenza virus using reverse transcription-polymerase chain reaction comparison of reverse transcription-pcr with tissue culture and other rapid diagnostic assays for detection of type a influenza virus infectious exacerbations of chronic obstructive pulmonary disease associated with respiratory viruses and non-typeable haemophilus influenzae rapid diagnosis of human parainfluenza virus type 1 infection by quantitative reverse transcription-pcr-enzyme hybridization assay detection of respiratory syncytial virus by rna-polymerase chain reaction and differentiation of subgroups with oligonucleotide probes simultaneous detection of influenza viruses a and b using real-time quantitative pcr respiratory viruses in exacerbations of chronic obstructive pulmonary disease requiring hospitalisation: a case-control study dual respiratory virus infections use of polymerase chain reaction for diagnosis of picornavirus infection in subjects with and without respiratory symptoms rhinovirus and coronavirus infection-associated hospitalizations among older adults centers for disease control and prevention. prevention and control of influenza: recommendation of the american committee on immunization practices influenza vaccine for patients with chronic obstructive pulmonary disease does influenza vaccination increase consultations, corticosteroid prescriptions, or exacerbations in subjects with asthma or chronic obstructive pulmonary disease? patients hospitalized for copd have a high prevalence of modifiable risk factors for exacerbation (efram study) risk factors for severe respiratory syncytial virus infection in elderly persons mortality associated with influenza and respiratory syncytial virus in the united states winter viruses: influenza-and respiratory syncytial virus-related morbidity in chronic lung disease respiratory syncytial and other virus infections in persons with chronic cardiopulmonary disease reverse transcription polymerase chain reaction (rt-pcr) for diagnosis of respiratory syncytial virus infection in adults: use of a single-tube 'hanging droplet' nested pcr a newly discovered human pneumovirus isolated from young children with respiratory tract disease human metapneumovirus: a new player among respiratory viruses human metapneumovirus infections in young and elderly adults virological features and clinical manifestations associated with human metapneumovirus: a new paramyxovirus responsible for acute respiratory-tract infections in all age groups prevalence and clinical symptoms of human metapneumovirus infection in hospitalized patients rapid detection of parainfluenza virus type 3 rna in respiratory specimens: use of reverse transcription-pcr-enzyme immunoassay coronavirus infects and causes demyelination in primate central nervous system neuroinvasion by human respiratory coronaviruses rapid typing of human adenoviruses by a general pcr combined with restriction endonuclease analysis detection of adenoviruses in stools from healthy persons and patients with diarrhea by two-step polymerase chain reaction this study was supported by research contract n01-ai-15103 from the division of microbiology and infectious diseases, national institute of allergy and infectious diseases, national institutes of key: cord-321989-1enjopig authors: li, yanpeng; deng, xilong; hu, fengyu; wang, jian; liu, ying; huang, huang; ma, jinmin; zhang, jianhui; zhang, fuchun; zhang, chiyu title: metagenomic analysis identified co-infection with human rhinovirus c and bocavirus 1 in an adult suffering from severe pneumonia date: 2017-10-31 journal: j infect doi: 10.1016/j.jinf.2017.10.012 sha: doc_id: 321989 cord_uid: 1enjopig • we first present a hrv-c and hbov1 co-infection in an adult woman with severe acute respiratory distress syndrome. • our study suggests that hbov1 co-infection might worsen the disease caused by hrv-c infection, and raises a question whether hrv-c-related severe pneumonia is often associated with co-infection of other respiratory viruses such as bocavirus. dear editor, human rhinovirus (hrv) and bocavirus (hbov) are two of the most common respiratory viruses associated with acute respiratory tract infections among children. 1, 2 recent articles in this journal have highlighted the role of bocavirus and rhinovirus in potentially severe pneumonia in adults and younger children. 3, 4 however co-infection with both viruses was very rare among adults. a previous study reported a 16-month child who was infected by hbov1 and also showed presence of hrv, developed severe pneumonia. 5 here we reported co-infection with hbov1 and hrv-c in an adult woman with acute respiratory distress syndrome (ards). a 26-year-old woman caught a cold on jan. 28, 2017, and developed symptoms of recurrent fever, cough, chills, expectoration, slight hemoptysis, muscle and joint pain in the following days (fig. 1a) . she felt chest tightness and shortness of breath on february 1, and visited guangzhou eighth people's hospital for further examination and treatment on february 3 since her condition continuously worsened with cyanotic lips and pararthria. the patient had fever of 39.3°c, heart rate of 144 beats/ min, respiratory rate of 38 breaths/min, blood pressure of 112/66 mmhg, and pulse oxygen saturation (spo2) of 85%. laboratory evaluation showed white blood cell count 2.2-3.77 × 10 9 /l(normal, 3.5-9.5 × 10 9 /l), lymphocyte 0.61 × 10 9 /l (normal, 1.1-3.2 × 10 9 /l), platelet count (plt) 72 × 10 9 /l (normal, 100-350 × 10 9 /l), c-reactive protein 89.5 mg/l (normal, <8.0 mg/l), hemoglobin (hb) 108 g/l (normal, 110-150 g/l), oxygenation index (oi) 105 mmhg (normal, 400-500 mmhg), cd3 t cell 197/mm 3 (normal, 500-1500/mm 3 ), cd4 t cell 197/mm 3 (normal, 300-1200/mm 3 ), and cd8 t cell 121/mm 3 (normal, 238-874/mm 3 ) (supplementary table s1 ). auscultation of the lungs revealed extensive moist rales and computed tomographic (ct) scan showed bilateral diffuse infiltration (fig. 1a) . she was diagnosed with severe pneumonia with ards, and admitted immediately to the icu with high flow oxygen to maintain the spo2 at approximately 90% on february 3. during this period, she showed polypnea, dysphoria, frequent cough and slight hemoptysis. abundant hemorrhagic secretions were observed through bronchofiberscope. she was treated with antiviral and antibiotic therapies in combination with ulinastatin ( fig. 1a) . antiviral drug peramivir was used in the first three days, followed by oseltamivir in another three days. antibiotics (imipenem, cilastatin and moxifloxacin) were given for 10 days. her body temperature returned normal on the fourth day and ct showed significant improvement in the patient's condition. she was switched to sequential non-invasive ventilation from february 10 to 12, and then transferred to general ward and treated with low-flow oxygen and prednisone. the patient was discharged from the hospital on february 17 and showed a sustained recovery in two subsequent visits on february 28 and march 15. throat swabs and sera were collected during her hospitalization. blood and respiratory secretions were negative for bacteria, and fungi in cultures, and g-test and gm-test, respectively. hiv, hbv, influenza viruses, sars-cov, mers-cov and other coronaviruses were negative by elisa and/or (rt-)pcr assays. pulmonary secretions from the first day of hospitalization were further analyzed using metagenomic sequencing as described previously. 6 of 64,024,857 total reads, 2166 (0.0034%) were of viral origin. two respiratory viruses, hrv (1309 reads, 60.4%) and hbov (197 reads, 9.1%) were found with high percentages (fig. 1b) . the presence of hrv and hbov were confirmed by specific (rt-)qpcr assays, and hbov1 rna was also detected at all sample points. hrv-c and hbov1 were determined by phylogenetic analyses of hrv vp4/vp2 and hbov vp1/vp2 fragments, respectively (fig. 1c) . specific igm and igg responses against hbov1 and hrv-c were further investigated by elisa (feiya biotech, china). the serum igg and igm levels of the patient showed 2.2 to 4.7 fold increases for hrv-c and 5.1 to 2.6 fold increases for hbov1 from february 4 to february 15, respectively (fig. 1d) . above results indicated an acute co-infection with hbov1 and hrv-c in the patient. we further characterized the dynamics of both viruses using (rt-)qpcr assays (fig. 1e) . after 2 days of treatment, the viral load of hrv-c and hbov1 among swabs showed a rapid decrease from 1.3 × 10 4 copy/ml to below the detection limit, and from 1.2 × 10 6 copy/ml to 2.7 × 10 4 copy/ml, respectively. then, hrv-c viral load remained undetectable among swabs and sera, while hbov1 viral load started a slowdown from 2.7 × 10 4 copy/ml to 1.2 × 10 3 copy/ml during whole treatment, indicating a persistent infection of hbov1. 5 accompanied by the antiviral treatment, the patient progressively recovered and bilateral lung opacity improved rapidly, indicating that the treatment was effective. it was difficult to determine which virus was mainly responsible for the case. but the recovery of the patient from the rapid elimination of hrv-c and a persistent presence of hbov1 among swabs and sera suggested that hrv-c may be the principal causative agent for the severe pneumonia. hrv was often associated as severe respiratory illness in children and adults. [7] [8] [9] as there are still questions about the role of hbov as a single causative agent, it is possible that the simultaneous hbov1 infections worsen the illness caused by preceding hrv-c infection. in this case, metagenomic sequencing showed a robust power to detect the causative agents of severe illness when the routine and traditional methods (e.g. culture, elisa, pcr, etc.) failed. after excluding bacterica, fungi, and major pneumonia-causing viruses (i.e. sars-cov, mers-cov, and h7n9), we applied metagenomic sequencing to obtain the dna and rna sequences present in the sample, and determined the potential pathogens by similarity search with the sequence database covering all known viruses. the candidate viruses were further confirmed by specific (rt-)qpcr assays, phylogentic analysis, as well as seroconversion of igm and igg responses. using this pipeline, hrv-c and hbov1 were identified as the causative agents of this index patient with ards. in conclusion, our study reports a life-threatening pneumonia case caused by co-infection with hrv-c and hbov1, and raises a question whether hrv-c-related severe pneumonia is often associated with co-infection of other respiratory viruses such as bocavirus. timeline of the patient's clinical course and evidence supporting the co-infection with hrv-c and hbov1. a, timeline of the patient's clinical course and outcome. b, viral reads distribution. viral reads were classified by aligning again the ncbi viral sequence database with blast-n and blast-x. c, maximum-likelihood phylogenetic trees were generated by mega6 based on partial vp1/vp2 sequences (~550 bp) of bocavirus (left) and partial vp4/vp2 sequences (~530 bp) of rhinovirus (right). d, antibody responses to hbov1 and hrv-c at two time-points. results are shown as absorbance values at 450 nm, cutoff 0.15. difference between different groups was determined by the student's t-test. ** p < 0.01. e, viral load dynamic of hbov1 and hrv-c in swabs and serums collected in different time-points. a high-resolution version of this slide for use with the virtual microscope is available as eslide: vm04609. no reported conflicts. human bocavirus: current knowledge and future challenges human rhinoviruses respiratory viruses and influenza-like illness: epidemiology and outcomes in children aged 6 months to 10 years in a multi-country population sample genetic characterization of human bocavirus among children with severe acute respiratory infection in china primary and secondary human bocavirus 1 infections in a family the eukaryotic gut virome in hematopoietic stem cell transplantation: new clues in enteric graftversus-host disease fatal respiratory infections associated with rhinovirus outbreak clinical and molecular epidemiology of human rhinovirus c in children and adults in hong kong reveals a possible distinct human rhinovirus c subgroup clinical and molecular characterization of rhinoviruses a, b, and c in adult patients with pneumonia guangzhou women and children's medical center supplementary data related to this article can be found online at https://doi.org/10.1016/j.jinf.2017.10.012. key: cord-331931-2et3gcb5 authors: zeng, jie; peng, shengkun; lei, yu; huang, jianxin; guo, yang; zhang, xiaoqin; huang, xiaobo; pu, hong; pan, lingai title: clinical and imaging features of covid-19 patients: analysis of data from high-altitude areas date: 2020-04-08 journal: j infect doi: 10.1016/j.jinf.2020.03.026 sha: doc_id: 331931 cord_uid: 2et3gcb5 nan sars-cov-2 has spread worldwide. we read wenjie's [1] report about the characteristics of the covid-19 outside hubei. due to the inclusion of the center, these data are basically from large cities. but about 100 million residents around the world live above 2500 meters all year around [2] , the residents in high altitude areas may have pathophysiology changes especially in cardiopulmonary function [3] . if they are combined with covid-19, these may cause more uncertainty to diagnosis and treatment. given the rapid spread of covid -19, we determined that an updated analysis of 68 cases from high altitude area from sichuan province, which might help the physicians to learn more about the covid-19. images from all cases were collected and analyzed by two radiologists. each segment of the lung was examined to determine whether there were ground glass opacities (ggo), consolidation, mix ggo and consolidation, reticular shadows, and the affected lung segment, range, location of the lesion, whether there was pleural effusion, and lymphadenopathy. we use semi-quantitative score system to evaluate the area of the lesion [4] . ct imaging features recorded from our cohort were summarized in supplementary table 2 . the infected people ranged in age from 3 to 77 years old. the patient under the age of 14 accounts for 7/68 (10%). most of the patients had no specific covid-19 symptoms, which is quite different from previous research. laboratory results suggest that lymphocytes had varying degrees decreased. crp was not too high (75% patients were in the normal range) and 79% patients with normal pct level. among all patients, 22 patients had negative initial ct and follow-up of lung ct, 5 patients had negative first ct scan, and positive after follow-up.the involvement range of the lung can be seen in the supplementary as table 2 . the total lung severity score of 46 ct-positive patients was 161, with an average score of 3.5 (range 1-10). pulmonary cavity and tree bud signs appeared in 3 patients with tuberculosis, and the other patients did not present similar lesions. there were 3 cases of pleural effusion (3/46) (small-medium amount of left pleural effusion). there were 7 / 46 patients with old tuberculosis signs. during the study period, 65 patients underwent a ct review. the average time between first chest ct and follow-up rescan was 3.7 days (2-10 days). 3 patients developed pleural effusion, but all of them had a history of tuberculosis. emphysema in 3 people. there were 7 patients with previous tuberculosis. sars-cov-2 is highly contagious and spread quickly among the population and spread to 79 countries worldwide in recent 3 months. some of the patient can progress to severe or critical condition which might need the oxygen supplementary or ventilation support. it was estimated that patients who were initial infected with sars-cov-2 due to exposure to wildlife. but in our study, none of the patients have been exposed to wildlife, nor have they been to wuhan since the outbreak. therefore, the cases mentioned in our study are all infections caused by imported epidemic areas. the oxygen content is low in high altitude areas. the incidence of sars-cov-2 infection in this region is unclear. this is the first report talking about the covid-19 from high altitude areas. in our study, the patient under the age of 14 accounts for 7/68 (10%), which is higher than previous reports, we thought that may be related to family clustering. this is different with previously reported clinical data that 2% of the 72314 infected patients were younger than 20 years old [5] it may be because these groups do not have such a large range of social activities and have fewer opportunities to contact the source of infection. the clinical symptoms of patients in this study group are quite different，most of the patients had no specific covid-19，they had significantly lower rate of fever symptoms than other studies present before [4, 6, 7] . we believe that most of our patients are not from the epidemic area, and the virus may have been passaged several generations. due to the strengthening of the epidemic prevention from the health department, they also received early diagnosis and treatment and changed the natural course development of covid-19. however, among these patients, 6 severe patientsstill need oxygen therapy to relieve the dyspnea symptoms. we analyzed that these patients were older and combined more comorbidities than mild patients. it is worth mentioning that a 77-years-old female patient had a history of tuberculosis, although only one lobe was involved with sars-cov-2, but still progresses to severe illness. therefore, the severity of covid-19 is closely related to the underlying lung disease. like clinical symptoms, the imaging of this group of patients is also diverse. among the patients in this group, 41 ct tests were positive at the initial scan. in terms of lesion distribution, most of the patients' lung ct lesions were distributed at subpleural site, and the right lower lung involvement was the most common (33/46). follow-up ct scans in 5 exacerbated patients indicated lung disease progression. there were 5 patients who had a negative initial ct scan but positive follow-up ct， which means that negative ct images cannot completely rule out covid-19. we can also found the pleural effusion in covid-19 patients, which was similar with previous research [8] , and the pleural effusion was completely absorbed after treatment. in terms of treatment, most patients have a negative nucleic acid review after 1-2 weeks. the respiratory symptoms relief, the severe patients are still being followed up. in conclusion, this is the first report of covid-19 in high altitude area in the world. in high altitude area, the population is generally susceptible, including children, so everyone should be well protected. residents high altitude area may face more complicated medical and social conditions (such as hypoxia, tuberculosis, and relative lack of medical resources), which may increase the complexity and severity of the disease. our research shows that patients who underwent early screening and intervention could significantly reduce the severity of the disease，and patients with pulmonary tuberculosis would significantly increase the severity of the disease. clinical characteristics and imaging manifestations of the 2019 novel coronavirus disease (covid-19):a multi-center study in wenzhou city acute high-altitude illnesses. the new england journal of medicine high-altitude adaptation in humans: from genomics to integrative physiology clinical features of patients infected with 2019 novel coronavirus in wuhan characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a report of 72 314 cases from the chinese center for disease control and prevention epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 50466 hospitalized patients with 2019-ncov infection radiological findings from 81 patients with covid-19 pneumonia in wuhan, china: a descriptive study key: cord-290385-0smnl70i authors: chan, jasper f.w.; choi, garnet k.y.; yip, cyril c.y.; cheng, vincent c.c.; yuen, kwok-yung title: zika fever and congenital zika syndrome: an unexpected emerging arboviral disease date: 2016-03-03 journal: j infect doi: 10.1016/j.jinf.2016.02.011 sha: doc_id: 290385 cord_uid: 0smnl70i unlike its mosquito-borne relatives, such as dengue, west nile, and japanese encephalitis viruses, which can cause severe human diseases, zika virus (zikv) has emerged from obscurity by its association with a suspected “congenital zika syndrome”, while causing asymptomatic or mild exanthematous febrile infections which are dengueor rubella-like in infected individuals. despite having been discovered in uganda for almost 60 years, <20 human cases were reported before 2007. the massive epidemics in the pacific islands associated with the zikv asian lineage in 2007 and 2013 were followed by explosive outbreaks in latin america in 2015. although increased mosquito breeding associated with the el niño effect superimposed on global warming is suspected, genetic changes in its rna virus genome may have led to better adaptation to mosquitoes, other animal reservoirs, and human. we reviewed the epidemiology, clinical manifestation, virology, pathogenesis, laboratory diagnosis, management, and prevention of this emerging infection. laboratory diagnosis can be confounded by cross-reactivity with other circulating flaviviruses. besides mosquito bite and transplacental transmission, the risk of other potential routes of transmission by transfusion, transplantation, sexual activity, breastfeeding, respiratory droplet, and animal bite is discussed. epidemic control requires adequate clearance of mosquito breeding grounds, personal protection against mosquito bite, and hopefully a safe and effective vaccine. globalisation and urbanisation with increasingly frequent and large-scale movements of humans, animals, and commodities by aviation and water transport has led to the spread of previously geographically-restricted microbes and vectors to distant and isolated places. 1 recent examples of emerging viruses that have spilled over to other continents from their original localities via exportation of travelrelated cases include coronaviruses (severe acute respiratory syndrome coronavirus and middle east respiratory syndrome coronavirus), influenza viruses, and ebola virus. 2e11 moreover, global warming and climate changes have redefined the geographical distributions of important vectors of arthropod-borne viruses (arboviruses), such as the aedes mosquitoes, and facilitated the global spread of these viruses. dengue virus (denv), west nile virus (wnv), and chikungunya virus (chikv), have been introduced (wnv in 1999 and chikv in 2013) and/or spread rapidly in the western hemisphere in the past two decades. 12 zika virus (zikv) is an arbovirus that was little known before it caused a large outbreak on yap island of the federated states of micronesia in 2007. 13 even then, zikv was not considered as an important emerging pathogen because clinical disease was generally mild. the recent report of a possible association between zikv infection and an epidemic of microcephaly among neonates in brazil has attracted global attention. 14 the rapid spread of zikv beyond africa and asia to the americas and europe, and the potentially novel "congenital zika syndrome" outbreak have led the world health organisation (who) to declare the zikv epidemic as a global public health emergency on 1 february 2016. 15 it would therefore be important to review the current knowledge on the epidemiology, virology, clinical manifestations, and laboratory diagnosis of zikv infection, and most importantly, to formulate clinical management options with special reference to perinatal care and control measures based on comparisons made with other mosquito-borne arboviruses. important historical and epidemiological events zikv (strain mr 766) was first isolated from the blood of a febrile sentinel rhesus monkey (macaca mulatta), rhesus 766, during a study on yellow fever virus (yfv) in zika forest of uganda in april 1947 (table 1) . 16 in 1948, zikv was isolated from aedes africanus mosquitoes caught in zika forest, suggesting that the virus might be mosquito-borne. in 1954, zikv was isolated from the serum of a 10-year-old nigerian girl who had fever and headache, implying its role as a possible human pathogen. 17 further virological and/or serological evidence of human zikv infection was reported in african (uganda, tanzania, egypt, central african republic, sierra leone, and gabon) and asian (india, malaysia, the philippines, thailand, vietnam, and indonesia) countries. 18e26 zikv infection remained relatively restricted geographically with less than 20 sporadic cases reported in these areas in the first 60 years after its discovery. 13, 27 in 2007, zikv emerged outside africa and asia for the first time and caused a major outbreak on yap island of the federated state of micronesia. 13 over 70% of the yap residents who were !3 years were infected within 4 months. 13 the attack rate of zikv infection in this outbreak was 14.6 per 1000 residents (range, 3.5e21.5 per 1000 residents). subsequently, another major outbreak was reported in french polynesia in october 2013. an estimated 30,000 humans (>11% of the french polynesian population) were infected by zikv. zikv infection then spread from french polynesia to other pacific islands including new caledonia, cook islands, vanuatu, and solomon islands. 27e30 the first cases of human zikv infection in the western hemisphere occurred on easter island, chile, in february 2014, possibly originating from french polynesia during the annual tapati festival. 31 phylogenetic analysis revealed that the ns5 gene sequence of the chilean strains had !99.8% nucleotide and 100% amino acid identity to the french polynesian strains. 31 the epidemic continued to expand rapidly and autochthonous human cases were reported in many latin american countries in the ensuing 2 years. 32 brazil stands out as the hardest hit latin american country with an estimated 500,000e1,500,000 cases of zikv infection since march 2015. 33 based on the close phylogenetic relationship between the south american strains and asian and oceanic strains of zikv, the virus might have been introduced into brazil by asian travellers during the world cup or participants from the oceanic countries of the va'a world sprint championship canoe race in the summer of 2014. 30,34e36 the climate changes associated with el niño in north and eastern south america in 2015 on the background trend of global warming might have facilitated the rapid spread of aedes mosquitoes and zikv. 37 currently, >30 countries in africa, asia, south america, oceania, and micronesia have reported autochthonous cases of human zikv infection. 38 travel-related cases from endemic and epidemic regions were also reported in europe, north america, australia, and japan. 39e50 more worryingly, the brazil health ministry reported the detection of an unusual increase in the number of cases of neonates with microcephaly in northeastern brazil in october 2015, coinciding with the expanding zikv infection epidemic. 14 over 3000 suspected cases including some fatal cases were reported during the second half of 2015 alone. 14 this represented a >20-fold increase in the rate of microcephaly as compared to previous years. 32 on 24 november 2015, the french polynesia health authorities also reported an unusual increase in the number of foetal and neonatal central nervous system malformations in 2014 and 2015. 51 like other flaviviruses, zikv is mainly transmitted by mosquitoes. in addition to the sylvatic (enzootic) transmission cycle between the haematophagous mosquito vectors and susceptible primary vertebrate hosts, the recent large-scale epidemics suggest that zikv is also adapting to an urban transmission cycle. 52, 53 among the various mosquito species, aedes (stegomyia) mosquitoes appear to be the most important vector for zikv transmission, although some anopheles, culex, eretmapodites, and mansonia species have also been proposed as possible vectors (table 2) . 25,52,54e69 the animal reservoirs of zikv are unclear. non-human primates including m. mulatta, cercopithecus aethiops, c. ascanius schmidti, c. mona denti, c. albigena johnstoni, chlorocebus sabaeus, colobus abyssinicus, erythrocebus patas, and pongo pygmaeus, and other mammals including zebras, elephants, and rodents, have been suggested as possible vertebrate hosts of zikv in africa and asia, based on virological and/or serological evidence of infection. 16,54,65,68,70e72 ae. africanus is the first mosquito species from which zikv was isolated, and is likely an important vector in the sylvatic transmission cycle of zikv. 16, 54 inoculation of unfiltered supernatant of zikv-infected ae. africanus into mice and rhesus macaques led to clinical disease and/or neutralising antibody response. 16, 54 ae. hensilli is the most commonly found mosquito species on yap island, but no virus isolate was made from field-collected mosquitoes to ascertain its role as a vector for zikv transmission during the outbreak. 67 ae. aegypti and ae. albopictus, which have much wider geographical distributions than other aedes mosquitoes, are considered to be more important vectors in the urban transmission cycle of zikv. 73 these aedes mosquitoes are highly susceptible to zikv infection in vitro with potential for further transmission after an extrinsic incubation period of 5e10 days. 58, 63 they bite both indoors and outdoors, and mostly during daytime. 74 non-vector-borne transmission routes of zikv have been proposed (table 3) . like other arboviruses, blood transfusion-related transmission of zikv is possible, especially in endemic regions or where blood products obtained from infected travellers immediately returning from endemic regions are used. zikv rna was detected in the blood of 2.8% of the donors in french polynesia during the epidemic. 75 sexual transmission of zikv appears highly probable, especially in patients presenting with haematospermia with infectious viral particles and rna in semen. 76, 77 notably, no other arboviruses have been associated with haematospermia or isolated from human semen. 76 this might further complicate the control of the zikv epidemic, since most infected patients are asymptomatic. inadvertent sexual transmission of zikv to the female partner may then lead to virus transmission to the foetus, which may be potentially associated with severe congenital anomalies. besides transplacental transmission, perinatal transmission of zikv may also occur during delivery, via breastfeeding, and/or close contact after birth via exchange of saliva and other bodily fluids. zikv rna could be detected in breast milk and saliva of infected women, although replicative virus particles have not been demonstrated 78, 79 perinatal transmission of other arboviruses, including denv, chikv, wnv, and yfv, has also been reported. 80e90 other suspected routes of transmission of zivk infection are those reported for other flaviviruses. these include mucocutaneous exposure to the virus in infected blood or via monkey bite, haemodialysis, or organ transplantation. 91e96 particularly, as zikv may be shed in the urine of infected patients for more than 30 days, the risk to recipients of donated kidneys from donors at or returning from endemic areas has to be considered. 40, 41, 49, 50, 97 it is unknown whether zikv could be transmitted via respiratory droplets as viral rna could occasionally be detected in nasopharyngeal swab and saliva samples. 40, 78, 79, 96 virology and pathogenesis zikv is an enveloped, positive-sense, single-stranded rna virus belonging to the genus flavivirus in the family flaviviridae. it is closely related to spondweni virus and the 2 viruses represent the only members of their clade within the mosquito-borne cluster of flaviviruses ( fig. 1) . 98, 99 phylogenetic analysis suggests that zikv has likely emerged between 1892 and 1943 in uganda. 66 the two major lineages of zikv are the african (subdivided into west and east african) and asian lineages, which are responsible for causing the majority of infections in africa and asia (as well as the pacific and americas), respectively. 34, 69, 100 the single-stranded rna genome of zikv has a size of 10,794 nucleotides encoding 3419 amino acids, with 2 flanking untranslated regions (5 0 and 3 0 utrs) and a single long open reading frame encoding a polyprotein, which is cleaved into capsid (c), precursor of membrane (prm), envelope (e), and 7 non-structural (ns) proteins 66, 101 reverse transcription-polymerase chain reaction (rt-pcr) using primers targeting the e or ns5 gene is a key laboratory diagnostic tool for zikv infection in the recent outbreaks. 13, 102, 103 the e protein is a major virion surface protein that is involved in receptor binding and membrane fusion. the domain iii of e protein contains a panel of antigenic epitopes that are important targets of serological assays, neutralising antibodies, and vaccines. 104, 105 loss of the n154 glycosylation site in the e protein may be associated with adaptation to mosquito vectors and thus facilitate transmission. 66 a single amino acid mutation in the e protein (e1-a226v) of chikv has been reported to be associated with increased fitness of the virus in ae. albopictus and allows chikv to disseminate in regions lacking the typical ae. aegypti vector. 106 the recent spread of the asian lineage of zikv to oceania and the americas may be associated with significant ns1 codon usage adaptation to human housekeeping genes, which could facilitate viral replication and increase viral titres. 107 mutations in the e and ns1 genes should be detected in zikv strains causing the current epidemic. when an infected aedes mosquito bites an infected patient, it ingests a blood meal containing zikv. as in other flaviviruses, zikv likely replicates in the midgut epithelial cells and subsequently the salivary gland cells. after an extrinsic incubation period of 5e10 days, zikv can be found in the mosquito's saliva which can then infect human. 58, 63 moreover, the virus can likely be vertically transmitted transovarially as other flaviviruses. when the mosquito's saliva containing zikv is inoculated into human skin, the virus can infect epidermal keratinocytes, skin fibroblasts in the subcutaneous layer, and the langerhans cells. 108 the keratinocytes and fibroblasts contain axl, tyro3, and tim-1, which can serve as attachment factors or receptors for zikv. the langerhans cells contain dc-sign, which can also serve as a receptor for virus entry. 108 zikv infection of primary skin fibroblasts is associated with the upregulation with tlr3 mrna expression, and enhanced transcription of rig-i and mda5, which are known innate immune responses to rna virus infection. this is followed by enhanced expression of interferon-alpha and -beta, and their downstream pathways of immune activation. both types i and ii interferons can suppress the viral load of infected cells. moreover, zikv is capable of increasing its replication by the induction of autophagy in host cells. thus, autophagy inhibitors can decrease the viral load of infected cells. 109 infected cells of human skin explant exhibits cytoplasmic vacuolation, pyknotic nuclei, and oedema in the stratum granulosum. 108 after replication in endemic/epidemic areas: + universal nucleic acid testing of blood donors. + temporary discontinuation of blood donation (importation of blood products from blood blank centres in non-endemic regions). non-endemic/epidemic areas: + pre-donation questionnaire to identify donors with recent travel history to endemic/epidemic areas. + deferral of blood donors who have travelled to endemic areas within the preceding !14 days. + self-reporting of symptoms after blood donation ( these local tissue cells and the regional lymph nodes, zikv may then disseminate from the lymphatics and bloodstream to reach other organs/tissues, including the central nervous system, the skeletal muscles, myocardium, and perhaps transplacentally to the foetus. zikv was highly neurotropic in infected suckling mice. the brains of infected suckling mice show neuronal degeneration, cellular infiltration, and softening in the brain with virus replication in astroglial cells and neurons on histopathological examination. 16, 54, 110 moreover, evidence of inflammation in skeletal muscles and myocardium has also been demonstrated in infected suckling mice. 111 axl and tyro3 are members of the tam family of receptor tyrosine kinases (rtks). they are also present in neurons and under the influence of gonadotropin releasing hormone (grh), which in turn may affect neuronal survival and migration. furthermore, flaviviruses such as yfv may persist for up to 159 days after intracerebral inoculation in rhesus macaques. 112 the neurotropism and persistence of zikv may therefore partially explain microcephaly and predominantly neurological complications and foetal anomalies in this suspected entity of congenital zikv infection. most patients with zikv infection are asymptomatic. in the outbreak of zikv infection on yap island, only 18% of cases were estimated to be symptomatic. 13 the incubation period of zikv infection is unclear, but is estimated to be similar to other mosquito-borne flaviviruses (2e14 days). 76, 113 the clinical syndromes of symptomatic zikv infection can be broadly divided into zika fever and congenital infection ("congenital zika syndrome") ( table 4 ). zika fever is an acute "dengue fever-like" illness characterized by low-grade fever (37.8e38.5 c), rash, retroorbital headache, bilateral non-purulent conjunctivitis, myalgia, and arthritis/arthralgia with periarticular oedema of the small joints of hands and feet. 13 the rash in zika fever is typically described as a generalized, erythematous, maculopapular rash that spreads downward from the face to the limbs. 30 less commonly, some patients may have more prominent systemic symptoms including high-grade fever, chills, rigours, sore throat, hypotension, and cervical, submandibular, axillary, and/or inguinal lymphadenopathies. 74 digestive tract symptoms including nausea, vomiting, diarrhoea, constipation, abdominal pain, and aphthous ulcers may also be present. 13, 17, 19 patients with genitourinary symptoms including haematuria, dysuria, perineal pain, and haematospermia often have detectable viral rna or infectious virus particles in urine and/or semen. 76, 77 haematological and biochemical laboratory parameters are usually normal. however, some patients may have transient and mild leucopenia, neutropenia, lymphopenia or activated lymphocytes, monocytosis, thrombocytopaenia, and elevated serum levels of lactate dehydrogenase, aspartate aminotransferase, g-glutamyl transferase, fibrinogen, ferritin, c-reactive protein, and erythrocyte sedimentation rate during the viraemic phase. 29 associated with restoration of normal number of peripheral immune cells and normal function of antigen-presenting cells. 45 notably, the clinical manifestations of zika fever are non-specific and may mimic those seen in infectious diseases caused by other arthropod-borne pathogens, especially denv and chikv. some suggest that zika fever may be distinguished from dengue fever and chikungunya fever by more prominent oedema of the extremities, less severe headache and malaise, and milder degree of thrombocytopaenia seen in the former. 13, 115 moreover, haemorrhagic complications seen in dengue fever have not been reported in zika fever, and arthralgia in zika fever is less severe than that in chikungunya fever. 13 however, none of these features are pathognomonic and laboratory confirmation is required to exclude co-infections with these arboviruses and other causes of acute febrile illness in returned travellers from endemic regions, such as malaria. zika fever is usually self-limiting with most clinical manifestations resolving completely within 3e7 days. 13, 49, 116 no death, hospitalisation, or haemorrhagic complication was reported during the outbreak on yap island. 13 however, some patients may experience more protracted symptoms and other non-haemorrhagic complications. zika fever-related rash usually resolve within the first week, but may last for up to 14 days and may be pruritic. 13 other exanthematous diseases, such as denv, chikv, rubella virus, measles virus, parvovirus b19, adenovirus, enterovirus, and rickettsial infection, should be excluded. the median duration of arthralgia is 3.5 days, but some patients may develop persistent or recurrent arthralgia for more than a month after symptom onset, mimicking the post-infectious chronic arthritis seen in chikungunya fever and lyme disease. 13, 76 lymphadenopathies may be present for 2 weeks after symptom onset, and alternative diagnoses such as infectious mononucleosis-like syndrome, streptococcus pyogenes infection, and toxoplasmosis should be considered in refractory cases. 45 a post-infection asthenia appears to be frequent and further investigations may be necessary to determine possible association between zikv infection and chronic fatigue syndrome. 13, 117, 118 immune-thrombocytopenic purpura and cardiac complication have also been reported in a few cases. 114 jaundice was observed in patients with virological and/or serological evidence of zikv infection in eastern nigeria in the 1950s who had co-infections (malaria and microfilaraemia) and a patient with sickle cell anaemia. 17, 119 a possible association between zikv infection and severe neurological complications has been proposed during the recent epidemics in oceania and south america, during which the incidence of guillainebarré syndrome has increased by 8e20 times in french polynesia. 115,120 74/ 8750 (0.8%) patients with suspected zikv infection in the french polynesia outbreak developed neurological syndromes after presenting with a zika fever-like illness. 120 forty-two of these 74 (56.8%) patients were diagnosed with guillainebarré syndrome. 12, 51, 121 similarly, guil-lainebarré syndrome has been reported among patients with zika fever-like illness in south america. 51, 121 other neurological complications potentially linked to zikv infection include encephalitis, meningoencephalitis, myelitis, paraesthesia, vertigo, facial paralysis, and 48,76,114,115,120e122 suspected fatalities due to zikv-related guillainebarré syndrome have been reported. 123 while the neurotropism of zikv may partially explain these neurological manifestations, more details and serial studies on their cerebrospinal fluid and magnetic resonance images by case-control studies are required to ascertain their association. zika fever-related death appears to be extremely rare but a number of probable cases have been reported, especially among immunocompromised patients and neonates with suspected congenital zikv infection. 51,119,121 a small number of patients with coinfection with denv or hiv did not appear to have more severe disease. 29, 124 further studies should be conducted to identify patients who are at risk of severe disease or death. microcephaly (head circumference !2 standard deviations below the mean for sex and gestational age at birth) is the most prominent and commonly reported clinical feature of suspected congenital zika syndrome. 14, 125 besides microcephaly, neonates and foetuses with suspected congenital zikv infection also had other malformations (table 4 ). general features included low birth-weight, redundant scalp skin, anasarca, polyhydramnios, and arthrogryposis. neurological abnormalities included cerebral lesions, polymalformative syndromes, brainstem dysfunction, and absence of swallowing. 51 ophthalmological defects included cataract, asymmetrical eye sizes, intraocular calcifications, macular atrophy (well-defined macular neuroretinal atrophy and/or macular pigment mottling and foveal reflex loss), optic nerve hypoplasia, iris coloboma, and lens subluxation. 122, 126, 127 notably, other features characteristic of intrauterine infections, such as hepatosplenomegaly, rash, and chorioretinitis have not been reported. 14 ultrasonographic examination revealed cerebral atrophy, intracranial calcifications especially over the white matter of frontal lobes, caudate, lentostriatal vessels, cerebellum, or around the lateral and fourth ventricles, dysgenesis of corpus callosum, vermia, and thalami, enlarged cisterna magna, asymmetrical cerebral hemispheres, severe unilateral ventriculomegaly, displacement of the midline, and thinning of the parenchyma on the dilated side, pons and brainstem. 14,51,121,128 zikv particles and rna may be detected by electron microscopy and rt-pcr, respectively, in autopsied samples. 125 two important questions concerning congenital zikv infection remain unanswered. the first question is whether zikv is indeed the cause of microcephaly and other congenital anomalies in these patients. severe consequences have been reported for materno-foetal transmission of other arboviruses, such as dengue virus (preterm delivery, foetal death, low birth-weight, prematurity, acute foetal distress during labour), wnv (chorioretinitis and focal cerebral destruction), and chikv (encephalopathy and haemorrhagic fever). 80, 82, 86 preliminary analysis in the current epidemic of microcephaly has not yet completely excluded other infectious or environmental aetiologies. 51 moreover, there is some virological evidence to support the association between congenital zikv infection and these anomalies. zikv rna has been detected by rt-pcr in the amniotic fluid of 2 pregnant women whose foetuses had ultrasonographic evidence of microcephaly, in the blood and foetal tissues of a neonate with microcephaly and other congenital anomalies who died within the first 5 min of birth, and in the neonatal brain tissues of a few cases of full-term miscarriages and neonates with microcephaly. 14, 51, 121, 125 however, there is still no large-scale prospective cohort or caseecontrol study to demonstrate a causal link between the presence of zikv in the foetus and the congenital anomalies after exclusion of other infectious and toxic causes. some have suggested that the apparent microcephaly surge might be attributable to the intense search for cases due to the heightened awareness of a possible association with the zikv outbreak or the use of larvicide. 129 furthermore, detailed investigations for exclusion of other pathogens associated with congenital malformations have only been reported in a small number of cases. 14, 125 microcephaly is well reported in congenital cytomegalovirus, rubella virus, and varicella zoster virus infection. chorioretinitis and intracranial calcifications are common in congenital cytomegalovirus infection and toxoplasmosis, but the latter is more commonly associated with hydrocephalus. cataract and cardiac anomalies are characteristic of congenital rubella syndrome, although cataract can also be found in congenital herpes simplex virus infection. thus, the diagnosis of congenital zika syndrome would depend on the exclusion of these "torch" infections in future studies using clinical criteria, histopathological findings, and serological, molecular and conventional cell culture techniques. if zikv is eventually confirmed to be the cause of these congenital anomalies, the second key question would be whether congenital zika syndrome actually comprises a wider spectrum of varying clinical severities than that seen in the reported cases. as with other congenital infections, it is possible that the reported cases of microcephaly represent only the tip the iceberg, focussing on the more severely affected patients, and that the timing of infection is likely to be important in determining the severity and outcome of the affected foetus. early infection during the first or even second trimester may be associated with congenital anomalies or even intrauterine death. 14, 125, 130 indeed, preliminary data suggested that the greatest risk of microcephaly or congenital anomalies in the affected neonates appears to be associated with zikv infection in the first trimester of pregnancy. 51 of 35 mothers with infants born with microcephaly, 57% and 14% had a rash during the first and second trimester of pregnancy, respectively. 14 besides neurological defects, cardiac and muscular abnormalities should also be excluded, as suckling mice infected with zikv developed evidence of central nervous system infection, myositis and myocarditis. 111 some suspected cases of congenital zika syndrome developed severe arthrogryposis. 14, 51, 121, 128 it is possible that intrauterine zikv infections that occur at a later stage of the pregnancy may present differently, either with less severe manifestations, such as mental retardation, sensorineural deafness, and/or ophthalmological lesions, or as full-term miscarriages. 14 neonates with probable perinatal transmission of zikv infection appear to have mild disease and favourable outcome. 78 further investigations should be conducted to better define the spectrum of manifestations in different gestational stages of congenital zikv infection. definitive diagnosis of zikv infection requires laboratory confirmation as there are no pathognomonic clinical, biochemical, or radiological features that reliably distinguish zika fever from other arboviruses, and congenital zikv infection from other infective, toxic, or genetic causes of congenital anomalies. successful isolation of zikv in viral culture, the gold-standard of laboratory diagnosis of viral infections, mainly depends on the timing of specimen collection and viral loads in the specimens. zikv has been isolated in vero and vero e6 cells inoculated with infected patients' serum, urine, and/or semen samples (table 5) . 40, 75, 77 however, infectious virus particles were not recovered by culture in most specimens with low viral loads. a positive serum immunoglobulin (ig) m or 4-fold rise in the titre of neutralising antibodies in paired serum samples collected approximately 2 weeks apart also establishes the diagnosis of zikv infection. igm may be detected by enzyme-linked immunoassay on as early as day 3 of symptom onset and may last for over 2 months. 40, 49 igm antibodies to denv and wnv usually persist for 3 months and 5 months, respectively. 131e134 the major limitation of these serological tests is possible cross-reactivity with other flaviviruses. neutralising antibodies detected by plaque-reduction neutralisation test may be more specific than igm detection by elisa for primary zikv infection, but may also have indeterminate results for secondary infection, including patients with previous vaccination against or exposed to other flaviviruses. 13, 40, 102, 120 this is especially problematic in areas where there is cocirculation of multiple flaviviruses with the same aedes mosquito vectors. 13, 64, 102, 120 patients with primary zikv infection and past denv infection are more likely to have higher titre (usually !4-fold) of igm and/or neutralising antibodies against zikv than against denv or other flaviviruses. 13, 102 a positive serum denv ns1 antigen test without serial increase in igm or the combination of a positive igm response to denv and lack of an igg seroconversion in the convalescent-phase serum sample should prompt the clinician to investigate for another flavivirus such as zikv. moreover, co-infections with other mosquito-borne arboviruses, such as denv, chikv, wnv, and japanese encephalitis virus, are always possible and should be excluded by more extensive laboratory testing if clinically indicated. rapid and accurate diagnosis of zikv infection during the recent epidemics has mainly been achieved by the application of rt-pcr using primers that target the e or ns5 gene of zikv. 13, 102, 103, 135, 136 alternatively, rt-pcr sequencing using universal primers that target the conserved regions in the genomes, such as the ns5 gene, of multiple flaviviruses, may allow simultaneous detection of >50 different flaviviruses. 137 serum samples should be collected in the early phase of the disease, because viraemia is usually shortlived (usually 5 days, rarely up to 11 days) and may be low-level ( 10 2 copies/ml). 97, 102 alternatively, urine and semen samples may have higher viral rna loads table 5 advantages, limitations, and uses of different diagnostic tests and types of specimens for laboratory diagnosis of zikv infection. 14,40,41,49,50,75e79,96,97,102,121,125,127,137e139,153 may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or atypical rash. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with persistent or recurrent arthritis. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. may be useful to exclude concomitant infections in patients with unusually persistent or severe cytopenia. other tissues brain, liver, spleen, and pooled visceral (kidney, lung, and heart) tissues were positive in a fatal case (an adult male with co-morbidities and immunosuppressive treatment). may be useful to exclude concomitant infections in patients with unusually severe or fatal infection. abbreviations: rt-pcr, reverse transcription-polymerase chain reaction; zikv, zika virus. (>10 6 copies/ml) than serum samples, and may be persistently positive for >30 days and !62 days after symptom onset, respectively. 97, 138 in a few cases, zikv rna has also been detected in saliva and nasopharyngeal swab samples of patients whose serum samples tested negative for zikv. these samples should therefore also be collected in suspected cases of zikv infection. 40, 78, 79, 96 collection of amniotic fluid should be considered in pregnant women with positive zikv test result or if the foetuses show ultrasonographic evidence suggestive of congenital zikv infection. 14, 102, 128 cerebrospinal fluid, placental, and/or umbilical cord tissues from neonates with suspected congenital zikv infection should be sent for virological and/or histopathological examinations to establish the diagnosis. 14,121,125 zikv rna may also be detected in organ tissues in the rare cases of suspected zikv-related deaths. 121 future studies should aim to better stratify the clinical use of these tests and to develop point-of-care tests (eg: antigen tests) that can be widely used in less developed regions without the facilities and expertise for molecular or serological tests. treatment is usually not required for patients with asymptomatic or uncomplicated zika fever. the mainstay of treatment is supportive as there are no specific anti-zikv antiviral agents. acetaminophen may be used to relieve fever and arthralgia. anti-histamines may help to control pruritus. adequate rehydration for fluid loss through sweating, vomiting, and insensible losses should be encouraged. aspirin should be avoided due to the risks of bleeding in those with thrombocytopaenia and developing reye's syndrome in children less than 12 years of age. nonsteroidal antiinflammatory drugs are also contraindicated in cases where denv and chikv infections cannot be confidently excluded in order to avoid haemorrhagic complications. potential neurological complications, especially guillainebarré syndrome, should be diagnosed promptly to allow early use of intravenous immunoglobulins and/or plasmapheresis. the risk of immune enhancement should also be considered if convalescent-phase plasma therapy with neutralising antibodies against zikv is used for treatment of severe cases. virological testing and foetal ultrasound to exclude zikv infection and foetal microcephaly or intracranial calcifications should be offered to pregnant women who develop zika fever-like symptoms during or within 2 weeks of travel to areas with zikv transmission. 139 besides collecting the appropriate specimens for virological tests, serial foetal ultrasound examinations should be performed every 3e4 weeks to monitor foetal anatomy and growth in suspected cases of congenital zikv infection. foetal ultrasound and/ or aminocentesis should also be offered to asymptomatic and seropositive pregnant women with history of travel to affected areas. after delivery, serum should be collected either from the umbilical cord or directly from the neonate within 2 days of birth for rt-pcr, igm and/ or neutralising antibodies against zikv. 140 comprehensive physical examination including measurement of the occipitofrontal circumference, length, and weight, evaluation for neurological abnormalities, dysmorphic features, hepatosplenomegaly, rash, ophthalmological lesions, and auditory defects, and laboratory testing for torch screening should be performed. 140 the affected child and the family should be managed and counselled by a multidisciplinary team consisting of paediatric neurologist, clinical geneticist or dysmorphologist, infectious disease specialist, medical social worker, and other relevant specialists. 140 long-term follow-up to monitor physical, intellectual, and functional progress of the child should be offered. 140 both vector control and personal preventive measures are important for interrupting the transmission of zikv. systematic mosquito surveillance and control programs should be established and coordinated by health authorities. 51 mass sanitation campaigns to eliminate mosquito breeding sites in household and high-risk areas such as garbage collection points, construction sites, illegal dumping grounds, and invalid car fields should be organised. mosquitoes should be removed with a radius of at least 400 m around areas with high population densities, such as schools, transport terminals, churches, and healthcare facilities. in areas where autochthonous or imported cases of zikv are detected, the use of adulticide through spraying to remove infected adult mosquitoes should be considered. residents in or travellers to affected areas should stay indoor with air conditioning, window and door screens if possible, wear long sleeves and pants, use permethrintreated clothing and gear, and use insect repellents when outdoor. 140 most environmental protection agency (epa)registered insect repellents, including n,n-diethyl-mtoluamide (deet), should be safe for pregnant and lactating women (20% deet), and children (10% deet) aged >2 months. 141 individuals returning from affected areas to non-affected regions should continue to use insect repellents for at least an additional 14 days to prevent local non-infected mosquitoes from the acquisition of virus from the asymptomatically infected returned travellers. this will serve to interrupt the mosquito-human-mosquito transmission chain. hospitalised laboratory-confirmed cases should be managed in designated wards to avoid mosquito bites. the effects of other novel mosquito-control measures, such as the wolbachia biological control approach, should be evaluated. 142 other animals such as rodents should also be investigated as potential animal reservoirs and controlled as findings indicate. non-vector-borne transmission of zikv may be prevented by specific measures (table 3) . concerning blood transfusion, universal nucleic acid testing of blood donors is recommended. the use of universal primers that can simultaneously detect multiple arboviruses such as denv and zikv should be considered. temporary discontinuation of blood donation should be considered during an outbreak situation. in non-endemic areas, pre-donation questionnaire to identify donors with recent travel history to regions with reported cases of zikv infection and deferral of blood donation from these donors until at least 14 days after returning from affected regions should be implemented. most transfusion-related transmissions of arboviruses are associated with asymptomatic infections, and symptomatic donors who were rt-pcr-positive for zikv usually developed symptoms between 3 and 10 days after blood donation. 143 newer pathogen reduction technologies for blood products should be considered. 143 similarly, donated organs, especially kidneys, from individuals with travel history to affected areas should be tested for zikv as the virus may persist in the genitourinary tract for an undetermined period. 40, 41, 49, 50, 97 barrier methods should be used to prevent sexual transmission through infected semen. male returned travellers should continue the use of condom with pregnant sex partner throughout the whole duration of pregnancy. future studies should evaluate the duration of virus shedding in semen and the infectiousness of rnapositive semen samples, in order to determine how long barrier methods should be used by men returning to nonendemic regions. some regional authorities have advised women to avoid pregnancy until the epidemic is over. 144 pregnant women or those planning for pregnancy should defer travelling to regions with reported cases of zikv infection. if such travel was unavoidable, they should strictly comply with personal protective measures to avoid mosquito bites. further studies are needed to determine the risk of zikv transmission by breast milk and saliva. other less common transmission routes, including mucocutaneous exposure to infected bodily fluid during laboratory and patient-care procedures, and bites by infected primates should be avoided with strict compliance to infection control measures. in the laboratory setting, zikv can be killed by potassium permanganate, ether, and heat (>60 c), but it is not effectively neutralised with low concentration (10%) of ethanol. 54 no zikv vaccine is available currently. because the moratorium for pregnancy may be impractical for some people, a safe and effective zikv vaccine is urgently needed. some realistic approaches include liveattenuated or killed vaccine from human cell lines (as in the case of yfv and japanese encephalitis vaccines), attenuated chimeric vaccine (denv vaccine using the yfv vaccine backbone, currently in phase iii clinical trial), dna and recombinant protein vaccine. 145 suitable animal models for evaluation of these potential vaccine candidates should be developed for zikv infection. the role of passive immunisation before and after exposure to zikv should also be assessed in future studies. the zikv 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of mosquito-borne disease zika virus and the never-ending story of emerging pathogens and transfusion medicine jamaica advises women to avoid pregnancy as zika virus approaches a review of successful flavivirus vaccines and the problems with those flaviviruses for which vaccines are not yet available arthropod-borne viral infections of man in nigeria dengue and other arboviral diseases in south-east asia zika virus, french polynesia, south pacific rapid spread of emerging zika virus in the pacific area first report of autochthonous transmission of zika virus in brazil zika virus spreads across americas as concerns mount over birth defects zika virus-related hypertensive iridocyclitis laboratory infection with zika virus after vaccination against yellow fever the study was partly supported by the consultancy service for enhancing laboratory surveillance of emerging infectious disease of the department of health, the food and health bureau, hong kong special administrative region, china; and by the donations of hui hoy and chow sin lan charity fund limited and mr. larry chi-kin yung. the authors declare no conflict of interest. key: cord-350159-i2ig6dtr authors: daunt, anna; perez-guzman, pablo n; liew, felicity; hauck, katharina; costelloe, ceire e; thursz, mark r; cooke, graham; nayagam, shevanthi title: validity of the uk early access to medicines scheme criteria for remdesivir use in patients with covid-19 disease date: 2020-06-21 journal: j infect doi: 10.1016/j.jinf.2020.06.049 sha: doc_id: 350159 cord_uid: i2ig6dtr nan however, given current limitations in drug supply, an interim risk score has been proposed to identify those patients thought most likely to benefit from the drug. the score includes eight variables: radiographic severity score > 3, male gender, non-white ethnicity, diabetes, hypertension, neutrophils >8.0 10/l, age >40 and crp >40. 1 it seems to have first been developed for a recently launched covid-19 immune modulator therapy trial (tactic-r), 2 based on an initial unpublished cohort of 200 patients. 3 an adapted version of the risk score, with twelve variables, was used to predict clinical deterioration (i.e. death or admission to critical care) in a cohort of 1,157 confirmed covid-19 patients in one london nhs trust. 4 this score showed good performance, with an area under the receiver operating curve (auroc) of 71.2% (test data). 4 the findings were published in this journal, which we read with interest. however, how the score proposed by eams performs in front-line settings and its implications for how many patients will likely receive remdesivir for covid-19 is currently unknown. we evaluated the performance of the eams criteria in a cohort of 517 patients covid-19 confirmed patients admitted to imperial college healthcare trust between the start of the pandemic and 5 th april 2020. 5 we found that 348 patients in our cohort would have met criteria to be considered for remdesivir therapy (i.e. age > 12, weight >=40kg, creatinine clearance >50ml/min and ast/alt <5 x uln or no history of childs pugh c liver cirrhosis). according to the eams score, 262 (75.3%) of the eligible patients in our cohort would have been classified as high-risk and 86 (24.7%%) as low-risk. the composite risk of death or itu admission was 2.58 times greater (95%ci 1.56-4.25, p < 0.001) for the high-compared to the low-risk group ( figure 1a ). the performance of the score was reasonable when considering the auroc of 71.1% (p<0.001) ( figure 1b) . however, the overall misclassification of outcome was of 45.7%, with 14 (4.0%) patients who deteriorated classified as low-risk and 145 (41.7%) who did not deteriorate as high-risk, which has potential implications for allocation of a scarce resource. common characteristics of those classified as low-risk that subsequently deteriorated included being female, white and having a nonsevere appearance by chest x-ray (table 1) . additionally, of the eight individual covariates in the full predictive model proposed by eams, only rale score and crp levels were statistically significant in predicting the composite outcome in our cohort ( table 2) . also of note, 169 patients in our cohort did not meet initial eams eligibility criteria for remdesivir. the majority (n=160) would have been excluded due to a creatinine clearance <50ml/min, 1 based on age, 1 for weight and 7 for known cirrhotic liver disease. the crude incidence rate of deterioration in this group was of 47.9% and, if the eams score would have been applied, it would not have differentiated the risk of deterioration between those classified as high or low-risk (rr 1.43, 95%ci 0.87-2.36, p=0.12). worryingly, their crude incidence rate of deterioration was similar to the high-risk group meeting inclusion criteria, at 42.0%. this highlights an important group of patients with renal impairment with poor covid-19 outcomes who are often excluded from clinical trials. we acknowledge the urgent need to be responsive to the rapidly changing context, given the enormous public health implications of treatment allocation decisions based on clinical criteria. nevertheless, the release of the eams criteria based on a single cohort of patients seems premature. while reassuring that the scoring system seems show reasonable performance in identifying most of those at high risk of adverse outcomes in our cohort, 11.3% of those who deteriorate and met inclusion criteria were missed by this score. moreover, amongst those not meeting inclusion criteria, the score was not able to accurately predict outcome, highlighting the urgent need to identify safe treatments for use in those with renal and/or hepatic impairment. importantly, in both the adapted risk criteria published previously in this journal and in those proposed by our group, hypalbuminaemia, reduced glomerular filtration and admission hypoxia (among other parameters) were also important predictors of worse hospitalisation outcomes. 4, 5 none of these parameters are included in the eams criteria, which could improve the misclassification issues observed. however, rationalising the number of parameters included for a scoring system intended for front-line clinical use should be carefully assessed to maximise its utility and limit increasing workload for already overstretched clinical teams. it has to be hoped that access to remdesivir for all who may benefit from it will be achievable in coming months as manufacturing capacity expands. in the meantime, criteria for eligibility should be refined based on data from a wide range of clinical settings and shared as quickly as possible to ensure a finite resource is used as rationally as possible. uk department of health & social care. early access to medicines scheme for remdesivir in the treatment of covid-19 multi-arm therapeutic study in pre-icu patients admitted with covid-19 early epidemiological and clinical analysis of the first 200 patients with covid-19 admitted via the emergency department in king's college hospital a clinical risk score to identify patients with covid-19 at high risk of critical care admission or death: an observational cohort study clinical characteristics and predictors of outcomes of hospitalised patients with covid-19 in a london nhs trust: a retrospective cohort study key: cord-354612-7f91l0n9 authors: villar, livia melo; da costa, vanessa duarte; marques, bianca leires; da silva, lucas lima; santos, alanna calheiros; da fonseca mendonça, ana carolina; marques, vanessa alves; do nascimento, giselle prado; lewis-ximenez, lia laura; de paula, vanessa salete title: usefulness of saliva samples for detecting sars-cov-2 rna among liver disease patients date: 2020-07-23 journal: j infect doi: 10.1016/j.jinf.2020.07.017 sha: doc_id: 354612 cord_uid: 7f91l0n9 nan first, we evaluated extraction method and limit of detection of artificially spiked sars-cov-2 saliva samples (estimated viral load: 10 3 , 10 2 , 10 1 , 10 0 copies/ml). saliva were collected using salivette device as previous described (3) both methods were feasible to extract sars-cov-2 rna saliva, however using m1 the detection limit was 10 copies/ml and m2 the limit of detection was 1 copy/ml. m2 was applied to extract rna from saliva and nps from 13 volunteers (5 hepatitis cases and 8 non hepatitis cases). volunteers gave saliva samples using salivette device after signing informed consent. a total of four individuals (two hepatitis cases and two without liver disease) were negative to sars cov-2 in nps and saliva (100% of specificity). the overall positivity was 9/13 (69.2%) lower than observed in saliva from ambulatory patients without liver disease (84.6%) (5). a total of 11/13 (84.6%) had concordant results in saliva and nps samples what is lower than observed by azzi and coleagues (1) and probably is the reflex of severity of disease among both studies. positive concordant results in nps and saliva were observed in seven individuals (two hepatitis cases and 5 without liver disease) until 7 days after onset of symptoms (100% of sensitivity). after 7 days of onset of symptoms, rna was detected in nps but it was not observed in paired saliva samples. this is the first report of sars cov-2 detection in saliva samples among liver disease patients showing best results until 7 days of beginning of symptoms. there is an urgency for alternative methods for sars-cov-2 rna detection to overcome swab availability and increase the access of diagnosis. saliva samples have been evaluated for sars cov-2 rna detection in severe cases or hospitalized patients, but there is a lack of data about theses samples in mild cases or a standard protocol for sample collection and viral detection. in addition, there is no information regarding the usefulness of saliva for detecting sars cov-2 rna in individuals presenting comorbidities, such as liver disease. the present study gives new information regarding the presence of sars cov-2 in saliva of liver disease patients. since saliva can be collected easily, sars cov-2 rna detection in saliva can be useful strategy to increase the access of sample collection for the diagnosis of covid-19 in patients with liver disease. saliva is a reliable tool to detect sars-cov-2 utility of oral fluid samples for hepatitis b antibody detection in real life conditions comparison of oral fluid collection methods for the molecular detection of hepatitis b virus centers for disease control and prevention (cdc). cdc 2019-novel coronavirus (2019-ncov) real-time rt-pcr diagnostic panel saliva as a noninvasive specimen for detection of sars-cov-2 figure 1. box plot graph of cycle threshold (ct) values in nasopharyngeal swabs and saliva specimens of positive samples for sars cov-2. vertical lines indicate range of values, and the median ct value is represented as black horizontal line within the box plot key: cord-312602-855n5av1 authors: chen, min; hong, nan; hu, shan; wang, peng; guan, hongzhi; xiao, meng; zhu, xinlin; al-hatmi, abdullah m.s.; zhou, zhe; gao, lei; boekhout, teun; xu, jianping; xu, yingchun; liao, wanqing; yang, ying title: molecular identification of cryptococcus gattii from cerebrospinal fluid using single-cell sequencing: a case study date: 2020-06-23 journal: j infect doi: 10.1016/j.jinf.2020.06.040 sha: doc_id: 312602 cord_uid: 855n5av1 a 31-year-old man presented with cryptococcal meningitis (cm) without typical clinical characteristics, but with abnormal walking, difficult leg lifting and frequent falling. he was admitted to a hospital in beijing for two months and then transferred to peking union medical college hospital. after multiple tests failed to identify the pathogen, single-cell sequencing (scs) was used to test the cerebrospinal fluid (csf). comparing the sequence obtained from single-cell sequencing with the reference database, it was found that the infection was caused by cryptococcus gattii sensu stricto (aflp4/vgi genotype). cryptococcus is difficult to cultivate from complex body fluids. the etiological agent of this patient was identified and the patient was treated. this case is the first case in which scs was used to detect and identify fungal pathogen after conventional testing failed to identify the cause of the disease. this report demonstrates that the scs approach can be used to generate fungal genome sequences directly from the csf of a cm patient. the scs technology could become a powerful tool to precise detect microscopically visible but uncultured pathogens in clinical samples. the etiological agent of this patient was identified and the patient was treated. this case is the first case in which scs was used to detect and identify fungal pathogen after conventional testing failed to identify the cause of the disease. this report demonstrates that the scs approach can be used to generate fungal genome sequences directly from the csf of a cm patient. the scs technology could become a powerful tool to precise detect microscopically visible but uncultured pathogens in clinical samples. infection is crucial for targeted treatment and patient survival [1, 2] . a considerable proportion of the etiologic agents of cns-related mycoses remains unidentified [3, 4] . cryptococcal meningitis (cm) is a life-threatening fungal infection associated with the human central nervous system (cns), which is mainly caused by yeasts of the basidiomycetous genus cryptococcus, particularly species that belong to the cryptococcus neoformans and cryptococcus gattii complexes [5] . the taxonomy and nomenclature of c. neoformans and c. gattii species complexes have undergone several changes and remain a subject of controversy [6] . compared to the high incidence rate of cm caused by c. neoformans sensu stricto, the incidence of the c. gattii complex is significantly less at a global level, but can be found more frequently in specific geographic or climatic zones [7, 8] . the five genotype groups within c. gattii sensu lato identified based on their amplified fragment length polymorphism (aflp) banding patterns were proposed as five separate species, including c. gattii (genotype aflp4/vgi), c. bacillisporus (genotype aflp5/vgiii), c. deuterogattii (genotype aflp6/vgii), c. tetragattii (genotype aflp7/vgiv), and c. decagattii (genotype aflp10/vgiv) [9, 10] . isolates of c. gattii s.s. and c. deuterogattii are the most frequently encountered globally, whereas c. bacillisporus and c. decagattii are mostly reported from the american continents, and c. tetragattii and vgv isolates of c. gattii seem to be restricted to southern africa and india, respectively [7, 8, 11] . clinical symptoms and radiological signs of cm are notoriously non-specific, variable, and often absent [12] . laboratory assays, such as indian ink staining and cryptococcal antigen (cr-ag) detection fulfill an important role for the diagnosis of cm in clinics worldwide [13] . however, these conventional diagnostic assays are generally used to target the cryptococcal capsule. this can be problematic if cm is caused by capsule-deficient cryptococcus isolates [13] [14] [15] . furthermore, the overwhelming majority of conventional assays on cm without pure cultures cannot distinguish members of the c. neoformans/c. gattii species complexes. as a result, proper treatments against c. gattii sensu lato may not be achieved [8, 16] . routine molecular methods, such as multiple pcr-based assays, have not shown advantages when compared to cr-ag detection to diagnose cm [17] . recently, nextgeneration sequencing (ngs) of csf has been shown some potential advantages over traditional methods to identify culture-negative organisms among patients with cns infections [3, 18] . however, the effectiveness of ngs for identification of fungal pathogens can be challenging. because fungi have hard cell walls different from other pathogens, making it difficult to extract their dna from small quantities of complex clinical specimens. this is especially the case when there are questions related to whether the identified microbe represents a true pathogen or a contaminant. in addition, ngs data require appropriately trained personnel to interpret the results [18] . although the majority of human fungal pathogens are culturable, clinical specimens often contain visible spores or hyphae, but may fail to yield viable cultures in many clinical cases [19] . thus, molecular identification of visible spores or hyphae from clinical specimens, such as csf, can provide critical information for a reliable diagnosis of cns-related mycoses. notably, single-cell sequencing (scs) recently has been shown to be a powerful approach for exploring biological systems with unprecedented resolution. for example, the scs technology has been successfully used to do pre-implantation genetic diagnosis and analysis of circulating tumor cells [20] . moreover, scs technology was used to analyze the convalescent patients' b cells and identify potent neutralizing antibodies against sars-cov-2 during the covid-19 pandemic in 2020 [21] . this is helpful for prescribing specific targeted therapy on diseases. although scs has demonstrated a broad potential, it has seldomly applied to detect pathogens. as mentioned above, due to the hard fungal cell wall, it is typically more difficult to extracted dna from a small number of pathogen cells than the mammalian cells associated with clinical specimens. here, we firstly used scs and laser dissection technology to directly identify c. gattii from csf from a cm patient with atypical clinical characteristics. an otherwise healthy 31-year-old man with a 1-month history of intermittent fever showed that the pathogen belonged to c. gattii sensu stricto. because the patient could not tolerate side effects of amb, the patient was treated with voriconazole (vor, 400 mg/day) and 5-fc (6.0 g/day) for nearly 7 months when both csf pleocytosis and head mri examinations showed that the patient was recovered. the details of this case history are summarized in figure 1 . for scs, we first isolated the yeast cells from the csf using laser microdissection. approximately 40 μl of the reaction solution was added to the amplification reaction mixture (prepared according to the instruction as specified in the repli-g single cell kit), which consisted of the following: 9 μl of ddh 2 o, 29 μl of the repli-gsc reaction buffer repli-g, and 2 μl of the repli-g scdna polymerase. following incubation for 8 h at 30°c, repli-g dna polymerase was inactivated by incubating at the trimming of reads and the removal of illumina adapters were performed by trimmomatic 0.36. the leading and trailing bases with a quality value below 5 were removed and a sliding window trimming was performed with a window size of 15 bp. an average quality value threshold of 4 and reads with length above 60 bp were kept for further analyses. after trimming and filtering, a total of 6,925,939 read pairs (94.95%, 6,925,939/7,294,295) fulfilled our criteria. the filtered reads were then assembled de novo using spades 3.10.1 with metagenomic assembly mode and kmers of 21, 33, and 55 were tested to achieve the best assembly effect [20] . the assembled contigs were then identified by kraken2 [22] using a combined genome database of archaea, bacteria, fungi, homo sapiens (grch38.p13), protozoa and viruses. the assembly sequence has been submitted to the ncbi sar database, with the accession number of srx6952669. we extracted the internal transcribed spacer (its) sequences from the obtained sequence and compared the its sequences with those present in genbank. our comparison showed that the yeast-like cells belonged to the human pathogenic the extracted sequences were concatenated by fasconcat-g v1.0 [24] and further aligned by mafft 7.271 [25] . a phylogenetic tree subsequently was constructed based on the alignment of the dna obtained in this study. the 10 reference strains were analyzed using mega 7.0.21 with the neighbor joining (nj) method with 1000 bootstraps [26] . a total of 12,384 contigs with a contig n50 of 1151bp were obtained by spades using kmer of 55. this has a better assembly effect than when using the kmers of 21 and table s1 . the complete kraken identification results are shown in table s2 . a total of 2,963 contigs in our assembly were found to have consistent matches with the 10 reference strain genomes. these contigs were further used to construct the phylogenetic tree. the 2,963 contigs accounted for 70.3% (3,004,566 bp / 4,274,048 bp) of the 4,575 contigs identified by kraken2 as c. gatti sensu stricto. the phylogenetic analysis revealed that the yeast-like cells in this patient belonged to the genotype aflp4/vgi of the c. gattii species complex, closely related to c. gattii sensu stricto. strain e566 (figure 2 ). following this deduction, we could identify the suspected pathogenic isolate as c. gattii sensu stricto with a high level of confidence. as mentioned above, the routine clinical diagnostic tests of our csf samples obtained initially from this patient failed to identify the etiological agent responsible for his condition. in contrast, scs in combination with a bioinformatics pipeline allowed us to confirm that the infection was caused by c. gattii sensu stricto which equals the the aflp4/vgi genotype. compared to cm caused by c. neoformans sensu stricto, cm caused by c. gattii sensu lato is often associated with cerebral cryptococomma [27] and elevated csf opening pressure [28, 29] . however, our case did not exhibit these clinical characteristics notably, the scs technology in our study effectively and accurately generated enough sequence reads to facilitate direct detection and phylogenetic analysis of the cells of c. gattii sensu stricto in clinical csf samples. the majority (approximately 82%) of the classified contigs (n=5,644) in our study were identified as fungi (81.73%, 4,438/5,644). among all the classified contigs, 4438 of them were identified as c. gattii sensu stricto. this accounted for approximately 83% of the total length of the classified contigs (83.23%, 4,274,048 bp/5,135,098 bp). in our opinion, the scs technology is more effective than routine metagenome sequencing for identifying unexpected pathogens in clinical specimens. this is particularly true for the biopsy containing microscopically visible cells that fail to grow viable colonies onto standard laboratory media. one key reason is that the laser microdissection and scs technologies allow the end users to isolate and analyze individual pathogen cells directly from the clinical samples. in contrast, metagenome sequencing only yields a small fraction of the obtained sequences from the putative pathogen, with the majority sequences coming from the host cells. we successfully isolated ample target cells following indian ink staining using laser capture microdissection (lcm) [30, 31] . this provided the basis for us to successfully extract cryptococcal genomic dna from the csf and generate a genomic dna library using a combination of snail enzyme, lysozyme and the repli-g single cell kit. in our opinion, the present study clearly provides a suitable basis to make additional refinements for extraction of fungal dna from other clinical sample types. this will allow molecular identification of the etiologic agents of cns-related and other invasive mycoses. efficient methods for the extraction of fungal dna from clinical specimens are urgently required for pathogen detection using molecular assays [32] . the generated sequence reads facilitated our phylogenetic analyses to identify the targeted yeast cells as a specific lineage of c. gattii sensu lato. these were derived from the genotype aflp4/vgi representing c. gattii sensu stricto, which is the primary dna of c. gattii sensu stricto in our study showed a close phylogenetic relationship with that of strain e566 originating from australia. this suggests that vgi isolates from china have a close genetic relationship with those from australia [33, 34] . the environmental source of them could be related to wood production and the introduction of foreign tree species, such as eucalyptus species, into china from australia [35, 36] . in summary, using a scs approach, we successfully identified a c. gattii sensu stricto strain that causes cns infection. to our knowledge, this is the first time that this technology has been used to diagnose a case of cns-related mycosis caused by pathogenic fungi that could not be cultured. after further optimization of clinical sample separation, the scs technology will have great potential to accurately identify the etiologic agents of cns-related mycoses, as well as other disease-causing fungal pathogens. bootstraps test. one reference c. deneoformans strain jec21 was used as outgroup. tab. s1: spades assembling effect with different kmers tab. s2: kraken identification report of all classified contigs acknowledgements: we thank dr. ferry hagen (westerdijk fungal biodiversity institute is the recipient of major infectious diseases such as aids and viral hepatitis prevention and control technology major projects (2018zx10712001 and 2018zx10101003). m.c. is the recipient of national natural science foundation of china neuroinfections caused by fungi the spectrum of fungi that infects humans. cold spring harbor perspectives in medicine actionable diagnosis of neuroleptospirosis by next-generation sequencing chronic meningitis investigated via metagenomic next-generation sequencing cryptococcus: from environmental saprophyte to global pathogen importance of resolving fungal nomenclature: the case of multiple pathogenic species in the cryptococcus genus global molecular epidemiology of cryptococcus neoformans and cryptococcus gattii: an atlas of the molecular types cryptococcus gattii infections recognition of seven species in the cryptococcus gattii/cryptococcus neoformans species complex. fungal genetics and biology a new lineage of cryptococcus gattii (vgv) discovered in the central zambezian miombo woodlands autochthonous and dormant cryptococcus gattii infections in europe clinical practice guidelines for the management of cryptococcal disease: 2010 update by the infectious diseases society of america. clinical infectious diseases : an official publication of the infectious diseases society of evaluation of a newly developed lateral flow immunoassay for the diagnosis of cryptococcosis difficulty in diagnosing chronic meningitis caused by capsule-deficient cryptococcus neoformans false-negative cerebrospinal fluid cryptococcal latex agglutination tests for patients with culture-positive cryptococcal meningitis antifungal therapy and management of complications of cryptococcosis due to cryptococcus gattii evaluation of five conventional and molecular approaches for diagnosis of cryptococcal meningitis in non-hiv-infected patients illuminating uveitis: metagenomic deep sequencing identifies common and rare pathogens british society for medical mycology best practice recommendations for the diagnosis of serious fungal diseases spades: a new genome assembly algorithm and its applications to single-cell sequencing potent neutralizing antibodies against sars-cov-2 identified by high-throughput single-cell sequencing of convalescent patients' b cells kraken: ultrafast metagenomic sequence classification using exact alignments bedtools: the swiss-army tool for genome feature analysis fasconcat-g: extensive functions for multiple sequence alignment preparations concerning phylogenetic studies mafft multiple sequence alignment software version 7: improvements in performance and usability mega7: molecular evolutionary genetics analysis version 7.0 for bigger datasets cryptococcus gattii genotype vgiia infection in man clinical and host differences between infections with the two varieties of cryptococcus neoformans clinical manifestations of cryptococcus gattii infection: determinants of neurological sequelae and death. clinical infectious diseases : an official publication of the infectious diseases society of future medical applications of single-cell sequencing in cancer laser microdissection of fungal septa as visualised by scanning electron microscopy. fungal genetics and biology enhancing molecular approaches for diagnosis of fungal infections epidemiology of fungal infections in china epidemiology of cryptococcus and cryptococcosis in china. fungal genetics and biology cryptococcus gattii infections in china: extent of the problem? taxonomic analysis of cryptococcus species complex strain s8012 revealed cryptococcus gattii with high heterogeneity on the genetics the authors declared that they have no conflicts of interest. key: cord-352111-frk319q1 authors: woodruff, amelita title: covid-19 follow up testing date: 2020-05-11 journal: j infect doi: 10.1016/j.jinf.2020.05.012 sha: doc_id: 352111 cord_uid: frk319q1 • positive cases of sars-cov-2 were seen in the mayo clinic fl covid virtual clinic. • 70% of patients met cdc guidelines for release from quarantine & still tested (+); • the average time from onset of symptoms to negative testing was 19 days.  positive cases of sars-cov-2 were seen in the mayo clinic fl covid virtual clinic.  70% of patients met cdc guidelines for release from quarantine & still tested (+)  the average time from onset of symptoms to negative testing was 19 days dear editor, there is some uncertainty regarding the incubation period of the sars-cov-2 virus. there is also some uncertainly on the proportion of infected individuals who are asymptomatic carriers, and the timeframe from when a patient is infectious until becoming non-infectious. 1 we provide care for covid-19 patients in the outpatient setting through a virtual clinic. our patients have tested positive via nasopharyngeal swabs and rna detection with rt-pcr. they are followed throughout their illness with visits at intervals based on the severity of their symptoms using telemedicine technology. the cdc has two strategies to determine when a patient with covid-19 can discontinue self-isolation. one is a "test-based" strategy, and the other is a "non-test-based" strategy. the non-test-based strategy recommends that covid-19 patients can discontinue self-isolation when they have been afebrile for 72 hours without anti-pyretic medications, have improvement in respiratory symptoms, and have at least 10 days elapse since symptoms started, recently increased from 7 days. the test-based strategy requires resolution of fever without the use of anti-pyretics, improvement of respiratory symptoms, and two consecutive negative covid-19 nasopharyngeal swabs collected ≥24 hours apart. 2 we decided as part of our covid-19 virtual clinic to use the test-based strategy for all of our patients to better ensure that they were not contributing to the spread of disease. our organization manufactures the test, so we had ample testing supplies and laboratory capacity. as this disease is a reportable condition, these patients were also followed by the respective county health departments. the county health departments were using the test-based strategy only for healthcare workers, or those with essential public service jobs. as of april 17, 2020, we have enrolled 97 patients in our covid virtual clinic. of these, 72 have been tested after being afebrile for at least 72 hours, and had 7 days pass since symptoms started, along with symptom improvement. that is, 72 patients met criteria for the original release from self-isolation with the non-test-based strategy, but were tested using the test-based strategy. of these, twenty-two (30.1%) tested negative upon the first two tests, while the vast majority of patients (69.9%) tested positive at this interval. of the 69.9% who failed, thirty-six (72%) were positive on the first test, while fourteen (28%) had a negative first test but were positive on the second test. in our patient population, the average time from the onset of symptoms to negative testing is 19 days. this data shows that the cdc non-test-based strategy may cause early release from isolation for covid-19 patients and result in additional community transmission. given this, it may be beneficial to prolong the self-isolation time to greater than 14 days after symptom onset. the incubation period of coronavirus disease from publicly reported confirmed cases: estimation and application discontinuation of isolation for persons with covid-19 not in healthcare settings mh&view=epic footnote: 1. the authors do not have a commercial or other association that might pose a conflict of interest (e.g., pharmaceutical stock ownership, consultancy, advisory board membership key: cord-315723-unhg20e9 authors: juan, hui-chun; chao, chien-ming; lai, chih-cheng; tang, hung-jen title: decline in invasive pneumococcal disease during covid-19 pandemic in taiwan date: 2020-09-19 journal: j infect doi: 10.1016/j.jinf.2020.09.018 sha: doc_id: 315723 cord_uid: unhg20e9 nan dear editor , we read with great interest lim et al's report, 1 which showed a decreased incidence of pneumococcal disease in singapore during the first 27 weeks in 2020 in the time of covid-19. although the collateral benefit of controlling covid-19 for other common respiratory infectious diseases, such as influenza and tuberculosis in taiwan have been demonstrated, 2 , 3 the impact of the infection control and policy to prevent covid-19 outbreak on pneumococcal disease remained unclear. therefore, this study was conducted to determine whether the incidence of pneumococcal disease in taiwan would be decreasing as lim et al's study in singapore. in taiwan, invasive pneumococcal disease (ipd) is a notifiable disease for which reporting is mandatory for all clinicians. therefore, this study can obtain the case number of patients with ipd used data from open data website of taiwan's cdc. 4 to compare the case number of ipd during the same period each year, we extracted the monthly cases between january and august from 2015 to 2020. first, a total of 162 ipd cases were reported during the first 8 months in 2020, by contrast, the accumulative case number within the 8 months ranged from 282 in 2019 to 400 in 2016, which were much higher than that in 2020 ( fig. 1 a) . second, in 2020, the case number of ipd was highest in january ( n = 62) and gradually decreased with time, which was lowest in may ( n = 7) ( fig. 1 b) . in this study, we found the similar phenomena that ipd was decreasing during covid-19 in taiwan, like lim et al's findings in singapore. 1 the possible explanation could be the strict performance of infection control and policy during covid-19 pandemic. since the first case of covid-19 was identified in january, a total of 498 covid-19 cases were reported till now and caused seven deaths. to prevent the outbreak of covid-19, taiwan authority immediately practiced many infection control measures, particularly mask wearing, hand hygiene and avoid visiting crowd area. 5 most of these interventions can help prevent the transmission of sars-cov-2 via respiratory droplets and may also provide additional benefit in the controlling other respiratory infectious diseases, such as pneumococcal disease, which was demonstrated here. although many confounding factors, such as vaccine strategy or under-report of ipd during covid-19 pandemic were not evaluated in this study, our findings was consistent with singapore's study 1 suggest that strictly performance of infection control and policy not only mitigate the threaten of covid-19 but also reduce the burden of other respiratory infections disease -invasive pneumococcal diseases. decline in pneumococcal disease incidence in the time of covid-19 in singapore the covid-19 pandemic and tuberculosis in taiwan one benefit of covid-19 measures in taiwan: the reduction of influenza infections and severe complications. influenza other respir viruses severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and coronavirus disease-2019 (covid-19): the epidemic and the challenges key: cord-328479-1tzysg7u authors: chen, jianjun; huang, chaolin; zhang, yanan; zhang, sai; jin, meilin title: severe acute respiratory syndrome coronavirus 2-specific antibodies in pets in wuhan, china date: 2020-06-21 journal: j infect doi: 10.1016/j.jinf.2020.06.045 sha: doc_id: 328479 cord_uid: 1tzysg7u nan (sars-cov-2), were first reported in wuhan, china (2) (3) (4) . since then, a large number of human covid-19 cases have been reported around the world, becoming a global pandemic as declared by the world health organization (who). experimental data has confirmed that the sars-cov-2 virus can use angiotensin-converting enzyme ii (ace2) as a receptor (2) . furthermore, animal models of sars-cov-2 infection have shown that ferrets and cats are susceptible to infection (5) , implying that companion animals might be infected after contact with individuals carrying covid-19. in this study, we collected swab and blood samples from pet cats and dogs in wuhan whose owners were confirmed to have covid-19. our results provide serological evidence for sars-cov-2 infection in pets. pet cats and dogs were recruited from covid-19 patients admitted to wuhan jinyintan hospital, one of the designated hospitals for covid-19. participation of each pet was granted by the verbal informed consent of the respective owner. sampling was conducted on 30 and 31 march 2020. whole blood, oral, and rectal swabs were collected, and the sex and age of the pets were recorded. swab samples were collected using sterile swabs and placed in a vial containing 2 ml of viral transport medium, stored at 2-8°c, and shipped to the laboratory within 12 h for further analysis. whole blood samples (0.5-1 ml) were drawn into evacuated tubes containing edta. plasma was separated, aliquoted, and stored at -40°c prior to processing. swab and whole blood samples were collected from 10 cats (four female, six male) and 9 dogs (four female, five male) (supplementary table 1 we then conducted telephone questionnaires with the owners of the three pets. all owners and their spouses were diagnosed with covid-19. all three pets were in close contact with the owners and their spouses when they developed covid-19 symptoms. during the period when the owners were admitted to hospital, cat 8 left the house and wandered outside for more than 1 month; dog 4 was fostered by a pet hospital for more than 1 month until the owner was discharged and back home; and cat 18 was quarantined at home with cat 17 and dog 16, and cared for by other family members once every 3 days; cat 17 and dog 16 tested negative for sars-cov-2 antibodies. questionnaire data suggests that pets acquired sars-cov-2 virus from their owners, while other source of infection could not be excluded. in this study, we conducted a survey for sars-cov-2 in pets whose owners were diagnosed with covid-19, in 15 communities in wuhan. serological data suggests that three pets (two cats, one dog) had been exposed to the virus, although viral rna detection was negative. prior to our study, a preprint of a research article posted online at biorxiv indicated that sars-cov-2-specific antibodies were detected in cats in wuhan at the time of the covid-19 epidemic (7) . in addition, pet dogs and cats in hong kong (8), whose owners had been diagnosed with covid-19, tested positive for sars-cov-2 rna. collectively, these results indicate the sars-cov-2 can be transmitted to companion animals, possible through contact with owners carrying covid-19. however, we were not able to determine whether, under natural conditions, pet cats and dogs can be readily infected with or transmit sars-cov-2. further studies will be needed to establish the role of pets in the current covid-19 epidemic. none. interplay of personal, pet, and environmental colonization in households affected by community-associated methicillin-resistant staphylococcus aureus a pneumonia outbreak associated with a new coronavirus of probable bat origin clinical features of patients infected with 2019 novel coronavirus in wuhan genomic characterisation and epidemiology of 2019 novel coronavirus: implications for virus origins and receptor binding susceptibility of ferrets, cats, dogs, and other domesticated animals to sars-coronavirus 2. science serological survey of sars-cov-2 for experimental, domestic, companion and wild animals excludes intermediate hosts of 35 different species of animals sars-cov-2 neutralizing serum antibodies in cats: a serological investigation infection of dogs with sars-cov-2 key: cord-337835-78i6j11i authors: alfaraj, sarah h.; al-tawfiq, jaffar a.; alzahrani, nojoom a.; altwaijri, talal a.; memish, ziad a. title: the impact of co-infection of influenza a virus on the severity of middle east respiratory syndrome coronavirus date: 2017-02-09 journal: j infect doi: 10.1016/j.jinf.2017.02.001 sha: doc_id: 337835 cord_uid: 78i6j11i nan ho and colleagues recently drew attention to the consequences of co-infection with influenza and hiv. 1 we present four cases of combined infection with influenza and middle east respiratory syndrome coronavirus (mers-cov) infection. nasopharyngeal swabs or tracheal aspirates were tested for mers-cov using real-time reverse-transcription polymerase chain reaction (rt-pcr). 2,3 samples were tested for influenza a, b and h1n1 by rapid molecular test (gen-exper for detection of flu a, b and 2009 h1n1, cepheid). in the first case, a 39 year-old male, health care worker, an engineer who became ill seven days before admission. he had fever >38 c, cough and sore throat. he had no nausea, vomiting, diarrhoea or shortness of breath (sob). he denied history of travel or contact with positive case or camels. he was febrile with a temperature of 39.5 c. chest x-ray showed non-homogenous opacity at the lower right lung zone. a nasopharyngeal swab was positive for mers-cov with ct value upe gene 34, and orf1a 34 ( table 1 ). the test was negative for influenza but a repeat swab after 48 h was negative for mers-cov and positive for h1n1. the patient received azithromycin, ceftriaxone and oseltamivir. the patient was discharged home after two negative swabs of mers-cov and being asymptomatic for 48 h. in the second case, a 61 year-old female with diabetes mellitus and dyslipidemia was admitted with a three-day history of shortness of breath and productive cough. she had no nausea, vomiting, or diarrhoea. she denied history of travel or contact with positive case or camels. she was afebrile with a temperature of 37 c. chest x-ray showed patchy opacities involving middle and lower zones of both lung fields. a nasopharyngeal swab was positive for mers-cov with ct value upe gene 34, orf1a 35 and negative for influenza. a repeat swab after 48 h was negative for mers-cov but positive for h1n1. she required bipap and she was subsequently intubated and was started on mechanical ventilation. she was extubated after 13 days. the patient received piperacillinetazobactam, and erythromycin. the patient was discharged home after she had 2 negative swabs of mers-cov and being asymptomatic for 48 h. in the third case, a 29 year-old housekeeper female was admitted with two days history of fever and cough. she had no nausea, vomiting, diarrhoea nor shortness of breathing. she had a history of contact with mers-cov positive case. she was afebrile with a temperature of 36.9 c. chest x-ray was normal. a nasopharyngeal swab collected upon presentation was positive for mers-cov with ct value upe gene 32 orf1a 32. the swab was negative for influenza. a repeated swab after 48 h was positive mers-cov and positive for h1n1. the patient received oseltamivir, azithromycin and ceftriaxone. the patient was discharged home after she had 2 negative swab of mers-cov and being asymptomatic for 48 h. in the fourth case, the patient was a 73 year-old female with a history of hypothyroidism, heart failure, lymphoma, and lung fibrosis. she has no history of travel or contact with positive case or camels. four days prior to her presentation, she had productive cough and shortness of breath. she had no fever, diarrhoea, vomiting or nausea. she was afebrile with a temperature of 36.7 c. chest x-ray showed bilateral diffuse infiltrate (fig. 1) . a nasopharyngeal swab was positive for mers-cov with ct value upe gene 37; orf1a 36 and negative for influenza. a repeat swab after 3 days was negative for mers-cov but positive for influenza a. the patient was treated with piperacillinetazobactam for six days and oseltamivir for 5 days. the patient was discharged home after two negative mers-cov and being asymptomatic for 48 h. these patients highlight the co-infection with mers-cov and influenza. the exact reason to have a negative influenza test at the time of positive mers-cov is not completely understood. it is possible that the presence of mers-cov inhibits the pcr reaction for influenza virus. however, an earlier case of mers-cov tested initially positive for influenza a(h1n1)pdm09. 4 on the other hand, the positivity of nasal swabs for influenza is specimen type and technique dependent. 5 thus, initially negative influenza tests could be a false test result. positive results for viral respiratory pathogens should not preclude testing for mers-cov because coinfection can occur. 6 only a small number of mers cases had co-infection with influenza a, parainfluenza, herpes simplex, and streptococcus pneumoniae. 7 in one case, a co-infection with herpes simplex virus type 1 dna13 and rhinovirus rna14 were detected by rt-pcr. 8 the investigation of the first 47 cases showed no co-infection with mers-cov. 2 there is a controversy regarding the risk of increased or decreased severity of co-infections. for example co-infections with respiratory syncytial virus (rsv) and human meta-pneumovirus (hmpv) causes more severe infection than either virus alone with longer hospitalization and oxygen requirement. 9 other studies did not demonstrate these effects. 10 the association and the impact of co-infection with mers-cov and influenza viruses deserve further evaluation and studies. all authors have no conflict of interest to report. completeness of case ascertainment and availability of environmental data in legionnaires' disease enhanced surveillance in england, 2012e2014 to the editor, bai et al. 1 previously highlighted the importance of effective communicable disease surveillance in china for the detection of outbreaks to inform infectious disease prevention and control. to achieve similar aims with relation to the control of legionnaires' disease in england a national enhanced surveillance scheme operates. surveillance data must be both complete and as timely as possible, as highlighted by freeman et al. in this journal. 2 therefore we sought to assess the completeness of case ascertainment in addition to the availability of environmental data reported to the national enhanced legionella surveillance scheme (nelss) for residents in england. the value of environmental data is particularly important for legionnaires' disease in order to inform the attribution of potential environmental sources of cases and clusters of this infection. in england, the national legionella surveillance team (nlst) leads on surveillance and control of legionnaires' disease, while field epidemiology services (fes) teams collect surveillance data and investigate sporadic cases, clusters and outbreaks in conjunction with local health protection teams (hpts). the nlst worked with the fes teams in the north west (nw) and west midlands (wmids) regions of england to audit the reporting of legionella cases with onset dates during each calendar year between 2012 and 2014, inclusive. eligible cases were those cases of legionella spp. infections resident in either of these regions in england, which were laboratory confirmed by urinary antigen testing, culture or serological testing. these cases were identified in the month of february following each calendar year by the nlst and were compared to those known by the fes teams for nw and wmids regions. completeness of case ascertainment was calculated as the proportion of cases recorded by fes teams for cases with onset of symptoms between 2012 and 2014 inclusive which were reported to the nlst. the availability of environmental data reported to the national enhanced surveillance scheme was calculated as the proportion of cases during the same time period with any environmental investigation data reported to the nlst. the results are summarised in table 1 ; in the nw region, a mean of 34 cases were reported per year between 2012 and 2014. in the wmids region, the mean number of cases per year was 46. both regions reported 100% of cases to the nlst. mean availability of environmental data reported was 80.8% in the nw and 42.8% in wmids. however, there was a the impact of hiv on the burden and severity of influenza illness in malawian adults: the bash-flu study epidemiological, demographic, and clinical characteristics of 47 cases of middle east respiratory syndrome coronavirus disease from saudi arabia: a descriptive study assays for laboratory confirmation of novel human coronavirus (hcov-emc) infections health protection agency (hpa) uk novel coronavirus investigation team. evidence of person-to-person transmission within a family cluster of novel coronavirus infections an adult returned traveler from dubai hospitalized with an influenza-like illness (ili): middle east respiratory syndrome (mers) or influenza? infection control implications from a near mers case saudi ministry of health. case definition and surveillance guidance for mers-cov testing in saudi world health organization. who guidelines for investigation of cases of human infection with middle east respiratory syndrome coronavirus (mers-cov) clinical features and virological analysis of a case of middle east respiratory syndrome coronavirus infection prospective study of human metapneumovirus infection in children less than 3 years of age the role of infections and coinfections with newly identified and emerging respiratory viruses in children key: cord-305583-p2jp5fiq authors: lalloo, david g.; shingadia, delane; bell, david j.; beeching, nicholas j.; whitty, christopher j.m.; chiodini, peter l. title: uk malaria treatment guidelines 2016 date: 2016-02-12 journal: j infect doi: 10.1016/j.jinf.2016.02.001 sha: doc_id: 305583 cord_uid: p2jp5fiq 1.malaria is the tropical disease most commonly imported into the uk, with 1300–1800 cases reported each year, and 2–11 deaths. 2. approximately three quarters of reported malaria cases in the uk are caused by plasmodium falciparum, which is capable of invading a high proportion of red blood cells and rapidly leading to severe or life-threatening multi-organ disease. 3. most non-falciparum malaria cases are caused by plasmodium vivax; a few cases are caused by the other species of plasmodium: plasmodium ovale, plasmodium malariae or plasmodium knowlesi. 4. mixed infections with more than one species of parasite can occur; they commonly involve p. falciparum with the attendant risks of severe malaria. 5. there are no typical clinical features of malaria; even fever is not invariably present. malaria in children (and sometimes in adults) may present with misleading symptoms such as gastrointestinal features, sore throat or lower respiratory complaints. 6. a diagnosis of malaria must always be sought in a feverish or sick child or adult who has visited malaria-endemic areas. specific country information on malaria can be found at http://travelhealthpro.org.uk/. p. falciparum infection rarely presents more than six months after exposure but presentation of other species can occur more than a year after exposure. 7. management of malaria depends on awareness of the diagnosis and on performing the correct diagnostic tests: the diagnosis cannot be excluded until more than one blood specimen has been examined. other travel related infections, especially viral haemorrhagic fevers, should also be considered. 8. the optimum diagnostic procedure is examination of thick and thin blood films by an expert to detect and speciate the malarial parasites. p. falciparum and p. vivax (depending upon the product) malaria can be diagnosed almost as accurately using rapid diagnostic tests (rdts) which detect plasmodial antigens. rdts for other plasmodium species are not as reliable. 9. most patients treated for p. falciparum malaria should be admitted to hospital for at least 24 h as patients can deteriorate suddenly, especially early in the course of treatment. in specialised units seeing large numbers of patients, outpatient treatment may be considered if specific protocols for patient selection and follow up are in place. 10. uncomplicated p. falciparum malaria should be treated with an artemisinin combination therapy (grade 1a). artemether–lumefantrine (riamet(®)) is the drug of choice (grade 2c) and dihydroartemisinin-piperaquine (eurartesim(®)) is an alternative. quinine or atovaquone–proguanil (malarone(®)) can be used if an act is not available. quinine is highly effective but poorly-tolerated in prolonged treatment and should be used in combination with an additional drug, usually oral doxycycline. 11. severe falciparum malaria, or infections complicated by a relatively high parasite count (more than 2% of red blood cells parasitized) should be treated with intravenous therapy until the patient is well enough to continue with oral treatment. severe malaria is a rare complication of p. vivax or p. knowlesi infection and also requires parenteral therapy. 12. the treatment of choice for severe or complicated malaria in adults and children is intravenous artesunate (grade 1a). intravenous artesunate is unlicensed in the eu but is available in many centres. the alternative is intravenous quinine, which should be started immediately if artesunate is not available (grade 1a). patients treated with intravenous quinine require careful monitoring for hypoglycemia. 13. patients with severe or complicated malaria should be managed in a high-dependency or intensive care environment. they may require haemodynamic support and management of: acute respiratory distress syndrome, disseminated intravascular coagulation, acute kidney injury, seizures, and severe intercurrent infections including gram-negative bacteraemia/septicaemia. 14. children with severe malaria should also be treated with empirical broad spectrum antibiotics until bacterial infection can be excluded (grade 1b). 15. haemolysis occurs in approximately 10–15% patients following intravenous artesunate treatment. haemoglobin concentrations should be checked approximately 14 days following treatment in those treated with iv artemisinins (grade 2c). 16. falciparum malaria in pregnancy is more likely to be complicated: the placenta contains high levels of parasites, stillbirth or early delivery may occur and diagnosis can be difficult if parasites are concentrated in the placenta and scanty in the blood. 17. uncomplicated falciparum malaria in the second and third trimester of pregnancy should be treated with artemether–lumefantrine (grade 2b). uncomplicated falciparum malaria in the first trimester of pregnancy should usually be treated with quinine and clindamycin but specialist advice should be sought. severe malaria in any trimester of pregnancy should be treated as for any other patient with artesunate preferred over quinine (grade 1c). 18. children with uncomplicated malaria should be treated with an act (artemether–lumefantrine or dihydroartemisinin-piperaquine) as first line treatment (grade 1a). quinine with doxycycline or clindamycin, or atovaquone–proguanil at appropriate doses for weight can also be used. doxycycline should not be given to children under 12 years. 19. either an oral act or chloroquine can be used for the treatment of non-falciparum malaria. an oral act is preferred for a mixed infection, if there is uncertainty about the infecting species, or for p. vivax infection from areas where chloroquine resistance is common (grade 1b). 20. dormant parasites (hypnozoites) persist in the liver after treatment of p. vivax or p. ovale infection: the only currently effective drug for eradication of hypnozoites is primaquine (1a). primaquine is more effective at preventing relapse if taken at the same time as chloroquine (grade 1c). 21. primaquine should be avoided or given with caution under expert supervision in patients with glucose-6-phosphate dehydrogenase deficiency (g6pd), in whom it may cause severe haemolysis. 22. primaquine (for eradication of p. vivax or p. ovale hypnozoites) is contraindicated in pregnancy and when breastfeeding (until the g6pd status of child is known); after initial treatment for these infections a pregnant woman should take weekly chloroquine prophylaxis until after delivery or cessation of breastfeeding when hypnozoite eradication can be considered. 23. an acute attack of malaria does not confer protection from future attacks: individuals who have had malaria should take effective anti-mosquito precautions and chemoprophylaxis during future visits to endemic areas. 7. management of malaria depends on awareness of the diagnosis and on performing the correct diagnostic tests: the diagnosis cannot be excluded until more than one blood specimen has been examined. other travel related infections, especially viral haemorrhagic fevers, should also be considered. 8 . the optimum diagnostic procedure is examination of thick and thin blood films by an expert to detect and speciate the malarial parasites. p. falciparum and p. vivax (depending upon the product) malaria can be diagnosed almost as accurately using rapid diagnostic tests (rdts) which detect plasmodial antigens. rdts for other plasmodium species are not as reliable. 9. most patients treated for p. falciparum malaria should be admitted to hospital for at least 24 h as patients can deteriorate suddenly, especially early in the course of treatment. in specialised units seeing large numbers of patients, outpatient treatment may be considered if specific protocols for patient selection and follow up are in place. 10 . uncomplicated p. falciparum malaria should be treated with an artemisinin combination therapy (grade 1a). artemetherelumefantrine (riamet ò ) is the drug of choice (grade 2c) and dihydroartemisinin-piperaquine (eurartesim ò ) is an alternative. quinine or atovaquone eproguanil (malarone ò ) can be used if an act is not available. quinine is highly effective but poorly-tolerated in prolonged treatment and should be used in combination with an additional drug, usually oral doxycycline. 11 . severe falciparum malaria, or infections complicated by a relatively high parasite count (more than 2% of red blood cells parasitized) should be treated with intravenous therapy until the patient is well enough to continue with oral treatment. severe malaria is a rare complication of p. vivax or p. knowlesi infection and also requires parenteral therapy. 12. the treatment of choice for severe or complicated malaria in adults and children is intravenous artesunate (grade 1a). intravenous artesunate is unlicensed in the eu but is available in many centres. the alternative is intravenous quinine, which should be started immediately if artesunate is not available (grade 1a). patients treated with intravenous quinine require careful monitoring for hypoglycemia. 13 . patients with severe or complicated malaria should be managed in a high-dependency or intensive care environment. they may require haemodynamic support and management of: acute respiratory distress syndrome, disseminated intravascular coagulation, acute kidney injury, seizures, and severe intercurrent infections including gram-negative bacteraemia/septicaemia. 14. children with severe malaria should also be treated with empirical broad spectrum antibiotics until bacterial infection can be excluded (grade 1b). 15 . haemolysis occurs in approximately 10e15% patients following intravenous artesunate treatment. haemoglobin concentrations should be checked approximately 14 days following treatment in those treated with iv artemisinins (grade 2c). 16 . falciparum malaria in pregnancy is more likely to be complicated: the placenta contains high levels of parasites, stillbirth or early delivery may occur and diagnosis can be difficult if parasites are concentrated in the placenta and scanty in the blood. 17 . uncomplicated falciparum malaria in the second and third trimester of pregnancy should be treated with artemetherelumefantrine (grade 2b). uncomplicated falciparum malaria in the first trimester of pregnancy should usually be treated with quinine and clindamycin but specialist advice should be sought. severe malaria in any trimester of pregnancy should be treated as for any other patient with artesunate preferred over quinine (grade 1c). 18 . children with uncomplicated malaria should be treated with an act (artemether elumefantrine or dihydroartemisinin-piperaquine) as first line treatment (grade 1a). quinine with doxycycline or clindamycin, or atovaquoneeproguanil at appropriate doses for weight can also be used. doxycycline should not be given to children under 12 years. 19 . either an oral act or chloroquine can be used for the treatment of non-falciparum malaria. an oral act is preferred for a mixed infection, if there is uncertainty about the infecting species, or for p. vivax infection from areas where chloroquine resistance is common (grade 1b). 20. dormant parasites (hypnozoites) persist in the liver after treatment of p. vivax or p. ovale infection: the only currently effective drug for eradication of hypnozoites is primaquine (1a). primaquine is more effective at preventing relapse if taken at the same time as chloroquine (grade 1c). 21 . primaquine should be avoided or given with caution under expert supervision in patients with glucose-6-phosphate dehydrogenase deficiency (g6pd), in whom it may cause severe haemolysis. 22 . primaquine (for eradication of p. vivax or p. ovale hypnozoites) is contraindicated in pregnancy and when breastfeeding (until the g6pd status of child is known); after initial treatment for these infections a pregnant woman should take weekly chloroquine prophylaxis until after delivery or cessation of breastfeeding when hypnozoite eradication can be considered. 23. an acute attack of malaria does not confer protection from future attacks: individuals who have had malaria should take effective anti-mosquito precautions and chemoprophylaxis during future visits to endemic areas. ª 2016 the british infection association. published by elsevier ltd. all rights reserved. malaria remains one of the most common imported infections in the united kingdom (uk). between 1300 and 1800 malaria cases are reported each year in the uk, although reviews of reporting suggest that this may represent about 65% of all cases that occur. 1 approximately three-quarters of reported infections are due to plasmodium falciparum and there are between 2 and 11 deaths annually. children under 16 account for around 10% of cases. 2 two thirds of cases occur in people of african or south asian ethnic origin and over half of the cases occur in those who had been visiting friends and family in endemic areas. 3 most patients with falciparum malaria acquire infection in africa and malaria accounts for about 20% of travellers from africa presenting to hospital with fever; west africa is the commonest geographical source. most plasmodium vivax infections are acquired in south asia. 2, 4 this document offers guidance for the management of both uncomplicated and complicated malaria in the uk. it complements existing public health england (phe) guidelines on the prevention of malaria in uk travellers. https:// www.gov.uk/government/publications/malariaprevention-guidelines-for-travellers-from-the-uk. it has been based on a review of the available evidence by the phe advisory committee on malaria prevention (acmp), with input from other experts and expert bodies, and incorporates international guidance including who guidelines on treatment and definitions of severe malaria. 5,6 these guidelines will specifically present a uk perspective on management. other non-endemic countries including the usa, canada and europe have also developed their own guidelines. 7e9 these uk guidelines have been developed specifically for use in a non-endemic setting, but necessarily depend heavily upon evidence obtained from studies in endemic areas. more detailed information about individual drug regimens and contra-indications can be found in the british national formulary (http://www.bnf.org/). a short summary of key points in the initial assessment and management, for use in emergency departments, is available from the british infection association website (http://www.britishinfection.org/). major recommendations have been graded using a modified grade approach which grades both the strength of the recommendation and the level of evidence for the recommendations. a grade 1 recommendation is a strong recommendation to do (or not do) something, where benefits clearly outweigh risks (or vice versa) for most patients. a grade 2 recommendation is a conditional recommendation, where the risks and benefits are more closely balanced or are more uncertain. grade a evidence means high-quality evidence that comes from consistent results from high quality randomised controlled trials (rcts). grade b evidence means moderate-quality evidence from randomised trials that suffers from flaws in conduct or design or methodologically strong observational studies with consistent effects and exclusion of most sources of bias. grade c evidence is low-quality evidence from controlled trials with serious limitations or inconsistent results, or observational studies with limited evidence on effects. grade d evidence is based only on case studies or expert judgement or poor quality observational studies. assessment of the patient with suspected malaria (boxes 1 and 2) the crucial issue in the management of malaria is consideration of the possibility of this diagnosis. malaria should be suspected in anyone with a fever or a history of fever who has returned from or previously visited a malaria endemic area, regardless of whether they have taken prophylaxis. the minimum incubation period for naturally acquired infection is six days. most patients with falciparum infection present in the first month or months after exposure; almost all present within six months of exposure. vivax or ovale infections commonly present later than six months after exposure and presentation may be delayed for years. there are no specific symptoms of malaria: most patients complain of fever, headache and general malaise. 10 gastrointestinal disturbances, jaundice or respiratory symptoms occasionally occur and are often responsible for misdiagnosis. most missed malaria infections are erroneously diagnosed as non-specific viral infections, influenza, gastroenteritis or hepatitis. children are less likely than adults to complain of chills, arthralgia/myalgia or headaches and more likely to present with non-specific symptoms (fever, lethargy, malaise, somnolence): gastrointestinal symptoms (nausea, abdominal pain, vomiting, diarrhoea) are particularly common. 11, 12 the physical examination of patients with uncomplicated malaria is often unremarkable apart from a fever which is not invariably present. most patients have no specific fever pattern. children are more likely to have hepatomegaly, splenomegaly and somnolence than adults. 11, 12 if the diagnosis of falciparum malaria has been delayed, severely ill patients may present with jaundice, confusion or seizures. if malaria is suspected, a blood test for malaria without delay is mandatory. unless rapid malaria testing can be achieved in primary care, the patient should be referred to hospital for testing. results should be communicated the same day: all positive tests should be telephoned back to the requesting doctor as soon as practicable and ideally within 4 h of the test reaching the laboratory. the most important test is examination of thick and thin blood smears by microscopy. this is highly sensitive and specific in expert hands. however, because of a lack of expertise in many uk labs, particularly out of hours, rapid diagnostic tests (rdts) based upon detection of parasite antigens, are now commonly used in addition to blood slides. although slightly less sensitive than good quality blood films examined by experienced microscopists, they are easier for the non-expert to use to detect falciparum infections and are useful as an initial screen if expertise in reading slides is not immediately available. rdts are generally not as specific and sensitive for the detection of non-falciparum infections, although newer generation rdts perform well in diagnosing vivax infection. 13, 14 rdts may be used in addition to, but not as a replacement for blood films and all patients with suspected malaria should have blood films prepared and examined as recommended in the british committee for standards in haematology guidelines. 15 if falciparum or knowlesi malaria is diagnosed, the percentage of red blood cells that are parasitized should be estimated. if there is clinical suspicion of malaria, but initial blood films are negative, repeat films, with or without rdt, should be examined after 12e24 h and again after a further 24 h. rdt use alone should be discouraged where good microscopy is available. thrombocytopaenia is suggestive of malaria in non-immune adults and children, both in nonfalciparum malarias and in p. falciparum, 16, 17 although it can also occur in a number of other imported infections. malaria is unlikely if three negative specimens have been examined by a competent microscopist. empirical therapy for malaria should not be given unless a patient with a convincing exposure history demonstrates features of severe malaria and expert advice has been taken. in pregnancy, thick films can be negative, despite the presence of parasites in the placenta. expert advice should be sought if malaria is suspected. pcr techniques are used in reference laboratories to determine the species of malaria, but are not sufficiently standardised or validated to use for routine clinical diagnosis. cases of malaria should be notified to public health authorities. in england and wales, thick and thin films and a blood aliquot should be sent to the malaria reference laboratory for confirmation (which is performed free of charge). the scottish parasite and diagnostic reference laboratory provides a reference service for scotland. if malaria is diagnosed in a returned traveller, other members of the family or travelling group should be warned that they may have shared the same exposure risk and that they should seek medical attention if they develop symptoms. malaria, particularly severe malaria, should always be managed in consultation with someone experienced in managing the disease. non-falciparum malaria the distinction between falciparum malaria and other species of malaria is important. malaria caused by plasmodium ovale, p. vivax, and plasmodium malariae rarely causes life-threatening disease and can usually be managed on an outpatient basis, unless the patient has other comorbidities or cannot tolerate oral medication. however, severe presentations of plasmodium knowlesi infection and p. vivax are well recognised and severe p. ovale can occur in exceptional circumstances and clinicians should look out for these rare cases. 20e23 box 1. important considerations in the assessment of a patient with possible malaria. malaria is a medical emergency and patients with suspected malaria should be evaluated immediately. symptoms of malaria are often non-specific: fever/sweats/chills, malaise, myalgia, headache, diarrhoea, and cough. falciparum malaria is most likely to occur within 3 months of return from an endemic area. the incubation period for malaria is at least 6 days. malaria caused by other species may present more than a year after return from an endemic area. a careful exposure history is necessary: country and area of travel, including stopovers, and date of return. consider what malaria prophylaxis was taken (i.e. drug, dose & adherence, premature cessation); appropriate prophylaxis with full adherence does not exclude malaria. consider other travel-related infections: e.g. typhoid, hepatitis, dengue or other arboviruses, avian influenza, mers-cov, hiv, meningitis/encephalitis and viral haemorrhagic fevers (vhf). 18 three negative diagnostic samples over a period of 24e48 h are necessary to exclude malaria. estimation of the haemoglobin concentration should be done, and in malaria caused by p. vivax or p. ovale, glucose-6-phosphate dehydrogenase (g6pd) activity should be measured, as concomitant primaquine therapy will be necessary to eliminate hypnozoites (dormant forms) from the liver. primaquine can cause haemolysis in patients with g6pd deficiency. patients with a mixed infection that includes falciparum parasites or with an infection with an unidentified species should be treated as though they had falciparum infection in the first instance. patients with falciparum malaria should usually be admitted to hospital initially because of the risk of deterioration even after effective treatment has been initiated. there is limited evidence that some patients with falciparum malaria can be managed safely as outpatients in units that see large numbers of patients and use well defined protocols for assessment and follow up. 24e27 certain factors such as age, ethnicity and parasite count can predict the likelihood of severe malaria, but even patients who might be expected to be semi-immune may deteriorate rapidly and require intensive care treatment. 28e31 outpatient management of malaria in adults should only be undertaken by clinicians experienced in managing malaria with clear protocols and systems for assessing the likely risk of severe malaria and for rapidly reassessing patients. children with falciparum malaria should be observed in hospital initially for at least 24 h, because of the possibility of rapid progression and also to ensure that they are tolerating oral therapies; treatment in children may be complicated by vomiting. 32, 33 patients should be observed closely; certain categories such as pregnant women, infants and the elderly are more likely to develop severe disease or to deteriorate rapidly. 29, 34, 35 the management of patients with falciparum malaria, especially if severe, should always be discussed with a specialist; mortality is higher in regions of the uk where malaria is less commonly managed. 29 26% of uk malaria cases are seen in centres with less than 10 cases a year. 36 patients with falciparum malaria (or a mixed infection which includes falciparum parasites) can be divided into those with uncomplicated and those with severe or complicated disease (box 3). assessment of the patient should include careful clinical evaluation and review of investigations for the features of severe malaria detailed below. a full blood count, urea, creatinine and electrolytes, liver function tests and blood glucose should be done routinely. thrombocytopaenia is common and in isolation does not reflect severe disease. in ill patients, blood gases, blood culture, lactate and clotting studies should be also performed. urine dipstick and culture, stool culture and chest x-ray may be appropriate. lumbar puncture to exclude meningitis should be considered in febrile patients with impaired consciousness or repeated seizures. the initial parasite count is helpful in estimating the potential future severity of disease. although highly dependent upon the stage of the infection, if more than 2% of red blood cells are parasitized, there is an increased chance of developing severe disease even if the patient initially box 2. common errors in diagnosis or management of malaria (adapted from beeching nj et al. 19 with permission). delayed patient presentation. failure of health care worker to take a travel history or consider diagnosis of malaria. belief that chemoprophylaxis prevents all malaria. belief that malaria is unlikely if patient does not remember being bitten by mosquitoes. belief that malaria presents with a classical fever pattern. failure to recognise nonspecific clinical presentations of malaria. failure to obtain immediate blood films or rdt. failure to repeat diagnostic tests if first tests are negative. failure to prescribe adequate and appropriate chemotherapy immediately. failure to anticipate or treat complications. impaired consciousness or seizures. renal impairment (oliguria <0.4 ml/kg bodyweight per hour or creatinine >265 mmol/l). acidosis (ph < 7.3). hypoglycemia (<2.2 mmol/l). pulmonary oedema or acute respiratory distress syndrome (ards). haemoglobin 80 g/l. spontaneous bleeding/disseminated intravascular coagulation. shock (algid malaria e bp < 90/60 mmhg). haemoglobinuria (without g6pd deficiency). parasitaemia >10%. appears well, and a 10% parasitaemia is considered to represent severe disease. other important poor prognostic factors are: the presence of peripheral blood schizonts of p. falciparum, pigment deposits in peripheral polymorphonuclear leucocytes on the blood film, 37,38 metabolic acidosis or an elevated lactate level, 39e41 older age, coma and renal impairment. 42, 43 treatment of uncomplicated falciparum malaria in adults there are now three main therapeutic options for the treatment of uncomplicated falciparum malaria in adults in the uk: artemisinin combination therapy (act), oral atovaquoneeproguanil or quinine plus doxycycline (or quinine plus clindamycin in certain circumstances) (see box 4 for details of doses). two acts are licenced for use in the uk; artemetherelumefantrine or dihydroartemisininpiperaquine. although mefloquine is an effective treatment, the side effects and high rate of non-completion of courses 44 means that we do not recommend this as therapy in the uk. acts are highly effective and clear parasites more rapidly than other options because they are effective throughout a broader range of the parasite life cycle. 45 for this reason, they are now considered to be the drugs of choice in uncomplicated malaria (grade 1a). studies comparing acts with oral quinine in africa have demonstrated greater efficacy using acts, driven partly by poor compliance with quinine. 46 there has been most experience with artemetherelumefantrine in a western setting and this seems to be well tolerated. 47, 48 artemetherelumefantrine is ideally taken with a high fat meal to maximise absorption and is considered the act of choice (grade 2c). both acts need to be given for only three days. although dha piperaquine (dha-ppq) has the advantage of only needing single daily dosing, there has been concern about the potential for qtc prolongation with dha-ppq. until further data become available, it is recommended that dha-ppq is taken more than 3 h after food and that patients should not eat for 3 h after doses of dhapiperaquine to prevent excessive peak piperaquine levels. dha-ppq should not currently be used in patients with previous arrhythmias, cardiac conditions that predispose to arrhythmia, or those taking drugs that prolong the qt interval. ecgs should be obtained early in the course of treatment with dha-ppq and before and after the last daily dose of dha-ppq; more frequent monitoring may be indicated in those taking drugs which inhibit cyp3a4 and potentially increase piperaquine levels, such as clarithromycin. atovaquoneeproguanil (malarone ò ) has been used extensively in some western settings with high levels of efficacy, although parasite clearance is relatively slow (66% at three days). 47, 49 almost a quarter of patients experienced gastro-intestinal side-effects and patients should be warned about these to ensure full adherence. in contrast to the three day regimens for acts and atovaquoneeproguanil, quinine needs to be taken for five to seven days, or until parasites have cleared, which requires daily monitoring. quinine is often associated with "cinchonism" (nausea, deafness and ringing in the ears), which may result in poor adherence. although international recommendations suggest that quinine should be taken for seven days in endemic areas, 5 uk experience suggests that five days treatment is adequate for the vast majority of cases when combined with a second drug (doxycycline for adults or clindamycin in pregnant women and young children) to ensure complete eradication of parasites. the second drug can be taken either simultaneously with quinine or sequentially after the quinine. in view of increasing failure rates of anti-folate drugs in most part of the world, sulfadoxine-pyrimethamine (fansidar ò ) should not be used routinely as a second drug to accompany quinine, except on specialist advice. chloroquine should not be used for the treatment of falciparum malaria. antibiotics, including tetracyclines, sulfa drugs, macrolides and clindamycin, should only be used in combination therapies and not used alone for the treatment of malaria. there is insufficient evidence to support the use of azithromycin either alone or in combination with other drugs for the treatment of malaria. 50 treatment of severe or complicated falciparum malaria antimalarial therapy urgent appropriate parenteral therapy with antimalarials has the greatest impact on prognosis in severe malaria. treatment should not be delayed in patients with proven or strongly suspected malaria. parenteral treatment is indicated in all patients with severe or complicated malaria, those at high risk of developing severe disease (box 5) or if the patient is vomiting and unable to take oral antimalarials. there is now substantial evidence for the superiority of intravenous artesunate over intravenous quinine with two large trials and a meta-analysis combining with other smaller trials demonstrating a mortality benefit in adults and children 51e53 for all patients with severe malaria. intravenous artesunate is therefore considered the treatment of choice for severe malaria. (grade 1a). the manufacturers of intravenous artesunate have not achieved good manufacturing practice (gmp) certification and artesunate is not licenced in the european union (2015). however, the factory manufacturing artesunate has achieved who prequalification standards and some importers of artesunate also carry out quality checks on imported batches. intravenous artesunate is now stocked by many infectious diseases units in the uk and can also be obtained from specialist tropical disease centres in london and liverpool (see below for contact details). we recommend that any centre regularly seeing patients with severe malaria should stock artesunate. however, treatment should never be delayed whilst obtaining artesunate: every patient with severe malaria should start quinine initially if artesunate is not immediately available as delay is very dangerous (grade 1a). there is no additional benefit from using artesunate in combination with quinine but it is safe to do so. 54 following a minimum of 24 h of intravenous artesunate, and once patients have improved and are able to take oral medication, a full course of an act (artemetherelumefantrine or dha-ppq) should be given. alternatively, a full course of quinine and doxycycline (clindamycin in children/ pregnant women) or atovaquoneeproguanil could be used. clearance of parasites should be checked by daily thick blood films. there has been increasing experience with the use of intravenous artesunate in western non-immune travellers, demonstrating rapid effectiveness and generally low adverse event rates compared to quinine. 55, 56 however, there is clear emerging evidence of delayed haemolysis (7e21 days post treatment) following intravenous artesunate in approximately 10e15% of adults or children, especially those with high parasite counts. 57e59 this appears to be self-limiting but patients should be warned to be aware of potential symptoms of anaemia and their haemoglobin level should be routinely checked approximately 14 days after completing artesunate (grade 2c). the emerging evidence of reduced susceptibility to artemisinin derivatives in se asia 60 does not alter our current recommendations for the use of artesunate as first line therapy for this region (grade 2c). however, as with all cases of severe malaria, monitoring of clearance of parasites and for recurrence of symptoms is recommended. artemether is an oil-based intramuscular artemisinin preparation, which is produced to gmp standards. however, despite generally favourable trends, several studies and meta-analyses have not shown a clear advantage of artemether over quinine in the management of severe malaria in either adults or children 61e63 and we do not recommend its use (grade 1b). intravenous quinine dihydrochloride is an alternative if artesunate is not immediately available. it should be given as an intravenous infusion with an initial loading dose of 20 mg/kg in 5% dextrose or dextrose/saline over 4 h to achieve high blood levels rapidly 64 (see box 6) . this should be followed by 10 mg/kg infused over 4 h every 8 h. a loading dose should not be given if quinine or mefloquine therapy has been taken within the previous 12 h (grade 2c). 6 caution should be exercised in older patients or those with cardiac disease, because of the potential for quinine to lead to arrhythmias. these patients should have ecg monitoring during intravenous quinine treatment. when the patient is well enough to take oral medication, treatment should be completed with a full course of an oral box 5. other indications for parenteral therapy in adults. parasitaemia >2% red blood cells parasitized. pregnant women following specialist advice. patients unable to swallow/retain tablets. box 6. drug treatment of severe or complicated malaria. artesunate regimen: 2.4 mg/kg given as an intravenous injection at 0, 12 and 24 h then daily thereafter. after completion of a minimum of 24 h therapy (maximum five days), a full course of an oral act should be taken when the patient can tolerate oral medication. quinine: loading dose of 20 mg/kg quinine dihydrochloride in 5% dextrose or dextrose saline over 4 h. followed by 10 mg/kg every 8 h for first 48 h (or until patient can swallow). frequency of dosing should be reduced to 12 hourly if intravenous quinine continues for more than 48 h. parenteral quinine therapy should be continued until the patient can take oral therapy when quinine sulphate 600 mg should be given three times a day to complete five to seven days of quinine in total. quinine treatment should always be accompanied by a second drug: doxycycline 200 mg (or clindamycin 450 mg three times a day for children or pregnant women), given orally for total of seven days from when the patient can swallow. agent (box 4). if quinine is used, a total combined iv and oral course of seven days is appropriate. if intravenous quinine needs to be continued for longer than 48 h, or the patient is in renal failure or has severe hepatic dysfunction, quinine doses should be reduced by a third. 5, 65 neither quinine nor artesunate levels are affected by haemofiltration; dose modification is not necessary. all patients with severe or complicated malaria should be managed in a high dependency unit. patients may deteriorate rapidly and close observation is vital. transfer to an intensive care unit should be considered for those with severe acidosis, high lactate levels, pulmonary oedema/acute respiratory distress syndrome, complicated fluid balance problems or renal impairment and those deteriorating despite appropriate treatment (box 7). careful fluid balance is important to avoid over-filling, which may exacerbate the increased pulmonary capillary permeability that occurs in severe malaria (grade 2c). there is evidence that measurement of the central venous pressure is not useful in predicting true volume status in severe malaria and much of the lactic acidosis seen in severe malaria may be due to microvascular obstruction rather than hypovolaemia. 67 hypoglycemia may occur in severe malaria, complicated by quinine-induced hyperinsulinemia which may develop late in the clinical course, even after the patient appears to be recovering. 68, 69 blood glucose levels (using a "stix" method) should be checked routinely every 4 h (grade 2c), two-hourly during quinine infusion (grade 2c) and at any time that reduced consciousness occurs (grade 1a). infusion of 10% dextrose may be necessary to correct hypoglycemia. haemoglobin, clotting, electrolytes (including calcium and sometimes magnesium) and renal function should be closely monitored. frequent parasite counts are not helpful in the early management of severe malaria; the peripheral parasite count will fluctuate according to the stage of parasite development and it is not uncommon for the parasite count to increase in the first 24e36 h of treatment: this does not indicate failure of therapy. 31 daily parasite counts are sufficient and are recommended to ensure clearance. some patients develop shock which may be secondary to complicating bacteraemia/septicaemia ("algid malaria"). patients with signs of shock should be treated with a broad spectrum antibiotic (grade 1c). platelet transfusion is not indicated even with low counts unless there is active bleeding. appropriate ventilatory support or renal replacement therapy should be initiated if clinically indicated: haemofiltration appears to be superior to peritoneal dialysis. 70 patients with impaired consciousness or coma should be managed appropriately. corticosteroids, mannitol, n-acetylcysteine and levamisole have all been shown to be ineffective as adjunctive therapies for the treatment of severe malaria. 71e75 the role of exchange transfusion in the management of severe malaria has always been controversial, with no clear evidence of benefit and potential risks, especially in individuals who may have haemodynamic instability. 76, 77 artesunate has its greatest mortality advantage in those with high parasite counts. the rapid action of artesunate in reducing parasite burden means that any benefit of exchange transfusion is likely to be substantially reduced. 78 current opinion is that exchange transfusion is now no longer indicated in severe malaria (grade 2c). routine blood transfusion may be indicated in those with symptomatic anaemia. there is clear evidence that risks of both falciparum and vivax malaria leading to mortality increase steadily with age over 65. 29 all elderly patients should be admitted, and monitored closely. malaria in pregnancy carries a higher risk of severe disease and is also associated with miscarriages or stillbirths. pregnant women with malaria require prompt treatment and should be managed in collaboration with the obstetric team. close observation in hospital, including uterine and foetal heart monitoring for development of complications, is necessary and early delivery of a near-term infant at risk may need to be considered. the rcog has produced specific guidelines for the treatment of malaria in pregnancy. 79 uncomplicated falciparum malaria. neither artemetherelumefantrine (riamet ò ), atovaquoneeproguanil (malarone ò ) or dha-ppq are licenced in pregnancy. there is no evidence of adverse effects of artemetherelumefantrine in the second and third trimester from a limited number of studies. 80 data on safety in the first trimester are even more limited but no evidence of harm has been detected. small studies on atovaquoneeproguanil have shown no evidence of adverse effects. artemetherelumefantrine is considered the treatment of choice in the second and third trimester (grade 2b) (information on dha-ppq is currently more limited). quinine (seven days) in combination with clindamycin (see dose above) can be used in all box 7. intensive care management of severe or complicated malaria. careful management of fluid balance to optimise oxygen delivery and prevent pulmonary oedema. regular monitoring for hypoglycemia. consider broad spectrum antibiotics if evidence of shock or secondary bacterial infection. haemofiltration for renal failure or control of acidosis or fluid/electrolyte imbalance. consider medication to control seizures. three trimesters. quinine can increase the risk of uterine contraction and hypoglycemia. severe malaria. severe malaria in pregnancy is associated with high case fatality rates, pregnancy loss and hypoglycemia and pulmonary oedema are particularly common. 6 there is little published evidence of the use or safety of intravenous artesunate in pregnant women, particularly in the first trimester. on balance of risk, artesunate is preferred to quinine on the basis of its likely higher effectiveness in reducing mortality (grade 1c). intravenous quinine (with clindamycin) is an alternative. oral quinine, atovaquoneeproguanil (malarone ò ), artemetherelumefantrine and dha-ppq can all be used for the treatment of uncomplicated malaria in children (box 8). there is limited experience in the use of artemisinin combination therapies in a non-endemic paediatric population, although acts are recommended as first-line treatment of uncomplicated malaria in children in malaria endemic regions. despite this, on the basis of evidence from endemic areas, the committee believes that an act should be first line therapy for children in the uk (grade 1a). the combination of oral quinine with seven days of clindamycin or doxycycline (for children greater than 12 years age) remains highly effective in the uk, with very low relapse rates in children. in contrast to the views of some authors, 6,81 we believe that oral quinine is usually well-tolerated by children and should be used for the treatment of uncomplicated falciparum malaria in the uk if an act is not available. 81, 82 tetracyclines should not be given to children under 12 years of age because of risk of dental hypoplasia and permanent discolouration of teeth. severe and complicated falciparum malaria in children (box 9) the main clinical presentations of severe malaria in children are cerebral malaria, severe anaemia and respiratory distress/acidosis. features of cerebral malaria include depressed conscious level, seizures, altered respiration and posturing (decorticate or decerebrate). hypoglycemia, metabolic acidosis, circulatory shock and electrolyte disturbance may also be present. prostration (the inability to stand or sit) is also an indicator of severe disease in children. management of severe or complicated malaria in children involves emergency assessment and provision of supportive care including respiratory and cardiovascular support as outlined by maitland et al. 81 children with severe or complicated malaria should be managed in a paediatric intensive care unit or high dependency unit together with support/advice from a paediatric infectious diseases/tropical medicine specialist who has experience in managing malaria. judicious and slow volume resuscitation is important in those children presenting with shock; the feast trial showed a detrimental effect of routine fluid bolus administration. 83 hypoglycemia is a common complication of severe malaria; serial blood glucose estimations must be performed, and hypoglycemia corrected using 5e10% glucose in maintenance fluid. 68 as it is often difficult to exclude or differentiate concurrent bacterial septic shock or meningitis from severe malaria, empirical broad spectrum antibiotics should be given to children with severe malaria until bacterial infection can be excluded 84 (grade 1b). management of seizures should follow the evidence-based guidelines advocated by the advanced paediatric life support group. 85 blood transfusions may be required for severe anaemia, although a cochrane review found that routine transfusion did not reduce mortality, but caused more adverse events. 86 clear evidence from a large randomised trial now shows that although quinine remains effective, artesunate is associated with a survival advantage (relative risk reduction of 22.5%) and a significant reduction in clinical complications (development of coma, convulsions and deterioration of coma score). 52 in line with the who guidelines, we recommend that iv artesunate should be used preferentially over quinine as the drug of choice for treatment of severe falciparum malaria in children (grade 1a). recent who guidelines suggest that a higher dose is required for children under 20 kg. 5 intravenous quinine is still indicated if artesunate is not immediately available and treatment should not be delayed whilst awaiting artesunate therapy. treating the acute infection the treatment of non-falciparum malaria consists of treating the erythrocytic asexual forms that cause symptoms and, for infections with p. vivax and p. ovale, also ensuring eradication of liver hypnozoites to prevent relapse of infection (box 10). if a mixed infection of either vivax or ovale with falciparum has been treated, there is no need for an additional drug to treat the blood forms of nonfalciparum infection, but relapse due to the liver forms will still need to be prevented. either an oral act or chloroquine (25 mg/kg in total over 3 days) can be used to treat the blood forms of all non-falciparum species (grade 1b). fever and parasite clearance times are faster with most act regimens than chloroquine in non-falciparum malaria; most of the evidence comes from studies in vivax. 87, 88 chloroquine is highly effective against p. malariae, p. ovale and p. knowlesi and is effective in most cases of vivax malaria. chloroquine resistance leading to poor clinical outcomes of chloroquine treatment has been recognised in vivax malaria since 1992. this is an uncommon but increasing problem, particularly in the regions of papua new guinea and indonesia. 52, 89, 90 chloroquine can still be used for vivax infections from such regions with appropriate follow-up but an act may be preferred (grade 1b). act regimens should be first line therapy if falciparum malaria cannot be reliably excluded or for treatment of mixed infections that include p. falciparum. they also provide an alternative for individuals with non-falciparum malaria who cannot tolerate chloroquine. cases of severe or complicated non-falciparum malaria should be treated with parenteral artesunate or quinine as for severe falciparum malaria. artesunate appears to be highly effective for severe p. knowlesi infections. 22 chloroquine is a safe option for treatment of nonfalciparum malaria throughout pregnancy. acts can be used in the second and third trimesters. quinine may be used in the first trimester if there is concern about resistant vivax. box 9. severe or complicated malaria in children. impaired consciousness or seizures. respiratory distress or acidosis (ph < 7.3). hypoglycemia (2.2 mmol/l). severe anaemia (<8 g/dl). prostration. parasitaemia >2% red blood cells parasitized. late presentation or relapse due to hypnozoites in the liver occurs in more than 25% of patients with vivax malaria treated with chloroquine alone. 91 blood schizonticides such as chloroquine, and all other drugs currently used for treating acute malaria, do not eliminate these liver stages, so a second drug is required to achieve "radical" cure. primaquine is the drug of choice for elimination of hypnozoites in ovale or vivax malaria (grade 1a). patients should be screened for g6pd deficiency before primaquine treatment, as primaquine may cause haemolysis in g6pd deficient individuals. the efficacy of primaquine in preventing relapse is highly dependent upon co-administration with chloroquine or an alternative drug to clear the red cell forms. 91, 92 primaquine also has intrinsic activity against asexual blood forms of p. vivax and p. ovale, and concomitant administration with chloroquine boosts blood primaquine levels. 91, 93 administration of the two drugs should therefore overlap (grade 1c). in centres that commonly see patients with vivax malaria, systems for rapid assessment of g6pd status should be set up. the standard therapeutic dose of 15 mg primaquine base/day for 14 days is appropriate for the radical treatment of p. ovale. however, certain geographical strains of p. vivax have long been recognised to be less sensitive to primaquine and to require higher doses of primaquine to prevent relapse. 94 there has also been increasing evidence of failure of standard dose primaquine from other geographical areas: clinical relapse occurs in the uk in more than 10% of patients with imported vivax treated with chloroquine followed by unsupervised primaquine 15 mg daily for 14 days. 95 higher dose primaquine 30 mg daily (0.5 mg/kg) is more effective than 15 mg daily in south asia, the source of most uk infections. 91, 96 administration of primaquine therapy for less than 14 days is associated with higher relapse rates than 14 day regimens. 97 we therefore recommend that in vivax malaria, primaquine should be given at a dose of 30 mg daily for 14 days to prevent relapse along with treatment with chloroquine.(grade 1c). in pregnant or breastfeeding women, weekly suppressive chloroquine prophylaxis (500 mg each week) should be given until primaquine can be given following delivery or completion of breastfeeding. expert opinion should be sought when treating patients with g6pd deficiency. in those with mild to moderate g6pd deficiency, alternative regimens of 45 mg (0.75 mg/kg) primaquine weekly for eight weeks may be effective and safely tolerated 98e101 (grade 2c). in some cases, particularly those who have previously suffered severe adverse effects of primaquine, it may be prudent to withhold primaquine treatment, but to treat relapses promptly. patients and their relatives should be provided with a full explanation of the condition and specific issues related to their therapy, including warnings about possible immediate and late complications of their treatment (box 11). although there are no systematic studies on the value of early follow-up of patients following treatment for imported malaria, the committee believes that it is good practice to repeat a blood film and full blood count approximately fourteen days after treatment, particularly if patients have had severe malaria or received artemisinin therapy. it also provides a further opportunity to reinforce the need for appropriate antimalarial precautions to be taken by the patient and his or her relatives next time they travel (https://www.gov.uk/government/ publications/malaria-prevention-guidelines-for-travellersfrom-the-uk). malaria reference laboratory https://www.gov.uk/ government/publications/malaria-reference-laboratorymrl-user-handbook. scottish parasite and diagnostic reference laboratory http://www.nhsggc.org.uk/about-us/professional-supportsites/scottish-microbiology-reference-laboratories/ scottish-parasite-diagnostic-reference-laboratory/ hospital for tropical diseases, uch, london. 020 3456 7890. tropical and infectious disease unit, royal liverpool university hospital. 0151 706 2000. estimating unreported malaria cases in england: a capture-recapture study malaria imported into the united kingdom imported malaria and high risk groups: observational study using uk surveillance data 1987e2006 falciparum malaria as a cause of fever in adult travellers returning to the united kingdom: observational study of risk by geographical area world health organization. guidelines for the treatment of malaria e world health organisation. management of severe malaria e a practical handbook canadian recommendations for the prevention and treatment of malaria among international travel-lerse2009 management of imported malaria in europe imported falciparum malaria in europe: sentinel surveillance data from the european network on surveillance of imported infectious diseases imported malaria in children. seminars imported malaria in children: a national surveillance in the netherlands and a review of european studies rapid diagnostic tests for diagnosing uncomplicated non-falciparum or plasmodium vivax malaria in endemic countries world health organisation. malaria rapid diagnostic test performance: results of who product testing of malaria rdts: round 5 (2013) guideline: the laboratory diagnosis of malaria. general haematology task force of the british committee for standards in haematology imported malaria is a major cause of thrombocytopenia in children presenting to the emergency department in east london clinical and laboratory predictors of imported malaria in an outpatient setting: an aid to medical decision making in returning travelers with fever fever in returned travellers presenting in the united kingdom: recommendations for investigation and initial management principles and practice of travel medicine plasmodium vivax malaria plasmodium ovale: a case of not-so-benign tertian malaria a prospective comparative study of knowlesi, falciparum, and vivax malaria in sabah, malaysia: high proportion with severe disease from plasmodium knowlesi and plasmodium vivax but no mortality with early referral and artesunate therapy severe vivax malaria: a systematic review and meta-analysis of clinical studies since 1900 treatment of imported malaria in an ambulatory setting: prospective study the selective outpatient treatment of adults with imported falciparum malaria: a prospective cohort study the case for outpatient treatment of plasmodium falciparum malaria in a selected uk immigrant population selective ambulatory management of imported falciparum malaria: a 5-year prospective study risk factors for severe disease in adults with falciparum malaria risk factors for mortality from imported falciparum malaria in the united kingdom over 20 years: an observational study falciparum malaria in malaria-naive travellers and african visitors assessing the severity of malaria review of 40 children with imported malaria acute uncomplicated malaria treatment in children in france in 2002 age as a risk factor for severe manifestations and fatal outcome of falciparum malaria in european patients: observations from tropneteurop and simpid surveillance data world health organisation. severe falciparum malaria geographical concentration of falciparum malaria treated in the uk and delay to treatment with artesunate in severe cases: an observational study relation of the stage of parasite development in the peripheral blood to prognosis in severe falciparum malaria intraleucocytic malaria pigment and prognosis in severe malaria pathophysiological and prognostic significance of cerebrospinal-fluid lactate in cerebral malaria the pathophysiologic and prognostic significance of acidosis in severe adult malaria the clinical spectrum of severe imported falciparum malaria in the intensive care unit: report of 188 cases in adults the relationship between age and the manifestations of and mortality associated with severe malaria prognostic indicators in adults hospitalized with falciparum malaria in western thailand treatment of imported malaria in adults: a multicentre study in france artemisininbased combination therapy for treating uncomplicated malaria effectiveness of quinine versus artemetherelumefantrine for treating uncomplicated falciparum malaria in ugandan children: randomised trial therapy of uncomplicated falciparum malaria in europe: malt treatment of acute uncomplicated falciparum malaria with artemetherelumefantrine in nonimmune populations: a safety, efficacy, and pharmacokinetic study atovaquoneeproguanil in the treatment of imported uncomplicated plasmodium falciparum malaria: a prospective observational study of 553 cases azithromycin for treating uncomplicated malaria artesunate versus quinine for treatment of severe falciparum malaria: a randomised trial artesunate versus quinine in the treatment of severe falciparum malaria in african children (aqua-mat): an open-label, randomised trial artesunate versus quinine for treating severe malaria a comparison of artesunate alone with combined artesunate and quinine in the parenteral treatment of acute falciparum malaria intravenous artesunate versus intravenous quinine in the treatment of severe falciparum malaria: a retrospective evaluation from a uk centre artesunate versus quinine in the treatment of severe imported malaria: comparative analysis of adverse events focussing on delayed haemolysis intravenous artesunate for severe malaria in travelers delayed hemolysis after treatment with parenteral artesunate in african children with severe malariaea double-center prospective study haemolysis associated with the treatment of malaria with artemisinin derivatives: a systematic review of current evidence artemisinin resistance in plasmodium falciparum malaria a controlled trial of artemether or quinine in vietnamese adults with severe falciparum malaria a trial of artemether or quinine in children with cerebral malaria the artemether-quinine meta-analysis study group. a metaanalysis using individual patient data of trials comparing artemether with quinine in the treatment of severe falciparum malaria quinine loading dose in cerebral malaria plasma quinine concentrations in falciparum malaria with acute renal failure management of severe malaria: interventions and controversies fluid resuscitation of adults with severe falciparum malaria: effects on acid-base status, renal function, and extravascular lung water hypoglycemia in african children with severe malaria severe hypoglycemia and hyperinsulinemia in falciparum malaria hemofiltration and peritoneal dialysis in infectionassociated acute renal failure in vietnam dexamethasone proves deleterious in cerebral malaria n-acetylcysteine as adjunctive treatment in severe malaria: a randomized, double-blinded placebo-controlled clinical trial brain swelling and mannitol therapy in adult cerebral malaria: a randomized trial high-dose dexamethasone in quininetreated patients with cerebral malaria: a double-blind, placebo-controlled trial randomized controlled trial of levamisole hydrochloride as adjunctive therapy in severe falciparum malaria with high parasitemia exchange transfusion as an adjunct therapy in severe plasmodium falciparum malaria: a meta-analysis severe falciparum malaria: predicting the effect of exchange transfusion manual blood exchange transfusion does not significantly contribute to parasite clearance in artesunate-treated individuals with imported severe plasmodium falciparum malaria the diagnosis and treatment of malaria in pregnancy a systematic review of the safety and efficacy of artemetherelumefantrine against uncomplicated plasmodium falciparum malaria during pregnancy management of severe malaria in children: proposed guidelines for the united kingdom childhood malaria in east london mortality after fluid bolus in african children with severe infection proposed guidelines for severe imported malaria in children need more evidence advanced paediatric life support: the practical approach blood transfusion for treating malarial anaemia (cochrane review) efficacy and safety of artemisinin combination therapy (act) for non-falciparum malaria: a systematic review artemisinin-based combination therapy for treating uncomplicated plasmodium vivax malaria plasmodium vivax resistance to chloroquine? chloroquine resistance in plasmodium vivax. antimicrob agents chemother primaquine therapy for malaria potentiation of the curative action of primaquine in vivax malaria by quinine and chloroquine pharmacokinetic interactions between primaquine and chloroquine. antimicrob agents chemother plasmodium vivax chesson strain treatment of plasmodium vivax malariaetime for a change? primaquine: report from cdc expert meeting on malaria chemoprophylaxis i primaquine for preventing relapse in people with plasmodium vivax malaria treated with chloroquine mitigation of the haemolytic effect of primaquine and enhancement of its activity against exoerythrocytic forms of the chesson strain of plasmodium vivax tolerability and safety of weekly primaquine against relapse of plasmodium vivax in cambodians with glucose-6-phosphate dehydrogenase deficiency a randomised trial of an eight-week, once weekly primaquine regimen to prevent relapse of plasmodium vivax in northwest frontier province chemotherapy of malaria revised 2nd edition plc is supported by the national institute for health research university college london hospitals biomedical research centre. none declared.box 11. what to tell the patient.reassure patients they are not infectious to others. discussion about informing fellow travellers of potential risk of having caught malaria and need to present early if symptoms develop. inform patient that he/she will be notified to public health authorities as part of routine national policy. warn patients about possible recrudescence or relapse of malaria following treatment and to report recurrence of fever to their general practitioner. warn patients treated with artesunate about possible haemolysis and the importance of attending for follow-up blood tests. at follow up, review results of any tests needed or performed for other travel related infections including hiv infection. discuss period of exclusion from blood donation after malaria e blood donors need to inform and discuss own situation with national blood service. reinforce need for up to date advice on malaria prevention during all future travels, for patient and family, and provide information on sources of advice. key: cord-309476-hel3h25h authors: brown, julianne r.; bharucha, tehmina; breuer, judith title: encephalitis diagnosis using metagenomics: application of next generation sequencing for undiagnosed cases date: 2018-01-02 journal: j infect doi: 10.1016/j.jinf.2017.12.014 sha: doc_id: 309476 cord_uid: hel3h25h background: current estimates suggest that even in the most resourced settings, the aetiology of encephalitis is identified in less than half of clinical cases. it is acknowledged that filling this gap needs a combination of rigorous sampling and improved diagnostic technologies. next generation sequencing (ngs) methods are powerful tools with the potential for comprehensive and unbiased detection of pathogens in clinical samples. we reviewed the use of this new technology for the diagnosis of suspected infectious encephalitis, and discuss the feasibility for introduction of ngs methods as a frontline diagnostic test. methods: a systematic literature review was performed, using mesh and text word searches for variants of “sequencing” and “encephalitis” in medline and embase, and searching bibliographies and citations using the web of science database. two authors independently reviewed, extracted and summarised data. findings: the review identified 25 articles reporting 44 case reports of patients with suspected encephalitis for whom ngs was used as a diagnostic tool. we present the data and highlight themes arising from these cases. there are no randomly controlled trials to assess the utility of ngs as a diagnostic tool. interpretation: there is increasing evidence of a role for ngs in the work-up of undiagnosed encephalitis. lower costs and increasing accessibility of these technologies will facilitate larger studies of these patients. we recommend ngs should be considered as a front-line diagnostic test in chronic and recurring presentations and, given current sample-to-result turn-around times, as second-line in acute cases of encephalitis. encephalitis is defined as inflammation of brain parenchyma associated with neurological dysfunction. 1, 2 it is strictly a pathological diagnosis. recent epidemiological studies suggest that the global burden of encephalitis has been grossly underestimated, with current incidence suggested to be over 6000 annual cases in the uk, and 500,000 worldwide. 3, 4 the syndrome encapsulates a myriad of diverse diseases, with distinct global distributions, presenting features and clinical courses. 4, 5 infections represent the most frequently identified aetiology, with data suggesting this accounts for 20-50% of cases. 2, 6, 7 hundreds of pathogens have been associated with encephalitis, with the most frequently identified including herpes simplex virus (hsv), varicella zoster virus (vzv), enteroviruses, measles morbillivirus, mumps virus, japanese encephalitis virus (jev), influenza viruses, adenoviruses and mycoplasma pneumoniae. 4 hsv, jev and rabies are the chief causes in europe, asia and africa respectively. the main alternative aetiology to infection is immune mediated, for which management includes immune suppression. it is critical to differentiate between autoimmune and infectious causes of encephalitis; immune suppression in cases where the cause is an undiscovered pathogen could be devastating. strikingly, more than one third of cases of encephalitis remain unidentified, even in the best-equipped medical centres. 3, 6, 8 there are well-recognised challenges and inadequacies in current diagnostics and treatment, and these correspond with the poor reported outcomes. [9] [10] [11] [12] overall mortality is estimated at 30%, but is highly variable and dependent on the aetiology and access to supportive care. a high proportion is left with complex disability. 11 the introduction of management guidelines for cases of acute encephalitis syndrome over the last decade has aimed to improve outcomes. 8, [13] [14] [15] [16] [17] [18] [19] notably, there are many differences between guidelines in the approach to diagnostic evaluation, such as standard and extended diagnostic testing, or whether to administer empirical aciclovir. 20 these variations may reflect geographically contrasting aetiologies, timing of publication and the rapid acceleration of technologies, as well as available resources. further, there is a lack of a systematic approach to access to pathogen discovery methods, discussed below. it is only the newest french guidelines in which ngs is mentioned at all, and the authors reasonably state that the clinical role is still to be evaluated. 19 current diagnostic techniques for suspected infectious cases rely on prior knowledge of the likely causative agent. informed by clinical presentation, epidemiological data, guidelines and local resources, a laboratory will perform targeted tests for a disease. these are largely confined to specific polymerase chain reaction (pcr) or serological assays. this approach has fundamental limitations, and contributes to the relatively high proportion of encephalitis cases that remain undiagnosed. aside from the difficulties of testing for the myriad of rare pathogens that might be expected to cause encephalitis, this approach does not permit the identification of new or unexpected pathogens. undeniably, methods for novel pathogen discovery such as electron microscopy and cell culture have existed for many years, however they are cumbersome, time-consuming, lack sensitivity and specificity and are often no longer routinely available. furthermore, there are groups of patients, such as immunosuppressed patients, who frequently present with subtle or non-specific symptoms and signs, are high-risk for encephalitis, infection with unexpected or unusual pathogens, and are seen to have more severe outcomes. 21 there is a need for improved diagnostic methods for encephalitis. a method which has recently been applied to pathogen detection in cases of encephalitis is metagenomic analysis using next generation sequencing (ngs). proof of concept for its use in the diagnosis of encephalitis has been demonstrated in the literature, however its suitability for routine diagnosis has not been assessed and is the subject of this review. ngs, also known as deep sequencing, generates a single sequence from each fragment of dna, or cdna, present in a specimen. downstream analysis allows differentiation between the origin of sequence fragments, for instance human, a specific bacterial species or a particular virus. this means mixed specimens, that contain host and microbial sequences, can be resolved (fig. 1 ). sequencing the total dna or rna (known as metagenomics) from a biopsy or body fluid allows the identification of genetic material from any microorganism present in the specimen, and thus potentially causing encephalitis. this approach overcomes the limitations of targeted diagnostic methods such as pcr as it requires no prior knowledge or assumptions about the type of pathogen causing infection therefore enabling detection of novel and unexpected pathogens. the majority of readily available sequencing methods to date are dna based, however sequencing only total dna would exclude detection of viruses with rna genomes. consequently, an alternative approach is to synthesise metagenomics for diagnosis of encephalitis complementary dna (cdna) from total rna, which will enable detection of viruses with rna genomes but also the rna transcripts of organisms with dna genomes. once sequences are generated, complex downstream bioinformatic analysis is required to identify the presence of any pathogen sequences. in brief, any reads mapping to the human genome are removed, after which all remaining nonhuman sequences are compared to a database of known sequences to identify the provenance of the unknown sequences (fig. 2) . the possibility of incorporating unbiased pathogen discovery technology into routine diagnostics for encephalitis would represent a paradigm shift in diagnostic algorithms. we and others 23 are validating the use of metagenomics for clinical use and prospective studies are already underway to examine whether application of ngs at the outset of management pathways improves patient outcome and costs, namely the precision diagnosis of acute infectious disease (pdaid) study. 24, 25 nonetheless, there is a paucity of evidence in this field which is largely limited to case reports. we aim to perform a rigorous summary and critical review of existing evidence, to assess the utility of ngs in diagnosis of encephalitis. only articles reporting application of ngs in csf or brain biopsies in suspected encephalitis and published in english between january 2008 and april 2017 were included. the web of science database was also used to search bibliographies and citations of relevant article. two authors independently reviewed, extracted and summarised included literature. data was extracted in the first instance by the first reviewer into a custom data extraction excel sheet with pre-defined data headings (supplementary file 1), designed for the purpose of this review; additional miscellaneous data or observations were also noted where relevant. the extracted data and each included study were independently reviewed by the second author, with a focus on technical and scientific aspects of each study. consensus extracted data was used in analyses; both review authors were in full agreement on the extracted consensus data. relevant manuscripts that were not identified through the initial search, but were identified in the reference list of included literature, were also included. the funding bodies had no role in the decision to write, the analysis, manuscript preparation or the decision to submit for publication. twenty-five articles were identified from the search (fig. 3) . all the included articles were case reports, or case series of 1-7 patients. altogether 44 cases were reported in which ngs provided a diagnosis in otherwise undiagnosed cases of encephalitis (table 1 ). an exponential temporal increase in cases has been observed over the last decade (fig. 4) . country of origin of cases included australia, china, france, germany, india, ireland, japan, poland, sri lanka, uk, usa, and vietnam. samples were analysed in laboratories largely in the usa and europe (uk, france, germany, poland), but also in china, japan and vietnam. among the 27 cases for which age was documented, the median age was 14 years (interquartile range 3-61). of the 22 cases that reported immune status of the patient, 73% (16/22) were immunocompromised. there was uniformly poor reporting of encephalitis or meningoencephalitis case definitions, and limited explanation of diagnostic assays performed and algorithms used for testing. none of the studies reported adherence to published or unpublished clinical guidelines. in 16 of the 44 known cases, well-established causes of encephalitis were detected which could have been identified by rapid and specific primary screening methods such as pcr. these organisms included hsv, coxsackievirus a9, measles virus, vzv, mumps virus, epstein-barr virus, jc virus and mycobacterium tuberculosis. however, in the remaining 28 cases novel (18/44), rare (5/44) or unexpected (5/44) organisms were detected which could not (in the case of novel organisms) or are unlikely (in the cases of rare and unexpected pathogens) to have been detected using specific pcr assays. the five unexpected cases were known human pathogens but novel causes of encephalitis. although diagnostic pcr assays may exist for some of these viruses, they are unlikely to have been considered in the differential diagnosis and therefore would not be routinely tested. this included two cases of human parvovirus 4 (parv4), 28 first described in 2005 when it was associated with a viraemic patient in whom an acute viral infection was suspected 49 ; one case of human coronavirus oc-43, 43 typically a human respiratory pathogen never previously described in a human case of encephalitis but known to cause encephalitis in mice 50 ; one case of human astrovirus mlb1, 46 of mumps vaccine virus in a child who was vaccinated prior to a primary immunodeficiency diagnosis. 48 the five cases in which rare causes of encephalitis were identified were brucella melitensis, candida tropicalis, leptospira santarosai and two cases of balamuthia mandrillaris. eighteen cases were considered to be novel pathogens. three (3/14) of the identified organisms were arenaviruses, three cases were a variegated squirrel bornavirus, four were a novel astrovirus (astrovirus va1/hmo-c), three were cycloviruses, three were gemycircuarlviruses and one was a densovirus. the three arenavirus cases occurred in three solid organ transplant recipients who all received organs from the same donor, who was later shown to be antiarenavirus igm and igg seropositive. the three cases of variegated squirrel bornavirus occurred in three breeders of variegated squirrel and was retrospectively detected in one of the breeder's squirrels. the novel astrovirus va1/hmo-c was initially detected in an adolescent with primary immunodeficiency; it has since been shown, through four further case reports identified by ngs, 31, 32, 35, 42 as an emerging under recognised cause of encephalitis in immunosuppressed patients that should be included in the differential diagnosis of encephalitis in this patient group. the clinical significance of the densovirus, cyclovirus and gemycircularviruses is doubtful, and discussed in detail further on in this review. an additional advantage of using ngs for the diagnosis of encephalitis is that, aside from pathogen identification, in instances where virus titre and read depth is high enough it is possible to generate partial or full genome sequences for the pathogen. pathogen sequences can be used for phylogenetic analysis to elucidate the strain 31, 43 or possible source of the organism, as was the case for morfopoulou et al., who demonstrated 99.5% homology between the mumps virus found in the brain of an encephalitic child with primary immunodeficiency and the vaccine batch used to immunise the child. 48 ngs is a powerful tool for pathogen detection, allowing us to detect organisms that may not previously have been described or associated with the disease in question. however, as with all molecular tools, detection of a microorganism does not prove causality. to provide further evidence for an aetiological role in encephalitis of the identified pathogen, a challenge reviewed in detail elsewhere, 2 some reports make use of additional clinical and laboratory indicators to exclude the possibility that the detected organism is an incidental finding. seroconversion to the pathogen in question is highly suggestive of etiological significance of an organism 51 however preinfection or follow-up serum samples are rarely available; of the 44 cases identified in this review seroconversion was demonstrated in only two. 26, 36 a further eight were able to demonstrate the presence of specific antibodies, but without knowledge of the sero-status prior to onset of symptoms. although detection of pathogen-specific intrathecal antibodies is also highly suggestive of a causal relationship and recommended by uk guidelines, 2 none of the cases identified in this review reported intrathecal antibody testing. in cases where encephalitis is caused by reactivation of a dormant pathogen, rather than primary infection, or where the pathogen does not cause a strong systemic antibody response, serology may not be useful. in the case of a novel or emerging cause of encephalitis for which the clinical significance may be unclear, proving causality is particularly important. in this instance organismspecific immunostaining or in situ hybridisation in affected tissues will provide additional evidence of the cellular distribution of infection and exclude the possibility of reagent or tissue contamination; 18 of the reviewed cases report confirmatory immunostaining or in situ hybridisation. in this context brain biopsies are a more useful specimen than cerebrospinal fluid (csf) since it allows immunostaining of the affected tissue. moreover, in encephalitis caused by mutated pathogens such as in subacute sclerosing panencephalitis (sspe) caused by chronic measles infection 52 or cases of mumps vaccine encephalitis 48 the pathogen may not be detected in csf but only in brain parenchyma. an alternative molecular method, such as pcr, can be used to confirm the presence of the detected organism and exclude the possibility that the identified organism is an artefact of the bioinformatics analysis. in this review 33/44 cases confirmed the presence of the organisms by pcr. pcr supports the identity of the organism sequenced by ngs, however does not contribute to proving causality. six cases identified novel small circular ssdna viruses in the csf of patients with encephalitis of unknown aetiology; three cycloviruses 29, 37 and three gemycircularviruses. 37 however, the clinical significance of these is doubtful. phan et al. 37 confirmed, via repeat dna extraction using an alternative method, that the source of the cyclovirus is not reagent contamination. nevertheless, whilst screening csf samples for cyclovirus by pcr, tan et al. 29 detected cyclovirus in the csf of patients in whom the aetiology of their central nervous system (cns) disease had already been confirmed as japanese encephalitis, dengue virus or bacterial meningitis. the only identified cellular host for gemycircularviruses is fungi. in the absence of other evidence of pathogenicity, such as seroconversion or demonstration of the pathogen within cells in the brain, the probability remains that detection of cyclovirus or gemycircularvirus in csf is an incidental finding. similarly the detection of densovirus, a small linear ssdna virus, in csf may be incidental since it was detected in a case with confirmed n-methyl d-aspartate (nmda)-receptor encephalitis. 44 autoimmune antibodies have previously been co-detected with herpesvirus dna in csf from cases of encephalitis, 53 however the host range of densoviruses is to date exclusively invertebrates; the authors suggest a possible explanation for detection in csf is the passive transfer of virus from an insect bite or csf contamination from skin flora or an environmental source. in these cases, further evidence is required before assigning a pathogenic role. as with other molecular tests, including pcr which has become the gold standard of virological diagnostics, results from metagenomics applied to cases of encephalitis should be interpreted in the context of other clinical and laboratory findings, particularly when a novel or unexpected organism is detected. the majority of reports concerning the use of metagenomics for diagnosis of encephalitis are comprised of single case reports, therefore it is difficult to assess the diagnostic yield (number of positive results/number of cases tested), and thus utility, of metagenomics for encephalitis. five reports included testing multiple cases of encephalitis; in these the diagnostic yield was 0% (0/36), 7 1.6% (2/125), 29 6% (4/62), 37 19% (3/16) 41 and 30% (3/10). 45 the first three studies with low diagnostic yield of 0-6% tested only csf supernatant which is cell-free, therefore only cell free viruses or cell-free microbial nucleic acid can be detected; moreover csf often contains a lower pathogen load then brain biopsies and so pathogen detection is more challenging. the aforementioned studies also included only samples for which primary routine diagnostic testing using standard methods was negative therefore the utility of ngs as a first-line test cannot be assessed. a higher diagnostic yield was reported where specimens were tested using metagenomics as a firstline screening tool; 19% using whole csf 41 and 30% using brain biopsies. 45 the use of brain tissue rather than csf may increase diagnostic yield. in three cases a pathogen was detected in brain biopsy but not in csf 32, 35, 48 while the opposite was not observed, although in one instance the proportion of pathogen reads was greater in csf than brain biopsy. 33 in our hands, the diagnostic yield for metagenomics in encephalitis is, to date, 50% (8/16) all of which were brain biopsies. the eight identified pathogens were coronavirus oc-43, 43 two cases of vaccine derived mumps virus, 48, 54 toxoplasma gondii (unpublished) and four cases of astrovirus va1/hmo-c 31,42 (two cases unpublished). all of our positive results were in immunocompromised patients and in the majority of cases there was a high index of suspicion of infection, suggesting metagenomics may be best applied to a targeted population in whom it will be most rewarding. quality assurance prior to providing a new diagnostic test, extensive validation must be undertaken to ensure the service is fit for purpose, such as determining the specificity and sensitivity of an assay, the purpose of which is to ensure a robust, accurate and reproducible result all of which is part of quality assurance. the regulatory requirements that should be fulfilled for the validation of metagenomics for pathogen detection is discussed in detail elsewhere, 23 however one aspect that must continue beyond the validation stage is the use of positive and negative controls. in the context of metagenomics for pathogen detection a positive control is a specimen (real or constructed) that is known to be positive for one or multiple organisms; a negative control is one that is known to be negative for any pathogens. these should be included in every sequencing run; if the positive control fails (i.e. the known pathogen/s is not detected) this invalidates the results of all clinical specimens processed in parallel for which no pathogen was identified. conversely if the negative control fails (i.e. an unexpected organism is identified in the sequence data) this could indicate reagent contamination or a problem with the analysis pipeline and thus positive results from samples processed in parallel are invalidated and should be repeated. this having been said, of 44 cases of encephalitis identified in this review (table 1) , only 5 included positive controls and 15 included negative controls. the accuracy of a positive result is critical for patient management; however a negative result can also be useful to exclude infection, particularly where anti-inflammatory and immunosuppressive treatments are being considered. consequently prior to provision of a clinical service appropriate controls must be in place to ensure results are reliable and therefore clinically actionable. the use of controls is aptly demonstrated by mongkolrattanothai et al., 47 who not only included positive and negative controls but also implemented defined criteria in their analysis pipeline that dictates any viruses detected in a clinical specimen should not be detected in the negative controls and, moreover, bacteria detected in a clinical specimen should only be reported as a significant finding if detected with a reads per million (rpm) ratio ≥10 (rpm ratio = rpm sample / rpm negative control). this approach overcomes the common problem of reagent contamination with microbial nucleic acids; of 33 cases in which the presence or absence of contaminating reads was reported, 22 cases reported the presence of environmental bacteria, plant viruses, bacteriophages and/or avian retroviruses. 24, 29, 31, 33, 35, 36, [38] [39] [40] [41] 43, 45, 47, 48 turn-around times only 5 cases reported the time from specimen collection to pathogen identification; for these the turn-around time was 2-6 days, with a median time of 6 days. due to limitations of currently available sequence library preparation methods and sequencing chemistries a sample-to-answer turn-around time as short as 48 hours 24 is only achievable with a fast downstream analysis pipeline; in the reported case analysis took only 97 minutes compared to up to two days in other, computationally intensive, pipelines. nevertheless it serves as proof of principle that relatively short turn-around times are achievable, even though up to 6 days is more common. the sample-to-answer turn-around time of specific realtime pcr, which is the current gold standard for diagnosis of viral infections, is often less than 12 hours in a clinical laboratory and potentially less than 4 hours without batch processing. consequently ngs cannot yet offer the same speed of result as pcr, which could delay the diagnosis in instances of encephalitis caused by well-known pathogens that are detectable by pcr. nonetheless with rapidly increasing library preparation and sequencing speeds, turn-around times are likely to significantly improve in coming years. metagenomics for pan-pathogen detection has the potential to revolutionise the diagnosis of encephalitis and other difficultto-diagnose infections; however, there are some limitations of the technique that should be considered. the sensitivity and limit of detection (lod) of metagenomics can be determined for model organisms that represent major pathogen groups, such as dna and rna viruses, gram positive and negative bacteria, fungi and parasites; however, the broad-range nature of the technique makes it impossible to determine the sensitivity or lod for every possible organism. this is also a recognised problem with pan-bacterial pcr detection which is used clinically, 55 however can be confounded in metagenomics by differences between specimens and specimen types (for instance tissue biopsies versus csf) in the quantity of genomic material, the ratio of host:pathogen sequences and, depending on the sequencing chemistry and degree of specimen multiplexing, the sequencing yield. some of these limitations may be overcome by the careful use of processing and sequencing controls, as discussed elsewhere. 23 whilst metagenomics will produce a sequence for every fragment of dna or rna in a specimen, only pathogens with homology to known organisms in the sequence database of choice will be identified. if an organism is missing from the database, or if the pathogen causing infection is novel with no homology to known organisms, it will not be identified. consequently, a negative result obtained by metagenomics, whilst reducing the likelihood of an infectious cause, cannot unequivocally exclude infection. sequencing total dna or rna will inevitably include sequencing host dna or rna transcripts which can result in >99% of the sequence data generated mapping to the human genome. the consequence of this is wasted cost, as the sequencing reaction is dominated by host rather than pathogen sequences, and also has implications for the turn-around times and sensitivity of pathogen detection. in order to detect pathogen sequences, which can be as few as nine in 68 million reads, 30 vast sequencing read depths are required; very high throughput sequencing platforms with only a very limited number of samples sequenced in parallel are required to achieve this. to overcome this, depletion of host dna or rna prior to sequencing is required; however the options for this are currently limited and are not all suitable for detection of viral pathogens. improved methods for host dna and rna depletion would considerably reduce the cost and time to result and improve the sensitivity of metagenomics for diagnosis of encephalitis. finally it is important to remember that in some cases of encephalitis, the pathology is due to the immune response and the pathogen may be rarely detected if at all. for example in japanese encephalitis virus (jev) infection, the commonest cause of encephalitis in asia, the most sensitive rt-qpcr detects rna in less than 10% of cases, and the mainstay of diagnosis is serology. 56 in these cases, ngs is unlikely to significantly improve the diagnostic yield. this systematic literature review and case series suggests there is preliminary evidence to support a role for ngs in the management of undiagnosed encephalitis. undeniably, the research is limited to case reports, with poor reporting of clinical case definitions of encephalitis, baseline tests performed, or adherence to clinical guidelines. nonetheless, current epidemiological data suggests that the cause of encephalitis remains unknown in 30-60% of cases 8, 21 ; and ngs has striking potential to identify undiagnosed pathogens and thus reduce the number of cases with unknown aetiology. ngs also has utility for pathogen detection in other clinical syndromes, such as respiratory infections, 57 therefore the implementation of this technique in clinical laboratories would have wider implications for diagnosis of infection beyond encephalitis. notably, current turn-around-times prevent the replacement of routine methods, such as pcr, for the diagnosis of acute encephalitis. for these reasons, the role of ngs in clinical algorithms is still to be delineated. at this point in time, we suggest ngs is routinely applied for the diagnosis of acute cases of encephalitis for which no cause is found after targeted investigations using pcr. recommendations for firstline targeted testing are discussed in detail elsewhere 14 but in the uk should include pcr for hsv, vzv and enteroviruses. however due to differing local epidemiology clinicians should consult the relevant national guidelines. 8, 13, 14, 16, 17 in immunocompromised patients however, metagenomics ought to be considered earlier; 73% of case reports in this review involved immunocompromised patients. this is the population at most risk of infection with novel and unexpected organisms and, moreover, may present with a more chronic or insidious clinical history 58 in which a one-week turn-aroundtime is more acceptable. given that the causative pathogen is not always detected in csf, in all cases of encephalitis in which diagnosis by ngs is being sought the preferred specimen type is brain biopsy. nevertheless csf samples are acceptable if it is the only specimen available. our recommendations for the use of ngs in diagnosis of microbial causes of encephalitis are summarised in fig. 5a , with the contrasting algorithm for targeted testing summarised in fig. 5b . this review was limited to pathogen detection by ngs in brain biopsies or csf. there may also be a role for testing other specimens, such as throat samples and urine. this was recently shown in an encephalitis case diagnosed by ngs of urine, identifying a case of japanese encephalitis virus. 59 it is expected that over the next few years, the cost and time-to-result of metagenomics will reduce, and with this, it is foreseen that it will be possible to offer this as the first-line diagnostic test. this depends on the ability to deplete host dna and rna prior to sequencing, reduced read depth requirement and faster sequencing and bioinformatics technologies. the role of autoimmune encephalitis, in some instances triggered by an infection, 60 is beyond the scope of this review. however, since up to 20% of cases of encephalitis are caused by autoimmune disorders, in which the immune system attacks specific host proteins, 21,61 a comprehensive diagnostic service should include antibody mediated, as well as infectious, causes of encephalitis. the clinical presentation of autoimmune (non-infectious) and infectious encephalitis are similar, 62 however the treatment is often opposing. non-infectious causes may require immunosuppressive therapy 63 ; however administering immunosuppressive therapy where the cause is infectious exacerbates the infection, with potentially fatal results. a conclusive diagnosis of the causative agent of encephalitis would improve differentiation between infectious and auto-immune causes thus appropriate management of immunosuppression. in addition to the direct impact on individual patients, improving the diagnosis of encephalitis will increase our understanding of the causes of encephalitis generally. this knowledge is critical for future development of fast point-of-care tests and to develop clinical algorithms that minimise the time to diagnosis and treatment, thus maximising the chances of recovery. tb and jrbrown conducted the systematic review and prepared the manuscript, figures and table. jbreuer contributed to manuscript preparation. case definitions, diagnostic algorithms, and priorities in encephalitis: consensus statement of the 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densovirus in cerebrospinal fluid from a case of anti-nmda-receptor encephalitis next-generation sequencing in neuropathologic diagnosis of infections of the nervous system acute encephalopathy in an immunocompromised boy with astrovirus-mlb1 infection detected by next generation sequencing neurobrucellosis: unexpected answer from metagenomic next-generation sequencing deep sequencing reveals persistence of cellassociated mumps vaccine virus in chronic encephalitis new dna viruses identified in patients with acute viral infection syndrome coronaviruses as encephalitis-inducing infectious agents viruses and koch's postulates subacute sclerosing panencephalitis and chronic viral encephalitis csf herpes virus and autoantibody profiles in the evaluation of encephalitis human ifnar2 deficiency: lessons for antiviral immunity development of broad-range 16s rdna pcr for use in the routine diagnostic clinical microbiology service japanese encephalitis surveillance standards. in: whorecommended standards for surveillance of selected vaccinepreventable diseases who/v&b metagenomic sequencing complements routine diagnostics in identifying viral pathogens in lung transplant recipients with unknown etiology of respiratory infection acute encephalitis in the immunocompromised individual central nervous system infection diagnosis by next-generation sequencing: a glimpse into the future? pediatric autoimmune encephalitis the spectrum of acute encephalitis: causes, management, and predictors of outcome encephalitis, myelitis, and acute disseminated encephalomyelitis (adem): case definitions and guidelines for collection, analysis, and presentation of immunization safety data a clinical approach to diagnosis of autoimmune encephalitis jr brown is supported by a paediatric research grant from the great ormond street hospital children's charity ("diagnosis of encephalitis by deep sequencing", v4317). jbreuer receives funding from the ucl/uclh nihr biomedical research centre.all research at great ormond street hospital nhs foundation trust and ucl great ormond street institute of child health is made possible by the nihr great ormond street hospital biomedical research centre. the views expressed are those of the authors and not necessarily those of the nhs, the nihr or the department of health.the corresponding author (jbrown) had access to all the data and had final responsibility for the decision to submit for publication. the authors declare no conflicts of interest. supplementary data related to this article can be found at https://doi.org/10.1016/j.jinf.2017.12.014. key: cord-328068-qoz85x3r authors: alanio, alexandre title: the presence of pneumocystis jirovecii in critically ill patients with covid-19 date: 2020-11-04 journal: j infect doi: 10.1016/j.jinf.2020.10.034 sha: doc_id: 328068 cord_uid: qoz85x3r nan 1 letters to the editor we read with interest the recent review on co-infections in coronavirus disease-2019 (covid-19) patients 1 and believe that fungal co-infections as evaluated from selected studies are underestimated. severe acute respiratory syndrome coronavirus-2 (sars-cov2) is still spreading pandemically. approximately 5-10% of covid-19 patients may require intensive care unit (icu) management and 30% may develop secondary pneumonia without identified etiology. 2 hospital-acquired bacterial or fungal superinfections, as described in critically ill patients with influenza virus, can be suspected. 3 since pneumocystosis is usually reported in patients with t-cell immunodepression, 4 less attention has been paid to pneumocystis jirovecii in non-immunocompromised icu patients although it accounts for 7% of the co-infections reported in those admitted with influenza. 5 interestingly, covid-19 patients may develop lymphocytopenia and acute respiratory distress syndrome (ards) requiring adjunctive steroids and/or immunomodulatory therapies, well-known susceptibility factors for developing pneumocystosis. 5 we designed this observational cohort study to investigate the prevalence of p. jirovecii acid nucleic detection in respiratory specimens sampled to identify co-infections in covid-19 patients in the icu. all consecutive patients admitted to the icu between 2020/03/15 and 2020/05/01 with a positive sars-cov-2 pcr (cobas ® sars-cov-2 test, roche, france) and ≥1 respiratory sample (bronchoalveolar lavage (bal), tracheal aspirate, sputum) sent to the mycology department. this study was part of the covid-icu and french covid-19 cohort registries. our institutional ethics committee approved the study (idrcb, 2020-a00256-33; cpp, 11-20-20.02.04.68737). when possible, signed informed consent was obtained from the patients or the next of kin. whenever possible, bronchoalveolar lavage (bal) was performed in the middle lobe by a trained pneumologist in the icu using at least 120 ml saline (yield, ~50%). upon reception, specimens including bal fluids and dtt-treated aspirations (dtt 1x at 37°c for 15min) were centrifuged, suspended in 200µl of water and submitted to extraction (whole nucleic acids extraction) using the genelead-viii extractor-thermocycler™ (precision system science, japan). p. jirovecii reverse transcriptase quantitative pcr (rtqpcr) was performed to amplify mtssu and mtlsu rna and dna of p. jirovecii using the new r-diapnj kit™ (diagenode, belgium). serum -d-glucan was tested using the fungitell kit™ (cape cod inc, us) as recommended by the manufacturer. data are presented as median [25 th -75 th percentiles] or percentages as appropriate. comparisons were performed using mann-whitney or exact fisher tests as required. p-values ≤0.05 were considered as significant. one hundred-and-eight successive hiv-negative covid-19 patients (male/female sex ratio, 4.4; age, 62 years [56-68]) with the usual risk factors for severe covid-19 presentation were included (table1). all except three patients were intubated on admission. thirty-four patients (31.4%) who developed ards received at least one day of corticosteroids before bal sampling. respiratory samples included 80 bals (74.1%), 22 tracheal aspirates (20.4%), 4 sputa (3.7%) and two bronchial aspiration fluids (1.9%). in 10/108 patients (9.3%), p. jirovecii rtqpcr was positive. median delay between sampling and icu admission was 2days [1] [2] . the median quantitative cycle value was 32.6 [30. 8-34.7] . serum -d-glucan were measured in nine patients and was negative (>80pg/ml) in seven patients. clinical characteristics of the patients carrying p. jirovecii did not significantly differ from the other patients except for lower plasma d-dimer (1,270ng/ml [750-2,390] vs 2,610ng/ml [1, ,700], p=0.03) and more frequent lopinavir/ritonavir administration (40.0% vs 12.2%, p=0.04), while long-term corticosteroid prescription tended to be more frequent (30.0% vs 8.2%, p=0.06). of note, among our 10 p. jirovecii carriers, five concomitantly met the criteria for covid-19-associated pulmonary aspergillosis. 6 out of these patients, four (40%) received co-trimoxazole as prophylaxis (80/400mg once daily) whereas six including four who rapidly improved did not. one co-trimoxazole-treated and two nontreated patients died while the seven remaining patients were discharged. mortality was similar in both groups. we found an unexpectedly high proportion of critically ill covid-19 patients detected with p. jirovecii (10/108 patients; 9.3%), similarly to previous findings in influenza patients (3/45; ~7%). 5 the presence of p. jirovecii in the healthy adult population has been measured using oropharyngeal wash samples obtained by gargling and examined by conventional or nested pcr methods. 7 however, experts agree that the reported prevalence (~20%) has been overestimated due to technical issues such as contamination with amplicons responsible for false positives. 4 in our center managing almost exclusively immunocompromised patients, prevalence of qpcr-positive respiratory specimens with fungal load as low as in our covid-19 patients, is ~13% (unpublished data), as reported elsewhere. 3 patients mostly exhibited marked lymphopenia and alterations in lymphocyte functions, 8 likely explaining the high-rate of p. jirovecii detection. since serum -d-glucan is advocated in pneumocystosis diagnosis, 4 we measured its concentrations in four of our five p. jirovecii rtqpcr-positive patients and obtained low values (<120pg/ml) in accordance with the low nucleic acids fungal loads in the lung alveoli. 9 of note, in two out of our nine tested p. jirovecii rtqpcr-negative patients, higher b-d-glucan concentrations (450 and 500pg/ml) lead to the diagnosis of pulmonary aspergillosis, another fungal infection of risk in covid-19 patients. 6 although a recent meta-analysis questioned its sensitivity in non-hiv patients, 10 -d-glucan has been widely used to rule out pneumocystosis because of its high negative predictive value. this finding may support the hypothesis that our patients were carrying p. jirovecii, yet not being infected per se. thus, although interesting in the context of invasive fungal infections diagnosis, serum -d-glucan should be interpreted with caution when excluding the diagnosis of pneumocystosis. here, four out of ten p. jirovecii rtqpcr-positive patients received co-trimoxazole as prophylactic regimen, based on the treating physician's decision. whether a positive result should be an indication to consider administering co-trimoxazole, at least at prophylactic dosage in covid-19 patients remains questionable. our study limitations include the relatively small number of patients, the bi-center setting, and the short study period. however, to the best of our knowledge, this is the first study evaluating the prevalence of p. jirovecii in covid-19 patients. because we focused on critically ill covid-19 patients, p. jirovecii prevalence in less severe patients remains to be determined. in conclusion, an unexpectedly high proportion of p. jirovecii-positive pulmonary samples is observed in critically ill covid-19 patients. based on our findings, we advocate systematically searching for p. jirovecii in deep respiratory specimens in these patients. we believe that this strategy may be useful in limiting enhanced inflammation due to the presence of p. jirovecii in the lung and avoiding inter-patient p. jirovecii transmission. the authors declare that they have no competing interests. this study was part of the french covid-19 cohort registry conducted by the reacting consortium dr. alanio has full access to all data and takes responsibility for the data integrity and its analysis accuracy. all the authors agree to publish. none. the case investigations, analysis, and manuscript preparation were completed as part of official duties at the university hospital. co-infections in people with covid-19: a systematic review and meta-analysis clinical features of patients infected with 2019 novel coronavirus in wuhan, china invasive aspergillosis in patients admitted to the intensive care unit with severe influenza: a retrospective cohort study ecil guidelines for the diagnosis of pneumocystis jirovecii pneumonia in patients with haematological malignancies and stem cell transplant recipients influenza virus and factors that are associated with icu admission, pulmonary co-infections and icu mortality prevalence of putative invasive pulmonary aspergillosis in critically ill patients with covid-19 pneumocystis jirovecii in general population longitudinal characteristics of lymphocyte responses and cytokine profiles in the peripheral blood of sars-cov-2 infected patients accuracy of β-d-glucan for the diagnosis of pneumocystis jirovecii pneumonia: a meta-analysis diagnostic accuracy of serum (1-3)-β-d-glucan for pneumocystis jirovecii pneumonia: a systematic review and meta-analysis the authors would like to thank mrs. alison good (scotland, uk) for her helpful review of the manuscript. key: cord-302855-wwg6x1df authors: ma, jia; yin, jing; qian, yu; wu, yuan title: clinical characteristics and prognosis in cancer patients with covid-19: a single center's retrospective study date: 2020-04-14 journal: j infect doi: 10.1016/j.jinf.2020.04.006 sha: doc_id: 302855 cord_uid: wwg6x1df • we describe the demographics, clinical characteristics, and prognosis of cancer patients with covid-19 infection. • cancer patients had a high infection rate of sars-cov-2 and were easy to become severe or critical cases. • among cancer patients with covid-19, the numbers of neutrophil, nlr(ratio of neutrophil to lymphocyte, il-6, ldh and pct in the severe/critical group were significantly higher than those in the mild group. we read with the interest the recent paper by chen et al. who described the clinical progression with covid-19 in shanghai, china, 1 which described ninety patients (36.1%) had one or more coexisting chronic medical conditions and one patient had malignant tumor (0.4%). a small amount of literatures have reported that the infection rate of covid-19 was higher in cancer patients compared with non-cancer patients. in addition, cancer patients are more likely to turn to severe covid-19 cases than non-cancer patients. [2] [3] [4] in this letter, we describe the demographics, clinical characteristics, and prognosis of cancer patients with covid-19 infection. a total of 1380 covid-19 patients admitted to renmin hospital of wuhan university from january 1, 2020 to march 30, 2020 were screened, and 37 cancer patients with covid-19 were enrolled. all the enrolled patients were confirmed covid-19 infected as positive in laboratory tests, including nucleic acid testing or antibody test for sars-cov-2. severe and critical patients are diagnosed according to the new coronavirus pneumonia prevention and control program (7th edition). 5 the study was approved by the institutional review board of renmin hospital of wuhan university. demographic information and laboratory tests including complete blood count test, liver and kidney function tests, coagulation and immune function tests results were collected upon admission. continuous variables were shown as median (interquartile range) and percentages were presented for categorical variables. mann-whitney u test was used to compare continuous variables. fisher's exact test was used to compare categorical variables. p < 0.05 was considered to be statistically significant. in our study, the infection rate of covid-19 among cancer patients in the single center was estimated to be 2.7% (37 of 1380 patients), which was 6 times higher than that in wuhan until mar 30, 2020 (0.45%, 50,006 of 11,081,000). the proportion of severe/critical covid-19 patients with cancer was 54.1%, which was also significantly higher than that of the general population. 6 as shown in table 1 , there were 20 male patients (54.1%), and the median age of the patients was 62 years old. according to the previous studies, cancer patients with covid-19 were much older. 6 , 7 the most common symptoms of onset were fever (75.7%) and cough (56.8%), complicated with dyspnea (32.4%) or fatigue, diarrhea and myalgia. in addition, cancer patients with covid-19 were divided into two groups, as mild group, and severe/critical group, according to the severity of covid-19. compared with the mild group, patients in the severe/critical group were more likely to have dyspnea (50%). among the 37 patients, thirteen patients had a history of anti-cancer therapy including surgery, radiotherapy, chemotherapy, targeted therapy, or immunotherapy within one month before hospitalization; six patients in the mild group and seven patients in the severe/critical group respectively. however, with/without the cancer therapy history did not differ significantly between the two groups, suggesting that anti-cancer therapy did not effect on the severity of covid-19 among these cancer patients. it is noteworthy that the most common cancer was colorectal cancer (29.7%), followed by lung cancer (21.6%) and breast (18.9%). among the severe/critical patients, there were seven colorectal cancer patients. previous studies indicated that lung cancer patients were the most common to be infected, which needs more multicenter retrospective studies with a larger sample size to identify. [3] [4] [5] studies have shown that the prognosis of covid-19 patients with underlying diseases was poor, while the prognosis of patients with two or more basic diseases would be even worse. 6 in our study, a total of twelve covid-19-infected cancer patients were complicated with underlying diseases, such as hypertension, diabetes, chronic obstructive pulmonary disease etc., accounting for 32.4%. in the severe/critical group, 8 wbc count, ×10 9 /l 3.5-9. derlying diseases, which was two times higher than that in the mild group, although there was no statistical difference between the two groups. moreover, five cancer patients died, with a mortality rate of 13.5%, all of which occurred in the severe/critical group. in terms of the results of laboratory tests, we interestingly found that the numbers of neutrophil, nlr, il-6, ldh and pct ( table 2 ) in the severe/critical group were significantly higher than those in the mild group. studies have shown that increased neutrophils and decreased lymphocytes will increase the risk of severe illness in covid-19 patients. 6 at the same time, the increase of nlr was also an independent prognostic factor in t1-2 rectal cancer patients. 7 in addition, ldh and pct were significantly higher in the severe/critical group than that in the mild group. these two indexes were also increased in the general population of patients required icu admission. 6 moreover, il-6 was known to participate in the storm of inflammatory factors caused by covid-19. the storm could be the main cause of death in infected patients. 8 therefore, we should pay more attention to the above laboratory indicators in order to identify early and reduce the severe ratio and mortality of cancer patients with covid-19. in conclusion, our report originally described and analyzed clinical characteristics and prognosis in cancer patients with covid-19. cancer patients had a high infection rate of sars-cov-2 and were easy to become severe or critical cases, which should be monitored closely during the covid-19 epidemic. more multicenter retrospective studies are needed to investigate the comprehensive demographics and risk factors for cancer patients with covid-19. all the authors have no conflicts of interest to declare. this study was supported by the national natural science foundation of china (no. 81903137 ). clinical progression of patients with covid-19 in shanghai cancer patients in sars-cov-2 infection: a nationwide analysis in china sars-cov-2 transmission in patients with cancer at a tertiary care hospital in wuhan, china clinical characteristics of covid-19-infected cancer patients: a retrospective case study in three hospitals within wuhan, china new coronavirus pneumonia prevention and control program clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china significance of neutrophil-to-lymphocyte ratio, platelet-to-lymphocyte ratio, lymphocyte-to-monocyte ratio and prognostic nutritional index for predicting clinical outcomes in t1-2 rectal cancer covid-19: consider cytokine storm syndromes and immunosuppression key: cord-334443-3pyu8ucs authors: he, yu; wang, zhengli; li, fang; shi, yuan title: public health might be endangered by possible prolonged discharge of sars-cov-2 in stool date: 2020-03-05 journal: j infect doi: 10.1016/j.jinf.2020.02.031 sha: doc_id: 334443 cord_uid: 3pyu8ucs • the published data, which showed the covid-19 patients with low digestive. • manifestation, might be misleading. case with negative urt test showed positive in. • rectal scarab which challenge the isolation protocol. • as fomite transmission caused clusters of infection of sars, adequate disinfection. • operations should be adopted in sars-cov-2 outbreak. according to a recent report, since december 8 2019, a novel identified coronavirus, sars-cov-2(previously named as 2019-ncov) is causing outbreak of pneumonia in wuhan, china and become the major concern throughout the world [1] . the world health organization has recently announced this disease to constitute a public health emergency of international concern and then named this disease as corona virus disease 2019 . by the 28 feb, 2020, more than 80,0 0 0 confirmed cases including over 2500 death cases were officially announced. till now, the infection control and surveillance focus on respiratory system. the ignorance of sars-cov-2 in digestive system may cause troubles in the disease control. gastrointestinal symptoms seem to be uncommon in patients with covid-19 when compared with severe acute respiratory syndrome (sars) ( table 1 ) [2 , 3] . however, they should not be ignored as the increasing rate of diarrhea occurs in confirmed covid-19 patients according to a recent report that 14 of 138 confirmed patients had diarrhea [4] . those early reports may not represent actual rate of gastrointestinal symptoms caused by sars-cov-2, because in early stages of the outbreak, the limited resources for detection were only provided to those patients with severe symptoms like respiratory distress syndrome. about 27 percent of sars patients have diarrhea and since full-length genome sequences identified that sars-cov-2 is 79.5% identical to sars-cov and share the same receptor angiotensin-converting enzyme 2 (ace2), it is estimated that rate of gastrointestinal symptoms would be higher in patients with covid-19 [5] . one possible route for the movement of sars-cov-2 into digestive system may be "trachea-esophagus-ileum-colon" as single-cell transcriptom analysis showed ace2, the entry receptor for sars-cov-2, highly expressed in lung at2 cells, esophagus upper and stratified epithelial cells and enterocytes from ileum and colon [6] . the evidence for this route is that all of the specimens including pharyngeal swab, esophageal biopsy, gastric mucosa, rectal mucosa, duodenal mucosa and stool tested positive to sars-cov-2 in two cases [7] . another route may be bloodstream infection since sars-cov-2 was directly detected in bleeding site in one case [7] . in addition, expression of ace2 in endothelial cells and macrophages, as well as the detection of sars-cov in plasma and blood lymphocytes also support the possibility of bloodstream infection of sars-cov-2 [8 , 9] . the discharge guideline depending on respiratory tract test also meets challenge. in cases, sars-cov-2 infected infant initially behaved as vomiting, diarrhea or feeding intolerance [15] . interestingly, while the virus test of nasopharyngeal swab switched from positive to negative after treatment, the rectal swab specimens still tested positive [10] . these cases remind the clinicians that the rectal swab may be equally important to the pharyngeal swab even the patient is asymptomatic which challenge the latest published guideline provided by national health commission of china that two successive negative of the respiratory tract tests are regarded as the standard for discharge and termination of compulsory isolation for covid-19 patients [11] . a famous well-described clusters of infection of sars in amoy gardens, hong kong drew the attention of health official on fomite transmission because two thirds of the confirmed sars patients in this amoy gardens had diarrhea [12] . as findings showed that patients with sars could discharge sars-cov in their stool up to 73 days after symptom onset, the stools with the virus became the resource of contamination of airdrops and a variety of environmental surfaces, which may contribute to the clusters of infection [13] . similarly, evidence showed that sars-cov-2 were identified in 4 stool specimens (4 out of 62), so fomite transmission should not be ignored in the transmission of sars-cov-2 since the virus may move from respiratory tract in to gastrointestinal tract the recovered patients may discharge the stool with virus for a long time [9] . according to a recent published report by cdc of china, the community acquired infections are becoming the predominant route in transmission [14] . based on these cases and the lessons from sars, we recommend 1) the attentions should be drawn in digestive symptoms and stool or rectal scwrab tests for patients with suspicion or confirmed sars-cov-2 infection, 2) preventive education and publicity on hands washing and bathroom infection, 3) compulsory isolation till scwrab tests switch to negative, 4) surveillance and adequate disinfection in latrines in areas with seversars-cov-2 infection to avoid fomite transmission. the authors declare no conflicts of interest. emergence of a novel coronavirus causing respiratory illness from wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong the digestive system is a potential route of 2019-ncov infection: a bioinformatics analysis based on single-cell transcriptomes clinical characteristics of coronavirus disease 2019 in china single-cell rna expression profiling of ace2, the putative receptor of wuhan 2019-ncov detection and monitoring of sars coronavirus in the plasma and peripheral blood lymphocytes of patients with severe acute respiratory syndrome first case of neonate infected with novel coronavirus pneumonia in china the sars epidemic in hong kong enteric involvement of severe acute respiratory syndrome-associated coronavirus infection team tncpere the epidemiological characteristics of an outbreak of 2019 novel coronavirus diseases (covid-19) in china sars-cov-2 infection with gastrointestinal symptoms as the first manifestation in a neonate key: cord-337557-ct43uoir authors: guetl, katharina; moazedi-fuerst, florentine; rosskopf, konrad; brodmann, marianne; krause, robert; eller, philipp; wilhelmer, patricia; eisner, florian; sareban, nazanin; schlenke, peter; kessler, harald h.; steinmetz, ivo; redlberger-fritz, monika; stiasny, karin; stradner, martin title: sars-cov-2 positive virus culture 7 weeks after onset of covid-19 in an immunocompromised patient suffering from x chromosome-linked agammaglobulinemia date: 2020-10-27 journal: j infect doi: 10.1016/j.jinf.2020.10.025 sha: doc_id: 337557 cord_uid: ct43uoir nan ) are considered to be without infectious potential beyond day 10 after the onset of symptoms. in contrast, immunocompromised and severe-to-critical patients may have prolonged viral shedding and, thus, may also provide prolonged infectiousness. data addressing prolonged viral shedding and potential spread of sars-cov-2 are matter of public concern. whereas isolation precautions as recommended by the united states centers of disease control and prevention (2) and the european health care authorities (3) are considered to fit for most sars-cov-2 infected patients, uncertainty remains in those patients with underlying immunodeficiency. walsh et al. included a total of 15 relevant studies, but only two of them identified immunosuppressed patients from whom sars-cov-2 was isolated for up to 20 days beyond onset of disease. (4, 5) here, we report sars-cov-2 positive viral culture 7 weeks after onset of covid-19 in a patient with an underlying immunosuppressive disorder, so-called x chromosome-linked agammaglobulinemia (xla), demonstrating the potential of prolonged sars-cov-2 spreading beyond widely accepted isolation precautions. our patient had been tested positive for sars-cov-2 ribonucleic acid (rna) by reverse transcription polymerase chain reaction (rt-pcr) from upper respiratory specimen first on march 11 2020. at this point of time, symptoms comprised fever and fatigue. in the patient's medical history, xla, obstructive respiratory disorder, impaired alveolar diffusion capacity and non-cystic fibrosis bronchiectasis were recorded. due to worsening of fever, cough and dyspnea, the patient required for hospitalization 16 days after the initial diagnosis of covid-19. the patient received antibiotic treatment with amoxicillin/clavulanic acid and azithromycin, was switched to piperacillin/tazobactam, followed by moxifloxacin and meropenem. based on local recommendations valid at this point of time, the patient was treated with hydroxychloroquine and then by an antiretroviral combination of lopinavir/ritonavir. furthermore, posaconazole was administered for aspergillus positive sputum culture and intravenous immunoglobulin substitution (ivig) was performed regarding the absence of endogenous antibody production in underlying xla. due to persistent fever up to 40.4°c, progressive respiratory insufficiency and deterioration of laboratory parameters expressing an increasing inflammatory activity, the patient was transmitted to the intensive care unit (icu) on april 9. interleukin-6 (il-6) receptor blockade by tocilizumab and convalescent plasma were administered on april 10. no adverse effects related to this treatment regimen were recorded. the rationale behind this approach was to restrain the inflammatory response by il-6 blockade and to provide neutralizing covid-19 immunoglobulins by convalescent plasma. afterwards, we observed a rapid recovery regarding clinical and laboratory parameters. ferritin and c-reactive protein (crp) significantly declined as compared to pretransfusion whereas the lymphocyte count returned to normal. we observed an increase in il-6 after treatment followed by a fast and almost complete decline within the next days. body temperature did not exceed a limit of 38°c as compared to measurements of up to 40.4°c pre-transfusion. oxygen demand decreased resulting in an increase of pao2/fio2 ratio (143 before versus 223 after treatment). a chest radiograph showed a significant decline in infiltrative opacities. on april 15, five days after tocilizumab and convalescent plasma administration and five weeks after the initial diagnosis of covid-19, sars-cov-2 rna was not detectable for the first time. the patient showed progressive clinical recovery, but an alternating course of three negative followed by three positive sars-cov-2 rt-pcr results was subsequently observed. convalescent plasma transfusion was showed 500 copies/ml in transport medium (containing the oropharyngeal swab) and the viral culture was negative. figure 1 presents an overview by timeline from the initial diagnosis of covid-19 up until negative viral culture. in summary, we have to assume that in our patient shedding of infectious sars-cov-2 stopped between week 7 and 10 of disease. our patient suffers from xla, also known as bruton's agammaglobulinemia, which is caused by a mutation in bruton's tyrosine kinase resulting in an inability of endogenous antibody production du to a developmental arrest of pre b cells. symptom-based strategy to discontinue isolation for persons with covid-19 guidance for discharge and ending isolation in the context of widespread community transmission of covid-19 -first update shedding of infectious virus in hospitalized patients with coronavirus disease-2019 (covid-19): duration and key determinants cov-2 shedding and mild course of covid-19 in a patient after recent heart transplantation virological assessment of hospitalized patients with covid-2019 presymptomatic sars-cov-2 infections and transmission in a skilled nursing facility x-linked agammaglobulinaemia: outcomes in the modern era a possible role for b cells in covid-19?: lesson from patients with agammaglobulinemia none to declare. none to declare. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-291561-sxvgue36 authors: haixu, liang; haibin, wang; lili, ren title: detection of 20 respiratory viruses and bacteria by influenza-like illness surveillance in beijing, china, 2016–2018 date: 2019-11-25 journal: j infect doi: 10.1016/j.jinf.2019.11.014 sha: doc_id: 291561 cord_uid: sxvgue36 nan in previous reports, workers have characterized the presentation of middle east respiratory syndrome (mers) 1 and severe acute respiratory syndrome (sars) 2 to aid clinical teams in the recognition, diagnosis and management of these cases. now with the emergence of a novel coronavirus (cov) from wuhan, china (tentatively named as 2019-ncov by the world health organization -who), 3 , 4 similar clinical, diagnostic and management guidance are required. available information at the time of writing indicates that the virus has now spread beyond china and infection has been confirmed in individuals without direct contact with the index wuhan wet market (huanan south china seafood market), where the sale of game meat from live animals was also available. this suggests that human-to-human transmission is not only possible but very likely. a full genome phylogenetic analysis of this 2019-ncov indicates that it is closely related to bat sars-like cov ( fig. 1 ) , compatible with a zoonotic origin for this virus, similar to sars-cov and mers-cov. 5 two cases have now been confirmed to date in thailand. the first was a 61-year old chinese woman travelling with 5 family members in a 16-member tour group. she developed symptoms of fever, chills, headache and sore throat on 5 january 2020 and flew from wuhan directly to thailand on 8 january, where she was diagnosed and isolated. she reported regular visits to wet markets in wuhan but not the index wet market from where most cases were reported. 6 the second case confirmed in thailand was that of a 74-year old chinese woman, who was laboratory-confirmed to be infected with the 2019-ncov on 17 january 2020. this second case was not linked epidemiologically to the first case, and she had not visited any market in wuhan. so far both cases are recovering well in the negative pressure isolation facilities at the bamrasnaradura institute in thailand, and may be discharged soon. 7 , 8 in addition, one case of 2019-ncov was confirmed in a male patient in his thirties in japan who was staying in wuhan during late december 2019 to early january 2020, and developed fever on 3 january. although he had not visited any wet or live animal markets during his stay in wuhan, he did report close contact with someone with pneumonia. on return to japan on 6 january he visited a local clinic where he tested negative for influenza. despite this, his symptoms of fever, cough, and sore throat continued, so he attended a local hospital on 10 january where he was admitted and found to have abnormal infiltrates on his chest x-ray. he remained febrile until 14 january and was eventually tested as positive for 2019-ncov on 15 january. 9 he became afebrile on the same day and was discharged home where he remains stable. this was the second 2019-ncov case to be confirmed outside of china (being identified between the two cases from thailand). thus, clinically, the symptoms of 2019-ncov infection appear very non-specific and may be very similar to influenza, including fever, cough, fatigue, sore throat, runny nose, headache and shortness of breath, with possible ground glass shadowing on the chest x-ray. importantly, such symptoms appear to persist longer in cases of 2019-ncov infection than in most cases of uncomplicated influenza. similar to sars and mers, there is still no specific, licensed antiviral treatment for covs and the clinical management is mainly supportive. infection control guidance will be likely based on existing guidance for sars and mers, perhaps with some additional heightened precautions due to the largely unknown nature of this new virus. also similar to the sars and mers cases, there is likely a lot of variability in the clinical presentation, including mild or asymptomatic cases that may never present to healthcare services. larger population level seroprevalence studies to test for past infection or exposure are required to determine how many such cases may exist. whether "super-spreaders" who are associated with multiple secondary infections, as has occurred most prominently with sars but also mers, will be (or indeed may have already been) a feature of the epidemiology of this virus is not yet known. no pediatric 2019-ncov infections have been diagnosed so far, and infections in other vulnerable patient groups, such as transplant and other immunocompromised patients, pregnant women and those with chronic diseases (diabetes, liver, kidney, heart disease, etc.) and extremes of the body-mass index (bmi) , are yet to be reported. further data are awaited on such cases. based on the current and limited data available and the likelihood that many milder or asymptomatic cases have not presented to healthcare services, it is too early to compare case-fatality rates with sars or mers. so far, there have been two deaths out of a total of 48 cases reported from wuhan and overseas, 7 which have been in older patients with various comorbidities. most recently, a mathematical modelling study from imperial college (london, uk) suggests that the number of unrecognized, undiagnosed cases could be as high as 440 0-450 0, though ∼170 0 may be more realistic -assuming that the model assumptions are reasonably accurate. 10 this situation is evolving and more updates will be forthcoming. sars was the first emerging infectious disease of the 21st century and it came and went quickly despite a tremendous global impact. mers in contrast remains largely confined to the middle east with occasional exported cases and has smouldered since 2012. we clearly have a lot to learn about these zoonotic bat coronaviruses (see fig. 1 ), but hopefully the scientific, medical and public health worlds are now much better prepared this time round to deal with this new emerging threat. recently, an outbreak of unusual viral pneumonia in wuhan city, china has sickened dozens of people. preliminary studies indicated a novel coronavirus as the likely cause of the outbreak. genetic recombination has been shown to contribute to the evolution of coronaviruses, including severe acute respiratory syndrome coronavirus (sars-cov) and the middle east respiratory syndrome coronavirus (mers-cov). recent papers in this journal also described the involvement of recombination in viral evolution. 1 , 2 here i use ferret coronaviruses (frcovs) as an example to show recombination in coronaviruses. coronaviruses (covs) are enveloped, single positive-stranded rna viruses that can infect a wide range of host species. cov was first reported to infect ferret and associate with epizootic catarrhal enteritis in the united states in 2006, referred to ferret enteric coronavirus (frecv). 3 5 this study aim to assess the genetic diversity and potential role of genetic recombination in the evolutionary dynamics of frcovs. genetic analyses were conducted with five complete genomes and 160 gene sequences of frcovs downloaded from the niaid virus pathogen database and analysis resource. 6 these sequences were analysed in combination with 36 representative genomes of covs from other host species. phylogenetic analysis of the complete genome confirmed the division of four genetic genera in covs. frcovs fall in alpha genera and most closely related to the cov from mink (fig. s1 ). frcovs from us and japan (ferrets imported from us) were more closely related to each other than a dutch strain. phylogenetic tree for n gene supported two geographically dependent lineages, european and american ( fig. 1 a) . the european lineage comprised frcovs from the netherlands and slovenia, while the american lineage comprised frcovs from us and japan. phylogenetic tree for rdrp gene showed that the american and japanese strains comprised the american lineage and are distant from the european lineage represented by the dutch strain (fig. s2 ). different grouping was observed in the phylogenetic tree for s gene. the european lineage was consistent with that observed in the n tree, however, the american lineage was further divided into two sub-groups: american-i and american-ii ( fig. 1 b) . the american-ii sub-group comprised four frcovs from america, but was more closely related to european lineage rather than the american-i sub-group. differences between the topologies of phylogenetic trees of s gene, complete genome, and n gene suggest the occurrence of potential recombination events ( fig. 1 a and b ). simplot and bootscan analyses of the five complete frcov genomes were performed to investigate the genetic variability in different parts of the genome and potential recombinations. 7 the fecv1(us) strain was used as the query and compared with four strains: frcov_nl_2010(nl), fscv6(us), ferret063(japan), and fr-cov4370(japan). higher genetic variability was observed in the s gene, particularly at the 3 terminal end, compared to other parts of the genome ( fig. 1 c) . the average shared sequence identity was 91.0% for the complete genome, 93.1% for n gene, 95.0% for rdrp gene, and 73.6% for s gene, respectively. the 3 terminal end frcovs from america and europe are indicated by red and blue boxes, respectively. two genetic groups identified for s gene of american frcovs are represented by american-i and american-ii. numbers at the nodes indicate bootstrap support evaluated by 500 replicates. recombination analyses of s gene of frcovs (c and d). the fecv1 strain from america was used as the query and compared with four strains: frcov_nl_2010 (nl), fscv6(us), ferret063(japan), and frcov4370(japan). (c) simplot shows the genetic distance between query sequence and each reference sequence in different parts of the genome. (d) bootscan plot shows phylogenetic relationship between query sequence and reference sequences in different parts of the s gene. potential region for recombination event is highlighted by dash lines. comparison of putative transcription regulatory sequences (trs) for orf1ab, spike, membrane, and nucleocapsid genes between five ferret coronavirus (frcov) strains. the core trs is indicated in bold. accession number and origin for each strain: frcov-nl-2010 (nc_030292, the netherlands), fecv1 (kx512809, the united states), fscv6 (kx512810, the united states), ferret063 (lc215871, japan), and frcov4370 (lc119077, japan). orf1ab fig. 1 d) . next, separate phylogenetic analyses were conducted for the recombination part and non-recombination part of the s gene. phylogeny for the recombination part showed that american-ii sub-group is closer to european lineage, whereas phylogeny for the non-recombination part showed that american-i and american-ii sub-groups are both separated from european lineage (fig. s3) . comparison of the putative transcription regulatory sequences (trs) for these five strains showed identical core trss ( table 1 ) . taken together, these results suggest potential genetic recombination event at the 3 terminal end of s gene between frcovs from european and american lineages. however, due to relatively low sequence identity in the s gene and small number of available sequences, other possibilities cannot be excluded. this study focused on the s gene, and recombination could happen in other regions of the genome. earlier studies have identified recombinations in the 3c and envelop genes. 8 , 9 the n and orf 7b genes of two japanese strains, no.22 and no.160, were completely different from those from other frcov strains. 9 the phylogeny of the n gene in this study supported the observation; phylogeny of the rdrp gene also showed that these two strains are distant from other strains ( fig. 1 and fig. s2 ). the sequence identities in the rdrp gene between these two strains and other frcovs are relatively low (87.5%). while the source of these two japanese strains are not clear, potential recombination, including recombination with other covs, could have contributed to the uniqueness of their genomes. a critical question yet to be answered is the molecular basis for pathotype switch between less pathogenic frecv and pathogenic frscv. recombination in the s gene has been suggested to associate with the transmissibility and virulence of coronaviruses. 10 it is reasonable to propose that frscv may have evolved from frecv through recombination. 8 , 9 although this study cannot establish a direct link between the detected recombination and change of pathogenicity, it is interesting to see that 3 out of 4 strains are frscv, including msu-s, wadl, and msu-1 ( fig. 1 b) . future in vivo experiments are needed to clarify the precise biological implications of this recombination in s gene. one major limit of this study is the small number of frcov sequences available in the public databases. considering the wide spread of frcov and extensive use of ferret as an animal model for influenza pathogenicity study, enhanced surveillance is required to monitor the spread and genetic diversity of frcov. the author declare no conflict of interest. supplementary material associated with this article can be found, in the online version, at doi: 10 recently, the emergence of african swine fever virus in china has raised great concern in this journal. 1 coronaviruses are a large family of viruses that cause respiratory illnesses. although coronaviruses (covs) have been known for decades, they did not raise great attention in human medicine until the outbreaks of severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers). 3 sars-cov first emerged in november 2002 in guangdong province of southern china and then rapidly spread to 29 countries and regions, infecting over 80 0 0 individuals with a death toll of nearly 800. 3 ten years after the sars, mers emerged in 2012, have caused 2494 human infections with 858 deaths (as of november 2019) and remains a disease of global, and particularly middle eastern, public health concern. in 2017, a novel hku2-related bat coronavirus, swine acute diarrhea syndrome coronavirus (sads-cov), caused the death of 24,693 piglets, 4 , 5 raising further concern about these coronavirus. in december 2019, a new coronavirus (2019-ncov), which is about 70% similar to sars-cov, was discovered in the central chinese city of wuhan, with 545 cases in 25 provinces being diagnosed ( fig. 1 a, january 22, 2020). according to the world health organization ( https://www.who.int ), in addition to mainland china, this virus has also been detected in japan, thailand, republic of korea and the united states in travelers from wuhan. the number of confirmed cases has been gradually increasing ( fig. 1 b) , which might be partially due to recent the establishment of relevant detection methods, but, at the same time, could signal that there could be a quick expansion of the epidemic. the three basic elements required for an infectious disease epidemic are source of the infection, route of transmission, and susceptible hosts (humans). eliminating the source of the infection and cutting off the transmission route are usually effective means to block the spread of an infectious diseases. the successful precedent of emergent cov containment based on the elimination of the primary reservoir is sars. bats are suggested to be the reservoir hosts of sars-covs. 6 , 7 however, without an intermediary host, bat derived covs cannot directly infect humans. the carnivoraintermediate amplifying host (civets) of sars-covs was found. 8 the quick control of the intermediate amplifying host by banning wild animal trade was the key factor in the effective control of sars. many of the 2019-ncov infected people had either worked at or frequently visited a seafood market in wuhan. apart from fish, the market also sold other live wild animals -sparking concerns that the virus might have been transmitted from an animal to humans, just like sars and mers. the wuhan covs cluster with sars/sars-like coronaviruses, and have about 70% overall genome sequence similarity. as this virus clusters with various bat betacoronavirus, it is reasonable to deduce that bats are the native host for the 2019-ncov. however, its origin (source) remains unknown, that is, the animals that are the origin and amplifying hosts for this virus. this makes the elimination of this disease from the source difficult. the control of the route of transmission is another effective means of epidemic control. infections of healthcare workers and family clusters suggest that 2019-ncov has the ability to spread from human to human. although the seafood market has been closed, the number of confirmed cases continues to gradually increase ( fig. 1 b) . this further supports the conclusion that this virus can spread by human-to-human contact. previous research has shown that coronaviruses usually have an ability to rapidly mutate. 9 we cannot exclude the possibility that some 2019-ncovs will mutate to become "super-spreaders", as seen in sars. frequent disinfection of people-intensive places and animal trading markets should help stop the spread of the virus. unfortunately, the outbreak has cast a shadow over the celebrations for the lunar new year, which falls on january 25. millions of people in china travel over the course of the spring festival, both within the country and overseas. in addition, millions of college students (wuhan has more than 1 million college students) will return to school after the winter vacation. this large-scale population migration could lead to further spread of this virus. therefore, it is possible that this epidemic will be more serious after the spring festival. at present, the national health commission of china ( http://www.nhc.gov.cn ) has listed 2019-ncov as class b infectious disease and managed as class a. measures including the temperature monitoring of passengers at railway stations, airports, terminals and other transportation hubs have been carried out, which should slow the spread of the virus to a certain extent. however these disease control policies are far from enough, more strict methods to control population flow should be on the table. it is increasingly recognized that a one health approach at the human-animal-ecosystem interface is needed for effective investigation, prevention and control of any emerging zoonotic disease. in the context of emerging zoonoses, human and veterinary medicine must work together. these viruses emerge from animal trading markets, where veterinary workers need to be vigilant and concerned whether these viruses have the potential to spread between animals and the impact the livestock industry. veterinary workers should also actively invest in epidemiological investigations to find the natural hosts of these viruses and develop corresponding detection methods. considering that the original source of the 2019-ncov is very likely related to a wild animal, additional strict laws to limit wildlife markets should be made, otherwise, more emerging zoonoses from wild animals will occurred in the near future. several recent studies in this journal have highlighted the threat posed by mosquito-borne viruses in china, such as zika virus and dengue virus. 1-3 getah virus (getv) is also a mosquitoborne virus that is classified in the alphavirus genus of the togaviridae family. the genome of getv is linear, positive-sense ssrna, encoding a total of nine viral proteins (nsp1-nsp4, e1-e3, c, and 6 k). getv was first isolated from culex mosquitoes collected in malaysia in 1955 and has since been found in mosquitoes in asian countries surrounded by the pacific ocean (china, japan, south korea, mongolia, russia, and india) based on viral isolation and/or molecular epidemiological investigations. 4 , 5 getv infection has been found in multiple vertebrates, including humans, monkeys, birds, pigs, horses, and other mammals, and infection of these species is essential for the maintenance of getv zoonotic transmission cycles in nature. however, getv has long been regarded as pathogenic only in pigs and horses. getv infection can cause fever, generalized rash, and leg edema in horses and is often associated with fetal death and reproductive disorders in pigs. several disease outbreaks caused by getv infection in pigs and horses have been reported in japan and india. 6 in china, the epidemic status of getv has become increasingly problematic recently, posing a great threat to animal and public health. initially, getv was detected in several new mosquito species. getv has been regarded as being primarily carried and spread by mosquitoes of the genera aedes and culex . in china, getv was first isolated from wild culex mosquitoes in hainan province in 1964 and was recently detected in and/or isolated from other mosquito species, including aedex vexans, armigeres obturbans, armigeres subalbatus , and anopheles sinensis . 4 , 5 , 7-9 unfortunately, these mosquito species are widely distributed in china with large populations. currently, getv has a wide distribution. before 2006, getv had been found in only six provinces in china. 8 however, the number of getv-affected provinces dramatically increased to fifteen in 2018 ( fig. 1 ) . 4 , 5 , 7 , 9 , 10 in addition, more mammalian species were found to be infected with getv. direct molecular evidence supporting getv infection in pigs in china was first obtained in taiwan in 2002 and was obtained in five other provinces in mainland china after 2011 ( fig. 1 ). in addition, horses infected with getv in china were first identified in 2018. 10 moreover, getv infection in fox and cattle was reported in northern china in 2019. 7 , 9 it should be noted that this was the first time that getv was isolated from these two animal species in the world, which undoubtedly expanded the host range of this virus. furthermore, getv was determined to be pathogenic in several more vertebrate species. neutralizing antibodies against getv have been identified in human sera in china. the anti-body titer in people with fever is significantly higher than that in healthy people, indicating that getv infection is possibly associated with disease in humans. getv infection in pigs and horses in china has been commonly associated with piglet death and fever, respectively. getv infection could result in fever, anorexia, depression, neurological symptoms, and even death in foxes and has been associated with fever, anorexia, and depression in cattle. moreover, the genetic complexity of getv in china is increasing. among the published getv strains, the majority were isolated in china ( fig. 1 ) . most of the chinese getv strains obtained in the field were isolated after 2010. getv strains worldwide can be classified into four groups, groups i-iv, based on the sequence of the e2 gene ( fig. 1 ) . 4 nearly all getv strains identified worldwide after the 1960s belong to group iii, except for one chinese strain (yn12031, which was isolated from mosquitoes in 2012) and one russian strain, which were classified as group iv. in addition, some chinese getv strains in group iii show special genetic characteristics. for example, the hnjz-s2 strain, which was isolated from pigs in 2015, has a closer relationship to japanese getv strains than other chinese getv strains. the jl1707 strain, which was isolated from mosquitoes in 2017, was clustered together with one mosquito-derived chinese getv strain in 2002 but showed a distant relationship with all six other chinese getv strains in 2017. getv is transmitted between individuals by mosquitoes, which serves as a vector, and the infected vertebrates act as amplifying hosts in getv transmission cycles. 5 china has a vast territory with a wide variety of mosquito and vertebrate species. in china, the numbers of newly discovered vector and infected vertebrate species of getv have increased rapidly in recent years. however, the threat posed by getv has not yet attracted enough attention in china. to prevent and control getv in china, it is necessary to take a series of related measures, such as increasing the popular knowledge of getv, decreasing the density of mosquitoes, improving biosafety awareness, and strengthening epidemiological surveillance. in addition, vaccination is regarded as an effective strategy for getv prevention and control. an inactive getv vaccine has been developed in japan to control the spread of this virus in horses ( http://www.jp-nisseiken.co.jp/en/products/ vaccine/index.html ). however, the vaccine antigen composition is based on a getv strain isolated in japan in 1978 and cannot provide adequate protection against infection with currently circulating getv strains, as shown by the getv outbreaks in japan in 2014. 6 therefore, there is an urgent need to develop a getv vaccine using a chinese strain that is prevalent in the field as the antigen component to vaccinate susceptible animals to reduce the increasing threat of getv to animal and public health in china. none. we recently published a case series of typically commensal neisseria spp. disease among eculizumab recipients. 1 eculizumab is a terminal complement inhibitor indicated for treatment of paroxysmal nocturnal hemoglobinuria, atypical hemolytic uremic syndrome, and certain patients with generalized myasthenia gravis or neuromyelitis optica spectrum disorder. 2 due to complement inhibition, many different neisseria spp. can cause invasive disease in eculizumab recipients 1 , 3 , 4 and eculizumab recipients are at an estimated 20 0 0-fold increased risk of meningococcal disease (caused by neisseria meningitidis ). all eculizumab recipients should receive meningococcal vaccinations prior to therapy; however, eculizumab recipients may develop meningococcal disease or other neisseria infections despite vaccine receipt. [4] [5] [6] for patients who cannot receive meningococcal vaccinations ≥2 weeks before starting eculizumab, u.s. eculizumab labeling recommends 2 weeks of antibiotic prophylaxis. 2 in july 2017, the centers for disease control and prevention (cdc) advised that prescribers could consider antibiotic prophylaxis in eculizumab recipients for the duration of eculizumab therapy. 4 there are no published studies evaluating the efficacy or safety of antibiotic prophylaxis in eculizumab recipients. here, we report on potential risks and benefits of antibiotic prophylaxis for the prevention of meningococcal disease in a case series of vaccinated eculizumab recipients who developed meningococcal disease. we included patients received eculizumab within the three months preceding a diagnosis of meningococcal disease, defined as a report of a symptomatic patient with a positive culture or other confirmatory test for n. meningitidis from any body site . faers cases were matched to cdc meningococcal disease surveillance data from the national notifiable diseases surveillance system, when possible, for confirmation of infection, serogroup, 1 and antibiotic susceptibility testing. time to onset (tto) was calculated from the date of first eculizumab dose (assumed to be the date of starting antibiotic prophylaxis) to the date of the patient's first meningococcal disease episode. for patients with multiple episodes of meningococcal disease, the calculations described below were performed using only the first meningococcal disease episode. the series included 47 patients, of whom 15 were taking antibiotic prophylaxis at the time of meningococcal disease onset and 32 were not ( table 1 ). there were four fatalities due to meningococcal disease (all among patients not taking prophylaxis). all 47 patients ✩ the views expressed are those of the authors and do not necessarily represent those of, nor imply endorsement from, the u.s. food and drug administration, the centers for disease control and prevention, or the u.s. government. 1 when serogroup was determined at cdc through multiple methods, slide agglutination results were used as the final serogroup. reportedly received ≥1 dose of a meningococcal vaccine. three of 47 patients had ≥1 episode of meningococcal disease (two patients were taking prophylaxis; one was not). tto of first episode of meningococcal disease was reported for 11 of 15 patients taking prophylaxis and 26 of 32 not taking prophylaxis. median tto of first episode of meningococcal disease was 835 days in prophylaxis recipients versus 333 days in patients not taking prophylaxis. the range of tto was large in both groups (3-1873 days in prophylaxis recipients vs. 8-2247 days in non-prophylaxis recipients). among patients with susceptibility results available, penicillin nonsusceptibility was reported more frequently in patients taking prophylaxis (5 of 6 isolates, 83%) than in patients not taking prophylaxis (2 of 9 isolates, 22%). with the limited data available in both the faers reports and published reports, it is not possible to determine whether antibiotic prophylaxis is effective in preventing meningococcal disease in eculizumab recipients. however, in this descriptive analysis we observed a prolonged tto of first meningococcal disease episode and a higher frequency of reduced penicillin susceptibility among prophylaxis users compared to non-prophylaxis users (all previously vaccinated). although these results suggest that antibiotic prophylaxis may delay tto, prescribers should interpret these findings conservatively, particularly given the wide range of tto and the substantial differences in patient age and country of residence between prophylaxis recipients and non-prophylaxis recipients. prescribers must weigh the potential risks of antibiotic prophylaxis, such as adverse events and antibiotic resistance, against the potential benefits. when considering the benefit-risk balance of prophylaxis use, the more frequent reports of reduced penicillin susceptibility among prophylaxis users deserve comment. if this is a true relationship, colonization by meningococcal isolates with reduced penicillin susceptibility may have precluded penicillin from preventing meningococcal disease. reduced penicillin susceptibility could be a consequence of selective antibiotic pressure. further complicating use of prophylaxis, eculizumab recipients are also at risk for disseminated neisseria gonorrhoeae , 3 an organism in which penicillin resistance is common. the analysis has several limitations inherent to the data source. cases were derived from spontaneous reports, which are subject to underreporting of outcomes, biased reporting, and variable quality. confounding of the relationship between antibiotic prophylaxis and tto is possible since patient characteristics, including age ( fig. 1 ) and country of residence, differed substantially between groups. ascertainment of prophylaxis exposure was challenging and inconsistent prophylaxis use could reduce differences in tto between groups. other important data deficiencies include missing information on meningococcal antimicrobial susceptibility testing results, methods, and breakpoint criteria. overall, the data are inconclusive. however, we did observe a trend towards prolonged tto among prophylaxis users but also towards increased penicillin non-susceptibility among prophylaxis recipients. validation of these potential associations in a larger sample, with systematic ascertainment of antibiotic exposure, could further elucidate the potential impact of prophylaxis on development of meningococcal disease among eculizumab recipients. healthcare professionals should remain vigilant for signs of meningococcal disease among eculizumab recipients, irrespective of the preventive measures in use. the authors have no potential conflicts of interest to disclose. table 1 characteristics of patients taking antibiotic prophylaxis and patients not taking antibiotic prophylaxis. note, one patient in the "prophylaxis group" was 18 years of age with a tto = 53 days, and one patient in the "no prophylaxis group" was also 18 years of age with a tto = 53. these data points overlap on the figure. none. we read with interest the recent report by lam and colleagues in this journal, who compared global rates of respiratory viruses (ref) (1) . we found that 20 respiratory pathogens circulated in beijing, china, and influenza virus, human rhinovirus (hrv) and mycoplasma (mp) were the major pathogens. this information needs to be considered by clinicians when treating patients presenting with influenza-like illness (ili). influenza viruses, other respiratory viruses and bacteria have been detected in patients with ili (2-4). these respiratory viruses and bacteria, including seasonal humans influenza virus a (sflua) and humans influenza virus b (sflub), human coronavirus (hcov-oc43,hcov-nl63,hcov-hku1 and hcov-229e), para-influenza virus (piv 1-4), adenovirus (adv), enterovirus (ev), hrv, respiratory syncytial virus (rsv), boca virus (bov), human metapneumovirus (hmpv), chlamydia (cm) and mp are well recognized. patients infected by these pathogens exhibit highly similar symptoms, rendering a clinical diagnosis unreliable and limiting aetiological and epidemiological studies. 2 elucidating the epidemiological characteristics and regularity of ili pathogens is of great significance in guiding clinical diagnoses and avoiding the abuse of antibiotics. we collected 6327 throat swabs. of the 6327 (female 2782 vs. male 3545) outpatients who sought treatment in 2016 to 2018, 1886 outpatients were confirmed as pathogen positive. in this study, we studied only ili outpatients who were single pathogen positive. overall, the most frequently detected agents were h3n2 (530/6327, 8.38%), h1n1 (285/6327, 4.50%), hrv (216/6327, 3.41%), and b-yamagata (195/6327, 3.08%). ( fig. 1 and table 1 ). in short, the positive rate of each year was basically the same as the total positive rate. the top three pathogens were mainly influenza viruses. influenza and other pathogens were present for most of the 3 years in this study. however, the same influenza subtype did not persist as the dominant strain; rather, a mixture of high-intensity peaks of single subtypes and the co-circulation of types and subtypes at variable intensities occurred. (fig. s1-3) . this was consistent with other ili surveillance data that demonstrated asynchronous peaks and the co-circulation of different pathogens. 5 of the 6327 ili, the overall prevalence of influenza was 18.90% (1196/6327). relatively low detection rates have even been reported in studies conducted in other geographical areas, such as gansu province in china, 6 the discrepancies in the influenza detection rates among patients with ili from different areas highlighted the geographical differences in virus burdens. however, these geographical differences in the detection rate may have been affected by several other factors, such as different technical approaches, sampling periods, study durations, or target populations (global population, paediatric population, etc.). 1 , 3 a pearson correlation analysis was performed considering the numbers of influenza-positive cases and non-influenza cases; the result showed that the former was not correlated with the latter (rs = −0.707, p < 0.05) (fig. s4) . moreover, among the noninfluenza respiratory pathogens, hrv and mp maintained high positivity levels, and the rsv infection rate increased annually ( table 1 ) . the results of this study are similar to those of ili in ho chi minh city 5 and zhuhai city. 2 it was hrv that had a detection rate of 3.41% (216/6327) in this study; hrv had a great impact on persons under 60 years old and was a major viral pathogen of ili during the study period (table 1 ). in many regions of the world, there have been reports of outbreaks caused by hrv, such as the uk (2009-2017) 8 and vietnam (2013-2015), 5 at 2.14% and 8.8%, respectively. our study showed that hrv infection occurred predominantly in april, july, and august, with the majority of cases in the 5-24 years age group ( table 1 ) . the virus was also highly associated with influenza and other pathogens. respiratory symptoms caused by hrv are complicated, easily leading to a misdiagnosis and delayed treatment. 9 in the future, it is necessary to strengthen the monitoring of hrv in ili patients, provide data support for the early warning and prevention of relevant epidemic situations, and avoid large-scale epidemics. this study also found that mp accounted for a large proportion of the pathogen spectrum of ili in beijing, with a detection rate of 2.01%. mp is a common pathogen associated with human respiratory infections that can cause endemic or even global outbreaks among people of all ages. 10 however, there has been few studies on mp in routine surveillance at home and abroad, and the inclusion of these additional pathogens in ili studies might greatly increase the positive detection frequency (2) . therefore, in an early epidemic of unexplained fever, it is necessary to be alert to the possibility of mp infection, especially when the epidemic occurs in a closed environment. the strength of this study was that there were no previous reports on the detection of these 20 pathogens, especially mp and cm, in ili patients during the influenza epidemic in beijing. our study showed that the data from this study people were important reference data, and that understanding the interactions between the different influenza subtypes and types and other respiratory pathogens is critical. in addition, we must improve prevention and management strategies for ili. these results demonstrate that a wide range of respiratory pathogens are circulating in beijing city and that h3n2, h1n1, b-yamagata, b-victoria, hrv and mp are the major pathogens. it is recommended that the trend of pathogen spectrum changes in patients with ili in the region should be continuously monitored. at the same time, the health department should also strengthen the analysis and utilization of monitoring data and track the activity level and variation in influenza virus rates to address fever outbreaks in a timely manner. none. this work was supported by the chn beijing chaoyang district science and technology plan project (grant no. cysf1721) and the national major science and technology project for control and prevention of major infectious diseases in china (2017zx10103004). the funder of the study had no role in study design, data collection, data analysis, data interpretation, or writing of the report. we are very grateful to the patients, clinical and laboratory staff at the two hospitals concerned and the numerous support staff in chinese academy of medical sciences and peking union medical college in china. all authors declare no competing interest. we read with great interest the report of the evaluating the use of apheresis for severe falciparum malaria, loiasis or babesiosis in a systemic review. 1 in this study, 1 odedra et al. demonstrated that suggests, that apheresis may be a useful adjunct in the treatment of babesiosis, and loiasis. in addition to these uncommon infectious diseases, we are much more concerning the efficacy of another modality -polymyxin b hemoperfusion (pmx-hp), which can reduce blood endotoxin levels in sepsis, on the clinical outcome of patients with sepsis and septic shock. therefore, we conducted a meta-analysis to investigate the clinical efficacy of pmx-hp on the mortality of patients with sepsis and septic shock. from the literature review using pubmed database, eight randomized clinical studies 2-9 compared the use of pmx-hp with standard therapy on the mortality of patients with sepsis and septic shock were identified. table 1 summarized the characteristics of these included eight randomized trials. [2] [3] [4] [5] [6] [7] [8] [9] two studies 4 , 9 were multinational study and japan was the most common country as study site. six trials were multicenter studies and two trials were single center study. in addition to nakamura et al.'s study, 5 all the other study initiated pmx-hp from the first day. finally, the duration and the uses of pmx-hp varied according to each study design. overall, a total 993 patients were enrolled. five hundred and ten and 483 patients were assigned to pmx-hp group and standard therapy group, respectively. those received pmx-hp and standard therapy had 28-day mortality rate of 36.3% (185/509) and 40.3% (195/483), respectively. no significant difference was observed between these two groups (risk ratio 0.76, 95% ci, 0.56-1.05, i 2 = 69%). in subgroup analysis of two studies which performed pmx-hp for four hours per session, 6 , 8 the patients receiving pmx-hp had lower mortality rate with 48.7% (38/78) than standard therapy with 83.8% (57/68) (risk ratio 0.51, 95% ci, 0.25-1.05, i 2 = 73%), but the difference did not reach statistical significance. other six studies 2-5 , 7 , 9 evaluating the effect of pmx-hp for two hours per secession also showed that there was no significant difference in mortality among those received pmx-hp and those received therapy (risk ratio 0.90, 95% ci, 0.65-1.25, i 2 = 51%). in conclusion, based on the findings of this meta-analysis, it indicated that the survival benefit does not justify the routine use of pmx-hp to treat patients with sepsis and septic shock. none. the asf-affected country name and the time when the first outbreak of asf was determined in that country are indicated. " * " demonstrates the total asf outbreaks / total animal losses in that country. for russia, only its geographical area within asia is shown. contagious hemorrhagic disease of domestic pigs with a high morbidity and mortality. however, there are currently no effective vaccines to prevent and control this disease. the causative agent of asf is asf virus (asfv), which was described and isolated for the first time in kenya in 1921. 3 asfv is endemic in most countries in sub-saharan africa and sardinia. after 1957, this virus spread into european and american countries, and recently, it spread to asia. in asia, except for russia, no countries had experienced asf outbreaks before 2018. in august 2018, china reported its first asf outbreak. 4 in one year, asfv quickly spread into all of the provinces in mainland china. at the time we submitted this manuscript (november 12, 2019), a total of 164 asf outbreaks in china had been recorded by oie ( fig. 1 ) ( https://www.oie. int/en/animal-health-in-the-world/information-on-aquatic-andterrestrial-animal-diseases/african-swine-fever/reports-on-asf/ ). in 2019, after mongolia reported its first outbreak in january, asf rapidly spread into nine other asian countries over the next eight months, including vietnam (february), cambodia (march), north korea (may), laos (june), the philippines (july), myanmar (august), russia (august), south korea (september), and timor-leste (september). as of now, a total of 6647 asf outbreaks affecting 4,306,568 animals have been reported in asia, resulting in great economic losses ( fig. 1 ) . moreover, the emergence and rapid spread of asfv in asia also poses a threat to unaffected countries. asf can be transmitted directly through contact between sick and healthy pigs or indirectly through untreated swill or other feed products. asia raises a large number of pigs, and pork is the main meat source for local people. among asian countries, china is responsible for about half of the global pig population. therefore, asia has a high pig density. it should be noted that backyard and small-scale farms with weak biosecurity systems comprise a very large proportion of pig herds in asian countries with a developing swine industry, such as in the as of yet asf-unaffected asian countries indonesia, india, and malaysia. asfv is stable and infectious for a long period of time in blood, feces, tissue, and other viral contaminated products. however, it is common to feed pigs untreated swill in asia. therefore, food is also a potential source for asf spread. shortly after the first asf outbreak in china, asfv was found in dried pig blood used in pig feed, which is an important protein source for pigs in china. the developing swine industry with a high pig density but low biosecurity systems in asia provides more opportunity for contact between healthy pigs and asfv-affected pigs/ contaminants and may contribute to the spread of asf in asia in the past/future. luggage/pork products/waste from aircrafts/vessels/passengers from asf-affected countries represent another important route for asf spread. japan and south korea reported that asfv was detected in pork products from china in april and august 2019, respectively. ( https://www.nippon.com/en/news/yjj2019040200923/ infectious-african-swine-fever-virus-found-in-japan-for-1st-time. html ). with the development of the global economy as a whole, international economic cooperation and exchange are increasingly frequent. countries with a developing/developed pig industry should implement enhanced national sanitary measures to mon-itor quarantine inspections of travelers from asf-affected asian countries. soft ticks of the genus ornithodoros are considered to be reservoirs of asfv and are widely distributed in asia. 5 , 6 several asfv epidemiological cycles involving these ticks have been identified. 7 surprisingly, a new asfv variant strain was recently identified in hard ticks ( dermacentor ) collected from sheep and bovines in china, expanding the vectors and hosts for asfv. 8 wild boar is also susceptible to asfv and plays an important role in asf persistence in endemic areas or in sporadic outbreaks. having no restricted movement area, wild boar is regarded as a source for further the geographic expansion of asf. 9 in november 2018, asfv was found in a dead wild boar at the border between china and north korea. 10 although there have been no further reports of asfv in wild boar in asia since then, considering wide geographical distribution of wild boar in both asf-affected andunaffected asian countries, its potential role in asf spread into unaffected counties should not be underestimated. it should be noted that the latest asf outbreak in asia occurred in timor-leste ( fig. 1 ) . timor-leste has a long geographical distance from the nearest asf-affected asian country, the philippines ( ∼ 2628 km), and is closer to australia ( ∼ 588 km). it is still unclear how asfv spread into this country. however, the asf outbreak in timor-leste is undoubtedly a great threat to pigs in australia. in summary, multiple factors are probably responsible for rapid spread of asfv in asia. to control the continuous spread of asfv in asia and reduce its potential threat to currently unaffected countries, there is a need for the joint updating of national policies and increasing international cooperation between different countries worldwide, such as increasing biosafety awareness, developing a modern pig industry, reducing the density of backyard pigs, strengthening the biosafety management of travelers and their luggage, and developing strategies for controlling related ticks and wild boar. none. recently, hu and colleagues declared in this journal that they had isolated two h3n2 avian influenza viruses (aivs) that were derived from recombination events. 1 aivs pose great challenges for disease control due to their rapid evolution. their viral genomes are comprised of eight negative-strand rna segments, and the lack of a proofreading mechanism during rna replication results in a high frequency of point mutations. in addition to generating genetic diversity by rapid mutation, if multiple aiv strains coinfect a single cell, then the eight segments of the aiv genome can reassort and yield progeny virions with novel combinations of segments, a process termed reassortment. this is a very frequent process. in addition, homologous recombination is an important evolutionary mechanism that drives the formation of genetic variation for viruses that allows them to overcome selective pressures and adapt to new environments and hosts. 2 , 3 however, since aiv rna is always encapsidated by a ribonucleoprotein complex, recombination in these viruses is very rare, 4-7 with some of the previously described putative recombination events proving to be false-positive signals. 8 thus, the two h3n2 recombination events suggested by the hu et al. study 1 are unexpected. to examine these potential recombination events in more detail, we collected all available complete avian h3-ha and n2-na aiv sequences from the public ncbi ( https://www.ncbi.nlm.nih. gov ), and gisaid ( https://www.gisaid.org ) databases. redundant sequences and laboratory strains were removed. finally, 2270 and 2883 unique h3-ha and n2-na, respectively, sequences were used for our recombination analyses. a number of statistical methods have been developed to detect recombination in sequences, with each of the different methods having distinct performance characteristics and efficiencies. 9 therefore, we performed recombination using the rdp4 program for the 3seq, bootscan, chimaera, genecov, lard, maxchi, rdp and siscan detection methods. 10 to avoid false positives, we recorded a recombination event only if it was detected to have a significant signal by at least three different methods. using this approach, we detected three h3-ha sequences and seven n2-na sequences that had strong signals for recombination ( table 1 ) , however, these sequences did not include the two h3n2 viruses (a/duck/guangdong/f138/2017 (h3n2) and a/duck/guangdong/f352/2018 (h3n2)) that were suggested to have recombination signals by hu et al. 1 to determine whether our strict criteria lead to the conflict with the result of hu et al., 1 we reanalyzed the data used by hu et al. for potential recombination signals with the simplot program 11 used in their study. our simplot graphs, which are the same as those in hu et al., 1 clearly show a high degree of similarity within the ha and na sequences ( figs. 1 a, b and 2 a, b) . we further used bootscan analyses to control the false positive signals. signals of greater than 70% of the observed permuted trees indicate potential recombination events. however, the bootscan analyses demonstrate that there are no significant signals of recombination in these sequences ( figs. 1 c, d and 2 c, d) . this suggests that hu et al. 1 did not use a bootscan analysis to avoid detecting false positive signals, and thus reached an incorrect conclusion that f352 and f138 h3n2 aivs were derived from recombination. 1 examination of the variable sites in the sequences of the ha and na genes of f138 and f352 ( figs. 1 e, f and 2 e, f) further suggest that the recombination regions by hu et al. 1 have only 1-12 variable sites. aivs have very high mutation rate. random mutations can cause false positive signals. in conclusion, we described an approach for the detection of recombination breakpoints in h3n2 aivs and show that homologous recombination is very rare. when bioinformatic analyses are used to detect the presence of recombination, an assessment of the nonrandomness of the signals is needed, and multiple methods must be used to avoid false positive results. the authors declare not conflict of interest. dear editor, diagnostic performance of the xpert mtb/rif ultra (xpert ultra) in comparison to xpert mtb/rif (xpert) assay for the detection of paediatric pulmonary tuberculosis has been documented in this journal. 1 end tuberculosis (tb) strategy aims to achieve 90% reduction in incidence and 95% reduction in mortality by 2035; obviously sensitive, rapid, accessible diagnostics is the most important factor to overcome tb which is one of the oldest and deadliest diseases of the mankind. 2 sputum smear microscopy is inexpensive, easy to perform and still the primary method for diagnosis of tb although maximum sensitivity has been found around 60% under optimal conditions. 3 cultivation and antimicrobial susceptibility are still considered as gold standard, however due to lack of access to mycobacteriology laboratory facilities point of care tests are required for early diagnosis and to prevent dissemination of drug resistance strains all over the world. xpert mtb/rif assay (cepheid, sunnyvale, ca, usa) is the most commonly used point-of-care assay for tuberculosis (tb) that was endorsed by who in december 2010. in 2014, this recommendation is expanded for use in all patients. since the end of march 2017, who has recommended the replacement of xpert by ultra (ultra, cepheid, sunnyvale, california). this advanced version has better tb detection capabilities and more definitive identification of rifampicin resistance especially important in problematic cases such as hiv coinfection, pediatric patients and extra pulmonary tb cases. 4 that increased sensitivity may predispose to false-positive results due to sample cross contamination, particularly in laboratories with heavy work load. 4 , 5 we have replaced xpert mtb/rif assay to ultra assay in december in our hospital that is localized in istanbul, turkey where 17 million inhabitants live. incidence of tuberculosis (per 10 0,0 0 0 people) in turkey was reported at 18 in 2015. we have processed 675 clinical materials from 515 patients by smear microscopy and culture and by xpert ultra assay between december 2018-may 2019. specimens were first digested with n-acetyl-lcysteine and sodium hydroxide and concentrated using standard methods. smear microscopy was done using ziehl-neelsen and auraminerhodamine staining . 0 ·5 ml of the resuspended pellet was inoculated into liquid culture using mycobacteria growth indicator tube (mgit) with a bactec 960 instrument (bd microbiology systems, sparks, md, usa), and 0 ·2 ml was inoculated on löwenstein-jensen medium. cultures positive for growth of acid-fast bacilli underwent confirmation of m. tuberculosis complex by mpt6 4/mpb6 4 antigen detection. xpert ultra assay was done by adding sample reagent to the first collected sputum specimen in a 2:1 dilution, and 2 ·0 ml of the resulting mixture was added to xpert ultra cartridge. respiratory specimens (sputum, brochoalveolar lavage, deep tracheal aspiration, gastric aspiration) was consisted 83.9% of the specimens where as 16.1% specimens were csf and urine ( table 1 ) . 217 (42, 1%) of the patients were women and mean age was 46, 33 years (1 month, 94 years of age) although 33 (4.9%) samples were pcr positive, we could not isolate m. tuberculosis from 11 samples. when repeated samples are excluded we analyzed 26 patients. six patients (3 trace positive and 3 very low positive) were from bronchoscopy unit and we reoverall 675 (100,0) 33 (4, 9) alized that positive results were detected following bronchoscopy of high positive patient. all of the patients were smear negative and clinician who performed the procedure was very anxious and wanted to know if they are false positive or not. we convinced her to wait for the results and we screened the unit by using clean-trace surface atp and clean-trace water atp tests (3 m, usa) and also obtained samples from bronchoscopes and the environment for smear examination and culture. 6 in our unit automated washing machines are used and high level disinfection is done by orthophthalaldehyde (opa). atp levels were in acceptable limits and mycobacteria is not detected in those samples. five of 6 patients remained culture negative and therapy was not given. among other culture negative patients, 3 of them were trace positive and we have learned that they have had tb treatment previously. in the other two patients, low level pcr positivitiy in bal samples was supported by clinical and radiological findings therefore they have received tb treatment. dorman et al. 5 enrolled 2368 participants for sputum sampling and the study was done at ten reference laboratories in eight countries (south africa, uganda, kenya, india, china, georgia, belarus, and brazil). the sensitivity of expert ultra was 63% for the 137 participants with smear-negative and culture-positive sputum. 19 (44%) of 43 participants with a positive xpert ultra test but no positive culture had trace and only 2 had m. tuberculosis identified on a follow-up culture . these results are in line with other studies show that xpert-positive, culture negative results were more common in individuals with a history of tuberculosis. 7 , 8 ultra provides a category which is defined as 'trace' that does not exist with xpert, corresponds to specimens positive for the pcr targeting the multicopy genes is6110 and is1810 and negative for the pcr targeting the single copy gene rpob. 4 positive trace result is related with the presence of a very low quantity of m. tuberculosis dna and should be evaluated carefully. inconsistent culture and pcr results is a big challenge for clinicians. false positive results can be related with amplification of dead bacilli from environmental contamination or previous tb treatment. 5 , 7 false positivity is related with endoscopic procedure in our study and despite high level disinfection the surface of the bronchoscope remained positive for mycobacterium dna. although the number of clinical samples is not high, every patient counts and we have had difficult time to evaluate our results to guide clinicians. arend et al., metaphorically speaking de-fines, xpert as a knife and xpert ultra is a sharper knife and hopes to hear additional studies and algorithms that assess xpert ultra's improved sensitivity and solutions to avoid the trap of treating patients for dead bacilli. 9 so far, it is for the best to combine pcr results with 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general station of forest and grassland pest management, national forestry and grassland administration continuous adaptation of the ha and na gene of h3n2 subtypes of avian influenza virus in south china recombination in viruses: mechanisms, methods of study, and evolutionary consequences mechanisms and consequences of positive-strand rna virus recombination genetics of influenza viruses phylogenetic analysis reveals a low rate of homologous recombination in negative-sense rna viruses homologous recombination is very rare or absent in human influenza a virus guidelines for identifying homologous recombination events in influenza a virus questioning the evidence for genetic recombination in the 1918 "spanish flu" virus evaluation of methods for detecting recombination from dna sequences: empirical data rdp4: detection and analysis of recombination patterns in virus genomes full-length human immunodeficiency virus type 1 genomes from subtype c-infected seroconverters in india, with evidence of intersubtype recombination xpert mtb/rif ultra assay for the diagnosis of pulmonary tuberculosis in children: a multicentre comparative accuracy study who's new end tb strategy microscopy as a diagnostic tool in pulmonary tuberculosis the new xpert mtb/rif ultra: improving detection of mycobacterium tuberculosis and resistance to rifampin in an assay suitable for point-of-care testing xpert mtb/rif ultra for detection of mycobacterium tuberculosis and rifampicin resistance: a prospective multicentre diagnostic accuracy study validation of adenosine triphosphate to audit cleaning of flexible endoscope channels xpert mtb/rif results in patients with previous tuberculosis: can we distinguish true from false positive results the good, the bad and the ugly of the next-generation xpert mtb/rif(®) ultra test for tuberculosis diagnosis performance of xpert mtb/rif ultra: a matter of dead or alive this work was supported by the national natural science foundation of china (grant no. 31822056 ), fund for the key program and creative research group of the department of education of guangdong province, and the 111 project (d20 0 08). this work was supported by the national natural science foundation of china ( 31702271 ), the guangdong provincial natural science foundation ( 2017a030310367 ), and donkey innovation team project of modern agricultural product technology system in shandong province (sdait-27). we thank amy blain for assistance matching faers reports with cdc meningococcal disease surveillance data. we would like to acknowledge the ministry of health for its support and all the sentinel site healthcare workers. supplementary material associated with this article can be found, in the online version, at doi: 10.1016/j.jinf.2019.11.014 . key: cord-336591-a31oe7kc authors: wong, martin; huang, junjie; teoh, jeremy; wong, sunny title: evaluation on different non-pharmaceutical interventions during covid-19 pandemic: an analysis of 139 countries date: 2020-06-21 journal: j infect doi: 10.1016/j.jinf.2020.06.044 sha: doc_id: 336591 cord_uid: a31oe7kc nan effectiveness to mitigate the covid-19 pandemic is yet to be testified in real-life settings and in different countries. we noticed an opportunity to examine the impact of these npis by a recently released index. the university of oxford has developed an oxford covid-19 government response tracker (oxcgrt) to offer a systematic way to follow the stringency of government responses to the pandemic across countries and time [2] . the stringency index consists of school closure (c1); workplace closure (c2); public event cancellation (c3); restrictions on gathering size (c4); public transport closure (c5); staying at home requirements (c6); restrictions on internal movement (c7); restrictions on international travel (c8); and public information campaigns (h1). we examined if the stringency of these containment measures could potentially reduce the number of confirmed infections. we extracted the rate of increase in cumulative incidence for each country between 15 april to 30 april, 2020 from the covid-19 data repository of the johns hopkins centre for systems science and engineering [3] . we computed the average of all stringency indices for each nation on or before 31 march 2020. a 14-day window period was applied between the closing date of the stringency index and the starting date of incidence change by making reference to recent literature [4] , and was determined by a panel of epidemiologists, physicians and public health practitioners. a linear regression model was constructed to examine the association between average stringency index and increase in incidence of covid-19 cases as the outcome variable. we controlled for the gross domestic product (gdp) [5] and the population density of each country as potential confounders [6] . the distribution of the government response stringency index in various countries shows its increase over time (01 march, 2020 to 31, march 2020) [7] , probably due to the increase in incidence in this period. in multivariaable regression analysis of data in 139 countries (table 1) , a higher stringency index was significantly associated with lower incidence increase between 15 april to 30 april, 2020 (β coefficient -0.03, 95% c.i. -0.05 to -0.01, p=0.014). three indicators also showed an inverse association with incidence increase, namely "school closing" (β coefficient -0.53, 95% c.i. -1.00 to -0.06, p=0.027), "workplace closing" (β coefficient -0.56, 95% c.i. -1.06 to -0.06, p=0.028) and "public information campaign with public officials urging caution about covid-19" (β coefficient -0.71, 95% c.i. -1.31 to -0.12, p=0.028). there are no interactions or multicollinearity detected among the predictors. the findings of this study showed that more stringent containment and control measures could potentially lead to better covid-19 pandemic control. in particular, closure of schools and workplace was found to be influential in mitigation of the disease. stopping schools and workplace attendance involves a substantial number of students and employees, and this could represent a significant containment measure that exerted material effects on the disease incidence. in addition, public information campaign urging caution against covid-19 was reported to be effective. this highlights the importance of communication among various stakeholders in the community during a pandemic [8] . one limitation of the study included the absence of control for some cofounders like personal hygienic measures, testing capability [9] and the government's public health resources [10] . also, our results represent preliminary findings that should be further examined by large-scale confirmatory studies. we recommend future evaluations to explore the effectiveness of these containment strategies in relation to different global health capacities in different countries. we declared no conflict of interests. none. the study was approved by the survey and behavioral research ethics committee of the chinese university of hong kong (sbre-19-592). modelling sars-cov2 spread in london: approaches to lift the lockdown variation in government responses to covid-19 the 2019 novel coronavirus covid-19 (2019-ncov) data repository by johns hopkins centre for systems science and engineering (csse). available at the incubation period of coronavirus disease covid-19) from publicly reported confirmed cases: estimation and application countries by density by population 2020 coronavirus government response tracker nonpharmaceutical interventions implemented by us cities during the 1918-1919 influenza pandemic strengthening early testing and surveillance of covid-19 to enhance identification of asymptomatic patients the potential impact of vulnerability and coping capacity on the pandemic control of covid-19 key: cord-338333-yvm3d6xy authors: tu, danna; shu, junhua; wu, xiaoli; li, heng; xia, zhi; zhang, yanfang; fang, yaohui; shen, shu; guan, wuxiang; wang, hualin; huang, zhaoxuan; wang, guirong; zhou, xiaoqin; deng, fei title: immunological detection of serum antibodies in pediatric medical workers exposed to varying levels of sars-cov-2 date: 2020-07-25 journal: j infect doi: 10.1016/j.jinf.2020.07.023 sha: doc_id: 338333 cord_uid: yvm3d6xy • pediatric healthcare workers are at risk for sars-cov-2 transmission from children and aerosols increase sars-cov-2 infection rate. • elisa and dual-target fluorescence accurately detect sars-cov-2 antibodies indicated that antibody detection should be considered as an auxiliary diagnosis of covid-19. • antibody positive subjects tested negative for sars-cov-2 neutralizing antibodies and sars-cov-2 antibodies diminish to near undetectable levels within two months. highlights pediatric healthcare workers are at risk for sars-cov-2 transmission from children and aerosols increase sars-cov-2 infection rate. elisa and dual-target fluorescence accurately detect sars-cov-2 antibodies indicated that antibody detection should be considered as an auxiliary diagnosis of covid-19. antibody positive subjects tested negative for sars-cov-2 neutralizing antibodies and sars-cov-2 antibodies diminish to near undetectable levels within two months. dear editor: since the initial reports of covid-19 disease outbreak in wuhan, china, it has continued to spread rapidly with cases identified in virtually all countries, worldwide [1] . the population is generally susceptible to sars-cov-2, including children and pregnant women, and medical staffs are a high-risk population for this disease. in this journal, chen et al. have reported the high sars-cov-2 antibody prevalence among healthcare workers exposed to covid-19 patients [2] . here we would like to share our finding about the serum antibodies analyzed in a special group of pediatric medical workers exposed to varying levels of sars-cov-2 after wuhan severe epidemic of covid-19. a preliminary study suggests children can be infected with sars-cov-2 like adults but are less likely to be symptomatic or develop severe symptoms [3, 4] . the asymptomatic or mildly symptomatic children might transmit the disease [5]. therefore they are tested for sars-cov-2 less often than adults, leading to an underestimate of the true numbers of children infected [6] . laboratory tests play a pivotal role in the diagnosis and management of covid-19; the current gold standard being real-time reverse transcription polymerase chain reaction (rrt-pcr) on respiratory tract specimens [7] . the measurement of specific covid-19 antibodies (both igg and igm) should serve as an additional, non-invasive tool for disease detection and management, especially in patients who present late, with a low viral load. due to the high infection rate of medical workers and the uncertainty of child-to-person transmission, we chose a special group of pediatric medical workers as the research subjects to investigate their infection status with sars-cov-2 and analyze possible causes. this study also helps clarify the potential of different immunological techniques for antibody detection as an auxiliary diagnosis of covid-19. on march 19-20, 2020, pediatric medical workers (n = 325) in one hospital but not the designated hospital for covid-19 in wuhan were recruited. they were divided into three groups depends on their level of contact with confirmed and/or suspected covid-19 cases during the outbreak: i. close contact group (contact with confirmed and/or suspected cases of covid-19), ii. non-close contact group (contact only with non-covid-19 patients), and iii. non-contact group (no contact with any patients). three different immunological detection methods were used to measure sars-cov-2 serum antibodies: colloidal gold-based detection, enzyme-linked immunosorbent assay (elisa), and dual-target immuno-fluorescence assay (dtfa) (details in the supplementary methods). the overall positive rate for sars-cov-2 igg and igm antibodies in the pediatric medical workers was 43.08 and 5.85%, respectively. for the close contact, non-close contact, and non-contact groups, respectively, the dtfa positive rates for igg were 41.36, 14.68, and 12.50% (p < 0.05), and the elisa positive rates for igg were 34.55, 10.91, and 4.17% (p < 0.05) and 8.38, 0.91, and 0% for igm (p < 0.05). colloidal gold detection results were negative for igg and only two participants tested positive for igm, both in the close contact group. it suggests the colloidal gold detection kit used in this research is not sensitive enough to be useful in accurate antibody detection, whereas the dtfa and elisa positive rate performed similarly. we further conducted a multivariate logistic regression analysis using antibody results as the independent variables to investigative the relationship of positive serum antibody results, with the performance of aerosol procedures, exposure levels to covid-19 cases, clinical symptoms (including fever, cough, headache, stuffy nose, runny nose, sneezing, pharyngalgia, diarrhea, fatigue, etc.), chest ct imaging changes, and age of participant ( table 2 ). the results showed that although we cannot clearly track antibody kinetics for asymptomatic infections, we can observe that the majority of participants with positive igg antibodies had a significant decline in antibody levels after one month. that means the sars-cov-2 antibodies diminish to near undetectable levels within two months. this research revealed that pediatric medical workers are a high-risk group for infection by sars-cov-2, and the higher the exposure levels to covid-19 patients and aerosol production, the greater chance of being infected. meanwhile, we found that pediatric workers had lower levels of igg antibodies than patients with covid-19 [2] . children-to-person transmission is almost inevitable, but pediatric medical workers often have no or only mild clinical symptoms and also cannot produce enough antibodies to neutralize the virus. the antibody protection that healthcare workers obtained after infection by sars-cov-2 in this study, could not be maintained for a long time and no nabs were detected to provide them with sufficient protection. none. table 1 test results of serum antibodies in pediatric medical workers exposed to different levels of sars-cov-2 from containment to mitigation of covid-19 in the us online ahead of print high sars-cov-2 antibody prevalence among healthcare workers exposed to covid-19 patients retracted: clinical and epidemiological characteristics of 34 children with 2019 novel coronavirus infection in shenzhen online ahead of print diagnosis and treatment recommendations for pediatric respiratory infection caused by the 2019 novel coronavirus coronavirus infections in children including covid-19: an overview of the epidemiology, clinical features, diagnosis, treatment and prevention options in children the novel coronavirus (2019-ncov) outbreak: think the unthinkable and be prepared to face the challenge. diagnosis (berl) key: cord-346967-br4kpm6i authors: bongiovanni, marco; zago, tiziano title: acute hepatitis caused by asymptomatic covid-19 infection date: 2020-09-03 journal: j infect doi: 10.1016/j.jinf.2020.09.001 sha: doc_id: 346967 cord_uid: br4kpm6i nan coronavirus disease (covid-19) is a novel enveloped rna beta-coronavirus that emerged from wuhan, china, in december 2019 and rapidly spread across europe, usa and south america, becoming a worldwide pandemic affecting more than 25 million people (1) (2) (3) . the most common clinical presentation included mainly respiratory symptoms such as shortness of breath, dyspnea, fever and cough, associated with radiological findings consistent with interstitial pneumonia (4) . at the moment, there are no antiviral drugs of proven efficacy against covid-19 and supportive therapy is the only method for the management of symptomatic subjects, many of whom require mechanical ventilation and other intensive care services. acute liver injury at admission is a quite common finding in subjects affected by covid-19 pneumonia (5) ; although the elevation of aminotransferases is usually mild, it seems associated with disease severity. in particular, it has been demonstrated that sars-cov-2 infection in the liver directly contributes to hepatic impairment in patients with covid-19 pneumonia (6) . a meta-analysis recently published into your journal by kunutsor sk et al. (7) assessed that liver enzyme abnormalities, acute hepatic injuries and hypoproteinaemia are frequent hepatic complication among patients hospitalized for covid-19 pneumonia. further, patients with pre-existing hepatic diseases appear to have worse outcome of covid-19 pneumonia. nevertheless, no data are available on liver enzyme abnormalities in asymptomatic subjects with covid-19 infection. we report here the case of a young woman diagnosed with covid-19 infection in absence of respiratory symptoms, presenting at the admission with significant elevation of liver function tests compatible with acute hepatitis. a 30-years old woman was admitted at the emergency department for mild fever, anosmia and dysgeusia from 10 days. she denied cough, sore throat, shortness of breath, diarrhea, nausea, vomiting, or abdominal pain. her parents and an uncle were diagnosed positive for covid-19 infection in the previous three days. she did not have any chronic disease and she was not taking any drug at the time of admission. a nasopharyngeal swab was promptly done and rt-pcr resulted positive for covid-19 infection. chest x-ray did not show findings compatible with interstitial pneumonia; arterial oxygen saturation was 99% on room air. on presentation, her temperature was 36 °c. there were no cutaneous manifestations, her lung examination was normal, and there was no jaundice, right upper quadrant tenderness, hepatomegaly, or splenomegaly. laboratory results were as follows: ast 1531 iu/l (normal value < 35), alt 893 iu/l (normal < 36), serum bilirubin 1.02 mg/dl (normal < 1.2), alkaline phosphatase 106 iu/l (normal 33-98), inr 1, gammaglutamiltransferase 1276 iu/l (normal < 40), white blood cells 4070 cells/mm 3 (normal 4000-10000), platelets 152000 cells/mm 3 (normal 150000-450000). she denied recent intake of reliever drugs as paracetamol or antibiotics in the previous weeks. the abdominal ultrasound did not show significant abnormalities of liver, gallbladder, kidneys, spleen, pancreas and abdominal vessels. the following serological tests were performed and all were negative: hepatitis a, b, c, e, cytomegalovirus, epstein-barr and respiratory viral panel. blood cultures for bacteria and fungi, and the screening for autoimmune diseases were also negative. she was then treated with infusion of saline solution 0.9% (1500 cc/daily) with progressive reduction of liver abnormalities. in particular, after 3 days laboratory results were: ast 111 iu/l, alt 89 iu/l, alkaline phosphatase 97 iu/l and gammaglutamiltransferase 246 iu/l. no respiratory symptom occurred during follow-up and the patient was discharged after 5 days of hospitalization, in good clinical condition and asymptomatic from both hepatic and respiratory point of view. at our knowledge, this is the first report of covid-19 infection presenting as acute hepatitis in absence of respiratory symptoms. our patient had very mild symptoms related to covid-19 infection and was only tested due to her familiar cluster. other possible causes of liver abnormalities were ruled out, therefore it seems likely that acute hepatitis was directly caused by covid-19. recently, wander et al. (8) described a non-icteric, acute hepatitis in an hiv-infected woman, but their patient developed overt respiratory symptoms in the hours immediately following diagnosis and also had other possible causes of liver tests abnormalities such as the use of concomitant drugs and a fair number of comorbidities. mild-to moderate liver test abnormalities are becoming a frequent finding in subjects admitted to hospital for covid-19 infection. patients with known risk factors for covid-19 infection presenting with acute hepatitis should be rapidly isolated and tested. in our patient, the abnormalities in liver function tests quickly normalized, in absence of specific therapy. the real meaning of liver tests transient alterations has yet to be determined in covid-19 infected subjects. with the future evolution of the pandemic, prospective observations could provide further information on this specific clinical issue. epidemiological and clinical characteristics of 99 cases of 2019 novel coronavirus pneumonia in wuhan, china: a descriptive study transmission of sars-cov-2 infection from an asymptomatic contact in germany first case of 2019 novel coronavirus in the united states clinical features of patients infected with 2019 novel coronavirus in wuhan, china acute liver injury in covid-19: prevalence and association with clinical outcomes in a large us cohort sars-cov-2 infection of the liver directly contributes to hepatic impairment in patients with covid-19 hepatic manifestations and complications of covid-19: a systematic review and metanalysis covid-19 presenting as acute hepatitis key: cord-347280-jpwf55l6 authors: skevaki, chrysanthi; fragkou, paraskevi c.; cheng, chongsheng; xie, min; renz, harald title: laboratory characteristics of patients infected with the novel sars-cov-2 virus date: 2020-06-21 journal: j infect doi: 10.1016/j.jinf.2020.06.039 sha: doc_id: 347280 cord_uid: jpwf55l6 a subgroup of covid-19 patients develop very severe disease with requirement for icu treatment, ventilation, and ecmo therapy. laboratory tests indicate that the immune and clotting system show marked alterations with hyper-activation, hyper-inflammation, cytokine storm development. furthermore, organ-specific biomarkers demonstrate the involvement of cardiac muscle, kidney, and liver dysfunction in many patients. in this article the use of laboratory biomarkers is discussed with regard to their use for diagnosis, disease progression, and risk assessment. although only a minority of covid-19 patients show critical disease progression from moderate to severe stages of the disease including requirement for ventilation and ecmo therapy, this subgroup of covid-19 patients requires particular attention. data collection from several regions of the world including china, europe, and the united states clearly demonstrate that covid-19 is not only a disease of the lung and the airways. many other organ systems are involved and contribute to disease variety and progression. with regard to the immune system, hyper-inflammation together with the development of exorbitant increased cytokine production represents a hallmark of severe patients requiring ventilation. some of these patients develop bacterial superinfections with increased levels of sepsis markers. another important systems which recently caused increased attention is the clotting system. this is particularly highlighted by the detection of increased levels of d-dimers. organ dysfunction has been reported in many patients including the heart (myocardial muscle damage), the kidney, and the liver. laboratory diagnostics play not only an important role in disease diagnosis, but also in assessing progression and severity in these patients. furthermore, laboratory diagnostics allow early detection of organ dysfunction in many cases. moreover, biotests are used to assess an increased mortality risk in severe lethal patients. in this article we summarize the most prominent findings in covid-19 patients and discuss the use of these markers for diagnosis, disease progression, and risk assessment. retrospective analyses from china demonstrated that leukocyte counts were higher among non-survivors compared to recovered patients 1, 2 ; in particular, zhou et al. reported that covid-19 patients who did not survive had a median of 9.8x10 9 /l wbc count compared to 5.2x10 9 /l among those who survived (p<0.0001), although the exact time point of measurement was not defined in their methods. furthermore, another study of 140 hospitalized patients in wuhan, demonstrated significantly higher leukocyte counts among those with severe covid-19 disease, compared to patients with milder infection (p=0.003) 3 . finally, a series from the same center, and possibly overlapped populations with the previous study, reported significantly higher wbc counts upon hospital admission among patients requiring critical care, although median values were within normal range (wbc count median 6.6x10 9 /l for icu vs 4.3x10 9 /l for non-icu admission, p=0.003) 4 . the observed leukocytosis is attributed to an elevation of neutrophils, as the other wbc populations seem to drop in severely ill and eventually fatal covid-19 cases 5 . absolute lymphopenia is commonly observed in patients with covid-19, but pronounced lymphocyte depletion is a cardinal marker of enhanced disease severity and an indicator of imminent death, that has been consistently depicted by almost all currently published reports, coming mainly from china 1-7 . importantly, not only the degree of lymphocyte drop, but also the persistence of low lymphocyte counts throughout the disease course have been associated with critical illness and death 1, 2, 4 . in contrast to previous reports for sars-cov, peripheral blood smears reveal the presence of reactive lymphocytes, including some lymphoplasmacytoids, in the majority of covid-19 patients [7] [8] [9] . severe sars-cov-2 infection depletes all lymphocyte subsets, including cd4+ t cells, cd8+ t cells, b cells and natural killer (nk) cells, but cd4+/ cd8+ ratio is not inverted as seen in other viral infections [10] [11] [12] [13] . not only the absolute numbers of t-cells are reduced, but also receptors suppressing their cytotoxic effects, like the cd94/nkg2a receptor, are up-regulated leading to diminished defense mechanisms against the virus 10 . monocyte, eosinophil and basophil counts are also decreased in covid-19, but the magnitude of this reduction has not been associated with disease severity, in currently published data from chinese centers 3,10,14 . moreover, pro-inflammatory cytokines are known to blunt erythropoiesis 15 . however, aside from one study that found significantly higher frequencies of decreased hemoglobin concentrations among severe (43.6%) and critical cases (37.2%) compared to mild/moderate ones (23.1%) (p<0.001), solid evidence of significant hemoglobin reduction in severe covid-19 has not been consistently reported as yet 5, 11, 16 . in one particular study, lower hemoglobin concentration was associated with increased odds for lack of disease improvement but not death (odds ratio 1.731, p=0.008) 16 . preliminary reports imply that high neutrophil counts and persistently deep lymphocyte nadir counts during hospitalization as well as high neutrophil to lymphocyte ratios (nlr) are indicators of adverse outcomes such as icu admission and death 10 . a retrospective chinese study reported that nlr, along with the sars-cov-2 igg levels, could be used as a simple discriminative tool for severity between covid-19 patients, and further predict the clinical outcome of these patients 14 . however, whether these indices can actually risk stratify patients and predict poor outcomes, most importantly at an early stage of the disease, remains to be addressed and validated in large prospective trials. the regulation of ferritin synthesis is cytokine-controlled 17 only scarce data have contextualized the erythrocyte sedimentation rate (esr) kinetic in patients with covid-19. one study reported that fatal cases had a tendency for higher esr compared to those who recovered (median esr 38.5 vs 28 mm/h) without reporting the statistical significance of the observed difference among the two groups 1 . a similar trend was also depicted for c-reactive protein (crp) concentration by the same study, with median levels being 4-fold higher among non-survivors (median concentration 113 vs 26.2 mg/l) 1 . between severe and non-severe cases, reported crp differences are not that striking (median (iqr): 47.6 mg/l (20.6-87.1) vs 28.7 mg/l (9.5-52.1), p<0.001), but significantly increased frequency of higher concentrations among severe and critical cases compared to mild/moderate ones are nevertheless evident (mild/moderate cases: 50.5%, severe cases: 79.2% and critical cases: 92%, p<0.001) 3, 16 . finally, one chinese study with 663 covid-19 patients reported that higher crp levels are inversely associated with disease improvement (odds ratio 4.697, p<0.0001) 16 . individual studies demonstrate that greater procalcitonin (pct) concentrations (usually ≥0.05 ng/ml) can significantly distinguish between non-severely from severely ill and fatal cases, thus possibly acting as a prognostic marker [2] [3] [4] 6, 18, 19 . however, a meta-analysis found that severe from non-severe covid-19 could be differentiated by a marginally higher pct (by 0.2 ng/ml) 5 . increments of both crp and pct may be associated, not only with the immense inflammatory response, but also with the higher frequency of bacterial superinfections among critically ill covid-19 patients (up to 50% rate among non-survivors) 5 albumin is a negative acute phase reactant whose synthesis is down-regulated by inflammatory cytokines 21 . therefore, it is not surprising that hypoalbuminemia (usually <30 g/l) has been persistently noticed among patients with severe or fatal covid-19 1, 2, 6, 11 . moreover, one study demonstrated that low albumin concentration was associated with lack of disease improvement (odds ratio 2.377, p<0.0001), while hypoalbuminemia was also introduced as a risk factor, among other parameters, in a proposed risk prediction nomogram for severe covid-19 16, 19 . serum amyloid a (saa) is another acute phase reactant inhibiting monocyte mobilization, platelet activation and various chemotactic pathways 21 . high concentrations of saa among all covid-19 patients have only been reported by zang et al., without a significant difference between severe and non-severe cases 3 . exuberant release of pro-inflammatory cytokines is associated with multi-organ injury and acute respiratory distress syndrome (ards), which is inevitably fatal if left untreated 22 . fulminant hypercytokinemia has been increasingly recognized among critically ill covid-19 patients. moreover, the connection of viral spike protein to ace2 receptor, down-regulates ace2 levels in lungs; this in turn, increases the angiotensin ii (angii) levels, reduces angiotensin 1-7 (ang-(1-7) ), and imbalances the renin-angiotensin system in the lung, leading to vasoconstriction 28 . these data are in concordance with a notably distinct type of ards with highly compliant lungs, which is seen in a major subset of covid-19 patients; this manifestation is quite possibly consistent with an underlying vasoconstriction and microvasculature injury leading to loss of lung perfusion regulation 29 . though neither histopathology specimens nor lung ace2 or angii levels are easily obtainable in daily clinical practice, they would definitely be useful in research settings in order to elucidate the disease's pathophysiology and may assist diagnosis in the future. cardiac troponin i and t are highly sensitive and specific biomarkers of myocardial injury which can be caused by myocardial ischemia, inflammation, immune response, and toxin 30 according to a cohort study 34 of 701 patients with covid-19, the proportion of proteinuria, hematuria, abnormal serum creatinine and urea nitrogen at admission and were 43.9, 26.7, 14.4 and 13.1%, respectively. in addition, there was a high prevalence (5.1%) of acute kidney injury (aki) during the study period. the result showed proteinuria, hematuria, and elevated serum creatinine/urea nitrogen at admission and acute kidney injury (aki) during hospitalization over stage 2 were associated with in-hospital death. however, the other largest retrospective study to date found that the prevalence of serum creatinine abnormalities and aki was only 1.6% and 0.5% 18 . this may be due to the different proportions of severe patients between the two studies and the different definitions of the normal reference range for serum creatinine. from the result of autopsy of 26 covid-19 patients 35 , the histopathology of the kidney revealed significant acute tubular injury and found that the tubular epithelial cells were directly infected by sars-cov2. therefore, sars-cov2 may cause kidney injury or exacerbate existing kidney disease. attention should be paid to monitoring renal function and the occurrence of aki. abnormal liver function tests, such as increased levels of alt, ast, tbil, ggt and decreased level of albumin were relatively common in patients with covid-19, and 10-33% of these patients had abnormal alt or ast 2,4,18,31,36 . although patients with severe covid-19 seem to have higher rates of liver dysfunction, it is reassuring that the levels of alt, ast, tbil, ggt in covid-19 patients were not significantly different in compared with hospitalized community-acquired pneumonia patients and even the median or average transaminase level in severe covid-19 patients was lower than twice upper reference limit 4, 36, 37 . therefore, the clinical effect of these elevated indicators may not be evident in covid-19 patients. liver dysfunction may be related to severe infection, inflammation induced liver injury, medication associated hepatotoxicity and hypoxia 38 . d-dimer is a degradation product of fibrin. elevated d-dimer levels were consistently reported in covid-19 patients with prevalence ranging from 43-68% 2, 3, 31 . d-dimer>1ng/ml at admission were associated with increased severity and odds of death with covid-19, and the gradual increasing of d-dimer during disease course was particularly associated with disease worsening and mortality 2, 4 . serum d-dimer can reflect fibrinolytic activities and is also an inflammatory biomarker. furtherly, recent studies found that severe cases of covid-19 were commonly complicated with thrombosis 39, 40 , markedly elevated d-dimer was related to thrombosis and poor prognosis of severe covid-19 patients. of fatal cases 45 . in fact, the fibrinogen would decrease when excessive consumption happened due to hypercoagulability or the worst disseminated intravascular coagulation occurred. hence, the abnormality of the coagulation profile should be interpreted individually. lactate dehydrogenase (ldh) is a cytoplasmic enzyme that is present in every tissue, and high serum concentrations indicate underlying organ damage. thus, ldh is expected to rise in severe covid-19 cases, where multi-organ damage occurs 23 . current data support that critically ill patients as well as fatal cases of covid-19 have significantly higher ldh levels (usually >320 u/l) compared to moderate infections 1, 2, 5, 6, 11, 16 . moreover, higher ldh quadruples the odds for lack of disease improvement (odds ratio: 4.381. p<0.0001) 16 . lastly, greater ldh concentrations upon admission correlate with a higher risk for serious covid-19, and therefore it has been added in a proposed early predictive tool for severe infection 19 . these data favor the utilization of ldh as a candidate prognostic marker for disease severity. hypertriglyceridemia is commonly encountered in hyperinflammatory states, like the css and the secondary hlh, due to the reduced lipoprotein lipase activity driven by the high tnf-α levels 46 bicarbonate concentration in patients who died, without reporting the statistical significance of this finding 1,2 . importantly, but not surprisingly, in the latter study more than 50% of the deceased patients had arterial partial pressure of oxygen (pao2) of <60mmhg (compared to 0% in the survivor group), while none in the same group had a partial pressure of oxygen to fraction of inspired oxygen ratio (pao2:fio2) of >300 1 . hence, arterial blood gases constitute important prognostic tools for disease severity and poor outcomes, as they are directly associated with the degree of functional lung damage. sars-cov-2 infection causes systemic disease, involving multiple organs and systems, including hyperactivation of the immune system, the nervous system and the clotting system. these in turn leading to pathologies in several organs, including the heart, liver and kidneys. in order to stratify patients at risk and to monitor high risk patients at intensive care units, tight laboratory diagnostics provide instrumental information. laboratory tests can be used as prognostic markers for increased risk and mortality. the spectrum of currently available biomarkers is sufficient to fullfill this purpose. a major limitation of available studies is that the time point of sampling/biomarker assessment since onset of symptoms and/or presentation at health care facilities is not clearly mentioned. furthermore, currently, there are no internationally acceptable criteria regarding disease severity, which renders evaluation of data quite subjective, depending on individual study investigations. over the next months and years, with the use of further knowledge on the pathogenesis of sars-cov-2 infections, an even more comprehensive list of suitable biomarkers will be developed. clinical characteristics of 113 deceased patients with coronavirus disease 2019, retrospective study clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china, a retrospective cohort study clinical characteristics of 140 patients infected with sars-cov-2 in wuhan clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china hematologic, biochemical and immune biomarker abnormalities associated with severe illness and mortality in coronavirus disease 2019 (covid-19), a meta-analysis clinical and immunological features of severe and moderate coronavirus disease 2019 hematologic parameters in patients with covid-19 infection haematological parameters in severe acute respiratory syndrome a major outbreak of severe acute respiratory syndrome in hong kong covid-19, immunopathology and its implications for therapy clinical features of patients infected with 2019 novel coronavirus in wuhan, china pathological findings of covid-19 associated with acute respiratory distress syndrome dysregulation of immune response in patients with covid-19 in wuhan, china immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid-19 anemia of inflammation risk factors for disease severity, unimprovement, and mortality of covid-19 patients in wuhan, china hyperferritinemia and inflammation clinical characteristics of coronavirus disease 2019 in china a tool to early predict severe corona virus disease 2019 (covid-19), a multicenter study using the risk nomogram in wuhan and guangdong, china blood biomarkers differentiating viral versus bacterial pneumonia aetiology, a literature review acute phase reactants. statpearls publishing. treasure island (fl) covid-19, consider cytokine storm syndromes and immunosuppression th17 responses in cytokine storm of covid-19, an emerging target of jak2 inhibitor fedratinib high il-6/ifn-γ ratio could be associated with severe disease in covid-19 patients effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 endothelial cell infection and endotheliitis in covid-19. the lancet complement associated microvascular injury and thrombosis in the pathogenesis of severe covid-19 infection, a report of five cases searching therapeutic strategy of new coronavirus pneumonia from angiotensin-converting enzyme 2, the target of covid-19 and sars-cov covid-19 does not lead to a "typical" acute respiratory distress syndrome how is cardiac troponin released from injured myocardium? risk factors for severity and mortality in adult covid-19 inpatients in wuhan cardiovascular implications of fatal outcomes of patients with coronavirus disease 2019 (covid-19) suspected myocardial injury in patients with covid-19, evidence from front-line clinical observation in wuhan, china kidney disease is associated with in-hospital death of patients with covid-19 renal histopathological analysis of 26 postmortem findings of patients with covid-19 in china liver impairment in covid-19 patients, a retrospective analysis of 115 cases from a single center in wuhan city covid-19 and the liver, little cause for concern liver injury in covid-19, management and challenges incidence of thrombotic complications in critically ill icu patients with covid-19 thrombotic events in sars-cov-2 patients, an urgent call for ultrasound screening fibrinogen gene regulation coagulation in sepsis covid-19 with different severity, a multi-center study of clinical features clinical features of 85 fatal cases of covid-19 from a retrospective observational study hemophagocytic lymphohistiocytosis, review of etiologies and management esicm. important announcement of new ssc guidelines -covid-19 pneumonia and the syndrome of inappropriate antidiuretic hormone secretion, don't pour water on the fire mechanism of hyponatremia in community-acquired pneumonia, does b-type natriuretic peptide play a causative role the syndrome of inappropriate antidiuretic hormone secretion, distribution and characterization according to etiologies clinical findings in a group of patients infected with the 2019 novel coronavirus (sars-cov-2) outside of wuhan, china, retrospective case series gm-csf: granulocyte-macrophage colony-stimulating factor, il: interleukin, ip10:interferon gamma-induced protein 10, k: potassium, masp 2: mannose binding lectin associated serine protease 2. mcp1 (ccl2): monocyte chemoattractant protein 1, mip-1α (ccl3): macrophage inflammatory protein 1-alpha), na: sodium,paco2:arterial carbon dioxide partial pressure, pao2: arterial oxygen partial pressure, siadh: syndrome of inappropriate antidiuretic hormone secretion inr, international normalized ratio hs-troponin i, high sensitivity troponin i key: cord-331929-rot2vgso authors: piaserico, stefano; meneguzzo, alberto; messina, francesco title: reply to: interleukin-17: a potential therapeutic target in covid-19 date: 2020-06-29 journal: j infect doi: 10.1016/j.jinf.2020.06.063 sha: doc_id: 331929 cord_uid: rot2vgso nan we read with great interest the paper by mendoza, which suggests the use of anti-il-17 therapy in coronavirus disease 2019 (covid-19) patients 1 . while agreeing on the potential role of anti-il-17 drugs in controlling the excessive production of inflammatory cytokines, we would like to emphasize another possible relevant benefit that could derive from using these treatments in such patients. as observed in the paper by mendoza, il-17 is thought to play a key role in the immune dysregulation observed in covid-19 and in other severe coronavirus-mediated respiratory syndromes, therefore its inhibition has been suggested to determine a better outcome in these patients. 1 basing on these assumptions, the use of the il-17 inhibitor ixekizumab is currently being investigated for the acute phase of covid-19. 3 however, covid-19, besides being associated with acute respiratory distress syndrome (ards), can also determine long-term sequelae. these will likely represent a considerable therapeutic challenge in the upcoming months, chronic lung fibrosis arguably being the most important 2 . il-17 is a cytokine with pleiotropic effects which might enhance the progression towards pulmonary fibrosis in covid-19 patients. il-17 has been shown to exert direct pro-fibrotic effects on fibroblasts isolated from mouse and human lung through the activation of nf-kb, promoting production of tgf-β and expression of αsmooth muscle actin and eventually leading to collagen deposition 5 . moreover, by acting on both epithelial cells and fibroblasts, il-17 promotes epithelial to mesenchymal transition, as well as myofibroblast differentiation and extracellular matrix production, resulting in pulmonary fibrosis 4 . intriguingly, permanent pulmonary damage appears to be more severe in ards patients who have been mechanically ventilated 5 . pulmonary fibrosis in sars patients may be the result of the high inflammatory burden related not only to the anti-viral immune response, but also to ventilatorinduced lung injury (vili). neutrophilic inflammation in response to higher levels of il-8 is a pivotal step in the pathogenesis of vili 5 and higher concentrations of il-6 and tnfα have been reported in the lungs of patients with vili 6 . although il-17 levels have not been evaluated in specific studies, il-17 represents a crucial molecule in il-8-mediated neutrophil recruitment and its interplay with il-6 and tnfα has been largely demonstrated 1 . moreover, il-17 has been suggested to be involved in the endothelial dysfunction and thrombophilia that has been observed in covid-19 7 . in fact, studies performed on both human patients and murine models have shown that il-17 correlates with vascular dysfunction and induces platelet aggregation, facilitating arterial thrombosis 7 . furthermore, il-17 activates endothelial cells, stimulating the production of tissue factor and modulating thrombomodulin expression 7 . in turn, the activation of coagulation might enhance the fibrotic process. in fact, in the bronchoalveolar lavage of interstitial lung disease patients, tissue factor has been shown to be more active than in healthy controls 8 . the pro-coagulative cascade following the activation of tissue factor has been pathogenetically linked to the establishment of pulmonary fibrosis 8 . notably, factor xa and thrombin seem to activate protease-associated receptors which induce myofibroblast differentiation in lung fibroblasts. moreover, anticoagulant therapies have been shown to ameliorate lung fibrosis in both murine models and human patients. 8 therefore, it is conceivable that il-17 inhibition could also contribute to fibrosis prevention in covid-19 by interfering with the coagulative pathways. currently, il-17 inhibitors like secukinumab and il-17 receptor inhibitors like brodalumab are used for the treatment of psoriasis 9 . in psoriatic patients, secukinumab has been demonstrated to decrease serum levels of krebs von der lungen-6 (kl6), a marker of lung fibrosis. 10 furthermore, in a case series of psoriatic patient with concomitant interstitial pneumonia, the inhibition of the il-23/il-17 pathway by ustekinumab, secukinumab or brodalumab correlated with a stabilization or an amelioration of lung fibrosis 9 . in conclusion, il-17 appears to be involved in several processes which might be relevant in the pathogenesis of covid-19-induced pulmonary fibrosis. notably, il-17 could act both directly, by activating fibroblasts, and indirectly, enhancing virus-mediated and ventilator-mediated inflammatory processes. furthermore, il-17 could stimulate fibrogenesis through the activation of pro-coagulative pathways. therefore, we suggest that il-17 inhibitors may be helpful not only for the acute phase of covid-19, but also for the prevention of its long-term fibrotic sequelae. interleukin-17: a potential therapeutic target in covid-19 pulmonary fibrosis and covid-19: the potential role for antifibrotic therapy chinese clinical trial register ministry of health (china) a randomized, blinded, controlled, multicenter clinical trial to evaluate the efficacy and safety of ixekizumab combined with conventional antiviral drugs in patients with novel coronavirus pneumonia il-17 in the lung: the good, the bad, and the ugly overview of ventilator-induced lung injury mechanisms interleukin-6 mediates pulmonary vascular permeability in a two-hit model of ventilator-associated lung injury interleukin-17a (il-17a), a key molecule of innate and adaptive immunity, and its potential involvement in covid-19-related thrombotic and vascular mechanisms correspondence and requests for materials should be addressed to stefano piaserico key: cord-350670-qk76szs0 authors: vrsalovic, mislav; presecki, ana vrsalovic title: cardiac injury and mortality in covid-19: a reappraisal date: 2020-06-30 journal: j infect doi: 10.1016/j.jinf.2020.06.068 sha: doc_id: 350670 cord_uid: qk76szs0 nan ng/ml, and >1 ng/ml), and the troponin cut-off (0.1 ng/ml) was approximately 5-6 times higher compared to other studies included in the meta-analysis. 3 in our meta-analysis, cardiac injury was defined as serum troponin levels above the 99th percentile upper reference limit. kollias et al. pooled the hazard ratios reported in the study, but the method of pooling the data to "study-unique" hr was not explained in the letter. meta-analysis is used to derive a consolidated estimate of individual studies, and inclusion of post hoc pooled estimates (instead of raw data analysis) may result in an increased heterogeneity. moreover, in the study of petrilli et al. a competing risk model for the mortality or hospice outcome was performed. hospital discharge was considered to be a competing risk, since mortality data were limited after that point unless the patient was readmitted into the hospital system. as such, the inclusion of this study in the meta-analysis (i.e. meta-analysis investigated the impact of cardiac injury in terms of mortality) can be questioned. patients. 4 the aforementioned cohort study included both non-severe and severe covid-19 patients. consequently, epidemiological, demographic and clinical characteristics of hospitalized patients with severe and non-severe disease were compared. however, the multivariable cox regression analysis that identified factors associated with death was performed in only 269 severe patients (median age 65 years, interquartile range 54-72 years), and the results cannot be applied to the entire cohort, as was stated in the letter. based on the aforementioned methodological issues we performed an additional sub-analysis that included the two largest studies from wuhan, china, which included 940 patients with covid-19 (table 1) , accounting for the fact that both studies analysed hospitalized cases with severe and critical illness. 4,5 a total of 149 (16%) patients died during in-hospital stay, and positive cardiac troponin was present in 200 (21%) patients. meta-analysis of those studies that reported adjusted hr (using the generic inverse variance method and fixed effects model), showed a significant association between elevated troponin values and mortality (hr = 3.08; 95% ci 1.95 -4.87) (fig. 1) , and no significant heterogeneity between studies was detected (cochran q = 0.44, p = 0.50). in conclusion, troponin positivity is common and has an independent prognostic role in hospitalised severe covid-19 patients. consequently cardiac injury may serve as an additional risk stratification tool in daily clinical setting. the meta-analysis was conducted using the generic inverse variance method, and pooled hr was reported with 95% confidence interval (ci). there was no significant heterogeneity observed across studies (cochran q = 0.44, p = 0.50). cardiac injury and prognosis in covid-19: methodological considerations and updated meta-analysis cardiac troponins predict mortality in patients with covid-19: a meta-analysis of adjusted risk estimates factors associated with hospital admission and critical illness among 5279 people with coronavirus disease 2019 in new york city: prospective cohort study risk factors for severity and mortality in adult covid-19 inpatients in wuhan characteristics and clinical significance of myocardial injury in patients with severe coronavirus disease the authors declare no conflict of interest.funding: this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. key: cord-322451-cwpz4akv authors: hsin, dena hsin-chen; macer, darryl r.j. title: heroes of sars: professional roles and ethics of health care workers date: 2004-07-27 journal: j infect doi: 10.1016/j.jinf.2004.06.005 sha: doc_id: 322451 cord_uid: cwpz4akv objectives. to examine the professional moral duty of health care workers (hcws) in the outbreak of severe acute respiratory syndrome (sars) in 2003. methods. descriptive discussion of media reports, analysis of ethical principles and political decisions discussed in the outbreak, with particular emphasis on the events in mainland china and taiwan. results. there were differences in the way that taiwan and mainland china responded to the sars epidemic, however, both employed techniques of hospital quarantine. after early policy mistakes in both countries hcws were called heroes. the label ‘hero’ may not be appropriate for the average hcw when faced with the sars epidemic, although a number of self-less acts can be found. the label was also politically convenient. conclusions. a middle ground for reasonable expectations from hcw when treating diseases that have serious risk of infection should be expected. while all should act according to the ethic of beneficence not all persons should be expected to be martyrs for society. it was too heavy to be called as a hero; i just do what i should do. -doctor facing sars in taiwan, may 2003. severe acute respiratory syndrome (sars) will go into the medical records as the first new panic disease that has swept international society in the 21st century. although the number of persons who died from the disease is currently less than a thousand, 1 it affected the lives of millions of persons in 2003. we want to discuss the important lessons that it raises for medical professionals-the 'heroes' of sars. the focus on sars was so high in the media that news of sars overshadowed the outbreak of another panic disease, ebola virus, that killed more than 100 persons in march 2003 in congo. the attention paid on sars meant less attention was given to disease outbreaks like ebola. 2 sars is the latest of more than 35 new or reemerged infectious diseases over the last 30 years. the difference was that most people in the world, especially in safe and secure social settings felt protected from ebola virus of africa, and even the global pandemic of hiv seems distant from most people who donned masks to avoid sars. sars infected and killed young and old, healthy and unhealthy, making everyone seem vulnerable. in taiwan, the sars outbreak started from the time a hospital was detected to have a widespread hospital infection. the hospital was sealed as an emergency and patients and staffs were all locked up inside the hospital building to isolate them from outside, to spread the disease. the quarantine order was announced without any warning and preparation, which caused a massive panic. 3 similar quarantine emergencies were reported in other places also. 4 hcws who were placed in working quarantine experienced fear, depression, anxiety, anger and frustration. there will be long-term psychological consequences for some of these persons. this paper discusses some of the ethical lessons of the first sars outbreak in early 2003, with a hope that lessons will be learnt in time for the next outbreak of sars, or similar new diseases that face those working in the field of infectious diseases control. during the international battle against sars one of the features was the proportion of frontline health care workers (hcw) who were infected and who died. according to the data compiled from the who until the 7 august 2003, 1 20% of all persons affected with sars were hcws (1725/8422). the percentage of hcw was highest in canada (43% with 3/41 deaths), and singapore (41%), higher than that reported for mainland china (19%), hong kong (22%) and taiwan (13% with 6/180 deaths). in the early stages of the outbreak, they had all unknowingly treated patients with sars. even for the latter stages in the outbreak in the first half of 2003, there were several hcw who became ill with sars in spite of 'full' precautions. when nurses and doctors see their colleagues being critically ill around them, dying or on ventilators, when just few days ago they seemed so robust and well, they realized the dangers of being a professional. despite the rapid advancement of knowledge with the intense research, and papers appearing in all major medical journals, 5 the threat of the disease to hcw will remain for some time. before a vaccine is made, there will always be a threat of being infected and killed on duty as a hcw. we witnessed a number of hcw who started to think about withdrawing from their post. it is ethically unacceptable to abandon patients. however, beyond the duty of a hcw, should we demand (or even expect) that hcw should be ready to sacrifice their lives for our society in severe pandemics, like the outbreak of sars. as one doctor in taiwan said: 'it too heavy to be called as a hero; i just did what i should'. we understand that even a virtuous doctor or nurse might not be willing to die for a patient. does the accepted norm of responsibility mean they must put their own lives at risk? one important point is that the social function of medical professionals can not be replaced by others. even in times of peace, we should always remember the reality that being a helper of sickness will always present a certain risk of being infected. medical professions have been well-rewarded by the society not only because they are competent in operating medicine but they chose the work of being a 'life saver'. in a number of countries in order to encourage hcw, the government and the public started to give the title of 'hero' to nurses and doctors who are working in the frontline of sars outbreak. on the other hand, some suggested to punish those who were afraid of treating sars patients. in taiwan this included threats of retracting their professional license. there are memories of the aids scars in the 1980s when some hospitals in asia refused to admit patients with hiv. in a 1992 survey by the japan hospital association it was found that 40% of hospitals refused hiv positive patients. 6 currently refusals are not permitted. can someone who makes an involuntary sacrifice be called as a hero? most nurses and doctors actually died from taking care of sars patients involuntarily. except for those on international teams who actively sought out sars patients, most did not choose to do so. in large scale hospital quarantines in beijing and taiwan the hospitals were encircled and no one could leave. many hcw in taiwan thus denied the title of hero. some said the more people call them in this way, the more they fear they are in danger. we could even imagine that the spiritual inspiration of being a 'hero' even lessens the implementation of good clinical precautions. in taiwan four nurses and two doctors sacrificed their life in taking care of sars patients. the numbers for other countries are not clear, but one can expect it to be higher for hong kong and mainland china, based on the proportion of infected hcws quoted above. in taiwan, a doctor died from giving airway intubations to a terminal old woman even with the protection of masks and glove; he was so young to graduate from medical school a few months ago and got married only 4 months before. a nurse died with her 7 month old fetus just because of a short contact with an undiagnosed sars patient in the emergency room. as those scary stories are repeated numerous times among hcws, can medical ethics overcome their emotions? in facing the crisis of sars, health professionals may make efforts to combat the enemy like in the war. however, could we regard the hcw as a soldier in an army, as quoted in a china daily report? 7 when the health staffs or soldiers decided to resign from their job just in the moment of crisis/war, some cultures attempt to remind the persons of their sworn duties to contribute to national security by giving the person a feather (sign of shame). while the ethical ideal of self-less sacrifice of life for curing disease is promoted in the public image and media, discussions with hcw in several countries suggests that being a hero is not what modern medical practice is for some hcws. most hcws in taiwan are working in the commercial hospital, where the hirer pushes them to focus their effort of work on business competition rather than the basic role of helpers to human's health. beside academic achievement, the profit they can make for their institute is the element to promote their position in their profession. it is easy to loss the ideas of being heroes that is part of the intrinsic nature of being doctors or nurses. most modern hospitals there are designed under the intention of attracting people to visit frequently. besides for a commercial hospital, the most effective way to limit the budget is to reduce labour costs. to use part time staffs saves much money from less benefit and salary pay. could we expect nurses who were called when they are needed and paid by working hour to devote themselves to the full professional code and make all efforts when their life is threatened? it is obvious that the feeling of belonging and sense of nobility are essential for a professional worker, the problem is do we really respect those health worker as health profession to supply the components to achieve the sense of a professional. many hcw in modern times have only faced remote fears of death, and it is a shock for many to realize that their own lives are in danger. when they considered that even with necessary precaution; they still had to run a certain amount of risk, their duties of being a wife/husband, mother/father or daughter/son will call them home. although sars was reported to have a relatively low mortality rate, it attacks the young and healthy as well as the old and frail. moreover, this is a totally new disease, we know very little about it. the fear and worry of being infected will always be a shadow to their care. will the public accept a health professional to exercise their right to remain off the job in this critical moment? every person of any profession has their personal role in a family to be a father, mother, spouse and child, in addition to their professional roles. the constitution of most countries respect a person's human rights and ego (beyond the superego) i.e. ego is the basic human nature which should be honored too. part of the love of life that makes a bioethics of an ethical person in ethical theories is self-love, not just love of others. those medical practitioners who stick to their post should be respected; however, those who need to take a break to recover themselves would also be acting within their human rights and what is expected of a reasonable citizen. there are cases recorded where doctors spent weeks continuously battling the disease, and there is need for a proper assessment of how fatigue may have led to mistakes in care for patients and mistakes in precautions of carers. there is a human limit for everyone to cope with. those who battled self-lessly are called exceptions, for example, ye xin, 47, head nurse at the guangdong hospital of traditional chinese medicine, died in march after contracting sars while treating patients infected with the virus. ye, together with nine other nurses, was posthumously awarded the florence nightingale prize by the international committee of the red cross in may for 'courage and dedication in the line of duty'. 8 in carrying out the responsibility of reporting the truth, the media created the sars panic. in most of the infected areas, the government had no control over the media. under the pressure of commercial competition, those narratives reported by media could be exaggerated which cause mass panic and changed the relationships between people. on the contrary there was a lack of information in mainland china especially until 20 april, when the government was attempting to limit panic by controlling the media. however, when the epidemic was revealed, the following month saw panic there also. everywhere society has to pay a price for liberty of the media, and to deal with the result of transparent reporting. there were also rumours spread through the internet that generated fear. 9 there are reports that in china 117 people were charged with spreading 'sars-related rumours', though the exact nature of the types of email they were sending is doubted. 10 however, without media reporting as a way to educate society, the death toll everywhere would have been higher. the media generated fear, stigma and discrimination, but also showed the evil sides of the panics. people who were subjects of discrimination included those working in the hospital or entering and coming back from infected areas, suspected sars patients and their family. even those with very common syndromes of cold (cough, fever, etc.) were psychologically and socially isolated by their friends and relatives. from the narratives reported by media, the mass panic caused by sars has changed the relationships between people. in certain cases by calling health professionals 'heroes' policy makers in government wanted to escape from their guilt of policy mistakes by giving ambiguous honors. governments had to face up to the mass fear that sars created, and any target could be chosen. there were scapegoats in mainland china on the 20 april with the firing of the health minister. in many countries affected by sars, and neighbours like japan, one of the usual targets for blame is foreigners. persons from distant lands have always been blamed in cases of disease. 11 in early april, a hong kong resident came to taiwan to visit his younger brother despite being supposed to be under home isolation in hong kong due to the spread of the disease in his apartment building, amoy garden. his brother was infected by him and it was the first fatal case of sars in taiwan. one woman, who took the same train with him, was highly suspected to be the source of a major hospital outbreak. at first, the doctors were not sensitive about her case since she had no contact history to match the susceptive criteria for sars. thus, she spread sars in the hospital before she was diagnosed. many hospital staff, patients and patients' families were infected at the same time. the hospital was able to detect this spread and sealed its premises entirely without good preparation. four thousand people were locked inside the building to prevent further spread of the disease; and more than 30 000 people were isolated in their homes. this strong measure resulted in a mass panic. but it was too late. the numbers of infected cases increased exponentially. inside china, the people in guangzhou province are blamed. when one of us was in beijing on 19 april, 2003, some experts said 'the people in guangzhou eat anything that moves. it is their fault.' people in hong kong may have blamed the chinese. people in taiwan or canada blamed those from hong kong. people in japan in may blamed a doctor who traveled from taiwan who later came down with sars. all the people they blamed were just being human, but foreigners are convenient targets. the chinese government was so concerned about the image of china that it is rumoured that persons were threatened by death in case they transmitted the disease to foreign countries. 10 if we view the work of medicine a sacred vocation, an inner calling to dedicate and care for the sick, it is contradictory to imagine some medical doctors may be accused of giving rise to a great loss of our society. taiwanese society decided to punish some of the head doctors who did not detect and report the hospital infection in the early stage. the final decision was to give the very tough punishment of retracting their professional license. ethically a wrong decision should not be punished as a crime of that magnitude unless the health profession had a criminal intention (motivation). although the consequences were a great loss for society it is not a wise move for the future to punish physicians for making mistaken decisions in emergency circumstances over public health if they were not intending any cover-up. there seems to be a threat of political scapegoats in every health crisis. we can expect future hcws to move for greater self-protection and hesitance in making decisions that are necessary for public health crises like emerging diseases. between the hero and the coward, there must have some space where people can be humane. it is normal and proper for people to be scared to die yet fulfill their duty in the frontline. except for special 'danger pay' or another kind of reimbursement, a calling for increased emphasis on workplace safety and a review of precautions is most important for this critical situation. a well trained and equipped health worker also needs to prepare for the frontline of possible bioterrorism, which may be similar to what we saw with sars. we need a well prepared expert more than a hero. there were cases reported where proper diagnosis of other diseases was hindered by the infection prevention measures being applied to treat all patients from sars infected areas as potential sars victims. 12 spiritual motivation should not slacken the implementation of sound precautionary measures. as the who director-general said 'the containment of sars required heroic efforts and extraordinary measures that are difficult to sustain over time'. 13 a taiwanese study of nurses in may-june 2003 found that nurses' agreement with the government control measures was a predictor of the extent to which they fulfilled their professional care obligation. 14 this suggests that having the support of the healthcare professions in policy is essential for everyone to work wholeheartedly. before the next outbreak, we need long-term planning and humane intervention to prepare a better response for the expected return of the disease. in the future if some repressive regimes are hit by sars they might employ brutal tactics to quarantine and isolate people, possibly sowing division among outside countries and multilateral organizations over how to respond to apparent human rights violations. 15 calls for development of effective centers for disease control in other regions of the world, including europe, have been made in response to the ways sars was fought. 16 the intention of calling for hcws to above all do good should not be blurred by wrong planning. with sars as with other severe infectious diseases that are readily transmitted, the manner that home and work isolation is handled is a key ethical issue. if people were diagnosed to be suspect or probable cases, all the people around them may as well be suspected of having been contaminated by them. consequently, the persons they have been in close contact with, for example, families, colleagues or schoolmates may be isolated to avoid further possible spread of the disease. such home isolation has serious social and psychological influences. in some cases, those who break home isolation may be punished by imposing a fine. people who were victims of sars in 2003 were accused of hindering the prevention or treatment of sudden disease outbreaks if they broke such isolation. it is normal to expect that quarantine will cause fears, and people will lose their patience and self-restraint, but we should assume that all people were originally infected by others with a few recent exceptions infected by laboratory medical research. persons should be taken care of better than being totally restricted to avoid them inflicting possible harm on others. with the policy of respecting human individuals complete needs for physical and mental well being, more people will be motivated not just to protect themselves, but to contribute to the macro wellbeing of the society. there are still lessons for all in society about the ethics of quarantine. modern society has forgotten the past risks of infectious disease outbreaks. we would suggest to respect health worker's autonomy of making their own choice to take a break from intensive physical or emotional loading or to accomplish the historical mission of coping with sars. a well developed society that we live in should have sufficient space to practice humanism to everyone in any kind of situation including a public health crisis. this is one of the lessons, we should learn in preparation for the next crisis of infectious disease. world health organization, summary table of sars cases by country an ebola epidemic simmers in africa sars: an asian catastrophe which has challenged the relationships between people in society-my experience in taiwan what have we experienced and learned from the outbreak of sars in beijing? flying publisher editorial: three lessons of sars too soon to celebrate the foreignness of germs: the persistent association of immigrants and disease in american society nurses' professional care obligation and their attitudes towards sars infection control measures in taiwan during and after the 2003 epidemic sars: down but still a threat. scope note (u) united states national intelligence center severe acute respiratory syndrome (sars): loud clang of the leper's bell key: cord-353608-de6rrf7v authors: saito, sho; asai, yusuke; matsunaga, nobuaki; hayakawa, kayoko; terada, mari; ohtsu, hiroshi; tsuzuki, shinya; ohmagari, norio title: first and second covid-19 waves in japan: a comparison of disease severity and characteristics: comparison of the two covid-19 waves in japan date: 2020-11-02 journal: j infect doi: 10.1016/j.jinf.2020.10.033 sha: doc_id: 353608 cord_uid: de6rrf7v • coronavirus disease has emerged as a global pandemic. • japan has experienced two waves of the disease. • the second wave had a lower proportion of severe cases on admission. has become a global pandemic, occurring in forming several peaks in waves 2, 3 . this study compared the severity and characteristics of the first and second waves in japan. we obtained the study data from the covid-19 registry japan (coviregi-jp). the coviregi-jp includes data from a observational cohort study using medical records in japan. the criteria for enrolment were (1) tests such as polymerase chain reaction (pcr) and loop-mediated isothermal amplification (lamp) that turned positive for severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and (2) inpatient treatment at a health care facility. we evaluated age, sex, comorbidities, disease severity at admission, supportive care, medications, and the outcome on discharge. a patient's condition was denoted as "severe" on fulfilment of one or more of the following criteria: the need for invasive or non-invasive mechanical ventilation, need for supplemental oxygen, an oxygen saturation (spo 2 ) of < 94% at room air, and tachypnoea (respiratory rate of >24 breaths per minute). patients who did not meet these criteria were classified as "non-severe" at admission. patients admitted between 26 january and 31 may 2020, were included in the first wave, and those admitted between 1 june and 31 july 2020, were included in the second wave 4 (frozen data as of 2 september 2020). continuous variables were expressed as medians and interquartile ranges, and categorical variables were expressed as numbers (%). all statistical analyses were conducted using r version 4.0.2 (r core team). data of 5194 cases from 327 facilities were included in the analysis: 3833 and 1361 cases from the first and second waves, respectively. at admission, the second wave had a smaller proportion of severe cases (12.0% vs 33.1%, fig. 1a ); the duration from onset to admission was also shorter (median, 4 vs 7 days) than that in patients in the first wave (fig. 1b, 1c) . patients in the second wave tended to be younger (median age, 37 vs 56 years), were less frequently transferred from other hospitals (3.8% vs 15.0%) and were less likely to have comorbidities such as cardiovascular diseases (1.9% vs 5.9%), and cerebrovascular disease (1.8% vs 6.1%). mortality (1.2% vs 7.3%) in hospitalized or discharged patients was also lower in the second wave; the same trend was observed on stratification according to age and severity at admission (table 1 ). our study showed that the proportion of cases involving severe disease at admission was smaller in the second wave. considering the lower percentage of patients transferred from other hospitals in the second wave, it is likely that the first wave had a more critical effect on the ability of healthcare institutions to receive patients. moreover, the number of pcr tests performed was greater in the second wave than in the first wave 5 . earlier admission of patients in the second wave may reflect the increase in the number of pcr tests performed and the number of beds available to covid-19 patients. data from the second wave indicated a demographic shift toward a younger population with fewer comorbidities, a lower proportion of severe patients at admission, and decreased mortality. however, the mortality was lower in second wave even if stratifying age and severity at admission. this may be because of the shorter time between disease onset and admission, differences in patient background, comorbidities, and advances in treatment methods. although this registry gathers information on a large number of patients, it does not cover all patients in japan, and data from the second half of the second wave was not included in this study; this may be a source of bias in this study. in addition, since data are updated daily, there is a possibility that future findings will differ from the current results. the findings of our study indicated that in the first wave, the medical system was under greater strain with more severe cases on admission. in the second wave, patients were younger with fewer underlying diseases and lower mortality rates. declarations of interest: none. this study was funded by health and labour sciences research grant, "research for risk assessment and implementation of crisis management functions for emerging and re-emerging infectious diseases (19ha1003)" e immunosuppression includes neutropenia (< 500 neutrophils/μl), use of glucocorticoids/steroids within 1 month (doses greater or equal to an equivalent of 20 mg of prednisone per day for at least 1 month), chemotherapy or radiation therapy or the use of immunosuppressants (such as antitumor necrosis factor-α therapy, anti-il-6 receptor/anti-cd20 monoclonal antibodies, selective t-cell co-stimulation blockers, methotrexate, tacrolimus) within the past 3 months, post hematopoietic stem cell transplantation, post organ transplantation, asplenia, and primary immunodeficiency syndrome or hiv infection. f patients who received these treatments at least once during their hospitalization were included. g data were counted only for patients who were alive at discharge. abbreviations: covid-19, coronavirus disease; ecmo, extracorporeal membrane oxygenation; iqr, interquartile range risk factors associated with disease severity and length of hospital stay in covid-19 patients characteristics and outcomes of covid-19 patients during initial peak and resurgence in the houston metropolitan area decreased case fatality rate of covid-19 in the second wave: a study in 53 countries or regions labour and welfare. the 6th advisory board for the control of covid-19 reference 3-1 we thank all the participating facilities for the provision of care to covid-19 patients and for cooperation regarding data entry. key: cord-348129-tph0d5fl authors: de deyn, michelle lee zhi qing; ng, qin xiang; loke, wayren; yeo, wee song title: a tale of two cities: a comparison of hong kong and singapore's early strategies for the coronavirus disease 2019 (covid-19) date: 2020-06-25 journal: j infect doi: 10.1016/j.jinf.2020.06.058 sha: doc_id: 348129 cord_uid: tph0d5fl nan in hksar, it is known that community-wide masking was practiced by the general population at an early stage of the pandemic 1 . based on news reports and official press releases, it is evident that many asian countries, which have successfully contained the first wave of infections, are now experiencing a second wave of imported cases from abroad and worsening local transmission 2 . as the authors rightly mentioned, singapore and hksar share several similarities in that they are both sprawling city states with developed economies, a high trade-to-gross domestic product (gdp) ratio, advanced infrastructure and healthcare systems, and they both had varying successes thus far in the management of the ongoing pandemic, albeit adopting different approaches 3, 4 . singapore saw her first imported case of the novel coronavirus infection on 23 january 2020. prior to this, the authorities had a working multi-ministry taskforce, issued travel advisories for china to the public, precautionary advisory to preschools (good hygiene, travel declarations and temperature screening for staff) and implemented temperature screening at land and sea checkpoints. given the evolving situation in china at that time and the high volume of international travel to singapore, the singapore government had accurately pre-empted the possibility of more suspect cases and imported cases 5 . after the 2003 severe acute respiratory syndrome-related coronavirus (sars-cov) outbreak, singapore authorities had put in place a multi-ministry taskforce and a disease outbreak response system condition (dorscon) framework that enables the whole-of-government to respond immediately to any disease outbreak and guide interventions 5 . while hong kong saw her first imported case on 22 january 2020 and had taken similar pre-emptive measures prior to this 6 . in terms of the alert level, singapore raised its risk assessment (in accordance with the dorscon framework) from dorscon yellow to dorscon orange on 6 february 2020 after the country saw an increased number of cases of local transmission. in hong kong, the serious response level was activated in public hospitals on 4 january 2020 and the hospital authority activated the emergency response level on 25 january 2020. both countries contained the first wave of imported cases well and boasted robust testing and contact-tracing capabilities. however, a key difference was that the hong kong government implemented social-distancing measures and partial closures earlier, extending the lunar new year holidays and keeping all schools and government offices closed after the holidays, while the singapore government had all schools (including preschools) running up till the implementation of a sweeping 'circuit breaker' measure on 7 april 2020, only allowing residents to leave their homes to exercise alone or purchase essential items. all private or public gatherings, non-essential services and sports and recreation facilities have ceased. although both countries did not advise compulsory mask-wearing hitherto, most members of public in hong kong chose to wear a mask when going outside anyway. the initial advice from the hong kong government was for people who have respiratory symptoms, visiting hospitals and those travelling to wear a mask (and continue to do so until 14 days after returning). the generally high mask usage amongst the public could be partly due to their previous experience with sars-cov or their distrust in the hksar central government 7 . although there are no official statistics, it was evident from media and news reports that most singaporeans still went about their daily activities without the use of a mask, unless they were unwell 8 . the singapore government has now dramatically reversed their recommendations on the use of masks because it is thought that they may confer additional protection against the covid-19, due to the variable incubation period for clinical disease and possibility of asymptomatic spreaders 9 . it is now mandatory to wear a mask when going out. tough laws have been put in place to enforce this; first-time offenders would be fined while egregious cases may be prosecuted in court 10 . the covid (temporary measures) act was passed by singapore parliament on 7 april 2020, and under the act, the government has the powers to make regulations to further prevent or control the incidence or transmission of covid-19. either way, it appears that hong kong has managed to flatten the coronavirus curve, while singapore is seeing a rapid upward trend (figure 1) , mostly due to an outbreak of cases amongst foreign workers living in the dormitories 11 . there are more than 250,000 foreign workers on 'construction work permits' in singapore, and the growing outbreak amongst workers living in the dormitories could be due to the limited space, their culture of communal cooking and sharing food, and communal toilets. given the above context and factors, it is difficult to precisely apportion the contribution of masking versus social distancing, rigorous contact tracing and other control measures to covid-19 containment. properly-worn face masks probably help stem the spread of the coronavirus, albeit other environmental and ambient factors such as temperature, wind velocity and humidity would also affect how the respiratory droplets travel. although the efficacy of wearing masks in public cannot be stated definitively, we believe the takeaway lessons are clear. caution and pre-emptive measures yield significant preventive benefits in this crisis. the public needs to act responsibly and pay heed to these advice and physical distancing measures. given the escalating medical and socioeconomic costs associated with this pandemic, the adage that 'prevention is better than cure' is especially relevant today. the role of community-wide wearing of face mask for control of coronavirus disease 2019 (covid-19) epidemic due to sars-cov-2 asia pacific steps up covid-19 efforts as second wave of infections strikes multinationals and the growth of the singapore economy. routledge city states in the global economy: industrial restructuring in hong kong and singapore. routledge confirmed imported case of novel coronavirus infection in singapore; multi-ministry taskforce ramps up precautionary measures macau confirms first case as new disease looms over hong kong. south china morning post flattened the curve for now but hong kong braces for a second coronavirus wave singapore u-turns on wearing masks as local virus cases climb asymptomatic transmission, the achilles' heel of current strategies to control covid-19 continued stringent implementation and enforcement of circuit breaker measures thousands of people in dorms pose new challenge to singapore virus fight. bloomberg the authors report no conflicts of interest. this research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors. the authors alone are responsible for the content and writing of the article. key: cord-307333-n6jc0jy3 authors: selvaggi, carla; pierangeli, alessandra; fabiani, marco; spano, lucia; nicolai, ambra; papoff, paola; moretti, corrado; midulla, fabio; antonelli, guido; scagnolari, carolina title: interferon lambda 1–3 expression in infants hospitalized for rsv or hrv associated bronchiolitis date: 2014-01-02 journal: j infect doi: 10.1016/j.jinf.2013.12.010 sha: doc_id: 307333 cord_uid: n6jc0jy3 objectives: the airway expression of type iii interferons (ifns) was evaluated in infants hospitalized for respiratory syncytial virus (rsv) or rhinovirus (hrv) bronchiolitis. as an additional objective we sought to determine whether a different expression of ifn lambda 1–3 was associated with different harboring viruses, the clinical course of bronchiolitis or with the levels of well established ifn stimulated genes (isgs), such as mixovirus resistance a (mxa) and isg56. methods: the analysis was undertaken in 118 infants with rsv or hrv bronchiolitis. nasopharyngeal washes were collected for virological studies and molecular analysis of type iii ifn responses. results: rsv elicited higher levels of ifn lambda subtypes when compared with hrv. a similar expression of type iii ifn was found in rsva or rsvb infected infants and in those infected with hrva or hrvc viruses. results also indicate that ifn lambda 1 and ifn lambda 2–3 levels were correlated with each other and with mxa and isg56-mrnas. in addition, a positive correlation exists between the ifn lambda1 levels and the clinical score index during rsv infection. in particular, higher ifn lambda 1 levels are associated to an increase of respiratory rate. conclusions: these findings show that differences in the ifn lambda 1–3 levels in infants with rsv or hrv infections are present and that the expression of ifn lambda 1 correlates with the severity of rsv bronchiolitis. keywords ifn lambda; il-28; il-29; rsv; hrv; mxa; isg56; viral load; bronchiolitis summary objectives: the airway expression of type iii interferons (ifns) was evaluated in infants hospitalized for respiratory syncytial virus (rsv) or rhinovirus (hrv) bronchiolitis. as an additional objective we sought to determine whether a different expression of ifn lambda 1e3 was associated with different harboring viruses, the clinical course of bronchiolitis or with the levels of well established ifn stimulated genes (isgs), such as mixovirus resistance a (mxa) and isg56. methods: the analysis was undertaken in 118 infants with rsv or hrv bronchiolitis. nasopharyngeal washes were collected for virological studies and molecular analysis of type iii ifn responses. results: rsv elicited higher levels of ifn lambda subtypes when compared with hrv. a similar expression of type iii ifn was found in rsva or rsvb infected infants and in those infected with hrva or hrvc viruses. results also indicate that ifn lambda 1 and ifn lambda 2e3 levels were correlated with each other and with mxa and isg56-mrnas. in addition, a positive correlation exists between the ifn lambda1 levels and the clinical score index during rsv infection. in particular, higher ifn lambda 1 levels are associated to an increase of respiratory rate. conclusions: these findings show that differences in the ifn lambda 1e3 levels in infants with rsv or hrv infections are present and that the expression of ifn lambda 1 correlates with the severity of rsv bronchiolitis. ª 2014 the british infection association. published by elsevier ltd. all rights reserved. bronchiolitis is a disorder most commonly caused in infants by viral lower respiratory tract infections; it is characterized by acute inflammation, edema and necrosis of epithelial cells lining small airways, increased mucus production, and bronchospasm. 1 the most common virus causing bronchiolitis is the respiratory syncytial virus (rsv). 2, 3 other viruses identified as causing bronchiolitis are rhinovirus (hrv), human metapneumovirus, bocavirus, and parainfluenza. 4 in particular, hrv has been recently shown to infect the lower airway as well 5 and confirmed to be the second most frequent cause of bronchiolitis. 6 it has been demonstrated that the combination of both host and viral factors profoundly influence the severity of viral associated bronchiolitis. 7e9 however, it is not yet clear whether the different subtypes of rsv (a and b) or hrv species (a, b, and c) cause different grades of bronchiolitis severity. 10, 11 furthermore, the role of the innate immune response, in the pathogenesis of severe rsv or hrv disease is still to be defined in detail. 7e9 among the main players of antiviral innate immune response, the type i interferons (ifns), ifn alpha and beta, are considered cytokines crucial for anti-viral resistance and represent an early antiviral host defense mechanism against viral infections. 12 in 2003, a novel class of antiviral cytokines was discovered, characterized and classified as type iii ifns: ifn lambda1/il-29, ifn lambda 2/il-28a, and ifn lambda 3/il-28b. 13 at the amino acid level ifn lambda2 and lambda3 are highly similar having 96% sequence identity while ifn lambda1 shares approximately 81% sequence identity with ifn lambda2 and lambda3. 13 the type iii ifns possess antiviral properties similar to those of type i ifns but appear to be expressed especially by epithelial cells and consequently exert host protection primarily at epithelial surfaces. 14e16 despite the fact that it is known that ifn lambda contributes to the control of viral infections in epithelial cells of respiratory tract 17e19 and that the presence of single nucleotide polymorphism around ifn lambda 3 (il-28b) can increase the risk of hospitalization for bronchiolitis at early age, 20 the ifn lambda 1e3 expression in the respiratory tracts of hospitalized infants with rsv or hrv infections has never been addressed. hence, considering the importance of the ifn lambda in protecting the airway tract from virus infections, 17e19 we hypothesized that the heterogeneity of ifn lambda 1e3 levels could, at least in part, explain the broad clinical spectrum of rsv or hrv bronchiolitis. therefore, we evaluated whether there was a difference in the gene expression of ifn lambda 1e3 subtypes between infants with a clinical diagnosis of rsv associated acute bronchiolitis and those with hrv infection. the same analysis was also performed between rsv or hrv subtypes. in addition, to characterize the activation of type iii ifns in the airway tract of infants with rsv or hrv infections, we evaluated whether there was a coordinate activation between ifns lambda and that of mxa and ifn-stimulated gene (isg) 56, which are well known markers of type i and iii ifn antiviral activity. 12 finally, to further characterize the above issues, we also assessed whether a correlation between ifn lambda 1e3 levels and demographic, virological and clinical parameters in rsv and hrv infected infants actually exist. a total of 118 infants with single rsv or hrv infection were retrospectively selected from a total of 250 infants admitted with a clinical diagnosis of acute bronchiolitis during three epidemic seasons (2008e2011) to the paediatric department of policlinico umberto i hospital. the study was approved by the ethics committees and informed consent was obtained from the infant's parents. bronchiolitis was diagnosed from the presence of a history of upper respiratory tract infection followed by the acute onset of respiratory distress with cough, tachypnea, retraction, and diffuse crackles on auscultation (wheezing alone was not considered sufficient cause for inclusion in the study). the exclusion criteria were underlying chronic disease (such as cystic fibrosis, chronic pulmonary disease, congenital heart disease, and immunodeficiency) and recurrent (more than one) wheezing episodes. 4, 21 the severity of the illness was assessed clinically on the following four indications, each of which was assigned a score within the range 0e8. 4 in particular, on admission to hospital, the clinical severity was assigned to each infant, based on respiratory rate (<45 breaths/min z 0, 45e60 breaths/min z 1, >60 breaths/min z 2) arterial oxygen saturation in room air (>95% z 0, 95e90% z 1, <90% z 2), presence of retractions (none z 0, present z 1, present þ nasal fare z 2), and ability to feed (normal z 0, reduced z 1, endovenous z 2). nasopharyngeal washings were collected in the first 48 h after admission to the hospital from infants suffering from acute bronchiolitis, and an aliquot was tested for viruses as previously described. 22 in particular nasopharyngeal washings were obtained with 3 ml of sterile saline physiological solution injected into each nostril and collected with a syringe. all samples were delivered on ice within 1e2 h to the virology laboratory and on arrival, if needed, they were vortexed with beads to solve mucus. they were divided into two aliquots: one was treated for nucleic acid extraction and viral detection; the second was centrifuged at 2000 rpm for 10 min, and each cell pellet was resuspended in 1 ml of phenol and guanidine isothiocyanate reagent (trizol, gibco-brl, ny) and frozen at à80 c for gene expression analysis. a panel of reverse transcription-pcr (rt-pcr) or nested pcr assays, some in a multiplex format, were used for the detection of 14 respiratory viruses, including rsv; influenza viruses a and b; coronaviruses oc43, 229e, nl63, and hku1; metapneumovirus; adenovirus; hrv; and parainfluenza virus types 1e3, human bocavirus as previously reported. 22, 23 the evaluation of the sensitivity of the rt-pcr or pcr tests for the 14 respiratory viruses was made as described in our previously published papers. 22, 23 in particular, the integrity of the extracted nucleic acid was tested by means of the amplification of the cellular gene beta actin. rsv or hrv-positive samples were typed as rsva-b or as hrv a-c respectively. the rsv fragment to be sequenced was obtained by re-extracting rna from rsv-positive samples and directly amplifying it using superscript one-step rt-pcr for long templates kit (life technologies, monza, italy) with two expressly designed forward primers, a-fseq 5 0 -aat gat ttt cac ttt gaa-3 0 ; b-fseq 5 0 -gat gat tac cat ttt gaa-3 0 , corresponding to g gene position 481e498 of rsv-a2 and to position 475e492 of the rsv-ba reference strains, respectively, and with one reverse primer targeting the fusion protein gene 5 0 end. 24 hrv-positive samples were retrospectively amplified with primers widely used for genotyping targeting 390 bases of the 5 0 untranslated region (5 0 utr) central portion. 25 the mrna copy content of ifn lambda 1e3, mxa and isg56 was measured by a real-time 5 0 exonuclease rt-pcr assay using the light cycler 480 sequence detector (roche, monza, italy). briefly, the total cellular rna was extracted from the cells collected from nasopharyngeal washings as described, using phenol and guanidine isothiocyanate reagent (trizol, gibco-brl, ny), by following the manufacturer's instructions, and was retro-transcribed as previously specified. 26 primer pairs and probes for ifn lambda 1 [forward primer, 5 0 -ggacgccttggaagagtcact-3 0 ; reverse primer, 5 0 -agaagcctcaggtcccaattc-3 0 ; probe, 5 0 fam-agttgcagctctcctgtcttccccg-3'tamra 27 ], ifn lambda 2e3 [forward primer, 5 0 -ctgccacatagcccagttca-3 0 ; reverse primer, 5 0 -agaagcgactcttctaaggcatctt-3; pro be, 5 0 fam-tctccacaggagctgcaggccttta-3'tamra 27 ], mxa (forward primer, 5 0 -ctgcctggcagaaaaacttac-3; reverse primer, 5 0 -ctctgttattctctggtgagtctcctt-3 0 ; probe, 5 0 fam catcacacatatctgtaaatctctgcccctgtt-3'tamra); isg56 [forward primer, 5 0 -tgaagaagctctagc-caacatgtc-3 0 ; reverse primer, 5 0 -gagctttatccaca-gagccttttc-3 0 ; probe: 6'fam 5 0 -tatgtctttcgatatg cagccaagttttaccg-3 0 tamra 28 ] were added to the universal pcr master mix (roche) at 600 and 400 nm, respectively, in a final volume of 20 ml. the coamplification of the beta-glucuronidase gene (forward primer, 5 0 -tctgtcaagggcagtaacctg-3; reverse primer, 5 0 -gcccacgactttgttttctg-3 0 ; probe, 5 0 fam-tcaagttggaagtgcgtcttttggatgc-3'tamra) was used to normalize the amount of total rna present using the threshold cycle relative quantification [the 2e(delta) ct] method according to the supplier's guidelines. all the determinations were performed in duplicate. a taqman-based real-time pcr technique for rsv 28 or hrv 29 rna quantification was performed on all nasopharyngeal washing specimens with positive rt-pcr results for rsv or hrv respectively. briefly, viral rna was extracted from nasopharyngeal washings npw that were positive for rsv or hrv, using a qiaamp viral rna mini kit (qiagen, milan, italy). the rna was dissolved in rnase-free water and the rsv quantification was performed by taqman assay after generation of cdna using a high capacity cdna archive kit (applied biosystems, monza, italy). type-specific primers and probes for n gene of both rsv a and b 30 or 5 0 utr region of hrv a-c strains 29 were added to the universal pcr master mix (roche) at 600 and 400 nm, respectively, in a final volume of 20 ml. the standards for rsv or hrv were obtained respectively by cloning the 82 bp of rsv n gene or 203 bp of the 5 0 utr hrv region into the pcr2.1 plasmid using a topo ta cloning kit (invitrogen corporation, san diego, ca, usa). a linear distribution (r z 0.99) was obtained between 10 1 and 10 8 copies of rsv or hrv-dna. viral load values were log transformed for analysis and data was expressed as the log number of rsv or hrv copies per ml of nasopharyngeal washings. all the determinations were performed in duplicate. all measurements are expressed median (range) or frequency (percentage), unless otherwise indicated. the demographic and clinical characteristics of infants suffering from rsv or hrv associated bronchiolits were compared using the mannewhitney test. differences in the clinical score index values were analyzed using student's t test. differences between infants with rsv or hrv infections and between rsv (a and b) or hrv (a and c) strains, in terms of the level of ifn lambda 1e3 measured in cells from nasopharyngeal washings, were compared using the man-newhitney test. spearman's rho coefficient was calculated in order to assess the correlation between the level of ifn lambda 1 and ifn lambda 2e3 and between ifn lambda 1e3 and isgs, demographic, clinical and rsv or hrv viral load. differences in the ifn lambda1 levels in rsv infected infants divided into 3 groups on the basis of the respiratory rate were evaluated by using kruskalewallis test. the significance was fixed at the 5% level. analysis was performed with spss v.17.0 for windows. one hundred and eighteen infants, admitted over a period of 3 years to the paediatric department of policlinico umberto i university hospital with a diagnosis of single rsv or hrv associated bronchiolitis were included ( table 1) . as far as virological characteristics are concerned, a total of 78 (66%) infants carried a single rsv infection the clinical severity was assigned to each infant with the range 0e8, based on respiratory rate (<45 breaths/min z 0, 45e60 breaths/ min z 1, >60 breaths/min z 2), arterial oxygen saturation in room air (>95% z 0, 95e90% z 1, <90% z 2), presence of retractions (none z 0, present z 1, present þ nasal fare z 2), and ability to feed (normal z 0, reduced z 1, endovenous z 2). whereas 40 (34%) had an hrv single infection. in particular 81% (63/78) of the rsv positive infants had a rsva infection and the remaining had a rsvb infection. among hrv infected infants, 45% (18/40) had an infection with hrva and 55% (22/40) had an hrvc infection. interestingly, no infants had an hrvb infection. when we analyzed the demographic and clinical parameters of infants with rsv or hrv infection, there were no significant differences between infants with rsv or hrv infection and between infants with different rsv (a vs b) or hrv (a vs c) strains (table 1) . on the contrary the differences between infants with rsv or hrv infection were statistical significant when the clinical score index, and the percentage of infants with fever were analyzed (table 1) . moreover, the number of eosinophils was lower in infants with rsv infection compared to those with hrv infection ( table 1) . the airway transcription levels of ifn lambda 1 and ifn lambda 2e3 were evaluated using real-time rt-pcr in cells from nasopharyngeal washings collected from infants with rsv infection (n z 78) or hrv infections (n z 40). the gene expression level of ifn lambda 1 and ifn lambda 2e3 showed high variability between infants with rsv or hrv infection [(coefficient of variation >100%), fig. 1 as reported in fig. 1 (panel a) the transcript levels of ifn lambda 1and ifn lambda 2e3 in infants suffering from rsv infection were higher than in those with hrv infection [ifn lambda 1 (rsv vs hrv): p z 0.029; ifn lambda 2e3(rsv vs hrv): p z 0.015]. furthermore, we found no differences between mrna levels of ifn lambda 1 and those of the ifn lambda 2e3 in rsv or hrv infections (fig. 1, panel a) . no significant differences in transcript levels of type iii ifn were observed between infants with different rsv (a vs b) or hrv (a vs c) strains (fig. 1 , panel bec). in order to characterize the activation of type iii ifn response during bronchiolitis, we also evaluated whether there was a coordinate activation of ifn lambda subtypes in the airway tract of infants suffering from bronchiolitis. results indicate that in the respiratory tract of infants with rsv or hrv the transcript levels of ifn lambda 1 were significantly correlated with those of ifn lambda 2e3 (table 2) . furthermore, considering that ifn lambda induces the expression of ifn-stimulated genes (isgs), we evaluated whether there was a correlation between the expression of ifn lambda subtypes and that of well established isgs, namely mxa and isg56. results indicate that type iii ifn mrna levels were significantly correlated with the transcript expression of mxa and isg56 in infants with rsv or hrv bronchiolitis ( table 2) . the relationship between patient data as independent variables and ifn lambda 1e3 mrna levels measured in nasopharyngeal washings in infants with rsv or hrv bronchiolitis was analyzed (table 3) . we found a significant positive correlation between the transcript level of ifn lambda 1 and the clinical score index in infants with rsv infection (r z 0.350, p z 0.025) but not in those hrv infected (table 3 ). in particular, ifn lambda 1 seems to be associated to an increase in the respiratory rate during rsv infection. indeed, as showed in fig. 2 , when rsv infected infants were divided into 3 groups on the basis of the respiratory rate (<45 breaths/min, 45e60 breaths/ min, and >60 breaths/min), there was a significant difference between the groups (p z 0.022). specifically, infants with >60 respiratory breaths per minute showed higher gene expression of ifn lambda 1 compared with infants with <45 or 45e60 respiratory breaths per minute. in contrast, we failed to detect any correlation between the ifn lambda 1e3 gene expression levels and age, weight, number of days of hospitalization, and several immunological and biochemical parameters (numbers of neutrophils, lymphocytes, eosinophils or platelets, and levels of glycemia, sodium, c-reactive protein and hemoglobin). in addition, no differences were detected in ifn lambda 1e3 gene expression for male and female and between infants with fever or without fever (data not shown). in an attempt to determine whether rsv or hrv load influences the gene expression of type iii ifn, levels of rsv-or hrv-rna were analyzed respect to the expression of ifn lambda subtypes. no significant correlations were observed between rsv or hrv load and the levels of ifn lambda 1e3 (table 3 ). it has been proposed that ifn lambdas are likely one of the main ifn produced during innate responses to respiratory viruses in the airway tract, 14, 17, 19, 31 in line with the observations that the expression of the ifn lambda receptor is limited primarily to epithelial surfaces including that of the lung. 14,32 however the in vivo role of these cytokines in the host innate immune response to rsv or hrv infection is yet to be defined. our study gave some significant new insights into this complex issue. in particular, we found that in the respiratory tract of infants infected with rsv or hrv, there is a coordinate expression of different subtypes of ifn lambda: such an expression parallel also with those of mxa and isg56, well established antiviral proteins induced by type i and iii ifns. 12 this data could suggest that the airway tract is responsive to type iii ifns and that rsv or hrv caused a coordinate induction of ifn lambda subtypes that in turn can regulate antiviral pathways. 12 however since both isgs analyzed are also regulated by ifn type i which is known to be produced during viral infections and to share the same pathways with ifn lambda, it remains unclear to which extent ifn lambda might specifically contribute to antiviral immunity against rsv or hrv in infants suffering from bronchiolitis. in this study we also compared the activation of ifn lambda 1e3 in the respiratory tract of infants suffering from rsv or hrv associated bronchiolitis. results demonstrated that in cells collected from nasopharyngeal washings of rsv positive infants there are higher mrna levels of type iii ifns compared to those observed in infants with figure 1 gene expression of ifn lambda 1e3 during rsv or hrv bronchiolitis. the levels of type iii ifns were evaluated by using rt-taqman based real time pcr assays in cells from nasopharyngeal washings collected from 118 infants suffering from rsv hrv infection. this is particularly interesting considering that it is known that the rsv ns1 and ns2 proteins can suppress in vitro the induction of ifn lambda. 33 however, whether such response reflects host reactions for counteracting the ns1/ns2 viral interference on ifn lambda induction is actually unknown. in our previous studies, we also observed a higher transcript level of isgs as well as of most pattern recognition receptors in infants with rsv compared to those with hrv infections. 26, 34 all these findings may suggest that rsv infection are generally associate to a more robust innate immune response compared to those caused by hrv. in agreement, garcia et al. reported that concentrations of several cytokines in nasal wash tend to be higher in children with rsv than in those with hrv. 35 in contrast, jartii et al. found that children with hrv-associated wheezing episodes show increased concentrations of th1 and th2 cytokines compared with those with rsv. 36 however they measured cytokines concentrations in serum rather than respiratory tract secretions during wheezing episodes. as far as hrv is concerned, there are no studies on the evaluation of ifn lambda expression during hrv bronchiolitis: however wheezing during the first hrv infections is considered a risk factor for subsequent asthma development. 37 interestingly, a deficient ifn lambda response to hrv infection has been reported in childhood in asthmatic subjects irrespective of their atopic status and in atopic patients without asthma. 38 furthermore, contoli et al. reported a deficient induction of ifn lambda by hrv in asthmatic primary bronchial epithelial cells and alveolar macrophages, which was highly correlated with severity of hrv-induced asthma exacerbation and virus load in experimentally infected human volunteers. 27 in apparent contrast, the presence of higher ifn lambda levels were recently associated with worsening illness of hrv associated asthmatic exacerbations in children. 39 undoubtedly, the complex correlation between cytokine responses and viral infections deserves more studies to be performed carefully monitoring the various components of innate immunity during the natural course of respiratory viral infections. in this study, we also found no differences in the expression of type iii ifns between infants infected with rsva or rsvb subtypes and between those infected with hrva or hrvc strains, suggesting that the specific strain of rsv or hrv would not affect diversely the rate of activation of antiviral response. as far as the influence of specific rsv or hrv strains on the clinical course of bronchiolitis is concerned, no significant differences in the clinical characteristics between rsv (a vs b) or hrv (a vs c) infected infants were also found. however it must be considered that the samples size of infants analyzed in this study when divided according to the specific rsv or hrv strain was too small. conflicting results have been published on rsv (a or b) and hrv (a-c) association with recurrent gravity of respiratory diseases. 10,11,40e43 in particular, many, but not all, of the published studies 11,40e43 found that hrv-c is associated with more severe lower respiratory disease and with wheezing, compared with hrv-a. nonetheless, because of the small size of hrv-positive specimens within each individual diagnosis category, most studies suffered from low power and were unable to detect significant differences among specific diagnoses, as discussed in one of the largest study ever. 43 moreover, few data are available on the comparison of the ability of rsv a or b to modulate innate immune responses 44 and no studies were performed on this issue for hrv a-c. therefore we retain that no definite conclusions can be drawn on such issues. interestingly, this study also demonstrated that levels of ifn lambda 1 seem to be associated with severity of respiratory disease in rsv but not in hrv infected infants. this observation is not unusual for rsv pathogenesis. in fact, several studies have described severe rsv disease in children who have high levels of inflammatory cytokines and chemokines produced by innate immunity in respiratory secretions. 45, 46 whether or not ifn lambda contributes to augmentation of inflammation in our rsv positive infants is currently unknown. however, in addition to their antiviral effects, type iii ifns have been shown to play a critical role in regulating the adaptive immune response by acting directly on th1/2 polarization and cytokine production. 47, 48 in particular, ifn lambda 1 has been shown to increase production of il-6, il-8, and il-10, with no concomitant increase in il-1 or tnf, suggesting it may not directly engender local tissue destruction, but could contribute to the inflammatory process during bronchiolitis. 47 a reciprocal control of ifn lambda 1 and th2-associated cytokines seems also to exist. 49 indeed, it has been shown that there is a reciprocal regulation between ifn lambda1 and il-4 or il-13 which are well established th2 cytokines associated with increased rsv disease severity. 49 in addition, although type iii ifns seems to be activated during in vivo rsv infection, we observed that enhancement was not related to viral loads. indirectly the lack of a direct effect of ifn lambdas on viral replication may reflect a more relevant role of the cross-talk between inflammatory citokines and type iii ifn subtypes in determining the bronchiolitis clinical course. intriguingly, there are also indications that ifn lambda signaling may be resistant to feedback mechanisms targeting ifn alpha 50 allowing it to be a more effective and durable antiviral and immunomodulator cytokine, at least under some circumstances, that could cause a prolonged, diffuse inflammatory response in the airway tract of rsv infected infants. the presence of this strong inflammatory response is consistent with the observation of the presence of string respiratory rates in rsv positive infants with increased ifn lambda1 levels. on the other hand, the reduced expression of ifn lambda subtypes observed in the airway tract of hrv infected infants compared to those with rsv infection, may not be enough to activate an excessive inflammatory response affecting the clinical course of bronchiolitis. in agreement with this hypothesis, wang q et al. found that mda5 deficient mice with reduced ifn lambda productions show less hrv induced airway inflammation. 51 a greater number of eosinophils was also observed in hrv infected infants than in those with rsv accordingly to our previous study. 52 in this regards it has been recently demonstrated that eosinophils may contribute to antiviral immunity and play a beneficial role in limiting viral respiratory lung dysfunction. 53 however, it is also believed that eosinophilia is one of the atopic features that may contribute to the higher risk to develop recurrent wheezing and asthma. 37 furthermore, in this study in line with previous studies 4,34,52 a greater clinical severity in rsv infected infants than in those infected with hrv has been observed. therefore it is conceivable that the presence of reduced airway ifn lambda response in hrv infected infants than in those with rsv infections could reflect the presence of a milder bronchiolititis clinical course caused by hrv compared to that associated to rsv. indeed, miller et al. reported that ifn lambda1 levels were higher in wheezing children infected with hrv compared with no-wheezing and increased with worsening symptoms. 39 alternatively, our data could indicate that an impaired ifn lambda production during hrv infection is present, not only in asthmatic subjects, 27, 54 but also in infants with bronchiolitis which can be associated with the inability to control early virus replication and to mount an adequate th1/ th2 immune response which in turn may impact on recurrent wheezing predisposition and an exacerbation pathogenesis. these results on the presence of a positive correlation between ifn lambda1 levels and the clinical score of bronchiolitis in rsv infected infants does not seem to be in agreement with those obtained previously on the evaluation of isgs levels in infants with bronchiolitis. 34 this discrepancy, although unexpected, might be explained considering that several host and viral factors independently from ifn lambda can regulate, directly or indirectly, the isgs production. 12, 55 in addition it has been shown that the signaling pathways which lead to the activation of ifn regulatory factor can induce transcription of ifnstimulated response elements without the involvement of ifns. 56e58 thus it remains plausible that two different players of the same biological system might exert an opposite, complementary or simply additive effect on the clinical outcome of rsv bronchiolitis. all these findings would be greatly strengthened by comparing the level of expression of mrnas encoding type iii ifns with the level of expression of other cytokines or antiviral responses (e.g. type i ifns) in the cells from nasopharyngeal washings derived from infants with rsv or hrv associated bronchiolitis. this analysis could allow us to deep understand the complex picture of the airway ifn lambda response as well as the intensity and the dynamic nature of the antiviral and inflammatory pathways associated to type iii ifn response during pediatric lower respiratory tract infections. unfortunately the collected material was just enough to perform the experiments shown in the present study and the above issues could not be addressed. another important analysis which should be, but for the above reason have not been made, is the separate analysis of ifn lambda 2 and ifn lambda 3 subtypes expression in order to characterize the distinct contribute of these subtypes in rsv or hrv bronchiolitis. further studies specifically aimed to address these important issues are needed. in conclusion, we have shown for the first time to our knowledge that ifn lambda 1e3 are expressed in infants suffering from bronchiolitis although their levels may be different on the basis of which virus, rsv or hrv, has been detected in the respiratory tract. in addition, we have demonstrated that ifn lambda 1 can influence the severity of bronchiolitis caused by rsv, but not by hrv, suggesting that the rate of activation of type iii ifn response may act as a double-edged sword in some circumstance during pediatric respiratory viral infections. however, it must be underlined that several factors associated or not with the ifn system may influence the clinical severity of bronchiolitis. 59 the latter issue is exemplified by our observations about the opposite effect exerted by isgs 34 and ifn lambda (this study) on the clinical outcome of bronchiolitis. all these findings highlight the importance of studying the interplay between the pathways of ifn lambda subtypes and those of other inflammatory cytokines or chemokines in order to deeply understand the influence of type iii ifn response on the clinical course of respiratory diseases. substantial variability in community respiratory syncytial virus season timing viral bronchiolitis for the clinician respiratory syncytial virus, human bocavirus and rhinovirus bronchiolitis in infants the role of rhinovirus in asthma exacerbations newly identified human rhinoviruses: molecular methods heat up the cold viruses current 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antiviral immunity and promote clearance of respiratory syncytial virus impaired innate interferon induction in severe therapy resistant atopic asthmatic children respiratory syncytial virus nonstructural proteins ns1 and ns2 mediate inhibition of stat2 expression and alpha/beta interferon responsiveness transcriptional profiling of interferon regulatory factor 3 target genes: direct involvement in the regulation of interferon-stimulated genes distinct and essential roles of transcription factors irf-3 and irf-7 in response to viruses for ifn-alpha/beta gene induction multiple signaling pathways leading to the activation of interferon regulatory factor 3 respiratory viral infections in infants: causes, clinical symptoms, virology, and immunology this work was supported by a grant from "sapienza" university of rome to carolina scagnolari ("ricerche universitarie", anno 2012, prot c26a12x2hp). key: cord-308501-z3eiac25 authors: zhu, chengliang; liu, weiyong; su, hanwen; li, sitong; shereen, muhammad adnan; lv, zhihua; niu, zhili; li, dong; liu, fang; luo, zhen; xia, yuchen title: nbreastfeeding risk from detectable severe acute respiratory syndrome coronavirus 2 in breastmilk date: 2020-06-04 journal: j infect doi: 10.1016/j.jinf.2020.06.001 sha: doc_id: 308501 cord_uid: z3eiac25 nan an emerging severe acute respiratory syndrome coronavirus 2 (sars-cov-2), causing coronavirus disease 2019 (covid-19) pandemic, imposes a great threat to global public health 1 . the transmission and pathophysiology of sars-cov-2 gradually known among various populations, but public health effects of covid-19 on women and their outcomes should not be ignored 1, 2 . in pregnant and perinatal women, vertical transmission of sars-cov-2 from infected mother to her newborn is a controversial issue [2] [3] [4] . sars-cov-2 was not detected in vaginal fluid from a women with covid-19 5 , however, no clear evidence regarding optimal delivery timing and safety of vaginal or cesarean delivery preventing sars-cov-2 vertical transmission has been reported 6 . thus, the management of in pregnancy based on obstetrical indications and maternal-fetal status is highly concerned. here, we represent clinical characteristics of covid-19 pneumonia in puerperal women and evidence of sars-cov-2 shedding in her breastmilk. between february 1 and march 25, 2020, five pregnant patients with covid-19 were included to analyze this study ( table 1 ). the mean age of five mothers was 32 years (range 27 to 34 years), with mean gestational age of 38 weeks plus 1 week (range 35 weeks to 40 weeks plus 1 week). all mother's main onset symptoms were fever (40%), cough (20%), nasal congestion (20%), rhinorrhea (20%), poor appetite (20%), chest distress (40%), dyspnea (40%), and diarrhea (20%), that is consistent with clinical signs and symptoms, as previously described 7 . chest ct scan of all patients (except patient 4) before delivery showed typical viral pneumonia, such as patchy and scattered ground-glass opacities, and blurred borders. table 1) . none of the patients had co-infection with other common respiratory viruses (enlisted in table 1 ). five (100%) nasopharyngeal swab samples from patients were tested positive for sars-cov-2 rt-pcr. all the available vaginal secretion samples were negative for sars-cov-2 rt-pcr test, that is similar as previously reported 5 . during follow-up, three of four (75%) available serum samples from patients had significant elevated concentrations of sars-cov-2 igm and igg (table 1) . more importantly, four out of five (80%) patient`s breastmilk samples were negative for sars-cov-2 rt-pcr, which is similar to previous observations 2, 8 , while one (20%) patient`s (patient 3) breastmilk showed sars-cov-2 rna test positive (table 1) . additionally, the breastmilk samples from patient 3 after delivery for two and three days, still remained positive for sars-cov-2 ( figure 1b) . of note, ct value of rt-pcr test results were relatively high as 38.2 and 38.5 ( figure 1b, a and collectively, we reported detectable sars-cov-2 in human breastmilk from a puerperal woman with covid-19. although our conclusions are limited by the small sample size, we believe our findings are important for the concern of sars-cov-2 infection risk in breastfeeding of mother with covid-19 to her neonate. the study was approved by the ethics committee and institutional review board of the renmin hospital of wuhan university (file no. wdry2020-k066), and the tongji hospital of huazhong university of science and technology (file no. tj-irb20200201). written informed consent was obtained from each enrolled patient. the data in this study can be provided after the article is published with the permission of the corresponding authors. we can provide participant data without names and identifiers, but not the study protocol, statistical analysis plan, or informed consent form though an appointed email address for communication. the corresponding authors have the right to decide whether to share the data or not regarding to the research objectives and plan provided. coronavirus disease 2019 (covid-19): we don't leave women alone. international journal of public health possible vertical transmission of sars-cov-2 from an infected mother to her newborn clinical characteristics and intrauterine vertical transmission potential of covid-19 infection in nine pregnant women: a retrospective review of medical records clinical infectious diseases: an official publication of the infectious diseases society of america sars-cov-2 is not detectable in the vaginal fluid of women with severe covid-19 infection. clinical infectious diseases: an official publication of the infectious diseases society of america expert consensus for managing pregnant women and neonates born to mothers with suspected or confirmed novel coronavirus (covid-19) infection. international journal of gynaecology and obstetrics: the official organ of the international federation of gynaecology and obstetrics clinical features and obstetric and na=not available; +=positive crp=c-reactive protein; pct=procalcitonin alt=alanine aminotransferase pcr, short for real-time pcr against sars-cov-2 nucleic acid; ct=curve threshold value of sars-cov-2 n gene hcov=human seasonal coronavirus; hmpv=human metapeumovirus; hpiv=human parainfluenza virus; hrsv=human respiratory syncytial virus we declare no competing interests. key: cord-322812-9u3ptqjs authors: wells, philippa m.; doores, katie j.; couvreur, simon; nunez, rocio martinez; seow, jeffrey; graham, carl; acors, sam; kouphou, neophytos; neil, stuart j.d.; tedder, richard s.; matos, pedro m.; poulton, kate; lista, maria jose; dickenson, ruth e.; sertkaya, helin; maguire, thomas j.a.; scourfield, edward j.; bowyer, ruth c.e.; hart, deborah; o'bryne, aoife; steel, kathyrn j.a.; hemmings, oliver; rosadas, carolina; mcclure, myra o.; capedevilla-pujol, joan; wolf, jonathan; ourselin, sebastien; brown, matthew a.; malim, michael h.; spector, tim; steves, claire j. title: estimates of the rate of infection and asymptomatic covid-19 disease in a population sample from se england date: 2020-10-15 journal: j infect doi: 10.1016/j.jinf.2020.10.011 sha: doc_id: 322812 cord_uid: 9u3ptqjs background: understanding of the true asymptomatic rate of infection of sars-cov-2 is currently limited, as is understanding of the population-based seroprevalence after the first wave of covid-19 within the uk. the majority of data thus far come from hospitalised patients, with little focus on general population cases, or their symptoms. methods: we undertook enzyme linked immunosorbent assay characterisation of igm and igg responses against sars-cov-2 spike glycoprotein and nucleocapsid protein of 431 unselected general-population participants of the twinsuk cohort from south-east england, aged 19-86 (median age 48; 85% female). 382 participants completed prospective logging of 14 covid-19 related symptoms via the covid symptom study app, allowing consideration of serology alongside individual symptoms, and a predictive algorithm for estimated covid-19 previously modelled on pcr positive individuals from a dataset of over 2 million. findings: we demonstrated a seroprevalence of 12% (51participants of 431). of 48 seropositive individuals with full symptom data, nine (19%) were fully asymptomatic, and 16 (27%) were asymptomatic for core covid-19 symptoms: fever, cough or anosmia. specificity of anosmia for seropositivity was 95%, compared to 88% for fever cough and anosmia combined. 34 individuals in the cohort were predicted to be covid-19 positive using the app algorithm, and of those, 18 (52%) were seropositive. interpretation: seroprevalence amongst adults from london and south-east england was 12%, and 19% of seropositive individuals with prospective symptom logging were fully asymptomatic throughout the study. anosmia demonstrated the highest symptom specificity for sars-cov-2 antibody response. funding: nihr brc, cdrf, zoe global ltd, rst-ukri/mrc the covid-19 pandemic has constituted an international emergency, accounting world-wide for greater than 600,000 deaths thus far. in order to understand the transmission of the virus through the population, and to estimate protection afforded to those post-infection, we must first understand the seroprevalence to sars-cov-2 with regards to demographics and clinical presentation. data on the rates of infection in the united kingdom (uk) come mainly from the office for national statistics (government ons) surveys. as of 29 th june 2020, the ons estimate 6.3% (95% ci 5 to 7.8) of the uk population to be seropositive for igg or igm s glycoprotein detected using enzyme linked immunosorbant assay (elisa) testing, based on blood tests of 885 individuals since 26 april 2020. 1, 2 in the 7 th july report from the ongoing ons household survey involving pcr swab testing of around 20,000 people, the authors reported an asymptomatic infection rate of 66%. 3 however, these surveys did not study the full range of symptoms associated with covid-19, and only assessed symptomatology at the time of swabbing, and therefore may have overestimated this rate. we undertook a population-based study of the humoral immune response to sars-cov-2, with regards to longitudinal clinical symptoms collected through a mobile phone app in a population-based sample of 431 twinsuk volunteers. 2 participants were members of the twinsuk cohort, the largest uk registry of adult twins. 4 participants were visited in their home to obtain saliva and serum samples to test for active infection and antibody response. the majority of participants had completed regular logging of symptoms, via the c-19 covid symptom study app 5 since, enabling measurement of antibody response to covid-19 with regards to clinical symptoms. participants were members of the twinsuk cohort, the largest uk registry of adult twins. 4 participants were visited in their home to obtain saliva and serum samples to test for active infection and antibody response. for three months prior to the visit, the majority of participants had completed regular logging of symptoms, via the c-19 covid symptom study app 5 , enabling measurement of antibody response to covid-19 with regards to clinical symptoms. the inclusion criteria for the study were residence within an 80-mile radius of the cohort headquarters st thomas' hospital in central london, active use of the covid symptom study app, and availability for visit between 27 th april and 2 nd june 2020. the exclusion criterion (for safety reasons) was report of recent symptoms indicating potential covid-19 at the time of the study or within 14 days prior, which participants were required to confirm via telephone consultation. ethics approval for the study was granted by nhs north west -liverpool east research ethics committee (rec reference 19/nw/0187), iras id 258513, and all participants gave written informed consent. 895 twins met the geographical and logging based inclusion criteria. of these, 231 could not be contacted, 154 declined a visit as they were either not interested or did not want a home visit due to being in the high risk category for covid-19, or because they or a member of their household were currently experiencing symptoms suggestive of covid-19 infection and were not able or willing to be seen later (n = 22). a further 85 could not be visited within the study period. visited participants comprised 431 twinsuk volunteers, aged between 19 and 86, of whom 367 (85%) were female. they were visited in their home for antibody testing between 27th april and 2nd june 2020, an average of 18 days from the first peak of the pandemic in the uk on the 3rd april 2020. the demographics of the study participants in the context of the wider geographical area in which they reside are summarized in table 1 serum samples obtained on home visits were tested for igg and igm binding to sars-cov-2 s and n proteins using enzyme linked immunosorbent assay (elisa) using serum diluted at 1:50. the elisa serology methods used in this study have been previously described. 9 briefly, the n/s elisa demonstrated 100 % specificity (as determined using 300 pre-covid-19 serum samples), and sensitivity was secondary to time post infection, improving in performance with increasing days from initial infection: after 10 days it was 84.7%. after 14 days it was 87.0% and at greater than 20 days it was 96.4% sensitive. a participant was considered seropositive if an igg response (optical density (od) value) to both n and s was detected that was 4-fold above the background of the assay. this cut-off is based on the analysis of 300+ pre-covid-19 serum samples. 9 ￼ the elisa was validated by a separate laboratory at imperial college london, who employed a hybrid double antigen bridging assay (daba) using immobilised s1 and hrplabelled s1 receptor binding domain (rbd) of known high specificity (>99.9%) to compare antibody reactivity in 50 unselected samples. of these, 10 were reactive for rbd using the hybrid daba whereas the n/s elisa method determined 9 of these to be seropositive. all samples found reactive in the n/s elisa were also shown to be reactive using the hybrid daba. thus, comparing to hybrid daba, our elisa showed sensitivity 90% (95% ci 60-99, and specificity 100% (95% ci 93-100), 95% cis were calculated using the clopper pearson method. 7 testing for active the presence of sars-cov-2 buccal swabs obtained during the home visits were used to test for current sars-cov-2 rna (and by inference active replication) using rt-pcr as previously described by lista et al. 10 briefly, nasopharyngeal swab samples were heat inactivated at 70 °c for 30 min and 140 µl were used to extract rna with the beckman coulter rnadvance blood kit, ending with elution in 50 µl of water. for qpcr, the us cdc designed primer/probe set were used for the n gene (n1 and n2) and rnasep with 5 µl of rna sample and taqman fast virus 1step master mix (thermofisher). longitudinal experience of potential covid-19 symptoms was prospectively logged by participants via the c-19 covid symptom study app, the primary app for self-report of symptoms during the pandemic in the uk. 5 the c-19 symptom study app has been developed by the health science company zoe in collaboration with king's college london. thus far, 4,007,688 participants have logged symptoms, providing invaluable epidemiologic information with regards to the pandemic. the symptoms which app participants were asked to record are listed in table 2 . algorithm for predicting prior covid-19 from symptoms reported a predictive algorithm for identification of covid-19 using longitudinal symptoms reported via the c-19 app was used to identify participants who were predicted to have had prior symptomatic infection, using the method recently described by menni et al. 11 briefly, the algorithm was developed from symptoms in people testing positive for sars-cov-2 using rt-pcr. the formula includes two core symptoms (anosmia and cough), two non-core symptoms (fatigue and skipped meals), in addition to participant age and sex. in addition, participants were asked whether they had experienced any symptoms suggestive of covid-19 prior to launch of the app on 24 th march 2020. consideration of serology in relation to longitudinal symptoms logs and statistical analysis 382 participants had regularly logged their symptoms prospectively from app launch on 24 th march, and also reported on symptoms retrospectively prior to this date. symptom profiling included core covid-19 symptoms and general symptoms, in addition to algorithm prediction of prior covid-19. participants were delineated according to antibody status in relation to reported symptoms and predicted covid-19 prior infection. using the status regarding prior sars-cov-2 infection as predicted by the algorithm, we calculated seroprevalence amongst those who were likely to have had covid-19. in these participants, we investigated association of demographic factors: age, sex, and bmi, with seroprevalence (student's t test for difference between groups). confidence intervals for proportions were calculated using the clopper pearson method. 7 all analyses were performed using the r software environment for statistical computing. in our sample of 431 adults aged 19-89 years living in london and south-east england, seroprevalence using our elisa assay was demonstrated to be 12% (51 participants, 95% ci = 9.1-15.2). during the study period, beginning 24 th march 2020, a total of 382 participants completed longitudinal logging of symptoms via the c-19 covid symptom study app, providing detailed insight into clinical presentation of this community sample. the median number of app entries to record symptoms, per individual, was 51 days (iqr 30 to 64). 48 (12%; 95% ci 9-16) were seropositive, of whom, 9/48 (19%; 95% ci 9-33) were completely asymptomatic, including no prior symptoms before the app launch. 27% reported only noncore symptoms, (i.e. neither fever, persistent cough, nor anosmia; table 3 ), and would have been reported as asymptomatic in other surveys. the symptom which most strongly predicted seropositivity was anosmia. of participants reporting anosmia, 47.9% had detectable igg to sars-cov-2. the specificity of anosmia for seropositivity was 95%, whereas for fever, cough, and anosmia combined was less at 85%. both were not highly sensitive with (anosmia 48%; all core symptoms together 73%). there were 34 individuals in the cohort who were predicted to have had a sars-cov-2 infection using the app based algorithm. the algorithm correlated better with serology than did core symptoms alone ( figure 1b; table 3 ). of the 34 individuals with predicted covid-19, 52% (18) were seropositive ( factors associated with seroprevalence amongst those with predicted covid-19 on comparison of the seropositive and seronegative participants with predicted covid-19 (n = 34, out of 381 participants who prospectively logged their symptoms), seropositive participants were older (median age seropositive 48, median age seronegative 36; p = 0.046). no difference in sex (% female of seropositive participants 72, and 87 for seronegative) or bmi (median 23.8 seropositive; 22.8 seronegative) was evident between the groups. understanding seroprevalence is important in order to estimate epidemiological spread of covid-19 through the population, to inform on the potential efficacy of vaccination, and for antibody fold change above background antibody fold change above background the concept of herd immunity which may eventually be achieved via a combination of vaccination and convalescence. 12 we estimate the seroprevalence rate within our sample to be 12%, higher than the ons estimate for the uk of 6% (studies summarized in supplementary table 1 ), but could be compatible given the potentially higher prevalence in london and the south-east and the fact that, unlike the ons surveys, we only included adults. our results indicate that a substantial portion (19%) of those who have detectable antibodies to sars-cov-2 were entirely asymptomatic, confirming that even those who have clinically very mild disease with no perceivable symptoms may produce antibodies. this frequency is substantially lower than most other estimates of asymptomatic covid-19, which we believe to be due to our more complete assessment of symptoms over time. indeed, this may still be an overestimate, as, at the start of the survey during the peak epidemic, we did not ask about rashes, which our data suggest are present in 8% of positive cases. 13 our estimate relates to asymptomatic development of specific antibodies, rather than asymptomatic carriage of sars-cov-2, as it has been reported that not all who have covid-19 develop detectable antibodies. 14, 15 in a population study by solbach et al. of 110 german participants who had had pcr confirmed prior covid-19 and who self-reported symptoms, there were ten asymptomatic individuals (11%). of these ten asymptomatic cases, four were seropositive, and the remainder had no detectable antibodies in two consecutive analyses. 15 it is clear that asymptomatic disease holds important implications with regards to transmission, as asymptomatic individuals are unaware of their infected status, and may support a significantly longer period of viral shedding, thereby exacerbating the potency of transmission potential. 16, 17 without parallel pcr testing alongside symptom tracking, we cannot be certain whether seronegative individuals reporting covid-19 symptoms either did not develop detectable sars-cov-2 antibodies, their antibody levels had declined to undetectable 18 or whether their symptoms related to another infection or condition. within the 34 people who were predicted to have had covid-19 using the app based algorithm, there was approximately a 50/50 likelihood of participants having a detectable antibody response (52% were seropositive, whilst 48% had no detectable antibodies). our understanding of the human immune response to sars-cov-2 continues to improve, and emerging evidence indicates that t cell mediated immunity plays an important role in the immune control of 20 for example, a small community-based study of patients and their household families showed than an unexpected six out of eight family members who had been infected demonstrated a covid-19-antigen specific t cell response, but had no detectable antibodies. 21 speculatively, therefore, t cell mediated responses may afford efficacious immunity in the absence of a detectable antibody response. in the participants with symptoms predictive of covid-19 using the algorithm, age differed by antibody response: seropositive individuals being older than those who were seronegative (p = 0.046). this may be secondary to increased shielding of older participants, however it is consistent with previous reports of a positive correlation of igg covid-19 antibodies with age. 14,17 whether t cell mediated reponses are present or more prominent in younger individuals is part of an ongoing follow-on of this study. using longitudinal symptoms logged using the app, we demonstrated that of all the individual clinical symptoms, anosmia was the strongest indicator of seropositivity; the specificity of anosmia was 95% and sensitivity was 48 %, whilst the specificity of all core symptoms combined was 85%. using data from the same covid symptom study app, our group was able to demonstrate previously that anosmia is a core symptom of covid-19, in addition to the prior recognised symptoms of fever and persistent cough. 8, 13 . these results further highlight anosmia as an important core covid-19 symptom which correlates strongly with both swab positivity and antibody response. this study, like many similar surveys has a number of limitations. an overarching consideration with regards to seroprevalence to sars-cov-2 is the longevity of seropositivity. in a longitudinal study of rt-pcr confirmed prior covid-19 cases by doores et al., individuals mounted a range of antibody responses, and a decline in levels and virus neutralisation was typically observable within three months of the onset of symptoms. 18 thus, it is plausible that antibody responses would have fallen below the level of detection by the time of assessment in some of our study participants. estimation of prior covid-19 infection was via a symptom based algorithm as pcr confirmation had not been undertaken, nevertheless, we have previously published that this algorithm had a high ppv of close to 80 percent and was trained on symptoms of 7178 swab positive individuals. 11 no volunteer cohort is fully representative of the general population, but we sampled a range of age and ethnicities and included people from a wide area of deprived and affluent neighbourhoods and with a range of bmi. despite this, the cohort is detectably more affluent and white,than the general population which would serve to reduce our estimate of prevalence, give the extra burden of disease shouldered by less affluent groups and people of black, asian and minority ethnic background. additionally we use the index of multiple deprivation which is an area-level indicator rather than individual level socioeconomicposition. the excess of females in our data set reflects the twinsuk cohort and, so far, differences in seropositivity between genders have been minor in other datasets. it is possible that women report symptoms more readily than men which means that the true assymtomatic rate may be higher. the data are from london and the south east and seroprevalence data from this study will therefore not be generalisable to the whole of the country or to children. further work is planned to extend the study using larger numbers from the 4 million app users who reported symptoms to address many of the underlying factors influencing swab positivity and antibody responses. the present study is underpowered in this regard with the asymptomatic seropositive group comprising 9 individuals out of a total of 48 seropositive participants. in conclusion, we estimated that 12% of the se england population were seropositive between 27 th april and 2 nd june, an average of 18 days from the peak of the epidemic in the uk. we estimate the asymptomatic rate to be 19%. anosmia was the symptom with the highest specificity for seropositivity. these data should be useful for both epidemiology and public health planning and reinforces the need to collect good symptom data as neither pcr testing nor antibody tests adequately capture all disease. ons diagnostic value of skin manifestation of sars-cov-2 infection convergent antibody responses to sars-cov-2 in convalescent individuals antibody profiling of covid-19 patients in an urban low-incidence region in northern germany clinical and immunological assessment of asymptomatic sars-cov-2 infections prevalence of asymptomatic sars-cov-2 infection longitudinal evaluation and decline of antibody responses in sars-cov-2 infection robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19 sars-cov-2-specific t cell immunity in cases of covid-19 and sars, and uninfected controls intrafamilial exposure to sars-cov-2 induces cellular immune response without seroconversion. infectious diseases (except hiv/aids) this study was supported by a covid urgent response grant from the chronic disease research foundation (cdrf). zoe global limited developed the app with the guidance of clinicians. investigators received support from the wellcome trust, the mrc/bhf, alzheimer's society, eu, nihr, cdrf, rst-ukri/mrc and the nihr-funded bioresource, clinical research facility and brc based at gstt nhs foundation trust in partnership with kcl. we thank the volunteers of twinsuk without whom this work would not be possible, and all participants who entered data into the c-19 covid symptom study app. we thank the staff of zoe global, the department of twin research at king's college london and the clinical and translational epidemiology unit at massachusetts general hospital for tireless work in contributing to the running of the study and data collection. we thank e. segal and his laboratory for helpful input. thank you to philip brouwer, marit van gils and rogier sanders (university of amsterdam) for the s protein construct, and leo james, jakub luptak and leo kiss (lmb) for the provision of purified n protein. cjs, ts & mhm designed and implemented the study. kjd led methods for serology. js, cg, sa, ab, kjas, oh and nh undertook the elisas, cr and mom undertook the dabas, and pmm, kp, mjl, red, hs, tjam and ejs performed the pcr work. sc analysed the elisa and app data. cjs supervised the analysis. pmw contributed to data analysis and drafted the manuscript. rb curated the demographic data. all authors contributed to reviewing of the manuscript. tds is a consultant to zoe global. jcp and jw are employees of zoe global. protein microarray using serum samples. testing of 7773 individuals in the usa.antigens: s1 glycoprotein, receptor binding domain (rbd), s2 glycoprotein and sars-cov2 using serum samples. .031) and were higher in participants with more than 10 days since onset of symptoms (p-value=0.022), and iga levels were higher in symptomatic than asymptomatic subjects (p-value=0.041).garcia-basteiro et al. supplementary table 1 summary of prior studies of the general population antibody response to sars-cov-2.supplementary figure 1 . antibody response in relation to symptom pattern. core symptoms are defined as fever, cough and/or anosmia. colour depicts symptom pattern of core symptoms (red), non-core symptoms (green), and asymptomatic (blue). shape depicts antibody status of detectable (positive; circle) or undetectable (negative; triangle). antibody level denotes fold change above background, and a threshold of <4 indicates seropositivity. predicted covid-19 (predcovid) is the algorithm score predicting which participants are likely to have had covid-19 using symptoms reported via the c-19 app. the threshold for predicted covid-19 of 0.5 is marked.visualisation of longitudinal symptom reporting using heat maps demonstrated that anosmia clearly delineated seropositive from seronegative individuals (supplementary figure 2) . key: cord-295074-fsbp4fky authors: broor, shobha; dawood, fatimah s.; pandey, bharti g.; saha, siddhartha; gupta, vivek; krishnan, anand; rai, sanjay; singh, pratibha; erdman, dean; lal, renu b. title: rates of respiratory virus-associated hospitalization in children aged <5 years in rural northern india date: 2013-11-21 journal: j infect doi: 10.1016/j.jinf.2013.11.005 sha: doc_id: 295074 cord_uid: fsbp4fky objectives: though respiratory viruses are thought to cause substantial morbidity globally in children aged <5 years, the incidence of severe respiratory virus infections in children is unknown in india where 20% of the world's children live. methods: during august 2009–july 2011, prospective population-based surveillance was conducted for hospitalizations of children aged <5 years in a rural community in haryana state. clinical data and respiratory specimens were collected. swabs were tested by rt-pcr for influenza and parainfluenza viruses, respiratory syncytial virus (rsv), human metapneumovirus, coronaviruses, and adenovirus. average annual hospitalization incidence was calculated using census data and adjusted for hospitalizations reported to occur at non-study hospitals according to a comunity healthcare utilization survey. results: of 245 hospitalized children, respiratory viruses were detected among 98 (40%), of whom 92 (94%) had fever or respiratory symptoms. rsv accounted for the highest virus-associated hospitalization incidence (34.6/10,000, 95% ci 26.3–44.7) and 20% of hospitalizations. there were 11.8/10,000 (95% ci 7.9–18.4) influenza-associated hospitalizations (7% of hospitalizations). rsv and influenza virus detection peaked in winter (november–february) and rainy seasons (july), respectively. conclusion: respiratory viruses were associated with a substantial proportion of hospitalizations among young children in a rural indian community. public health research and prevention in india should consider targeting rsv and influenza in young children. rates of respiratory virus-associated hospitalization in children aged <5 years in rural northern india introduction acute respiratory infections are recognized as an important cause of mortality, hospitalization, and healthcare utilization in young children globally. 1e3 respiratory virus infections are increasingly recognized as major contributors to the burden of severe acute respiratory illness in many countries due to expanding global surveillance and the advent of improved molecular diagnostic testing for respiratory viruses. 4e8 in studies of respiratory virus detection among children hospitalized with respiratory illness from different parts of the world, rsv and influenza are frequently associated with a substantial proportion of hospitalizations. 9e14 these findings are of public health importance because effective and safe influenza vaccines are available but not widely used in many countries, and development of respiratory syncytial virus (rsv) vaccines continues to be an area of intense study although no licensed rsv vaccine is available. 12, 15, 16 understanding the incidence of respiratory virus-associated severe illness and the timing of respiratory virus circulation is critical to inform research priorities and policy decisions about introduction of available respiratory virus vaccines for children. india is the second most populous country in the world with 20% of the world's population of children aged <5 years, and almost a third of global pneumonia cases among children aged <5 years are thought to occur in india. 3 public health policies that effectively address causes of severe respiratory illness among children in india would have a substantial impact on global child morbidity and mortality. however, the burden of respiratory virus-associated severe illness among young children in india is unknown. in addition, the few studies that evaluated respiratory viral etiologies of severe illness among children in india were conducted before the advent of newer and more sensitive diagnostic tests, including tests to detect more recently discovered viruses. 17e20 using data from population-based surveillance of approximately 9500 children for hospitalizations for acute medical illness in rural northern india and concomitant testing for respiratory viruses by real-time reverse transcription polymerase chain reaction (rrt-pcr), we estimate the incidence of respiratory virus-associated hospitalizations among children aged <5 years. we also describe the timing of virus circulation and clinical presentation of children hospitalized with predominant viruses. the comprehensive rural health services project (crhsp), ballabgarh study site includes a 323 square kilometer area in haryana state, about 40 km south of new delhi. the climate is temperate with a defined colder winter season during novemberemarch and rainy season during julyeseptember. as part of the crhsp, a health and demographic surveillance system (hdss) was maintained under which all residents of the crhsp study site were enrolled in a computerized database with unique identification numbers. the hdss tracked major events such as births, deaths, marriages and migrations. during the study period, the crhsp study site included a population of approximately 88,000 persons including approximately 9500 children aged <5 years in 28 villages. the main providers of inpatient care to the crhsp population are a government-funded secondary level facility with 60 beds that provides outpatient and inpatient care and serves 15e20% of the crhsp population, two other government-funded secondary level facilities, and a large number of private health facilities (ranging in size from 5 to 35 beds) that provide inpatient and outpatient health services. health facilities are largely situated outside crhsp villages within a range of 3e20 km and largely accessible by two/three wheelers. most facilities have the resources to care for patients requiring supplemental oxygen but transfer patients requiring mechanical ventilation and intensive level to tertiary care facilities outside the district. hospitalized patients were enrolled from the three secondary level facilities and 18 private facilities in ballabgarh and faridabad towns where patients from the crhsp area were likely to seek inpatient care corroborated by health utilization survey. patients were eligible for enrollment if they were residents of the crhsp area and were being hospitalized overnight with any acute medical illness, excluding hospitalizations for the following conditions assumed to be unlikely to be related to respiratory infection: trauma, diarrhea without fever, elective surgery, accidental poisonings, elective blood transfusions, or orthopedic or ophthalmologic conditions. during august 2009ejuly 2011, patients were prospectively enrolled in the study as previously described. 21 data on demographic characteristics, medical history, and clinical symptoms were obtained by interview of patients' caregivers. data on clinical signs were abstracted from the medical record using a standardized data collection form. respiratory specimen samples were collected by study doctors from enrolled patients within 24 h of admission using polyester swabs. during augustedecember 2010, study investigators visited all houses in the 28 villages of the crhsp area to conduct a healthcare utilization survey using a standardized questionnaire that was field testing in previous years; 90% of households in the area completed the survey. the survey asked whether any member of the household had been admitted to a hospital for an overnight stay during the preceding year (using a reference period of august 1, 2009ejuly 31, 2010 . for each reported hospitalization, the survey asked about the location and reason for hospitalization, and field workers attempted to validate reports with any available documentation related to the hospitalization. combined throat and nasal swabs were collected from each participant (nasal swabs alone in infants). the swabs were transported in viral transport media on ice to the all india institute of medical sciences (aiims) laboratory within 24 h. samples were then divided into aliquots for respiratory virus detection by rrt-pcr. testing for influenza viruses and influenza virus subtyping was conducted at aiims using us centers for disease control and prevention (cdc) protocols 22 and after laboratory staff received cdc-sponsored training and the laboratory underwent quality control assessment by cdc. testing for non-influenza respiratory viruses was also conducted at aiims using highly sensitive rrt-pcr assays for respiratory syncytial virus (rsv), human metapneumovirus (hmpv), human parainfluenza viruses 1e3 (piv 1e3), adenovirus, and human coronavirus 229e using cdc protocols 23e25 ; protocol are available from cdc upon request. baseline characteristics, clinical symptoms and signs, and length of hospitalization were compared between children with and without respiratory virus detection using bivariate analysis. frequencies of clinical symptoms and signs and median length of hospitalization were also calculated for children with rsv and influenza, the most commonly detected viruses. tachypnea was defined based on integrated management of childhood illness criteria for fast breathing as 60 breaths per minute in children aged <2 months, 50 in children aged 2e11 months, and 40 in children aged 1e5 years. 26 hypoxia was defined as an oxygen saturation of <92% by pulse oximetry at admission. chisquared test or fisher's exact test was used to calculate pvalues for categorical variables and the wilcoxon test was used to compare continuous variables (sas, version 9.2, sas institute inc., cary, nc). since all children hospitalized with acute medical illness were enrolled, the proportion of respiratory virus detections among children with and without fever or key respiratory signs or symptoms was evaluated. fever was defined as either measured temperature >38.0 celsius at admission or parental report of fever because antipyretic use was common in the study community. key respiratory symptoms or signs were defined as parental report of cough or fast breathing or physician exam findings of tachypnea, crepitations, wheezing, nasal flaring, chest indrawing, grunting, or stridor. inclusion of nasal discharge did not change the proportion of children meeting criteria for key respiratory symptoms or signs. all children, regardless of symptoms or signs, were included in all subsequent analyses. using data from the healthcare utilization survey, the proportion of hospitalizations among children in the study community that occurred at study facilities was calculated as reported hospitalizations at study facilities divided by total reported hospitalizations; 95% confidence limits were calculated using the wald method. average annual cumulative incidences of hospitalizations associated with detection of each respiratory virus were calculated for children aged <1 year, 1e4 years, and <5 years. incidences were also calculated for children aged <6 months for rsv and influenza since maternal immunization with rsv and among the 98 children with respiratory virus-associated illness, history of fever (82%) and cough (69%) were the most commonly reported symptoms. parental report of cough, nasal discharge or congestion, and fast breathing and exam findings of hypoxia, crepitations, wheezing, and increased work of breathing were more common in children with respiratory virus-associated hospitalizations than those without (table 2) ; the median length of hospitalization was similar in both groups (4 vs. 3 days, p z 0.05). none of the children died. a larger proportion of children with rsv detection compared to children with other respiratory viruses had a reported history of fast breathing (51% vs. 26%, p z 0.01) and exam findings of increased work of breathing (32% vs. 6%, p < 0.01), crepitations (52% vs. 21%, p < 0.01), and wheezing (40% vs. 10%, p < 0.01). of 9509 children aged <5 years residing in crhsp area, 8773 (92%) were included in the health utilization survey, and 75% (95% ci 74e76%) of all acute medical hospitalizations reported on the health utilization survey occurred at study facilities ( respiratory viruses were detected almost year-round among children hospitalized with acute medical illnesses (fig. 1) . rsv detection occurred from november through may with a longer period of detection during 2009e2010 than during 2011 (fig. 1) . in contrast, influenza virus detection peaked during the rainy season in juneejuly when influenza accounted for 20e42% of monthly acute medical illness hospitalizations. detection of other respiratory viruses occurred sporadically throughout the year. based on surveillance of approximately 9500 children, this study is the first prospective, population-based study to measure the incidence of respiratory virus-associated hospitalizations among children aged <5 years in rural india. we found that respiratory virus infections were associated with 40% of acute medical illness hospitalizations among children aged <5 years, and one in five children hospitalized for acute medical illness had rsv infection. consistent with prior studies, we also found that virus-associated hospitalization rates were highest among children aged <1 year. rsv was the predominant virus identified among children aged <1 year. among children aged 1e4 years, incidence rates of rsv and influenza viruses were similar. rsv and influenza viruses circulated with clearly defined but different seasonality and were infrequently detected among children without fever or respiratory symptoms or signs, similar to prior studies. 9, 10, 30 the incidence of rsv-associated hospitalizations in our study community was substantial, with an incidence of 35 per 10,000 child-years among children aged <5 years and 99 per 10,000 child-years among children aged <1 year. many studies have reported rsv as the most common respiratory virus resulting in hospitalization in young children, but relatively few have estimated the incidence of rsvassociated hospitalizations. rsv-associated hospitalization rates per 10,000 children ranged from 109 to 274 among children aged <1 year in studies from rural thailand, indonesia, the united kingdom, and the united states; 25e30 among children aged <5 years in hong kong and the united states; and 194 and 450 among children aged <5 years without and with hiv infection in south africa. our estimates are similar to those of prior studies from the united states, europe and asia. given the high incidence of rsv-associated hospitalization in our study, availability of an effective and accessible rsv vaccine for young children in india would profoundly impact hospitalization rates in this age group. because the incidence of rsv-associated hospitalization is highest in infants aged <6 months in whom maternal antibody interference with immune responses may make vaccination challenging, rsv vaccination 0.7 (0e3.9) 0.9 (0e5.5) a adjusted to account for hospitalizations that occurred at non-study facilities based on data from healthcare utilization survey. b children with co-detection of more than 1 respiratory virus were included in the incidence estimate of each respiratory virus detected. c among infants aged <6 months, the adjusted incidence of hospitalization for rsv was 136.8 hospitalizations/10,000 (95% ci 91.1e207.6). d among infants aged <6 months, the adjusted incidence of hospitalization for influenza was 17.8 hospitalizations/10,000 (95% ci 6.3e51.9). of pregnant women has been suggested as another potential vaccination strategy. 27e29 though licensed rsv vaccines are not currently available, candidate vaccines are under development 15, 16 and data on rsv disease burden will be important for policy decisions about rsv vaccine introduction if a licensed vaccine becomes available. although we found that influenza viruses were associated with a lower incidence of hospitalization than rsv among children aged <5 years, influenza-associated hospitalizations accounted for 7% of all acute medical hospitalizations and up to 42% of monthly hospitalizations during peak circulation. a similar population-based study conducted in western india during the same time period as ours found similar influenza-associated hospitalization rates among children aged <1 year and higher rates among children aged 1e4 years. 31 the contribution of influenza to severe respiratory illness among children in india deserves attention because influenza vaccines are not currently recommended and are not widely available in india. both traditional inactivated influenza vaccines 32e34 and live attenuated influenza vaccines 35e38 have been shown to be effective against influenza in children in high-income countries. additional studies are needed to further develop the evidence base for policy makers in india to decide whether to support influenza vaccination recommendations for children. these studies should include data on the incidence of severe influenza in other regions of india, the effectiveness of influenza vaccine among indian children, and the costs of influenza in india. a study of influenza vaccine effectiveness among children aged <5 years in rural india is underway. 39 the timing of respiratory virus circulation varies globally. rsv and influenza viruses circulate during the colder winter months in some but not all temperate countries. however, influenza viruses circulate year-round in some tropical countries and both rsv and influenza virus circulation seems to coincide with increased rainfall in some places. 40, 41 india is a large and geographically diverse country with temperate, sub-tropical and tropical climates. in our study, conducted in a temperate area, we found that rsv and influenza circulated during clearly defined but different periods. studies from western india suggest that influenza virus circulation is less defined there, with peaks during the rainy season but perennial influenza virus circulation. 20, 31 additional studies of influenza virus seasonality in other regions of india are needed to determine optimal timing for influenza vaccination and other prevention measures. the advent of highly sensitive pcr assays for respiratory viruses allows easier detection of a wider array of viruses but also presents the challenge of interpreting positive test results. prior studies have shown that some respiratory viruses are frequently detected by pcr among children without fever or respiratory symptoms. 9, 10, 30 respiratory virus detection in these children may reflect acute infection with atypical symptoms or prolonged shedding of virus from a prior infection highlighting the utility of a control group of children without acute illness to aid interpretation of test results. our study did not have a control group, but the large majority of children with respiratory virus detection had fever or respiratory findings that support the clinical diagnosis of respiratory infection. our estimates of rsv and influenza-associated hospitalization incidence are supported by prior studies of hospitalized children in which detection of these viruses was rare from control groups comprised of hospitalized children without fever or respiratory symptoms or non-hospitalized children. though adenoviruses, coronaviruses, human metapneumovirus, and parainfluenza viruses have all been previously implicated in the pathogenesis of severe illness, our hospitalization incidence estimates for these viruses should be interpreted with more caution as detection of these viruses has varied among control groups in other studies. 9, 10, 30 several points should be considered when interpreting our findings. first, our study was conducted at primary and secondary level health facilities without capacity to provide mechanical ventilation and intensive care for critically ill children. thus, we may have missed hospitalizations of children with more severe illness who were directly admitted to tertiary care hospitals outside the study area. our study community may also have been too small to evaluate the frequency of rare but severe outcomes such as death. second, although we attempted to collect the discharge diagnosis of all enrolled children, these data were largely absent from the medical record and therefore could not be used to aid interpretation of respiratory virus testing results. third, we included testing results for only a select group of respiratory viruses previously identified as being associated with a large proportion of respiratory illnesses in children. therefore, our results reflect the burden of respiratory virus-associated hospitalizations caused by this select group of viruses. we did not include test results for rhinoviruses because interpretation of rhinovirus detection in the absence of a control group is difficult, although rhinovirus was the most commonly detected virus in children with respiratory illness in some prior studies. additionally, we did not conduct testing for coronaviruses other than coronavirus 229e, and may have underestimated the incidence of coronavirus-associated hospitalization in the study community. lastly, respiratory virus circulation and incidence is known to vary by year. our study was conducted over a two year period and we present average annual cumulative incidences of virus-associated hospitalization. additional studies of respiratory virusassociated disease burden during additional years will be useful for comparison with our findings. the findings and conclusions in this report are those of the authors and do not necessarily represent the views of the centers for disease control and prevention. this study was supported in part by cooperative agreements u01 ip000206 from the centers for disease control and prevention, atlanta, us. the authors do not have any relevant conflicts of interest to declare. global, regional, and national causes of child mortality: an updated systematic analysis for 2010 with time trends since global and regional burden of hospital admissions for severe acute lower respiratory infections in young children in 2010: a systematic analysis global burden of childhood pneumonia and diarrhoea global burden of respiratory infections due to seasonal influenza in young children: a systematic review and meta-analysis viral infections of the lower respiratory tract 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respiratory syncytial virus (rsv) vaccines for infants: safety, immunogenicity, and efficacy an evaluation of the emerging interventions against respiratory syncytial virus (rsv)-associated acute lower respiratory infections in children etiology of acute respiratory infections in children in tropical southern india etiology of acute lower respiratory tract infection viral etiology of acute respiratory infections in south indian children respiratory viruses in acute respiratory tract infections in western india validity of clinical case definitions for influenza surveillance among hospitalized patients: results from a rural community in north india. influenza and other respiratory viruses world health organization. cdc protocol of realtime rtpcr for swine flu influenza a (h1n1) the burden of hospitalized lower respiratory tract infection due to respiratory syncytial virus in rural thailand pring-akerblom p. rapid and quantitative detection of human adenovirus dna by real-time pcr human coronavirus infections in rural thailand: a comprehensive study using real-time reverse-transcription polymerase chain reaction assays. journal of infectious diseases who guidelines approved by the guidelines review committee pocket book of hospital care for children: guidelines for the management of common childhood illnesses effectiveness of maternal influenza immunization in mothers and infants maternal immunization against viral disease immunopathology of rsv infection: prospects for developing vaccines without this complication. reviews in medical virology viral respiratory infections in hospitalized and community control children in alaska burden of seasonal and pandemic influenza-associated hospitalization during and after 2009 a(h1n1)pdm09 pandemic in a rural community in india vaccine effectiveness against laboratoryconfirmed influenza in children 6 to 59 months of age during the 2003e2004 and 2004e2005 influenza seasons vaccine effectiveness against medically attended, laboratory-confirmed influenza among children aged 6 to 59 months influenza vaccine effectiveness among children 6 to 59 months of age during 2 influenza seasons: a case-cohort study prevention of otitis media in children with live attenuated influenza vaccine given intranasally efficacy and safety of 1 and 2 doses of live attenuated influenza vaccine in vaccine-naive children efficacy and safety of a live attenuated, cold-adapted influenza vaccine, trivalent against cultureconfirmed influenza in young children in asia. pediatric infectious disease safety, efficacy, and effectiveness of coldadapted influenza vaccine-trivalent against communityacquired, culture-confirmed influenza in young children attending day care design and initiation of a study to assess the direct and indirect effects of influenza vaccine given to children in rural india seasonality, timing, and climate drivers of influenza activity worldwide epidemiology and seasonality of respiratory tract virus infections in the tropics. paediatric respiratory reviews the authors wish to acknowledge the division of viral diseases, centers for disease control and prevention for providing assay kits used for laboratory testing in this study. key: cord-301491-frv4c5ny authors: ran, jinjun; zhao, shi; han, lefei; chen, dieyi; yang, zuyao; yang, lin; wang, maggie h; he, daihai title: the ambient ozone and covid-19 transmissibility in china: a data-driven ecological study of 154 cities date: 2020-07-08 journal: j infect doi: 10.1016/j.jinf.2020.07.011 sha: doc_id: 301491 cord_uid: frv4c5ny • we quantified the covid-19 transmissibility by the basic reproductive number. • the covid-19 transmissibility could be negatively associated with ambient ozone. • the daily 1-h maximum ozone might cover 7.6% of the transmissibility variability. • it echoes a previous study of negative effects of ozone on the flu transmission. • the ground-level ozone may be a “double-edged sword” to public health. the coronavirus disease 2019 (covid-19) has triggered a pandemic and is still spreading around the world. exploring the environmental factors that associate with the prevalence and transmission would improve the understanding of covid-19 and contribute to the long-term control strategies of the outbreak. 1,2 besides the meteorological factors 3 , to what extend do common air pollutants may affect the covid-19 transmission remains unclear. to fill this knowledge gap, we examined the associations between common air pollutants and covid-19 transmissibility in chinese cities. we obtained the daily count of covid-19 cases for each chinese city from the chinese provincial health agencies and china national health commission (cnhc). 4 we quantified the transmissibility of covid-19 by using the basic reproduction number (r 0 ), a unit-free measure of infectivity that is commonly used in infectious disease epidemiology. then, we filtered out a case series of each city from the first-case occurrence to the following 16 days for estimating the r 0. 5 we calculated the r 0 for each chinese city with a gamma distribution having mean (±sd) values of 5.5 (±3.3) days for the serial interval averaged from previous estimations. the calculation in detail was shown in supplementary materials. as r 0 is a measurement regarding the population as a whole, the demographic heterogeneities across each city could be thus neglected. we obtained air pollutants monitoring data in 1,642 stations from china national environmental center between december 10, 2019 and february 29, 2020, including ozone (o 3 ) with three metrics daily average, daily 1-h maximum, and daily 8-h maximum, as well as other criteria pollutants. after calculating their average values across the time period, we computed a raster for each pollutant by the kriging interpolation. we then extracted and linked pollutants' values to each chinese city. the spatial distribution of o 3 (1-h maximum) was shown in fig. 1a . the corresponding meteorological factors were addressed and matched by the same method. we adopted the nonlinear (i) univariable, and (ii) multivariable, i.e., adjusted by temperature and absolute humidity, regression analyses to explore the association between r 0 and each pollutant. we carried out the fitting procedure considering two weighting schemes that are (i) equal-weighted, and (ii) weighted by the total number of cases from each city. in addition, we employed the spline regression and permutation and perturbation analysis as sensitivity analysis for validation. a total of 154 chinese cities were detected with the covid-19 outbreak (r 0 > 1) and were included in the regression analysis. the maximal r 0 was estimated at 2.5 (95%ci: 2.4−2.6) in wuhan (table 1) , which is largely consistent with previous estimates. 6, 7 their spatial distribution was shown in fig. 1b . we found that the r 0 of covid-19 was negatively associated with the daily 1-h maximum o 3 concentration for both univariable and multivariable analyses and both weighting schemes significantly and consistently ( fig. 1c and fig. s1 ). by contrast, associations with other pollutants failed to reach statistical significance. the average concentration of daily 1-h maximum o 3 across the 154 cities has a median at 73.1 µg/m 3 and ranges from 51.6 µg/m 3 to 106.7 µg/m 3 (table 1) . the spearman's ranked correlation coefficient between daily 1-h maximum o 3 and r 0 was −0.21 (95% ci: −0.33, −0.02) (table s1 ). we estimated that the ambient o 3 could solely explain 7.6% of the variability in the r 0 of covid-19 in terms of mcfadden's pseudo-r-squared for the univariable analysis, and this term increased to 9.3% in the multivariable analysis. a similar protective association between o 3 and the transmissibility of influenza was found in a previous study. 8 the spline regression validated the negative association (fig. s2) , and the permutation and perturbation tests represented that the observed relationship was unlikely due to chance. to our best knowledge, this is the first study on the association of covid-19 transmissibility with ambient ozone. ambient ozone was associated in reducing covid-19 transmissibility probably due to its virucidal activity and possible impact on host defense. ozone gas is highly effective in broadspectrum disinfection and sterilization against many repertory infections, such as sars and influenza viruses. 9 ozone-primed immunity against viral infection might also play a critical role in the reduction of covid-19 infectivity. specifically, exposure to ambient ozone may trigger slight allergic reactions to human, which may enhance pulmonary innate immunity. two significances of the current study were emphasized here. first, as the weather getting warmer in the northern hemisphere, the ambient ozone concentration in many places will gradually escalate, and this could be a good sign for the covid-19 control regionally. second, as a highly reactive oxidant air composition, ground-level ozone is also a "double-edged sword" to public health. we advocate that more attention on the good side of ambient ozone and the trade-off of ambient ozone control to avoid over-vigilance. regional air pollution persistence links to covid19 infection zoning the role of the environment and its pollution in the prevalence of covid-19 no association of covid-19 transmission with temperature or uv radiation in chinese cities open-source analytics tools for studying the covid-19 coronavirus outbreak the role of absolute humidity on transmission rates of the covid-19 outbreak preliminary estimation of the basic reproduction number of novel coronavirus (2019-ncov) in china, from 2019 to 2020: a data-driven analysis in the early phase of the outbreak early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia ambient ozone and influenza transmissibility in hong kong il-33 isoforms: their future as vaccine adjuvants? il-33 drives augmented responses to ozone in obese mice dynamical resonance can account for seasonality of influenza epidemics 2020 by spatial interpolation with inverse distance weighting. (b) spatial distribution of basic reproductive number (r 0 ) across 154 chinese cities. (c) estimated nonlinear relationships and corresponding confidence intervals between the r 0 and daily 1-h maximum ozone concentrations in the multivariable regression analysis with two weighting schemes (equal-weighted and weighted by the number of cases). the violin plot on the left of the panel c indicates the distribution of r 0 and the plot in the bottom shows the distribution of ambient 1-h maximum ozone concentrations key: cord-303904-8ubm0kig authors: verhagen, lilly m.; gómez-castellano, keyla; snelders, eveline; rivera-olivero, ismar; pocaterra, leonor; melchers, willem j.g.; de waard, jacobus h.; hermans, peter w.m. title: respiratory infections in eñepa amerindians are related to malnutrition and streptococcus pneumoniae carriage date: 2013-06-22 journal: j infect doi: 10.1016/j.jinf.2013.06.010 sha: doc_id: 303904 cord_uid: 8ubm0kig objectives: high acute respiratory tract infection (arti) rates are observed in indigenous populations. we assessed the role of viral infections and nasopharyngeal bacterial carriage in artis in eñepa amerindians from venezuela. methods: in 40 children aged 0–10 years with artis, healthy nearest-age sibling controls and their mothers the presence of streptococcus pneumoniae, haemophilus influenzae, staphylococcus aureus, moraxella catarrhalis, mycoplasma pneumoniae, chlamydophila pneumoniae/psittachi and 15 respiratory viruses was investigated. results: s. pneumoniae was the most frequently detected pathogen, with carriage rates of 75% and 38% in children and mothers respectively. in children, s. pneumoniae carriage was associated with arti risk in multivariate analysis (or 14.1, 95% ci 1.4–137.7). viral infections were not associated with arti risk. s. pneumoniae carriage was common in children of all ages while viral co-infections were more frequently present in children under 4 years compared to older children (46% vs. 17%, p < 0.01). an increase of one unit height-for-age z score (i.e. improved chronic nutritional status) was associated with decreased odds of s. pneumoniae colonization in multivariate analysis (or 0.66, 95% ci 0.44–0.99). conclusions: in eñepa children high s. pneumoniae carriage rates associated with a poor nutritional status contribute to the development of artis. acute respiratory tract infections (artis) are among the leading causes of childhood mortality worldwide, responsible for about two million child deaths annually. 1 pneumonia rates are especially high in lowincome countries and underlying malnutrition is a major risk factor for pneumonia in children from developing countries. 2 recent estimates suggest that streptococcus pneumoniae and haemophilus influenzae together account for more than 50% of childhood pneumonia deaths each year. 3, 4 acquisition of nasopharyngeal carriage of these bacteria is an initial step in the process leading to invasive bacterial diseases. 5 the increased ability to simultaneously test for multiple pathogens has highlighted the potential role of co-colonization with multiple respiratory tract bacteria and coinfection with viral infections in progression to disease after colonization. 6e9 in indigenous children artis, in particular acute lower respiratory tract infections (alrtis), are more common and associated with higher morbidity than among nonindigenous age-matched counterparts in the same region. in a population-based birth cohort study performed in australia, pneumonia rates in aboriginal children were 13.5 times higher than in non-aboriginal children (95% ci 12.8e14.4). 10 in the united states, the alrti-associated hospitalization rate was 3-fold higher for american indian/alaska native children than for the general u.s. population in retrospective analyses. 11, 12 the high prevalence rates of artis, including alrtis and acute otitis media (aom), in australian and north american native children have been associated with increased carriage of viral and bacterial respiratory tract pathogens compared to non-natives. 13e18 while in australia, canada and the u.s. native populations make up only two to four percent of the population, ten percent of the south american population consists of indigenous people. 19e21 there is a lack of health research reports concerning the principal clinical presentations and infectious etiologies of artis in indigenous people from the south american region. 19 the eñepa (or panare) amerindians inhabit the cedeño municipality of the venezuelan state of bolívar, characterized by a forest-savanna landscape. they live in around 40 isolated communities where they have very little interaction with other indigenous or non-indigenous venezuelan populations. pneumococcal vaccinations have not been introduced in this population. we investigated bacterial nasopharyngeal carriage, viral infections and nutritional status in eñepa amerindian children aged 0e10 years with and without artis and their mothers. in august 2011, during the rainy season, five geographically isolated eñepa communities (biscochuelo, colorado, el guamal, macanilla, quebrada seca) were visited for primary health care services. during these visits, all inhabitants were registered. of the 145 children aged 0e10 years present in the 5 communities at the time of survey, 40 (28%) were diagnosed with an arti, including aom. nasopharyngeal samples were taken of these children and of 40 nearest-age full sibling controls aged 0e10 years and within 5 years of patient age. when several siblings fulfilled these eligibility criteria, the sibling whose age was nearest to that of the case was included. when no siblings fulfilled these criteria (n z 3), the nearest-age cousin living in the same household was included as a matched control. nasopharyngeal samples were also taken of mothers of the included children (n z 43). as nasopharyngeal samples alone may be insufficient to detect colonization by s. pneumoniae and h. influenzae, 22 oropharyngeal swabs for bacterial isolation were taken as well. nasopharyngeal samples for bacterial and viral isolation were obtained with a flexible swab (copan italia). oropharyngeal samples were obtained by use of rigid cotton-tipped applicators. swabs were transported at 4 ce7 c within 4 h after sampling, in stgg medium 23 for bacterial isolation and in te (10 mm trisehcl, 1 mm edta, ph 8) for virus isolation, to a à20 c freezer. within 7 days, swabs were transferred to à70 c where they were stored until microbiological and virological analyses. physical examinations, including ear examination by pneumatic otoscopy and anthropometric measurements of children and mothers, were performed and documented on a standardized data collection sheet. artis were classified as upper respiratory tract infection (urti) or alrti. a diagnosis of urti was made when at least 2 common cold symptoms (fever, rhinorrhea, sore throat, headache, cough, muscle aches) with at least 1 symptom involving the respiratory tract (rhinorrhea, sore throat, cough) in the absence of an increased respiratory rate, chest indrawing, or auscultatory findings such as crepitations or rhonchi, were present. alrtis were classified as follows: pneumonia: the presence of (1) a history of cough or difficulty in breathing and (2) chest indrawing or increased respiratory rate (!60 breaths/min for children <2 months of age, !50 breaths/min for children 2e11 months of age, !40 breaths/min for children 1e5 years of age and !30 breaths/min for children 6e10 years of age). 24, 25 acute bronchitis: productive cough of <3 weeks' duration following a urti, without wheezing, and no parental history of recurrent wheezing. 25 acute bronchiolitis: an upper respiratory prodrome followed by wheezing and increased respiratory effort, manifested as tachypnoea or chest indrawing, in children <24 months of age. 25, 26 acute wheezing: an attack of wheezing preceded by upper respiratory tract symptoms in children !24 months of age, with or without a parental history of recurrent wheezing. diagnosis of aom was based on the combined presence of acute symptoms (fever, irritability, pulling at the ears), at least two signs of middle-ear effusion detected by means of pneumatic otoscopic examination (bulging, decreased or absent mobility, abnormal color or opacity not due to scarring, air-fluid interfaces) and at least one sign of middle-ear inflammation (erythema, increased vascularity over full, bulging or yellow tympanic membrane). 27 a history of breastfeeding was determined by parent's response and defined as breastfeeding at the time of survey in children aged 12 months and breastfeeding during the first year of life in children aged >12 months. swabs in stgg were plated on blood and chocolate agar plates and cultured for s. pneumoniae, h. influenzae, moraxella catarrhalis and staphylococcus aureus in the laboratory of the 'instituto de biomedicina' in caracas, venezuela, using standard methods. 28 virological analysis was performed in the radboud university medical centre in nijmegen, the netherlands, using multiplex pcr as previously described. 29 briefly, respiratory swabs were dissolved in te and spiked with equine arthritis virus (eav) as an internal control. 30 dna/rna was isolated using the magna pure 96 dna and viral na small volume kit (roche diagnostics) and the viral na plasma sv protocol. realtime multiplex pcr for detection of the atypical bacteria mycoplasma pneumoniae (mp) and chlamydophila pneumoniae/psittachi (cp) and for respiratory syncytial virus (rsv), influenza a (flua), influenza b (flub), human rhinovirus (hrv), human metapneumovirus (hmpv), parainfluenza 1, 2, 3 and 4 (piv1-4), human coronaviruses oc43 and 229e (hcov), enteroviruses (ev), human parechoviruses (hpev), human bocavirus (hbov) and human adenoviruses (adv) was performed on the roche lightcycler ò 480 system. anthropometric measurements were transformed into weight-for-height, height-for-age, and body mass index (bmi)-for-age z scores based on who standard reference populations 31,32 using who anthro software. 33 children under 5 years of age with weight-for-height or height-for-age z scores < -2 standard deviations (sd) were defined as malnourished. children aged 5e10 years with bmi-for-age or height-for-age z scores < -2 sd were defined as malnourished. weight-for-height and bmi-for-age z scores are indicators of wasting (acute malnutrition) in respectively children under 5 years and those aged 5 years and above. the height-for-age z score is an indicator for grading stunting (chronic malnutrition) in children of all ages. 34e36 mothers with a bmi <18.5 were classified as malnourished. 37 before the start of the survey, meetings were held with village elders and members of the communities to explain to them in spanish and/or in their native language the nature and objectives of the study. children were included on the basis of written informed consent from parents. mothers also provided written informed consent. the ethical committee of the instituto de biomedicina (comité de bioética, estado portador nasofaríngeo de s. pneumoniae en adultos y niños de la poblaci on indígena panare y relaci on con neumonía-cb-2011-07) and the regional health services approved the study protocol. the mc nemar test was used to assess the association of viral and bacterial pathogens with arti univariately. for univariate analysis of the association of continuous variables with arti the paired student's t test or nonparametric wilcoxon signed rank test was used, depending on whether or not the variables were normally distributed (kolmogorovesmirnov's test, p > 0.05). odds ratios (ors) and 95% confidence intervals (95% cis) were calculated by means of multivariate conditional logistic regression analysis. those pathogens and variables associated with arti with a p-value 0.20 in univariate analyses were entered into the multivariate model. sex and indicators of nutritional status (weight-for-height, bmi-for-age and height-for-age z scores) were retained in the multivariate model, irrespective of their p-values in univariate analysis. determinants of viral infection and bacterial colonization were identified univariately using chi-square test or fisher's exact test, as appropriate, for categorical variables. for continuous variables, the unpaired student's t test or nonparametric mannewhitney's test was used depending on whether or not the variables were normally distributed (kolmogorovesmirnov's test, p > 0.05). variables associated with s. pneumoniae colonization with a p-value 0.20 in univariate analysis were entered into a multivariate logistic regression model. age, sex and indicators of nutritional status were retained in the multivariate model, irrespective of their p-values in univariate analysis. statistical significance was set at p < 0.05. the spss program for windows version 20.0 (spss inc, chicago, il) was used for statistical analyses. samples for viral and bacterial analyses were taken in 79/80 children (99%). of the 43 included mothers, samples for bacterial analyses were taken in 42 mothers (98%), while swabs for viral analyses were taken in all mothers. most of the 40 cases were classified as having alrti (58%, n z 23), further differentiated as pneumonia (15%, n z 6), acute bronchitis (33%, n z 13), acute bronchiolitis (3%, n z 1) and acute wheezing (8%, n z 3). eleven children (28%) were classified as urti. twenty-one children (53%) were diagnosed with aom, of which 18 were unilateral and three were bilateral. fifteen (71%) of the children with an aom were also diagnosed with an arti at the time of the survey. characteristics of cases and controls are shown in table 1 . cases were significantly more often malnourished compared to controls (83% vs. 58%, p z 0.013). in particular indicators of acute malnutrition, i.e. weight-for-height and bmi-for-age z scores, were lower in cases compared to controls ( table 1) . viral or atypical bacterial pathogens were detected in 24/ 79 (30%) of the children. the most commonly identified viruses were hrv (10%), ev (6%), flua h1n1 (6%), and hbov (3%). adv, hmpv and rsv were all detected in 1% of the children. of the atypical bacterial pathogens, cp was detected in 1 child (1%); mp was not detected. in three mothers a viral infection was detected, either flua h1n1, hrv or hbov. no children or mothers were infected with more than one virus. fifty-nine children (75%) were colonized with s. pneumoniae, 17 (22%) were colonized with h. influenzae, 17 (22%) were colonized with s. aureus and 22 (28%) were colonized with m. catarrhalis. nasopharyngeal colonization rates of all four bacteria in children of different ages were approximately equal ( table 2 ). for mothers, colonization rates were lower but still considerable; respectively 38%, 2%, 12% and 2% for s. pneumoniae, h. influenzae, s. aureus and m. catarrhalis. thirty-two children (41%) and one mother (2%) were colonized with multiple bacterial pathogens. in 12% of the children and in 81% of the mothers that were colonized with s. pneumoniae, pneumococci were detected only in the oropharyngeal sample and not in the nasopharyngeal sample. in contrast, no h. influenzae was isolated from the oropharyngeal sample alone. s. pneumoniae carriage rates were significantly higher in cases compared to controls (90% vs. 59%, p < 0.01). in contrast, m. catarrhalis was less often detected in cases compared to controls, but this was not statistically significant (18% vs. 36%, p z 0.14). colonization with h. influenzae or s. aureus was not significantly associated with arti (p z 1.0 and p z 0.58 respectively). none of the detected viral infections showed a significant association with arti with p-values varying from p z 0.38 to p z 0.73. in multivariate conditional logistic regression analyses, s. pneumoniae colonization was a significant determinant of arti risk (or 14.1, 95% ci 1.4e137.7). furthermore, the indicator of acute malnutrition (weight-for-height and bmi-for-age z score in respectively children under 5 years of age and those aged 5 years and above) was associated with arti in multivariate analysis: an increase of one unit z score (i.e. improved acute nutritional status) was significantly as the prevalence of s. pneumoniae colonization was high in both children and mothers, we determined factors associated with carriage in both groups (table 3) . no significant differences in sex or number of people living in the household between s. pneumoniae colonized and non-colonized children were seen. also, age was not significantly correlated with s. pneumoniae colonization. children colonized by s. pneumoniae were significantly more often malnourished than non-colonized children (76% vs. 50%, p z 0.027). in particular the indicator of chronic malnutrition, height-forage z score, was lower in children colonized with s. pneumoniae compared to non-colonized children (à2.08 vs. à1.03, p z 0.056). in multivariate analysis including all variables with a p 0.20 an increase of one unit z score of heightfor-age (i.e. improved chronic nutritional status) was significantly associated with decreased odds of s. pneumoniae colonization (or 0.66, 95% ci 0.44e0.99). the absence of a statistically significant relationship between age and s. pneumoniae colonization was observed in both cases and controls. of the cases aged 0e5 years 92% was colonized by s. pneumoniae while 78% of cases aged 6e10 years were s. pneumoniae positive (p z 0.30). in controls, s. pneumoniae was observed in 70% of children aged 0e5 years vs. 50% of children aged 6e10 years (p z 0.23). children with a viral infection were younger than children without a viral infection, but this was not statistically significant (3.3 vs. 4.5 years, p z 0.090). sex and indicators of nutritional status were not significantly associated with detection of a respiratory virus. in mothers, s. pneumoniae colonization was significantly associated with a lower mean bmi (20.9 vs. 23.4, p z 0.049 in colonized vs. non-colonized mothers, table 3 ). furthermore, the median number of people living in the household was significantly lower in mothers colonized with s. pneumoniae compared to mothers not colonized with s. pneumoniae (4.5 vs. 5, p z 0.041). s. pneumoniae colonization rates in mothers of control children were not significantly different from rates observed in mothers of cases (36% vs. 46%, p z 0.72). there were no significant associations between bacterial colonization in mothers and children. s. pneumoniae was more often detected in mothers of children colonized by s. pneumoniae than in mothers of children not colonized by s. pneumoniae, but this was not statistically significant (39% vs. 20%, p z 0.098, table 3 ). the effect of the co-occurrence of bacteria and viruses on the risk of arti was investigated by dividing the data according to whether at least one vs. no bacterial pathogen was detected. this showed no significant difference in the prevalence of a viral infection in addition to bacterial colonization between cases and controls (31% vs. 26%, p z 0.80). children that suffered from a viral infection in addition to colonization with a bacterial pathogen were significantly younger than children in which a bacterial pathogen alone, without viral co-infection, was detected (3.4 years vs. 4.8 years, p z 0.043). of the children younger than 4 years of age with bacterial colonization 46% suffered from a viral co-infection compared to 17% of the children aged 4e10 years (p < 0.01). in eñepa amerindian children, acute malnutrition and s. pneumoniae colonization were significantly associated with the presence of an arti in multivariate analysis. s. pneumoniae carriage rates were high in children up to 10 years of age and chronic malnutrition was significantly associated with an increased risk for s. pneumoniae colonization. a viral infection in addition to colonization by respiratory tract bacteria occurred significantly more often in children under 4 years of age. to our knowledge, this is the first report describing the detection of both respiratory viruses and bacteria in south american indigenous children. in caseecontrol studies performed in vietnam, turkey and greenland a significant association of s. pneumoniae colonization with artis was also observed. 7, 38, 39 in healthy children, s. pneumoniae carriage rates observed in studies performed in industrialized areas before the introduction of pneumococcal vaccines decrease from 30% to 40% in the first 5 years of life to below 20% in children aged 6e10 years. 40e42 in a study performed in another venezuelan amerindian population, the warao people, s. pneumoniae carriage rates in children under 2 years of age were higher compared to carriage rates in children older than 2 years of age (50% vs. 25%). 43 however, in other tropical rural areas s. pneumoniae carriage rates up to 40% have been observed in healthy children between 5 and 10 years of age. 44e46 we observed a s. pneumoniae carriage rate of 50% in healthy controls older than 5 years of age. the high s. pneumoniae colonization rate in eñepa amerindians increases the size of the pneumococcal reservoir in these communities. as in these populations approximately half of the population is less than 15 years of age, those children aged 5e10 years represent a sizeable fraction of the total population and one that is likely to interact frequently with young children who are most susceptible to the development of disease after nasopharyngeal colonization. 5 carriage rates of s. pneumoniae in eñepa mothers were extremely high (38%) compared to the carriage rates of 7%e 9% that are observed in mothers and other close adult contacts in cross-sectional studies from industrialized as well as rural areas. 45e47 a previously performed study in the eñepa amerindian population during the dry season showed a prevalence of s. pneumoniae colonization in mothers of children under 5 years of age of only 11%. 48 this suggests that the high prevalence observed in our study was due to seasonal influences. longitudinal studies have demonstrated seasonal variations in the rate of pneumococcal colonization and rainy season has been identified as a significant risk factor for s. pneumoniae carriage in children and adults. 16, 44, 49, 50 however, the prevalence of s. pneumoniae colonization observed in healthy eñepa children under 5 years of age in the previous study (69%) was similar to the rate we observed in healthy controls up to 5 years of age (70%). another possible explanation for the high carriage rate in mothers in our study is the observation that in 81% of the s. pneumoniae positive mothers, pneumococci were only isolated from the oropharyngeal sample. in the previously performed study, oropharyngeal samples were not taken. 48 the percentage of s. pneumoniae positive children in which s. pneumoniae was isolated from the oropharyngeal sample alone was lower (12%). the importance of oropharyngeal sampling for detection of s. pneumoniae and h. influenzae in children and mothers was previously demonstrated by greenberg et al. 22 however, in contrast to their findings, in our study no h. influenzae was isolated from the oropharyngeal sample alone. this might be due to the high number of children with arti in our study, as h. influenzae detection in the nasopharyngeal swab increased during illness in the study of greenberg et al. 22 we did not study the molecular characteristics of identified pathogens. as hrv was the viral infection with the highest prevalence in our study population, it would be of interest to perform phylogenetic analysis of the hrv strains circulating in this population in order to compare the strains detected in this area with published hrv sequences from other parts of the world. 51 respiratory viruses were, however, not significantly associated with artis in our study. s. pneumoniae carriage was significantly associated with artis in our study and further characterization of s. pneumoniae isolates by serotyping, molecular typing and susceptibility testing is recommendable to obtain insight into resistance patterns and pneumococcal vaccination coverage in this population. carriage rates of s. pneumoniae in children included in our study were significantly increased in chronically malnourished children. due to the caseecontrol study design, it was unknown whether chronic malnutrition is a risk factor for s. pneumoniae colonization or whether s. pneumoniae carriage affects growth leading to growth deficits and chronic malnutrition. in a longitudinal study including south indian infants, s. pneumoniae carriage was significantly associated with increased odds of chronic malnutrition (or 3.1, 95% ci 1.3e7.4), suggesting that s. pneumoniae colonization affects growth. 52 in bangladesh, immunization of infants with a pneumococcal conjugate vaccine (pcv7) was independently associated with an increase in height-for-age. 53 acute malnutrition was significantly associated with arti risk in our study. a significant relationship between acute malnutrition and respiratory tract infections has also been observed in other studies and undernutrition, either acute or chronic, negatively affects disease outcome. 1,54e56 while bacterial colonization was not associated with the age of the child in our study, children with viral-bacterial co-occurrence were significantly younger than children with bacterial colonization alone. a mixed nasopharyngeal flora containing respiratory viruses and bacteria was also significantly more often observed in children under 4 years of age compared to children aged 5e8 years in greenland. 39 interestingly, we did not observe a significant association of respiratory viral infections with arti risk. in studies performed in developed countries, viruses have been identified as the potential etiological cause in more than half of the arti cases. 57, 58 it is important to know the relative contributions of viruses and bacteria to the burden of arti and aom in different populations to ensure appropriate population-specific case management and preventive strategies, including vaccination. conclusions from the present study must be tempered by an appreciation of limitations. as we matched cases with sibling controls, we may underestimate the effect of factors related to residence and household exposure. an example of a factor related to residence that has been associated with artis is exposure to biomass smoke. 55, 59 larger crosssectional studies are needed to examine the associations between household-related factors and arti risk. additionally, the magnitude of the associations between demographic and nutritional variables and s. pneumoniae colonization in our study may differ from the effect magnitude in the general population as our study population was not a representative sample of the general population of eñepa amerindian children due to the caseecontrol study design. finally, confidence intervals of the or determined in multivariate analyses of factors associated with arti and s. pneumoniae colonization were wide due to the small sample size of our study. rural areas are generally difficult to reach during the winter season due to heavy rain falls, limiting possibilities for field workers and researchers. the communities included in our study were very isolated communities that could be reached only after several hours of walking through an undulating forest-savanna area as heavy rains had flooded area roads. as most respiratory viruses circulate in the rainy season, 60,61 this is the preferred period for research focusing on the correlation of respiratory tract bacteria with viral infections. in conclusion, our study shows that high s. pneumoniae carriage rates in eñepa amerindian children up to 10 years of age are a risk factor for arti while viral infections were not significantly associated with arti risk. these data highlight the 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pediatric academic society meeting in vancouver pneumonia in severely malnourished children in developing countries -mortality risk, aetiology and validity of who clinical signs: a systematic review correlates of acute lower respiratory tract infections in children under 5 years of age in india the evaluation of delayed-type hypersensitivity responsiveness and nutritional status as predictors of gastro-intestinal and acute respiratory infection: a prospective field study among traditional nomadic kenyan children enteroviruses as major cause of microbiologically unexplained acute respiratory tract infections in hospitalized pediatric patients etiology of bronchiolitis in a hospitalized pediatric population: prospective multicenter study prevalence of acute respiratory infections in women and children in western sierra leone due to smoke from wood and charcoal stoves comparative dynamics, morbidity and mortality burden of pediatric viral respiratory infections in an equatorial city seasonal patterns of invasive pneumococcal disease the authors thank the participating families and the field workers involved in the recruitment of participants, in particular the medical students of the escuela de medicina josé maría vargas of the universidad central de venezuela.this work was partly supported by 'integrated microsystems for biosensing (3e-01), fes0901:fes htsm', a project of nanonextnl, a micro and nanotechnology consortium of the government of the netherlands and 130 partners. additionally, the study was funded by the fundacion para la investigaci on en micobacterias (fundaim), caracas, venezuela. key: cord-008695-y7il3hyb authors: nan title: pandemic flu: clinical management of patients with an influenza-like illness during an influenza pandemic date: 2007-01-25 journal: j infect doi: 10.1016/s0163-4453(07)60001-2 sha: doc_id: 8695 cord_uid: y7il3hyb nan • this document is intended for use in the uk in the event that the world health organisation declares that an influenza pandemic has started 1 , and the department of health in england (uk-wide lead agency on pandemic influenza, including the devolved administrations) has declared uk pandemic alert level 2 (cases of pandemic influenza identified within the uk). • these guidelines are not relevant for the management of patients affected by seasonal/interpandemic influenza, lower respiratory tract infections, community-acquired pneumonia or exacerbations of chronic obstructive pulmonary disease (copd). • once an influenza pandemic is under way, users are strongly urged to ensure that they refer to the most up-todate version of these guidelines (from web-based access points). synopsis 1. clinical management of adults referred to hospitals s1.1. severity assessment in hospital • patients with uncomplicated influenza infection would be expected to make a full recovery and do not require hospital care. • in uncomplicated infection, the illness usually resolves in seven days although cough, malaise and lassitude may persist for weeks. • patients with worsening of pre-existing co-morbid medical conditions should be managed according to best practice for that condition with reference to published disease-specific guidelines, if available, for example, the national institute of clinical excellence's copd guideline. • in hospital, patients with influenza-related pneumonia and who have a curb-65 score of 3, 4 or 5 (see box a) are at high risk of death and should be managed as having severe pneumonia. • patients with bilateral lung infiltrates on chest radiography consistent with primary viral pneumonia should be managed as having severe pneumonia regardless of curb-65 score. • patients who have a curb-65 score of 2 are at increased risk of death. they should be considered for short stay inpatient treatment or hospital supervised outpatient treatment. this decision is a matter of clinical judgment. • patients who have a curb-65 score of 0 or 1 are at low risk of death. they can be treated as having non-severe pneumonia and may be suitable for home treatment. • patients with primary viral pneumonia or a curb-65 score of 4 or 5 should be considered for hdu/icu transfer. • general indications for hdu/icu transfer include: (1) persisting hypoxia with pao 2 < 8 kpa despite maximal oxygen administration (2) progressive hypercapnia (3) severe acidosis (ph < 7.26) (4) septic shock • patients with influenza admitted to intensive care unit should be managed by specialists with appropriate training in intensive care, respiratory medicine and/or infectious diseases. pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s3 s1.4. general investigations • the following investigations are recommended in patients referred to hospital: who this applies to full blood count all patients urea and electrolytes all patients liver function tests all patients chest x-ray all patients pulse oximetry all patients. if <92% on air, then arterial blood gases. patients with cardiac and respiratory complications or co-morbid illnesses. c-reactive protein if influenza-related pneumonia is suspected • in those patients who are subsequently followed up in a hospital outpatient clinic or by a general practitioner a repeat chest x-ray should be obtained at around six weeks if respiratory symptoms or signs persist or where there is a higher risk of underlying malignancy (especially smokers and those over 50 years of age). • further investigations including a ct thoracic scan and bronchoscopy should be considered if the chest x-ray remains abnormal at follow up. s1.5. microbiological investigations s1.5.1. early in a pandemic (uk alert levels 1, 2 and 3) • virology all patients (1) nose and throat swabs in virus transport medium. (2) if presentation is more than seven days after onset of illness, an 'acute' serum (5 10 ml clotted blood) should be collected and a 'convalescent' sample (5 10 ml clotted blood) obtained after an interval of not less than seven days. • bacteriology patients with influenza-related pneumonia (1) blood culture (preferably before antibiotic treatment is commenced) (2) pneumococcal urine antigen (20 ml urine sample) (3) legionella urine antigen (20 ml urine sample) (4) sputum gram stain, culture and antimicrobial susceptibility tests on samples obtained from patients who: (i) are able to expectorate purulent samples, and (ii) have not received prior antibiotic treatment. (5) paired serological examination for influenza/other agents. acute serum should be collected and a 'convalescent' sample obtained after an interval not less than seven days (both 5 10 ml clotted blood). • virology not routinely recommended • bacteriology patients with influenza-related pneumonia in accordance to the severity of illness. (a) non-severe pneumonia (curb-65 score 0, 1 or 2) no routine testing. in patients who do not respond to empirical antibiotic therapy, sputum samples should be sent for gram stain culture and antimicrobial susceptibility tests. b severe pneumonia (curb-65 score 3, 4 or 5, or bilateral cxr changes) blood culture, preferably before antibiotic treatment is commenced pneumococcal urine antigen (20 ml urine) sputum gram stain, culture and antimicrobial susceptibility tests on samples obtained from patients who are able to expectorate purulent samples, and have not received prior antibiotic treatment. paired serological examination for influenza/other agents. 'acute' serum should be collected and a 'convalescent' sample obtained after an interval not less than seven days (both 5 10 ml clotted blood). tracheal or endotracheal aspirate samples, if available, should be sent for gram stain, culture and antimicrobial susceptibility testing. s1.6. general management s1. 6 • hypoxic patients should receive appropriate oxygen therapy with monitoring of oxygen saturations and inspired oxygen concentration with the aim to maintain pao 2 8 kpa and sao 2 ges; 92%. high concentrations of oxygen can safely be given in uncomplicated pneumonia. • oxygen therapy in patients with pre-existing chronic obstructive pulmonary disease complicated by ventilatory failure should be guided by repeated arterial blood gas measurements. non-invasive ventilation may be helpful. • in patients without pre-existing copd who develop respiratory failure, niv may be of value as a bridge to invasive ventilation in specific circumstances when level 3 beds are in high demand. respiratory and/or critical care units experienced in the use of niv are best placed to ensure the appropriate infection control measures are adopted at all times. • patients should be assessed for cardiac complications and also volume depletion and their need for additional intravenous fluids. • nutritional support should be given in severe or prolonged illness. • temperature, respiratory rate, pulse, blood pressure, mental status, oxygen saturation and inspired oxygen concentration should be monitored and recorded initially at least twice daily and more frequently in those with severe illness or requiring regular oxygen therapy. an early warning score system is a convenient way to perform this. • in patients who are not progressing satisfactorily a full clinical reassessment and a repeat chest radiograph are recommended. • patients should be reviewed 24 hours prior to discharge home. those with two or more of the following unstable clinical factors should consider remaining in hospital: (1) temperature >37.8ºc (2) heart rate >100/min (3) respiratory rate >24/min (4) systolic blood pressure <90 mmhg (5) oxygen saturation <90% (6) inability to maintain oral intake (7) abnormal mental status • follow up clinical review should be considered for all patients who suffered significant complications or who had significant worsening of their underlying disease, either with their general practitioner or in a hospital clinic. • at discharge or at follow up, patients should be offered access to information about their illness, take home medication and any follow up arrangements. • it is the responsibility of the hospital team to arrange the follow up plan with the patient and the general practitioner. • individuals should only be considered for treatment with antivirals (neuraminidase inhibitors) if they have all of the following: (1) an acute influenza-like illness (2) fever (>38ºc) and (3) been symptomatic for two days or less. • treatment schedule: adults oseltamivir 75 mg every 12 hours for five days. (dose to be reduced by 50% if creatinine clearance is less than 30 ml/minute, i.e. 75 mg od). • patients who are unable to mount an adequate febrile response, e.g. the immunocompromised or very elderly, may still be eligible for antiviral treatment despite lack of documented fever. • hospitalised patients who are severely ill, particularly if also immunocompromised, may benefit from antiviral treatment started more than 48 hours from disease onset, although there is no evidence to demonstrate benefit, or lack of it, in such circumstances. s1.8. antibiotic management s1.8.1. influenza • previously well adults with acute bronchitis complicating influenza, in the absence of pneumonia, do not routinely require antibiotics. • antibiotics should be considered in those previously well adults who develop worsening symptoms (recrudescent fever or increasing dyspnoea). • patients at high risk of complications or secondary infection (appendix 2) should be considered for antibiotics in the presence of lower respiratory features. • most patients can be adequately treated with oral antibiotics. • the preferred choice includes co-amoxiclav or a tetracycline. • a macrolide such as clarithromycin (or erythromycin) or a fluoroquinolone active against streptococcus pneumoniae and staphylococcus aureus is an alternative choice in certain circumstances. • most patients can be adequately treated with oral antibiotics. • oral therapy with co-amoxiclav or a tetracycline is preferred. • when oral therapy is contra-indicated, recommended parenteral choices include intravenous co-amoxiclav, or a second or third generation cephalosporin (cefuroxime or cefotaxime). • a macrolide (erythromycin or clarithromycin) or a fluoroquinolone active against s. pneumoniae and staph. aureus is an alternative regimen where required eg. for those intolerant of penicillins. currently levofloxacin and moxifloxacin are the only recommended fluoroquinolones licensed in the uk. • antibiotics should be administered within four hours of admission. • patients with severe pneumonia should be treated immediately after diagnosis with parenteral antibiotics. • an intravenous combination of a broad spectrum b-lactamase stable antibiotic such as co-amoxiclav or a second (e.g. cefuroxime) or third (e.g. cefotaxime) generation cephalosporin together with a macrolide (e.g. clarithromycin or erythromycin) is preferred. • an alternative regimen includes a fluoroquinolone with enhanced activity against pneumococci together with a broad spectrum b-lactamase stable antibiotic or a macrolide. currently levofloxacin is the only fluoroquinolone with an intravenous formulation licensed in the uk. • patients treated initially with parenteral antibiotics should be transferred to an oral regimen as soon as clinical improvement occurs and the temperature has been normal for 24 hours, providing there is no contra-indication to the oral route. • for most patients admitted to hospital with non severe and uncomplicated pneumonia, seven days of appropriate antibiotics is recommended. • for those with severe, microbiologically undefined pneumonia, ten days treatment is proposed. this should synopsis 1. clinical management of adults referred to hospitals synopsis of main recommendations pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s5 be extended to 14 to 21 days where staph aureus or gram negative enteric bacilli pneumonia is suspected or confirmed. • for those with non-severe pneumonia in hospital on combination therapy, changing to a fluoroquinolone with effective pneumococcal and staphylococcal cover is an option. • adding further antibiotics effective against mrsa is an option for those with severe pneumonia not responding to combination antibiotic therapy. • high fever (>38.5ºc) and cough or influenza-like symptoms. these children should seek advice from a community health professional. if there are no features that put them at high risk of complications they should be treated with oseltamivir, and given advice on antipyretics and fluids. children aged <1 year and those at risk of complications (appendix 2) should be seen by a gp. • high fever (>38.5ºc) and cough or influenza-like symptoms, plus at risk group. these children should be seen by a gp or in a&e. children may be considered at increased risk of complications if they have cough and fever (or influenza-like illness) and temperature >38.5ºc, plus either chronic co-morbid disease or one of following features: breathing difficulties severe earache vomiting >24 hours drowsiness these patients should be offered an antibiotic as well as oseltamivir (in those >1 year of age) and advice on antipyretics and fluids. children aged <1 year with none of the above features should be treated with antipyretics and fluids with a low threshold for antibiotics if they become more unwell. • indicators for hospital admission are: (1) signs of respiratory distress. markedly raised respiratory rate grunting intercostal recession breathlessness with chest signs (2) cyanosis (3) severe dehydration (4) altered conscious level (5) complicated or prolonged seizure (6) signs of septicaemia extreme pallor, hypotension, floppy infant • most children admitted to hospital are likely to need oxygen therapy and/or intravenous support as well as antibiotics and oseltamivir. • indications for transfer to high dependency or intensive care are: (1) failure to maintain sao 2 > 92% in fio 2 > 60% (2) the child is shocked (3) severe respiratory distress and a raised paco 2 (>6.5 kpa) (4) rising respiratory rate and pulse rate with clinical evidence of severe respiratory distress with or without a raised paco 2 (5) recurrent apnoea or slow irregular breathing (6) evidence of encephalopathy • when there are no picu beds available, children will have to be triaged on the basis of the severity of their acute and co-existing disease, and the likelihood of their achieving full recovery. • a full blood count with differential, urea, creatinine and electrolytes, liver enzymes and a blood culture should be done in all severely ill children. • a cxr should be performed in children who are hypoxic, have severe illness or who are deteriorating despite treatment. • pulse oximetry should be performed in every child being assessed for admission to hospital with pneumonia. • virology all children (1) nasopharyngeal aspirate or nose and throat swabs (2) if presentation is more than 7 days after onset of illness, an 'acute' serum (2 5 ml clotted blood) should be collected and a 'convalescent' sample (2 5 ml clotted blood) obtained after an interval of not less than 7 days. • bacteriology children with influenza-related pneumonia (1) blood culture (before antibiotic treatment is commenced) (2) sputum samples obtained from older children (3) paired serological examination for influenza/other agents. • virology not routinely recommended • bacteriology children with influenza-related pneumonia (1) blood culture (before antibiotic treatment is commenced) (2) sputum samples obtained from older children • patients whose oxygen saturation is 92% or less while breathing air should be treated with oxygen given by nasal cannulae, head box, or face mask to maintain oxygen saturation above 92%. • when children are unable to maintain oral intake, supplementary fluids should, when possible, be given by the enteral route. intravenous fluids in those with severe pneumonia should be given at 80% basal levels. • children can be safely discharged from hospital when they: (1) are clearly improving (2) are physiologically stable (3) can tolerate oral feeds (4) have a respiratory rate <40/min (<50/min in infants) (5) have an awake oxygen saturation of >92% in air. • in the setting of a pandemic, children should only be considered for treatment with antivirals if they have all of the following: (1) an acute influenza-like illness (2) fever (>38.5ºc) and (3) been symptomatic for two days or less • oseltamivir is the antiviral agent of choice. • in children who are severely ill in hospital oseltamivir may be used if the child has been symptomatic for <6 days (but there is no evidence to demonstrate benefit, or lack of it, in such circumstances). • children (a) who are at risk of complications of influenza or (b) with disease severe enough to merit hospital admission during an influenza pandemic should be treated with an antibiotic that will provide cover against s. pneumoniae, staph. aureus and h. influenzae. • for children under 12 years co-amoxiclav is the drug of choice. clarithromycin or cefuroxime should be used in children allergic to penicillin. for children over 12 years doxycycline is an alternative. • oral antibiotics should be given provided oral fluids are tolerated. • children who are severely ill with pneumonia complicating influenza should have a second agent added to the regime (e.g. clarithromycin or cefuroxime) and the drugs should be given intravenously to ensure high serum and tissue antibiotic levels. to facilitate preparedness planning, this document has been written in advance of the emergence of the next influenza pandemic, at a time when the identity of the causative virus remains unknown. these guidelines are based on the best evidence available from previous pandemic and interpandemic influenza periods. the guidance may evolve as clinicopathological information on the eventual pandemic virus emerges. once an influenza pandemic is under way, users are strongly urged to refer to the most up-todate version of these guidelines (from web-based access points). seasonal influenza is a familiar infection in the uk, especially during winter. every year strains of influenza (type a or b) circulate, giving rise to clinical consultations in primary care (age-specific impact varies by season), episodes of hospital treatment (mainly in older persons and young children, but occasionally in working age adults), and deaths (mainly in the elderly). treatment in primary care and hospital may be required due to the direct effects of influenza virus infection or its possible complications, most commonly secondary bacterial pneumonia. increases in gp consultations for influenza-like illness and winter bed pressures are frequently associated with periods of known community influenza activity 6 . pandemic influenza occurs when a new influenza a virus subtype emerges which is markedly different from recently circulating subtypes and strains, and is able to: • infect humans; • spread efficiently from person to person; • cause significant clinical illness in a high proportion of those infected. because the virus is novel in humans, a high proportion of the population will have little or no immunity, producing a large pool of susceptible persons; accordingly the disease spreads widely and rapidly. influenza pandemics occur sporadically and unpredictably. in 1918, a devastating and unusual pandemic caused by influenza a/h1n1 ('spanish flu') killed between 20 and 40 million people worldwide. other pandemics that followed had a less devastating impact but were nevertheless severe. influenza a/h2n2 ('asian flu') emerged in 1957, and h3n2 ('hong kong flu') in 1968; both produced roughly 1 million excess deaths worldwide 7 . the circumstances still exist for a new influenza virus with pandemic potential to emerge and spread, and the longest interval so far recorded between pandemics is 39 years (1918 1957) . the unpredictability of the timing of the next pandemic is underlined by the occurrence of several large outbreaks of highly pathogenic avian influenza associated with epizootic transmission to humans 8 . by far the most serious has been the massive and unprecedented outbreak of highly pathogenic influenza (a/h5n1) affecting poultry in east and south east asia in late 2003, which is still continuing. this outbreak has so far been associated with a small number of human cases but a high proportion of deaths. recently, epidemiological and virological changes have been reported from northern vietnam which may indicate that the virus is beginning to adapt to humans 9 . although the emergence of an a/h5n1 strain with capacity to spread efficiently between humans is neither inevitable nor imminent, international concern has increased regarding the possibility that avian influenza a/h5n1 may evolve to produce the next pandemic. other events and developments that inform the creation of this guidance are the development and licensing of a new class of drug (neuraminidase inhibitors) active against influenza, and uk government's announcement of plans to procure 14.6 million treatment courses of oseltamivir (tamiflu ® ) 10 for use in the uk in the event of a pandemic. be involved in the management of patients with influenza. it is intended that these guidelines also be of value to health-care practitioners who do not usually manage patients with influenza but may be called upon to do so in a pandemic situation. modification of some recommendations at a local level may be necessary in specific instances. these guidelines are not relevant for the management of patients affected by seasonal influenza, sporadic acute exacerbations of chronic obstructive pulmonary disease (aecopd), lower respiratory tract infections (lrtis) or community-acquired pneumonia (cap). at the primary care level, a national operational plan including the following three broad areas is deemed important: (a) clinical management of patients with influenza (b) management of patient demand, including patients who do not have influenza (c) health service delivery plans these guidelines cover the first of these areas and will serve as the source document for the primary care operational plan. the primary care operational plan will incorporate all three areas within a single reference and is being developed by the dh in collaboration with the rcgp and the bma. even though it is impossible to predict with certainty the impact of the next pandemic, based upon the available epidemiological and modelling information, it is clear that it will generate demands for health care which may saturate or overwhelm normal nhs acute services for a period of time, perhaps several weeks or months. accordingly, it should be anticipated that the nhs (in common with all health systems around the world) will need to revert to emergency arrangements. these are laid out in further detail in operational guidance for health service planners 4 , the uk operational framework for stockpiling, distributing and using antiviral drugs in the event of pandemic influenza 5 and in the primary care operational plan. with regard to the delivery of medical care for patients with influenza this is normally achieved through: • gp treatment of community patients 'well' enough to be managed in the community • hospital care in acute medicine for persons considered too ill to be managed at home. in the event of a pandemic, the following additional care settings may have to be considered as the threshold for hospital admission rises: • treatment of patients in the community (who would normally receive care from a gp) by other health-care professionals (nurses, paramedics, pharmacists etc.) following treatment guidance laid out in this publication and using prescription-only medicines according to patient group directives (pgds). • treatment of patients in their own homes or in temporary intermediate care facilities by a gp, following treatment guidance laid out in this publication when, under normal circumstances, such patients would have been admitted for hospital care. • treatment of severely ill patients in hospital by medical and nursing teams who do not normally manage patients with influenza or community-acquired pneumonia, in areas of the hospital not normally used for providing medical care (for example, surgical teams and bed space diverted from routine elective work towards pandemic response). the recommendations offered in the current guidelines are based on a matrix of evidence centred mainly around seasonal influenza, expert opinion and group consensus. grading of these recommendations based on the strength of the evidence base was deemed inappropriate. section 2. epidemiology and health impact projections (1) the scale and severity of illness (and hence consequences) caused by pandemic influenza generally exceed those of even the most severe winter epidemics. (2) mortality in the uk is likely to exceed 50,000 deaths, possibly appreciably higher. (3) besides the elderly, excess mortality is also likely in younger adults and children. (4) modelling studies suggest that after a case occurs in hong kong, because of international travel, it will take less than one month for the virus to reach the uk. (5) once cases begin to occur in the uk it will take only two to three weeks before activity is widespread and roughly a further three weeks (six weeks after initial cases in uk) until activity peaks. (6) it is possible that there will be more than one epidemic wave (with an interval of several months) and, if a second wave occurs, it may be more severe than the first. (7) cumulative clinical and serological attack rates across all waves together may be in the order of 25% and 50% respectively. (8) increases in demand for health-care services are likely to be very substantial in both primary care and hospital settings. when an influenza pandemic occurs, a substantial proportion (possibly all) of the population is likely to be non-immune, producing a large pool of susceptible persons. in past pandemics, the scale and severity of illness (and hence consequences) have been variable but broadly of a §2. epidemiology and health impact projections introductory observations higher order than even the most severe winter epidemics. it is reasonable to expect this to be the case with the next pandemic as well. excess mortality due to influenza occurs in most winter seasons but is especially marked during epidemics. the average annual excess mortality attributable to influenza in recent years is around 12,000 deaths per annum in england and wales 11 , although there is considerable yearly variation and some years are notably much higher than the average (est. 26,000 in 1989/90 epidemic). excess mortality in england and wales associated with the three pandemics of the twentieth century has also varied widely; this was estimated at 198,000 civilians in 1918/19, and 37,500 in 1957/58. in 1968/69 and 1969/70 (both seasons considered to be associated with the influenza a/h3n2 pandemic), there were an estimated 31,000 and 47,000 deaths respectively 7 . therefore the extent of mortality associated with the next pandemic cannot be reliably predicted although it is reasonable to plan for a scenario worse than a severe winter epidemic of normal influenza. typically, there are changes in the age-distribution of cases compared with seasonal influenza. mortality, which in typical seasonal influenza is usually confined to age groups over 65 years, tends to be increased in younger age groups. the size of any increase in morbidity and mortality and the extent to which a shift in age distribution occurs depend on a variety of factors including the nature of the pandemic virus and pre-existing immunity but appears to be a consistent phenomenon 12 . therefore, clinicians can expect to see relatively larger amounts of influenza-related illness in younger adults compared with normal winter activity. at least one third of all excess deaths may be expected in persons under 65 years of age. virological and clinical surveillance of influenza have improved markedly since the last pandemic in 1968. however, the extent of international travel has also grown. modelling studies using transmission characteristics based on the 1968/69 pandemic and international air-traffic data from 2002 indicate that the approximate delay between a first case in hong kong and first introduction to uk will be less than one month 13 . in terms of the spread within the uk, it will probably take only two to three weeks from the initial introduction(s) until activity is widespread and a further three weeks (six weeks from initial uk cases) until activity peaks. the temporal and spatial spread of a pandemic strain is important, particularly in terms of the demand placed on health-care services. pandemic activity taking the form of a brief but severe peak in cases will be more difficult for all services to cope with, compared with an identical number of cases distributed over a longer time course. for example, during the a/h3n2 pandemic a long first wave occurred in the winter of 1968/9 with morbidity and mortality approximately at the same level as the previous seasonal influenza; but in the following winter of 1969/70 a short and more severe epidemic occurred with a threefold higher peak in general practice consultation rates and a four-fold higher peak in mortality attributed to influenza, bronchitis and pneumonia. the high peak in consultation rates is well illustrated in fig. 2 .1. in 1918/19, the a/h1n1 pandemic occurred in three distinct epidemic waves: early spring 1918, autumn 1918 introductory observations §2. epidemiology and health impact projections s10 provisional guidelines from bis/bts/hpa in collaboration with the department of health, version 11 (2 october 2006) and late winter 1919. the second wave was by far the largest and case-fatality rates were also higher than in the first wave. the a/h3n2 pandemic caused an epidemic wave in the winter of 1968/69 but a more severe one in 1969/70. in contrast, the second wave of the 1957/58 pandemic in the uk was very small in comparison to the first 7 . thus it should be considered a possibility that more than one wave of influenza will occur within a few months of the emergence of a pandemic virus and a subsequent wave could be worse than the first. it is impossible to predict reliably with precision the level of excess mortality that will be experienced in the next pandemic. however, table 2 .1 illustrates the broad range of excess mortality that it is reasonable to consider, based on various realistic combinations of case fatality rate and clinical attack rates derived from previous pandemics and epidemics. a case fatality rate of 0.37% corresponds to the aggregate rate observed in recent epidemic seasons (1989/90, 1991/92, 1993/94, 1995/96, 1996/97, 1997/98 and 1998/99) and the 1957 pandemic, although the overall case-fatality rate observed in the 1918 19 pandemic was in the region of 1 2%. a clinical attack rate of around 25% corresponds to the approximate clinical attack rate seen in all three previous pandemics of the twentieth century. thus, a figure of at least 50,000 excess deaths is likely. using mathematical projections, it is possible to illustrate the potential impact of the next pandemic, but these do not amount to accurate predictions. table 2 .2 summarises the number of events that might be expected by a gp with 1000 patients on his/her list and by a pct serving a population of 100,000 persons. using the same assumptions, table 2 .3 illustrates the number of events by week over an assumed 15-week (single wave) pandemic period in a typical pct population of 100,000. most major acute trusts receive patients from a catchment area spanning several pcts and the figures below require pro-rata adjustment before applying to individual hospitals. section 3. clinical features in adults (1) influenza is clinically defined as the presence of fever and new (or, in those with chronic lung disease, worsening) cough of acute onset in the context of influenza circulating in the community. this clinical definition may be modified once a pandemic occurs. (2) the spectrum of clinical disease associated with a pandemic strain cannot be forecast. (3) pneumonia, either primary viral or secondary bacterial, is the commonest complication of influenza in adults. (4) neurological complications are rare in adults. the clinical manifestations of infection by influenza viruses are diverse, ranging from asymptomatic infection to fulminant respiratory distress leading to respiratory failure and death. furthermore, the presence of an influenza-like illness (ili) comprising of a combination of fever, cough, sore throat, myalgia and headache is not specific for influenza infection. other respiratory pathogens that may present with an ili include viruses such as respiratory syncytial virus (rsv), adenovirus, rhinovirus and parainfluenza virus, as well as bacterial pathogens such as chlamydia pneumoniae, legionella sp., mycoplasma pneumoniae and streptococcus pneumoniae [14] [15] [16] . studies that have examined the value of a clinical definition of ili in the diagnosis of influenza infection have not always used the same clinical definition for an ili and have included different study populations, making comparison between studies complicated. a systematic review of the literature in this area identified the threefold combination of the presence of fever, cough and acute onset to be the most predictive clinical features. the accuracy of this clinical definition was higher in persons aged 60 years and above compared to patient groups without age restrictions [positive likelihood ratio (95% ci) 5.4 (3.8 7.7) vs 2.0 (1.8 2.1)] 17 . the probability of influenza infection also increases with increasing level of fever 18, 19 . importantly, the predictive value of clinical definitions based on an ili increases when influenza virus is known to be circulating in the community 15, 17, 20 . in cohort studies, correlation of ili with laboratory-confirmed influenza infection ranges from 25% to 45% while in clinical trials, rates of 70% have been consistently reported 15, [21] [22] [23] .. these findings relate to influenza infections during interpandemic periods. during a global influenza pandemic, when a pandemic strain is known to be circulating locally in an immunologically susceptible population, the presence of an ili would be expected to be highly predictive for influenza infection. (however, the extent to which a clinical diagnosis of ili becomes predictive during a pandemic will also be determined by the behaviour of the public. if many who would not normally present to a health professional are prompted to present, then the predictive value of a clinical diagnosis of ili will be reduced.) the following description will relate mainly to interpandemic influenza a infections. influenza b and c are not considered pandemic threats. different strains may be associated with different clinical presentations and disease severity. for instance, there is evidence to suggest that the h3n2 subtype causes more severe disease than h1n1 subtype 24 . the spectrum of clinical disease associated with a new influenza a subtype (eg. a pandemic strain) cannot be determined currently and may differ from that described for interpandemic influenza. the incubation period prior to the onset of symptoms is commonly two to four days (range 1 7 days). in adults, the illness typically presents as an abrupt onset of fever accompanied by a range of other symptoms as listed in box 3.2 [25] [26] [27] [28] [29] . fever is the paramount symptom and may reach 41ºc although more usually it ranges between 38ºc and 40ºc. the peak occurs within 24 hours of onset and lasts typically for three days (range 1 5 days) [25] [26] [27] [28] [29] . the cough is generally dry although in up to 40% of cases it may be productive. a productive cough together with chest tightness and substernal soreness is more common in patients with underlying chronic lung disease. myalgia affects mainly the back and limbs. gastrointestinal symptoms such as vomiting and diarrhoea are uncommon (<10%) in adults. abdominal pain is rare. clinical findings include a toxic appearance in the initial stages, hot and moist skin, a flushed face, injected eyes and hyperaemic mucous membranes around the nose and pharynx. tender cervical lymphadenopathy is found in a minority (~10%) of cases. wheezing or lung crackles are recognised findings (~10%) more commonly noted in patients with coexisting chronic lung disease. although the overall clinical picture of uncomplicated influenza in any specific age group is similar for different influenza a subtypes, the frequency of certain symptoms may vary. for instance, during the 'asian' pandemic of 1957 (h2n2), headache and sore throat were frequent initial symptoms 30 . in uncomplicated infection, the illness usually resolves in seven days although cough, malaise and lassitude may persist for weeks. influenza virus infection has been associated with worsening in the clinical condition of patients with a range of existing medical conditions, such as, heart failure, diabetes, coronary heart disease, asthma and chronic obstructive airways disease (copd). in addition, specific complications associated with influenza infection regardless of co-existing medical conditions are recognised (table 3 .1). based on data from interpandemic influenza, certain persons are identified as being at high risk from influenza-related complications. such patients are similar to the group currently recommended for influenza vaccination by the department of health. these include those of all ages with chronic respiratory disease including asthma, chronic heart disease, chronic renal disease, chronic liver disease, immunosuppression due to disease or treatment, or diabetes mellitus, and all those aged 65 years or older, or those in long stay residential care (see appendix 2). in the course of a pandemic, it may emerge that the patient group at high risk of complications differs from the group currently identified. in such circumstance, details of the 'high risk' patient group will be altered according to relevant clinico-epidemiological data. the incidence of pneumonia (defined as a combination of respiratory symptoms and signs supported by chest radiographic changes consistent with infection) complicating influenza infection varies widely, from 2% to 38%, and is dependent on viral and host factors [25] [26] [27] . pneumonia generally occurs more frequently and with greater severity in patients with pre-existing chronic cardiac and respiratory conditions. patients who develop pneumonia may present with symptoms and signs indistinguishable from pneumonia related to other viral and bacterial pathogens. in the context of an influenza pandemic, the presence of an ili and new or worsening dyspnoea should prompt a careful examination for the presence of complicating pneumonia. two main types of influenza-related pneumonia are recognised: primary viral pneumonia and secondary bacterial pneumonia [25] [26] [27] [28] . patients with primary viral pneumonia typically become breathless within the first 48 hours of onset of fever. an initially dry cough may become productive of blood-stained sputum. cyanosis, tachypnoea, bilateral crepitations and wheeze on chest examination and leucocytosis are usual. the commonest chest radiographic abnormality is of bilateral interstitial infiltrates predominantly in the mid-zones, although focal consolidation is also well recognised. rapid clinical deterioration with respiratory failure may ensue 31 . the mortality in hospitalised patients is high (>40%) despite maximum supportive treatment on intensive care [25] [26] [27] [28] . in the majority of fatal cases, death occurs within seven days of hospital admission. secondary bacterial pneumonia is more common (up to four times) than primary viral pneumonia. typically, symptoms and signs of pneumonia develop during the early convalescent period (four to five days from onset of initial symptoms). in others, symptoms of pneumonia blend in with the initial symptoms of influenza. chest radiography usually demonstrates a lobar pattern of consolidation. mortality rate ranges from 7% to 24% [25] [26] [27] [28] [29] 32 , although some small studies report higher mortality rates. the spectrum of pathogens implicated is similar to that observed in cap and includes streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae and groups a, c and g b-haemolytic streptococci 27, 28, [33] [34] [35] . different pathogens have predominated at different times. for instance, in the 1918 pandemic, h. influenzae, b-haemolytic streptococci and s. pneumoniae were the predominant pathogens isolated. in 1968, s. pneumoniae was the predominant pathogen (48%) followed by staph. aureus (26%) and non-typeable h. influenzae (11%) 34 . notably, staph. aureus was identified two and a half times more frequently during the 1968 pandemic compared to pneumonia occurring in the interpandemic period 34, 36 . secondary staphylococcal pneumonia is associated with a higher incidence of lung abscess formation (14% vs 2%) and carries a poorer prognosis compared to non-staphylococcal pneumonias (mortality 47% vs 16%) 25, 29, 32, 37 . during the 1957 pandemic, staph. aureus was the predominant bacterial pathogen isolated in fatal cases of influenzarelated pneumonia (up to 69% of cases in some series) 25 . bacterial and viral pneumonia can occur concurrently. in these instances, the chest radiograph may demonstrate lobar consolidation superimposed on bilateral diffuse lung infiltrates. the mortality rate in mixed viral bacterial pneumonia is high (>40%), as for primary viral pneumonia [25] [26] [27] [28] . minor abnormalities on ecg such as st segment deviation, t wave changes and rhythm disturbances have been described in uncomplicated influenza illness. they have been reported in up to 81% of patients hospitalised with influenza 25 . most do not have cardiac symptoms. myocarditis and pericarditis are occasionally encountered in severe illness 38, 39 . post mortem evidence of necrotising myocarditis has been reported in patients without clinically significant myocarditis in the antemortem period. in contrast with myalgia affecting the back and limbs which is common on initial presentation, myositis generally develops after the subsidence of the acute upper respiratory tract symptoms. the gastrocnemius and soleus muscles are typically involved with pain and tenderness to palpation. complete recovery usually occurs in three days. elevation in serum creatine phosphokinase is recognised 40, 41 . rarely, this is associated with myoglobinuria and renal failure 42, 43 . myositis is more commonly described in children than in adults. central nervous system (cns) involvement in adults is uncommon. most reports originate from japan and occur in children 44, 45 . the main clinical syndrome is an encephalitis or encephalopathy manifesting in the form of decreased consciousness and seizures about three days (range 0 7 days) following the onset of upper respiratory tract symptoms. focal neurological signs such as paresis, aphasia, choreoathetosis and cranial nerve palsies are less common. cerebrospinal fluid (csf) examination may be normal or reveal an elevation in protein or white cell count. imaging by ct or mri may be normal and if so, is indicative of a good prognosis and full recovery may be anticipated 46 . young age and abnormal ct/mri findings are associated with a poor outcome including death or recovery with severe neurological sequelae. [a fuller description is given in section 4.2. 6.] acute necrotising encephalopathy is a rare fulminant syndrome associated with multifocal brain lesions that is described mainly in japan 46 . other rare manifestations include transverse myelitis and guillain barré syndrome 47,48 . reye's syndrome, characterised by an encephalopathy, acute fatty liver, association with aspirin use and high mortality (~40%), is a special situation that is almost exclusively seen in children and adolescents 46 . nevertheless, physicians managing adults are advised to be aware of this complication. [a fuller description is given in section 4.2.6.1.] other complications rarely encountered in adults with influenza a infection include toxic shock syndrome in conjunction with secondary staph. aureus infection 49,50 and parotitis 51 . otitis media is more commonly encountered in children than adults. human infections have been caused by different avian influenza a viruses in the past, including h9n2, h7n7, h7n3 and h7n2. in recent years, outbreaks of human infections by a novel strain of avian influenza a (h5n1) have raised particular concerns globally regarding the risk of a human pandemic 52 . these concerns have been due in part to recognition that (a) avian influenza a (h5n1) can pass directly from birds to humans and (b) once in humans, avian influenza a (h5n1) causes severe disease with a high mortality. the full spectrum of human illness associated with avian influenza a (h5n1) infection is not completely known. descriptions of the clinical features of influenza a (h5n1) infection in humans are based largely on case series of hospitalised patients. subclinical infections, mild illnesses and atypical presentations of influenza a (h5n1) infections in humans have been reported, but the frequency of such infections is difficult to determine [53] [54] [55] . in hospitalised patients, an ili similar to that associated with seasonal influenza a (h1n1 or h3n2) infection is recognised. gastrointestinal symptoms are present in a relatively large proportion of both adult and paediatric cases, in contrast to the relatively low incidence of gastrointestinal symptoms in seasonal influenza. the majority of patients develop a severe primary viral pneumonia usually associated with lymphopenia, thrombocytopenia and deranged liver function tests. renal failure and multiorgan failure may develop subsequently. mortality is high. a more detailed description is given in appendix 10. should influenza a (h5n1) acquire efficient humanto-human transmission capabilities, it may result in an influenza pandemic. in such an event, the clinical features of human h5n1 disease may alter. (1) the commonest presenting features of influenza during an epidemic are fever, cough and rhinorrhoea. in infants, fever with non-specific symptoms or diarrhoea and vomiting is common; in older children pharyngitis and headache are frequent. (2) the clinical features of influenza in children during a pandemic cannot be forecast. (3) children with underlying respiratory or cardiac disease, immune compromise or who are nonambulant are more likely to be severely affected. (4) the younger the child the more likely hospital admission will be needed. the clinical features of influenza presenting in a pandemic cannot be predicted as they appear to be dependent on the strain of influenza and, in some respects, the host. a new strain of influenza a responsible for an epidemic or pandemic may result in a different spectrum of clinical features than previous strains 56, 57 . common features during previous epidemics have been described and depend on the age of the child. the studies of clinical features are hospital based and are therefore likely to reflect more severe illness. these are nevertheless informative as one of the main issues in a pandemic is which patients require hospital admission. in young children presenting to primary care in a non-pandemic influenza season there are no specific clinical features that distinguish influenza from other winter viruses 58 . neonates may present with non-specific signs of sepsis such as pallor, floppiness, (poor peripheral circulation, poor tone), lethargy, poor feeding, episodes of apnoea 59 . fever may be the only presenting feature. a north american study identified influenza as the most common reason for children aged 0 60 days being admitted to hospital during an epidemic with fever as the only clinical feature 60 . fever may be the only presenting feature in this age group too. they may also be irritable and toxic and are more likely than older children to present with gastrointestinal symptoms such as diarrhoea and vomiting. febrile convulsions, particularly repeated convulsions, are positively associated with influenza a 61 . otitis media is also a common complication in children 62 . admission rates for under two year olds are 12 times higher than for children aged 5 17 years 63 . the presentation does not differ significantly from adults. common features are sudden onset of high fever, chills (76 100%), cough, headache, sore throat, fatigue (51 75%), nasal stuffiness and conjunctivitis (26 50%) . fever tends to settle two to four days later though a dry cough and clear nasal discharge last for one to two weeks 59 . a clinical prediction model from north america for influenza in children has shown that the triad of cough, headache and pharyngitis had a sensitivity of 80% and a specificity of 78% for a positive viral culture for influenza 64 . the subjects, mean age six years, presented during an epidemic to a suburban emergency department with a febrile respiratory illness and one or more symptoms of influenza. a finnish retrospective study of children referred to hospital from 1980 to 1999 with influenza confirmed by antigen testing reported that the median age for those with influenza a was two years. the most common features were cough, fever and rhinorrhoea 62 . these were also the commonest features reported in a chinese study where the mean age of the subjects with influenza a was four years 65 . conditions these children 66 (table 4 .1) and those who are not ambulant 67 experience substantial morbidity during influenza seasons, with a disproportionate number requiring inpatient care and ventilatory support. of the 22% of (table 4 .2) as in adults, influenza can present with either primary viral pneumonia or bacterial pneumonia most commonly caused by s. pneumoniae or staph. aureus. there is much less published about pneumonia complicating influenza in children. an outbreak of severe pneumococcal pneumonia in children occurred in iowa in the winter of 1995 96. this was coincident with an epidemic of influenza (h1n1). compared with controls, patients were 12 times more likely to have rare experienced a recent influenza-like illness. they were also more likely to have family members with the illness and to have positive serology in the convalescent period. many of these patients required chest drainage 69 . another study in 2002 of 202 children with proven influenza reported that 78 who had chest radiographs had either radiographic evidence of viral pneumonia or normal radiographs. no child had lobar pneumonia reported 70 . evidence from recent outbreaks of avian influenza (h5n1) in hong kong and vietnam suggests that while some children had mild disease 71 , others appeared to have multi-organ disease including acute respiratory distress syndrome (ards) 57 . all children who developed progressive pneumonia with ards died. there were no reports of bacterial pneumonia. there is no reason to believe that, apart from ards, pneumonia complicating influenza presents differently from community-acquired pneumonia in children 72 . the general clinical indicators for severity assessment of lower respiratory tract infection are summarised in the bts guidelines 72 (appendix 8). failure to improve following 48 hours of antibiotics, or deterioration including a new, distinct spike of fever, should also be treated as severe and further complicating factors sought. the clinical course of croup caused by influenza appears to be more severe than croup caused by the more common parainfluenza virus 73 . it is more likely to be complicated by bacterial tracheitis 62 . influenza is a well recognised cause of otitis media 74 . it is the commonest bacterial superinfection of influenza and is reported in approximately 25% of patients aged <5 years 75 . influenza ranks second only to respiratory syncytial virus as a cause of bronchiolitis 76 . the clinical features are the same 77 . children with influenza may present with febrile convulsions. in a community study in the netherlands, recurrent febrile seizures were positively related to influenza a. it was recommended that children who have had a previous febrile convulsion should be immunised against influenza a 61 . these complications are described in small case series. this is defined as depressed or altered level of consciousness including lethargy and/or extreme irritability in younger children or significant change in personality or behaviour persisting beyond 24 hrs or confusion (older children). encephalopathy usually presents as seizures within several days of the onset of fever 78 . seizures at this point are usually the first symptom of involvement of the central nervous system. febrile convulsions, which are more likely to be repeated with influenza than with other causes of fever, generally occur with the onset of fever. disturbances of behaviour and neurological deficit have been reported. a rapid and severe clinical course is usual with encephalopathy and is thought to be due to brain oedema mediated by cytokines rather than by direct invasion of the brain. steroids are therefore considered. 202 children with encephalopathy were recognised in japan between 1997 and 2001. death occurred in 31%, residual neurological deficit in 26% and full recovery in 43% 79 . this is a rare childhood acute encephalopathy associated with liver dysfunction. the cause is unknown but it typically follows viral illness and there is a clear association with aspirin therapy: thus an innate susceptibility coupled with aspirin taken for relief of viral symptoms. influenza (particularly influenza b) is commonly implicated 80 . there was a dramatic fall in incidence following warnings about aspirin use in children 81 . it is possible that children on long term aspirin treatment for medical conditions may be at increased risk if they develop influenza infection. reye's syndrome is characterised by protracted vomiting and encephalopathy in afebrile patients with minimal or absent jaundice, and hepatomegaly in 50% of patients. it comprises: • acute non-inflammatory encephalopathy with an altered level of consciousness • elevation of ammonia levels 24 48 hours after the onset of mental status changes (the most frequent laboratory abnormality) • hepatic dysfunction with a liver biopsy showing fatty metamorphosis or a more than three-fold increase in alanine aminotransferase (alt), aspartate aminotransferase (ast) neurological symptoms usually occur 24 48 hours after the onset of vomiting. lethargy is usually the first neurological manifestation. diarrhoea and hyperventilation may be the first signs in children younger than two years. other investigations: head ct scanning may reveal cerebral oedema but results are usually normal. an electroencephalogram (eeg) may reveal slow wave activity in the early stages and flattened waves in advanced stages. cerebrospinal fluid may or may not have increased opening pressure with white blood cells (wbcs) fewer than 9/ml 3 (usually lymphocytes). there is no specific treatment for reye's syndrome. key aspects of management are correction of metabolic imbalance and reduction of intracranial pressure. advice should be requested from a specialist in metabolic medicine. many children have an underlying inborn error of metabolism. mortality has fallen from 50% to less than 20% as a result of earlier diagnosis and more aggressive therapy. acute necrotising encephalopathy (ane): this occurs mainly in japan where it was first described in 1995. an estimated 100 deaths per annum are related to central nervous system complications of influenza in japan 82 . this suggests either a genetic predisposition for this complication or a variation in the strains of influenza circulating in japan. ane is characterised by high fever, convulsions and coma in children aged one to five years. the onset is two to four days after the respiratory symptoms, and fewer than 10% of patients survive 83 . there are no specific markers although some patients have raised liver transaminases. in many, the csf is normal. symmetrical multi-focal brain lesions are seen and bilateral thalamic involvement is characteristic and may be demonstrated on mri 83 . this is defined as encephalopathy plus two of the following: fever of 38ºc or higher, seizures, focal neurological findings, wbc >5 cells/ml in csf, eeg findings consistent with encephalitis, abnormal neuro-imaging 84 . these must be considered when a child presents with altered level of consciousness or irritability. there is good evidence of an increased risk of meningococcal disease following influenza infection 85 . during a pandemic, the focus will be on diagnosing influenza-related illness. other neurological conditions or drug toxicity, for example, may be missed. a literature review of 316 cases of myositis 86 suggested that this was a complication mainly of schoolchildren. the calf muscles are predominantly affected. rhabdomyolysis and renal failure are rare. these are also rare complications but have been described in children with underlying medical conditions 62 . section 5. general management and investigations in primary care with widespread concern during a pandemic, a significantly increased demand for advice and consultation should be anticipated. there are likely to be significantly higher consultation rates for all types of respiratory tract infections including those which are normally managed well at home using over-the-counter remedies (e.g. febrile colds, sore throat with temperatures). consequently, demand management in both the practice and the pct will be crucial to avoid the service's capacity to triage care being overwhelmed. guidance on demand management and health service delivery is given in the primary care operational plan (see section 1.4) 87 . management decisions of patients with influenza should be based primarily on: • an assessment of illness severity • identification of whether the individual is in an 'at risk' group • current advice from doh/local public health officials based on the epidemiology of the pandemic patients who are not considered to be at high risk and who have no features suggesting severe disease or complications may not need to be seen in face to face consultations by a primary care clinician. all patients presenting in general practice with symptoms suggestive of influenza (except perhaps those in whom urgent admission is required) should be given general advice and advice on symptomatic treatment. it is important that clinicians identify and address individual concerns and expectations, provide information about the illness, and provide information about what patients can do to help themselves and when they should seek further help. some useful facts that can be provided to the patient are included in box 5.1. there is little scientific evidence for most symptomatic and self-help treatment, but experience suggests that some of the following may help, and are unlikely to cause harm: • treat fever, myalgias and headache with paracetamol or ibuprofen • rest • drink plenty of fluids • avoid smoking • consider: short course of topical decongestants, throat lozenges, saline nose drops many infants and children will have coughs and mild fevers which may be due to other infections such as respiratory syncytial virus, especially over the winter months. these children should be managed in the usual way at home by parents with antipyretics and fluids. (note: aspirin should not be used in children.) management of these children is determined by disease severity (see appendix 5). the principles of symptomatic management are similar to those for adults. box 5.1. information about influenza to provide to patients • influenza is caused by a number different types of 'influenza' viruses. • the incubation period is typically one to four days and infected adults are usually contagious from the day of illness onset to five days after. children are typically contagious for seven days, although sometimes for longer. • fever usually declines after two to three days and normally disappears by the sixth day. • cough, weakness and fatigue can persist for one to two weeks and up to six weeks. • antibiotics do not benefit most people with influenza but are sometimes needed to treat secondary infections. (important note: this information may be modified once a pandemic occurs) • fever for four to five days and not starting to get better (or getting worse) • started to feel better then developing high fever and feeling unwell again • if taking antiviral drugs (eg. oseltamivir), symptoms should start to improve within two days. lack of any improvement after two days from starting antiviral drugs is an indication to re-consult. (important note: this information may be modified once a pandemic occurs) • children under one year of age year and those at high risk of complications (see appendix 2) should be seen and assessed by a gp or at the a&e department. • children over one but under seven years of age may be seen by a nurse or a gp and those aged seven years and above may be seen by a member of the community health team (e.g. community pharmacist). • all children (and parents) should be given advice on antipyretics and fluids. • aspirin is contraindicated in children (aged under 16 years). examples of what should prompt a patient to re-consult are given in box 5.2. patients who are started on antiviral agents (see section 7 for indications for antiviral use) would be expected to begin to improve within 48 hours of starting treatment. failure to improve two days after starting an antiviral agent is an indication to re-consult. at the time of re-consultation, an alternative diagnosis should be considered as well as the occurrence of any influenzarelated complications. • any rapid deterioration following first consultation should prompt a patient to re-consult. • failure to improve two days after starting an antiviral agent is an indication to re-consult. • if the first consultation did not involve contact with a physician, re-consultation should preferably involve a physician, usually a gp. 5.5. what general investigations should be done in the community? • general investigations, including a chest x-ray, are not necessary for the majority of patients managed in the community. the aim of microbiological investigations early in a pandemic (uk alert levels 1, 2 and 3) will be to confirm that influenza a is circulating in the local community. once a pandemic is established (uk alert level 4), microbiological investigations are not recommended routinely or likely to be available readily. routine testing for bacterial pathogens is not recommended at any stage. • where possible, early in a pandemic (uk alert levels 1, 2 and 3), nose and throat swabs, or nasopharyngeal swabs (in children), in virus transport medium should be submitted to the local laboratory. • once a pandemic is established (uk alert level 4), microbiological investigations are not recommended. section 6. criteria for hospital referral 6 .1. which adults require hospital referral? adults with uncomplicated influenza infection usually do not require hospital referral. patients who might require hospital admission fall into two main groups: those with worsening of a pre-existing medical condition and those with an influenza-related complication. patients who experience a worsening or clinical deterioration of pre-existing medical problems due to influenza infection should be managed according to recommended best practice for the medical condition in question. for instance, a patient with an acute exacerbation of copd triggered by influenza infection should be managed according to current nice guidelines for copd 88 . those with a worsening of a pre-existing condition are likely to be in a group at 'high risk' of influenzarelated respiratory complications and consequently at risk §6. criteria for hospital referral part 1. clinical management in primary care pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s21 . this group should be promptly reassessed if the illness is getting worse to consider hospital referral. pneumonia is the commonest influenza-related complication requiring hospital admission. patients complaining of new or worsening dyspnoea should be carefully assessed for signs of pneumonia. if pneumonia is diagnosed, disease severity assessment is recommended and hospital referral made accordingly. there is no validated severity assessment tool developed specifically for influenza-related pneumonia. the crb-65 score (table 6 .1) is a well validated severity assessment tool developed for patients with community-acquired pneumonia (cap) 89, 90 and recommended in the british thoracic society cap guidelines 2004 for use in the community setting 72 . it is offered as an example of an assessment tool for influenza-related pneumonia. the use of any severity assessment tool does not replace clinical judgement. a patient's social circumstances should also always be taken into account. in view of the rapid and fulminant course of primary viral pneumonia, patients with pneumonia who have bilateral chest signs (crackles) should be considered for hospital referral. other influenza-related complications are uncommon. there are no specific recommendations relating to criteria for hospital admission or disease severity assessment in these cases. • patients with clinically defined uncomplicated influenza infection would be expected to make a full recovery. they require good symptomatic management, access to antiviral treatment, information about the natural history, and advice as to when to re-consult. • patients with new or worsening symptoms particularly shortness or breath or recrudescent fever not responding to treatment should be examined to assess the presence and severity of influenza-related pneumonia. • patients with worsening of pre-existing co-morbid medical conditions should be managed according to best practice for that condition with reference to published disease-specific guidelines, if available. • in patients with influenza-related pneumonia clinically, hospital referral and assessment should be considered for patients with a crb-65 score of 1 or 2 (particularly score 2) and urgent admission for those with crb-65 score of 3 or more. • patients with bilateral chest signs of pneumonia should be referred to hospital for further assessment regardless of crb-65 score. • the crb-65 score does not replace clinical judgment. • the antiviral treatment of choice is oseltamivir (tamiflu tm ). this is given as a five-day course of oral tablets; 75 mg twice daily for adults. liquid suspension is available for children from the age of one year upwards. (see table 7 .1.) from clinical trial data accrued to date and based on seasonal, interpandemic influenza, the anticipated positive effect of antivirals in a pandemic will be: (a) a reduction of illness duration by 24 hours, and therefore more rapid mobilisation of affected individuals including essential workers (b) a possible reduction in hospitalisation of infected individuals (c) a reduction of subsequent antibiotic use by infected individuals the evidence accrued to date does not suggest there will be a reduction of overall mortality, nor does it rule it out. 7.4. who should receive antiviral drugs? • ideally, antiviral treatment should be offered to every patient who is over one year of age who (a) has an acute influenza-like illness (b) has fever ( 38ºc in adults, or 38.5ºc in children) and (c) presents within 48 hours of the onset of symptoms. • exceptions: (i) patients who are unable to mount an adequate febrile response, e.g. the immunocompromised or very elderly, may still be eligible for antiviral treatment despite the lack of documented fever. (ii) immunosuppressed patients, including those on long-term corticosteroid therapy, may suffer more prolonged viraemia, and could possibly benefit from antiviral therapy commenced later than 48 hours after the onset of ili. (iii) patients who are severely ill, but who have not been hospitalised due to non-clinical reasons, may benefit from antiviral therapy commenced later than 48 hours after the onset of ili. there is no strong evidence to support antiviral use in these exceptional situations. the commonest adverse effect of oseltamivir is nausea in about 10% of patients. this can be managed with mild anti-emetic medication. other side-effects are listed in appendix 9. national distribution arrangements are laid out in the uk operational framework for stockpiling, distributing and using antiviral drugs in the event of pandemic influenza 5 and the primary care operational plan. the drug will be made available through these arrangements to pharmacies, pcts and/or gp surgeries. • pcts are encouraged to plan for the delivery of antivirals to the large numbers of previously healthy persons with an ili via community health professionals, including community pharmacists. • gps should focus their efforts on assessment and management of those persons at high risk of complications (see appendix 2) and patients developing complications. section 8. antibiotic use in primary care the use of antibiotics in adults with influenza not complicated by pneumonia is determined by (a) the presence of any co-morbid illnesses and (b) the timing of first consultation with respect to the onset of symptoms. features of an acute bronchitis, with cough, retrosternal discomfort, wheeze and sputum production are an integral part of the influenzal illness. in previously well individuals who do not have pneumonia or new focal chest signs, antibiotics are not indicated. if the patient is seen later in the course of the illness and the illness is worsening, for instance with recrudescent fever or increasing breathlessness, a worsening bacterial bronchitis §8. antibiotic use in primary care part 1. clinical management in primary care pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s23 or developing pneumonia is possible and the use of antibiotics should be considered. in selected patients, a delayed antibiotic prescription may be offered at first consultation. the antibiotic prescription should come with clear instructions that the antibiotics should be used if the illness is not starting to settle after two days or if there is worsening of symptoms. the potential advantage of this approach of delayed antibiotic prescription is to minimise rates of reconsultation 91 . there are no robust data regarding the effect of such an approach on the incidence of influenzarelated complications. those at high risk of influenza-related complications because of (a) chronic obstructive pulmonary disease (copd) and/or (b) other severe co-morbid diseases should be strongly considered for antibiotics at first consultation. if, having started antibiotics, patients do not begin to improve over the next 48 hours of antibiotic treatment (or if they get worse) they should be advised to re-contact their gp for assessment of pneumonia and its severity (see sections 3 and 6). antibiotics should cover the likely bacterial pathogens including s. pneumoniae, h. influenzae, m. catarrhalis and staph. aureus. the preferred first choice of antibiotic for nonpneumonic bronchial infections, including those patients with copd, should include an effective oral b-lactamase stable agent such as a tetracycline (e.g. doxycycline) or co-amoxiclav. a macrolide (e.g. erythromycin or clarithromycin) is an alternative for those intolerant of the preferred first choices, whilst remembering the possibility of antimicrobial resistance. clarithromycin has better activity against h. influenzae than azithromycin. further details regarding the principles of antibiotic use including antibiotic resistance patterns are given in section 14. • patients without severe pre-existing illnesses and who have uncomplicated influenza, or simple bronchitis, do not routinely require antibiotics. • patients without severe pre-existing illnesses who are seen later in the course of illness and who have developed significant worsening of symptoms (particularly recrudescent fever or increasing breathlessness) should be considered for antibiotics. • patients with copd and/or other severe pre-existing illnesses, and who are therefore at high risk of influenzarelated complications, should be strongly considered for antibiotics at first consultation. • most patients can be adequately treated with a week's course of oral antibiotics. • the preferred choice of antibiotic needs also to cover infection with staph. aureus for example either doxycycline or co-amoxiclav (see table 8 .2). • a macrolide (e.g. erythromycin or clarithromycin) is an alternative choice in certain circumstances. the principles of antibiotic selection for patients with influenza-related pneumonia who can be managed in the community are similar to those for the management of sporadic community-acquired pneumonia in general except that adequate cover for staph. aureus, in addition to cover for s. pneumoniae, should be included in any empirical regimen. for this reason a tetracycline, such as doxycycline or oral co-amoxiclav, is the preferred regimen (table 8 .2). a macrolide (e.g. erythromycin or clarithromycin) is an alternative for those intolerant of the preferred first choices. macrolide (erythromycin 500 mg qds po or clarithromycin 500 mg bd b po) a an alternative regimen is provided for those intolerant of or hypersensitive to the preferred regimen. b clarithromycin may be substituted for those with gastrointestinal intolerance to oral erythromycin and also has the benefit of twice daily dosage and better cover against h. influenzae. abbreviations: od, once daily; bd, twice; tds, 3 times; qds, 4 times. secondary bacterial infections particularly pneumonia and otitis media are common in children with influenza. s. pneumoniae, staph. aureus and h. influenzae are the most common pathogens encountered during influenza outbreaks. • children in any one of the following groups should be treated with an antibiotic that will provide cover against s. pneumoniae, staph. aureus and h. influenzae: (1) those at risk of complications of influenza (see appendix 2). (2) those with one or more of the following adverse features: (a) breathing difficulties (b) severe earache (c) vomiting for more than 24 hours (d) drowsiness. part 2. clinical management of adults referred to hospital section 9. severity assessment of adults referred to hospital 9.1. what severity assessment strategy is recommended for patients referred to hospital with influenzarelated pneumonia? there is no validated severity assessment tool developed specifically for influenza-related pneumonia. the curb-65 severity assessment tool as described in the bts cap guidelines 2004 is recommended for the stratification of hospitalised patients with influenza-related pneumonia into disease severity groups 72 (table 9 .1). in addition, the presence of diffuse bilateral lung infiltrates on chest radiography consistent with primary viral pneumonia is an adverse prognostic feature. such patients should be treated as for severe pneumonia. in all instances, clinical judgement is essential when assessing disease severity. • patients with bilateral lung infiltrates on chest radiography consistent with primary viral pneumonia should be managed as having severe pneumonia regardless of curb-65 score. • in hospital, patients with influenza-related pneumonia who have a curb-65 score of 3 or more are at high risk of death and should be managed as having severe pneumonia. • patients who have a curb-65 score of 2 are at increased risk of death. they should be considered for short stay inpatient treatment or hospital-supervised outpatient treatment. this decision is a matter of clinical judgement. • patients who have a curb-65 score of 0 or 1 are at low risk of death. they can be treated as having non-severe pneumonia and may be suitable for home treatment. 9.2. when should transfer to a high dependency unit (hdu) or intensive care unit (icu) be considered? the indications for transfer to hdu or icu are no different in patients with influenza infection compared to other patients. most patients who might require hdu/icu care will have influenza-related pneumonia or a severe exacerbation of underlying comorbid illness, e.g. exacerbation of copd. in a pandemic situation when hdu/icu beds may not be readily available, prioritisation of patients on an individual basis matched against available resources will be expected. • patients with primary viral pneumonia or a curb-65 score of 4 or 5 should be considered for hdu/icu transfer. • general indications for hdu/icu transfer include: (1) persisting hypoxia with pao 2 < 8 kpa despite maximal oxygen administration (2) progressive hypercapnia (3) severe acidosis (ph < 7.26) (4) septic shock • patients with influenza admitted to an intensive care unit should be managed by specialists with appropriate training in intensive care, respiratory medicine and/or infectious diseases. in acute uncomplicated influenza the chest x-ray is usually normal. when primary viral pneumonia occurs as a complication, particularly in elderly adults the chest x-ray often shows multiple infiltrates or consolidation. cavitations or pleural changes suggest bacterial superinfection. in combined viral-bacterial pneumonia, the clinical features typically appear later than primary viral pneumonia and the chest x-ray often shows cavitation or pleural effusions. secondary bacterial pneumonia usually occurs after apparent improvement from the viral infection; the chest x-ray may show consolidation. • a chest x-ray should be obtained during assessment of a suspected case of influenza seen in the hospital setting (accident and emergency department or acute admissions ward). • in those patients who are subsequently followed up in a hospital outpatient clinic or by a general practitioner a repeat chest x-ray should be obtained at around six weeks if respiratory symptoms or signs persist or where there is a higher risk of underlying malignancy (especially smokers and those over 50 years of age). • further investigations including a ct thoracic scan, and bronchoscopy should be considered if the chest x-ray remains abnormal at follow up 72 . in those patients with illness severe enough to present to secondary care the following tests may be useful: full blood count: a leucocytosis with left shift may occur in those with primary viral pneumonia, mixed viralbacterial pneumonia or secondary bacterial pneumonia. (lymphopenia has been noted in human cases of severe avian h5n1 influenza.) urea and electrolytes may reveal evidence of hypo or hypernatraemia or renal impairment. liver function tests are usually normal. creatine kinase (ck) may be elevated in those with severe myalgia. c-reactive protein (crp) is unlikely to be helpful except where superimposed bacterial infection is suspected 72 . however, the diagnostic value of crp in lower respiratory tract infections remains controversial 92 . • the following blood tests should be obtained in patients admitted to hospital: (1) full blood count; (2) urea, creatinine and electrolytes; (3) liver function tests; (4) creatine kinase (if myositis is suspected). • in patients with suspected secondary bacterial infection, the c-reactive protein (crp) level may aid diagnosis. in acute uncomplicated influenza larger airway function remains normal. however, there is often an increase in bronchial reactivity which may persist for many weeks after resolution of the infection 93 . lung function tests are unnecessary in most patients. section 11. microbiological investigations for adults in hospital 11 .1. introduction the guidelines provided below are based on the assumption that when cases are first occurring in the uk as part of a global pandemic, it will be possible to perform full microbiological investigations in all new cases of influenzalike illness and influenza-related pneumonia. as case numbers rise, possibly to pandemic levels, full or indeed any microbiological investigation will become increasingly difficult. thus, data on the relative frequency of different bacterial causes of influenza-related pneumonia and their antimicrobial susceptibilities amongst investigated cases gathered earlier in the pandemic should be available to guide and refine empirical antimicrobial therapy choices for cases occurring later in the pandemic. the most likely pathogens implicated in influenzarelated pneumonia are streptococcus pneumoniae, staphylococcus aureus, haemophilus influenzae and to a lesser extent b-haemolytic streptococci (see section 3.3). in the early phases (uk alert levels 1, 2 and 3 see appendix 1) of a pandemic, microbiological diagnostic approaches should focus on confirming influenza as the primary illness, defining bacterial causes of influenza-related pneumonia, and optimizing both specific (for individual patients) and general (for populations) antimicrobial treatment recommendations. in later pandemic phases (uk alert level 4) with the much higher caseloads anticipated, microbiological investigation should be focused on patients with severe influenza-related pneumonia unresponsive to empirical antimicrobial therapy. actual and practical local level transition to less intense microbiological investigation may occur at uk alert level 3 in some regions as the number of local cases is likely to vary between regions. §11. microbiological investigations for adults in hospital part 2. clinical management of adults referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s27 it will be necessary to perform full microbiological investigations on all hospitalised cases, including patients with severe and non-severe influenza-related pneumonia, in order to: confirm influenza as the primary infection, optimize treatment options for the patients investigated and define the most common bacterial causes of influenzarelated pneumonia and their antimicrobial susceptibility patterns. the latter data will help to inform empirical antimicrobial therapy of subsequent cases for which microbiological investigation may not be undertaken fully, or at all. in influenza, rapid virological tests, viral culture and pcr of respiratory samples will yield positive results between one and seven days after illness onset. however, if presentation is more than seven days after the onset of influenzalike illness then such sampling and testing is unhelpful. instead, serum samples for serological testing for evidence of recent influenza infection are recommended. specific detailed microbiological guidance for taking and handling specimens from individuals at risk of avian influenza prepared by prof maria zambon of health protection agency (hpa) centre for infections is available at: www.hpa.org.uk/infections/topics_az/avianinfluenza/ guidance/microbiological_guidance.htm bacteriological investigations are only recommended in patients with influenza-related pneumonia. legionella pneumophila infection is not normally associated with influenza-related pneumonia. despite this, legionella urine antigen tests should be performed on severe cap cases in the early stages of an outbreak/incident in order to confirm legionella infection is not the reason for a local increase in pneumonia admissions. these recommendations are modified from those contained in the british thoracic society community acquired pneumonia (bts cap) guidelines 2001 [thorax 2001;56(suppl iv), see sections 5.7, 5.8 and 5.9 (pp. iv23 iv28)] and the 2004 update (see pages 4 5), both available at: www.brit-thoracic.org.uk/ iqs/bts_#guidelines_pneumonia_html. sputum investigative efforts must be focused on quality samples (i.e. those from patients who are able to expectorate purulent samples, and have not received prior antibiotic treatment) and not dissipated on large numbers of poor quality samples. it is important to acknowledge that the criteria for quality samples may only be met for a minority of admissions. laboratories should offer a reliable sputum gram stain for appropriate samples, as on occasions this can give immediate indication of likely pathogens. the most likely influenzarelated pneumonia pathogens are s. pneumoniae, staph. aureus and h. influenzae, all of which may present a characteristic appearance on gram stain of purulent sputum. laboratories performing sputum gram stains should adhere to strict and locally agreed criteria for interpretation and reporting of results. a. virology all patients: • nose and throat swabs in virus transport medium should be collected from all patients and submitted to the local laboratory. the relevant laboratory should be notified of the suspected diagnosis and there should be close liaison over sample collection, handling and transport. • rapid testing by direct immunofluorescence or rapid eia test, virus culture and/or pcr should be undertaken according to local availability and/or referred to an appropriate laboratory • during uk alert level 1, when the uk is on high alert for the first cases of pandemic influenza, suspected cases are likely to be investigated by local health protection teams from the health protection agency and its partner organisations in the devolved administrations. • during uk alert levels 1 and 2, clinicians dealing with suspected cases of pandemic influenza should ensure that the local health protection team is informed and involved from the outset. • the health protection agency and its partner organisations in the devolved administrations have established a network of more than 20 laboratories across the uk which have been proficiency tested in molecular diagnosis of influenza a/h5n1. access to this service should be via local health protection teams. • if presentation is more than seven days after onset of illness, an 'acute' serum (5 10 ml clotted blood) should be collected and a 'convalescent' sample (5 10 ml clotted blood) obtained after an interval of not less than seven days. the two sera should be examined serologically for evidence of recent influenza infection. b. bacteriology patients with influenza-related pneumonia: • the following bacteriological tests should be performed: (1) blood culture (preferably before antibiotic treatment is commenced) (2) pneumococcal urine antigen (20 ml urine sample). agents. acute serum should be collected and a 'convalescent' sample obtained after an interval not less than seven days (both 5 10 ml clotted blood) and the two sera stored for subsequent testing. once a pandemic is established, virological investigations are not recommended routinely and in a pandemic situation may not be readily available. the diagnosis of influenza will be based on clinical findings. if influenza-related pneumonia is present, the degree of microbiological investigation will be directed by disease severity and the presence of co-morbidities. in influenza-related pneumonia, examination of sputum should be considered for patients who do not respond to empirical antibiotic therapy. this will be particularly relevant if staph. aureus is identified as a common influenza-related pneumonia pathogen during the early phase of the pandemic as, in contrast to s. pneumoniae and h. influenzae, antimicrobial susceptibilities of this organism are less predictable and empirical choices more speculative. a. virology not routinely recommended. b. bacteriology patients with influenza-related pneumonia: (i) non-severe pneumonia (curb-65 score 0, 1 or 2) • sputum samples should be sent for gram stain culture and antimicrobial susceptibility tests in patients who do not respond to empirical antibiotic therapy. (ii) severe pneumonia (curb-65 score 3, 4 or 5) • specific investigations should include: (1) blood culture, preferably before antibiotic treatment is commenced. (2) pneumococcal urine antigen (20 ml urine). (3) sputum gram stain, culture and antimicrobial susceptibility tests on samples obtained from patients who: (i) are able to expectorate purulent samples, and (ii) have not received prior antibiotic treatment. sputum specimens should be transported rapidly to the laboratory. (4) paired serological examination for influenza/ other agents. 'acute' serum should be collected and a 'convalescent' sample obtained after an interval not less than seven days (both 5 10 ml clotted blood) and the two sera stored for subsequent testing. (5) tracheal or endotracheal aspirate samples, if available, should be sent for gram stain, culture and antimicrobial susceptibility testing. section 12. general management of adults admitted to hospital 12 initial management will depend on the assessment of the reason for admission, the presence of complications, and the impact of the influenza on any pre-existing disease, or psychosocial factors. for instance, some elderly patients may require admission for social reasons. in broad terms, the most likely clinical reasons for admission will be (in order of frequency): • lower respiratory tract complications: non pneumonic bacterial exacerbation of chronic lung disease such as copd (possibly with a mixed viral infection) secondary bacterial pneumonia mixed bacterial and viral pneumonia primary viral pneumonia • cardiac complications: exacerbation of pre-existing cardiac disease with cardiac failure and/or arrhythmia primary myocarditis • other complications: exacerbation of other pre-existing disease, such as diabetes mellitus neurological complications rhabdomyolysis severe sinusitis the initial management is likely to most usually involve that of respiratory and cardiac complications, especially pneumonia and these are discussed below. management of other less common primary influenzal complications (such as rhabdomyolysis, encephalopathy) is not covered. all influenza patients admitted to hospital with abnormal cardiorespiratory symptoms and signs, including influenzarelated pneumonia, should have a chest radiograph and an electrocardiogram and should have oxygenation assessed by pulse oximetry, preferably whilst breathing air (see section 10). those with sao 2 < 92% should have arterial blood gas measurements, as should all patients with features of severe illness. knowledge of the inspired oxygen concentration is essential to the interpretation of blood gas measurements and should be clearly recorded with the blood gas result. continuous oxygen therapy is indicated for those patients with pao 2 < 8 kpa, hypotension with systolic bp <100 mmhg, metabolic acidosis with bicarbonate <18 mmol/l or respiratory distress with respiratory rate >30/min 94 . the aim of oxygen therapy should be to maintain pao 2 at >8 kpa or sao 2 > 92%. unless complicated by severe chronic obstructive pulmonary disease with ventilatory failure, high concentrations of oxygen of 35% or greater are indicated and can be safely used. high concentration oxygen therapy given to patients with pre-existing chronic obstructive pulmonary disease §12. general management of adults admitted to hospital part 2. clinical management of adults referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s29 who may have co 2 retention can reduce hypoxic drive and increase ventilation-perfusion mismatching. in such patients initial treatment with low oxygen concentrations (24 28%) should be progressively increased on the basis of repeated arterial blood gas measurements, the aim being to keep sao 2 > 90% without causing a fall in arterial ph below 7.35, in line with the management strategy recommended in the nice copd guidelines 95 . non-invasive ventilation (niv) may be of value in patients with copd who are in acute hypercapnic respiratory failure 72,96 . the use of niv in patients with respiratory failure due to severe pneumonia but without co-existing copd has not been shown to influence mortality 72,96 . nevertheless, during an influenza pandemic when critical care level 3 beds 97 are in high demand, niv may be of value as a bridge to invasive ventilation in specific circumstances. in all instances, the risks of infection due to the dissemination of respiratory droplets related to the use of niv must be taken into account when deciding on management strategies. respiratory and/or critical care units experienced in the use of niv are best placed to ensure the appropriate infection control measures are adopted and observed at all times, including the use of personal protection equipment (ppe) (see uk infection control guidance for pandemic influenza) 3 . all patients should be assessed for volume depletion and may require iv fluids. the potential for influenza to cause cardiac decompensation, either through exacerbation of pre-existing cardiac disease or from a primary myocarditis, should be borne in mind, with any complicating heart failure and arrhythmias being managed in the usual way. physiotherapy may be of benefit in selected patients with excess bronchial secretions, particularly those with concurrent chronic obstructive pulmonary disease. in cases of severe illness requiring prolonged hospital admission, increased nutritional support whether enteral, parenteral or via naso-gastric feeding should be arranged. • hypoxic patients should receive appropriate oxygen therapy with monitoring of oxygen saturations and inspired oxygen concentration with the aim to maintain pao 2 > 8 kpa and sao 2 > 92%. high concentrations of oxygen can safely be given in uncomplicated pneumonia. • oxygen therapy in patients with pre-existing copd complicated by ventilatory failure should be guided by repeated arterial blood gas measurements. non-invasive ventilation may be helpful. • in patients without pre-existing copd who develop respiratory failure, niv may be of value as a bridge to invasive ventilation in specific circumstances when critical care level 3 beds are in high demand. respiratory and/or critical care units experienced in the use of niv are best placed to ensure the appropriate infection control measures are adopted at all times. • patients should be assessed for cardiac complications and also volume depletion and their need for additional intravenous fluids. • nutritional support should be given in severe or prolonged illness. 12.3. what monitoring should be conducted during a hospital stay? pulse, blood pressure, respiratory rate, temperature, oxygen saturation (with a recording of the inspired oxygen concentration at the same time) and mental status should be measured initially at least twice daily. this is most conveniently performed using an early warning score (ews) chart, which all ward staff should be familiar with. those with severe illness, requiring continuous oxygen or cardiovascular support, should be monitored more frequently. failure to improve clinically within 48 hours should result in a full clinical reassessment and failure to improve over 4 days is an indication to repeat the chest radiograph. • temperature, respiratory rate, pulse, blood pressure, mental status, oxygen saturation and inspired oxygen concentration should be monitored and recorded initially at least twice daily and more frequently in those with severe illness or requiring regular oxygen therapy. • an early warning score system is a convenient way to perform this. • in addition to a full clinical reassessment, a chest radiograph should be repeated in patients who are not progressing satisfactorily. there will be considerable pressure to discharge patients early during a pandemic. the type and availability of out-of-hospital facilities will dictate hospital discharge decisions. some guidance regarding simple parameters to review when considering hospital discharge can be obtained from a recent us prospective, multi-centre, observational cohort study of 680 patients admitted to hospital with cap 98 , and is offered as advice for all patients admitted with influenza-related respiratory complications. • patients should be reviewed before 24 hours of discharge home. those with two or more of the following unstable clinical factors should be considered for continued hospital management: (1) temperature > 37.8ºc, (2) heart rate >100/min, (3) respiratory rate >24/min, (4) systolic blood pressure <90 mmhg, (5) oxygen saturation <90%, (6) inability to maintain oral intake, it is usual practice to arrange 'routine' hospital clinic follow up and repeat the chest radiograph at around six weeks after discharge for acute respiratory illness such as pneumonia. however, there is no evidence on which to base a recommendation regarding the value of this practice in patients who have otherwise recovered satisfactorily. it is also not known whether there is any value in arranging clinical follow up in a hospital clinic rather than with the patient's general practitioner. during an influenza pandemic situation, it is likely that only patients who developed complications or who had significant worsening of their underlying disease will be offered clinical review at one or other venue. at discharge, patients should be offered access to information about their take-home medication, smoking and lifestyle advice as appropriate, potential future complications and action to take in the event of a relapse of symptoms. • follow-up clinical review should be considered for all patients who suffered significant complications or who had significant worsening of their underlying disease, either with their general practitioner or in a hospital clinic. • at discharge or at follow up, patients should be offered access to information about their illness, take-home medication and any follow-up arrangements. • it is the responsibility of the hospital team to arrange the follow-up plan with the patient and the general practitioner. section 13. use of antivirals in hospitalised adults 13 .1. what drugs should be used for antiviral treatment during a pandemic? oseltamivir (neuraminidase inhibitor) will be the mainstay for therapy in the pandemic. the m2 inhibitors, amantadine and rimantadine, are unsuitable for use for treatment due to the rapid emergence of resistance together with sideeffects. from clinical trial data accrued to date and based on seasonal, interpandemic influenza, the anticipated positive effect of antivirals in a pandemic will be: (a) a reduction of illness duration by 24 hours, and therefore more rapid mobilisation of affected individuals including essential workers; (b) a possible reduction in hospitalisation of infected individuals; (c) a reduction of subsequent antibiotic use by infected individuals. there is insufficient evidence accrued to date to determine the effect of antivirals, if any, on overall mortality. therefore the major utility of antivirals will be to maintain the essential workforce, and reduce hospitalisation and antibiotic treatment of complications. (neuraminidase inhibitors) during a pandemic? • individuals should only be considered for treatment with neuraminidase inhibitors if they have all of the following: (1) an acute influenza-like illness (2) fever (>38ºc) and (3) been symptomatic for two days or less • treatment schedule: adults: oseltamivir 75 mg every 12 hours for 5 days. dose to be reduced by 50% if creatinine clearance is less than 30 ml/minute. • exceptions: (i) patients who are unable to mount an adequate febrile response, e.g. the immunocompromised or very elderly, make still be eligible despite lack of documented fever. (ii) hospitalised patients who are severely ill, particularly if also immunocompromised, may benefit from antiviral treatment started more than 48 hours from disease onset. this advice reflects the lack of robust evidence to guide the use of antivirals in these exceptional circumstances and places a high value on the potential benefits of antiviral therapy. drugs available for treatment and prevention of infection by influenza are summarised in table 13 .1. there are four drugs available, the older agents amantadine and rimantadine and the neuraminidase inhibitors oseltamivir and zanamivir. older agents: the older agents, amantadine and rimantadine (rimantadine is not currently licensed in the uk), are related substances that act by blocking the ion-channel function of the influenza virus m2 protein. this protein, although a minor surface constituent of the influenza virus particles, is essential for virus replication. these agents are only active against influenza type a. amantadine is not recommended by nice for treatment and/or prophylaxis of interpandemic influenza, so in the absence of national stockpiling, supplies of amantadine can be expected to be very low. h5 viruses in south east asia are resistant to amantadine, so this agent may play no role at all depending on the nature of the pandemic strain. two neuraminidase inhibitors so far have been developed to the level of entry into the formulary: zanamivir is a modification of neu5ac2en, a dehydrated neuraminic acid derivative. oseltamivir is a similar molecule except it has a cyclohexene ring and replaces a polyglycerol moiety with lipophilic sidechains. oseltamivir can be taken by mouth, whereas zanamivir must be inhaled, using a diskhaler device. an intravenous formulation of zanamivir has been developed but its efficacy has not been established. this may be relevant for the management of ventilator cases. both drugs are active against influenza type a as well as type b viruses. older agents. both amantadine and rimantadine are effective for the treatment of type a influenza virus infection if treatment is begun within 48 hours of the onset of illness 99 . historical data show that they can shorten the illness by approximately one day but their efficacy in preventing complications, hospitalisations, or deaths has never been established. although these drugs are effective, their use in clinical influenza treatment has been limited as a result of their proclivity to induce viral resistance, and their side-effect profile. several large clinical trials have demonstrated the utility of zanamivir and oseltamivir in treatment of adults with influenza in the community ( virtually all studies on the efficacy of neuraminidase inhibitors to reduce complications have been conducted with oseltamivir, and this drug has been shown to have some effect on outcomes other than time to recovery. in a meta-analysis of adults and adolescents with a virologically proven influenza illness, oseltamivir treatment reduced overall antibiotic use for any reason by 26 105 . so far, the neuraminidase inhibitors have not been extensively investigated in patients who are at the highest risk of serious complications of influenza. such patients include the elderly and those with serious cardiopulmonary illness, such as chronic obstructive pulmonary disease. the neuraminidase inhibitors have not been associated with a reduction in mortality, but the clinical trials conducted so far have not been appropriate to measure this. it is not known for certain whether the neuraminidase inhibitors will be effective in pandemic influenza because their use has only been assessed in inter-pandemic influenza, where the virulence is moderate and there is some degree of host immunity. the antiviral activity is likely to be adequate; in vitro, all neuraminidase inhibitors have been demonstrated to have a broad spectrum of activity against multiple avian influenza viruses 106 . the older agents, rimantadine and amantadine, were studied in both the 1968 hong kong pandemic and again when h1n1 influenza appeared in a pandemic in 1977. their efficacy has been reviewed by hayden 99 . when the older agents were given for four to eight week periods as prophylaxis in a community setting, their protective efficacy against influenza illness averaged 70% compared with placebo. this compares with 80 90% efficacy observed with the same agents in studies during the interpandemic period. when amantadine or rimantadine are used to treat patients, resistant viruses emerge rapidly and approximately 30% of treated children or adults will shed resistant variants starting two to five days after the onset of treatment 104 . the resistant viruses shed from these patients retain full virulence, infectivity and transmission potential. when contacts of cases treated with amantadine or rimantadine are given post-exposure prophylaxis with these older agents, the reduction in secondary cases is minimal 107 . in contrast, the frequency of emergence of resistance during treatment with the neuraminidase inhibitors is reported to be low. however, during studies of experimentally induced influenza a/h1n1 infection in healthy adults, 4% of participants shed viruses with a histidine to tyrosine substitution at position 274 within the binding site of oseltamivir 108 . in these cases the volunteers had increased influenza viral load within the nasopharynx but there was no deterioration of symptoms. so far, there have been no proven instances of transmission of oseltamivir or zanamivir-resistant variants in field clinical trials, but the experience is relatively small currently. sequence analysis of h5n1 human isolates from north vietnam have revealed virus with a 274 y (resistant) sequence. although the isolate was not fully resistant, its ic50 for oseltamivir was shifted upwards and it is therefore less susceptible to oseltamivir than other h5n1 isolates that had been tested from the region. the patient from whom the virus was isolated was concurrently being treated with oseltamivir. both amantadine and rimantadine can cause nausea and vomiting in a small percentage of individuals receiving them (table 13 .1). unfortunately amantadine is also associated with very unpleasant central nervous system side-effects including anxiety, depression, insomnia and hallucinations. the side-effects are dose-related and do resolve with discontinuation of the drug. in the case of the neuraminidase inhibitors, both drugs appear relatively safe. zanamivir has very few side-effects, but can result in bronchospasm which might be potentially serious in patients with asthma. oseltamivir requires dose-reduction in patients with low creatinine clearance (<30 ml/min). nausea occurs in 5 15% of oseltamivir recipients but is seldom severe enough to lead to drug discontinuation (see table 13 .3). antimicrobial chemotherapy will be indicated primarily for respiratory complications due to secondary bacterial infections, principally influenza-related pneumonia. the majority of patients with exacerbations of chronic obstructive pulmonary disease (copd) and other chronic lung conditions due to secondary bacterial infections, such as bronchiectasis, will also require antimicrobial chemotherapy, as will some patients with severe sinusitis. few pneumonias and lower respiratory tract infections are defined microbiologically at initial assessment and hence most prescribing is empirical. in broad terms the antimicrobial management of these patients should follow the guidance offered in relevant national guidelines for the management of community-acquired pneumonia and copd, but modified in the light of the different range of pathogenic bacteria that may be implicated, specifically staph. aureus infection. in the minority of cases, the aetiology may be determined after hospital admission, thereby permitting modification of the initial empirical regimen. although the pathogens responsible for communityacquired pneumonia are diverse, in the case of bacterial pneumonia complicating influenza the principal pathogens which should be covered by any initial empirical antimicrobial therapy include s. pneumoniae, h. influenzae and staph. aureus. the latter is said to be more common with combined viral bacterial pneumonia, as some strains of staphylococci have synergistic effect with the virus. gram-negative enteric bacillary infection is also sometimes seen. exacerbations of copd will be largely associated with s. pneumoniae, h. influenzae, and moraxella catarrhalis. severity assessment and the association of pre-existing co-morbid disease is essential in predicting prognosis and in turn determines management, choice of antibiotic therapy and its method of administration (see section 9). during an influenza pandemic this will be principally related to concerns about the local pattern of antimicrobial resistance of staph. aureus, and assessing the possibility of methicillin-resistant s. aureus (mrsa) being present locally. clinicians should be kept closely informed of any local shift in antimicrobial resistance patterns, both at the start and during a pandemic. staphylococcus aureus is widely resistant to penicillin 109 and an increasing number are now methicillin-resistant (mrsa); when occurring in the community this generally reflects hospitalisation within the recent past or residence within a nursing home 110 . hence, b-lactamase unstable penicillins (penicillin g, aminopenicillins) and, in the case of mrsa, isoxazolyl penicillins (flucloxacillin, cloxacillin) and cephalosporins, are inappropriate for such infections. the true incidence of resistance among pathogens in the community is difficult to estimate since most laboratory samples come from selected populations. with this limitation in mind, the presence of b-lactamase production among h. influenzae varies geographically but ranges from 2% to 17% 111, 112 in various parts of the uk. m. catarrhalis has a high rate of b-lactamase production. antibiotic resistance among s. pneumoniae is of concern world wide, owing to the dominance of this organism as a cause of community-acquired pneumonia and because penicillin and macrolide resistance are frequently linked 112, 113 . however, to date it is not a common enough problem in the uk to influence initial antimicrobial management decisions. recent data provided by the hpa of antimicrobial sensitivities of respiratory pathogens isolated from blood and respiratory samples during the last three to four years (robert george, personal communication) found macrolide resistance amongst about 10 14% methicillinsensitive staphylococcus aureus (mssa) isolates and 12 19% of s. pneumoniae. macrolides, apart from clarithromycin, have poor in vivo activity against h. influenzae. by contrast, tetracycline resistance was around 5 8% for s. pneumoniae, 3% for h. influenzae and 2 8% for mssa. fluoroquinolones have activity against methicillinsensitive staphylococcus aureus (mssa), with mic 90 figures of 1.0 mg/l for ciprofloxacin, 0.5 mg/l for levofloxacin and 0.12 mg/l for moxifloxacin 114 . modern fluoroquinolones (oral moxifloxacin and oral and iv levofloxacin currently licensed in the uk) are therefore a possible choice for secondary bacterial infections following influenza where mssa is a likely pathogen. a recent pharmacokinetic and pharmacodynamic in vitro study indicated that moxifloxacin 400 mg od had advantages over ciprofloxacin 500 mg bd or levofloxacin 500 mg od in antimicrobial effects against staph. aureus 115 . the quinolones, levofloxacin or moxifloxacin, also provide cover against s. pneumoniae and h. influenzae. mrsa is an unlikely pathogen in the uk in the context of community-acquired respiratory bacterial infection following influenza, and fluoroquinolones are not sufficiently active against mrsa. there are no robust research studies available to provide evidence-based guidance on the best empirical choice of antimicrobial therapy for bacterial complications of influenza. for these reasons the recommendations for treatment have been made on the basis of assessing a matrix of laboratory, clinical, pharmacokinetic and safety data, interpreted in an informed manner and taking account of other published guidelines 116 . in those with chronic lung disease, particularly copd, bacterial exacerbation will be the commonest cause of admission. it is likely that all such patients sufficiently ill to require hospital admission with an exacerbation will require antibiotics. management of their underlying macrolide (erythromycin 500 mg qds po or clarithromycin 500 mg bd b po) or fluoroquinolone with enhanced pneumococcal activity (e.g. levofloxacin 500 mg od po or moxifloxacin 400 mg od po c ) if iv needed: co-amoxiclav 1.2 g tds iv or cefuroxime 1.5 g tds iv or cefotaxime 1 g tds iv macrolide (erythromycin 500 mg qds iv or clarithromycin 500 mg bd b iv) or levofloxacin 500 mg od iv c 3. hospital-treated, severe pneumonia co-amoxiclav 1.2 g tds iv or cefuroxime 1.5 g tds iv or cefotaxime 1 g tds iv plus macrolide (erythromycin 500 mg qds iv or clarithromycin 500 mg bd b iv) fluoroquinolone with some enhanced pneumococcal activity (e.g. levofloxacin 500 mg bd iv, po c plus, either macrolide (erythromycin 500 mg qds iv or clarithromycin 500 mg bd b iv) or b-lactamase stable antibiotic (co-amoxiclav 1.2 g tds iv or cefuroxime 1.5 g tds iv or cefotaxime 1 g tds iv) a an alternative regimen is provided for those intolerant of or hypersensitive to the preferred regimen. b clarithromycin may be substituted for those with gastrointestinal intolerance to oral erythromycin and also has the benefit of twice daily dosage and better cover against h. influenzae. c levofloxacin and moxifloxacin are the only currently uk-licensed fluoroquinolones with enhanced activity against s. pneumoniae, in addition to cover for staph. aureus. levofloxacin comes in an oral and a parenteral formulation and is licensed for severe pneumonia. moxifloxacin comes in an oral formulation only in the uk and is not licensed for severe pneumonia. in the future, other fluoroquinolones such as gemifloxacin and gatifloxacin are likely to extend this choice, when licensed in the uk. abbreviations: od, once daily; bd, twice; tds, 3 times; qds, 4 times: iv, intravenous; po, oral. switch from parenteral drug to the equivalent oral preparation should be made as soon as clinically appropriate, in the absence of microbiologically confirmed infection. in the case of the parenteral cephalosporins, the oral switch to co-amoxiclav 625 mg tds is recommended rather than to oral cephalosporins. condition, such as copd, should follow standard guidelines, including the use of corticosteroids if indicated. antibiotics should cover the likely bacterial pathogens, including s. pneumoniae, h. influenzae, m. catarrhalis and staph. aureus. oral therapy should be sufficient for those without adverse severity features and who are able to take oral medication. the preferred first choice of antibiotic for nonpneumonic bronchial infections should include an effective oral b-lactamase stable agent such as co-amoxiclav, or a tetracycline, such as doxycycline. a macrolide is an alternative for those intolerant of the preferred first choices, whilst remembering the possibility of antimicrobial resistance. clarithromycin has better activity against h. influenzae than azithromycin. a newer-generation fluroquinolone (e.g. levofloxacin or moxifloxacin) with enhanced activity against s. pneumoniae is an alternative choice if there is increased likelihood of resistance or local issues that dictate such a choice. • previously well adults with acute bronchitis complicating influenza, in the absence of pneumonia, do not routinely require antibiotics. • antibiotics should be considered in those previously well adults who develop worsening symptoms (recrudescent fever or increasing dyspnoea). • patients at high risk of complications or secondary infection (appendix 2) should be considered for antibiotics in the presence of lower respiratory features. • most patients can be adequately treated with oral antibiotics. • the preferred choice includes co-amoxiclav or a tetracycline. • a macrolide such as clarithromycin (or erythromycin) or a fluoroquinolone active against s. pneumoniae and staph. aureus is an alternative choice in certain circumstances. patients will be suffering from primary viral pneumonia, or combined viral bacterial pneumonia, or secondary bacterial pneumonia. the features of each of these are covered in section 3. all patients with pneumonic involvement should receive antibiotics. the principles of antibiotic selection for nonsevere influenza-related pneumonia is similar to those for the management of sporadic community-acquired pneumonia in general 72 , except that adequate cover for staph. aureus should be included in any empirical regimen. it is also not felt necessary to routinely provide cover for atypical pathogens (mycoplasma pneumoniae, chlamydia sp., coxiella burnetti, legionella sp.) during a pandemic as the large majority of patients will be hospitalised as a direct result of influenza and its complications caused by bacterial infection. for these reasons oral co-amoxiclav or a tetracycline such as doxycycline is the preferred regimen (table 14 .1). when oral therapy is inappropriate, parenteral coamoxiclav or a second-or third-generation cephalosporin is offered as an alternative. based on in-vitro data, the activity of selected cephalosporins against mssa in the uk in descending rank order is cefuroxime (mic90 1 2 mg/l) > cefotaxime (mic90 2 mg/l) > ceftriaxone (mic90 16 mg/l) [robert george, personal communication]. only cefuroxime and cefotaxime are recommended as cephalosporins offering adequate mssa cover within an empirical regimen. a macrolide or one or the new fluoroquinolones are identified as alternatives in hospitalised patients, in specific circumstances. these include those intolerant of penicillins or where local microbiological surveillance suggests they are better choices. at the time of completing these guidelines, only levofloxacin and moxifloxacin are licensed and available in the uk for pneumonia. flucloxacillin is not recommended as part of an empirical regimen because its activity against a narrow spectrum of pathogens (predominantly staph. aureus) would require it to be used in combination with more than one other antibiotic. it is offered as the antibiotic of choice in confirmed methicillin-sensitive staph. aureus (mssa) infection. regardless of the regimen selected it is critical that the antibiotics be administered promptly (within four hours of admission), and in the case of the patient with severe pneumonia without delay, by the admitting doctor in the admissions ward or by the general practitioner if delays are expected in the hospital admission process. delays in administration of antibiotics are related adversely to mortality in some studies, particularly when managing elderly patients 117, 118 . following initial assessment and empirical therapy, progress should be monitored carefully. the route and choice of antibiotic treatment will require adjustment, either by stepping up and broadening the spectrum of microbiological activity in the light of clinical deterioration or as a result of positive microbiological information, or stepping down with improvement as discussed below. • most patients can be adequately treated with oral antibiotics. • oral therapy with co-amoxiclav or a tetracycline is preferred. • when oral therapy is contra-indicated, recommended parenteral choices include intravenous co-amoxiclav, or a second or third generation cephalosporin (cefuroxime or cefotaxime respectively). • a macrolide (erythromycin or clarithromycin) or a fluoroquinolone active against s. pneumoniae and staph. aureus is an alternative regimen for those intolerant of penicillins. currently levofloxacin and moxifloxacin are the only recommended fluoroquinolones licensed in the uk. • antibiotics should be administered within four hours of admission. mortality is greatly increased in those with severe pneumonia (section 9). the illness may progress before microbiological information is available. preferred and alternative initial treatment regimens are summarised in table 14 .1. the recommendation of broadspectrum b-lactam regimens plus a macrolide in those with severe influenza-related pneumonia is based on the following rationale: (a) while s. pneumoniae and staph. aureus remain the predominant pathogens, gram-negative enteric bacilli, although uncommon, carry a high mortality 119 . (b) the recommended empirical regimen will offer double cover for the likely pathogens implicated in influenzarelated pneumonia and there is some evidence to indicate that combination therapy is associated with better outcomes in severe pneumonia 120 . (c) although there is no evidence of an increased incidence of infection by atypical pathogens in influenzarelated pneumonia, in severe pneumonia it is felt necessary to include cover for atypical pathogens, particularly legionella sp. as it may not be possible at the outset to distinguish between patients with sporadic severe community-acquired pneumonia in whom legionella infection is important, and influenzarelated pneumonia. parenteral administration of antibiotic is recommended in those with severe community-acquired pneumonia regardless of the patient's ability or otherwise to take oral medication. this is to ensure prompt, high blood and lung concentrations of antibiotic. a fluoroquinolone is offered as an alternative, despite limited data on their use in severe pneumonia 121 . at the time of writing, levofloxacin is the only licensed and available agent in the uk for severe pneumonia. it is marketed in parenteral and oral formulations. however, until more clinical experience is available we recommend combining it with another agent active against s. pneumoniae and staph. aureus such as a broad-spectrum b-lactam or macrolide when managing severe influenzarelated pneumonia. • patients with severe pneumonia should be treated immediately after diagnosis with parenteral antibiotics. • an intravenous combination of a broad-spectrum b-lactamase stable antibiotic such as co-amoxiclav or a second-(e.g. cefuroxime) or third-(e.g. cefotaxime) generation cephalosporin together with a macrolide (clarithromycin or erythromycin) is preferred. • an alternative regimen includes a fluoroquinolone with enhanced activity against pneumococci together with a broad-spectrum b-lactamase stable antibiotic or a macrolide. currently levofloxacin is the only such fluoroquinolone licenced in the uk. • patients who have been in hospital within the last few months have a higher chance of carrying mrsa as opposed to patients who have not been hospitalised recently. therefore due consideration should be given to the possibility of mrsa if they are known or suspected to have a staphylococcal pneumonia and/or are not responding to empirical therapy. 14.7. when should the iv route be changed to oral? there can be no rigid recommendation concerning the timing of transfer to oral therapy and further studies of this area are needed 122 . any decision must be individualised on the basis of assessing all factors, including the absence of any contraindications to oral administration, the availability of any microbiological information regarding aetiology of the infection and clear evidence that the patient is responding to initial therapy. the recommended guideline is that oral therapy be considered in a patient who has shown clear evidence of improvement and whose temperature has resolved for a period of 24 hours. • patients treated initially with parenteral antibiotics should be transferred to an oral regimen as soon as clinical improvement occurs and the temperature has been normal for 24 hours, providing there is no contraindication to the oral route. 14.8. for how long should antibiotics be given? until there are more precise methods to reliably identify microbiological and clinical end-points, the duration of therapy will remain subject to clinical judgement and custom. for these reasons the duration of therapy will vary by individual patient, disease severity and speed of resolution. • for most patients admitted to hospital with nonsevere and uncomplicated pneumonia, seven days of appropriate antibiotics is recommended. • for those with severe, microbiologically undefined pneumonia, ten days treatment is proposed. this should be extended to 14 21 days where s. aureus or gramnegative enteric bacilli pneumonia is suspected or confirmed. 14.9. failure of initial empirical therapy in those patients who fail to respond to initial empirical therapy, several possibilities need to be considered, the first of which is whether the correct diagnosis has been made. radiographic review is recommended for the community-and hospital-managed patient. this may also indicate complications of pneumonia such as pleural effusion/empyema, lung abscess or worsening pneumonic shadowing, which will be more common in the presence of staphylococcal infection. the initial empirical antibiotic regimen may need to be reassessed. however, compliance with, and adequate absorption of an oral regimen should first be considered. microbiological data should be reviewed and further specimens examined, with a view to excluding staph. aureus and gram-negative bacillary infection. in the hospital-managed, non-severely ill patient, changing to a new fluoroquinolone such as levofloxacin provides a second alternative. in the severely ill patient already receiving a b-lactam/ clarithromycin regimen, it is recommended that further staphylococcal cover is added to include cover for mrsa 123 . in addition, urgent referral to a respiratory physician should be made for clinical assessment including the possible need for bronchoscopic sampling. other rapid mrsa diagnostic techniques are in the evaluation stage. • for those with non-severe pneumonia in hospital on combination therapy, changing to a fluoroquinolone with effective pneumococcal and staphylococcal cover is an option. • adding further antibiotics effective against mrsa is an option for those with severe pneumonia not responding to combination antibiotic therapy. specific pathogen-directed antibiotic therapy 14.10 . what are the optimum antibiotic choices when specific pathogens have been identified? when a pathogen has been identified, specific therapy as summarised in table 14 .2 is proposed. in transferring patients from empirical to pathogen-targeted therapy, the regimen and route of administration will be determined by the continued need for parenteral therapy and known drug intolerance. these recommendations are again based on a synthesis of information, which includes in vitro activity of the drugs, appropriate pharmacokinetics and clinical evidence of efficacy gleaned from a variety of studies. the choice of agent may be modified following the availability of sensitivity testing or following consultation with a specialist in microbiology, infectious disease or respiratory medicine. close liaison with the local microbiology service will be essential during a pandemic. currently s. pneumoniae highly resistant to penicillin (mic 4 mg/l) is uncommon in the uk. however, it is important that the situation is monitored and in future §14. use of antibiotics in hospitalised adults part 2. clinical management of adults referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s37 either ciprofloxacin 400 mg bd iv or piperacillin 4 g tds iv ± gentamicin or tobramycin (dose monitoring) higher doses of penicillins or alternative regimens may need to be considered. staphylooccus aureus is an uncommon cause of sporadic community-acquired pneumonia in the uk, but will assume much greater potential importance during a pandemic. most community isolates are methicillin-sensitive although the recent increase in mrsa in hospitalised patients may result in subsequent readmission with an mrsa infection, secondary to influenza. options for methicillin-sensitive and -resistant infections are based on parenteral administration in view of the serious nature of staphylococcal pneumonia. • if a specific pathogen has been identified, the antibiotic recommendations are summarised in children with high fever (>38.5ºc) and cough or influenzalike symptoms will be seen by a community health professional (a nurse or doctor if under seven years of age). if there are no features that put them at high risk of complications they should be treated with oseltamivir, and given advice on antipyretics and fluids. children under one year of age and those at risk of complications (appendix 2) should be seen by a gp. children may be considered at increased risk of complications if they have: cough and fever (or influenza-like illness) and temperature >38.5ºc and either (i) chronic co-morbid disease (see appendix 2) or (ii) one of the following features • breathing difficulties • severe earache • vomiting > 24 hours • drowsiness these patients should be offered an antibiotic as well as oseltamivir (in those over one year of age) and advice on antipyretics and fluids. children under one year of age with none of the above features should be treated with antipyretics and fluids with a low threshold for antibiotics if they become more unwell. the most severely ill children should be referred for assessment for admission. in a pandemic situation, paediatric high dependency and intensive care beds are likely to fill quickly and will be insufficient to meet demand. children will have to be triaged by the senior paediatrician on duty in consultation with tertiary specialists in respiratory medicine, paediatric intensive care or paediatric infectious diseases. triage will be on the basis of the severity of the child's (a) acute and (b) co-existing disease and the likelihood of the child achieving full recovery. where admission is not possible §16. general investigations for children in hospital part 3. clinical management of children referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s39 the tertiary specialists will provide advice and support on management to the general paediatrician. in the h5n1 cases reported from vietnam 125 all seven children had wbc < 4.0 (mean 2.44) and 6/7 had a lymphopenia <1.0 (mean 0.66). six of the seven children died. in contrast, only two of the seven children reported from hong kong died but they were both leukopenic and lymphopenic. the survivors had a mean wbc of 12.44 and lymphocyte count of 3.11 126 . four of five cases reported from thailand were lymphopenic 127 . in influenza a thrombocytopenia (<100) is found in 5 7% 71, 84 . thrombocytopenia was found in four out of seven cases of h5n1 infection in vietnamese children 125 . liver transaminases are raised in 27% of influenza a patients 124 and were raised in six out of six of those measured in the hong kong h5n1 outbreak 126 and five out of six in those measured in vietnam 125 . c-reactive protein (crp) is unhelpful in influenza with values <10 in 55% 84 ; <20 in 72% 62 and >80 in only 5% 62 . the cd4/cd8 ratio was inverted in the two children and three adults in whom it was measured in the vietnam outbreak (mean 0.7; range 0.59 1.08). two of these patients survived 125 . • a full blood count with differential, urea, creatinine and electrolytes and liver enzymes and a blood culture should be done in all severely ill children. one of the largest studies of the value of chest radiography was undertaken in children aged between two months and five years with community-acquired pneumonia managed as outpatients with time to recovery as the main outcome 128 . chest radiography did not affect the clinical outcome in these children with acute lower respiratory infection. this lack of effect was independent of clinicians' experience. there are no clinically identifiable subgroups of children within the who case definition of pneumonia who are likely to benefit from a chest radiograph. the authors concluded that routine use of chest radiography was not beneficial in ambulatory children aged over two months with acute lower respiratory tract infection (lrti). clinicians basing the diagnosis of lower respiratory infections in young infants on radiographic diagnosis should be aware that there is variation in intraobserver and interobserver agreement among radiologists on the radiographic features used for diagnosis. there is also variation in how specific radiological features are used in interpreting the radiograph. a recent study on standardization of cxr interpretation in paediatric pneumonia illustrates the importance of standardised training 129 . the cardinal finding of consolidation for the diagnosis of pneumonia appears to be highly reliable 130 and reasonably specific for bacterial pneumonia (74% of 27 patients with alveolar shadowing had bacterial proven pneumonia) 131 but overall chest radiography is too insensitive to be useful in differentiating between patients with bacterial pneumonia and those whose pneumonia is nonbacterial 132, 133 . in the context of an influenza pandemic, a cxr will not distinguish viral pneumonia from viral illness with bacterial superinfection, and all children with signs of pneumonia should be treated with antibiotics. • a cxr should be performed in children who are hypoxic, have severe illness or who are deteriorating despite treatment. oxygen saturation (sao 2 ) measurements provide a noninvasive estimate of arterial oxygenation. pulse oximetry will be a key tool in assessment and management and it is essential that it is used correctly and that users are aware of the possibility of artefactually low readings. the oximeter appears easy to use and requires no calibration. however, it requires a pulsatile signal from the patient. it is also highly subject to motion artefacts. to obtain a reliable reading: (1) the child should be still and quiet. (2) when using paediatric wrap around probes, the emitting and receiving diodes need to be carefully opposed. (3) a good pulse signal (plethysmograph) should be obtained. (4) once a signal is obtained, the saturation reading should be watched over at least 30 seconds and a value recorded once an adequate stable trace is obtained. • pulse oximetry should be performed in every child being assessed for admission to hospital with pneumonia. to be read in conjunction with the corresponding section for adults (section 11 in part 2). as with adults, the extent of virological and microbiological investigations undertaken in children should vary according to the stage of the pandemic and additionally according to the severity of an individual case. it should be noted however, that the clinical features of influenza in children are less characteristic than in adults (see section 4) and the need for special diagnostic tests is therefore greater 62, 134, 135 the utility of rapid influenza tests has been demonstrated in studies where rapid knowledge of a diagnosis of influenza (within ten minutes) has been shown to have an impact on clinicians' behaviour with respect to antibiotic use, performance of other tests and admission to hospital 136, 137 . it may be imagined that in a pandemic situation such a test could result in earlier use of antiviral therapy and a more rational approach to hospital admission and to prophylaxis of contacts. however, using a molecular reference standard, one test was shown to have low sensitivity (44%) but high specificity (97%) suggesting that its role might better be to 'rule in' influenza rather than 'ruling it out' 138 . similar conclusions have been made with other commercial rapid tests 139, 140 . as a reflection of this, rapid antigen tests were positive in only two of six patients with avian influenza a (h5n1) 125 . the need for bacteriological tests in cases of influenza with pneumonia is also logical and the range of pathogens similar to adults 36,69,141-145 except that legionella infection is extremely unlikely to occur in a previously healthy child and legionella-specific antigen testing is therefore unnecessary. the urinary pneumococcal antigen tests in children may lack both sensitivity and specificity and should be interpreted with care 146, 147 . sputum collection in children is also unreliable although in older children (e.g. over 12 years of age) it may be possible and should be handled as indicated for adults. a. virology all children: during an influenza pandemic children are likely to be admitted to hospital because of the severity of their disease and its complications or because of the impact of influenza on pre-existing disorders such as cardiac, respiratory or neurological disease. management of preexisting disorders is outside this guideline. • the most common reason for admission is likely to be: (1) lower respiratory tract disease with either a viral or bacterial or mixed pneumonia. • other reasons for admission include: (2) severe gastroenteritis (3) cardiac disease viral myocarditis (4) encephalitis children should be triaged to ward or hdu/picu after severity assessment (section 15). an influenza pandemic is likely to occur in the winter months when other winter viruses responsible for paediatric morbidity and hospital admission are circulating (such as rsv and adenovirus). particularly in the early stages of a pandemic (uk alert levels 1 3) it will be important to use rapid virological tests in an attempt to cohort influenzapositive and rsv-positive infants separately and to separate from other patients (see uk infection control guidance for pandemic influenza) 3 . hypoxic infants and children may not appear cyanosed. agitation may be an indication of hypoxia. patients whose oxygen saturation is less than 92% while breathing air should be treated with oxygen given by nasal cannulae, head box, or face mask to maintain oxygen saturation above 92%. nasal cannulae do not deliver a fio 2 more than around 40% even at flow rates of 2 l/min in infants and 4 l/min in older children. alternative methods of delivering higher concentrations of humidified oxygen such as a head box or a venturi face mask may be necessary. if sao 2 > 92% cannot be maintained with an fio 2 of 60% then additional support such as cpap, bipap or intubation and ventilation should be considered. • patients whose oxygen saturation is 92% or less while breathing air should be treated with oxygen given by nasal cannulae, head box, or face mask to maintain oxygen saturation above 92%. children who are unable to maintain their fluid intake due to breathlessness, fatigue or gastroenteritis need fluid therapy. where possible additional fluid should be by the enteral route, and where nasogastric tube feeds are used, the smallest tube should be passed down the smallest nostril to minimize effects on respiratory status. severely ill children may need intravenous fluids, and if the child is in oxygen therapy intravenous fluids should be given at 80% basal levels (to avoid complications of inappropriate adh secretion) and serum electrolytes should be monitored. the monitoring will depend on the child's condition. severely ill children will need continuous monitoring of heart rate, respiratory rate, oxygen saturation and neurological status. all children on oxygen therapy should have four-hourly monitoring including oxygen saturation. chest physiotherapy is not beneficial in previously healthy children with pneumonia. children with underlying conditions such as cystic fibrosis or neuromuscular weakness will benefit from intensive physiotherapy. children with influenza are generally pyrexial and may have some pain, including headache, chest pain, arthralgia, abdominal pain, and earache from associated otitis media. pleural pain may interfere with depth of breathing and may impair the ability to cough. antipyretics and analgesics can be used to keep the child comfortable and to help coughing. 18.9. when can children be safely discharged from hospital? in a pandemic situation there will be great pressure on hospital beds. all children should be assessed for discharge at least twice daily. children should not remain in hospital if they are receiving therapy that could be given in the community. in previously healthy children suitable discharge criteria would be: (1) child is clearly improving (2) child is physiologically stable (3) child can tolerate oral feeds (4) respiratory rate is <40/min (<50/min in infants) (5) awake oxygen saturation is >92% on air. most children will make an uneventful recovery and not require follow up. those with a prolonged illness may be followed up by their general practitioner. only children with severe disease and/or at high risk of sequelae need hospital follow up. children with lobar collapse should have a follow-up cxr. follow-up cxrs after acute uncomplicated pneumonia are of no value where the patient is asymptomatic 148, 149 . to be read in conjunction with the corresponding section for adults (section 13 in part 2) five antiviral agents are theoretically available for the therapy of influenza in children: the m2 ion channel inhibitors amantadine and rimantadine (both administered orally and for influenza a only), the neuraminidase inhibitors oseltamivir (administered orally) and zanamavir (administered through an inhaler), and ribavirin (aerosolised). the limitations of amantadine and rimantadine are detailed in section 13, particularly in the context of a pandemic where resistance may already be present 150 . both have been shown to be effective in the treatment of influenza a in children 151 . concerns exist about the development of resistance during therapy for both agents 151, 152 . a household study showed that treatment and prophylaxis with rimantadine resulted in rapid selection and transmission of drug resistant virus 153 . in a double-blind randomised, placebo controlled study, 217 children (1 12 years of age) received oseltamivir with a resultant reduction in the median duration of illness, incidence of otitis media as a complication of influenza (12% vs 21%) and the need for antibiotic prescriptions in those with influenza (68 of 217, 31% vs 97 of 235, 41%; p = 0.03) compared to placebo 103 . the most common sideeffect was vomiting (5.8%). a systematic review and meta-analyses published in 2003, which included studies up to december 2001, included only two studies of zanamivir and one study of oseltamivir 103 in which these drugs were administered for treatment of influenza a or b in children under 12 years of age 154 . the reduction in the median time to alleviation of symptoms for influenza-positive children when compared with placebo was 1.0 day (95% ci: 0.4 1.6) for zanamivir and 1.5 days (0.8 2.2) for oseltamivir. across all ages a 29% (10 44%) relative reduction in complications requiring antibiotics was observed for zanamivir, and for children specifically a 35% relative reduction was observed for oseltamivir. this was updated through to december 2002 in a cochrane review 155 . using its search criteria it identified two trials of oseltamivir (one in healthy children 103 and one in children with asthma which was later published 156 and only one with zanamivir. its conclusions were therefore the same with respect to median illness duration in healthy children. a significant reduction in complications (otitis media) was noted for oseltamivir while a trend to benefit was seen for zanamivir 155 . vomiting was significantly more common among oseltamivir recipients than placebo recipients (15% vs. 9%). the review noted that there may be a difference in efficacy according to serotype, with oseltamivir showing a significant reduction in time to resolution for influenza a (34%) but not b (8.5%) 155 . with respect to children with asthma there was a trend to reduction in time to freedom from illness for oseltamivir recipients but this did not reach statistical significance. oseltamivir appeared to result in a more rapid improvement in pulmonary function, and was well tolerated in children with asthma 155, 156 . the cochrane review concluded that oseltamivir was the preferred drug as it has shown a benefit with regard to secondary complications. it also concluded that there was no evidence of benefit in at-risk children (i.e. asthma). from the perspective of pandemic use however, it should be noted that there was no evidence of harm in this group. with regard to dosing of oseltamivir, pharmacokinetic studies have suggested that young children clear the drug faster than older children, adolescents and adults and therefore need higher doses 157, 158 . the major practical issue with regard to zanamivir is its mode of administration limiting its use to children over the age of five years (fda guidance: over seven years of age) 155 . the development of resistance to oseltamivir in children may be more common than appreciated and more common than seen in adults. in one study resistance mutations were documented in 18% of 50 children 158 . this has implications for widespread use in a pandemic situation. one particular issue with regard to paediatric use of oseltamivir is the apparent age limitation on its license (i.e. not for children under one year of age). this is particularly important because during epidemic years, of all children with influenza, it is children under six months of age who are most likely to be hospitalised 159 . the basis for this exclusion appears to be that rat data have shown high mortality in infant rats at seven days of age when given a dose of 1000 mg/kg together with high brain levels of oseltamivir, assumed to reflect the immature blood brain barrier at this age. this is reflected in product literature and an fda alert although there are no published data. as a result, there are few human data in this age group as it was felt that it would be difficult to monitor cns toxicity in this age group. however, because of a fear of encephalopathy due to influenza in young children, japanese paediatricians §19. use of antivirals in hospitalised children part 3. clinical management of children referred to hospital pandemic flu. clinical management of patients with an influenza-like illness during an influenza pandemic s43 have been using it in infants and data on 102 consecutive infants from japan revealed no encephalopathy or mortality in recipients 160 . a second japanese report where 47 children under one year were treated (4 mg/kg/day) showed similar efficacy for fever to a group of older children and no serious adverse effects 161 . there are no data on the effectiveness of oseltamivir if given more than two days from onset of illness. it is likely to be less effective and in particular to have little or no effect after five to six days of illness unless the child is immunosuppressed. giving oseltamivir to sick hospitalised patients is theoretically likely to decrease their infectivity and so may be useful but there are no data to support this. in a double blind placebo controlled study children hospitalized with influenza who had been ill for 48 hours or less and who had a temperature of 37.8ºc or more were randomised to receive either ribavirin or placebo. sixtytwo patients (35 in the placebo group, 27 in the ribavirin group) had a confirmed diagnosis of influenza. the time to reduction of temperature to 38.3ºc or less for the ribavirin group was 8.9 hours compared with 22.6 hours for the placebo group (p = 0.04). there were no other differences detected between groups 162 . there have been no further published studies in the 11 years since this report, thus ribavirin cannot be recommended at this time. • in the setting of a pandemic, children in the community should only be considered for treatment with antivirals if they have all of the following: (1) an acute influenza-like illness (2) fever (>38.5ºc) and (3) been symptomatic for two days or less. • oseltamivir is the antiviral agent of choice. • treatment schedule for children over one year: body weight 15 kg, i.e. <3 years: 30 mg every 12 h body weight >15 23 kg, i.e. 3 7 years:45 mg every 12 h body weight 24 kg, i.e. >7 years: 75 mg every 12 h • in children who are severely ill in hospital oseltamivir may be used if the child has been symptomatic for less than six days. • oseltamivir may be considered for the treatment of infants under one year of age, especially those with severe influenza. this would need to be done following appropriate discussion with the parents highlighting the concerns from the animal data and the relative paucity of human data in this age group. section 20. use of antibiotics in hospitalised children 20 .1. who should get antibiotics? secondary bacterial infections, particularly pneumonia and otitis media, are common in children with influenza. a case control study during an outbreak of severe pneumococcal pneumonia demonstrated that patients with severe pneumonia were 12 times more likely to have had an influenza-like illness and four times more likely to have positive influenza serology than controls 69 . infections with staph. aureus and h. influenzae are also more common during influenza outbreaks. a randomized controlled trial of antibiotics in 85 children aged four months to 11 years presenting with influenzalike symptoms during an influenza epidemic showed a decreased incidence of pneumonia in the antibiotictreated group (2.4% vs 16.3%, p = 0.031) 163 . there was no change in duration of fever or incidence of acute otitis media. interestingly only one out of seven of the cases of pneumonia in the placebo group was thought to be bacterial. the authors postulated that as bacterial proteases facilitate propogation and pathogenesis of influenza in a mouse model, decreasing bacterial numbers and hence protease levels in the lung may decrease viral pneumonia. another randomized trial of cephalosporins vs macrolides in 365 japanese children with influenza-like symptoms showed faster alleviation of fever (3.8±1.4 vs 4.3±1.4 days, p = 0.006) in the macrolide group and a decrease in number with cxr evidence of pneumonia (2 vs 13 cases, p = 0.002; 14/15 had interstitial changes) 164 . the authors postulate that anti-inflammatory effects of macrolides may be responsible. • children who (a) are at risk of complications of influenza or (b) have disease severe enough to merit hospital admission during an influenza pandemic should be treated with an antibiotic that will provide cover against s. pneumoniae, staph. aureus and h. influenzae. the antibiotics of choice must cover the likely pathogens as above. rarely a blood culture or pleural tap will provide the pathogen. the antibiotics should then be specifically tailored, e.g. iv benzylpenicillin or oral amoxicillin for s. pneumoniae and flucloxacillin or clindamycin for staph. aureus. part 3. clinical management of children referred to hospital §20. use of antibiotics in hospitalised children s44 provisional guidelines from bis/bts/hpa in collaboration with the department of health, version 11 (2 october 2006) a recent randomized controlled trial of the equivalence of oral amoxicillin vs iv benzylpenicillin in 252 children admitted to hospital with community-acquired pneumonia showed no difference in duration of illness or complications 165 . oral antibiotics should be given provided oral fluids are tolerated. 20.5. antibiotic choice for severe or complicated pneumonia? children who are severely ill with pneumonia complicating influenza should have a second agent which provides good cover for gram positive organisms added to the regime (e.g. clarithromycin or cefuroxime) and the drugs should be given intravenously to ensure high serum and tissue antibiotic levels. section 21. acknowledgements, committee members and affiliations 21 chronic obstructive pulmonary disease (copd) including chronic bronchitis and emphysema, and such conditions as bronchiectasis, cystic fibrosis, interstitial lung fibrosis, pneumoconiosis and bronchopulmonary dysplasia (bpd). asthma requiring continuous or repeated use of inhaled or systemic steroids or with previous exacerbations requiring hospital admission. children who have previously been admitted to hospital for lower respiratory tract disease. chronic heart disease congenital heart disease, hypertension with cardiac complications, chronic heart failure and individuals requiring regular medication and/or follow-up for ischaemic heart disease. chronic renal disease nephrotic syndrome, chronic renal failure, renal transplantation. chronic liver disease cirrhosis, inflammatory bowel disease diabetes and chronic metabolic disorders diabetes mellitus requiring insulin or oral hypoglycaemic drugs. immunosuppression and malignancy due to disease or treatment: asplenia or splenic dysfunction, hiv infection at all stages, malignancy. patients undergoing chemotherapy leading to immunosuppression. individuals on or likely to be on systemic steroids for more than a month at a dose equivalent to prednisolone at 20 mg or more per day (any age) or for children under 20 kg a dose of 1 mg or more per kg per day. long-stay residential care homes residents this does not include prisons, young offender institutions, university halls of residence. others doctors retain discretion in identifying additional individual patients who they recognise as at high risk of serious complications should they develop influenza; for example patients with haemoglobinopathies, neurological diseases with muscle weakness, cerebral palsy or children on long-term aspirin who are at increased risk of reye's syndrome. a the high-risk groups described in this appendix are largely based on data from interpandemic influenza. during the course of a pandemic, the definition of 'high-risk groups' may differ. if so, details of the 'high-risk' patient group will be altered according to relevant clinico-epidemiological data. users are strongly advised to refer to the latest version of these guidelines at all times. treat as severe pneumonia antibiotics not indicated <8.5 kg -7.5 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guillain-barre syndrome: a casecontrol study myelitis associated with influenza a virus infection toxic shock syndrome complicating influenza a infection: a two-case report with one case of bacteremia and endocarditis toxic shock syndrome following influenza a infection acute parotitis associated with influenza type a: a report of twelve cases avian influenza: assessing the pandemic threat. who atypical avian influenza (h5n1) fatal avian influenza a (h5n1) in a child presenting with diarrhea followed by coma avian influenza a (h5n1) infection in humans human disease from influenza a (h5n1) avian influenza virus infection of children in vietnam and thailand prospective study of the viral aetiology and clinical course among children in the community with cough and fever nosocomial influenza in children recurrence of febrile seizures in the respiratory season is associated with influenza a influenza a and b virus infections in children influenza in children clinical predictors of 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(ii) severely ill and immunosuppressed patients, including those on long-term corticosteroid therapy, may benefit from antiviral therapy commenced later than 48 hours after the onset of ili. (iii) severely ill children <1 year old. (parents must be informed that oseltamivir is not licensed for children <1 year old.) the first recorded instance of human infection by avian influenza h5n1 occurred in may 1997 in hong kong. the first patient was a 3-year old child who presented initially with symptoms of fever, sore throat and abdominal pain. he later developed reye's syndrome, ards, multi-organ failure and eventually died 166 . a total of 18 persons were subsequently infected before the outbreak ended in december 1997 126, 167 . half the patients were aged 18 years and below and only two were aged over 50 years. abdominal symptoms, such as diarrhoea, vomiting and abdominal pain, were described in ten (56%) patients. eleven (61%) had a severe illness characterised by pneumonia occurring within 14 days of symptom onset, lymphopenia, deranged liver function tests and a high mortality [six (55%) of 11 patients with pneumonia]. secondary bacterial infections were not identified as the cause of the pneumonias.the most recent human outbreak of influenza a (h5n1) infection began in december 2003. the clinical features of hospitalised patients infected by the re-emergent avian influenza a (h5n1) in 2004 were similar to those described in patients in 1997 (table a10 .1). children and young adults were the main groups affected. gastrointestinal symptoms were common. the presence of lymphopenia and deranged liver function tests was again associated with a poorer prognosis 125 .since december 2003, over 150 cases had been reported to the who 168 . the mortality rate among hospitalised patients has been generally high (>40%). death has occurred an average of ten days after the onset of illness and most patients have died of progressive respiratory failure.there has been a review of avian influenza a (h5n1) infection in humans up until september 2005 55 . updated information can be found at www.who.int/csr/disease/avian_influenza/en/. 