key: cord-262693-z9dolxky authors: nishiura, hiroshi; linton, natalie m.; akhmetzhanov, andrei r. title: initial cluster of novel coronavirus (2019-ncov) infections in wuhan, china is consistent with substantial human-to-human transmission date: 2020-02-11 journal: j clin med doi: 10.3390/jcm9020488 sha: doc_id: 262693 cord_uid: z9dolxky reanalysis of the epidemic curve from the initial cluster of cases with novel coronavirus (2019-ncov) in december 2019 indicates substantial human-to-human transmission. it is possible that the common exposure history at a seafood market in wuhan originated from the human-to-human transmission events within the market, and the early, strong emphasis that market exposure indicated animal-to-human transmission was potentially the result of observer bias. to support the hypothesis of zoonotic origin of 2019-ncov stemming from the huanan seafood market, the index case should have had exposure history related to the market and the virus should have been identified from animals sold at the market. as these requirements remain unmet, zoonotic spillover at the market must not be overemphasized. the clinical summary of the earliest cases of 2019 novel coronavirus (2019-ncov) infections in wuhan, china was recently published [1] , showing the majority of cases were exposed to the huanan seafood market, which also had wild animals, suggesting the possibility of zoonotic transmission in the market. this suggestion of zoonotic spillover was quoted by international organizations, including the world health organization (who), and as a result early research focused on zoonotic rather than direct human-to-human transmission of 2019-ncov. however, the index case had no exposure history related to the seafood market, indicating that huanan seafood market-related zoonotic spillover may have been an overblown hypothesis. here, we reanalyze the epidemic data of the initial cluster of cases with 2019-ncov infections to demonstrate that the epidemic curve is consistent with substantial human-to-human transmission in december 2019. three important arguments are made here with respect to epidemiological interpretation of the epidemic dataset. first, figure 1a shows the epidemic curve of cases in wuhan, distinguishing case generations by color. the index case developed symptoms on 1 december 2019, with cases 2-4 having onset nine days later, and cases 5-6 five days after that. together, these intervals indicate a possible serial interval (si)-the time between illness onset in an earlier case to that in a secondary case-with a mean of 7.4 days, consistent with the mean si of severe acute respiratory syndrome [2] . the latter is also consistent with the mean si estimate of 7.5 days presented in the preliminary epidemiological study [3] . although it is possible that the sis are shorter than quoted here [4] , the epidemic curve is still in agreement with the existence of asymptomatic and unascertained mild cases between diagnosed cases. epidemic curve is still in agreement with the existence of asymptomatic and unascertained mild cases between diagnosed cases. the expected number of cases in each subsequent generation was assumed to follow a poisson distribution, and the 95% confidence intervals of the reproduction number (whiskers) were derived from the profile likelihood. second, assuming a constant si of 8 days, the epidemic curve of cases by the date of illness onset can be transformed to that by generation of cases. the number of cases in each generation is therefore 1, 3, 4, 27, and 6 cases, respectively. these numbers allow for the estimation of generation-dependent reproduction numbers-the average number of secondary cases per primary case for each generation [5] ( figure 1b ). assuming that the offspring distribution is poisson distributed, the reproduction numbers can be estimated at 3.0 (95% confidence interval (ci): 0.75, 7.8), 1.3 (95% ci: 0.4, 3.1), 6.7 (95% ci: 4.5, 9.6), and 0.2 (95% ci: 0.1, 0.5)-broadly in line with preliminary basic reproduction number estimates of 1.5-3.5 quoted by the who and presented elsewhere [6, 7] . third, the common exposure supports secondary transmission events taking place in the market. although the virus has been identified in market environmental samples [8] , this does not exclude the likelihood of secondary transmission. that is, it is possible that the common exposure history at the huanan seafood market in wuhan originated from the human-to-human transmission events within the market. unfortunately, early emphasis that market exposure implied animal-to-human transmission considerably delayed global recognition of exportation of the virus from wuhan, especially during the first half of january [9, 10] . the emphasis on market-based zoonotic transmission may have been the result of observer bias-i.e., the bias that originates from having preconceptions or subjective feelings about what is being studied that could influence epidemiological observation and even recording information. for example, the zoonotic origin of another relatively recently emerged coronavirus with predominantly zoonotic transmission-the virus causing middle east respiratory syndrome (mers)-may have served as a strong reference for reducing concern about epidemic levels of sustained human-to-human transmission. in conclusion, we believe that zoonotic spillover at the market should not be overemphasized, because the epidemic curve is consistent with substantial human-to-human transmission in december 2019. there are two important take homes for any future investigations that begin with a similar scenario: first, to verify that zoonotic spillover is related to the exposure in question, the index case must be verified to have that exposure history. second, without identifying the virus in second, assuming a constant si of 8 days, the epidemic curve of cases by the date of illness onset can be transformed to that by generation of cases. the number of cases in each generation is therefore 1, 3, 4, 27, and 6 cases, respectively. these numbers allow for the estimation of generation-dependent reproduction numbers-the average number of secondary cases per primary case for each generation [5] ( figure 1b) . assuming that the offspring distribution is poisson distributed, the reproduction numbers can be estimated at 3.0 (95% confidence interval (ci): 0.75, 7.8), 1.3 (95% ci: 0.4, 3.1), 6.7 (95% ci: 4.5, 9.6), and 0.2 (95% ci: 0.1, 0.5)-broadly in line with preliminary basic reproduction number estimates of 1.5-3.5 quoted by the who and presented elsewhere [6, 7] . third, the common exposure supports secondary transmission events taking place in the market. although the virus has been identified in market environmental samples [8] , this does not exclude the likelihood of secondary transmission. that is, it is possible that the common exposure history at the huanan seafood market in wuhan originated from the human-to-human transmission events within the market. unfortunately, early emphasis that market exposure implied animal-to-human transmission considerably delayed global recognition of exportation of the virus from wuhan, especially during the first half of january [9, 10] . the emphasis on market-based zoonotic transmission may have been the result of observer bias-i.e., the bias that originates from having preconceptions or subjective feelings about what is being studied that could influence epidemiological observation and even recording information. for example, the zoonotic origin of another relatively recently emerged coronavirus with predominantly zoonotic transmission-the virus causing middle east respiratory syndrome (mers)-may have served as a strong reference for reducing concern about epidemic levels of sustained human-to-human transmission. in conclusion, we believe that zoonotic spillover at the market should not be overemphasized, because the epidemic curve is consistent with substantial human-to-human transmission in december 2019. there are two important take homes for any future investigations that begin with a similar scenario: first, to verify that zoonotic spillover is related to the exposure in question, the index case must be verified to have that exposure history. second, without identifying the virus in animals sold at the market, it is difficult to conclude with certainty that any zoonotic transmission occurred at the market. author contributions: h.n. conceived the study, and all authors participated in the study design. h.n. collected the data and a.r.a. and h.n. analyzed the data. all authors jointly drafted the manuscript. all authors gave comments on the earlier versions of the manuscript. all authors have read and agreed to the published version of the manuscript. the authors declare no conflicts of interest. clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet transmission dynamics and control of severe acute respiratory syndrome early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia serial interval of novel coronavirus (2019-ncov) infections assessing dengue control in tokyo transmissibility of 2019-ncov real time estimation of the risk of death from novel coronavirus (2019-ncov) infection: inference using exported cases china detects large quantity of novel coronavirus at wuhan seafood market the extent of transmission of novel coronavirus in wuhan, china, 2020 the rate of underascertainment of novel coronavirus (2019-ncov) infection: estimation using japanese passengers data on evacuation flights key: cord-325093-g2llk2p0 authors: pomara, cristoforo; li volti, giovanni; cappello, francesco title: covid-19 deaths: are we sure it is pneumonia? please, autopsy, autopsy, autopsy! date: 2020-04-26 journal: j clin med doi: 10.3390/jcm9051259 sha: doc_id: 325093 cord_uid: g2llk2p0 the current outbreak of covid-19 severe respiratory disease, which started in wuhan, china, is an ongoing challenge, and a major threat to public health that requires surveillance, prompt diagnosis, and research efforts to understand this emergent pathogen and to develop an effective response. due to the scientific community’s efforts, there is an increasing body of published studies describing the virus’ biology, its transmission and diagnosis, its clinical features, its radiological findings, and the development of candidate therapeutics and vaccines. despite the decline in postmortem examination rate, autopsy remains the gold standard to determine why and how death happens. defining the pathophysiology of death is not only limited to forensic considerations; it may also provide useful clinical and epidemiologic insights. selective approaches to postmortem diagnosis, such as limited postmortem sampling over full autopsy, can also be useful in the control of disease outbreaks and provide valuable knowledge for managing appropriate control measures. in this scenario, we strongly recommend performing full autopsies on patients who died with suspected or confirmed covid-19 infection, particularly in the presence of several comorbidities. only by working with a complete set of histological samples obtained through autopsy can one ascertain the exact cause(s) of death, optimize clinical management, and assist clinicians in pointing out a timely and effective treatment to reduce mortality. death can teach us not only about the disease, it might also help with its prevention and, above all, treatment. the coronavirus disease 2019 (covid-19) pandemic is an ongoing challenge, a threat to global health that requires surveillance, prompt diagnosis, and research efforts to understand this emergent pathogen and to develop effective countermeasures. due to scientific community's efforts, there is an increasing body of published studies describing the covid-19's biology, its transmission and diagnosis, its clinical features, its radiological findings, and the development of candidate therapeutics and vaccines. vice versa, very few autopsy-based data are yet available. are we sure that is it correct to treat covid-19 as a severe pneumonia? are we sure people are dying "with" and not "because of" covid-19? we have only one instrument in medicine to answer to these crucial questions: autopsy, autopsy, autopsy! despite the decline in postmortem examination rate, it remains the gold standard to determine why and how death happens. defining the pathophysiology of death is not only limited to forensic considerations, it may also provide useful clinical and epidemiologic insights [1, 2] . selective approaches to postmortem diagnosis, such as limited postmortem sampling over full autopsy, can also be useful in the control of disease outbreaks and to provide valuable knowledge for managing appropriate control measures [3, 4] . collecting cadavers' samples or biological fluid swabs can be also useful in the control of epidemics, as shown during previous infectious disease outbreaks. during west africa ebola epidemic and for the ebola virus disease (evd) surveillance strategy, the rna virus was isolated in body fluids days or months after the onset of the disease from any living or deceased individual who had, or had had, clinical symptoms compatible with evd. thanks to this procedure, it was possible to monitor the number of infected patients in order to recognize new sources of transmission and to control the epidemic phenomenon [5] [6] [7] [8] [9] [10] . many physicians are wondering these days if we are not facing a systemic pathology that affects the vessels of different anatomical districts, not only the lung but the heart, kidney, liver, intestine, brain, and even the skin. this hypothesis is supported by the fact that angiotensin-converting enzyme 2 (ace2), and putatively also sialic acids, the supposed "doors" by which covid19 enters into endothelial cells and pericytes, are almost ubiquitarian, and not only present in the endothelial cells of alveolar membrane [11, 12] . how can we ever answer these colleagues without doing a good number of autopsies? lack of data from autopsies might result in incomplete or even incorrect postmortem diagnosis during current covid-19 outbreak. however, it is likely that the suddenness of the outbreak, the number of patients in hospitals, the shortage of healthcare personnel, and the high rate of transmissions [13] may significantly reduce the number of autopsies and sampling from cadavers. clinical value of autopsy is also supported by several studies demonstrating that despite the introduction of more modern diagnostic techniques and of intensive and invasive monitoring, the number of missed major diagnoses has not essentially changed over the past 20 to 30 years; autopsies revealed ante mortem diagnostic errors or ante mortem unrecognized diagnoses in about 30% of cases [2, 14] . we cannot, though, underestimate the importance of autopsy as a diagnostic tool to understand the underlying mechanisms behind death. in accordance with the world health organization, postmortem examination for deceased persons infected with covid-19 should be consistent with those used for any autopsies of people who have died from an acute respiratory illness, following the recommended safety procedures. in this scenario, we strongly recommend performing full autopsies on patients who died with suspected or confirmed covid-19 infection, particularly in the presence of several comorbidities. only working a complete set of histological samples obtained through autopsy could help to ascertain the exact cause(s) of death, optimizing clinical management and assisting clinicians in identifying a timely and effective treatment to reduce mortality. moreover, the identification of the exact cause of death could be valuable in the near future, preventing legal and civil disputes for hospital personnel. reviving the practice of autopsy can provide useful information to match with clinical data in order to achieve a better understanding of the pathogenesis of this novel coronavirus disease. as a scientific community, we are called to face this global threat and to defeat it with all available tools needed, new and old, as the new and the old represent a proper union for continued progress in medicine. death can teach us not only about the disease, it might help with its prevention and, above all, treatment. author contributions: conceptualization, c.p., g.l.v., and f.c.; writing-original draft preparation, c.p., g.l.v., and f.c.; writing-review and editing, c.p., g.l.v., and f.c. all authors have read and agreed to the published version of the manuscript. the authors declare no conflict of interest. autopsy findings and clinical diagnoses: a review of 1,000 cases comparison of premortem clinical diagnoses in critically ill patients and subsequent autopsy findings learning from the dead outbreak of influenza a viral infection in ghana: a consideration of autopsy findings and a mini-review of the literature ebola virus shedding and transmission: review of current evidence validity of a minimally invasive autopsy for cause of death determination in adults in mozambique: an observational study a case of severe ebola virus infection complicated by gram-negative septicemia severe ebola virus infection complicated by gram-negative septicemia ebola virus disease complicated with viral interstitial pneumonia: a case report persistence of ebola virus in ocular fluid during convalescence functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses structural basis of receptor recognition by sars-cov-2 pulmonary pathology of early-phase 2019 novel coronavirus (covid-19) pneumonia in two patients with lung cancer autopsy-detected diagnostic errors over time in the intensive care unit key: cord-289422-5z012sr6 authors: kuniya, toshikazu title: prediction of the epidemic peak of coronavirus disease in japan, 2020 date: 2020-03-13 journal: j clin med doi: 10.3390/jcm9030789 sha: doc_id: 289422 cord_uid: 5z012sr6 the first case of coronavirus disease 2019 (covid-19) in japan was reported on 15 january 2020 and the number of reported cases has increased day by day. the purpose of this study is to give a prediction of the epidemic peak for covid-19 in japan by using the real-time data from 15 january to 29 february 2020. taking into account the uncertainty due to the incomplete identification of infective population, we apply the well-known seir compartmental model for the prediction. by using a least-square-based method with poisson noise, we estimate that the basic reproduction number for the epidemic in japan is [formula: see text] ([formula: see text] ci, [formula: see text] – [formula: see text]) and the epidemic peak could possibly reach the early-middle summer. in addition, we obtain the following epidemiological insights: (1) the essential epidemic size is less likely to be affected by the rate of identification of the actual infective population; (2) the intervention has a positive effect on the delay of the epidemic peak; (3) intervention over a relatively long period is needed to effectively reduce the final epidemic size. in december 2019, the first case of respiratory disease caused by a novel coronavirus was identified in wuhan city, hubei province, china. the outbreak of the disease is ongoing worldwide and the world health organization named it coronavirus disease 2019 (covid-19) on 11 february 2020 [1] . in japan, the first case was reported on 15 january 2020 and the number of reported laboratory-confirmed covid-19 cases per week has increased day by day (see table 1 ). as seen in table 1 , the number of newly reported cases per week has increased and a serious outbreak in japan is a realistic outcome. one of the greatest public concerns is whether the epidemic continues until summer so that it affects the summer olympics, which is planned to be held in tokyo. the purpose of this study is to give a prediction of the epidemic peak of covid-19 in japan, which might help us to act appropriately to reduce the epidemic risk. the epidemic data as shown in table 1 would have mainly twofold uncertainty. the first one is due to the fact that asymptomatic infected people could spread the infection [3] . the second one is due to the lack of opportunity for the diagnostic test as sufficiently simple diagnostic test kits have not been developed yet and the diagnosis in the early stage in japan was mainly restricted to people who visited wuhan [4] . in this study, taking into account such uncertainty, we apply a simple and well-known mathematical model for the prediction. more precisely, we assume that only p (0 < p ≤ 1) fraction of infective individuals can be identified by diagnosis. we apply the following well-known seir compartmental model (see, e.g., [5] ) for the prediction. where s(t), e(t), i(t) and r(t) denote the susceptible, exposed, infective and removed populations at time t, respectively. β, ε and γ denote the infection rate, the onset rate and the removal rate, respectively. note that 1/ε and 1/γ imply the average incubation period and the average infectious period, respectively. let the unit time be 1 day. based on the previous studies [6, 7] , we fix 1/ε = 5, and thus, ε = 0.2 and γ = 0.1, respectively. we fix s + e + i + r to be 1 so that each population implies the proportion to the total population. we assume that one infective person is identified at time t = 0 among total n = 1.26 × 10 8 number of people in japan [8] . that is, denotes the number of infective individuals who are identified at time t. thus, we obtain i(0) = 1/(p × 1.26 × 10 8 ). we assume that there is no exposed and removed populations at t = 0, that is, e(0) = r(0) = 0, and hence, it was estimated in [9] that 77 cases were confirmed among the possible 940 infected population in february in hokkaido, japan. based on this report, we assume that p ranges from 0.01 to 0.1. the basic reproduction number r 0 , which means the expected value of secondary cases produced by one infective individual [10] , is calculated as the maximum eigenvalue of the next generation matrix fv −1 [11] , where thus, we obtain (2) it is obvious that the basic reproduction number r 0 is independent from the onset rate ε. the sensitivity of r 0 to other parameters β, γ and p are calculated as follows: where a β , a γ and a p denote the normalized sensitivity indexes with respect to β, γ and p, respectively. we see from equation (3) that the k time's increase in β (resp. γ) results in the k (resp. k −1 ) time's increase in r 0 . in particular, we see from the third equation in equation (3) this implies that the identification rate p in a realistic range almost does not affect the size of r 0 . let y(t), t = 0, 1, . . . , 45 be the number of daily reported cases of covid-19 in japan from 15 january (t = 0) to 29 february (t = 45) 2020. we perform the following least-square-based procedure with poisson noise to estimate the infection rate β. (p1) fix β > 0 and calculate the numerical value of y(t), t = 0, 1, . . . , 45 by using model equation (1). where (t), t = 0, 1, . . . , 45 denote random variables from a normal distribution with mean zero and variance 1 [12] . note that for the reason stated above, the value of 0.01 ≤ p ≤ 0.1 does not affect this estimation procedure. by (p1)-(p6), we obtain a normal distribution with mean 0.26 and standard derivation 0.01. thus, we obtain an estimation of β as 0.26 (95%ci, 0.24-0.28) (see figure 1) . moreover, by equation (2), we obtain an estimation of r 0 as 2.6 (95%ci, 2.4-2.8) (see table 2 ). we define the epidemic peak t * by the time such that y attains its maximum in 1 year, that is, y(t * ) = max 0≤t≤365 y(t). we first set p = 0.1. in this case, we obtain the following figure on the long time behavior of y(t) for β = 0.28, 0.26 and 0.24. we see from figure 2 that the estimated epidemic peak is t * = 208 (95%ci, 191-229). that is, starting from 15 january (t = 0), the estimated epidemic peak is 10 august (t = 208) and the uncertainty range is from 24 july (t = 191) to 31 august (t = 229). we next set p = 0.01. in this case, we obtain the following figure. we see from figure 3 that the estimated epidemic peak is t * = 179 (95%ci, . that is, starting from january 15 (t = 0), the estimated epidemic peak is july 12 (t = 179) and the uncertainty range is from june 28 (t = 165) to july 30 (t = 197). in contrast to r 0 , the epidemic peak and the (apparent) epidemic size are sensitive to the identification rate p. note that the essential epidemic size, which is characterized by r 0 , is almost the same in both of p = 0.1 and p = 0.01. we next discuss the effect of intervention. in japan, school closure has started in almost all prefectures from the beginning of march [13] and many social events have been cancelled off to reduce the contact risk. however, the exact effect of such social efforts is unclear and might be limited as the proportion of young people to the whole infected people of covid-19 seems not so high (2% of 72, 314 reported cases in china [14] ). in this simulation, we assume that such social efforts successfully reduce the infection rate β = 0.26 to 75% during a period from 1 march (t = 46) to a planned day (t = t ≥ 47). in what follows, we fix p = 0.01. first, we set t = 77, that is, the intervention is carried out for 1 month (from 1 march to 1 april). in this case, the epidemic peak t * is delayed from 179 (12 july) to 190 (23 july). however, the epidemic size is almost the same. on the other hand, if t = 220, that is, the intervention is carried out for 6 months (from 1 march to 1 september), then the epidemic peak t * is delayed from 179 (12 july) to 243 (14 september) and the epidemic size is effectively reduced (see figure 4 ). more precisely, we see from figure 5a that the epidemic peak t * is delayed almost linearly for 47 ≤ t ≤ 239 and fixed to t * = 237 for t ≥ 240. this implies that the intervention has a positive effect on the delay of the epidemic peak, which would contribute to improve the medical environment utilizing the extra time period. on the other hand, we see from figure 5b that the number of accumulated cases at t = 365, which is calculated as pr(365) × 1.26 × 10 8 , is monotonically decreasing and converges to 0.99 × 10 6 as t increases. however, it almost does not change for small t ≤ 180. this implies that the intervention over a relatively long duration is required to effectively reduce the final epidemic size. in this study, by applying the seir compartmental model to the daily reported cases of covid-19 in japan from 15 january to 29 february, we have estimated that the basic reproduction number r 0 is 2.6 (95%ci, 2.4-2.8) and the epidemic peak could possibly reach the early-middle summer. of course, this kind of long range peak prediction would contain the essential uncertainty due to the possibility of some big changes in the social and natural (climate) situations. nevertheless, our result suggests that the epidemic of covid-19 in japan would not end so quickly. this might be consistent with the who's statement on 6 march 2020 that it is a false hope that covid-19 will disappear in the summer like the flu [15] . the estimated value of the basic reproduction number r 0 in this study is not so different from early estimations: 2.6 (95%ci, 1.5-3.5) [ [20] . in addition, in this study, we have obtained the following epidemiological insights: the essential epidemic size, which is characterized by r 0 , would not be affected by the identification rate p in a realistic parameter range 0.01-0.1, in particular, p ≥ 1.0 × 10 −6 . the intervention exactly has a positive effect on the delay of the epidemic peak, which would contribute to improve the medical environment utilizing the extra time period. intervention over a relatively long period is needed to effectively reduce the final epidemic size. the first statement implies that underestimation of the actual infective population would not contribute to the reduction of the essential epidemic risk. correct information based on an adequate diagnosis system would be desired for people to act appropriately. available online covid-19) situation reports transmission of 2019-ncov infection from an asymptomatic contact in germany japan sets up emergency measures for coronavirus age-structured populatin dynamics in demography and epidemiology incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data tracking and predicting covid-19 epidemic in china mainland population estimates monthly report japan's hokkaido may have 940 infected, researcher says on the definition and the computation of the basic reproduction ratio r 0 in models for infectious diseases in heterogeneous populations reproduction numbers and sub-threshold endemic equilibria for compartmental models of disease transmission parameter estimation and uncertainty quantification for an epidemic model the japan times. nearly all prefectures in japan shut schools amid coronavirus outbreak characteristics of and imoprtant lessons from the coronavirus disease 2019 (covid-19) outbreak in china it's a 'false hope' coronavirus will disappear in the summer like the flu, who says report 3: transmissibility of 2019-ncov novel coronavirus 2019-ncov: ealry estimation of epidemiological parameters and epidemic predictions 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 the reproductive number of covid-19 is higher compared to sars coronavirus the author would like to thank the associate editor and the anonymous reviewers for their helpful comments that allowed me to improve the manuscript. the author declares no conflict of interest. key: cord-264616-l8bv5t3o authors: zhao, shi; musa, salihu s.; lin, qianying; ran, jinjun; yang, guangpu; wang, weiming; lou, yijun; yang, lin; gao, daozhou; he, daihai; wang, maggie h. title: estimating the unreported number of novel coronavirus (2019-ncov) cases in china in the first half of january 2020: a data-driven modelling analysis of the early outbreak date: 2020-02-01 journal: j clin med doi: 10.3390/jcm9020388 sha: doc_id: 264616 cord_uid: l8bv5t3o background: in december 2019, an outbreak of respiratory illness caused by a novel coronavirus (2019-ncov) emerged in wuhan, china and has swiftly spread to other parts of china and a number of foreign countries. the 2019-ncov cases might have been under-reported roughly from 1 to 15 january 2020, and thus we estimated the number of unreported cases and the basic reproduction number, r(0), of 2019-ncov. methods: we modelled the epidemic curve of 2019-ncov cases, in mainland china from 1 december 2019 to 24 january 2020 through the exponential growth. the number of unreported cases was determined by the maximum likelihood estimation. we used the serial intervals (si) of infection caused by two other well-known coronaviruses (cov), severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) covs, as approximations of the unknown si for 2019-ncov to estimate r(0). results: we confirmed that the initial growth phase followed an exponential growth pattern. the under-reporting was likely to have resulted in 469 (95% ci: 403–540) unreported cases from 1 to 15 january 2020. the reporting rate after 17 january 2020 was likely to have increased 21-fold (95% ci: 18–25) in comparison to the situation from 1 to 17 january 2020 on average. we estimated the r(0) of 2019-ncov at 2.56 (95% ci: 2.49–2.63). conclusion: the under-reporting was likely to have occurred during the first half of january 2020 and should be considered in future investigation. a novel coronavirus (2019-ncov) infected pneumonia infection, which is deadly [1] , was first identified in wuhan, china in december 2019 [2] . the virus causes a range of symptoms including fever, cough, and shortness of breath [3] . the cumulative number of reported cases slowly increased to cumulative 41 cases by 1 january 2020, and rapidly increased after 16 january 2020. as of 26 january 2020, the still ongoing outbreak had resulted in 2066 (618 of them are in wuhan) confirmed cases and 56 (45 of them were in wuhan) deaths in mainland china [4] , and sporadic cases exported from wuhan were reported in thailand, japan, republic of korea, hong kong, taiwan, australia, and the united states, please see the world health organization (who) news release via https://www.who.int/csr/don/en/ from 14 to 21 january 2020. using the number of cases exported from wuhan to other countries, a research group at imperial college london estimated that there had been 4000 (95%ci: 1000-9700) cases in wuhan with symptoms onset by 18 january 2020, and the basic reproduction number (r 0 ) was estimated at 2.6 (95%ci: 1.5-3.5) [5] . leung et al. drew a similar conclusion and estimated the number of cases exported from wuhan to other major cities in china [6] , and the potentials of travel related risks of disease spreading was also indicated by [7] . due to an unknown reason, the cumulative number of cases remained at 41 from 1 to 15 january 2020 according to the official report, i.e., no new case was reported during these 15 days, which appears inconsistent with the following rapid growth of the epidemic curve since 16 january 2020. we suspect that the 2019-ncov cases were under-reported roughly from 1 to 15 january 2020. in this study, we estimated the number of unreported cases and the basic reproduction number, r 0 , of 2019-ncov in wuhan from 1 to 15 january 2020 based on the limited data in the early outbreak. the time series data of 2019-ncov cases in mainland china were initially released by the wuhan municipal health commission from 10 to 20 january 2020 [8] , and later by the national health commission of china after 21 january 2020 [9] . the case time series data in december 2019 were obtained from a published study [3] . all cases were laboratory confirmed following the case definition by the national health commission of china [10] . we chose the data up to 24 january 2020 instead of to the present study completion date. given the lag between timings of case confirmation and news release of new cases, the data of the most recent few days were most likely to be tentative, and thus they were excluded from the analysis to be consistent. we suspected that there was a number of cases, denoted by ξ, under-reported from 1 to 15 january 2020. the cumulative total number of cases, denoted by c i , of the i-th day since 1 december 2019 is the summation of the cumulative reported, c i , and cumulative unreported cases, ξ i . we have c i = c i + ξ i , where c i is observed from the data, and ξ i is 0 for i before 1 january and ξ for i after 15 january 2020. following previous studies [11, 12] , we modelled the epidemic curve, i.e., the c i series, as an exponential growing poisson process. since the data from 1 to 15 january 2020 appeared constant due to unclear reason(s), we removed these data from the fitting of exponential growth. the ξ and the intrinsic growth rate (γ) of the exponential growth were to be estimated based on the log-likelihood, denoted by , from the poisson priors. the 95% confidence interval (95% ci) of ξ was estimated by the profile likelihood estimation framework with cutoff threshold determined by a chi-square quantile [13] , χ 2 pr = 0.95, df = 1 . with γ estimated, the basic reproduction number could be obtained by r 0 = 1/m(−γ) with 100% susceptibility for 2019-ncov presumed at this early stage. here, the function m(·) was the laplace transform, i.e., the moment generating function, of the probability distribution for the serial interval (si) of the disease [11, 14] , denoted by h(k) and k is the mean si. since the transmission chain of 2019-ncov remained unclear, we adopted the si information from severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers), which share the similar pathogen as 2019-ncov [15] [16] [17] . we modelled h(k) as gamma distributions with mean of 8.0 days and standard deviation (sd) of 3.6 days by averaging the si mean and sd of sars, mean of 7.6 days and sd of 3.4 days [18] , and mers, mean of 8.4 days and sd of 3.8 days [19] . we were also interested in inferring the patterns of the daily number of cases, denoted by ε i for the i-th day, and thus it is obviously that c i = c i−1 + ε i . a simulation framework was developed for the iterative poisson process such that e[ denoted the expectation. the simulation was implemented starting from 1 january 2020 with a cumulative number of cases seed of 40, the same as reported on 31 december 2019. we conducted 1000 samples and calculated the median and 95% ci. the number of 2019-ncov unreported cases was estimated at 469 (95% ci: 403-540), see figure 1a , which was significantly larger than 0. this finding implied the occurrence of under-reporting between 1 and 15 january 2020. after accounting for the effect of under-reporting, the r 0 was estimated at 2.56 (95% ci: 2.49-2.63), see figure 1b , which is consistent with many existing online preprints with range from 2 to 4 [5, [20] [21] [22] . with the r 0 of 2.56 and ξ of 469, the exponential growing framework fitted the cumulative total number of cases (c i ) remarkably well, see figure 1c iterative poisson process such that denoted the expectation. the simulation was implemented starting from 1 january 2020 with a cumulative number of cases seed of 40, the same as reported on 31 december 2019. we conducted 1000 samples and calculated the median and 95% ci. the number of 2019-ncov unreported cases was estimated at 469 (95% ci: 403−540), see figure 1a , which was significantly larger than 0. this finding implied the occurrence of under-reporting between 1 and 15 january 2020. after accounting for the effect of under-reporting, the r0 was estimated at 2.56 (95% ci: 2.49−2.63), see figure 1b , which is consistent with many existing online preprints with range from 2 to 4 [5, [20] [21] [22] . with the r0 of 2.56 and ξ of 469, the exponential growing framework fitted the cumulative total number of cases (ci) remarkably well, see figure 1c , referring to mcfadden's pseudo-r-squared of 0.99. show the exponential growth fitting results of the cumulative number of cases (ci) and the daily number of cases (εi) respectively. in panels (c) and (d), the gold squares are the reported cases, the blue bold curve represents the median of the fitting results, the dashed blue curves are the 95% ci of the fitting results, and the purple shading area represents the time window from 1 to 15 january 2020. in panel (c), the blue dots are the cumulative total, i.e., reported and unreported, number of cases. in panel (d), the grey curves are the 1000 simulation samples. our estimation of r0 rely on the si of 2019-ncov, which remains unknown as of 26 january 2020. in this work, we employed the sis of sars and mers as approximations to that of 2019-ncov. the determination of si requires the knowledge of the chain of disease transmission that needs a sufficient number of patient samples and periods of time for follow-up [23] , and thus this is unlikely to be achieved shortly. however, using sis of sars and mers as approximation could provide an panels (a,b) , the green shading area represents the 95% ci (on the horizontal axis), and the vertical green line represents the maximum likelihood estimate (mle) of the number of unreported cases. with the mle of r 0 at 2.56, panels (c,d) show the exponential growth fitting results of the cumulative number of cases (c i ) and the daily number of cases (ε i ) respectively. in panels (c,d), the gold squares are the reported cases, the blue bold curve represents the median of the fitting results, the dashed blue curves are the 95% ci of the fitting results, and the purple shading area represents the time window from 1 to 15 january 2020. in panel (c), the blue dots are the cumulative total, i.e., reported and unreported, number of cases. in panel (d), the grey curves are the 1000 simulation samples. our estimation of r 0 rely on the si of 2019-ncov, which remains unknown as of 26 january 2020. in this work, we employed the sis of sars and mers as approximations to that of 2019-ncov. the determination of si requires the knowledge of the chain of disease transmission that needs a sufficient number of patient samples and periods of time for follow-up [23] , and thus this is unlikely to be achieved shortly. however, using sis of sars and mers as approximation could provide an insight into the transmission potential of 2019-ncov at the early outbreak. we note that slightly varying the mean and sd of si would not affect our main conclusions. the r 0 of 2019-ncov was estimated at 2.56 (95% ci: 2.49-2.63), and it is generally in line with those of sars, i.e., 2-5 [19, 24, 25] , and mers, i.e., 2.7-3.9 [26] . for the simulated daily number of cases (ε i ), see figure 1d , we found that ε i matched the observed daily number after 17 january 2020, but was significantly larger than the observations from 1 to 17 january 2020. this finding implied that under-reporting was likely to have occurred in the first half of january 2020. we estimated that the reporting rate after 17 january 2020 increased 21-fold (95% ci: [18] [19] [20] [21] [22] [23] [24] [25] compared to the situation from 1 to 17 january 2020 on average. one of the possible reasons was that the official diagnostic protocol was released by who on 17 january 2020 [27] , and the diagnosis and reporting efforts of 2019-ncov infections probably increased. thereafter, the daily number of newly reported cases started increasing rapidly after 17 january 2020, see figure 1d . we conducted additional sensitivity analysis by varying the starting date of the under-reporting time window, e.g., 1 january 2020 in the main results, from 2 december 2019 to 3 january 2020, and we report our estimates largely hold. the exact value of the reporting rate was difficult to determine due to lack of serological surveillance data. the reporting rate can be determined if serological surveillance data are available for a population; we would know who was infected (seropositive) and who was not (seronegative), with high confidence. the reporting rate is the ratio of reported cases over the number of seropositive individuals. it was statistically evident that increasing in reporting was likely, and thus it should be considered in the future investigation of this outbreak. previous preprint suggested cumulative cases of 1723 (95% ci: 427-4471) as of 12 january 2020, and 4000 (95% ci: 1000-9700) as of 18 january 2020 based on the aggregated international export cases [5] . our analysis yielded cumulative cases of 280 (95% ci: 128-613) as of 12 january 2020, and 609 (95% ci: 278-1333) as of 18 january 2020 based on the exponential growing mechanistic in the early outbreak. although our estimate case number appeared to have a lower mean than those estimated by imai et al. [5] , they are not statistically different. this study applied a different screening effort to detect the 2019-ncov cases from that in imai et al. [5] . imai et al. assumed the average screening effort at overseas airports that covered travelers arriving from wuhan. whereas we assumed a constant screening effort applied in wuhan at the same point of time, and then a number of cases (i.e., ξ) should have been reported yet failed to be reported in the first half of january 2020 due to all sorts of reasons. it is not surprising that different assumptions yielded different results, and this difference in screening effort also partly explained why the detected cases out of china mainly presented mild symptoms. thus, it was reasonable that our estimates appeared lower than those estimated by imai et al. [5] . it must be emphasized that such a gap in the knowledge would be resolved by serological survey study (for a large population to approximate the actual positive rate) or an explicit estimation of the actual reporting rate. under-reporting was likely to have occurred and resulted in 469 (95% ci: 403-540) unreported cases from 1 to 15 january 2020. the reporting rate after 17 january 2020 was likely to have increased 21-fold (95% ci: 18-25) compared with the situation from 1 to 17 january 2020 on average, and it should be considered in future investigation. we estimated the r 0 at 2019-ncov to be 2.56 (95% ci: 2.49-2.63). author contributions: all authors conceived the study, carried out the analysis, discussed the results, drafted the first manuscript. all authors have read and agreed to the published version of the manuscript. real-time tentative assessment of the epidemiological characteristics of novel coronavirus infections in wuhan, china, as at 22 clinical features of 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future modeling the spread of middle east respiratory syndrome coronavirus in saudi arabia laboratory testing for 2019 novel coronavirus (2019-ncov) in suspected human cases this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-262996-zxn86z6k authors: störmann, philipp; klug, alexander; nau, christoph; verboket, rené d.; leiblein, max; müller, daniel; schweigkofler, uwe; hoffmann, reinhard; marzi, ingo; lustenberger, thomas title: characteristics and injury patterns in electric-scooter related accidents—a prospective two-center report from germany date: 2020-05-22 journal: j clin med doi: 10.3390/jcm9051569 sha: doc_id: 262996 cord_uid: zxn86z6k since the introduction of rental e-scooters in germany in mid-june 2019, the safety of this new means of transport has been the subject of extensive public debate. however, valid data on injuries and usage habits are not yet available. this retrospective two-center study included a total of 76 patients who presented to the emergency department following e-scooter-related accidents. the mean age was 34.3 ± 12.4 years and 69.7% of the patients were male. about half of the patients were admitted by ambulance (42.1%). fractures were found in 48.6% of patients, and 27.6% required surgical treatment due to a fracture. the upper extremities were the most commonly affected body region, followed by injuries to the lower extremity and to the head and face. only one patient had worn a helmet. in-hospital treatment was necessary for 26.3% of the cases. patients presented to the emergency department mainly during the weekend and on-call times. this is the first report on e-scooter-related injuries in germany. accidents with e-scooters can cause serious injuries and, therefore, represent a further burden to emergency departments. the use of e-scooters appears to be mostly recreational, and the rate of use of protective gear is low. at the end of 2017, rental electrically powered scooters (e-scooters) were first introduced in the usa as a new, nationwide means of transport [1] . in germany, approval for the use of e-scooters in public road traffic was granted on june 15, 2019. since then, e-scooters have been distributed throughout several metropolitan regions and large cities in germany, such as frankfurt am main, primarily via rental companies. in germany, the maximum speed of the scooter is limited to 20 km/h, the rated power of the electric motor may not exceed 500 watts, and the maximum weight of the scooter is 55 kg. in addition to the existing insurance obligation, e-scooters must also be equipped with front and rear lights, as well as two separately functioning brakes. use is permitted from a minimum age of 14 years. however, helmets are not compulsory. in germany, the e-scooter is equivalent to a bicycle in terms of traffic law, and thus the use of sidewalks is officially prohibited. currently, there are around 2,000 e-scooters distributed throughout frankfurt via four major providers. according to press and police reports, traffic offences, such as the use of sidewalks, have occurred regularly since these vehicles were introduced. given that this is a new means of transport, meaning that riders may have insufficient experience in handling the scooters, an increased number of injured persons was expected in germany. however, reliable national data on e-scooter-related injuries are not yet available. likewise, worldwide data on injury patterns related to the use of e-scooters are sparse, mainly due to the short time period since their introduction to the public. in the usa, e-scooters have been approved since september 2017, and the first scientific studies in this area are now available. in these reports, the majority of injured persons were involved as scooter drivers, whereas about 10% were hit as pedestrians [2] . approximately 40% of patients suffered craniocerebral trauma, while fractures of the extremities were the second most frequent injuries (31%) [3, 4] . despite only including a small number of cases, another american study described a relevant increase in the number of cases of e-scooter-related injury and, in particular, reports of severe craniocerebral trauma as a relevant injury [1] . these figures are in line with an observational study performed in new zealand, where scooters have been registered since september 2018. this study also reported a relevant increase in serious injuries, particularly to the extremities, but also to the axial skeleton [5] . despite frequent reports of accidents in the german media, there are no valid figures on injury patterns related to e-scooter accidents available to date. likewise, the international literature is also very sparse. however, this information is important to estimate the burden these injuries pose to emergency departments and to our health care system. we hypothesize that e-scooter-related accidents result in typical high-energy injuries and that the rate of use of protective gear is low. the aim of this study was therefore to identify injury patterns following e-scooter accidents and to evaluate the need for in-hospital and surgical treatment associated with these specific injuries. the two largest level 1 trauma centers in frankfurt, both located within the city, participated in this retrospective analysis. the university hospital frankfurt is located in the south of the city, while the bg trauma center is located in the north of the city. frankfurt has about 750,000 inhabitants, and due to the numerous commuters from the surrounding area, the number of people present in the city on working days is over 1,000,000. the two participating trauma centers are the largest in the city with together approximately 42,000 patient presentations in the emergency departments every year. the inclusion criteria were patients over 14 years of age who suffered e-scooter-related injuries. all patients involved in an accident with an e-scooter who presented by ambulance or independently to the emergency department of one of the two hospitals were included in this prospective observational study. data on usage habits (e.g., wearing a helmet, using the scooter alone), injury pattern, clinical care (surgical vs. non-surgical treatment) and outcome (outpatient vs. in-hospital care) were prospectively collected, starting by the emergency department doctor during the initial treatment and after informed consent was obtained from the patient. thereafter, the patient's clinical course was prospectively followed, and all related data were collected by the study coordination team. each chronical illness was registered as co-morbidity, as well as each permanent medication (except oral contraception) was registered as pre-existing medication. the primary outcome included all injuries diagnosed during the clinical course. these injuries were categorized as serious (severe traumatic brain injury, fractures) or minor (contusions, lacerations). secondary outcomes included need for surgery, in-hospital and intensive care unit treatment. this study was approved by the local ethics committee of the johann-wolfgang goethe university (ev 19/408). values are reported as mean ± standard deviation (sd) for continuous variables and as percentages for categorical variables. the p-values for categorical variables were derived from the chi-square or two-sided fisher's exact test. all analyses were performed using statistical package for social sciences software (spss for mac; version 24.0; spss inc., chicago, il, usa). over the 9-month study period, a total of 76 patients were included. the mean age of patients was 34.28 ± 12.4 years (range 15-67 years). two patients were younger than 18 years of age and 69.7% were male. of the 76 patients, 13.2% (n = 10) stated that they suffered from a pre-existing morbidity, and 10.5% (n = 8) were on permanent medication of any kind prior to the accident. in total, 70 patients (92.1%) suffered from an accident without any external influence, whereas five patients (6.6%) were admitted after a collision with a car and one patient (1.3%) collided with a forklift truck. all patients used the e-scooter alone, with a rate of first use of 32.9%. self-admission was registered in 44 patients (57.9%), and 32 patients (42.1%) were admitted by ambulance. in 27.6% of the cases, the accident occurred on wet ground. only one patient was using a helmet while using the e-scooter. nine patients (11.8%) were initially unconscious; however, endotracheal intubation was not necessary in any of the patients, neither preclinically nor in-hospital. no differences with regard to usage habits, accident characteristics or preinjury medications and comorbidities were found between male and female patients ( table 1 ). table 2 presents the injury pattern. overall, 43 patients (56.6%) suffered from at least one serious injury, with two patients (2.6%) suffering two serious injuries. a total of 21 patients (27.6%) required surgical management for their injuries. the upper extremities were the main body region affected, with a total of 36 injuries (47.4%), of which 26 were considered serious. thirteen patients (17.1%) had to undergo surgical procedures for their upper extremity injury. the second most common injury location was the head and face (n = 29, 38.2%), followed by injuries of the lower extremities (n = 28, 36.8%). injuries of the chest were registered in 9.2% of patients (n = 7). no abdominal injuries were found. most accidents were registered during summer (august/september), with lower numbers observed in winter. due to restrictions imposed during the sars-cov-2 pandemic, numbers for march 2020 were the lowest (figure 1 overall, 73.7% of the patients (n = 56) were treated as outpatients. of those, despite having an indication for in-hospital treatment, four patients (equal to 5.3% of all patients) discharged themselves from the hospital against medical advice. in total, 26.3% of the patients (n = 20) required in-hospital treatment with a mean length of stay of 5.1 ± 4.5 days (minimum 1 day, maximum 15 days). of those 20 patients, four required intensive medical care. in all cases, the reason for intensive care unit admission was severe traumatic brain injury including intracerebral bleeding (n = 1), subdural hematomas (n = 2) and subarachnoidal bleeding (n = 2). the mean intensive care unit length of stay was 3.3 ± 2.2 days. no fatalities were registered. to the best of our knowledge, this is the first study of e-scooter-related injuries in germany. the first worldwide introduction of rental e-scooters took place in 2017 in san francisco, usa. since then, public discussion focused on increasing accident numbers and the involvement of e-scooters in traffic accidents has increased [6] [7] [8] . however, due to the short time period since the introduction of this overall, 73.7% of the patients (n = 56) were treated as outpatients. of those, despite having an indication for in-hospital treatment, four patients (equal to 5.3% of all patients) discharged themselves from the hospital against medical advice. in total, 26.3% of the patients (n = 20) required in-hospital treatment with a mean length of stay of 5.1 ± 4.5 days (minimum 1 day, maximum 15 days). of those 20 patients, four required intensive medical care. in all cases, the reason for intensive care unit admission was severe traumatic brain injury including intracerebral bleeding (n = 1), subdural hematomas (n = 2) and subarachnoidal bleeding (n = 2). the mean intensive care unit length of stay was 3.3 ± 2.2 days. no fatalities were registered. to the best of our knowledge, this is the first study of e-scooter-related injuries in germany. the first worldwide introduction of rental e-scooters took place in 2017 in san francisco, usa. since then, public discussion focused on increasing accident numbers and the involvement of e-scooters in traffic accidents has increased [6] [7] [8] . however, due to the short time period since the introduction of this new means of transport, data on injury characteristics and prevention are still scarce. only a few articles reporting e-scooter-related injury patterns and their outcomes have been published so far [9] . in a study performed in new zealand over a 4-month period, mayhew et al. recorded 46 patients who were admitted to a level 1 center after falling from an e-scooter [5] . a study by trivedi et al. reported on 90 patients who presented with e-scooter-related trauma in texas over a 7-month period. ishmael and colleagues recently analyzed the surgical procedures that have become necessary following an e-scooter accident [4] . comparing these publications with our results, a similar patient picture emerges from all studies. the gender distribution is comparable, with about two-thirds being male, and the patients are young to middle aged. although e-scooter use by minors is permitted in all countries, the proportion of juvenile patients was low in all investigations. in our study, peaks of accident-related emergency department presentations were observed at weekends and in the late evening and night hours. similarly, in the studies by mayhew et al. and ishmael et al., the majority of emergency department presentations occurred in the summer months, as well as during on-call times [4, 5] . this accumulation of patients outside regular working hours places further strain on the already scarce resources in emergency departments. moreover, the observed usage characteristics support the assumption that e-scooters are more likely used as leisure equipment and not-as hoped by politicians-as an additional means of transport to a workplace or to bridge the last distance between home and the local public transport stop. in this context, a recent study from california highlighted that e-scooters are particularly popular with tourists [4] . the limited data available to date suggest that a high percentage of accidents involving e-scooters will result in serious injuries, usually to the head and extremities. this finding is further substantiated by our investigation, as we found serious injuries such as traumatic brain injury and fractures in 56.6% of patients. the severity of the head injuries varied, but all intensive care stays were nevertheless attributable to traumatic brain injury. due to the relatively high speed on small wheels, which is comparable to that of cyclists, and the low fall height with a short reaction time, the extremities, especially the upper extremities, and head are the most commonly affected body areas [2, 6, 10] . as a consequence, the risk for relevant long-term functional limitations following e-scooter accidents should not be underestimated. complex articular fractures and ligamentous injuries, in particular to the elbow joint, may result in permanent instability and a reduced range of motion [11] . the unbraked impact of the head without a protective helmet may also cause permanent disability and significant restrictions of the individual's preinjury lifestyle. furthermore, the high number of midface injuries and tooth fractures might ultimately lead to a cosmetically unfavorable outcome. these significant injury patterns are not only due to the high speed and short reaction time associated with e-scooter use, as mentioned above, but also due to the very low rate of use of protective measures, such as helmets. in all available studies, including ours, the use of e-scooters without a helmet was found in almost 100% of cases. using a helmet might probably have reduced the rate of concussions and severe traumatic brain injury, even if the face is not protected by a classic bicycle helmet. in addition, in the context of leisure use, a high degree of inexperience in handling an e-scooter has to be assumed, which further increases the risk of being involved in an accident. in this respect, the current literature demonstrates a high rate of intoxicated patients, ranging from 17.8% up to 36.6% in different studies [3, 10] . unfortunately, due to restrictions imposed by our local ethics committee, we were not able to measure the blood alcohol level of our patients. nevertheless, from a clinical standpoint, a high percentage of patients appeared to be intoxicated to some degree at the time of presentation. a comparison of the e-scooter accident mechanism with other sports is rather difficult due to the special combination of high speed and proximity to the ground. it is noteworthy, however, that the injury patterns appear largely similar to those observed after skateboarding, skiing and snowboarding accidents [12, 13] . in all of these sport activities, the fall height is low, the speed is high, and the reaction time is short. skateboarding accidents, for example, did not only show a high percentage of traumatic brain injuries, but also a high rate of injury to the extremities. in contrast, injuries of the chest and abdomen were more rarely found. as a result of snowboarding and skiing accidents, glenohumeral dislocations-also seen in our study-are frequently observed [14] . the treating emergency department teams should therefore be aware of these injury patterns, which are otherwise only known from high speed or extreme sports. furthermore, similar injury patterns have also been described for accidents involving hoverboards, which are another newer means of transport. here, the use of helmets and wrist guards have been strongly recommended [15] . these safety measures could similarly reduce the number of significant injuries after e-scooter accidents. both in our study and in the available literature on e-scooter-related accidents to date, the use of helmets is virtually non-existent. in the present study, only a single injured person was wearing a helmet, even though the benefit of helmets in preventing traumatic brain injury has been well analyzed and proven in the past [16] [17] [18] . considering the rate of head injuries following e-scooter accidents, the use of a helmet should therefore be strongly recommended. additionally, adapted protective equipment may be needed to protect the face and extremities. as only level 1 centers took part in the present study, there was incomplete coverage of the city, as patients with minor injuries may have independently visited level 2 and 3 centers in the city. it can be assumed, however, that patients with multiple and/or serious injuries are primarily assigned to one of the maximum care centers. the data presented are also influenced by population density, topography of the city, the public transport system and other parameters, which should be taken into account when comparing the data with future studies. due to the short investigation period, the present study provides only a first overview of the injury patterns that should be expected and the burden that e-scooter-related injuries pose for emergency departments. for example, in the current literature, no fatalities have been documented so far, although these have to be expected in the case of further use in road traffic. in the future, multicenter studies should be carried out to evaluate the injury patterns and outcomes more precisely. electric scooter-related accidents are associated with a significant number of serious injuries. these injuries include fractures and lacerations of the midface, as well as fractures and dislocations of the upper extremities, which often require surgical treatment. in light of the significant rate of severe injuries, the use of protective clothing, especially helmets, is strongly recommended. the authors declare no conflict of interest. emergency department visits for electric scooter-related injuries after introduction of an urban rental program injuries associated with standing electric scooter use craniofacial injuries seen with the introduction of bicycle-share electric scooters in an urban 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of helmet use with traumatic brain and cervical spine injuries following bicycle crashes helmet use and bicycle-related trauma injury outcomes key: cord-354717-4vrqzbof authors: linton, natalie m.; kobayashi, tetsuro; yang, yichi; hayashi, katsuma; akhmetzhanov, andrei r.; jung, sung-mok; yuan, baoyin; kinoshita, ryo; nishiura, hiroshi title: incubation period and other epidemiological characteristics of 2019 novel coronavirus infections with right truncation: a statistical analysis of publicly available case data date: 2020-02-17 journal: j clin med doi: 10.3390/jcm9020538 sha: doc_id: 354717 cord_uid: 4vrqzbof the geographic spread of 2019 novel coronavirus (covid-19) infections from the epicenter of wuhan, china, has provided an opportunity to study the natural history of the recently emerged virus. using publicly available event-date data from the ongoing epidemic, the present study investigated the incubation period and other time intervals that govern the epidemiological dynamics of covid-19 infections. our results show that the incubation period falls within the range of 2–14 days with 95% confidence and has a mean of around 5 days when approximated using the best-fit lognormal distribution. the mean time from illness onset to hospital admission (for treatment and/or isolation) was estimated at 3–4 days without truncation and at 5–9 days when right truncated. based on the 95th percentile estimate of the incubation period, we recommend that the length of quarantine should be at least 14 days. the median time delay of 13 days from illness onset to death (17 days with right truncation) should be considered when estimating the covid-19 case fatality risk. as of 31 january 2020, mainland china reported 11,791 confirmed cases of novel coronavirus infections, causing 259 deaths [1] . initially, these infections were thought to result from zoonotic (animal-to-human) transmission; however, recently published evidence [2] and the exponential growth of case incidence show compelling evidence of human-to-human secondary transmission fueled by travel, with many cases detected in other parts of the world [3] . this geographic expansion beyond the initial epicenter of wuhan provides an opportunity to study the natural history of covid-19 infection, as these migration events limit the risk of infection to the time during which an individual traveled to an area where exposure could occur [4] . the incubation period is defined as the time from infection to illness onset. knowledge of the incubation period of a directly transmitted infectious disease is critical to determine the time period required for monitoring and restricting the movement of healthy individuals (i.e., the quarantine period) [5, 6] . the incubation period also aids in understanding the relative infectiousness of covid-19 and can be used to estimate the epidemic size [7] . time-delay distributions including dates of hospital admission (for treatment and/or isolation) and death also inform the temporal dynamics of epidemics. a published clinical study on the covid-19 epidemic has already shown that the average time delay from illness onset to hospital admission is approximately 7 days [8] , but this distribution has yet to be explicitly estimated. the time from hospital admission to death is also critical to the avoidance of underestimation when calculating case fatality risk [9] . using publicly available data from the ongoing epidemic with known case event dates, the present study aimed to estimate the incubation period and other time intervals that govern the interpretation of epidemiological dynamics of covid-19 infections. we retrieved information on cases with confirmed covid-19 infection and diagnosis outside of the epicenter of wuhan, china, based on official reports from governmental institutes, as well as reports on deceased cases from both in and outside of wuhan. we aggregated the data directly from government websites or from news sites that quoted government statements. the data were collected in real time, and thus may have been updated as more details on cases became available. the arranged data include a selection of cases reported through 31 january 2020 and are available as supplementary tables s1 and s2 . specifically, we collected the dates of exposure (entry and/or exit from wuhan or dates of close contact with a wuhan resident/known epidemic case), illness onset, earliest healthcare seeking related to infection, hospital admission (for treatment and/or isolation), and death. cases included both residents from other locations who travelled to wuhan, as well as individuals who lived, worked, or studied in wuhan (hereafter: wuhan residents) but who were diagnosed outside of wuhan and reported by the governments of the locations where their infection was detected. we thus estimated the incubation period by (i) excluding wuhan residents and (ii) including wuhan residents. the former may be more precise in defining the interval of exposure, but the sample size is greater for the latter. more detailed information about the criteria used for the estimation of each defined time interval and the data used are described in supplementary text s1. we used the dates of three critical points in the course of infection-illness onset, hospital admission, and death-to calculate four time intervals: the time from (a) exposure to illness onset (i.e., the incubation period), (b) illness onset to hospital admission, (c) illness onset to death, and (d) hospital admission to death. we used a doubly interval-censored [10] likelihood function to estimate the parameter values for these intervals, written as: here, in the case of (a) g(.) is the probability density function (pdf) of exposure following a uniform distribution, and f (.) is the pdf of the incubation period independent of g(.). d represents a dataset among all observed cases i, where exposure and symptom onset fall within the lower and upper bounds (e l , e r ) and (s l , s r ). we fit the pdf f (.) to lognormal, weibull, and gamma distributions. to address the selection bias in the dataset due to the continued growth of the outbreak (i.e., cases with shorter incubation periods are more likely to be included in the dataset), we also accounted for right truncation using the formula: here, r is the exponential growth rate (estimated at 0.14 [11] ), t is the latest time of observation (31 january 2020), and f(.) is the cumulative density function of f (.). in both cases, we used bayesian methods to infer parameter estimates and obtain credible intervals. we selected the best fit model by using the widely applicable information criterion (waic). we also verified that the bayesian estimates were in line with pointwise estimates derived by maximum likelihood estimation (mle). as the formulation of the likelihood with right truncation (1)(2) contained the function f and was dependent on both the time interval (s − e) and time of exposure e, we generalized a previously obtained result for doubly interval-censored likelihood with f (s − e, e) ≡ f (s − e) [10] . the data were processed using r version 3.6.2 [12] , mle was computed using julia version 1.3 [13] , and the markov chain monte carlo (mcmc) simulations were performed in stan (cmdstan version 2.22.1 [14] ). all code is freely available at the github repository: http://github.com/aakhmetz/ wuhanincubationperiod2020. the ratio of male to female cases among living cases resembled [2] , at 58%, with most 30-59 years of age (information missing for 9 cases). the deceased cases were more predominantly male (70%) and older (85% were 60 years of age or older). table 1 shows estimates for the various time intervals without right truncation. for the incubation period estimates, the lognormal distribution provided the best fit to the data, both when excluding and including wuhan residents. the mean incubation period was estimated at 5.0 days (95% credible interval [ci]: 4.2, 6.0) when excluding wuhan residents (n = 52) and 5.6 days (95% ci: 5.0, 6.3) when including wuhan residents (n = 158). the median time from illness onset to hospital admission was estimated at 3.3 days (95% ci: 2.7, 4.0) among living cases and 6.5 days (95% ci: 5.2, 8.0) among deceased cases using the gamma distribution, which provided the best fit for both sets of data. figure 1a shows the corresponding pdfs. data from the time from illness onset and hospital admission to death best fit lognormal and weibull distributions, respectively, as presented in figure 1b , c. the mean time from illness onset to death was 15.0 days (95% ci: 12.8, 17.5) and from hospital admission to death was 8.8 days (95% ci: 7.2, 10.8). table 2 shows estimates for the fit of the lognormal distribution for each interval when accounting for right truncation. the mean incubation period was 5.6 days (95% ci: 4.4, 7.4) when excluding wuhan residents-slightly larger than the estimate without right truncation. the mean estimate for illness onset to hospital admission was 9.7 days (95% ci: 5.4, 17.0) for living cases and 6.6 days (95% ci: 5.2, 8.8) for deceased cases, with the former nearly 2.5 times the length of its untruncated version. illness onset to death and hospital admission to death were likewise longer than their non-truncated counterparts, at 20.2 days (95% ci: 15.1, 29.5) and 13.0 days (95% ci: 8.7, 20.9), respectively. figure 2 shows the cumulative distribution function of the incubation period with and without right truncation. the 5th and 95th percentiles are shown in addition to the median. the 95th percentiles were estimated at 10.6 days (95% ci: 8.5, 14.1) and 10.8 days (95% ci: 9.3, 12.9) for non-truncated data excluding and including wuhan residents and 12.3 days (95% ci: 9.1, 19.8) when applying right truncation and excluding wuhan residents. the respective median values for these cdfs were 4.3 days (95% ci: 3.5, 5.1), 5.0 days (95% ci: 4.4, 5.6), and 4.6 days (95% ci: 3.7, 5.7). the present study advances the public discussion on covid-19 infections by presenting explicit estimations of the incubation period and other epidemiologic characteristics using publicly available data. our estimated mean incubation period of approximately 5 days is comparable to known mean values of the incubation period for severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) [9, [15] [16] [17] , as well as other recent estimates of the incubation period for covid-19 [17] . in addition to empirically showing the comparability of covid-19 to other disease-causing coronaviruses, the present study has also shown that the 95th percentile of the incubation period is around 10-14 days, indicating that a 14-day quarantine period would largely ensure the absence of disease among healthy exposed individuals. wuhan residents have a less precisely defined exposure period compared to travelers and secondary cases from known human to human transmission events. however, our calculations have shown that adding more cases to the dataset even with uncertainty reduces both the variance the present study advances the public discussion on covid-19 infections by presenting explicit estimations of the incubation period and other epidemiologic characteristics using publicly available data. our estimated mean incubation period of approximately 5 days is comparable to known mean values of the incubation period for severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) [9, [15] [16] [17] , as well as other recent estimates of the incubation period for covid-19 [17] . in addition to empirically showing the comparability of covid-19 to other disease-causing coronaviruses, the present study has also shown that the 95th percentile of the incubation period is around 10-14 days, indicating that a 14-day quarantine period would largely ensure the absence of disease among healthy exposed individuals. wuhan residents have a less precisely defined exposure period compared to travelers and secondary cases from known human to human transmission events. however, our calculations have shown that adding more cases to the dataset even with uncertainty reduces both the variance the present study advances the public discussion on covid-19 infections by presenting explicit estimations of the incubation period and other epidemiologic characteristics using publicly available data. our estimated mean incubation period of approximately 5 days is comparable to known mean values of the incubation period for severe acute respiratory syndrome (sars) and middle east respiratory syndrome (mers) [9, [15] [16] [17] , as well as other recent estimates of the incubation period for covid-19 [17] . in addition to empirically showing the comparability of covid-19 to other disease-causing coronaviruses, the present study has also shown that the 95th percentile of the incubation period is around 10-14 days, indicating that a 14-day quarantine period would largely ensure the absence of disease among healthy exposed individuals. wuhan residents have a less precisely defined exposure period compared to travelers and secondary cases from known human to human transmission events. however, our calculations have shown that adding more cases to the dataset even with uncertainty reduces both the variance of the estimates and selection bias, improving the fit of the distribution mean. our estimates are in agreement with the report of li et al. [2] . a recent study by backer et al. [17] noted a similar finding in their analysis of the incubation period for 88 cases (including 63 wuhan residents). however, the estimates of backer et al. for the model that included wuhan residents were subject to overestimation as the lower bounds for wuhan residents-who had unknown left exposure dates-were fixed in their analysis. in contrast, we considered the left exposure dates for wuhan residents as parameters to be fitted-see [14] for details. notably, our results demonstrated the overall benefit of using additional case data, even when some of exposure values were not precisely known. the time from the illness onset to death is also comparable to sars [15] , and the 15-20-day mean delay indicates that a crude estimation of the ratio of the cumulative number of deaths to that of confirmed cases will tend to result in an underestimation of the case fatality risk, especially during the early stage of epidemic spread [18, 19] . during the sars epidemic in hong kong, 2003, the time from illness onset to hospital admission was shown to have shortened as a function of the calendar time, gradually reflecting the effects of contact tracing [9] . it remains to be seen if this will be the case for covid-19 as well. the time delay distribution between illness onset and hospital admission may also be negatively associated with the basic reproduction number, i.e., the average number of secondary cases generated by a single primary case in a fully susceptible population [18] . the median time from illness onset to hospital admission was approximately 4 days among cases not known to be deceased at the time of the case report, and 6 days among cases reported as deceased. the reasons for this difference are not altogether clear. however, the living cases include persons who were isolated-in some cases more for reducing transmission than for treatment purposes-while all deceased cases were admitted for treatment. in addition, deceased cases for whom information was available had onset dates closer to the beginning of the outbreak compared to the living cases, who mostly had onset in the latter two-thirds of january 2020. the time delay distributions from illness onset to hospital admission for cases reported later in the epidemic, when there was a more widespread recognition of the virus and a more prevalent social imperative for those with symptoms to seek healthcare, may differ from those of early cases [2] . several limitations of the present study exist. first, the dataset relies on publicly available information that is not uniformly distributed (i.e., collected from various sources), and therefore the availability of dates relevant to our analyses is limited to a small, selective sample that is not necessarily generalizable to all confirmed cases. moreover, given the novelty of the covid-19 pneumonia, it is possible that illness onset and other event data were handled differently between jurisdictions (e.g., was illness onset the date of fever or date of dyspnea?). second, our data include very coarse date intervals with some proxy dates used to determine the left and/or right hand dates of some intervals. third, as the sample size was limited, the variance is likely to be biased. fourth, we were not able to examine the heterogeneity of estimates by different attributes of cases (e.g., severity of disease) [16] . lastly, as we only have information on confirmed cases, there is a bias towards more severe disease-particularly for earlier cases. this study presents the estimates of epidemiological characteristics of covid-19 infections that are key parameter for studies on incidence, case fatality, and epidemic final size, among other possibilities [7, 11] . from the 95th percentile estimate of the incubation period we found that the length of quarantine should be at least 14 days, and we stress that the 17-24-day time delay from illness onset to death must be addressed when estimating covid-19 case fatality risk. this study was made possible only through open sharing of case data from china and other countries where cases were diagnosed. continued communication of dates and other details related to exposure and infection is crucial to furthering scientific understanding of the virus, the infections it causes, and preventive measures that can be used to contain and mitigate epidemic spread. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/2/538/s1, table s1 : event dates for exported cases included in the analysis, table s2 : event dates for deceased cases included in the analysis, text s1, estimation of the time interval distribution using doubly interval-censored likelihood, estimation of the time interval distributions using bayesian framework, and data cleaning rules implemented for the various time intervals. the authors declare no conflicts of interest. update on pneumonia of new coronavirus infection as of 21:00 on early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia european centre for disease prevention and control data estimates of shortand long-term incubation periods of plasmodium vivax malaria in the republic of korea new york city department of health and mental hygiene swine influenza investigation team. outbreak of 2009 pandemic influenza a (h1n1) at a new york city school determination of the appropriate quarantine period following smallpox exposure: an objective approach using the incubation period distribution the extent of transmission of novel coronavirus in wuhan, china, 2020 clinical features of patients infected with 2019 novel coronavirus in wuhan epidemiological determinants of spread of causal agent of severe acute respiratory syndrome in hong kong estimating incubation period distributions with coarse data real time estimation of the risk of death from novel coronavirus (2019-ncov) infection: inference using exported cases r: a language and environment for statistical computing a fresh approach to numerical computing. siam rev stan modeling language users guide and reference manual, version 2.22.0 incubation periods of acute respiratory viral infections: a systematic review association between severity of mers-cov infection and incubation period the incubation period of 2019-ncov infections among travellers from wuhan, china. medrxiv 2020 who rapid pandemic assessment collaboration. pandemic potential of a strain of influenza a (h1n1): early findings methods for estimating the case fatality ratio for a novel, emerging infectious disease this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-290135-ax5ck4qw authors: urbano, nicoletta; scimeca, manuel; di russo, carmela; mauriello, alessandro; bonanno, elena; schillaci, orazio title: [(99)mtc]sestamibi spect can predict proliferation index, angiogenesis, and vascular invasion in parathyroid patients: a retrospective study date: 2020-07-13 journal: j clin med doi: 10.3390/jcm9072213 sha: doc_id: 290135 cord_uid: ax5ck4qw the aim of this study was to evaluate the possible association among sestamibi uptake and the main histopathological characteristics of parathyroid lesions related to aggressiveness such as the proliferation index (ki67 expression and mitosis), angiogenesis (number of vessels), and vascular invasion in hyperparathyroidism patients. to this end, 26 patients affected by primary hyperparathyroidism subjected to both scintigraphy with [(99)mtc]sestamibi and surgery/bioptic procedure were retrospectively enrolled. hyperfunctioning of the parathyroid was detected in 19 patients. our data showed a significant positive association among the sestamibi uptake and the proliferation index histologically evaluated both in terms of the number of ki67 positive cells and mitosis. according to these data, lesions with a higher valuer of l/n (lesion to nonlesion ratio) frequently showed several vessels in tumor areas and histological evidence of vascular invasion. it is noteworthy that among patients with negative scintigraphy, 2 patients showed a neoplastic lesion after surgery (histological analysis). however, it is important to highlight that these lesions displayed very low proliferation indexes, which was evaluated in terms of number of both mitosis and ki67-positive cells, some/rare vessels in the main lesion, and no evidence of vascular invasion. in conclusion, data obtained on patients with positive or negative scintigraphy support the hypothesis that sestamibi can be a tracer that is capable of predicting some biological characteristics of parathyroid tumors such as angiogenesis, proliferation indexes, and the invasion of surrounding tissues or vessels. recent data indicate a continuous increase in hyperparathyroidism (php) incidence [1] . it affects the patient's quality of life by dysregulating calcium homeostasis and thereby inducing various multiple organ complications. currently, surgery represents the unique definite treatment for php patients [1] . the "policlinico tor vergata" ethical committee approved this protocol with the reference number #129. 18 . in addition, all methodologies and experimental procedures here described were achieved in agreement with the last helsinki declaration. exclusion criteria: a second cancer and neoadjuvant hormonal or radiation therapy prior to surgery. according to these criteria, we retrospectively enrolled 26 consecutive patients with parathyroid dysfunction (64.81 ± 2.56 years; range 33-82 years; 20 women and 6 men), who underwent both [ 99 mtc]tc-spect with sestamibi and a parathyroid bioptic procedure from january 2018 to december 2019. for each of them, histological diagnosis and immunohistochemical investigations were performed. early acquisition was performed at 15 min after the intravenous injection of 740 mbq tc-sestamibi (bristol-myers squibb pharma, bruxelles, belgium) according to the recommendations of the european association of nuclear medicine [15] . planar images of the neck and chestwere were obtained in a 256 × 256 matrix, with a 20% energy window centered at a 140 kev photopeak using a high-resolution spect system (millenium vg & hawkeye; general electric medical systems, milwaukee, wi, usa) equipped with low-energy high-resolution parallel-hole collimators. patients were positioned supine, with the neck supported in an extended position and arms lowered alongside the body. a step-and-shoot protocol was used that consisted of 20 s per frame with a total of 64 frames. transverse, coronal, and sagittal spect images were generated by using a gaussian 2.0 prefilter, and they were post-processed by using fast low-angle shot three-dimensional iterative reconstruction (four iterations, eight subsets). an attenuation correction factor of 0.15/cm was applied with the chang method. all 26 patients had biopsy. [ 99 mtc]sestamibi spect was performed before biopsy in 15 patients and after biopsy in 11 patients. when [ 99 mtc]sestamibi spect was performed after biopsy, the minimum interval between biopsy and imaging was 7 days in an effort to avoid the effects of post-biopsy inflammation as much as possible. for qualitative analysis of [ 99 mtc]sestamibi spect, two investigators classified positive and negative findings. lesions with no demonstrable uptake and those with diffuse heterogeneous or minimal patchy uptake were considered negative, whereas lesions with scattered patchy uptake, partially focal uptake, or any other focal uptake were regarded as positive. irregular-shaped regions of interest (rois) were used to encase the lesions. the evaluation of the lesion to nonlesion ratio (l/n) was estimated according to our previous study [12] . for the patients who underwent [ 99 mtc]sestamibi spect before biopsy (n = 15), the [ 99 mtc]sestamibi spect-guided biopsy procedure was performed. semiquantitative analysis of the [ 99 mtc]sestamibi was performed. parathyroid bioptic samples were formalin fixed and embedded in paraffin [16] . serial sections were used for both hematoxylin-eosin (h&e) and immunohistochemicalstaining for ki67. for each sample, three h&e serial sections were used to evaluate the number of mitosis and the number of vessels on 10 high power field (hpf; 40 × magnification) randomly selected cancer areas. in all sections, the vascular and cap invasion was also assessed. all morphological evaluations were performed by using digital slides (iscan coreo, ventana, tucson, az, usa). immunohistochemistry was used to study the proliferation index by ki67 expression. three-µm-thick paraffin sections were treated with citrate buffers ph 6.0 for 30 min at 95 • c to antigen retrieval reaction. afterwards, sections were incubated with pre-diluted anti-ki67 rabbit monoclonal antibody (clone 30-9, ventana, tucson, az, usa). washings were performed with pbs/tween20 ph 7.6. reactions were detected by using an hrp-dab detection kit (ucs diagnostic, rome, italy). a digital scan was used to evaluate the immunohistochemical reactions (iscan coreo, ventana, tucson, az, usa). specifically, ki67 was calculated in terms of percentage of positive parathyroid cells. reactions have been set up by using specific positive and negative control tissues. specifically, negative controls were perfomed on serial paraffin section without using primary antibody, wherear positive controls were performed by investigated the ki67 expression on thymus paraffin sections. in order to evaluate the possible association among sestamibi uptake, the age, percentage of ki67 positive cancer cells, number of mitosis, number of vessels, and vascular invasion linear regression analyses were performed. one-way anova was used to evaluate the l/n ratio in parathyroid histotypes (3 groups) . the difference between groups was considered statistically significant at p < 0.05. [ 99 mtc]sestamibi spect analyses showed sestamibi uptake in 19 patients (l/n max 2.78; min 0.85) ( figure 1a) . conversely, no sestamibi uptake was observed in 7 patients ( figure 1b) . no significant differences were observed by comparing l/n ratio and parathyroid histotypes. j. clin. med. 2020, 9, x for peer review 4 of 10 in order to evaluate the possible association among sestamibi uptake, the age, percentage of ki67 positive cancer cells, number of mitosis, number of vessels, and vascular invasion linear regression analyses were performed. one-way anova was used to evaluate the l/n ratio in parathyroid histotypes (3 groups) . the difference between groups was considered statistically significant at p < 0.05. [ 99 mtc]sestamibi spect analyses showed sestamibi uptake in 19 patients (l/n max 2.78; min 0.85) ( figure 1a) . conversely, no sestamibi uptake was observed in 7 patients ( figure 1b) . no significant differences were observed by comparing l/n ratio and parathyroid histotypes (data not shown). a parathyroid carcinoma (0.6 cm) was identified after the surgery by histological analysis. (b) to evaluate the parathyroid sestamibi uptake, that of the thyroid has been subtracted (c) image displays no [ 99 mtc]sestamibi uptake in a 68-year-old woman with primary hyperparathyroidism. a parathyroid hyperplasia (0.2 cm) was identified after the surgery by histological analysis. (d) to evaluate the parathyroid sestamibi uptake, that of the thyroid has been subtracted. parathyroid biopsies were classified according to the world health organization [17] . in particular, we found 8/26 hyperplasia, 8/26 parathyroid adenoma, and 10/26 parathyroid carcinoma. no secondary, mesenchymal, and other tumors were observed. interestingly, parathyroid tumors (1 parathyroid adenoma and 1 parathyroid carcinoma) were detected in 2 patients with no sestamibi parathyroid biopsies were classified according to the world health organization [17] . in particular, we found 8/26 hyperplasia, 8/26 parathyroid adenoma, and 10/26 parathyroid carcinoma. no secondary, mesenchymal, and other tumors were observed. interestingly, parathyroid tumors (1 parathyroid adenoma and 1 parathyroid carcinoma) were detected in 2 patients with no sestamibi uptake. no association was found between sestamibi uptake and parathyroid histotypes (hyperplasia l/n 1.62 ± 0.36; parathyroid adenoma l/n 1.85 ± 0.84; parathyroid carcinoma l/n 2.02 ± 1.29; p = 0.678). to investigate the possible association between sestamibi uptake and cells proliferation in parathyroid lesions, linear regression analyses were performed ( figure 2 ). interestingly, positive significant associations were found by comparing the l/n ratio with both ki67 index (p = 0.0003; r 2 0.4657) and the number of mitosis (p = 0.0002; r 2 0.4720) (figure 2a ,b,e-j). it is important to note the high concordance between the value of mitosis and the percentage of ki67 positive cells. to exclude the influence of age on both sestamibi uptake and proliferation index, linear regression analyses were performed between age and both l/n ratio and ki67 value ( figure 2c uptake. no association was found between sestamibi uptake and parathyroid histotypes (hyperplasia l/n 1.62 ± 0.36; parathyroid adenoma l/n 1.85 ± 0.84; parathyroid carcinoma l/n 2.02 ± 1.29; p = 0.678). to investigate the possible association between sestamibi uptake and cells proliferation in parathyroid lesions, linear regression analyses were performed ( figure 2 ). interestingly, positive significant associations were found by comparing the l/n ratio with both ki67 index (p = 0.0003; r 2 0.4657) and the number of mitosis (p = 0.0002; r 2 0.4720) (figure 2a ,b,e-j). it is important to note the high concordance between the value of mitosis and the percentage of ki67 positive cells. to exclude the influence of age on both sestamibi uptake and proliferation index, linear regression analyses were performed between age and both l/n ratio and ki67 value ( figure 2c linear regression analysis was performed to study the possible association between sestamibi uptake and the number of vessels in the tumor area. it is noteworthy that a positive significant association was observed (p = 0.0148 r 2 0.2513) (figure 3) . in order to establish the capability of [ 99 mtc]sestamibi spect analyses to predict the aggressiveness of parathyroid carcinomas, we subdivided selected lesions according to the presence of vascular invasion, which was evaluated in terms of the presence of cancer cells in at least 2 vessels. our data showed a significant increase in sestamibi uptake in lesions characterized by vascular invasion as compared to lesions without any histological evidence of vascular invasion (p = 0.0377) (figure 3 ). it is important to note that biopsies of patients affected by hyperplasia have been excluded from the analyses of vessels and vascular invasion. of interest, no vascular invasion was observed in both parathyroid adenoma and parathyroid carcinoma of patients with negative [ 99 mtc]sestamibi spect. linear regression analysis was performed to study the possible association between sestamibi uptake and the number of vessels in the tumor area. it is noteworthy that a positive significant association was observed (p = 0.0148 r 2 0.2513) (figure 3) . in order to establish the capability of [ 99 mtc]sestamibi spect analyses to predict the aggressiveness of parathyroid carcinomas, we subdivided selected lesions according to the presence of vascular invasion, which was evaluated in terms of the presence of cancer cells in at least 2 vessels. our data showed a significant increase in sestamibi uptake in lesions characterized by vascular invasion as compared to lesions without any histological evidence of vascular invasion (p = 0.0377) (figure 3 ). it is important to note that biopsies of patients affected by hyperplasia have been excluded from the analyses of vessels and vascular invasion. of interest, no vascular invasion was observed in both parathyroid adenoma and parathyroid carcinoma of patients with negative [ 99 mtc]sestamibi spect. php represents the most common disorder of the endocrine system, with a prevalence of up to 1% and increased incidence in women and with advanced age [1] clinical studies reported several co-morbidities related to php such as musculoskeletal, neuropsychiatric, gastrointestinal, renal, and cardiovascular disorders. thus, the occurrence of php is associated to both a significant reduction of the patient's quality of life and an increase of risk for morbidity [18] . in addition, several studies reported an increase of the risk of parathyroid carcinoma occurrence in patients affected by php [19, 20] . indeed, despite parathyroid carcinoma being described as an uncommon malignancy, its incidence significantly increases in patients affected by php [20] . nevertheless, the pathogenesis of parathyroid carcinoma is not fully understood yet. therefore, the diagnosis of these tumors is considered a diagnostic challenge due to the absence of peculiar characteristics that allow a definite distinction of malignant from benign disease. concerning the therapy, currently, surgery remains the only curative approach for both php and parathyroid carcinoma, also allowing the identification of the histological and molecular characteristics of these lesions. php represents the most common disorder of the endocrine system, with a prevalence of up to 1% and increased incidence in women and with advanced age [1] clinical studies reported several co-morbidities related to php such as musculoskeletal, neuropsychiatric, gastrointestinal, renal, and cardiovascular disorders. thus, the occurrence of php is associated to both a significant reduction of the patient's quality of life and an increase of risk for morbidity [18] . in addition, several studies reported an increase of the risk of parathyroid carcinoma occurrence in patients affected by php [19, 20] . indeed, despite parathyroid carcinoma being described as an uncommon malignancy, its incidence significantly increases in patients affected by php [20] . nevertheless, the pathogenesis of parathyroid carcinoma is not fully understood yet. therefore, the diagnosis of these tumors is considered a diagnostic challenge due to the absence of peculiar characteristics that allow a definite distinction of malignant from benign disease. concerning the therapy, currently, surgery remains the only curative approach for both php and parathyroid carcinoma, also allowing the identification of the histological and molecular characteristics of these lesions. from a diagnostic point of view, parathyroid scintigraphy is often used to detect a hyperfunctioning parathyroid tissue in patients with php prior to surgery [10, 21, 22] . in this context, [ 99 mtc]sestamibi is the main radiotracer employed in parathyroid scintigraphy, since this molecule remains longer in the mitochondria of the parathyroid rather than thyroid, where it is washed out quickly [23, 24] . despite this, the predictive role of sestamibi uptake in the occurrence and progression of parathyroid lesions, as well as the association with histopathological characteristics, represents an open question in the management of patients affected by php and/or parathyroid carcinoma. starting from these considerations, the aim of this study was to evaluate the possible association among sestamibi uptake and the main histopathological characteristics of parathyroid lesions related to aggressiveness such as proliferation index (ki67 expression and mitosis), angiogenesis (number of vessels), and vascular invasion in php patients. to this end, 26 php patients subjected to both scintigraphy with [ 99 mtc]sestamibi and surgery/bioptic procedure were retrospectively enrolled. hyperfunctioning of the parathyroid was detected in 19 patients. our data showed a significant positive association among the sestamibi uptake and the proliferation index evaluated both in terms of the number of ki67 positive cells and mitosis. this can be explained by (1) the capability of sestamibi to remain in the mitochondria after passive diffusion [14] , (2) the increased uptake of sestamibi in mitochondria with high membrane potential [12] , and (3) the role of mitochondria and their membrane potential in the cell proliferation process [25] . of note, we also found that age affects neither the sestamibi uptake nor proliferation index in our case selection. according to data of the association between proliferation index and sestamibi uptake, we observed that lesions with a higher l/n value (lesion to nonlesion ratio) frequently showed several vessels in tumor areas and histological evidence of vascular invasion. therefore, the uptake of sestamibi increased in metabolically active lesions characterized by tumors cells proliferation, angiogenesis, and invasion. these characteristics are strongly related to the capability of tumors to grow and invade surrounding tissues [26, 27] . in particular, the angiogenesis phenomenon has been associated to metastatic spread in parathyroid cancers by garcia de la torre and colleagues [26] . it is noteworthy that among patients with negative scintigraphy, 2 patients showed a neoplastic lesion after surgery (histological analysis). however, it is important to highlight that these lesions displayed very low proliferation indexes, which were evaluated in terms of the number of both mitosis and ki67 positive cells, some/rare vessels in the main lesion, and no evidence of vascular invasion. thus, our data seem to indicate that scintigraphy with [ 99 mtc]sestamibi could underestimate the presence of parathyroid lesions with a greater morphological aspect of neoplasia but low aggressiveness. nichols et al. demonstrated that most frequently, false negative scintigraphy with [ 99 mtc]sestamibi occurs in the presence of parathyroid lesions contiguous with the upper or lower poles of the thyroid gland [28] . however, to the best of our knowledge, no study investigated the possible association between negative scintigraphy with [ 99 mtc]sestamibi and histopathological characteristics of parathyroid lesions. in this study, for the first time, a relationship between sestamibi uptake and the histopathological characteristics of parathyroid tumors was shown. both data obtained on patients with positive and negative scintigraphy support the hypothesis that sestamibi can be a tracer that is capable of predicting some biological characteristics of parathyroid tumors such as angiogenesis, proliferation indexes, and the invasion of surrounding tissues or vessels. the possibility of detecting these characteristics by in vivo analysis opens new perspectives in the management of php patients. indeed, our data, if confirmed on a large cohort of patients, could be used to develop diagnostic protocols that are capable of stratifying php patients according to prognostic and predictive information generally provided by histological and immunohistochemical analysis. in addition, our approach can be used for other diseases, thus expanding the diagnostic "equipment" available to nuclear physicians. the capability of sestamibi to identify malignant lesions by spect analysis has been shown for several human diseases such as hearth injury, breast cancer renal carcinomas, and php [29] [30] [31] [32] [33] . indeed, numerous investigations demonstrated a close association between positive [ 99 mtc]sestamibi analysis and the severity of disease [29] [30] [31] [32] [33] . however, few studies correlated the sestamibi uptake with the histopathological characteristics of human lesions [34, 35] . therefore, despite preliminary findings, the results of this study can support the physicians in the evaluation of [ 99 mtc]sestamibi spect in php patients. in general, the association between nuclear medicine and anatomic pathology data could provide the scientific rationale for developing new in vivo diagnostic methods that are capable of predicting prognosis or response to therapy for human cancers. oxyphil parathyroid adenoma: a malignant presentation of a benign disease the pathophysiologic basis of nuclear medicine an unusual ectopic location of a parathyroid carcinoma arising within the thyroid gland the endocrine system profiling analysis of long non-coding rna and mrna in parathyroid carcinoma parathyroid neoplasms: immunohistochemical characterization and long noncoding rna (lncrna) expression micrornas in parathyroid physiopathology detection rate of 99 mtc-mibi single photon emission computed tomography (spect)/ct in preoperative planning for patients with primary hyperparathyroidism: a meta-analysis 99 mtc labelled cationic lipophilic complexes in malignant and benign tumors: the role of spet and pinhole-spet in breast cancer, differentiated thyroid carcinoma and hyperparathyroidism snm practice guideline for parathyroid scintigraphy 4.0 breast-specific gamma imaging with [ 99 mtc]tc-sestamibi: an in vivo analysis for early identification of breast cancer lesions expressing bone biomarkers 99 mtc sestamibi spect: a possible tool for early detection of breast cancer lesions with high bone metastatic potential 99 mtc-sestamibi breast imaging: current status, new ideas and future perspectives eanm parathyroid guidelines programmed death ligand 1 expression in prostate cancer cells is associated with deep changes of the tumor inflammatory infiltrate composition iarc who classification of tumours of endocrine organs the american association of endocrine surgeons guidelines for definitive management of primary hyperparathyroidism parathyroid carcinoma: challenges in diagnosis and treatment role of pre-operative imaging using 99mtc-mibi and neck ultrasound in patients with secondary hyperparathyroidism who are candidates for subtotal parathyroidectomy evaluation of single isotope technetium 99m-sestamibi in localization efficiency for hyperparathyroidism detection and localization of parathyroid adenomas in patients with hyperparathyroidism using a single radionuclide imaging procedure with technetium-99m-sestamibi (double-phase study) parathyroid imaging with technetium-99m-sestamibi: preoperative localization and tissue uptake studies tca cycle and mitochondrial membrane potential are necessary for diverse biological functions angiogenesis and lymphangiogenesis in parathyroid proliferative lesions immunohistochemistry in diagnostic parathyroid pathology analysis of causes of false negative mibi spect studies in pts with single gland disease primary hyperparathyroidism (sgphp) visual evaluation and differentiation of renal oncocytomas from renal cell carcinomas by means of 99m tc-sestamibi spect/ct. ejnmmi res semi-quantitative analysis of 99mtc-sestamibi retention level for preoperative differential diagnosis of parathyroid carcinoma breast-specific gamma imaging or ultrasonography as adjunct imaging diagnostics in women with mammographically dense breasts diagnostic performance of mri, molecular breast imaging, and contrast-enhanced mammography in women with newly diagnosed breast cancer lung signal as a hint of covid-19 infection on tc-99m-sestamibi myocardial perfusion scintigraphy funding: this research received no external funding. the authors declare no conflict of interest. j. clin. med. 2020, 9, key: cord-284301-fg3hk94b authors: umemura, yutaka; yamakawa, kazuma; kiguchi, takeyuki; nishida, takeshi; kawada, masahiro; fujimi, satoshi title: hematological phenotype of covid-19-induced coagulopathy: far from typical sepsis-induced coagulopathy date: 2020-09-05 journal: j clin med doi: 10.3390/jcm9092875 sha: doc_id: 284301 cord_uid: fg3hk94b background: blood coagulation disorders commonly occur with severe coronavirus disease 2019 (covid-19). however, there is only limited evidence on differentiating the pattern of the hemostatic parameters from those of typical sepsis-induced coagulopathy (sic). methods: to elucidate the specific pattern of coagulopathy induced by covid-19 pneumonia, this retrospective, observational study targeted consecutive adult patients with covid-19-induced acute respiratory distress syndrome (ards) and compared hemostatic biomarkers with non-covid-19-induced septic ards. multilevel mixed-effects regression analysis was performed and kaplan–meier failure curves were constructed. results: we enrolled 24 patients with covid-19-induced ards and 200 patients with non-covid-19-induced ards. platelet count, antithrombin activity, and prothrombin time in the covid-19 group were almost within normal range and time series alterations of these markers were significantly milder than the non-covid-19 group (p = 0.052, 0.037, and 0.005, respectively). however, fibrin/fibrinogen degradation product and d-dimer were significantly higher in the covid-19 group (p = 0.001, 0.002, respectively). covid-19 patients had moderately high levels of thrombin–antithrombin complex and plasmin-alpha2-plasmin inhibitor complex but normal plasminogen activator inhibitor-1 level. conclusions: the hematological phenotype of covid-19-induced coagulopathy is quite different from that in typical sic characterized by systemic hypercoagulation and suppressed fibrinolysis. instead, local thrombus formation might be promoted in severe covid-19. the novel corona virus infection (coronavirus disease 2019: , originating in wuhan, china, has rapidly spread worldwide [1] . as of july 15, 2020, more than 13.8 million cases and approximately 590,000 deaths were reported from all over the world. along with acute respiratory distress syndrome (ards), coagulation disorders are reported to be induced by severe covid-19 pneumonia and to be associated with increased risk of death [2] [3] [4] [5] . besides, a previous study reported that severe acute respiratory syndrome coronavirus 2 (sars-cov-2) this was a single-center, retrospective, observational study conducted at a tertiary care hospital in japan. all adult patients who were admitted to the icu with covid-19-induced ards and required mechanical ventilation during march 1 through april 30 in 2020 were consecutively enrolled in this study. as a control group, we included consecutive adult patients who were admitted to the icu with the diagnosis of septic ards induced by non-covid-19 community-acquired bacterial pneumonia and required mechanical ventilation from january 2013 to february 2020. the exclusion criteria included the use of warfarin/acetylsalicylic acid/thrombolytic therapy before study entry; the limitation of sustained life care or post-cardiopulmonary arrest resuscitation status; history of fulminant hepatitis, decompensated liver cirrhosis, or other serious liver disorder; history of hematologic malignant disease; and other conditions increasing the risk of thrombosis at study entry. this study followed the principles of the declaration of helsinki. the protocol was approved by the institutional review board for clinical research of osaka general medical center (irb no. s202004004). the diagnosis of covid-19 was performed according to world health organization interim guidance [12] and confirmed by rna detection of sars-cov-2 in a clinical laboratory of the osaka institute of public health. in this study, sepsis was diagnosed based on sepsis-3 criteria proposed in 2016 [13] . ards was diagnosed according to the berlin definition [14] as fulfilling the following criteria: (1) onset within 1 week after predisposing diseases, (2) bilateral infiltration on chest roentgenogram, (3) pao 2 /fio 2 (p/f ratio) ≤ 300 with peep ≥ 5 cm h 2 o, and (4) no clinical signs of cardiac failure or fluid overload. we defined the first day of mechanical ventilation as "day 1" both in the covid and non-covid pneumonia groups. patients were followed up until hospital discharge or death. patient information collected included demographic characteristics, pre-existing comorbidities, laboratory tests, severity scores, and therapeutic interventions. laboratory tests included several hemostatic biomarkers, such as platelet count, prothrombin time (pt), fibrinogen level, fibrin/fibrinogen degradation products (fdps), d-dimer level, antithrombin activity, thrombin-antithrombin (tat) complex, plasmin-a2-plasmin inhibitor complex (pic), and plasminogen activator inhibitor (pai)-1. tat, pic, and pai-1 were evaluated only on day 1 in the covid-19 group; however, the other biomarkers were measured in both groups at each time point from day 1 to day 7. severity of illness was evaluated according to the sequential organ failure assessment (sofa) score and the acute physiology and chronic health evaluation (apache) ii score. the apache ii score was evaluated on day 1, and the sofa score was evaluated at each time point from day 1 to day 7. the incidence of disseminated intravascular coagulation (dic) was evaluated at each time point from day 1 to day 7 based on the international society on thrombosis and hemostasis (isth) overt dic and the japanese association for acute medicine (jaam) dic criteria. we also evaluated the incidence of sic at each time point [15] . the isth overt dic scoring system was adopted as proposed by the scientific subcommittee on dic of the isth for platelet counts, pt, fdp, and fibrinogen level [16] . to calculate the isth overt dic score, fdp values were chosen as the fibrin-related marker and scored according to the cut-off levels and ranges previously published by gando et al. [17] . the jaam dic scoring system consists of the sirs (systemic inflammatory response syndrome) score and global coagulation tests including platelet counts, pt, and fdp/d-dimer levels. [18] we used prophylactic low-dose unfractionated heparin for both the covid-19 and control groups when patients had a high risk of venous thromboembolism (vte), such as severe obesity, cancer, orthopedic surgery, and prior history of vte. the aim of this study was to reveal the specific pattern of coagulopathy induced by severe covid-19 pneumonia by comparing the hemostatic parameters chronologically with those in patients with ards induced by non-covid-19 pneumonia. therefore, to assess the time series variation in the hemostatic parameters, we fitted multilevel mixed-effects regression models with fixed effects assigned to patient categorization (covid-19 or non-covid-19), time points (from day 1 to day 7), and two-way interaction term for these independent variables, and random effects assigned to individual identification numbers. we also performed multilevel mixed-effects regression analysis to evaluate the time series differences during the first seven days in other organ dysfunction parameters, including p/f ratio, serum creatinine level, serum bilirubin level, glasgow coma scale, and sofa subscore for the cardiovascular component between the covid-19 and non-covid-19 groups. kaplan-meier failure curves were constructed to evaluate the cumulative incidence of jaam dic, isth overt dic, and sic over time (from day 1 to day 7). log rank tests were conducted to compare the kaplan-meier curves between two groups. descriptive statistics were calculated as medians (interquartile range) or proportions (numbers), as appropriate. univariate differences between groups were assessed using the mann-whitney u test or chi-square test, as appropriate. missing values were not imputed in any of the regression models. all statistical inferences were performed with a two-sided p at the 5% significance level. because of the underpowered nature of the interaction analysis, we used a two-sided significance level of 20% with statistical inferences for the interaction analyses [19] . all statistical analyses were conducted using stata data analysis and statistical software version 15.0 (statacorp, college station, tx, usa). baseline characteristics, laboratory tests, and illness severity scores on day 1 in the two groups are 141 shown in table 1 . age, sex, and pre-existing comorbidities were similar between the groups. although 142 the platelet counts on day 1 were similar between the two groups, there were statistically significant 143 differences in other hemostatic biomarkers. pt (%), fibrinogen level, and antithrombin activity in the 144 covid-19 group were almost within the normal range and were significantly higher compared with 145 those in the non-covid-19 group. 146 baseline characteristics, laboratory tests, and illness severity scores on day 1 in the two groups are shown in table 1 . age, sex, and pre-existing comorbidities were similar between the groups. although the platelet counts on day 1 were similar between the two groups, there were statistically significant differences in other hemostatic biomarkers. pt (%), fibrinogen level, and antithrombin activity in the covid-19 group were almost within the normal range and were significantly higher compared with those in the non-covid-19 group. there were no significant differences between groups in mental status and respiratory function, as indicated by the glasgow coma scale and p/f ratio, respectively. dic scores and sic score were significantly lower in the covid-19 group compared with those in the non-covid-19 group. a multilevel mixed-effects regression model suggested that the changes in platelet counts over time during the first seven days were significantly different between the two groups (p for interaction = 0.052), and the covid-19 group had constantly higher platelet counts after day 2 ( figure 2) . similarly, we detected significant time series differences in pt, fdp, d-dimer, and antithrombin activity between the two groups (p for interaction = 0.005, 0.001, 0.002, and 0.037, respectively). the covid-19 group had higher levels (almost within normal range) of pt and antithrombin activity at all day points but higher levels of fdp and d-dimer after day 2. we also show the levels of several hemostatic molecular biomarkers, such as tat, pic, and pai-1, in the covid-19 group in figure 3 . the median levels of tat and pic were 6.8 ng/ml and 2.0 µg/ml, respectively, and tended to be higher compared with the upper limits of normal range for these two biomarkers (3.0 ng/ml and 0.8 µg/ml, respectively). however, the median level of pai-1 was 28.0 ng/ml and was within the normal range (<50.0 ng/ml). we show the time series differences in several parameters related to organ dysfunction other than coagulation in figure 4 . although the p/f ratio in the non-covid-19 patients improved with time, that in the covid-19 group worsened with time, and there were significant time series differences between the two groups (p for interaction < 0.001). in contrast, serum levels of creatinine tended to be lower at all time points in the covid-19 group. only slight differences were detected in serum bilirubin level and the sofa subscore for the cardiovascular component between the covid-19 and non-covid-19 groups. we also show the levels of several hemostatic molecular biomarkers, such as tat, pic, and pai-167 1, in the covid-19 group in figure 3 . the median levels of tat and pic were 6.8 ng/ml and 2.0 g/ml, 168 respectively, and tended to be higher compared with the upper limits of normal range for these two 169 biomarkers (3.0 ng/ml and 0.8 g/ml, respectively). however, the median level of pai-1 was 28.0 170 ng/ml and was within the normal range (< 50.0 ng/ml). 171 we show the time series differences in several parameters related to organ dysfunction other than 177 coagulation in figure 4 . although the p/f ratio in the non-covid-19 patients improved with time, that 178 in the covid-19 group worsened with time, and there were significant time series differences between 179 the two groups (p for interaction < 0.001). in contrast, serum levels of creatinine tended to be lower at 180 the cumulative incidence curve constructed with the kaplan-meier method showed that there 193 were no statistically significant differences in the incidence of jaam dic and isth-overt dic during 194 the first seven days between the groups (log rank test, p = 0.587 and 0.101, respectively). however, the 195 incidence of sic in the covid-19 group during the first seven days was only 8.5% (2 of 24 patients) 196 and was significantly lower compared with that in the non-covid-19 group (log rank test, p = 0.003, 197 figure 5 ). one of the two sic patients survived to hospital discharge, however, another patient 198 developed multiple organ dysfunction syndrome and died despite full intensive care support. 199 the cumulative incidence curve constructed with the kaplan-meier method showed that there were no statistically significant differences in the incidence of jaam dic and isth-overt dic during the first seven days between the groups (log rank test, p = 0.587 and 0.101, respectively). however, the incidence of sic in the covid-19 group during the first seven days was only 8.5% (2 of 24 patients) and was significantly lower compared with that in the non-covid-19 group (log rank test, p = 0.003, figure 5 ). one of the two sic patients survived to hospital discharge, however, another patient developed multiple organ dysfunction syndrome and died despite full intensive care support. to date, several studies have reported on covid-19-related coagulopathy [2, 5, 20, 21] ; however, in 209 terms of the phenotypes or incidence of dic, there remains little evidence about whether it can be 210 differentiated from overall sic. in the present study, we compared the pattern and incidence of 211 to date, several studies have reported on covid-19-related coagulopathy [2, 5, 20, 21] ; however, in terms of the phenotypes or incidence of dic, there remains little evidence about whether it can be differentiated from overall sic. in the present study, we compared the pattern and incidence of coagulopathy over time between severe covid-19 pneumonia and non-covid-19-induced severe pneumonia using multilevel time series analyses and the kaplan-meier method. a recent study reported that the baseline levels of platelet count, pt, antithrombin activity, and fibrinogen were significantly higher in covid-19 patients compared to patients with non-covid-19 ards [22] . a similar pattern of hemostatic markers was observed in the present analysis at day 1: pt, antithrombin activity, and fibrinogen in the covid-19 group were almost within normal range and significantly higher compared with the values in the non-covid-19 group. furthermore, we showed that the chronological alterations of platelet count, antithrombin activity, and pt in the covid-19 group were significantly milder, whereas elevations of fdp and d-dimer were significantly higher in the covid-19 group. decreased platelet counts and prolonged prothrombin time are the most common hematologic signs induced by systemic hypercoagulation and therefore are involved in all widely used dic criteria [15, 16, 18] . antithrombin activity is also known to be markedly decreased in sepsis-induced coagulation, due to consumption as a result of ongoing thrombin generation [23] . however, elevations of fdp and d-dimer are generally within a mild range in the early phase of sic, mainly due to the impaired fibrinolysis driven by an increase in pai-1 [24, 25] . high levels of fdp and d-dimer with normal or mild alterations of other coagulation markers are typical in local thrombus formation, such as pulmonary embolism and deep venous thrombosis [26, 27] . indeed, several studies have reported high incidences of pulmonary embolism in covid-19 patients with or without underlying deep venous thrombosis [28, 29] . according to these insights, our results suggested that systemic hypercoagulation was hardly induced by covid-19 infection, but the risk of local thrombus formation increased in the acute phase of severe covid-19 pneumonia. we also evaluated several hemostatic biomarkers and found that covid-19 patients had moderately high levels of tat and pic but a normal level of pai-1. vascular endothelial cell dysfunction is an essential feature in the pathogenesis of sic. because the secretion of pai-1 is mainly regulated by endothelial cells, sepsis-related endothelial cell dysfunction causes a marked increase in the pai-1 level leading to disrupted fibrinolysis, and this key event represents the typical feature of the thrombotic type of dic [30] . therefore, our results regarding the levels of pai-1 suggested that vascular endothelial cell dysfunction and the thrombotic type of dic were hardly induced by severe covid-19 pneumonia. then, what phenotype of coagulopathy is induced by covid-19 pneumonia? severe coagulopathy can be classified into three common phenotypes according to the underlying disease: (1) enhanced fibrinolytic phenotype typically induced by acute promyelocytic leukemia, (2) balanced fibrinolytic phenotype typically induced by cancer, and (3) suppressed fibrinolytic phenotype typically induced by sepsis. in the present study, patients with covid-19 pneumonia had moderate elevations of tat and pic and a normal level of pai-1, which are characteristic of the balanced fibrinolytic phenotype [10] . one possible explanation for this specific phenotype of coagulopathy is that a pulmonary-restricted intravascular coagulopathy initially occurs due to the extensive alveolar and interstitial inflammation that occurs in patients with covid-19 pneumonia, and this causes the expression of active tissue factor leading to balanced fibrinolytic coagulopathy [31] . as mentioned above, the marked elevation of fibrin degradation products was a main characteristic of covid-19-induced coagulopathy. therefore, the incidence of jaam dic and isth overt dic involving the component of fibrin degradation products was equal between the two groups during the first seven days, whereas the incidence of sic, which does not involve the component of fibrin degradation products, was much lower in the covid-19 group. these findings suggested that the typical coagulopathy in covid-19 was distinct from sic, even though it could meet several of the dic criteria. we acknowledge several limitations of our study. first, the single-center design and short study duration resulted in a relatively small sample size, which may have influenced the precision of our findings. second, we enrolled patients with different pathophysiology (bacterial ards) as the control group for the study purpose of evaluating the specific phenotype of severe covid-19-induced coagulopathy by comparing it to other types of sepsis. as a result, there were no differences in baseline characteristics and pre-existing comorbidities, but the pattern of organ dysfunction between the groups reflected the unique clinical manifestation of severe covid-19 pneumonia. however, we are not confident that the effect of potential ascertainment bias can be completely excluded despite robust adjustment with regression models. third, the long-term design of the control group might be another source of bias. although the type and severity of patients and the key concepts for the management of ards were not greatly changed, physician staffing and several supportive therapies, such as antimicrobials and nutrition, might change according to the time course, which possibly influenced the study findings. finally, the control group included only bacterial sepsis and did not include non-covid-19 viral infections. further investigation is thus required to compare the coagulation disorders between covid-19-induced ards and non-covid-19-viral-infection-induced ards. in the present study, the pattern of coagulopathy in severe covid-19 pneumonia was quite different from that in other severe pneumonias. systemic hypercoagulation, suppressed fibrinolysis, and vascular endothelial cell dysfunction, typically observed in sepsis-induced dic, might be hardly induced; instead, local thrombus formation was possibly triggered by pulmonary-restricted intravascular coagulopathy in covid-19 pneumonia. further investigations are required to confirm our findings and establish appropriate management for severe covid-19 pneumonia. a novel coronavirus from patients with pneumonia in china abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia epidemiological and clinical characteristics of 99 cases of 2019-novel coronavirus (2019-ncov) pneumonia in wuhan clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet. 2020 more on covid-19 coagulopathy in caucasian patients endothelial cell infection and endotheliitis in covid-19 endotheliopathy in covid-19-associated coagulopathy: evidence from a single-centre, cross-sectional study tissue plasminogen activator (tpa) treatment for covid-19 associated acute respiratory distress syndrome (ards): a case series anticoagulant treatment is associated with decreased mortality in severe coronavirus disease 2019 patients with coagulopathy classifying types of disseminated intravascular coagulation: clinical and animal models efficacy and safety of anticoagulant therapy in three specific populations with sepsis: a meta-analysis of randomized controlled trials clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance the third international consensus definitions for sepsis and septic shock acute respiratory distress syndrome scientific and standardization committee on dic, and the scientific and standardization committee on perioperative and critical care of the international society on thrombosis and haemostasis diagnosis and management of sepsis-induced coagulopathy and disseminated intravascular coagulation scientific and standardization committee communications: towards a definition, clinical and laboratory criteria, and a scoring system for disseminated intravascular coagulation natural history of disseminated intravascular coagulation diagnosed based on the newly established diagnostic criteria for critically ill patients: results of a multicenter, prospective survey* a multicenter, prospective validation of disseminated intravascular coagulation diagnostic criteria for critically ill patients: comparing current criteria* oxidative stress and inflammation are associated with adiposity in moderate to severe ckd coagulopathy and antiphospholipid antibodies in patients with covid-19 covid-19-related severe hypercoagulability in patients admitted to intensive care unit for acute respiratory failure clinical research in intensive care and sepsis trial group for global evaluation and research in sepsis); et al. high risk of thrombosis in patients with severe sars-cov-2 infection: a multicenter prospective cohort study platelet drop and fibrinolytic shutdown in patients with sepsis fibrin-related markers in patients with septic shock: individual comparison of d-dimers and fibrin monomers impacts on prognosis review of d-dimer testing: good, bad, and ugly laboratory biomarkers for venous thromboembolism risk in patients with hematologic malignancies: a review autopsy findings and venous thromboembolism in patients with covid-19 high incidence of venous thromboembolic events in anticoagulated severe covid-19 patients advance in the management of sepsis-induced coagulopathy and disseminated intravascular coagulation immune mechanisms of pulmonary intravascular coagulopathy in covid-19 pneumonia author contributions: y.u. conceived and designed this study; contributed to acquisition, analysis, and interpretation of the data; and was responsible for drafting, editing, and submission of the manuscript. k.y. contributed to the study design; acquisition, analysis, and interpretation of the data; and drafting of the manuscript. t.k., t.n., m.k., and s.f. had a significant influence on the interpretation of the data and critical appraisal of the manuscript. all of the authors contributed to the acquisition of data and reviewed, discussed, and approved the final manuscript. all authors have read and agreed to the published version of the manuscript funding: this research received no external funding. the authors thank all of the emergency medical service personnel, nurses, and physicians who have confronted the covid-19 outbreak, and the patients who contributed to this study. the authors declare no conflict of interest. key: cord-274563-jimw6skv authors: fiumara, agata; lanzafame, giuseppina; arena, alessia; sapuppo, annamaria; raudino, federica; praticò, andrea; pavone, piero; barone, rita title: covid-19 pandemic outbreak and its psychological impact on patients with rare lysosomal diseases date: 2020-08-22 journal: j clin med doi: 10.3390/jcm9092716 sha: doc_id: 274563 cord_uid: jimw6skv background: lysosomal storage disorders (lsds) are rare, chronic, progressive multisystem diseases implying severe medical issues and psychological burden. some of these disorders are susceptible to a treatment, which is administered weekly or every other week, in a hospital. during the covid-19 (corona virus disease 2019) pandemic lockdown, patients with lsds on enzyme replacement therapy (ert) missed their scheduled access to the day hospital to get their treatment. methods: based on the feeling that our patients were experiencing profound distress, we designed a structured telephone interview with the aim to evaluate how, and to which extent, the pandemic outbreak was changing their behavior and feelings about their chronic disease, the impact on therapies, and future expectations. the same interview was administered to an age-matched control group. results: all interviewed people experienced an increase of anxiety, worries, and uncertainty fostered by incessant media updates. moreover, a striking similarity emerged between the groups regarding forced home reclusion and the profound feeling to be excluded by normal life, well-known to those affected by a chronic rare disease. conclusions: although no statistically significant difference was found compared to controls, we felt that the reactions were qualitatively different, underlining the fragility and isolation of such patients. since march 2020, the dramatic outbreak of corona virus disease 2019 in italy has changed our lifestyle as individuals, physicians, and patients. despite the evidence of minor involvement of children [1] , pediatric units also had to deal with healthcare crises. as a referral centre for inborn errors of metabolism (iem), we had to face an unexpected restriction concerning daily normal activity with lowering of programmed admissions for diagnosis and follow-up visits. our concern was especially directed to those patients with lysosomal storage disorders (lsds), which are rare, chronic, progressive, multisystem diseases associated with serious medical issues, physical disability, and psychological burden [2] . in the last decade, some of the lsds became treatable by pharmacological therapy, such as intravenous (iv) enzyme replacement therapy (ert) and oral substrate reduction therapy (srt), or chaperones. during the covid-19 alert, patients with lsds, under regular treatment with ert, failed their usual compliant behavior, missing scheduled infusions. based on the feeling that our patients were experiencing profound distress, we designed a structured interview [3, 4] with the aim to evaluate how, and to which extent, the covid-19 pandemic was changing our patients' behavior and feelings about their chronic disease, the impact on therapies, and their future expectations. we emphasize the importance to investigate attitudes and behavior with respect to health treatment, especially among people with rare diseases, such as patients with lsd. they represent a group with increased vulnerabilities to covid-19; thus, we felt the need to attempt any possible solution that would let them maintain treatment protocols and minimize disease progression. at our regional referral centre for metabolic diseases, pediatric clinic, department of clinical and experimental medicine, patients with different types of iem are followed. at the time of the study, 33 of them were affected by a treatable lsd and thus were regularly admitted to the day hospital with a personal schedule of ert (weekly or every other week) or followed-up every 3-6 months because of treatment at home. in this study, we included 15/33 patients who accepted to undergo our interview. there were 9 females and 6 males with age ranging from 3 to 40 years. seven of them were younger than 12 years. ten (66%) had pompe disease (pd; 2 early infantile type (eopd) and 8 late-onset type (lopd)). the sample also included 2 patients with mucopolysaccharidosis type iv (mps iv), 2 pediatric patients with gaucher disease, and 1 adult subject with fabry disease. all participants were receiving iv ert (alglucosidase alfa, elosulfase alfa, imiglucerase, or agalsidase beta, according to their disease). at the beginning of covid-19 emergency, study patients with gaucher disease or fabry disease were on home therapy. an ad hoc structured interview was developed and administered by phone and when possible by video calls (table 1) during the third week of lockdown. the interview investigated personal feelings, familial relationship, degree of faith in others, and future perspectives and was inspired and developed in light of this extraordinary, life-threatening event. quantitative data were obtained from dichotomous questions (yes/no) used for a clear distinction of respondents' opinions. according to the age, we got direct information from 8 subjects, while for 7 pediatric patients, the parents were asked to respond to the interview. a psychologist (gl) from the centre contacted the patients or their caregivers to assess how the covid-19 emergency modified the daily life of patients and their family, which changes were due to the resulting government restrictions, how these were felt, and if any change had occurred with the personal therapy schedule. moreover, we gathered information about the mood of the patients, their families, and social relationships, the need for psychological support, and their expectations for the future. since we thought and developed the interview in light of this extraordinary event, the tool could not have been previously validated. to overcome this issue, a group of healthy volunteers was carefully selected for comparison. the control group included 15 healthy subjects matching one-to-one with the patient and caregiver sample, in terms of age, social condition, instruction level, and family composition. data were presented as absolute frequencies and/or percentages for categorical variables. a comparison of proportions between groups was conducted by chi square test with yates' correction. differences with p ≤ 0.05 were considered to be significant. data were analyzed using the spss software, v. 23. (sps, bologna, italy) relations with family members appeared to be felt positively in 54% of patients stating that, being at home, they were closer and linked to each other in a co-working and beloved environment. on the contrary, before the lockdown, family members were less involved; moreover, the use of video calls and socials allowed contact with less frequently seen relatives and increased reciprocal affection and the feeling to be part of the same family. in the control group, a positive evaluation was found only in 30%, although they also stated to have rediscovered human values and lost values. a negative feeling was reported by 33% of our patients: they described intolerance, impatience, discomfort, distress, constriction, and impairment of contact with close relatives, if not by video calls. in contrast, 60% of the control group described a negative feeling of familial interrelationship because of isolation, uncertainty, fear, difficulties in handling children, and anxiety for older relatives with whom it was hard to communicate. a small percentage of investigated patients (13%) and 10% of the control group denied significant changes, stating that they were used to this aloneness and isolation. as a whole, no significant differences were observed in the rate of subjects experiencing positive, negative, or unchanged familial relationships between groups (x 2 (2, n = 25) = 1.7, p = 0.413). patients revealed a strong inclination to feel "others" negatively (87%), as other people were considered to be disrespectful, self-oriented, or dangerous and were to be avoided. thus, relationships were commonly seen as characterized by lack of empathy, indifference, and detachment. in the control group, we also found a clear tendency to perceive other people negatively (80%). however, in the control group, the image of "others" was that of insecure, frightened, suspicious, avoidant, and elusive people, although considered only slightly inaccessible and deserving of being turned away. a small percentage (6%) of our patients, on the contrary, stressed the empathic attitude toward others who were then sharing the common fragility state. in the control group, 20% reported social relations positively stating how useful it was to protect each other by avoiding contact and discovering new ways of social interaction even with neighbors. only 7% of patients stated that they did not feel significant changes. the proportion of participants who experienced social relationships as dangerous or positive was not significantly different between groups (x 2 (1, n = 25) = 0.7, p = 0.068). no significant differences between groups were observed in the rate of participants experiencing negative or positive reactions to modified daily activities (x 2 (1, n = 25) = 0.3, p = 0.802). sixty percent of patients described boring moments, monotony, weakness, and stress for web lessons, limitation of normal activities, prohibitions in moving to familial places and seeing relatives, and the need for repetitive hand hygiene procedures. likewise, the majority of control subjects (80%) demonstrated a negative reaction regarding the monotony of daily life, which was felt as difficulty in commitment to following rules, in the need for space and temporal organization, and in the occurrence of sleep-awake rhythm problems. on the contrary, 20% of patients stated that they felt more relaxed and helped by the family dedicating more time to them. in addition, 20% of controls felt the changes positively, having more free time for themselves and for their domestic activities. among patients, 20% felt that there were no changes in their daily routine. mostly negative feelings were encountered in our patients' sample; 67% experienced fear, distress, anger, frustration, impotence, negative mood, and feeling of neglection; and 33% showed ambivalent emotions, with co-existence of astonishment, confusion, doubt, curiosity, and uncertainty alongside the need to protect their beloved ones. seventy percent of controls manifested aloneness, anxiety, concern, fear of the unknown, fear of contamination, sharing difficulties, pessimism, mood depression, sadness, and feeling of being in a surreal condition. on the contrary, 20% had a positive mood characterized by adaption, respect, positive dependence, and ability to find incitements and new energies; the remaining 10% showed ambivalent aspects with a hard and pessimistic approach, despite a feeling of well-being. in sum, the proportion of subjects suffering negative or positive feelings was not significantly different in the two groups (x 2 (1, n = 25) = 0.2, p = 0.902). patients with lsds expressed their belief in state, regional, and hospital institutions in 67% of cases; 20% declared to be not confident; and 13% were uncertain because of discordant news and lack to timely assured protection devices. similar results were obtained in the control group with 70% manifesting faith, 20% manifesting diffidence and the feeling to be abandoned, and 10% showing an uncertainty to judge about the emergency-handling strategies (x 2 (1, n = 25) = 0.1, p = 0.986). defense mechanisms adopted by patients and controls during the covid-19 emergency were analyzed: both groups tried to use mature psychological defenses (33% versus 36%, respectively) or denied any concern (11% of patients versus 7% of controls); annihilation was encountered in 11% of patients and 15% of controls; a tendency to discredit others was present in 11% of patients and 14% of controls; some of the patients (17%) activated distressing and pacifying actions; this was also seen in 7% of controls. a small percentage of patients (11%) showed a passive mood, demonstrating lack of affective interactions. almost half of the patients thought that, from this experience, they learnt something positive (47%) such as the real meaning of relationships, gratitude, and the ability to accept and respect others and to identify priorities. this feeling was even stronger in the control group (70%), stating that some positive aspects were coming from the actual situation as the discovery and enforcement of community spirit, sense of belonging to the same community and nation, values of life, solidarity, and the ability to face hard tasks and overcome limits gave a look inside themselves. twenty percent of patients stated that they were living this experience in a negative way, learning disillusion, frustration, and resignation to death; 10% of controls lived this dramatic situation as subverting daily routine and forcing to reschedule life; 33% of patients stated that there was nothing to learn by this situation, but to just wait for improvement; and 20% of controls were not able to cope with the actual moment. the proportion of subjects who reported to have learnt positively or negatively from this experience did not differ between the groups (x 2 (1, n = 25) = 1.3, p = 0.5118). almost half of the patients (53%) manifested negative anticipations, forecasting more preventive precautions, difficulties, limitations, and discomfort than those that had already suffered because of the disease, thus passing to resignation and unavoidable acceptance. such a negative future perception was present in 50% of the control group, forecasting a sad, stressing, and financially hard future, with consequences on work and relationships. nevertheless, 34% of patients had a positive vision of the future, including the opportunity to come back to normal life, due to a profound faith in scientific research. fifty percent of controls prefigured the return to normal daily life, although with unavoidable changes in physical relationship and environment. among the patients' group, 13% had a passive, static attitude without changes in future perspectives. no significant differences were observed between groups (x 2 (1, n = 25) = 1.3, p = 0.5118). most patients (60%) refused to regularly come to the hospital for their therapies because they feared that they would be infected. the remaining 40% respected their scheduled infusion in the hospital, although they expressed their fear to be infected and thus showed a strict adhesion to hygiene procedures. all patients asked to be treated at home, except for a child that was severely affected with pompe disease, whose parents felt safer coming to day hospital , but accurately checked the personnel health state. the fear of contamination was also observed in patients who had already been treated with home therapy as they were scared to allow people to come home. the italian government's emergency declaration on 9 march, 2020, drastically changed our lives. every action, behavior, or even gesture was filtered by the covid-19 alert. "stay at home" was mandatory for all people, except for medical doctors, all health operators, and patients needing urgent medical care (www.salute.gov.it). this study emerged from the observation of different reactions in patients with lysosomal disorders dealing with pandemic outbreak. lsds are genetic, multisystem diseases [2] . to date, some of the rare lsds are susceptible to ert, which has been shown to at least delay progression, allowing a better quality of life in terms of disabilities. although, we offered a regular and covid19-controlled service for these patients, who deserved to be regularly treated despite the emergency period, we observed that patients and their parents were extremely scared and worried about coming to the hospital, fearing a higher risk for covid contamination. thus far, most of them missed their scheduled ert. conversely, those patients who had already been treated with home therapy refused treatment by the dedicated team because they felt that this could represent a potential source of the infection. in this regard, sechi et al. (2020) analyzed data collected by a questionnaire from 102 patients on ert therapy for lsds in italy [5] . they found that almost 50% of patients who were receiving therapy at a hospital (61.8%) had disruptions, especially for personal feelings (fear of infection). in the present study, we analyzed behavioral and emotional profiles of our patients with lsds during the pandemic, compared to healthy controls. for this reason, a structured interview was created and administered online by a trained psychologist, already known to all patients as working in the centre. since we thought and developed the interview in light of this extraordinary event, the tool could not have been previously validated. the interview was made during the first week of lockdown, when both patients and controls were experiencing the same uncertainty resulting from the special situation. this study certainly may have some limits due to the small sample size, but this is the rule dealing with patients with rare diseases who are referred to a single center. although no significant quantitative differences were observed in the type of response between clinical and control groups, there were some qualitative differences between the two groups in all investigated areas. in general, all people forced to stay at home and freeze their jobs and who were far from common relationships, but too close to the familial nucleus members, experienced an increase in anxiety, worries, and uncertainty fostered by incessant media updates on virus lethality and virulence, underlining its invisible presence in our environment and in our lives. nevertheless, peculiar differences emerged in social relations and perception of "others". the clinical group, always accustomed to dealing with diversity, exclusion, and unawareness of others with respect to their lives of illness, perceived "others" mostly in a negative way, as dangerous, disrespectful, and not empathetic and to be avoided. the control group, although having the same negative perception, experienced "others" as frightened, insecure, suspicious, and elusive. in the clinical group, daily routine was marked from the issues of illness and its treatment, while for the control group, main problems were related to difficulty in maintaining, according to an orderly sequence, rules, spaces, times, and sleep-wake rhythm. on the other hand, it is singular that, in the percentage of positive representation of the routine changes, subjects in the clinical group believed that the positive element was the opportunity of a better relationship with family members, while among controls, positive elements relied on the possibility of being alone and doing something for themselves. data about future perception evidenced that, while the control group directed its attention to a future focused on economic, social, and environmental issues, patients prefigured a future always oriented by their critical situation being aware of their limits, in terms of treatment opportunities and life expectation. we focused on the covid-19 pandemic effects on medical care and health status of patients with lsds. the real risk of contagion once again highlighted the vulnerability of patients with chronic rare diseases such as lsds, the difficulty of coping with his or her defenses, and the need to trust and rely on others. in this case, the subjects preferred to refuse treatment, the only chance to improve their condition. they gathered all their defenses and tried to put in place all the resources and strategies available, such as resist and wait, for example, for home treatment, rather than face unarmed an unknown, enigmatic, and dangerous "enemy". in our sample, a striking similarity emerged between the two groups, equally forced to stay at home and experience the same profound feeling to be excluded (isolation and inclusion, the lancet psychiatry 2020) [6] by normal life. the pandemic, which represented a scary event, suddenly occurring in the daily life, destabilizing, and giving rise to uncertainty, had the same impact as a diagnosis of a chronic rare disease. the control group experienced the feeling to be involved in a mutual fight against a common enemy, thus enhancing brotherhood with others. the patients with lysosomal disorders and their families felt that covid-19 opened the curtains, revealing their human condition of chronic exclusion and impairing their liberty to go out, walk, meet others, love, and breath without fear of death as all the others normally do. especially the mothers of our patients reacted with strength and determination, feeling that other people can know understand their withdrawn lives to assist their sons. they could now teach others how to face isolation with dignity, aware of fragility, as they usually do: "i can't help from sadly smiling when my neighbors complain because their children are sad as they cannot go outside . . . don't say it to me, please, my child and i do not go out since he was born and he is 4 years old. now we are all the same, all confined at home . . . you are not different from us, you also are vulnerable, now we all fear death". the outbreak of covid-19 evidenced the vulnerability of the patients with such rare diseases and their needs in terms of adhesion to the therapy schedule. thus far, a special license for home therapy was approved by aifa (agenzia italiana del farmaco (italian medicines agency), det. 341/2020), including those drugs prescribed and dispensed only at hospitals. this determination allowed home treatment for most of our patients. the covid-19 emergency revealed lsd patients' strength in terms of improved relationships, such as adhesion to the patients' group, family members, and community and their observance of imposed rules and precepts, trust in authority and doctors, and hope for improvement. systematic review of covid-19 in children shows milder cases and a better prognosis than adults lysosomal storage diseases research in psychology: a practical guide to research methods and statistics qualitative interviewing: the art of hearing data impact of covid-19 related healthcare crisis on treatments for patients with lysosomal storage disorders, the first italian experience isolation and inclusion. lancet psychiatr. 2020 the authors wish to thank the patients, their families, battista mangani, and nefri basile for their collaboration. the family association "bacodirame" is deeply acknowledged for financing the university research grant for the psychologist. the authors declare no conflict of interest. key: cord-264355-9quf59td authors: jung, sung-mok; kinoshita, ryo; thompson, robin n.; linton, natalie m.; yang, yichi; akhmetzhanov, andrei r.; nishiura, hiroshi title: epidemiological identification of a novel pathogen in real time: analysis of the atypical pneumonia outbreak in wuhan, china, 2019–2020 date: 2020-02-27 journal: j clin med doi: 10.3390/jcm9030637 sha: doc_id: 264355 cord_uid: 9quf59td virological tests have now shown conclusively that a novel coronavirus is causing the 2019–2020 atypical pneumonia outbreak in wuhan, china. we demonstrate that non-virological descriptive characteristics could have determined that the outbreak is caused by a novel pathogen in advance of virological testing. characteristics of the ongoing outbreak were collected in real time from two medical social media sites. these were compared against characteristics of eleven pathogens that have previously caused cases of atypical pneumonia. the probability that the current outbreak is due to “disease x” (i.e., previously unknown etiology) as opposed to one of the known pathogens was inferred, and this estimate was updated as the outbreak continued. the probability (expressed as a percentage) that disease x is driving the outbreak was assessed as over 29% on 31 december 2019, one week before virus identification. after some specific pathogens were ruled out by laboratory tests on 5 january 2020, the inferred probability of disease x was over 49%. we showed quantitatively that the emerging outbreak of atypical pneumonia cases is consistent with causation by a novel pathogen. the proposed approach, which uses only routinely observed non-virological data, can aid ongoing risk assessments in advance of virological test results becoming available. a cluster of cases of atypical pneumonia with unknown etiology in wuhan, china attracted global attention towards the end of 2019 [1, 2] . an impressive series of rapid virological examinations ruled out common pneumonia-causing viruses such as influenza viruses, adenoviruses, and the coronaviruses associated with middle east respiratory syndrome (mers) and severe acute respiratory syndrome (sars) [2] [3] [4] [5] . early in the outbreak, the causative agent was suspected to be a coronavirus of non-human origin [5, 6] . the coronavirus was subsequently found to be a relative of sars and named the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [7, 8] . while examination of the viral genome was critical for identifying the pathogen, information made publicly available in real time describing clinical characteristics and other outbreak-related factors also allowed experts to consider the etiology and thereby differential diagnoses. for instance, most cases shared a history of visiting or working at a seafood market in wuhan [3] , where exposure to the novel coronavirus is suspected to have occurred with no evidence of direct human-to-human transmission [2] , although human-to-human transmission was found later to be common. observed characteristics of the outbreak led us to believe that the cluster of cases was due to "disease x" (i.e., an infectious disease of previously unknown viral etiology). however, rigorous quantitative assessment based on these characteristics of the chance that the manifestations of atypical pneumonia were in fact disease x has not previously been undertaken. the present study addresses this, demonstrating that non-virological information can lead to an objective classification of disease x, using a simple statistical model that exploits the well-known bayes' theorem. as the outbreak unfolded, we calculated in real-time the probability that the pathogen responsible for the atypical pneumonia cases was novel (disease x), as opposed to the outbreak instead being generated by a previously known pathogen that can cause atypical pneumonia. our analysis began on 30 december 2019, when the wuhan municipal health commission announced that there had been a surprisingly large number of atypical pneumonia cases. at that time, we assumed the causative agent could have been one of eight known viral or three known bacterial pathogens, along with the chance that it was instead disease x. we tracked two active medical social media sites (promed [9] and flutracker [10] ) that collected reports of the non-virological characteristics of the outbreak as it progressed. these characteristics were basic observations from the outbreak and do not necessarily represent the features that were causing symptoms. given these characteristics, we then calculated the probability that the ongoing outbreak was due to a known pathogen or unknown disease x. on the first day of calculation (i.e., 30 december 2019)-the day that we became aware of the outbreak-the only explanatory factor we included was diagnosis of atypical pneumonia, which was common to all pathogens considered in our dataset. our analysis represents simple logical deductions from the limited data that were available during the outbreak in a quantitative manner and was updated to reflect new information about the outbreak as it became available in real time. table 1 shows the information compiled about the current outbreak, and the dates on which each of these characteristics were discovered. each characteristic listed was assigned a value of zero or one, denoting whether or not the outbreak characteristic was likely in general (rather than for individual cases) for the emerging outbreak, and the equivalent values for outbreaks of previously observed pathogens were also noted. we note that some information believed at the time was later found to be untrue; for example, it was believed that human-to-human transmission was infrequent. consequently, inclusion of a large number of characteristics is important for our analysis. once pathogens were ruled out as the causative agent of the current outbreak, they were removed from our analysis: for example, highly pathogenic avian influenza (hpai; h5n1) was confirmed not to be the causative agent by laboratory testing on 3 january 2020. hence, we omitted this pathogen from our analysis from that date onwards. we performed two versions of our analysis to demonstrate how our results might change with the inclusion of different outbreak characteristics. in the first, all characteristics in table 1 were included in the analysis. in the second, information about the exposure location (i.e., exposure at a wet market) was excluded from the analysis. zeros represent characteristics that are unlikely for outbreaks of that pathogen, and ones represent characteristics that occur. dates and characteristics for the ongoing outbreak were obtained from two online information systems [7, 8] , and information for other pathogens was summarized from the pathogen-specific pages on the who and cdc websites. to assess the probability that the emerging outbreak was caused by a known pathogen, we first calculated the distance between the set of characteristics of the ongoing outbreak and those of previously known pathogens. the distance between the characteristics of the ongoing outbreak and cases due to pathogen j is denoted by d j. we assumed that the probability that the outbreak is due to a variant of pathogen j decreased exponentially with distance d j . then, by bayes' theorem, pr(pathogen j | observed characteristics) = pr(observed characteristics pathogen j)q j i pr(observed characteristics | pathogen i)q i (1) in which the sum in the denominator is over all possible pathogens i (i.e., each of the columns of table 1 , including the column describing the current outbreak). the constants q i represented a priori probabilities that the outbreak is due to pathogen i [11, 12] . we set uninformative priors for all pathogens considered, so that q i was simply the reciprocal of the number of pathogens being considered (including disease x) on each date in our analysis. we initially estimated the distance between observed characteristics of the outbreak and each known candidate pathogen using the hamming distance (i.e., the sum of squares differences between the entries in the columns of table 1 corresponding to the disease x and the candidate pathogen). then, we assumed that the probability that the outbreak is driven by pathogen j was governed by a negative exponential function, where d j is the calculated hamming distance, although in principle any decreasing relationship, and any metric describing the distance between two vectors, could have been used. we also repeated our analysis using an alternative measure of the distance between observed characteristics of the outbreak and each known candidate pathogen, namely the euclidean distance (i.e., the square root of the hamming distance). in each case, we assumed that the importance of each characteristic had an identical weight in our analysis, so that a simple quantitative assessment could be obtained in a probabilistic manner without the need for subjective judgement. combining equations (1) and (2), and assuming uninformative priors for q i , gives, the probability that the outbreak was driven by disease x corresponds to the distance d x = 0, and represents a risk score taking values between the reciprocal of the number of candidate pathogens including disease x itself and one: if there are n known pathogens that can potentially cause atypical pneumonia, the probability of observing disease x without any information would be identical to the probability of observing any other listed pathogen (i.e., 1/(1 + n)). as pathogens were ruled out by laboratory testing, that uninformative probability increased (i.e., 1/12 until 2 january 2020, 1/8 from 3 january 2020 and 1/6 from 5 january 2020 in the current outbreak). in addition, if the probability of observing disease x according to equation (3) takes a value close to the probability of observing other candidate pathogens, the overall probability that the outbreak is due to a novel pathogen should be interpreted as being low. a result of significant practical importance, however, is when the probability of observing disease x is close to one or much larger than the probability corresponding to each previously observed candidate pathogen. in that case, all candidate pathogens are not similar to the causative agent of the ongoing outbreak, and so the outbreak is likely to be due to a novel pathogen. we converted the probability of disease x into the equivalent percentage value (so that, for example, a result of 0.8 in equation (1) is assumed to mean an 80% probability) and refer to the percentage value as the "probability of disease x" hereafter. we show temporal changes in estimates of the probability that the ongoing outbreak is driven by each candidate pathogen in figure 1 . because the only information on 30 december 2019 was that cases had symptoms of atypical pneumonia, the distances between the ongoing outbreak and the eleven known pathogens were all zero; thus, all eleven candidate pathogens initially showed an identical probability of 8.3% (i.e., 1/12, when the possibility of disease x is accounted for). if no further information had become available during the outbreak, other than the gradual ruling out of candidate pathogens through laboratory tests, then the inferred uninformative probability for each candidate pathogen would have been given by the dotted gray lines in figure 1 . we converted the probability of disease x into the equivalent percentage value (so that, for example, a result of 0.8 in equation (1) is assumed to mean an 80% probability) and refer to the percentage value as the "probability of disease x" hereafter. we show temporal changes in estimates of the probability that the ongoing outbreak is driven by each candidate pathogen in figure 1 . because the only information on 30 december 2019 was that cases had symptoms of atypical pneumonia, the distances between the ongoing outbreak and the eleven known pathogens were all zero; thus, all eleven candidate pathogens initially showed an identical probability of 8.3% (i.e., 1/12, when the possibility of disease x is accounted for). if no further information had become available during the outbreak, other than the gradual ruling out of candidate pathogens through laboratory tests, then the inferred uninformative probability for each candidate pathogen would have been given by the dotted gray lines in figure 1 . real-time estimation of the probability that the ongoing pneumonia outbreak is driven by each candidate pathogen, given available information on different days. the probability that the outbreak is due to an unknown pathogen (disease x) increases as more information becomes available, for two reasons: (i) the current outbreak can be seen to exhibit characteristics that are not similar to those observed in previous outbreaks, and; (ii) previously observed pathogens are ruled out by laboratory test results. arrows indicate new information available on each date. results are shown for different metrics describing the distance between characteristics of the ongoing outbreak and each candidate pathogen, and either including or excluding initial exposure information for the current outbreak (i.e., worked at/visited a wet market), specifically: (a) hamming distance (the sum of squares difference between the entries in the columns of table 1 corresponding to the ongoing outbreak and the candidate pathogen considered) with wet market exposure; (b) euclidean distance (the square root of the hamming distance) with wet market exposure; (c) hamming distance without wet market exposure; (d) euclidean distance without wet market exposure. dashed grey lines show the probability for every pathogen (including disease x) if the only information included is the ruling out of different pathogens through laboratory tests (i.e., a probability of 1/(1 + number of candidate pathogens remaining on that day)). note that the probability corresponding to different pathogens can be identical, for example, severe acute respiratory syndrome (sars) and mycoplasma pneumoniae were assessing as being equally likely as the causative pathogen from 30 december to 4 january, and legionellosis and chlamydia pneumoniae had equal probability from 30 december to 12 january (details in supplementary material s1). real-time estimation of the probability that the ongoing pneumonia outbreak is driven by each candidate pathogen, given available information on different days. the probability that the outbreak is due to an unknown pathogen (disease x) increases as more information becomes available, for two reasons: (i) the current outbreak can be seen to exhibit characteristics that are not similar to those observed in previous outbreaks, and; (ii) previously observed pathogens are ruled out by laboratory test results. arrows indicate new information available on each date. results are shown for different metrics describing the distance between characteristics of the ongoing outbreak and each candidate pathogen, and either including or excluding initial exposure information for the current outbreak (i.e., worked at/visited a wet market), specifically: (a) hamming distance (the sum of squares difference between the entries in the columns of table 1 corresponding to the ongoing outbreak and the candidate pathogen considered) with wet market exposure; (b) euclidean distance (the square root of the hamming distance) with wet market exposure; (c) hamming distance without wet market exposure; (d) euclidean distance without wet market exposure. dashed grey lines show the probability for every pathogen (including disease x) if the only information included is the ruling out of different pathogens through laboratory tests (i.e., a probability of 1/(1 + number of candidate pathogens remaining on that day)). note that the probability corresponding to different pathogens can be identical, for example, severe acute respiratory syndrome (sars) and mycoplasma pneumoniae were assessing as being equally likely as the causative pathogen from 30 december to 4 january, and legionellosis and chlamydia pneumoniae had equal probability from 30 december to 12 january (details in supplementary materials table s1 ). however, additional characteristics of the ongoing outbreak were observed on 31 december 2019. these characteristics allowed the ongoing outbreak to be distinguished from outbreaks due to previous pathogens, and consequently the inferred probability that the outbreak was driven by a novel pathogen increased substantially to 54.3% and 33.8% for hamming and euclidean distance metrics, respectively ( figure 1a,b) . if instead the exposure characteristic (i.e., exposure at a wet market) was excluded from the analyses, the probability of observing disease x given observed characteristics was still as high as 41.3% and 29.1% for the hamming and euclidean distance metrics ( figure 1c,d) . adenoviruses, hpai (h5n1 and h7n9) and other influenza viruses were ruled out on 3 january 2020, leading to an estimated probability that the outbreak was due to disease x of 80.8% and 50.2% for the hamming and euclidean distance metrics when all factors were considered. excluding the characteristic corresponding to wet market exposure, the probability that the outbreak was due to disease x was assessed to be 60.7% and 42.7% for the hamming and euclidean distance metrics, respectively. sars and mers coronaviruses were ruled out as possible causative agents on 5 january 2020, leading to a very high estimate for the probability that the outbreak was caused by a novel pathogen once all information was collected. on 12 january 2020, the probability the outbreak was due to disease x was estimated to be 82.2% and 56.5% according to the model considering all the characteristics (again, for the hamming and euclidean distances, respectively), while the model excluding the characteristic of exposure at the wet market suggested probabilities of 62.9% and 48.6%. in this analysis, we showed how the outbreak of pneumonia cases in wuhan was assessed in early january 2020 as being caused by a novel pathogen. this was demonstrated using a series of clinical, occupational, and behavioral observations extracted from fragmented reports describing the cases as these reports became available in real time [3, 6] . although virological investigation is the gold standard for pathogen identification, and the virus has now been confirmed to be a novel coronavirus that is a relative of sars, laboratory-based outcomes can only be obtained after successfully sequencing the novel virus, which can sometimes be a lengthy process. at the time of writing, it still remains for the microbiological causal link to be established, for instance by ensuring that koch's postulates are met (as seen, e.g., in a study of zika virus [13] ). in the ongoing outbreak, the provisional identification of a novel coronavirus was performed on 7 january 2020 and announced formally on 9 january 2020 [2] . we have shown that non-virological information can indicate that the cause of the outbreak is likely to be a novel pathogen ("disease x"), and that this conclusion was obtained before virological test results were announced. disease x was inferred to be very likely on all dates from 31 december 2019 onwards-the date on which descriptions of outbreak characteristics began to emerge. when sufficient clinical details of cases (e.g., complete blood cell counts) are available, the number of causative pathogens considered can be limited to a reasonable number. in this instance, atypical pneumonia combined with reduced white blood cell counts and the lack of response to antibiotics indicated that the pathogen was consistent with viral rather than bacterial infection. with such information, non-virological data can be used for convenient quantification of the probability that the outbreak was due to a novel pathogen, while awaiting the results of virological tests. we believe that the proposed approach can improve risk assessment practices across the world. it is important to consider two issues about the compilation of table 1 . first, a critical underlying assumption is that table 1 represents general outbreak characteristics of the ongoing outbreak and previously known outbreaks. the representation does not reflect observations from all confirmed cases nor epidemiological findings from a case control study (e.g., statistically significant risk factors). rather, zeros and ones in the table were defined in a phenomenological manner, and values may change as the ongoing outbreak continues. depending on the opinions of different experts (e.g., [14] ), the defined nominal values could have been different to those shown in table 1 ; in this study, we are simply demonstrating how such an approach might work in practice. second, as we have shown, quantitative estimates depend on the precise characteristics that are used. we showed results including and not including information on wet market exposure. in table 1 , infections due to previously observed pathogens other than hpai were assumed not to be associated with exposure to wet markets. since this assumption was not derived from empirical observations, it could be debated. in the past, descriptive outbreak information has been used to generate outbreak case definitions, and causative agents have been pinpointed without using statistical methods in combination with epidemiological observations. in the present study, we have shown that such assessments can be made quantitatively using a simple statistical model, allowing for comparisons between the possible causative agents among different candidates. when outbreak characteristics are shared and updated in real-time (table 1) , these data can contribute to efforts to narrow down the possible range of causative agents. in the case of the outbreak in wuhan, our calculation of the probability that each pathogen is the causative agent indicates that virological exclusion of influenza viruses, adenoviruses and known virulent coronaviruses associated with sars and mers on 3 and 5 january 2020 can be regarded as an "unsurprising" finding. as important limitations, the precision and credibility of the input data and the method for calculating the distance between the candidate pathogens and the observed outbreak, must be refined in future. first, our proposed approach used very limited data in table 1 for logical quantification of the probability that each pathogen was the causative agent. however, with more clinical data, the binary characteristics could be replaced by continuous frequencies (e.g., the proportion of cases experiencing coughing and/or breathing difficulties). second, with sufficient data it would also be possible to estimate the probability that each pathogen is the causal agent (equation (1)) not by arbitrarily measuring the distance but by using classification models involving regression or more sophisticated machine learning approaches. third, the erroneous input of incorrect information may be a challenge in real time analyses. the veracity of the sources of information for future analyses could have an impact on the resulting probability calculations. fourth, the estimated probability that an outbreak is driven by a novel pathogen might be slightly over-or underestimated due to limited information about the mode of transmission and small numbers of observed cases. of note, the respiratory syncytial virus (rsv) was not completely ruled out as a candidate pathogen in our real-time analysis. however, rsv was an unlikely candidate since the majority of cases in the ongoing outbreak are adults [15] while most rsv infections are observed in infants and young children. finally, we had to restrict ourselves to assuming the a priori probability that the ongoing outbreak driven by each candidate pathogens (q i ) is identical for each pathogen. however, since no alternative information was available, we believe such uninformative priors to be the optimal choice. despite the future improvements to our statistical modelling framework that are required, including the need to test our approach using data from outbreaks of previously known pathogens, this short study demonstrated clearly that the ongoing outbreak is consistent with causation by a novel pathogen, "disease x". we reached this conclusion after only a few days of the outbreak had passed. attention has now rightly turned towards identifying the pandemic potential of this outbreak [16] [17] [18] , as well as planning control interventions within china and elsewhere [19, 20] . however, at the start of the next outbreak of an unknown pathogen, virological testing and quantitative analyses of clinical data are two complementary methods that can be used. thus, analyses of the type conducted in this study can greatly support efforts to characterize causal agents in future outbreaks, with the benefit that analyses like this one can be carried out extremely quickly. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/3/637/s1, table s1 : estimated values of the probability of disease x, given available information at different timepoints using hamming distance and including wet market exposure, table s2 : estimated values of the probability of disease x, given available information at different timepoints using euclidean distance and including wet market exposure, table s3 : estimated values of the probability of disease x, given available information at different timepoints using hamming distance and excluding wet market exposure, table s4 : estimated values of the probability of disease x, given available information at different timepoints using euclidean distance and excluding wet market exposure. wuhan municipal health commission's briefing on the current pneumonia epidemic who statement regarding cluster of pneumonia cases in wuhan wuhan municipal health commission. wuhan municipal health and health committee's report on unexplained viral pneumonia novel human virus? pneumonia cases linked to seafood market in china stir concern mystery virus found in wuhan resembles bat viruses but not sars pneumonia cases possibly associated with a novel coronavirus in wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia severe acute respiratory syndrome-related coronavirus: the species and its viruses-a statement of the coronavirus study group incubation period as part of the case definition of severe respiratory illness caused by a novel coronavirus probabilistic differential diagnosis of middle east respiratory syndrome (mers) using the time from immigration to illness onset among imported cases who zika causality working group. zika virus infection as a cause of congenital brain abnormalities and guillain-barre syndrome: systematic review initial cluster of novel coronavirus (2019-ncov) infections in wuhan, china is consistent with substantial human-to-human transmission clinical features of patients infected with 2019 novel coronavirus in wuhan, china. lancet pandemic potential of 2019-ncov nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study real-time estimation of the risk of death from novel coronavirus (covid-19) infection: inference using exported cases novel coronavirus outbreak in wuhan, china, 2020: intense surveillance is vital for preventing sustained transmission in new locations estimation of the transmission risk of the 2019-ncov and its implication for public health interventions the authors declare no conflicts of interest. key: cord-289832-092dtzrd authors: villard, orianne; morquin, david; molinari, nicolas; raingeard, isabelle; nagot, nicolas; cristol, jean-paul; jung, boris; roubille, camille; foulongne, vincent; fesler, pierre; lamure, sylvain; taourel, patrice; konate, amadou; maria, alexandre thibault jacques; makinson, alain; bertchansky, ivan; larcher, romaric; klouche, kada; le moing, vincent; renard, eric; guilpain, philippe title: the plasmatic aldosterone and c-reactive protein levels, and the severity of covid-19: the dyhor-19 study date: 2020-07-21 journal: j clin med doi: 10.3390/jcm9072315 sha: doc_id: 289832 cord_uid: 092dtzrd background. the new coronavirus sars-cov-2, responsible for the covid-19 pandemic, uses the angiotensin converting enzyme type 2 (ace2), a physiological inhibitor of the renin angiotensin aldosterone system (raas), as a cellular receptor to infect cells. since the raas can induce and modulate pro-inflammatory responses, it could play a key role in the pathophysiology of covid-19. thus, we aimed to determine the levels of plasma renin and aldosterone as indicators of raas activation in a series of consecutively admitted patients for covid-19 in our clinic. methods. plasma renin and aldosterone levels were measured, among the miscellaneous investigations needed for covid-19 management, early after admission in our clinic. disease severity was assessed using a seven-category ordinal scale. primary outcome of interest was the severity of patients’ clinical courses. results. forty-four patients were included. at inclusion, 12 patients had mild clinical status, 25 moderate clinical status and 7 severe clinical status. in univariate analyses, aldosterone and c-reactive protein (crp) levels at inclusion were significantly higher in patients with severe clinical course as compared to those with mild or moderate course (p < 0.01 and p = 0.03, respectively). in multivariate analyses, only aldosterone and crp levels remained positively associated with severity. we also observed a positive significant correlation between aldosterone and crp levels among patients with an aldosterone level greater than 102.5 pmol/l. conclusions. both plasmatic aldosterone and crp levels at inclusion are associated with the clinical course of covid-19. our findings may open new perspectives in the understanding of the possible role of raas for covid-19 outcome. a new coronavirus called sars-cov-2 is responsible for the pandemic of covid-19, which has led to tens of thousands of deaths around the world so far [1] . briefly, the disease develops in two phases: the first one is linked to the viral invasion, and the second one consists of a severe acute inflammatory immune response, including a "cytokine storm", which results in severe morbidity and mortality, mainly related to lung injury [2] . in this context, the intensity of the inflammatory process contributes to the disease severity and the plasmatic level of c-reactive protein (crp) (a biomarker of systemic inflammation) could represent a marker of poor outcomes in covid-19 patients [3] [4] [5] [6] . as observed with the sars-cov responsible for sars 2003 [7] , sars-cov-2 uses the angiotensin converting enzyme type 2 (ace2) as a cellular receptor to infect cells. ace2 is a physiological inhibitor of the renin angiotensin aldosterone system (raas) through the catabolism of angiotensin type 2 (ang2) into angiotensin (1-7) peptide [8] . ang2 can induce pro-inflammatory responses through its receptor at1r, while ace2 reduces anti-inflammatory reactions through its receptor masr. the assessment of raas involvement in the course of covid-19 in humans is not easy, due to the poor value of ang2 and angiotensin (1-7) peptide assays in peripheral blood to investigate raas and ace2 in affected patients. therefore, we explored the levels of plasma renin and aldosterone as indicators of raas activation in a series of consecutively admitted patients for covid-19 in our clinic. relationships with the severity of disease course were investigated to assess whether raas activation could be considered as a biomarker of covid-19 outcomes. in a series of consecutive patients with covid-19 diagnosis, hormonal assays including plasma renin and aldosterone levels were performed among the miscellaneous investigations needed for the management of covid-19, early after admission in our clinic. this study is called dyhor-19 (dysfunctional hormone regulation during covid-19) and its protocol was reviewed and approved by the university hospital of montpellier institutional review board (irb-mtp_2020_04_202000441, clinicaltrials.gov identifier: application in process). the disease severity was assessed using a seven-category ordinal scale (os) [9] , as follows: 1-not hospitalized, no limitation on activities; 2-not hospitalized, limitations on activities; 3-hospitalized, not requiring supplemental oxygen; 4-hospitalized, requiring supplemental oxygen; 5-hospitalized, on non-invasive ventilation or high flow oxygen devices; 6-hospitalized, on invasive mechanical ventilation or extracorporeal membrane oxygenation (ecmo) and 7-death. the primary outcome of interest was the severity of patients' clinical courses during hospitalization, defined as severe for an ordinal scale higher than 4 and corresponding to the transfer to the intensive care unit and death from all-causes. clinical status using the seven-category ordinal scale was assessed at three different time points: (i) early after admission corresponding to the day of the biological test (day 0), (ii) two days later (day 2) and (iii) considering the maximum ordinal scale during the overall period of hospitalization (os max). the past medical history, clinical manifestations, comorbidities, treatment strategies, radiologic assessments and laboratory testing on admission were extracted from the electronic medical records. disease severity was assessed by the os, as indicated above. diagnosis of covid-19 was considered as suspected in patients with typical lung ct-scan lesions and negative sars-cov-2 pcr. laboratory variables were tested with conventional methods, including routine blood tests: blood count, renal function, inflammatory markers. determination of crp was performed using a cobas8000/e502 ® analyzer (roche diagnostic, meylan, france) using the immunoturbidimetric method with reagents from roche (total cv imprecision results in the laboratory = 3%). renin and aldosterone were determined on an ids-isys multi-discipline automated system (immunodiagnosticsystem, boldon, united kingdom) using kits from ids (total cv imprecision results in the laboratory 5% and 6% for renin and aldosterone respectively). a cutoff of 102.5 pmol/l was the lower limit of plasma aldosterone detection in our condition. plasma cortisol and adrenocorticotropic hormone (acth) levels were measured by automated electrochemiluminescence assays (cobas 8000, roche, basel, switzerland). laboratory confirmation of sars-cov-2 infection was determined by reverse transcription-pcr from nasopharyngeal swab specimens. categorical variables were described as frequency rates and percentages, analyzed using the chi-squared test or fisher's exact test. continuous variables were described using mean and standard deviation (sd). means for continuous variables were compared using student t-test or mann-whitney test according to the data distribution. due to skewed distribution, biological variables were presented with median (min-max), and median difference (hodges-lehamann estimator). a logistic regression was used for the analysis of the main criteria with odds ratio of disease severity adjusted on the delay from admission. covariates were selected in a backward selection procedure if p < 0.15 in the univariate analysis and then presented as adjusted odds ratios (ors). potential confounding factors were investigated by testing differences between groups. studying the relationship between variables was done using spearman correlation. statistical analyses were performed using sas enterprise guide, version 7.3 (sas institute, cary, nc, usa) and graphpad prism, version 8.4.2 for mac os (graphpad software, san diego, ca, usa). forty-four patients were included in the study during the period from 26 march to 20 april 2020. clinical characteristics of the patients are described in table 1 at inclusion, on day 0, patients with severe clinical course (os max ≥ 5) had more frequently a thyroid or chronic kidney disease, and a concomitant acute bacterial disease, compared to patients with mild or moderate course (os max ≤ 4). a history of hypertension was present in 25 patients (56.8%). among antihypertensive treatment, the use of raas blockers (angiotensin-converting enzyme inhibitors or angiotensin-receptor blockers) was not different between mild or moderate and severe clinical course and concerned 11 (38%) and 5 patients (33%) respectively. beta-blockers and loop diuretics were more frequently used in patients with severe clinical progression (os max ≥ 5) than in those with mild to moderate clinical course (os max ≤ 4). the most common symptoms on admission were fever (86.4%) and cough (65.1%) with no differences found between patients with mild or moderate and those with severe clinical course. as expected, treatments during hospitalization including corticosteroid therapy, antibiotics and vasoactive drugs, which were significantly more frequently delivered in patients with severe compared to those with mild or moderate clinical courses. laboratory findings according to disease severity during the overall period of hospitalization are described in table 3 between mild/moderate (os max ≤ 4) versus severe (os max ≥ 5) groups. in univariate analyses, aldosterone levels at inclusion were significantly higher in patients with severe clinical course (os ≥ 5) (median (min-max), 304.7 (102.5-1360.1) pmol/l) as compared to those with mild or moderate course (os ≤ 4) (102.5 (102.5-540.2) pmol/l) (p < 0.01). of note, at inclusion, potassium levels and aldosterone/renin ratios were not different between these two groups ( figure 1a ,c), but in some cases, we observed a trend toward an association between higher levels of aldosterone and lower renin and potassium levels ( figure 1b,d) . concerning cortisol and acth levels, no difference was observed between groups. among common hematological and inflammatory markers at baseline (including lymphocyte, monocyte and eosinophil counts, fibrinogen and d-dimers), crp at inclusion was significantly higher in patients with severe clinical course (152 (34-389) mg/l) compared to those with mild or moderate course (83 (3-298) mg/l, p = 0.03). in multivariate analyses including coexisting conditions, long-term anti-hypertensive treatments, care during hospitalization and laboratory findings at inclusion, only aldosterone (or = 1.07 (1.01-1.14), p = 0.033) and crp (or = 1.11 (1.01-1.22), p = 0.024) remained positively associated with the severity of clinical course. table 3 . biological findings at inclusion (on day 0) according to disease severity of patients with covid-19 infection classified in two groups: mild/moderate (os max ≤ 4) and severe (os max ≥ 5). number in addition, the plasma aldosterone and crp levels were examined according to the clinical status at three different time points (figures 2 and 3a) : (i) at inclusion (day 0), (ii) two days later (day 2) and (iii) at the maximum ordinal scale during the overall period of hospitalization (os max). at inclusion, aldosterone levels were not clearly associated with a specific clinical status (p = 0.61) (figure 2a) . however, higher aldosterone levels at inclusion were observed in patients with os at day 2 or os max ≥ 5 (p = 0.006 and p = 0.0013 respectively) (figure 2a) . moreover, aldosterone levels were also gradually and significantly increased when we compared clinical status of patients in the three following categories: mild (os ≤ 3), moderate (os = 4) and severe (os ≥ 5) on day 2 and at os max (analysis of variance, p = 0.001 and p = 0.006, respectively) ( figure 2b) . notably, similar findings were observed when patients receiving a raas blocker were excluded from the analysis on day 2 and at os max (analysis of variance, p = 0.008 and p = 0.025, respectively) (supplemental figure s1) . similarly, these findings were also observed when patients receiving beta-blockers were excluded from the analysis on day 2 and at os max (analysis of variance, p = 0.002 and p = 0.01, respectively). notably, we also investigated the effects of age and sex on our findings and did not observe any significant differences between groups. an additional analysis with crp level at baseline found concordant results. as compared to patients with mild or moderate clinical status, crp levels were significantly higher in patients with os ≥ 5 on day 2 or at os max (p = 0.01 and p = 0.02, respectively) ( figure 3a ). considering the hypothesis that aldosterone may be involved in inflammatory damages of covid-19, we searched for a relationship between the aldosterone and crp levels. we conducted this further analysis independently from disease severity (as assessed by the os), after having excluded the patients who had developed a documented acute bacterial infection in the days close to the biological investigation. finally, we observed a correlation between aldosterone and crp levels among patients with an aldosterone level greater than 102.5 pmol/l. in this group of 19 patients, aldosterone level was positively correlated with crp level at baseline (spearman coefficient r (95% ci) 0.61 (0.2-0.84), p = 0.006) ( figure 3b ). in the present study based upon data collected in the real-life settings of the brutal sars-cov-2 outbreak, we report an association between the plasma levels of aldosterone close to admission and the severity of covid-19 course, as defined by the ordinal scale grade. indeed, the most severe an additional analysis with crp level at baseline found concordant results. as compared to patients with mild or moderate clinical status, crp levels were significantly higher in patients with os ≥ 5 on day 2 or at os max (p = 0.01 and p = 0.02, respectively) ( figure 3a ). considering the hypothesis that aldosterone may be involved in inflammatory damages of covid-19, we searched for a relationship between the aldosterone and crp levels. we conducted this further analysis independently from disease severity (as assessed by the os), after having excluded the patients who had developed a documented acute bacterial infection in the days close to the biological investigation. finally, we observed a correlation between aldosterone and crp levels among patients with an aldosterone level greater than 102.5 pmol/l. in this group of 19 patients, aldosterone level was positively correlated with crp level at baseline (spearman coefficient r (95% ci) 0.61 (0.2-0.84), p = 0.006) ( figure 3b ). in the present study based upon data collected in the real-life settings of the brutal sars-cov-2 outbreak, we report an association between the plasma levels of aldosterone close to admission and the severity of covid-19 course, as defined by the ordinal scale grade. indeed, the most severe patients, who required at least intensive care (os ≥ 5), had significantly higher plasma levels of aldosterone when admitted than those hospitalized in medical units, with (os = 4) or without (os = 3) oxygen support. this association appears to be relevant both when considering the os 2 days after admission and according to the maximal os during the overall period of hospitalization. in most patients, aldosterone levels remained within a physiological range, but the significant differences observed between groups according to severity were independent of the renin levels and aldosterone/renin ratio. such a hormonal profile is suggestive of a renin-independent hyperaldosteronism [10] , which could be a hallmark of some patients with the most severe forms of covid-19. conversely, low aldosterone levels were observed in those with a less severe disease (os = 3 or 4). this could be related either to a failure of the aldosterone assay to discriminate within the minimal values or reflect a tendency to adrenal insufficiency. however, this latter hypothesis is not supported by the plasma cortisol levels. as previously reported [3] [4] [5] , crp levels were coherent with the severity of covid-19, which is characterized by a severe inflammatory syndrome. interestingly, patients with aldosterone levels higher than 102.5 pmol/l exhibited a linear relationship between crp and aldosterone levels. this further finding is in line with the suspected role of the viral load in the ace/ace2 imbalance, which occurs before the onset of the cytokine storm [2, 11, 12] . indeed, sars-cov-2 could disrupt the raas through its binding to ace2, which is the negative regulator of the system [8] . hence, the defective inactivation of ang2 could lead to the activation of raas, including an increased secretion of aldosterone. the role of ang2 in the severity of lung inflammatory damage in covid-19 is supported by previous investigational reports. first, imai et al. [13, 14] demonstrated in several animal models of acute lung injury (acid inhalation, sepsis or pneumonia) that ang2 can worsen pulmonary lesions (including inflammatory infiltrates) through the stimulation of the ang2 type 1 receptor (at1r). conversely, ace2 and ang2 type 2 receptor (at2r) can down-regulate these deleterious effects, whereas abrogated ace2 expression can induce severe respiratory failure in mice models. in addition, the levels of ang2 are increased in these mice, which exhibit severe lung involvement partially reversible with the pharmacological inhibition of the at1r [13, 14] . during sars-cov-1 infection, ace2 knockout mice were resistant to virus infection and their lung samples were devoid of inflammation [15] . in contrast, the binding of the sars-spike protein to ace2 downregulates this regulator pathway, leading to severe lung injury and acute respiratory failure, as illustrated in a mouse model by kuba and coll [15] . in their study, blocking the raas limited the lung injury. these findings are in line with the concept that raas disruption could trigger inflammation in covid-19. furthermore, beside coronavirus infections, the potentially deleterious effects of raas have been documented in several tissues (including heart and lung) and medical conditions (such as hypertension, heart failure, obesity, etc.) [16] and have been also documented beyond the regulation of sodium, extracellular volume and blood pressure. the mechanisms leading to raas toxicity also include (i) modulation of the production of pro-inflammatory cytokines (such as tnf alpha and il-1 by ang2 [17] and il-6 by aldosterone [18] ), leading potentially to recruitment of mono/macrophages; (ii) induction of fibrosis (through at1r) [19] ; and (iii) induction of vascular toxicity [20] and modulation of angiogenesis [21, 22] . in the context of ace2 neutralization by sars-cov-2, all these mechanisms could be exacerbated, while their clinical consequences are more limited in classical conditions of raas hyperactivation (such as chronic heart failure, etc. [16] ). importantly, the pathogenic mechanisms of covid-19 are concordant with autoptic observations and biological findings, which include the cytokine storm (with il6, il1, tnf, etc.) [2, 11, 12] , fibrosis [23] , endothelitis and modulated angiogenesis [24, 25] . in addition, the key role of raas toxicity could be also corroborated by the promising beneficial effects observed with anti-aldosterone and raas blocker treatments in several experimental conditions of pulmonary diseases [26] . in covid-19, these protective effects are extensively debated [8, 27, 28] . finally, the potentially deleterious effects of raas may take place in the pathophysiology of covid-19. from this point of view, our findings suggest that both crp and aldosterone levels may impact the clinical status. further studies are required to document and confirm the suspected role of raas in covid-19. our study has limitations due to the collection of plasma samples for hormonal assays in an emergency setting related to the admission for covid-19 acute infection. hence, optimal standardized conditions for assessing plasma renin and aldosterone levels were not met, and multiple confounding factors could be involved in the modulation of plasma aldosterone secretion. however, when we adjusted for all of these confounding parameters, plasma aldosterone levels remained significantly associated to disease severity. in the present study, higher plasmatic aldosterone and crp levels at inclusion are associated with severe clinical course of covid-19 in hospitalized patients, and both parameters appear to be correlated. our results suggest that aldosterone levels may reflect the severity of covid-19, but this remains to be demonstrated at a larger scale. our findings open new perspectives into the understanding of the contribution of raas in covid-19 and its possible role in the outcomes of covid-19. further investigations are awaited to explore more thoroughly the association between increased aldosterone levels, ace/ace2 imbalance, inflammatory biomarkers and the severity of the covid-19 course. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/7/2315/s1, figure s1 : patients with no raas blocker therapy: clinical status course according to aldosterone level at inclusion, * p < 0.05 (mann-whitney test) (a) and laboratory findings of raas explorations by severity of clinical status during the overall period of hospitalization (b), table s1 : clinical characteristics of patients with covid-19 according to disease severity classified in three groups: mild (os max ≤ 3), moderate (os max = 4) and severe (os max ≥ 5). author contributions: o.v.: data collection, design and conceptualization of the study, statistical analysis, drafting and reviewing of the manuscript; d.m.: data collection, investigation, software, design and conceptualization of the study, statistical analysis, supervision, drafting and reviewing of the manuscript; n.m.: methodology, statistical analysis, drafting and reviewing of the manuscript; i.r.: design and conceptualization of the study, drafting and reviewing of the manuscript; n.n.: design and conceptualization of the study, drafting and reviewing of the manuscript; j.-p.c.: data collection, drafting and reviewing of the manuscript; b.j.: data collection, investigation, drafting and reviewing of the manuscript; c.r.: data collection, investigation, drafting and reviewing of the manuscript; v.f.: data collection, investigation, drafting and reviewing of the manuscript; p.f.: data collection, investigation, drafting and reviewing of the manuscript; s.l.: data collection, investigation, drafting and reviewing of the manuscript; p.t.: data collection, investigation, drafting and reviewing of the manuscript; a.k.: data collection, investigation, drafting and reviewing of the manuscript; a.t.j.m.: design and conceptualization of the study, drafting and reviewing of the manuscript; a.m.: data collection, investigation, drafting and reviewing of the manuscript; i.b.: data collection, investigation, drafting and reviewing of the manuscript; r.l.: data collection, investigation, drafting and reviewing of the manuscript; k.k.: data collection, investigation, drafting and reviewing of the manuscript; v.l.m.: data collection, investigation, drafting and reviewing of the manuscript; e.r.: design and conceptualization of the study, methodology, drafting and reviewing of the manuscript; p.g.: design and conceptualization of the study, methodology, statistical analysis, supervision, drafting and reviewing of the manuscript. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. acknowledgments: this work was supported by montpellier university hospital, montpellier university. the authors declare no conflict of interest. clinical characteristics of coronavirus disease 2019 in china severe sars-cov-2 infections: practical considerations and management strategy for intensivists predictive factors for disease progression in hospitalized patients with coronavirus disease 2019 in wuhan viral and host factors related to the clinical outcome of covid-19 phenotypic characteristics and prognosis of inpatients with covid-19 and diabetes: the coronado study early predictors of clinical deterioration in a cohort of 239 patients hospitalized for covid-19 infection in lombardy angiotensin-converting enzyme 2 is a functional receptor for the sars coronavirus renin-angiotensin-aldosterone system inhibitors in patients with covid-19 a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 syndromes that mimic an excess of mineralocorticoids cytokine release syndrome in severe covid-19 immunology of covid-19: current state of the science angiotensin-converting enzyme 2 protects from severe acute lung failure angiotensin-converting enzyme 2 (ace2) in disease pathogenesis a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury abu-izneid, t. renin-angiotensin-aldosterone (raas): the ubiquitous system for homeostasis and pathologies angiotensin ii: its effects on fever and hypothermia in systemic inflammation interleukin-6 plays a critical role in aldosterone-induced macrophage recruitment and infiltration in the myocardium il-6 trans-signalling contributes to aldosterone-induced cardiac fibrosis the renin-angiotensin-aldosterone system in vascular inflammation and remodeling angiotensin ii and aldosterone in retinal vasculopathy and inflammation aldosterone inactivates the endothelin-b receptor via a cysteinyl thiol redox switch to decrease pulmonary endothelial nitric oxide levels and modulate pulmonary arterial hypertension dying with sars-cov-2 infection-an autopsy study of the first consecutive endothelial cell infection and endotheliitis in covid-19 pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 targeting the renin-angiotensin system as novel therapeutic strategy for pulmonary diseases drugs and the renin-angiotensin system in covid-19 renin-angiotensin-aldosterone system inhibitors and risk of covid-19 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-282384-qbcqbhk4 authors: savastano, alfonso; crincoli, emanuele; savastano, maria cristina; younis, saad; gambini, gloria; de vico, umberto; cozzupoli, grazia maria; culiersi, carola; rizzo, stanislao title: peripapillary retinal vascular involvement in early post-covid-19 patients date: 2020-09-08 journal: j clin med doi: 10.3390/jcm9092895 sha: doc_id: 282384 cord_uid: qbcqbhk4 the ability of severe acute respiratory syndrome coronavirus 2 (sars-cov-2′s) to cause multi-organ ischemia and coronavirus-induced posterior segment eye diseases in mammals gave concern about potential sight-threatening ischemia in post coronavirus disease 2019 patients. the radial peripapillary capillary plexus (rpcp) is a sensitive target due to the important role in the vascular supply of the peripapillary retinal nerve fiber layer (rnfl). eighty patients one month after sars-cov-2 infection and 30 healthy patients were selected to undergo structural oct (optical coherence tomography) and octa (optical coherence tomography angiography) exams. primary outcome was a difference in rpcp perfusion density (rpcp-pd) and rpcp flow index (rpcp-fi). no significant difference was observed in age, sex, intraocular pressure (iop) and prevalence of myopia. rpcp-pd was lower in post sars-cov-2 patients compared to controls. within the post-covid-19 group, patients with systemic arterial hypertension had lower rpcp-fi and age was inversely correlated to both rpcp-fi and rpcp-pd. patients treated with lopinavir + ritonavir or antiplatelet therapy during admission had lower rpcp-fi and rpcp-pd. rnfl average thickness was linearly correlated to rpcp-fi and rpcp-pd within post-covid-19 group. future studies will be needed to address the hypothesis of a microvascular retinal impairment in individuals who recovered from sars-cov-2 infection. coronaviruses are a subfamily of single-stranded positive-sense rna viruses widely diffused among animal species. their genome length (26-30 kb) is one of the largest known sequences among rna viruses. four known genera of coronaviruses (alfacoronaviruses, betacoronaviruses, gammacoronaviruses, and deltacoronaviruses) exist, but only seven species have the ability to infect humans: 229-e, nl-63, oc-43, hk-u1, mers-cov, sars-cov-1, and sars-cov-2 [1] . the earliest reports of endemic human cov date back to the 1960s, when hcov-oc43 and -229e were described. hcov-nl63 and -hku1 were discovered only in 2004 and 2005, respectively. in addition to these four patients who adhered to the study underwent a clinical and instrumental evaluation. patients from the post-covid-19 group were recruited at 1 month from hospital discharge. each patient underwent a comprehensive ophthalmologic examination, including best corrected visual acuity, slit lamp anterior segment observation (sl9900 slit lamp, cso, florence, italy), iop measurement (goldman tonometry) and dilated fundus inspection and photography (cobra hd fundus camera, cso, florence, italy). structural oct and octa analysis were performed by an expert physician using spectral domain zeiss cirrus 5000-hd-oct angioplex (sw version 10.0, carl zeiss, meditec, inc., dublin, ca, usa). one eye for each patient was chosen randomly to undergo the examination. in the case of unilateral eye disease, the other eye was selected. group attribution was blinded to the examiner performing the oct. structural oct images consisted of the optic disc cube 200 × 200, and macular cube 512 × 128 patterns. the subfoveal choroidal thickness (sct) was manually measured on cross-sectional oct b-scans [26] . two independent masked graders individually assessed all choroidal thickness measurement in the fovea region, from the rear edge of the rpe to the choroid-sclera junction. the agreement between the two observers was determined through bland-altman plot. octa scan protocol was 4.5 × 4.5 mm centered into the disc in healthy and post-covid-19 patient's eyes. two-dimensional en face oct angiograms of the rpc layer were generated with automated segmentation software (cirrus 10.0), with the rpc defined as the segment extending superficially from the inner limiting membrane to the posterior surface of the rnfl. en face images were processed using custom software with an interactive interface [26] . the software used a method combining a global threshold, hessian filter, and adaptive threshold to generate binary vessel maps, which were used to calculate quantitative indices of blood flow in matlab (r2017a; mathworks, inc., natick, ma, usa). the peripapillary flow index was defined as the average decorrelation value in the peripapillary region of the en face retinal angiogram. the peripapillary vessel density was defined as the proportion of the total area occupied by vessels. the blood vessels were defined as the pixels with decorrelation values over the threshold in the noise region, which were two standard deviations higher than the mean decorrelation value. the avascular zone of the onh was manually selected to establish baseline background noise level for global thresholding, and the onh was excluded from quantification [27] . finally, rpcp perfusion density (rpcp-pd) and rpcp flow index (rpcp-fi) were collected and used for analysis. the primary endpoint was a difference in the rpcp-fi and rpcp-pi. the following parameters were chosen as secondary outcome measures: gcc average thickness, rnfl average thickness, disc area, cd ratio, central foveal thickness, choroidal thickness. furthermore, we performed an additional analysis within the post-covid-19 group correlating the primary outcome measures with the other examined variables to detect potential risk factors for rpcp impairment in post sars-cov-2 patients. potential confounders taken into account were: age, systemic autoimmune and inflammatory diseases, axial myopia > 1d (dichotomous variable) [28] , systemic arterial hypertension, and diabetes. the sample size calculation was performed using g*power (3.1.9.7 software, düsseldorf, germany). statistical analysis was conducted using spss software (ibm spss statistics 26.0, ontario, canada). alpha and beta error were established at 5% and 20%, respectively. the following variables were considered as continuous quantitative variables: age, central foveal thickness, choroidal thickness, gcc average thickness, rnfl average thickness, disc area, cd ratio, rcp perfusion density, and rcp flow index. assimilability to normal distribution was evaluated using shapiro-wilk test. univariate comparison between the two groups was performed using t-test for independent groups. linear correlations between quantitative variables were performed using spearman's test. the remaining variables were considered as qualitative variables. the univariate comparison between the groups was performed by means of a chi 2 test. logistic regression analysis was performed to evaluate the actual strength of the associations detected by the univariate analysis. a bonferroni corrected p value < 0.01 was considered to establish the statistical significance of the results. demographic and anamnestic data were collected by the same physician performing the visit and are reported in table 1 . results from the descriptive analysis in post-covid-19 group are summarized in table 2 . the mean age in the group was 52.9 ± 13.5 years with 57.5% male patients. the prevalence of systemic arterial hypertension, diabetes, and autoimmune or inflammatory systemic diseases was 23.8%, 42.5%, and 23.8%, respectively. almost 14% of the patients presented ≥ 1d of axial myopia. mean iop at the visit was 16.2 ± 1.5 mmhg. data collected from the hospital admission for sars-cov-2 infection revealed that only 6.25% of the patients spent part of their recovery in the intensive care unit (icu) and 8.8% required the support of noninvasive ventilation (niv) during the hospital stay. the mean duration of the icu recovery was 3.2 days. medical therapy was administered as follows: 68.8% were treated with hydroxychloroquine, 33.8% with lopinavir + ritonavir, 43.8% with darunavir + ritonavir, 41.3% with anticoagulant therapy (heparin), and 35% with azithromycin. other drugs used for systemic support were antiplatelet therapy (aspirin or clopidogrel) in 7.5% of the patients and corticosteroids in 5% of the patients. finally, 45% of the post-covid-19 group reported tearing, dry eye, or red eye during the infectious period. in the control group, mean age was 48.5 ± 13.4 years, and 43.3% (13/30) were males and 56.6% (17/30) females. few patients (10%) reported systemic arterial hypertension while none of them was affected by diabetes or systemic autoimmune or inflammatory diseases. axial myopia ≥ 1d was present in 13.3% of the subjects. mean iop was 14.4 ± 2.1 mmhg. no statistically significant differences between the 2 groups were detected in terms of age, gender, iop at the visit or prevalence of axial myopia ≥ 1d. the post-covid-19 group showed a higher prevalence of systemic arterial hypertension (p < 0.03), diabetes (p < 0.001) and autoimmune or inflammatory systemic diseases (p < 0.001). one of the most significant differences between the two groups was observed in the rpcp-pd analysis. (figure 1 ) indeed, lower rpcp-pd value in post-covid-19 group compared to the control group (p < 0.04) was observed ( figure 2 ). no statistically significant differences between the 2 groups were detected in terms of age, gender, iop at the visit or prevalence of axial myopia ≥ 1d. the post-covid-19 group showed a higher prevalence of systemic arterial hypertension (p < 0.03), diabetes (p < 0.001) and autoimmune or inflammatory systemic diseases (p < 0.001). one of the most significant differences between the two groups was observed in the rpcp-pd analysis. (figure 1 ) indeed, lower rpcp-pd value in post-covid-19 group compared to the control group (p < 0.04) was observed ( figure 2 ) this difference was further confirmed by the binary logistic regression analysis including all potential confounders (p < 0.039). none of the other outcome measures showed statistically significant differences between the two groups (table 3) . bland-altman analysis of subfoveal choroidal thickness measurement revealed a good agreement between graders (bias = 1.23, ci = 0.94-1.41, la = 28.3%). this difference was further confirmed by the binary logistic regression analysis including all potential confounders (p < 0.039). none of the other outcome measures showed statistically significant differences between the two groups (table 3) . bland-altman analysis of subfoveal choroidal thickness measurement revealed a good agreement between graders (bias = 1.23, ci = 0.94-1.41, la = 28.3%). within the post-covid-19 group, patients affected by systemic arterial hypertension were characterized by a statistically relevant reduction of the rpcp-fi (p < 0.001). moreover, age distribution showed an inverse linear correlation with both rpcp-fi (p < 0.001) and rpcp-pd (p < 0.01). furthermore, patients treated with lopinavir + ritonavir during sars-cov-2 infection showed both a lower rpcp-fi (p < 0.01) and a lower rpcp-pd (p < 0.01) compared to the other patients in the post-covid-19 group. a similar result was demonstrated in patients treated with antiplatelet therapy during hospital recovery. indeed, in these patients, rpcp-fi and rpcp-pd were statistically lower than those not treated (respectively p = 0.004 and p = 0.003). a detailed description of the within post-covid-19 group analysis is available in table 4 . spearman's test revealed a statistically significant linear correlation between rnfl average thickness and both rpcp perfusion density (p < 0.001) ( figure 3 ) and rpcp flow index (p < 0.001) (figure 4) within the post-covid-19 group. contrarily, sct showed no significant linear correlation with rpcp parameters ( covid-19 caused by sars-cov-2 evolved into a severe pandemic moving the whole of humanity into jeopardy. while the main manifestation has been observed in the respiratory tract, multi-systemic organ involvement has been observed. according to our results, post-covid-19 patients have a lower rpcp pd and a normal rpcp fi compared to the general population. these covid-19 caused by sars-cov-2 evolved into a severe pandemic moving the whole of humanity into jeopardy. while the main manifestation has been observed in the respiratory tract, multi-systemic organ involvement has been observed. according to our results, post-covid-19 patients have a lower rpcp pd and a normal rpcp fi compared to the general population. these findings suggest an impairment in the blood supply to the peripapillary rnfl in patients who recovered from sars-cov-2 infection. it is, to our knowledge, the first published study to detect this potential threat. moreover, rpcp microvascular impairment is more evident in older patients (rpcp pd and rpcp fi are both inversely correlated with age in post-covid-19 group) and patients affected by systemic arterial hypertension (lower rpcp fi compared to the general population). in addition, patients treated with antiplatelet therapy or lopinavir + ritonavir during admission are more susceptible to rpcp impairment after sars-cov-2 infection. in addition, both rpcp pd and rpcp fi are linearly correlated to average rnfl thickness in early post sars-cov-2 patients. none of the structural oct parameters proved to be significantly different between the study groups. recent research has focused on sars-cov-2 s ability to damage the vascular endothelium causing irreversible ischemic damage to multiple organs; this microcirculatory impairment leading to functional disorders in all inner organs is believed to be the ultimate cause for the high mortality and morbidity rate [29] . indeed, macro-and microvascular thrombotic processes in severe sars-cov-2 infection cases cause a high burden of complications [30, 31] . several elements contribute to endothelial disruption during sars-cov-2 infection, such as complement activation, hypoxia, platelets, and thyroxin kinases [32] . endothelial dysfunction together with a generalized inflammatory state and complement elements may contribute to the overall pro-coagulative state described in covid-19 patients, leading to occlusions in veins and arteries [33] . due to this phenomenon, covid-19 has been shown to cause rare clinical events such as atypical thromboses (renal veins, uterine veins, and mesenteric vessels) and myocardial micro-thrombotic vessels. endothelial derangement and increased permeability are also reported to be early hallmarks of organ damage in patients with covid-19 [32] . in this panorama, our study investigated the involvement of the retinal capillary microcirculation focusing on the radial peripapillary capillary plexus, which is considered to be crucial for the homeostasis and function of the retinal ganglion cells and their axons. rpcp density is highly correlated to rnfl thickness and visual field index in glaucoma patients [33, 34] . moreover, a reduction in rpcp density has been demonstrated to be an early sign of glaucoma [35, 36] . rpcp density and flow index reduction are also correlated to visual acuity and visual field loss in non-arteritic ischemic optic neuropathy [37, 38] . on the contrary, a rpcp flow and density impairment can be the consequence of retinal neural remodeling secondary to optic nerve axonal degeneration [39] . our study examined this aspect outlining the correlation of the rpcp perfusion density and rpcp flow index with the rnfl average thickness also in early post-covid-19 patients. in our opinion, the conflicting results deriving from the comparison of post-covid-19 patients with the healthy controls on the field of rpcp integrity could be attributable to the characteristics of the examined groups. first, our shortage of healthy control patients led to an asymmetry in the sample sizes of the two groups. furthermore, our patients recruited in the post-covid-19 group manifested a mild to moderate variant of the infection: only 6.25% of the subjects required admission to the icu department and the cases of pulmonary and venous thromboembolism were only 4 in 80 patients. in this perspective, our results are in agreement with those of mazzaccaro et al. [40] , as we analyzed a cohort of covid-19 patients with mild disease progression. moreover, within the post-covid-19 group, both rpcp-fi and rpcp-pd are linearly inversely correlated with age. in addition, patients affected by systemic arterial hypertension showed a statistically lower rpcp-fi compared to other patients in the post-covid-19 group. in this regard, it is interesting to notice that patients in the post-covid-19 group showed a lower mean age, a lower prevalence of diabetes and systemic arterial hypertension, and a higher prevalence of females (typically affected by milder manifestations of the disease) compared to the reported sars-cov-2 epidemiologic data [38] . these data additionally confirm our hypothesis of an altered group composition being responsible for a mild significance of the result. curiously, our results show that patients treated with antiplatelet therapy during hospitalization were characterized by lower rpcp fi and lower rpcp pd. the role of platelets in inducing or amplifying the endothelial damage in covid-19 patients is still a matter of discussion. a low platelet count, possibly due to destruction, bone marrow infection, or autoimmune phenomena, was reported to cause a five-fold mortality rate increase in covid-19 patients and the rates reported were very heterogeneous among the analyzed studies [41, 42] . however, the opposite is more common in covid-19 patients: usually the platelet count is higher than in patients with sepsis or ards. increased serum levels of thrombopoietin caused by pulmonary inflammation have been supposed to explain this phenomenon [43, 44] . we hypothesize that this finding in our study could be due to the administration of adjunctive drugs in patients with more severe clinical condition, causing more systemic microvascular damage. this occurrence could possibly explain another unexpected finding of our study: the use of lopinavir + ritonavir during recovery was associated with lower rpcp-fi and rpcp-pd. another possible explanation can be related to antiviral drug that may induce endothelial damage. endothelial damage, secondary to medications, is reported in the literature for several substances: the damage caused by ponatinib, for example, is mediated by notch1 hyperactivation, but also propranolol and sirolimus inhibit endothelial proliferation [45] . similarly, carteolol induces apoptosis in corneal endothelial cells by caspase-and mitochondria-dependent pathways [46] . ace2, a sars-cov-2 target, inhibits proliferation of endothelial cells; however, it also reduces endothelial inflammation [47] . finally, steroids induce apoptosis in bone endothelial cells causing osteonecrosis, but this effect has not been proven in retinal capillary cells [48] . nowadays, no report of lopinavir + ritonavir induced retinal endothelial damage has been described. in conclusion, it is important to highlight how differential analysis of risk factors for microvascular peripapillary involvement in post-covid-19 infection represents a valuable tool for personalized medicine. despite being to our knowledge the first study to address a potential rpcp impairment in patients who recovered from sars-cov-2 infection, the results are undermined by some limitations. first, the selected sample of post-covid-19 patients is not fully representative of the average post-covid-19 population of patients. moreover, a larger cohort of healthy controls would be needed to increase the power of the study. future studies will be needed to address the question of a potential difference in rpcp perfusion between healthy subjects and individuals who recovered from sars-cov-2 infection. likewise, another future prospective will be to investigate whether peripapillary vascular damage can be a reversible occurrence in these patients. oct angiography provided several information of rpcp circulation. rpcp-pd was lower in post sars-cov-2 patients compared to controls. patients treated with lopinavir + ritonavir or antiplatelet therapy during admission had lower rpcp-fi and rpcp-pd. within the post-covid-19 group, patients with systemic arterial hypertension had lower rpcp-fi and age was inversely correlated to both rpcp-fi and rpcp-pd. rnfl average thickness was correlated to rpcp-fi and rpcp-pd within post-covid-19 group. future studies will be needed to confirm our hypothesis of a microvascular retinal impairment in individuals who early recovered from sars-cov-2 infection. emerging coronaviruses: genome structure, replication, and pathogenesis hosts and sources of endemic human coronaviruses summary table of sars cases by country n-a; world health organisation situation report 32; world health organisation emergency committee regarding the outbreak of novel coronavirus (2019-ncov); world health organisation director-general's opening remarks at the media briefing on covid-19; world health organisation covid-19 and chronic diseases: current knowledge, future steps and the 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apoptosis in vitro key: cord-332180-dw4h69tp authors: cheng, fu-yuan; joshi, himanshu; tandon, pranai; freeman, robert; reich, david l; mazumdar, madhu; kohli-seth, roopa; levin, matthew a.; timsina, prem; kia, arash title: using machine learning to predict icu transfer in hospitalized covid-19 patients date: 2020-06-01 journal: j clin med doi: 10.3390/jcm9061668 sha: doc_id: 332180 cord_uid: dw4h69tp objectives: approximately 20–30% of patients with covid-19 require hospitalization, and 5–12% may require critical care in an intensive care unit (icu). a rapid surge in cases of severe covid-19 will lead to a corresponding surge in demand for icu care. because of constraints on resources, frontline healthcare workers may be unable to provide the frequent monitoring and assessment required for all patients at high risk of clinical deterioration. we developed a machine learning-based risk prioritization tool that predicts icu transfer within 24 h, seeking to facilitate efficient use of care providers’ efforts and help hospitals plan their flow of operations. methods: a retrospective cohort was comprised of non-icu covid-19 admissions at a large acute care health system between 26 february and 18 april 2020. time series data, including vital signs, nursing assessments, laboratory data, and electrocardiograms, were used as input variables for training a random forest (rf) model. the cohort was randomly split (70:30) into training and test sets. the rf model was trained using 10-fold cross-validation on the training set, and its predictive performance on the test set was then evaluated. results: the cohort consisted of 1987 unique patients diagnosed with covid-19 and admitted to non-icu units of the hospital. the median time to icu transfer was 2.45 days from the time of admission. compared to actual admissions, the tool had 72.8% (95% ci: 63.2–81.1%) sensitivity, 76.3% (95% ci: 74.7–77.9%) specificity, 76.2% (95% ci: 74.6–77.7%) accuracy, and 79.9% (95% ci: 75.2–84.6%) area under the receiver operating characteristics curve. conclusions: a ml-based prediction model can be used as a screening tool to identify patients at risk of imminent icu transfer within 24 h. this tool could improve the management of hospital resources and patient-throughput planning, thus delivering more effective care to patients hospitalized with covid-19. with more than 3 million cases and 200,000 deaths [1] by the end of april 2020, the covid-19 pandemic has rapidly emerged as a serious global health emergency [2] , testing the ability of health care systems to respond. the burden on health care systems emanates both from the high incidence of covid-19 and the fact that 20% to 30% of patients experience a moderate-to-severe form of the disease-with multi-organ failure, prolonged periods of morbidity and hospitalization, and high mortality [3] . moreover, from 5% to 12% of all patients diagnosed with covid-19 and up to 33% of hospitalized patients require supportive critical care in an intensive care unit (icu) [3] [4] [5] . these estimates indicate that the rate of icu transfer of hospitalized patients with covid-19 is significantly higher than the icu transfer rates of 11% reported for other hospitalized patients [6, 7] . furthermore, the need for icu care may be even higher in specific high-risk groups with covid-19, such as older individuals [3] or those with pre-existing comorbidities [8] . for example, over 75% of covid-19 patients admitted to the icu have one or more pre-existing comorbid conditions [9] . according to an estimate by the american hospital association, there are just under 100,000 icu beds in the united states [10] , with over 67% occupancy under normal circumstances [11] -a potential constraint on resources during a surge in cases. moreover, constraints in the availability of trained manpower [12] may occur with a rapid surge in covid-19 hospitalizations. covid-19 patients admitted to non-icu units often experience rapid clinical deterioration [13] and, therefore, require frequent clinical assessments. however, with resources stretched thin, frequent assessment is difficult and can increase the risk of exposure among frontline personnel. to efficiently manage these finite resources and personnel, optimal prioritization of patients and efficient use of hospital resources are necessary. icu care may be needed for supportive management of severe covid-19-associated pneumonia, acute respiratory distress (ards), sepsis, cardiomyopathy, arrhythmia, and acute renal failure. icu care also may become necessary to manage prolonged hospitalization-associated complications, such as coagulopathy [14] , secondary infections, gastrointestinal bleeding, and other problems [13] . determining whether an individual's dynamic risk of clinical deterioration warrants an icu transfer may require analyses of temporal changes in patients' conditions and key indicators of imminent complications of covid-19. supervised machine learning approaches may be useful to (a) analyze and interpret patients' clinical and laboratory values and their temporal changes, and (b) quantify their dynamic risk of clinical deterioration and the need for icu transfer. the primary aim of this study is to develop a novel supervised machine learning classifier for predicting the risk of icu transfer within the next 24 h for covid-19 patients using hospital emr data. we applied a random forest (rf) [15] approach, which has proven promising in analyzing complex clinical data of multiple types [15] , has high model generalizability [15] , and can elucidate high-order interactions between variables without compromising predictive accuracy [16] . we describe the development and validation of such a model, its predictive performance, and the interpretation of our results. this study was approved by the mount sinai health system institutional research board (irb protocol number: 18-00581); the need for informed consent was waived. the study cohort was comprised of patients 18 years or older who had a covid-19 diagnosis and were admitted to the mount sinai hospital in non-icu general in-patient beds between 26 february and 18 april 2020. the diagnosis was based on a clinical conclusion of an infectious disease specialist or a positive pcr test (initial or repeat testing). the following data were retrospectively collected from the mount sinai health system covid-19 registry, sourced from an epic ehr system: demographic information, time-series of the admission-discharge-transfer events, structured and semi-structured clinical assessments, vital signs from nursing flowsheets, and laboratory and electrocardiogram (ecg) results. given the crisis nature of the pandemic, clinicians caring for this cohort collected data such as vital signs, diagnostic labs, ecgs, and nursing assessments based on clinical judgment and resource availability rather than a standard protocol. thus, to create time-series data for each observational variable, we included the three most recent assessments available when the feature vector was created. feature vectors were created daily during each covid-19 patient's non-icu general bed stay until discharge, icu transfer, or death. missing values for each variable were imputed by using the median value across the cohort [17] . the primary outcome of this study was icu transfer within 24 h from the time of prediction. labeling of feature vectors followed the following logic: (1) if the icu transfer was within 24 h of the feature vector creation, we labeled the feature vector as positive; (2) if the icu transfer occurred after 24 h from the creation of the feature vector, we labeled the feature vector as negative; (3) if the icu transfer did not occur during the patients' stay, then all feature vectors for that admission were labeled as negative. this process is depicted in figure 1 . the study cohort data were randomly split into a training set used for training the prediction model, and a test set used for testing the model's performance. the training set consisted of 70 percent of the full cohort, and the test cohort consisted of the remaining 30 percent. we randomly split our cohort so that patients were only included in the training or the test set. the non-icu bed to icu transfer rate in our cohort was 3.7 percent, which created an extreme class imbalance between the majority class (feature vectors without the occurrence of icu transfer within 24 h) and the minority class (feature vectors with icu transfer within 24 h). we performed random under-sampling [18, 19] on the training data set for balancing the majority class (negative label) until both classes were equally balanced. the rf model was trained with 10-fold cross-validation. the open-source apache spark project machine-learning library [20] was used. the features included in this study were based on clinical judgments and reports in the covid-19 literature. we included periodic monitoring of vital signs [21] , complete blood count, serum biochemical tests [22] , coagulation profile [14] , and electrocardiogram results [23] as relevant input variables. the full list of features used in modeling is provided in table s1 . features were ranked by using the gini importance [20] . the model performance was evaluated on the test set. rf model-derived class probabilities [20] were used to predict icu transfer within 24 h with a default threshold of ≥0.5. predictions less than the default threshold were categorized as negative. sensitivity, specificity, accuracy, and area under the receiver operating curve (auc-roc), along with 95% ci, were estimated for evaluating the screening tool's performance [24] . performance metrics were computed in the r environment [25] by using custom scripts and r packages-prroc (v.1.3.1) [26] , proc (v. 1.15) [27] , and epir (v. 1.0.4) [28] . cohort characteristics are shown in table 1 . the study cohort yielded 9639 feature vectors, which contained data from each day of non-icu hospital stay for 1987 unique patients. each individual vector, generated 24 h apart, represented a day of in-patient stay in a non-icu bed for each patient. the split cohort resulted in 5548 and 2386 feature vectors created from the stays of 1168 and 521 patients in the training and test datasets, respectively. after performing majority-class under-sampling, the final training set consisted of 2008 feature vectors, representing each non-icu stay of 401 unique patients. the median time to icu transfer from the time of admission was 2.45 days. the study cohort included a higher proportion of women, and about two-thirds of the cohort was between 18 and 65 years old. the median duration of hospital stay was 4.2 days and ranged between 1 to 43.6 days. about one-quarter of the patients in the cohort had more than one comorbidity, including copd, diabetes, hypertension, obesity, or cancer. a total of 31 variables (comprising 99 features) had predictive value using the gini importance metric in training the rf model. hyper-parameters used in the final model are provided in table s1 . the top 20 predictive variables are summarized in figure 2 . model input variables with their respective sources are listed in table s2 . our model identified a series of features related to progressive respiratory failure (respiratory rate, oxygen saturation), markers of systemic inflammation (c-reactive protein, white blood cell count), shock (systolic and diastolic blood pressures), renal failure (blood urea nitrogen, anion gap, and serum creatinine), and the pathophysiology of covid-19 (lymphocyte count). respiratory rate (the earliest recorded value of the latest three assessments) had the highest predictive value in the rf model, and white blood cell count was the second highest. variables included in the final model reflected the importance of temporal changes in vital signs, markers of acid-base equilibrium and systemic inflammation, and predictors of myocardial injury and renal function. the predictive performance of the rf-based model on the test dataset is presented in table 2 . of 2386 feature vectors, 89 represented patient-days where icu transfer occurred within 24 h of the prediction time point. the auc-roc of the prediction model is shown in figure 3 . our model provides a tool for dynamic risk quantification for icu transfer within the next 24 h. clinical management of covid-19 requires frequent monitoring and re-assessment among patients who may suffer rapid deterioration. although deterioration may be evident by corroboration of changes in vital signs, laboratory results, electrocardiograms, and information in nursing notes, frequent review of these important parameters might not be feasible in crisis situations. using machine learning, we developed a model for identifying deteriorating patients in need of icu transfer by using data routinely collected during inpatient care. this model could be easily automated as an alternative to manual clinical review. furthermore, inspection of important features in the model can provide insight into predictors and their plausible links to the pathophysiology of clinical deterioration among patients with covid-19. a key advantage of using an rf-based model is that the relative importance of predictive features is available for end users to interpret. our finding that lymphocyte count is a significant predictor of icu transfer correlates with previous reports that identified lymphopenia as a predictor of severe disease and poor prognosis [29, 30] . although age is clearly identified as a risk factor for needing icu care among patients with covid-19 [3] , patients above 65 years old have lower rates of icu transfer, despite higher mortality [5] , possibly reflecting a greater preference for palliative or less aggressive care in older patients. we believe that the relatively low rank of age as a risk factor in our model could mean that our model incorporates actual patient data and patterns of clinical practice into its predictions. acute worsening of respiratory rate and oxygen saturation are used for identifying covid-19 patients at risk of developing acute respiratory distress syndrome [31, 32] . the model ranks oxygen saturation with a significantly lower predictive value than respiratory rate. a significant proportion of covid-19 patients who are hospitalized need supplemental oxygen support. one possible our model provides a tool for dynamic risk quantification for icu transfer within the next 24 h. clinical management of covid-19 requires frequent monitoring and re-assessment among patients who may suffer rapid deterioration. although deterioration may be evident by corroboration of changes in vital signs, laboratory results, electrocardiograms, and information in nursing notes, frequent review of these important parameters might not be feasible in crisis situations. using machine learning, we developed a model for identifying deteriorating patients in need of icu transfer by using data routinely collected during inpatient care. this model could be easily automated as an alternative to manual clinical review. furthermore, inspection of important features in the model can provide insight into predictors and their plausible links to the pathophysiology of clinical deterioration among patients with covid-19. a key advantage of using an rf-based model is that the relative importance of predictive features is available for end users to interpret. our finding that lymphocyte count is a significant predictor of icu transfer correlates with previous reports that identified lymphopenia as a predictor of severe disease and poor prognosis [29, 30] . although age is clearly identified as a risk factor for needing icu care among patients with covid-19 [3] , patients above 65 years old have lower rates of icu transfer, despite higher mortality [5] , possibly reflecting a greater preference for palliative or less aggressive care in older patients. we believe that the relatively low rank of age as a risk factor in our model could mean that our model incorporates actual patient data and patterns of clinical practice into its predictions. acute worsening of respiratory rate and oxygen saturation are used for identifying covid-19 patients at risk of developing acute respiratory distress syndrome [31, 32] . the model ranks oxygen saturation with a significantly lower predictive value than respiratory rate. a significant proportion of covid-19 patients who are hospitalized need supplemental oxygen support. one possible explanation underlying the lower predictive value of oxygen saturation is that in patients with progressive hypoxia, a progressively greater fraction of inhaled oxygen (fio 2 ) is delivered to maintain adequate percutaneous oxygen saturation (spo 2 ) until the patient can no longer maintain normal oxygen saturation despite support from high-flow nasal oxygen or non-invasive ventilation. this makes spo 2 a less sensitive reflection of disease progression until severe respiratory decompensation occurs. we propose to include fio 2 , level of respiratory support, and spo 2 as variables in future versions of this model. c-reactive protein has been reported as a marker of disease severity in early phases of covid-19 infection and is positively correlated with covid-19 pneumonia [33] . patients' vital signs (e.g., pulse rate, blood pressure, and temperature) are among the top 20 predictors in this model and are widely accepted as identifying patients in critical condition who are at risk of deterioration [34] . hematologic parameters such as red blood cell count, hemoglobin, platelet count, and white blood cell count are conventionally used markers of sepsis in critical care settings [35] ; thus, it is not surprising that they were predictive of covid-19 in our model also. abnormalities in potassium, sodium, and calcium also have been associated with severe covid-19 [36] . our model has strengths in terms of methodology, utility, and scalability. the labeling approach of feature vectors-using the last 3 observations, rather than the earliest or latest-made it easier to minimize chances of over-fitting despite the low sample size for training. the cohort is diverse in distribution of key variables such as age, race, ethnicity, and length of hospitalization, supporting the generalizability of the model. the model uses input variables mainly comprised of routine laboratory and clinical data, which are commonly available in most streaming data models across the u.s. furthermore, the model can be adopted to different frequencies of assessments and different common input variables. it can be adjusted to use streaming data from the emr and provide frequent predictions for real-time risk prioritization. we use the fast healthcare interoperability resources (fhir) format for facilitating data exchange and retrieval from an epic-supported emr system. this can help to improve the model's scalability in other hospital settings. clinical judgment and resources can play a significant role in data availability. in addition, clinical documentation may not be perfect during crises, when normal documentation standards are relaxed due to the high work burden of clinicians. therefore, unavailable data (as in our case) may be the consequence of either clinical judgement on need for specific assessments or imperfect clinical documentation. despite the non-random pattern of data availability for specific variables, the imputation strategy and the rf model had reasonably high sensitivity. this supports previous reports that found rf models to be highly suitable in situations with missing data [17] , complex non-linear relationships among input variables, and their potential higher-level interactions [16] ; thus, an ensemble-based classification approach minimizes overfitting [15] . an additional asset of this model is that, unlike other models, key discriminatory variables underlying each prediction can be provided. low sample size and class imbalance resulting from low icu transfer rates are major limitations to this version of the model, which resulted in low precision. therefore, we recommend using this version of the model as a prioritization tool, not a tool for clinical decision support. since the model is based on data from a single hospital, its case mix may not be easily generalizable to other settings. for example, in this cohort, rates of hypertension and diabetes were lower than in others reported [4, 5, 37] . variables related to systemic inflammation and the coagulation cascade (e.g., d-dimer, fibrinogen, ferritin, and lactate dehydrogenase) were not available for modeling when this model was generated. while our model provides high sensitivity, we believe that inclusion of these other markers, which have predictive and/or prognostic value [38] , could improve subsequent iterations of the model. while spo 2 without assessments of fio 2 and level of oxygen support may not be sufficient to capture signs of progressive hypoxia, the inclusion of all three variables in subsequent versions of the model could also further improve its performance. however, given the low sample size of a single medical center in the acute phase of a pandemic, it may be difficult to generate a model with both high sensitivity and precision (positive predictive value). as a screening tool for development of critical illness, this model has multiple opportunities for clinical use. in addition to identifying patients with a potentially increased need for icu transfer within 24 h, the tool can also be used for improving the coordination of patient transfers to the icu. the tool can be used to inform clinicians of patients at higher risk of a greater need for frequent assessments, and thereby can facilitate inclusion of clinicians less familiar with critical care medicine. earlier identification of high-risk patients could potentially reduce the use of invasive mechanical ventilation [39] , sparing patients from avoidable morbidity and lowering mortality from complications. given the sensitivity of the model, it can effectively identify patients who are likely to be transferred to icu within 24 h, reducing the chance of missing the patients in need of icu care. moreover, clinical implementation of the tool can increase the rates of early icu transfers, which can potentially translate into reduced mortality and shorter lengths of icu stay [40, 41] , with favorable consequences on other complications affecting patient outcomes, such as delirium and sleep disorders [42, 43] . however, its positive predictive value and precision are limited, and it is not practical to perform labor-intensive interventions for all patients whom the model predicted are at high risk. nonetheless, our model has clinical utility in the setting of a pandemic. the high negative predictive value suggests that those identified as unlikely to require critical care in the next 24 h may be considered for a lower level of monitoring. our rf-based tool can reliably be used for prioritizing covid-19 patients not in the icu but at risk for deterioration and requiring icu transfer within 24 h. the model shows the importance of respiratory failure, shock, inflammation, and renal failure in the progression of covid-19. such a predictive tool may have wide implications and utility in clinical practice and hospital operations. further refinement of the model will yield even higher precision while maintaining sensitivity. more studies are needed to identify other ways to improve patient outcomes by early identification of covid-19 patients at risk of deterioration. implementing machine learning models has the potential to build capacity within a hospital's continuous learning and quality improvement environment. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/6/1668/s1, table s1 : hyperparameters used in the final model, table s2 world health organization. who covid-19 dashboard covid-19-navigating the uncharted cdc covid-19 response team. severe outcomes among patients with coronavirus disease 2019 (covid-19)-united states characteristics of hospitalized adults with covid-19 in an integrated health care system in california presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the active bed management by hospitalists and emergency department throughput evaluating implementation of a rapid response team: considering alternative outcome measures estimates of the severity of coronavirus disease 2019: a model-based analysis preliminary estimates of the prevalence of selected underlying health conditions among patients with coronavirus disease 2019-united states aha annual survey trends in critical care beds and use among population groups and medicare and medicaid beneficiaries in the united states united states resource availability for covid-19 interim clinical guidance for management of patients with confirmed coronavirus disease (covid-19) abnormal coagulation parameters are associated with poor prognosis in patients with novel coronavirus pneumonia iterative random forests to discover predictive and stable high-order interactions an analysis of four missing data treatment methods for supervised learning the class imbalance problem: significance and strategies enhancing the prediction of clinical deterioration in admitted patients through a machine learning model mllib: main guide-spark 2.3.0 documentation national institute for the infectious diseases "l. spallanzani" irccs. recommendations for covid-19 clinical management clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study cardiovascular implications of fatal outcomes of patients with coronavirus disease screening tests: a review with examples r: a language and environment for statistical computing computing and visualizing precision-recall and receiver operating characteristic curves in r proc: an open-source package for r and s+ to analyze and compare roc curves epir: an r package for the analysis of epidemiological data evaluation and treatment coronavirus dysregulation of immune response in patients with coronavirus covid-19 in critically ill patients in the seattle region-case series treatment for severe acute respiratory distress syndrome from covid-19 c-reactive protein levels in the early stage of covid-19 monitoring vital signs using early warning scoring systems: a review of the literature the hematologic system as a marker of organ dysfunction in sepsis electrolyte imbalances in patients with severe coronavirus disease 2019 (covid-19) laboratory abnormalities in patients with covid-2019 infection lower mortality of covid-19 by early recognition and intervention: experience from jiangsu province association between intensive care unit transfer delay and hospital mortality: a multicenter investigation an examination of early transfers to the icu based on a physiologic risk score sleep deprivation in intensive care unit-systematic review covid-19: icu delirium management during sars-cov-2 pandemic we acknowledge susan usyal for her editorial assistance. the authors declare no conflict of interest. key: cord-273351-vq3budip authors: farré, núria; mojón, diana; llagostera, marc; belarte-tornero, laia c.; calvo-fernández, alicia; vallés, ermengol; negrete, alejandro; garcía-guimaraes, marcos; bartolomé, yolanda; fernández, camino; garcía-duran, ana b.; marrugat, jaume; vaquerizo, beatriz title: prolonged qt interval in sars-cov-2 infection: prevalence and prognosis date: 2020-08-21 journal: j clin med doi: 10.3390/jcm9092712 sha: doc_id: 273351 cord_uid: vq3budip background: the prognostic value of a prolonged qt interval in sars-cov2 infection is not well known. objective: to determine whether the presence of a prolonged qt on admission is an independent factor for mortality in sars-cov2 hospitalized patients. methods: single-center cohort of 623 consecutive patients with positive polymerase-chain-reaction test (pcr) to sars cov2, recruited from 27 february to 7 april 2020. an electrocardiogram was taken on these patients within the first 48 h after diagnosis and before the administration of any medication with a known effect on qt interval. a prolonged qt interval was defined as a corrected qt (qtc) interval >480 milliseconds. patients were followed up with until 10 may 2020. results: sixty-one patients (9.8%) had prolonged qtc and only 3.2% had a baseline qtc > 500 milliseconds. patients with prolonged qtc were older, had more comorbidities, and higher levels of immune-inflammatory markers. there were no episodes of ventricular tachycardia or ventricular fibrillation during hospitalization. all-cause death was higher in patients with prolonged qtc (41.0% vs. 8.7%, p < 0.001, multivariable hr 2.68 (1.58–4.55), p < 0.001). conclusions: almost 10% of patients with covid-19 infection have a prolonged qtc interval on admission. a prolonged qtc was independently associated with a higher mortality even after adjustment for age, comorbidities, and treatment with hydroxychloroquine and azithromycin. an electrocardiogram should be included on admission to identify high-risk sars-cov-2 patients. previous reports have highlighted the potential risk of cardiac complications and arrhythmias in patients with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection [1] . the presence of a prolonged qt interval can further worsen prognosis. however, most of the information about the prognostic role of qt interval in sars-cov-2 infection has been derived from studies analyzing the effects of the treatment with hydroxychloroquine and azithromycin [2] [3] [4] [5] [6] , a treatment associated with qt interval prolongation. the benefits of these treatments on prognosis are currently controversial. baseline qt interval abnormalities in the setting of sars-cov-2 infection can be secondary to the viral infection per se, the inflammatory state associated with sars-cov-2 infection, and ischemia or hypoxia [1] . indeed, several viral infections like human immunodeficiency virus (hiv) and dengue have been independently associated with a prolonged qt interval [7] [8] [9] . interestingly, acute coronavirus infection has been associated with a prolonged qt interval in rabbits [10] , which suggests that the virus might have a direct effect on the heart. on the other hand, in the absence of infection, systemic inflammation and elevated c-reactive protein (crp) have also been associated with qt prolongation [11] [12] [13] [14] [15] [16] . these associations seem to be mediated, at least in part, by elevated interleukin-6 (il-6) levels. treatment with tocilizumab, an anti-il-6 receptor antibody, has been associated with qt interval shortening [12, 13] . intriguingly, in men with hiv infections, those with elevated il-6 had more prolonged qt [17] , suggesting a potential additive effect of infection and inflammation on the qt interval. thus, the presence of a prolonged qt interval on admission might be a marker of worse prognosis irrespective of the treatment the patients receive. therefore, the aim of this study was to test the hypothesis that the presence of prolonged qt on admission is an independent factor for mortality in patients with sars-cov-2 infection. a single-center cohort study conducted at hospital del mar, barcelona, spain, from 27 february to 7 april 2020. patients were followed up until 10 may 2020. all consecutive patients with laboratory-confirmed covid-19 by means of polymerase-chain-reaction (pcr) test were included in the study. we collected demographic data, laboratory findings, comorbidities, and treatment received. baseline electrocardiogram (ecg) was defined as the ecg taken within the first 48 h after laboratory-confirmed covid-19 diagnosis and always before the administration of any medication with a known effect on the qt interval. qt was automatically calculated as the time from the start of the q wave to the end of the t wave and corrected for heart rate by the bazett formula (qtc). all ecgs were done with the philips pagewriter tc30 cardiograph (koninklijke philips, eindhoven, the netherlands). prolonged qtc was defined as a qtc > 480 milliseconds (ms) [18] . although ecg was recommended in all patients, and especially in those who would receive medication that potentially modifies the qt interval, the decision to order the ecg was left to clinicians and adapted to the logistic capabilities of the center during the pandemic. therefore, in the current analysis, we focused on patients who had a baseline ecg ( figure 1 ). however, patients who had a baseline ecg were also compared to those who did not have a baseline ecg. when patients had more than one ecg during hospitalization, maximum qtc interval was also collected. qtc prolongation was defined as an increase of at least one millisecond in qtc compared to baseline qtc. according to the protocol at our center at the time of the study, treatment with hydroxychloroquine and azithromycin was recommended to all patients. azithromycin was given once a day (500 mg) for three days and hydroxychloroquine was given five days at a dose of 400 mg twice a day the first day and 200 mg twice a day the following four days. this treatment was contraindicated when qtc was longer than 550 ms. if qtc was longer than 500 ms, a daily ecg was mandatory. the use of tocilizumab was decided based on the presence of pulmonary infiltrates on chest x-ray or worsening of previous infiltrates, pao2/fio2 <300, and at least one of these parameters: il6 ≥ 40 ng/l (or pcr ≥ 100 mg/l), d dimer ≥ 1000 ng/ml, or ferritin ≥ 700 ng/ml. the primary endpoint was all-cause death at 30 days after covid-19 diagnosis. this study was performed in accordance with the provisions of the declaration of helsinki, iso 14155 and clinical practice guidelines. the study protocol was approved by the institutional ethics committee and the hospital's research commission (number ceim 2020/9178). oral informed consent was obtained, but the need for written informed consent was waived in light of the infectious disease hazard. according to the protocol at our center at the time of the study, treatment with hydroxychloroquine and azithromycin was recommended to all patients. azithromycin was given once a day (500 mg) for three days and hydroxychloroquine was given five days at a dose of 400 mg twice a day the first day and 200 mg twice a day the following four days. this treatment was contraindicated when qtc was longer than 550 ms. if qtc was longer than 500 ms, a daily ecg was mandatory. the use of tocilizumab was decided based on the presence of pulmonary infiltrates on chest x-ray or worsening of previous infiltrates, pao2/fio2 <300, and at least one of these parameters: il6 ≥ 40 ng/l (or pcr ≥ 100 mg/l), d dimer ≥ 1000 ng/ml, or ferritin ≥ 700 ng/ml. the primary endpoint was all-cause death at 30 days after covid-19 diagnosis. this study was performed in accordance with the provisions of the declaration of helsinki, iso 14155 and clinical practice guidelines. the study protocol was approved by the institutional ethics committee and the hospital's research commission (number ceim 2020/9178). oral informed consent was obtained, but the need for written informed consent was waived in light of the infectious disease hazard. categorical variables were summarized as number and percentages, and continuous variables were summarized as the mean and standard deviation (sd), or the median and interquartile range (iqr), depending on the variable distribution. patients' characteristics were compared between prolonged qtc (cut-off point > 480 ms) and outcome status categories (death) by student's t-test or mann-whitney u test for continuous variables, and by pearson's chi-squared test for categorical variables. kaplan-meier survival estimates were used to calculate the 30-day observed cumulative incidence of death, and statistical significance was tested by the log-rank test. the adjusted hazard ratio (hr) of death for qtc status was analyzed using cox proportional hazard models. the models were adjusted for potential confounders selected by stepwise forward inclusion, among patient characteristics that were significantly associated with a prolonged qtc status as well as with the composite endpoint (death). because the number of end-points was low, it was not possible to include all variables with p < 0.05. we chose the variables with p-value < 0.001 and prevalence >5%, therefore moderate to severe valve heart disease was not included in the model (overall prevalence categorical variables were summarized as number and percentages, and continuous variables were summarized as the mean and standard deviation (sd), or the median and interquartile range (iqr), depending on the variable distribution. patients' characteristics were compared between prolonged qtc (cut-off point > 480 ms) and outcome status categories (death) by student's t-test or mann-whitney u test for continuous variables, and by pearson's chi-squared test for categorical variables. kaplan-meier survival estimates were used to calculate the 30-day observed cumulative incidence of death, and statistical significance was tested by the log-rank test. the adjusted hazard ratio (hr) of death for qtc status was analyzed using cox proportional hazard models. the models were adjusted for potential confounders selected by stepwise forward inclusion, among patient characteristics that were significantly associated with a prolonged qtc status as well as with the composite endpoint (death). because the number of end-points was low, it was not possible to include all variables with p < 0.05. we chose the variables with p-value < 0.001 and prevalence >5%, therefore moderate to severe valve heart disease was not included in the model (overall prevalence 3.7%). the variables included in the model were age, baseline qtc > 480 ms, chronic kidney disease, treatment with azithromycin and hydroxychloroquine, ischemic chronic disease, atrial fibrillation or flutter, heart failure, and the presence of any cardiovascular risk factor. we acknowledge that there might be a survival bias associated with treatment (or an immortal time bias) wherein you must survive long enough to be treated. however, since the treatment with hcq and azm are known to prolong qt and might predispose to ventricular arrhythmias, we thought that the inclusion of treatment in the model was warranted. however, in order to minimize the bias, we created a model with the same variables except did not include the treatment received. second, we also did a sensitivity analysis excluding patients who died during the first 48 h of admission. finally, standardized differences were calculated, and a difference >0.10 was considered clinically significant. in addition, p-values < 0.05 were considered statistically significant. all tests were performed with spss version 25 (ibm spss versión 25, armonk, ny, usa). sixty-one patients (9.8%) had prolonged qtc on admission. only 20 patients (3.2%) had a baseline qtc > 500 ms. baseline characteristics are described in table 1 . briefly, patients with prolonged qtc were older and had more comorbidities. moreover, they had higher levels of c-reactive protein, leucocytes, lactate, and procalcitonin. similar results were seen in patients who died (table 2) . only 245 patients (39% of the cohort) had a follow-up ecg during hospitalization. of those, 77 patients (31.4%) had the longest qtc interval on admission, whilst 68.6% had qtc prolongation during hospitalization. baseline characteristics, treatment, and prognosis of patients who had qtc prolongation on follow-up ecg during hospitalization are described in table 3 . interestingly, both baseline qtc duration (441.75 ± 38.5 ms vs. 435.38 ± 31.6 ms, p = 0.17) and the percentage of patients with baseline qtc > 480 ms (10.4% vs. 11.9%, p = 0.73) were similar in those who prolonged qtc during hospitalization compared with those who did not. as expected, patients with qtc interval prolongation during hospitalization had higher prescription of hydroxychloroquine and azithromycin. in-hospital treatment and prognosis are shown in tables 4 and 5 . there were no episodes of ventricular tachycardia or ventricular fibrillation during hospitalization. when analyzed by sex, the presence of qtc ≥ 480 ms was associated with a higher mortality in both sexes. in women, mortality was 56.7% (17/30) in those with qtc ≥ 480 ms compared with 8.4% (20/237) in the non-prolonged qtc interval, p < 0.001. similar results were seen in men: mortality was 25.8% (8/31) in the prolonged qtc interval group vs. 8.9% (29/325) in the non-prolonged qtc interval group, p = 0.003. this cut-off was independently associated with death in women (univariable hr 8.53 (95% ci: 4.45-16.36), p < 0.001, multivariable hr 4.04 (1.98-8.27), p < 0.001), whereas there was a strong tendency in the same direction in men (univariate hr 2.27 (95% ci: 0.99-5.23), p = 0.053). mortality rate was much higher in patients with prolonged qtc at admission (41.0% vs. 8.7%, p < 0.001), as shown in table 6 and the kaplan-meier survival curves in figure 2 . a baseline qtc > 480 ms was independently associated with higher mortality (hr 2.68 (1.58-4.55), p < 0.001). this result was similar when treatment was not included in the model (hr 2.78 (95% ci 1.66-4.66), p < 0.001). in a sensitivity analysis excluding patients who died during the first 48 h of admission (18 patients, 24.3% of all patients who died), the results were also similar (hr: 2.073 (95% ci: 1.073-4.005), p = 0.03). the baseline characteristics, prognosis, and presentation of patients without a baseline ecg are summarized in table 7 . this group of patients was younger and had less cardiovascular risk factors and comorbidities. the clinical presentation was less severe and 24% were not treated with the baseline characteristics, prognosis, and presentation of patients without a baseline ecg are summarized in table 7 . this group of patients was younger and had less cardiovascular risk factors and comorbidities. the clinical presentation was less severe and 24% were not treated with hydroxychloroquine or azithromycin. death rate was similar to those with a baseline ecg (10.8 vs. 11.9, p = 0.66). in this study, we found that a prolonged qtc interval at admission is present in almost 10% of patients with sars-cov-2 infection. even though these patients had more comorbidities and worse clinical profile at presentation, the presence of a prolonged qtc was independently associated with increased mortality. the mean baseline qtc interval in our study was 437.0 ± 34.5 ms. several studies in sars-cov-2 infection have reported similar baseline qtc intervals, with mean values ranging from 415 to 455 ms [2] [3] [4] [5] [6] . there are different definitions of prolonged qtc and the use of any of them might have affected the results of our study. we chose the cut-off value of 480 ms following the esc guidelines [18] . although the prevalence of qtc > 480 ms was 9.8%, the prevalence of very prolonged qtc (qtc > 500 ms) was very low, only affecting 3.2% of patients and similar to other studies [6] . therefore, using this restrictive cut-off as a screening tool would have had limited clinical value. the majority of studies have focused on qtc interval and risk of arrhythmias, especially in the setting of hydroxychloroquine treatment. this treatment (with or without azithromycin) is associated with a prolongation of the qt interval in 2.8 to 18.9% of patients [2, 4, 6, 19, 20] . however, these results depend on the definition of qt prolongation used and the dose of hydroxychloroquine. interestingly, the risk of ventricular arrhythmias was very low and, consistent with our results, several studies did not show any episode of torsade de pointes or arrhythmic death [2, 4, 6, 20, 21] . in rheumatologic disease studies, the use of hydroxychloroquine has also been associated with qtc interval prolongation but not to increased mortality [22] . thus, if randomized controlled trials were to show increased survival in sars-cov-2 infection with this treatment, data available show that the fear of malignant arrhythmias should not be a deterrent to its use with proper qt interval monitoring. however, the interest in the prognostic value of qtc interval goes beyond its potential interaction with treatment. the electrocardiogram (ecg) is a cheap non-invasive tool that can be found in all healthcare settings, from local clinics to tertiary hospitals. however, ecg is an underused tool in risk stratification. in our cohort, all-cause death was higher in patients with prolonged qtc (41.0% vs. 8.7%, p < 0.001, multivariable hr 2.68 (1.58-4.55), p < 0.001). as expected from previous research, age and comorbidities were associated with prolonged qtc interval and worse prognosis [23] [24] [25] [26] . some studies have shown that almost 20% of patients with chronic kidney disease (ckd) have a prolonged qtc interval than patients without ckd, and the presence of a prolonged qtc interval in this group is associated with increased cardiovascular and all-cause mortality [24] . moreover, age per se is associated with a prolonged qt interval [23, 27] . in patients with acute heart failure, the qtc interval has been associated with 5-year all-cause mortality in j-shape with nadir of 440 to 450 ms in male and 470 to 480 ms in female, although its significance decreased in females [28] . similar results are seen in chronic heart failure, where the presence of prolonged qtc is also associated with higher mortality (41% vs. 14%, p = 0.001) [29] . in patients with prior cardiovascular disease, both cardiovascular mortality and sudden death were higher in patients with prolonged qtc, with relative risks that ranged from 1.1 to 3.8 for total mortality, from 1.2 to 8.0 for cardiovascular mortality, and from 1.0 to 2.1 for sudden death [30] . there are well documented sex-dependent differences in normal qt interval and ageand sex-specific cut-offs for prolonged qtc (>450 ms for men and >470 ms for women) have been proposed [27] . therefore, by using a cut-off of 480 ms, it is possible that high-risk men were not identified. when analyzed separately by sex, we saw that patients with prolonged qtc had higher mortality (56.7% vs. 8.4% in women, p < 0.001, and 25.8% vs. 8.9% in men, p = 0.003). the 480 ms cut-off was independently associated with death in women, whereas there was a strong tendency in the same direction in men (univariate hr 2.27 (95% ci: 0.99-5.23), p = 0.053). it is worth mentioning that the number of events was very low in men (only eight patients in the qtc ≥ 480 ms died) and that might explain the lack of statistical significance in men. although the use of a different cut-off according to sex could be useful, using several cut-off points depending on sex might not be feasible in clinical practice when different types of healthcare professionals at several levels of complexity are involved. the fact that patients with prolonged qtc had higher immune-inflammatory parameters and cardiac biomarkers (i.e., c-reactive protein, white blood cell count, serum lactate, procalcitonin, lactate dehydrogenase, d-dimer, troponin t, and ntprobnp) is intriguing. although these differences could be due to a more severe presentation in a group of elderly comorbid patients, sars-cov-2 infection could be the cause of this prolonged qtc interval, either as a direct effect of the virus or through systemic inflammation. studies done in rabbits showed that coronavirus infection was associated with qt interval prolongation [10] , and coronavirus infection caused right and left ventricular dilation, myocardial fibrosis, and myocarditis [31, 32] . similarly to what had been observed in the animal model, echocardiograms done in patients with sars-cov-2 infection have shown a predominant right ventricular dilation, which was associated with increased troponin levels and worse prognosis [33] . on the other hand, several studies have described abnormal immune-inflammatory response to sars-cov-2 infection. a recent study has shown that levels of interleukin (il)-1β, il-6, il-8, il-10 and soluble tnf receptor 1 (stnfr1) were all increased in patients with sars-cov-2 infection compared to healthy volunteers and cytokine ratios may predict outcomes in this population [34] . a recent meta-analysis has shown that other immune-inflammatory parameters, such as c-reactive protein, white blood cell count, and procalcitonin, were higher in severe sars-cov-2 infection compared to milder presentations [35] . given that inflammation can also lead to qt interval prolongation [14, 15] , it is possible that sars-cov-2 infection prolongs a qtc interval through an inflammatory response. hence, a prolonged qtc interval in sars-cov-2 infection could be the result of direct virus activity or be mediated by inflammation, which would help explain why a prolonged qtc is independently associated with 30-day mortality. there are some limitations to our study. first, asymptomatic patients were not included in this registry, which confers a selection bias. second, although this is the largest study assessing qtc prognostic value in sars-cov-2 infection, this is a single-center study with a limited number of patients. third, data on prolongation of qtc during hospitalization should be viewed with caution because only 39% of patients had a repeated ecg during hospitalization, hence the risk of bias is potentially high. fourth, the measurement of the qt interval can be difficult [27, 36] . previous studies have shown that only 60% of physicians were able to accurately measure a sample qt interval, even though the majority stated that their area of specialization was cardiology [37] . several studies have shown that automated qtc measurements are accurate in comparison with manual qtc measurements [6, 22, 38] . therefore, the use of automated ecg measurement is likely to offer greater accuracy and allow a wider use of this tool in all healthcare levels than the manual assessment. finally, we cannot exclude that some of the deaths might be due to ventricular tachycardia or ventricular fibrillation that went unnoticed and were ultimately attributed to other causes. up to 10% of patients with sars-cov-2 infection had a prolonged qtc interval (i.e., >480 milliseconds) on admission. a prolonged qtc was independently associated with a higher risk of mortality even after adjustment for age, comorbidities, and treatment with hydroxychloroquine and azithromycin. thus, the qtc interval should be measured in all patients with sars-cov-2 infection as a non-invasive and low cost tool for 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2017, 4, e000683 acquired long qt syndrome in chronic kidney disease patients heart rate-corrected qt interval is an independent predictor of all-cause and cardiovascular mortality in individuals with type 2 diabetes: the diabetes heart study how to measure it and what is "normal j-curve relationship between corrected qt interval and mortality in acute heart failure patients prognostic significance of qt interval prolongation in adult nigerians with chronic heart failure-pubmed prolonged qtc interval and risks of total and cardiovascular mortality and sudden death in the general population: a review and qualitative overview of the prospective cohort studies an experimental model for dilated cardiomyopathy after rabbit coronavirus infection an experimental model for myocarditis and congestive heart failure after rabbit coronavirus infection the spectrum of cardiac manifestations in coronavirus disease 2019 (covid-19)-a systematic echocardiographic study characterization of the inflammatory response to severe covid-19 illness immune-inflammatory parameters in covid-19 cases: a systematic review and meta-analysis what clinicians should know about the qt interval knowledge deficits related to the qt interval could affect patient safety assessment of reproducibility-automated and digital caliper ecg measurement in the framingham heart study key: cord-320823-a1fq6mno authors: moula, amalia ioanna; micali, linda renata; matteucci, francesco; lucà, fabiana; rao, carmelo massimiliano; parise, orlando; parise, gianmarco; gulizia, michele massimo; gelsomino, sandro title: quantification of death risk in relation to sex, pre-existing cardiovascular diseases and risk factors in covid-19 patients: let’s take stock and see where we are date: 2020-08-19 journal: j clin med doi: 10.3390/jcm9092685 sha: doc_id: 320823 cord_uid: a1fq6mno patients with pre-existing cardiovascular disease (cvd) might be more susceptible to infection from severe acute respiratory syndrome coronavirus 2 (sars-cov-2) and have higher mortality rates. nevertheless, the risk of mortality has not been previously quantified. the aim of this meta-analysis is to quantify the risk of mortality in coronavirus disease 2019 (covid-19) patients. a meta-analysis was conducted analyzing the impact of (1) sex, (2) age, (3) cvd with coronary artery disease (cad), (4) cad alone, (5) cvd without cad, (6) hypertension, (7) cerebrovascular diseases, and (8) diabetes on mortality. relative risk was assessed for dichotomous variables, mean difference for continuous variables. twenty-six studies were included, encompassing 8497 patients. males had 16% higher risk of mortality than females (p < 0.05) and elderly patients had higher chance of dying than younger patients (p < 0.0001). patients with overall cvd have a 1.96-fold higher mortality risk (p < 0.0001). cad increases risk of mortality by 1.90-fold (p < 0.05). cvd-cad were found to increase risk up to 2.03-fold (p < 0.05). hypertension, cerebrovascular disease and diabetes increase the risk of death up to 1.73-fold, 1.76-fold and 1.59-fold, respectively (p < 0.0001, p < 0.0001, p < 0.05, respectively). sex, age, presence of cad and/or other types of cvd, hypertension, cerebrovascular diseases and diabetes mellitus increase mortality in patients with covid-19. coronavirus disease 2019 (covid19) is an infectious disease caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the virus, first identified in 2019 in china, has a positive sense single stranded rna and seems to be of zoonotic origin. the virus is most likely airborne and highly contagious. it spreads via contaminated droplets that pass from one human to another while in close contact [1] . the recent global pandemic ignited by the covid-19 has had a considerable impact on many healthcare systems around the world [2] . for this reason, the disease has received increasing attention by the scientific community. previous literature suggests that patients with pre-existing cardiovascular disease (cvd) might be potentially more susceptible to infection from sars-cov-2 [3] . nevertheless, the exact mechanisms by which covid-19 affects the cardiovascular system and mortality are not yet well understood, despite accumulating evidence that such a connection exists [4, 5] . however, to the best of our knowledge the risk of death in relation to sex, age and cvd has not been quantified in large cohorts of patients. moreover, the association between covid-19, cvd and patient mortality has not yet been fully elucidated, setting the need for additional confirmation of the association between these two ailments as well as the impact of shared risk factors on such a relation. therefore, the present meta-analysis is aimed to quantify the risk of mortality in relation to sex, age and pre-existing cvd in covid-19 patients, and attempt to identify the potential factors involved in such a causation. the literature search was conducted in accordance with the principles of the preferred reporting items for systematic reviews and meta-analyses (prisma) [6] and the cochrane handbook [7] . two authors established the search strategy (aim and lrm) and the decisions were approved by a third author (sg). one investigator performed the literature search (aim), which was limited to articles published from 1 december 2019 until 18 may 2020. an unrestricted literature search was performed using pubmed, adopting the following search terms: "severe acute respiratory syndrome coronavirus 2" [supplementary concept] or "covid-19" [supplementary concept] or "spike glycoprotein, covid-19 virus" [supplementary concept] or "covid-19" or "covid 19" or "covid19" or "sars-cov-2" or "novel coronavirus" and epidemiology or comorbidities or heart or cardiovascular or myocardial or "cardiovascular diseases" [mesh] or heart or myocardium or stemi or infarction or arrhythmia or hypertension. the articles were selected based on the following inclusion criteria: (1) human studies; (2) full articles about covid-19 disease containing separate data for patients that survived and patients that did not; (3) analyses of fatality cases; (4) studies including at least 10 patients; (5) articles published from december 2019 and (6) articles in english language. the exclusion criteria used to reject articles were: (1) non-human studies; (2) case reports; (3) previous reviews and/or meta-analyses; (4) editorials; (5) comments; (6) studies without separate data on surviving and non-surviving patients; (7) studies in languages other than english. two reviewers (am and lm) independently assessed the risk of bias for the included studies. the robins-i tool (risk of bias in no-randomized studies of interventions) was used for the assessment of bias at the individual study level [7] . disagreements were resolved by discussion or by involving a third reviewer (sg). the domains assessed were (1) bias due to confounding; (2) bias in selection of participants into the study; (3) bias in classification of interventions; (4) bias due to deviations from intended interventions; (5) bias due to missing data; (6) bias in measurement of outcomes; (7) bias in selection of the reported result; and (8) overall bias assessment. the evaluation of the aforementioned domains was conducted with the aid of cochrane handbook [7] . furthermore, the generation of the plot for robins-i was achieved the software robvis [8] . the primary endpoint of this meta-analysis was to identify comorbidities and pre-existing cardio-metabolic diseases that could predict mortality in patients with covid-19. the risk factors and comorbidities taken into consideration were: (1) sex, (2) age, (3) cvd, (4) coronary artery disease (cad), (5) hypertension, (6) cerebrovascular disease, and (7) diabetes mellitus. to evaluate the impact of the several types of cvd and cad separately, first we conducted an analysis of patients with cvd including cad ("cvd with cad") vs. patients without cvd; then a separate analysis only of patients with cad ("cad") vs. patients without cad; and finally, an analysis of patients with cvd excluding cad ("cvd without cad") vs. patients without cvd. the meta-analysis was conducted using v. 3.6.1 (r foundation for statistical computing, vienna, austria). relative risk (rr) and 95% confidence interval (ci) were used as index statistics for dichotomous variables. for continuous variables, mean difference and 95% ci were calculated. in both cases the random effects model was adopted because heterogeneity among studies was anticipated. heterogeneity was assessed with the statistical inconsistency higgin's i 2 test [7] . i 2 values < 40% were considered having low heterogeneity, i2 values > 75% were considered having high heterogeneity [7] . publication bias was evaluated using egger's test of the intercept. p values < 0.05 were considered statistically significant. the initial search retrieved 1719 articles. after screening for the inclusion and exclusion criteria, 78 articles that included patient demographic data for covid-19 and mortality were found. after rejecting articles without separate data for patients that survived and non-survivors, a total of 22 articles were found to fulfil the criteria. four additional articles were added from the references of the articles found through the search on pubmed (figure 1 ). at the end of the selection process, 26 studies were included in the analysis . eight out of the twenty-six studies were analyses of fatality cases [11, 12, 15, 18, 21, 26, 33, 34] . there were twenty papers from china [9] [10] [11] [12] [13] [14] [15] [16] 18, [22] [23] [24] [25] [26] [27] [28] [29] [30] [31] [32] , two papers from italy [17, 21] , two papers from korea [33, 34] , one from iran [20] and one from the united states of america [19] . in total, the patient cohort included of 8497 patients, of whom 5121 (60.3%) were male and 3376 (39.7%) were female. non-survivors among males accounted for 25.0% of the patients (1280 individuals out of 5121), whereas non-survivors percentage among females was 19.5% (659 individuals out of 3376). the studies that were included in the meta-analysis and the characteristics of the patients with covid-19 are shown in table 1 . the animal trial was approved by the rusvm iacuc under number 19.05.18. figure 2 shows the "risk of bias" graph. low bias due to confounding was absent in all papers, as confounding was expected in all of them, although some of them [10, [14] [15] [16] 19, [21] [22] [23] [24] [25] 29, 33, 34] controlled for confounding through either multivariable and multivariate analysis, adjustment and stratification of patients. twelve papers [9] [10] [11] 13, 14, 24, 25, [27] [28] [29] 31, 32] had low bias due to selection of participants, as all patients eligible were included in the study and were enrolled in the same short period of time. no paper had low bias for classification of interventions as intervention status was not well defined. in addition, for the bias due to deviations from intended intervention, no study had low bias mainly due to the fact that in the majority this information was missing. two papers [11, 29] show low bias due to missing data because they had complete data. fifteen papers [9] [10] [11] 13, 14, 16, 17, [24] [25] [26] [27] [28] [29] [30] [31] had low bias in measurement of the outcome. eight studies [16, 19, [23] [24] [25] 30, 31] had low bias in the reported results. no study was overall lowly biased. incidence rate ration (irr), heterogeneity test and egger's test results are summarized in table 2 . the analysis revealed that men have 16% higher risk of mortality than women, as shown in figure 3a (rr: 1.16 [95% ci: 1.05, 1.27], p = 0.003; funnel plot in figure s1a ). as shown in figure 3b , we found that age is another predictor of mortality, as older patients had significantly higher chance of dying than younger patients (mean difference: −15.72 [95% ci: −18.62, 12.81], p < 0.0001; funnel plot in figure s1b ). furthermore, the analysis revealed that patients with cvd with cad have a 1.96-fold [95% ci: 1.51, 2.54] higher risk of mortality than patients without cvd, as presented in figure 4a (p < 0.0001; funnel plot in figure s1c ). in particular, the presence of cad increased the risk of mortality by 1.90-fold [95% ci: 1.32, 2.74] when compared to other cvds, as described in figure 4b (p = 0.0005; funnel plot in figure s1d ). however, by conducting an analysis on cvd excluding cad we found that the presence of other cvds is a strong predictor of death, since patients with cvd (excluding cad) had 2.03-fold [95% ci: 1.41, 2.92] higher risk of mortality compared to patients without cvd ( figure 4c , p = 0.0002; funnel plot in figure s1e ). we found that non-survivors were more likely affected by hypertension, having a 1.73-fold greater risk of mortality than patients without hypertension ( figure 4d , rr: 1.73 [95% ci: 1.37, 2.19], p < 0.0001; funnel plot in figure s1f ). patients with pre-existing cerebrovascular disease tend to die 1.76-fold more than patients without cerebrovascular disease, suggesting that cerebrovascular disease is a strong predictor of death ( figure 4e , rr: 1.76 [95% ci: 1.25, 2.50], p < 0.0001; funnel plot in figure s1g ). similarly, as pictured in figure 4f , patients with diabetes had 1.59-fold higher chance of dying than patients without diabetes (rr: 1.59 [95% ci: 1.25, 2.02], p < 0.0001; funnel plot in figure s1h ). in our analysis, we quantified the risk of death in almost 8500 covid-19 patients in relation to sex, age, pre-existing cvd and cardiovascular risk factors. to the best of our knowledge, this has not been previously done, especially in cohorts encompassing large numbers of patients. interestingly, our updated report shows that men still are more prone to dying but the effective increase in risk in males compared to females is lower than previously reported (around 16%). an increased risk of mortality for male covid-19 patients (2.4 times that of women) has been widely reported. this disproportionate death ratio in men was explained by a higher incidence of pre-existing disease, higher risk behaviors, occupational exposure, high levels of androgens in men, and behavioral/social differences that favor women [35] . androgens increase the expression of the transmembrane protease serine 2 (tmprss2) [36] . tmprss2 is a critical protease that enables the entry of sars-cov-2 in angiotensin-converting enzyme 2 (ace2) receptors, explaining why men tend to die more from covid-19 [36, 37] . a treatment against androgens that could theoretically interfere with the course of the disease is still debated [36] . however, it must be considered that although higher male-to-female death ratio was confirmed in all the countries with available data, the united states with the largest reported outbreak of covid-19 in the world provided only partial sex-disaggregated data and this might have biased the overall estimation of sex-related risk distributions. this warrants a careful epidemiological analysis to assess whether there has really been a turn in sex-specific differences with a rising incidence of death in women [38] . in contrast, the association with age was confirmed, with older patients being more vulnerable to die from covid-19 [39] . primarily responsible for the increased age-related susceptibility are the ace2 receptors and cd26; both overexpressed in senescent cells. both ace2 receptors and cd26 are targets for coronaviruses, and their overexpression in older patients might mediate the increased fatality rate in covid-19 patients [40] . ace2 is abundantly distributed in the lungs but also in the heart, kidneys, guts, and the pancreas [41] . ace2 is pivotal for the entry mechanism of the sars-cov-2 as it is harnessed by the virus as an entry point, whereas cd26 interacts with the s1 domain of the virus affecting virulence [42] [43] [44] . another mechanism contributing to the increased mortality in elderly patients is immunosenescence, in which naïve t and b cells are produced in lower quantities, and dendritic cells do not effectively differentiate after t cell interaction [39] . the third finding of our meta-analysis is that the presence of cardiovascular diseases, is associated with a higher risk of mortality when compared to covid-19 patients without pre-existing cvd. our outcomes are in contrast with the results of a previous meta-analysis conducted on three studies, which found no correlation between the history of cvd and mortality but revealed an association between cvd and enhanced disease severity [5] . it is possible that such a discrepancy could be due to the difference in terms of number of studies included in the analysis. previous literature already suggests that cvd might be involved in promoting death in covid-19 patients [45] . another recent meta-analysis conducted on six papers reported that among covid-19 patients admitted to the intensive care unit, 17.1% had hypertension and 16.4% cardiac and/or cerebrovascular diseases [3] . wu et al. [46] also reported that patients with cvd, hypertension and diabetes tend to die more often. the cause of such an association might be complex and multifactorial. cardiopathic patients with ventricular hypertrophy, diastolic dysfunction and heart failure tend to develop acute pulmonary hypertension while being affected by covid-19. this can result in pulmonary edema [41] . if sars-cov-2 causes sepsis, then acute respiratory distress syndrome (ards) can occur which per se aggravates the edema, and can become the cause of death in these patients [41, 47] . additionally, when infection by sars-cov-2 occurs, the virus is internalized and this triggers the activation of adam metallopeptidase domain 17 (adam17). adam17 causes cleavage of the ace2 receptors making them unresponsive to the negative feedback exerted by the activation of the renin-angiotensin-aldosterone system. this is ultimately responsible for further production of cytokines, which aggravate the inflammation [48] . in the presence of pre-existing cvd, the cytokine storm can exacerbate underlining diseases by aggravating pre-existing heart failure, causing depression of myocardial activity, increasing the oxygen demand/supply ratio and endothelial dysfunction [22, 48] . in addition, 17% patients with covid-19 had pre-existing cad and this raises the risk of death, especially when it is associated with potential hypercoagulability deriving from the febrile state [41] . nonetheless, in our analysis, cvd with or without cad showed very close rrs of death. in other words, although the presence of cad alone raised the risk of death by 90%, the presence of coronary disease did not increase death rr of patients with other cvds that increases the risk > 100%. moreover, we have found that covid-19 patients with hypertension had a 73% higher rr than those without high blood pressure. unfortunately, due to the lack of specific information, it was not possible to compare subgroups to study the true incremental risk associated with hypertension in covid-19 patients with cvd. however, it has been proposed that when patients with heart failure and hypertension receive ace inhibitors and type-i receptor blockers (arbs), these agents contribute to the upregulation of ace2 receptors. this increases susceptibility to contracting covid-19. the mechanisms mentioned help explain their vulnerability to mortality. for this reason, some authors have suggested the use of alternative antihypertensive medication during the pandemic, such as calcium channel blockers [49] . the fourth finding of our analysis is the increased fatality rate in patients with diabetes. according to the meta-analysis conducted by li et al. [3] , patients with diabetes represented 9.7% of the covid-19 patients in intensive care unit (icu). susceptibility of diabetic patients towards covid-19 and their increased chance of dying derives from the overexpression of ace2, impaired innate immunity and delayed th1 cell-mediated responses. these factors predispose to cytokine storm, with adverse outcomes. furthermore, while on one hand, insulin reduces ace2 expression, on the other, hypoglycemic drugs and statins upregulate ace2 [50] . in addition, diabetic patients might need additional administration of insulin or secretagogues, as the viral infection can stimulate cortisol release and thus increase of blood glucose levels. however, these drugs alter water and sodium reabsorption and increase the risk of developing pulmonary edema in cardiopathic patients, especially if sepsis causes renal dysfunction. therefore, intravenous fluids administration should also receive attention by clinicians. in this situation, concomitant treatment with ace inhibitors can aggravate the load on the respiratory system. for this reason, some authors suggest careful evaluation of the status of the lungs and interruption of ace inhibitors and arbs, if necessary before ards manifests worsening the prognosis of patients [41] . this study has some limitations. first, the majority of the studies were retrospective, predisposing to the risk of bias. second, some of the studies included were analyses of fatality cases. third, heterogeneity between studies was high in all the endpoints analyzed, because of a great variety in baseline characteristics. fourth, it is possible that the definition of cvd could be different in the different hospital settings/countries, with most of the papers that were included not including detailed description of the type of cardiovascular disease of the patient. fifth, it would have been of great interest to study the interaction between cvd and single risk factors in predicting death. unfortunately, within the papers data were not split into sub-groups to allow these analyses. finally, in the reports there was reported age cut-off, therefore it was not possible to examine the rr increase with age. the only data attainable was the difference in age between survivors and not survivors. our results provide a quantification of mortality risk in covid-19 patients with pre-existing cardiovascular comorbidities. our results demonstrate that sex, age, presence of cad and/or other types of cvd, hypertension, cerebrovascular diseases and diabetes mellitus increase mortality in patients with covid-19. in particular, cad and/or other types of cvd, hypertension, cerebrovascular diseases almost double the risk of mortality. further research to identify the underlining mechanisms of such an association is warranted. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/9/2685/s1, figure s1 : funnel plot of (a) sex; (b) age; (c) overall cvd (cvd + cad) vs. patients without cvd; (d) patients with cad vs. patients without cad; (e) patients with cvd (and no cad) vs. patients without cvd-cad; (f) patients with hypertension vs. patients without hypertension; (g)patients with cerebrovascular diseases vs. patients without cerebrovascular diseases; and (h) patients with diabetes mellitus vs. patients without with diabetes mellitus. funding: this research received no external funding. covid-19 infection: origin, transmission, and characteristics of human coronaviruses health system, public health, and economic implications of managing covid-19 from a cardiovascular perspective prevalence and impact of cardiovascular metabolic diseases on covid-19 in china covid-19 and cardiovascular disease association of cardiovascular disease with coronavirus disease 2019 (covid-19) severity: a meta-analysis the prisma extension statement for reporting of systematic reviews incorporating network meta-analyses of health care interventions: checklist and 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characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china extracorporeal membrane oxygenation for adults with refractory septic shock angiotensin converting enzyme 2: a double-edged sword are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? covid-19 pandemic, coronaviruses, and diabetes mellitus this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank judith wilson for english editing of the manuscript. the authors declare no conflict of interest. key: cord-325307-agaau27o authors: giavedoni, priscila; podlipnik, sebastián; pericàs, juan m.; fuertes de vega, irene; garcía-herrera, adriana; alós, llúcia; carrera, cristina; andreu-febrer, cristina; sanz-beltran, judit; riquelme-mc loughlin, constanza; riera-monroig, josep; combalia, andrea; bosch-amate, xavier; morgado-carrasco, daniel; pigem, ramon; toll-abelló, agustí; martí-martí, ignasi; rizo-potau, daniel; serra-garcía, laura; alamon-reig, francesc; iranzo, pilar; almuedo-riera, alex; muñoz, jose; puig, susana; mascaró, josé m. title: skin manifestations in covid-19: prevalence and relationship with disease severity date: 2020-10-12 journal: j clin med doi: 10.3390/jcm9103261 sha: doc_id: 325307 cord_uid: agaau27o background: data on the clinical patterns and histopathology of sars-cov-2 related skin lesions, as well as on their relationship with the severity of covid-19 are limited. methods and materials: retrospective analysis of a prospectively collected cohort of patients with sars-cov-2 infection in a teaching hospital in barcelona, spain, from 1 april to 1 may 2020. clinical, microbiological and therapeutic characteristics, clinicopathological patterns of skin lesions, and direct immunofluorescence and immunohistochemical findings in skin biopsies were analyzed. results: fifty-eight out of the 2761 patients (2.1%) either consulting to the emergency room or admitted to the hospital for covid-19 suspicion during the study period presented covid-19 related skin lesions. cutaneous lesions could be categorized into six patterns represented by the acronym “grouch”: generalized maculo-papular (20.7%), grover’s disease and other papulo-vesicular eruptions (13.8%), livedo reticularis (6.9%), other eruptions (22.4%), urticarial (6.9%), and chilblain-like (29.3%). skin biopsies were performed in 72.4%, including direct immunofluorescence in 71.4% and immunohistochemistry in 28.6%. patients with chilblain-like lesions exhibited a characteristic histology and were significantly younger and presented lower rates of systemic symptoms, radiological lung infiltrates and analytical abnormalities, and hospital and icu admission compared to the rest of patients. conclusion: cutaneous lesions in patients with covid-19 appear to be relatively rare and varied. patients with chilblain-like lesions have a characteristic clinicopathological pattern and a less severe presentation of covid-19. coronavirus disease 2019 , caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has rapidly spread to acquire pandemic proportions since the first outbreak was declared in wuhan, the capital of hubei province, china, in december 2019 [1] . although the most common manifestations of covid-19 are fever and respiratory symptoms such as cough and shortness of breath, other manifestations are also relevant, and subacute manifestations such as organizing pneumonia and decreased pulmonary function, or drug interactions and side effects are increasingly gaining attention as the knowledge on covid-19 pathophysiology and natural history accumulates [2] [3] [4] [5] [6] [7] [8] [9] . recently, skin lesions have been described as potential manifestations of covid-19. the skin changes reported to date include erythematous rash, urticaria, livedo reticularis, vesicular lesions, and chilblain-like lesions [10] [11] [12] [13] [14] [15] . however, there are insufficient data on the prevalence and histopathology of skin lesions associated with covid-19, as well as the associated clinical, analytical, and radiological findings. this study was aimed to characterize the prevalence, clinical features, and histopathology findings of covid-19-associated skin manifestations and their relationship with other covid-19 clinical-epidemiological features. single-center prospective cohort study carried out from 1 april to 1 may 2020. this study was performed following the strobe guidelines [16] . hospital clínic de barcelona is an 800-bed tertiary university hospital providing care to 600,000 people in the metropolitan area of barcelona, spain. consecutive patients with diagnosis of covid-19 presenting new-onset of skin lesions. all consultations to the dermatology department originated from the emergency department, hospital wards, or intensive care units. the performance of skin biopsies was assessed in all patients and performed reaching a consensual agreement between the patient and the treating physician. histologic studies requested included hematoxylin and eosin (he) stain, direct immunofluorescence (dif), and immunohistochemistry (ihc). dif was performed on cryostat sections using fitc-conjugated antibodies to igg, igm, iga, c3 and fibrinogen. in addition, we tested complement c9 expression by immunohistochemistry on paraffin sections of formalin-fixed tissue. assays for detection of sars-cov-2 in skin samples were not done. real-time polymerase chain reaction (rt-pcr) from nasopharyngeal swabs was carried out amplifying the betacoronavirus e gene and the specific sars-cov-2 rdrp gene (roche ® ; sensitivity 90% and specificity 100%). a serology kit was set up in our immunology laboratory for the detection of iga, igm or igg sars-cov-2-specific antibodies (sensitivity 97% for iga and igg, 75% for igm, and specificity of 100% for igg and igm and 98% for iga). diagnosis of covid-19 included confirmed cases with positive microbiological tests and highly suspected cases, which were managed according to our institutional protocols as being infected (isolation measures ± antiviral therapies) based on highly suggestive epidemiological, clinical, radiological, and analytical features. see in supplementary material. the hospital clínic ethical board approved this study (hcb/2020/0581) and waived the requirement for informed consent due to the ongoing situation of infectious disease emergency. a descriptive analysis of the characteristics of patients presenting skin lesions associated with covid-19 was carried out, as well as a univariate analysis of selected categories comparing patients with chilblain-like lesions and patients with other type of lesions. categorical variables were summarized as percentages and compared through the pearson chi-square test or fisher exact test when appropriate. continuous variables were summarized as median and interquartile ranges (iqr) and compared using anova test or t test. p values < 0.05 were considered statistically significant. computing environment r (v 4.0.0, foundation for statistical computing, vienna, austria) was used. during the study period, a total of 2761 patients either consulted to the emergency department (593) or were admitted to the hospital (2168) with a covid-19 suspicion ( figure 1 ). in the same period, the dermatology department received 110 consultations, of which 70 had initial suspicion of covid-19 related skin lesions. the hospital clínic ethical board approved this study (hcb/2020/0581) and waived the requirement for informed consent due to the ongoing situation of infectious disease emergency. a descriptive analysis of the characteristics of patients presenting skin lesions associated with covid-19 was carried out, as well as a univariate analysis of selected categories comparing patients with chilblain-like lesions and patients with other type of lesions. categorical variables were summarized as percentages and compared through the pearson chi-square test or fisher exact test when appropriate. continuous variables were summarized as median and interquartile ranges (iqr) and compared using anova test or t test. p values < 0.05 were considered statistically significant. computing environment r (v 4.0.0, foundation for statistical computing, vienna, austria) was used. during the study period, a total of 2761 patients either consulted to the emergency department table 1 shows a summary of the patients' characteristics, both general and according to the clinicopathological patterns (see table s1 (supplementary material) for an individually based account of patients' features, including histologic findings). median age was 54.8 years (iqr, 38.7-69) and 46.6% were female. the chronological onset of cutaneous symptoms with respect to other type of symptoms and treatments are shown in figure 2 . 1 (6%) 10 (83%) 6 (75%) 4 (100%) 4 (100%) 11 (85%) 36 (62%) anti-inflammatory treatment, n (%) 1 (6%) 10 (83%) 6 (75%) 4 (100%) 4 (100%) 11 (85%) 36 (62%) hospitalization required 2 (11.8%) 10 (83.3%) 6 (75%) 4 (100%) 4 (100%) 12 (92.3%) 38 (65.5%) icu required 1 (6%) 4 (33%) 3 (37%) 3 (75%) 2 (50%) 6 (46%) 19 (33%) in-hospital mortality 0 0 6 (75%) 0 0 2 (5%) 2 (3%) rt-pcr: real-time polymerase chain reaction; dif: direct immunofluorescence; icu: intensive care unit. * this group includes: pressure-induced ischemic necrosis in prolonged coma patient (n = 2), hematoma (n = 1), lichen planus (n = 1), contact dermatitis (n = 2), psoriasis (n = 1), generalized fixed drug eruption (n = 1), benign familial pemphigus (n = 1), chronic graft-versus-host disease (n = 1), stasis dermatitis (n = 1), dermatophytosis (n = 1), eruptive cherry angiomas (n = 1). table s1 . painful chilblain-like lesions on toes; (b) lichenoid dermatitis with perivascular and periadnexal lymphocytic infiltration on superficial and deep dermis. (hematoxylin and eosin stain, original magnification ×100); (c) granular igm deposition in dermal vessels (direct immunofluorescence, original magnification ×200); (d) c9 reactivity in dermal vessels (immunohistochemistry, original magnification ×100); (e) patient 8 table s1 . chilblain-like violaceus lesions on toes; (f) perivascular and perianexial lymphocytic infiltration on superficial and mid dermis. (hematoxylin and eosin stain, original magnification ×100); (g) granular c3 deposition in superficial dermal vessels (direct immunofluorescence, original magnification ×200); (h) c9 reactivity in dermal vessels (immunohistochemistry, original magnification ×100). table 2 shows a comparison of the characteristics of patients with chilblain-like versus the other types of cutaneous lesions. patients with chilblain-like lesions were significantly younger; had significantly lower percentages of microbiological confirmation, respiratory and systemic symptoms, and radiological and analytical abnormalities; were less frequently treated; and had lower hospital and icu admission rates. in all cases except one, skin lesions appeared after the classic symptoms of covid-19 ( figure 2 ). no specific association with autoantibodies was found. histologic examination could be done in 12 of the 17 (70.6%) patients with chilblain-like lesions. in addition, 11 (64.7%) of table s1 . chilblain-like violaceus lesions on toes; (f) perivascular and perianexial lymphocytic infiltration on superficial and mid dermis. (hematoxylin and eosin stain, original magnification ×100); (g) granular c3 deposition in superficial dermal vessels (direct immunofluorescence, original magnification ×200); (h) c9 reactivity in dermal vessels (immunohistochemistry, original magnification ×100). skin biopsies were performed in 42 (72%) patients and included dif in 30 (52%), and ihc in 35 (60%). table 2 shows a comparison of the characteristics of patients with chilblain-like versus the other types of cutaneous lesions. patients with chilblain-like lesions were significantly younger; had significantly lower percentages of microbiological confirmation, respiratory and systemic symptoms, and radiological and analytical abnormalities; were less frequently treated; and had lower hospital and icu admission rates. in all cases except one, skin lesions appeared after the classic symptoms of covid-19 ( figure 2) . no specific association with autoantibodies was found. histologic examination could be done in 12 of the 17 (70.6%) patients with chilblain-like lesions. in addition, 11 (64.7%) of them underwent dif: in 7 of them (63.6%), deposition of igm or igg, c3, and fibrinogen on dermal blood vessels suggested a vasculopathic pattern. this was confirmed by the presence of vascular c9 deposits by ihc observed in most of these patients (90.9%, 10 of the 11 studied cases). pcr tests on chilblains patients were performed in all cases when the "other symptoms associated with covid-19" had already been resolved. in patients who presented without other symptoms associated with covid-19, pcr was performed at least one week after the chilblains' appearance. in cases where serology was performed, it was done at the same time as pcr. twelve patients presented with generalized maculo-papular eruptions ( figure s1 ), and histology showed spongiotic dermatitis, interface dermatitis, or mixed patterns (interface plus spongiotic dermatitis). in 4 patients with urticarial eruptions, histology was compatible with urticaria ( figure s2 ). these 2 clinicopathological groups of patients were frequently taking many medications, and therefore, the differential diagnosis between skin lesions related to covid-19 infection or a drug eruption can very difficult, if not impossible, to do with the exception of the few patients that were not taking any medication. eight patients presented with papulo-vesicular eruptions on the trunk. in three of them, histology showed intraepidermal vesicles with suprabasal acantholysis with the presence of dyskeratotic keratinocytes, typical of grover's disease ( figure s3 ). in addition, there were 2 patients with chickenpox, 1 patient with herpes zoster, and 2 patients with pityrosporum folliculitis. the time from the beginning of respiratory symptoms to the appearance of infectious lesions was variable. four and 11 days for the two patients with folliculitis, 14 days for the patient with shingles, and 14 days for one of the patient's chickenpox. the other patient with chickenpox started with skin lesions at the same time as with respiratory symptoms. among the 58 patients included, 39 presented cough and dyspnea. regarding these respiratory symptoms, which were the most frequently found about the covid-19 infection, skin lesions' time of appearance was variable. acral lesions (n = 6) appeared in a median time of 10.5 days (range: 4-26.8 days) after respiratory symptoms. in the rash maculopapular group (n = 8), these lesions appeared in a median of 18 days (range 13.2-24.5 days). papulo-vesicular lesions (n = 7) were observed in a median of 11 days (range 7-18 days). the group of urticarial lesions (n = 3) appeared in a median of 28 days (range 28-35.5 days). lesions of livedo-reticularis (n = 4) appeared in a median of 23 days (range 13.8-29.5 days), and in the group of other lesions, they appeared in a median of 19 days (range 9.5-33.5 days). our findings suggest that skin lesions are a relatively uncommon manifestation of covid-19 and emphasize the importance of histopathology in the characterization of skin lesions during the covid-19 pandemic. the availability of skin biopsies, together with the specific traits of chilblain-like patterns in covid-19, appears to be the cornerstone of covid-19-associated skin lesions, allowing for their characterization and particularly their differentiation from other entities. moreover, differentiating chilblain-like lesions from idiopathic and lupus-associated chilblain lesions, and other types of lesions such as generalized maculopapular or urticarial eruptions from drug reactions, is essential, given the frequency and characteristics of the former and the difficulties posed by the latter due to the large number of drugs usually concomitantly received by covid-19 patients. inspired by galván-casas et al. proposed classification [17] , we divided the cutaneous lesions into the following six groups: (1) chilblain-like lesions, (2) generalized maculopapular eruptions, (3) grover's disease and other papulo-vesicular eruptions, (4) livedo reticularis, (5) urticarial eruptions, (6) other eruptions: lesions that did not meet any of the previous criteria. these skin manifestations are represented by the acronym "grouch": generalized maculo-papular; grover's disease and other papulo-vesicular; livedo reticularis; other eruptions; urticarial; and chilblain-like. this study provides a comprehensive picture of skin lesions of covid-19 supported by histopathology studies, including dif and ihc. compared to other studies published to date, ours has four main strengths. first, our study provides histopathology data in the majority of cases. to date, just short series of cases have provided data on biopsy findings in all patients, whereas others only provided this information in a small proportion of patients [15, 17, 18] . second, we provide an accurate description of the prevalence of skin lesions in covid-19 patients. third, all the cases included in our study had microbiologically-confirmed or highly-suspected covid-19, whereas the vast majority of prior reports, with some exceptions such as that of marzano and colleagues' study [18] , included a notable proportion of patients that might not have been infected by sars-cov-2. fourth, the detailed information on clinical manifestations other than cutaneous, radiological findings, analytical parameters, and particularly comprehensive histopathology data including immunofluorescence and ihc allowed us to better characterize the chilblain-like pattern as having largely specific features. a large proportion of patients in our study did not fall in any of the 5 categories described by galván-casas and colleagues [17] and nonetheless presented interesting features that might be related to specific pathophysiological pathways triggered by sars-cov-2. for instance, four of these patients presented with acro-ischemia, and in two of them, these changes seemed to be related to vasoactive drug use; clear predisposing factors were not found in the other two cases. in a series of seven cases with acral necrosis, alterations in coagulation were observed, as well as four specific criteria of disseminated intravascular coagulation [19] . zhang and colleagues described acro-ischemia in the context of antiphospholipid syndrome triggered by covid-19 [20] . we found other types of vasculopathy in our series, as one case of retiform purpura with necrotic areas and three cases of livedo reticularis. cases of transient livedo reticularis have been described in patients with covid-19, but histologic studies were not performed [21] . livedo reticularis can be idiopathic or associated with neoplasms, autoimmune or infectious diseases, among others, and it is also frequently observed in states of hypercoagulability [22] . one of the patients in our series presented concurrent pulmonary embolism and cutaneous intravascular thrombi, whereby dif showed the deposition of igm, c3, and fibrinogen within superficial-to-deep dermal blood vessel walls. in addition, c9 deposition was also demonstrated on the vessel walls by ihc [23] . chilblain-like lesions related to covid-19 infection have been mostly described in children and young adults [12, 15, 24, 25] . in line with previous reports, patients with chilblain-like lesions in our series were younger, rarely presented systemic symptoms, and presented significantly fewer blood tests and radiological abnormalities compared with patients presenting other type of skin lesions. in addition, we found that these patients rarely required hospital admission and only exceptionally icu admission. in our series, 41% of the patients with chilblain-like lesions had a confirmed diagnosis of covid-19 whereas most cases reported elsewhere did not report this information. it is noteworthy that many of the chilblain's patients did not have positive rt-pcr or serology. rt-pcr can be negative because chilblain occurs several days after systemic symptoms (when rt-pcr could have been negative). in other cases, chilblains appear in isolation without other symptoms, and these patients may not already have the virus present in their pharynx. serology is negative because perhaps these clinical manifestations are mediated by cellular immunity and do not produce circulating antibodies against covid-19. all these are hypotheses that have not been demonstrated. we believe that more studies are needed to understand the physiopathology of cashew nuts related to covid-19. in one study, the dermatological characteristics of 132 acral lesions in patients with suspected covid-19 were described; skin biopsies, however, were not performed, and only 18.1% of patients had a definite covid-19 diagnosis [14] . it has been hypothesized that these lesions begin as erythematous-violaceous macules-papules that evolve to produce subsequent blisters or digital swelling; we did not, however, observe this evolutionary pattern. the performance of histologic studies in patients with chilblain-like lesions in our series showed characteristic features in covid-19-related chilblain-like lesions. in the he examination, patients with chilblain-like lesions showed intense perivascular (lymphocytic vasculitis) and peri-eccrine lymphocytic infiltrations that, in many cases, also affected the subcutaneous tissue, as previously described [24, 25] . in addition, prominent lichenoid dermatitis and abundant dermal and hypodermal mucin deposition could be seen. dif showed immunoglobulin m (or g), complement, and fibrinogen deposits in the dermal blood vessels; and vascular c9 deposits were observed by ihc in most patients. in addition, cd3/cd4 positive lymphocytes with small aggregates of cd123 positive cells within inflammatory infiltrates could be observed in some of the cases. many of these histological findings can be found both in idiopathic chilblain or in chilblain lupus, but the presence of abundant mucin deposition is more suggestive of chilblain lupus [25] . however, the climatological conditions (spring) were very unusual for chilblain, and none of these patients either had other clinical manifestations compatible with lupus nor positive anti-ro antibodies, therefore suggesting that these lesions were indeed induced by sars-cov-2. remarkably, in the histologic study on necropsies of patients who died due to covid-19, varga and colleagues found viral particles inside endothelial cells along with an accumulation of inflammatory cells [26] . in our study, we found that most chilblain-like lesions had a vasculopathic pattern, with dif and ihc findings suggestive of complement pathway activation. we can hypothesize that in the setting of covid-19 these changes may be secondary to the arrival of viral particles to the distal circulation. [27, 28] another interesting observation is that patients with monogenic type i interferonopathies (familial chilblain lupus, aicardi-goutières syndrome) that lead to type i interferon overproduction develop chilblain-like lesions [29] . therefore, an exciting hypothesis to explain this type of lesions in patients with covid-19 would be that specific immunologic repertoires present prior to sars-cov-2 could lead to different clinicopathological presentations. patients who are able to develop an intense interferon response to the virus will develop mild systemic infection and chilblain-like lesions, while patients who are unable to produce interferon will develop a severe infection [27, 30] . finally, changes suggesting viral infection have been found in previous reports of papulo-vesicular lesions in covid-19 patients [14, 15, 18] . in our series, in three of these patients, histology was suggestive of grover's disease, while the others had chickenpox, herpes zoster or pityrosporum folliculitis. we believe that a proportion of covid-19-associated skin lesions presenting a papulo-vesicular pattern correspond to the "pseudo-herpetic" variant of grover's disease [31] [32] [33] . this is particularly relevant in the differential diagnosis with other viral-induced vesicular lesions. the main limitation of the study was a short inclusion period, therefore preventing us to provide a complete epidemiology description of covid-19-associated lesions. moreover, although this study provides detailed histology data in a larger proportion of patients with covid-19 and cutaneous lesions, skin biopsy was not performed in all patients. furthermore, a potential selection bias should be considered, since this is a single-center study carried out in a tertiary reference hospital providing care to adults but not patients in pediatric ages. patients with skin lesions potentially related to covid-19 with milder clinical presentations might have not been detected. nevertheless, most patient with symptoms suggesting covid-19 were seen at the hospital rather than the primary care during the peak of the pandemic in barcelona, and almost all private dermatology practice was discontinued during this period as well. in addition, chilblain-like lesions associated with covid-19 are more frequent in children and young adults; hence, the prevalence of chilblain-like lesions in our series might be lower than in the community. finally, isolation and contact prevention measures in the hospital's routine practices during the pandemic caused difficulties with complete skin exams. consequently, some asymptomatic or pauci-symptomatic skin lesions might have passed unnoticed. in conclusion, our data showed that skin lesions affect around 2% of patients with covid-19 and can present with various patterns that can be summarized by the acronym grouch. notably, patients with chilblain-like lesions have distinctive clinical and histological features and have less severe manifestations of the disease. the different clinico-pathologic patterns observed in the present study may be due to specific immunologic repertoires. further studies are required to better define the histopathology traits, including the presence of viral particles and genetic material, as well as the immunological blueprint of covid-19-associated skin lesions. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/10/3261/s1, figure s1 : patient 21 from table s1, figure s2 : patient 35 from table s1, figure s3 : patient 32 from table s1, figure s4 : patient 35 from table s1; table s1 : case-by-case description of the 58 patients included in the study. 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 covid-19: from epidemiology to treatment management of covid-19 respiratory distress covid-19 and thrombotic or thromboembolic disease: implications for prevention, antithrombotic therapy, and follow-up abnormal pulmonary function in covid-19 patients at time of hospital discharge pharmacologic treatments for coronavirus disease 2019 (covid-19): a review clinical characteristics of coronavirus disease 2019 in china presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the new york city area cutaneous manifestations in covid-19: a first perspective petechial skin rash associated with severe acute respiratory syndrome coronavirus 2 infection digitate papulosquamous eruption associated with severe acute respiratory syndrome coronavirus 2 infection chilblain-like lesions during covid-19 epidemic: a preliminary study on 63 patients characterization of acute acro-ischemic lesions in non-hospitalized patients: a case series of 132 patients during the covid-19 outbreak acral cutaneous lesions in the time of covid-19 strengthening the reporting of observational studies in epidemiology (strobe): explanation and elaboration classification of the cutaneous manifestations of covid-19: a rapid prospective nationwide consensus study in spain with 375 cases varicella-like exanthem as a specific covid-19-associated skin manifestation: multicenter case series of 22 patients coagulopathy and antiphospholipid antibodies in patients with covid-19 a dermatologic manifestation of covid-19: transient livedo reticularis livedo reticularis: an update retiform purpura as a dermatological sign of covid-19 coagulopathy clustered cases of acral perniosis: clinical features, histopathology and relationship to covid-19 comparative analysis of chilblain lupus erythematosus and idiopathic perniosis: histopathologic features and immunohistochemistry for cd123 and cd30 endothelial cell infection and endotheliitis in covid-19 the differing pathophysiologies that underlie covid-19 associated perniosis and thrombotic retiform purpura: a case series sars-cov-2 endothelial infection causes covid-19 chilblains: histopathological, immunohistochemical and ultrastructural study of seven paediatric cases dermatologic and dermatopathologic features of monogenic autoinflammatory diseases type i ifn immunoprofiling in covid-19 patients clinico-pathological study of 4 cases of pseudoherpetic grover disease: the same as vesicular grover disease reply to "clinical and histological characterization of vesicular covid-19 rashes: a prospective study in a tertiary care hospital": pseudoherpetic grover disease seems to appear in patients with covid-19 infection grover disease: a reappraisal of histopathological diagnostic criteria in 120 cases this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license to the entire dermatology team of the hospital clínic de barcelona, who worked intensively during the covid-19 pandemic: mercè alsina, josep malvehy, encarnación martín, paula aguilera, lorena barboza-gudagnini, sara gómez-armayones, javiera pérez-anker, natalia espinosa, and teresa estrach. the patients in this manuscript have given written informed consent to publication of their case details. the authors declare no conflict of interest. key: cord-336810-77wq9laa authors: klocperk, adam; bloomfield, marketa; parackova, zuzana; zentsova, irena; vrabcova, petra; balko, jan; meseznikov, grigorij; casas mendez, luis fernando; grandcourtova, alzbeta; sipek, jan; tulach, martin; zamecnik, josef; vymazal, tomas; sediva, anna title: complex immunometabolic profiling reveals the activation of cellular immunity and biliary lesions in patients with severe covid-19 date: 2020-09-17 journal: j clin med doi: 10.3390/jcm9093000 sha: doc_id: 336810 cord_uid: 77wq9laa this study aimed to assess the key laboratory features displayed by coronavirus disease 2019 (covid-19) inpatients that are associated with mild, moderate, severe, and fatal courses of the disease, and through a longitudinal follow-up, to understand the dynamics of the covid-19 pathophysiology. all severe acute respiratory syndrome coronavirus 2 (sars-cov-2)-positive patients admitted to the university hospital in motol between march and june 2020 were included in this study. a severe course of covid-19 was associated with an elevation of proinflammatory markers; an efflux of immature granulocytes into peripheral blood; the activation of cd8 t cells, which infiltrated the lungs; transient liver disease. in particular, the elevation of serum gamma-glutamyl transferase (ggt) and histological signs of cholestasis were highly specific for patients with a severe form of the disease. in contrast, patients with a fatal course of covid-19 failed to upregulate markers of inflammation, showed discoordination of the immune response, and progressed toward acute kidney failure. covid-19 is a disease with a multi-organ affinity that is characterized by the activation of innate and cellular adaptive immunity. biliary lesions with an elevation of ggt and the organ infiltration of interleukin 6 (il-6)-producing cells are the defining characteristics for patients with the fulminant disease. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is a novel human coronavirus that has caused a swiftly spreading disease named covid-19, which was defined as a pandemic by the world health organization in february 2020 [1, 2] . millions have been infected worldwide and hundreds of thousands have died, with the global estimated totals changing rapidly over time. most people (about 80%) who acquire covid-19 experience mild to moderate symptoms and recover without special treatment [3] . however, a subgroup of patients develops a severe form of the disease with a high mortality rate, which is hallmarked by severe respiratory distress syndrome, sepsis, coagulation disorder, or even multiple organ failure [4] [5] [6] [7] . although the exact pathogenesis of the virus-induced damage is not yet known, several mechanisms have been proposed. the surface spike protein of sars-cov-2 binds to the angiotensinconverting enzyme-2 (ace2) receptors [8] [9] [10] expressed in the alveolar epithelia of the lungs, kidneys, hepatocytes, epithelial cells of the bile ducts, the vascular endothelium, and other cells [11] [12] [13] [14] . other potential sars-cov-2 receptors, such as cd147 or cd26, have also been identified and are expressed in immune cells [15] . accordingly, the sars-cov-2 organotropism extends beyond the respiratory tract [16] . endothelitis, alveolar damage, and thrombotic microangiopathy have been described in the lungs and kidneys, which is accompanied by the infiltration of mononuclear cells and macrophages [12, 17, 18] . an efficient, well-coordinated host immune response is a crucial first-line antiviral defense. in severe covid-19 patients, several studies have documented various degrees of immune dysregulation that affect both innate and adaptive immunity, which may result in immune-mediated tissue injury [19, 20] . the recruitment and activation of immune cells, particularly neutrophils, is accompanied by an exuberant release of pro-inflammatory cytokines and chemokines-a so-called "cytokine storm" [4, 21, 22] . along with a simultaneous decrease in the monocytes, eosinophils, and basophils [20, 23] , marked lymphopenia and the functional exhaustion of cd8 t cells and natural killer (nk) cells have been associated with a severe course of the disease [24] [25] [26] [27] . various prognostic markers for the increased severity and mortality in adult covid-19 disease have been proposed in several heterogeneous studies, including male sex; older age; pre-existing lung, cardiac, renal, and liver disease; hypertension; obesity [28] [29] [30] [31] . individually, laboratory abnormalities have been reported in covid-19 patients, including an elevation of inflammatory markers and liver enzymes, abnormal renal function tests, and an elevated serum soluble interleukin 2 (il-2) receptor (sil2r) and il-6. coagulopathy associated with elevated d-dimers has also been frequently observed among severe covid-19 patients [4, 32] . the current clinical knowledge pool for research on covid-19 disease relies on largely heterogeneous cohort studies of various scales and individual objectives. therefore, we chose to prospectively follow all patients with a verified sars-cov-2 infection admitted to our hospital and construct a rich dataset derived from a single-center cohort of patients that was stratified based on disease severity. the dataset also featured key clinical information and a complex high-parametric laboratory profile of all patients spanning metabolic, hematologic, and immune parameters. the cohort was followed longitudinally throughout the disease. the studied parameters were selected based on previously published covid-19 data and the best local clinical practice, spanning both features important for disease pathogenesis and markers helpful for the clinical management of the patients. all patients admitted to the university hospital in motol, prague, czech republic, between march and may 2020 who tested positive for the presence of sars-cov-2 rna in a nasopharyngeal swab using a reverse real-time polymerase chain reaction (rtpcr) were included in this study. patients were retrospectively divided into subcohorts based on the severity of the disease course as follows: patients with a moderate course of the disease had clinical signs of pneumonia (cough and auscultation) and verified infiltration on a chest x-ray or computed tomography; patients with a severe course of the disease required mechanical ventilation; patients with a mild course of the disease did not fulfill any of the above criteria, but had a positive sars-cov-2 nasopharyngeal swab rtpcr; patients with a fatal course of the disease died during the study. patients included in the severe cohort were only included in the study if they exhibited a stable remission of symptoms allowing for their transfer from the intensive care unit. a summary of the overall cohort, including the cohort size, age, and sex, as well as the basic clinical characteristics of each subcohort, is given in table 1 . this study was carried out following the recommendations of the ethical committee of the second faculty of medicine, charles university in prague and the university hospital in motol, czech republic. the protocol was approved by the ethical committee. all subjects gave written informed consent following the declaration of helsinki. routine in-house methods were used for an evaluation of all laboratory parameters included in this study. details concerning individual laboratory methods are available from the authors upon request. for an evaluation of the serum anti-sars-cov-2 antibodies, the edi™ novel coronavirus covid-19 immunoglobulin m (igm) or igg elisa kits (edi epitope diagnostics, inc., san diego, ca, usa) were used and the data were acquired using a quanta-lyser 3000 (inova diagnostics, san diego, ca, usa). lymphocyte subsets were evaluated using flow cytometry. full blood was drawn into ethylenediaminetetraacetic acid (edta)-coated tubes and then stained according to the manufacturer's instructions using the dryflowex asc screen kit, the dryflowex act t screen kit, and the excellyse i lysing kit (all from exbio, prague, czech republic). data were acquired on a bd lsr ii fortessa (bd biosciences, franklin lakes, nj, usa) and analyzed using flowjo software (version 10; treestar, ashland, or, usa). tissue samples were fixed in neutral buffered 4% formaldehyde and embedded in paraffin. for the immunohistochemistry, 3 µm thin histological sections were used. an anti-cd8 antibody (clone c8/144b, agilent, santa clara, ca, usa, dilution 1:200, pre-treatment: heating in a buffer at ph9 in a water bath) and anti-il-6 antibody (monoclonal antibody against recombinant full-length protein corresponding to human il-6 aa 29-212, the clone was not specified by the antibody producer, abcam, cambridge, uk, dilution 1:2000, pre-treatment: heating in a buffer at ph6 in a water bath) were employed. detection was performed using a one-step micropolymeric non-biotin system (bio sb, santa barbara, ca, usa) with a peroxidase complex and 3,3 -diaminobenzidine tetra-hydrochloride (dab). the nuclei were counterstained with hematoxylin. a sample from lung transplantation donor lungs was used as a healthy control for the lung necropsy. a sample from a healthy liver biopsy was used as a healthy control for the liver necropsy. in the boxplots used throughout the manuscript, boxes depict the 25th and 75th percentiles (first and third quartile, respectively) and whiskers depict the 2.5-97.5th percentiles. student's t-tests with holm's multiple comparison adjustment were used for an assessment of the differences between groups. in the correlation graphs in figure 4 and the correlation matrices in figure 5 , spearman's correlation was used. statistical analyses and the generation of graphs were performed in the statistical language and environment r, version 3.6.3, using the "ggplot2," "ggpubr," and "corrplot" packages; graphpad prism figure 2 were constructed using the spice 6 software [33] . of the 37 patients included in this study, 10 (27%) had a mild course of covid-19 characterized by a few clinical symptoms, particularly a fever, myalgia, arthralgia, or a general malaise. patients with a moderate course of the disease (n = 13, 35%) were chiefly characterized by a cough, dyspnea, and the necessity of oxygen therapy; however, they did not require mechanical ventilation. patients who suffered from a severe course of the disease (n = 7, 19%) required admission to an intensive care unit and mechanical ventilation, and in several cases, developed systemic inflammation with multi-organ failure. finally, seven patients (19%) suffered from a fatal course of the disease after an average of 6.8 ± 10.4 days (mean ± sd) following their admission to the hospital. the specific characteristics of the cohort, found in table 1 , show that the trend of a severe course of the disease mostly occurred in the elderly, while the younger patients experienced predominantly mild symptoms. while patients with a mild course of the disease only rarely showed an overt elevation of inflammatory markers, such as the c-reactive protein (crp), procalcitonin, or ferritin ( figure 1a ), crp and ferritin were markedly elevated in the moderate, severe, and fatal subcohorts. high serum il-6 levels reaching thousands of picograms per milliliter and high procalcitonin were characteristic of severe patients who required mechanical ventilation and had multi-organ involvement. interestingly, patients with a fatal course of covid-19 failed to display an inflammatory response at similarly high levels, which may have contributed to their eventual demise; however, they averaged exceedingly elevated sil2r levels. crp was the highest at the beginning of the disease and decreased rapidly in the first 20 days from the onset of symptoms ( figure 1b) ; it flared up with multi-organ involvement in the delayed phase of severely ill patients. in contrast, sil2r remained mostly constant throughout the disease, regardless of the intermittent elevations in crp levels. therefore, we observed a gradual increase of crp, procalcitonin, ferritin, and serum il-6 corresponding to the severity of the disease; however, these markers displayed a relative failure to upregulate in patients with a fatal course, who instead displayed high sil2r and d-dimers ( figure 1c ). the hepatotrophic affinity of sars-cov-2 and its hepatopathic qualities have been demonstrated [34] . correspondingly, we observed an elevation of liver enzymes, i.e., aspartate transaminase (ast), alanine transaminase (alt), lactate dehydrogenase (ld), or bilirubin, throughout our cohort (figure 2a ). the elevation of alt peaked at around day 15 from the onset of symptoms and then gradually subsided ( figure 2b) . additionally, we also registered a significant elevation of gamma-glutamyl transferase (ggt) and alkaline phosphatase (alp) that was the most pronounced in patients with severe covid-19 ( figure 2a ) and had a more delayed onset than alt, starting after day 20 on average ( figure 2b ). indeed, while even patients with a mild course of the disease showed some elevation of liver enzymes above a healthy age-and sex-matched reference range ( figure 2d ), five out of seven patients with a severe course displayed a consistent elevation of all four enzymes and fulfilled the more stringent laboratory criteria for either a biliary lesion (defined as the elevation of ggt or alp > 2× the healthy age-and sex-matched reference range values) or both biliary and hepatic (ast or alt > 3× the reference value) damage ( figure 2e ). of all the enzymes, the elevation of ggt was the most significant and characteristic for severe, but non-fatal covid-19, with an average of 15 times the healthy age-and sex-matched reference range ( figure 2f ). cholestasis, which is the collateral feature of biliary injury, was indeed apparent in a liver autopsy from a patient with a fatal course of covid-19 ( figure 2g ), including a clot in the biliary tract ( figure 2h ), along with substantial steatosis, despite no previous history of liver disease. discrete production of il-6 was detected in the liver ( figure 2i) , which was not present in the liver of a non-covid-19 control (supplementary figure s1b) , suggesting a role for il-6 in tissue inflammation and the resulting damage. the activation of innate immunity is the body's first-line defense against all types of infectious pathogens, including viruses, although the functional integrity of adaptive immune cells, such as cytotoxic cd8 t cells and nk cells, is the principal component for the final clearance of viral infections. similar to previous studies [4] , we found a stark elevation of neutrophils in patients with moderate and especially severe courses of the disease, with a significantly elevated proportion of immature granulocytes ( figure 3a ). of note, eosinophils were also elevated in several patients with a severe course of the disease. we did not note a major difference in total serum igg levels between the subcohorts ( figure 3b ); however, patients with a fatal course of covid-19 exhibited significant igg hypergammaglobulinemia. the temporal development of specific anti-sars-cov-2 antibodies was apparent throughout the disease. specific igm antibodies appeared in the first 10 (5-15) days from the onset of symptoms and disappeared after day 15 (13) (14) (15) (16) (17) (18) (19) (20) (21) (22) (23) (24) , although, in some patients, they remained present for over 30 days ( figure 3c ). virtually concurrent igm and igg seroconversions were apparent in all patients, where igg antibodies showed a better persistence and even gradual increase over time. lymphopenia is a well-described negative prognostic factor associated with a severe course of covid-19 [4] . as part of the lymphopenia in our patients, we specifically noted a decrease of t cells and cd8 t cells in patients with severe and fatal disease courses ( figure 3d ). these cd8 t cells were highly activated, co-expressing the surface markers cd38 and human leukocyte antigen -dr isotype (hla-dr), and were significantly correlated with serum il-6 levels and the marker of biliary damage, namely, ggt ( figure 3e ). while numerous cd8 t cells were found to infiltrate the lungs with histologic signs of interstitial pneumonia in one patient who died from respiratory insufficiency, no such infiltration was found in his liver ( figure 3f ), despite the cholestasis and steatosis shown in figure 2g . whereas the humoral immune response displayed within the first 20 days from the onset of the disease led to the fast decrease of plasmablasts detected in the peripheral blood (cd45+, cd19+, cd27hi, cd38hi), the activation of cd8 t cells persisted for over 40 days ( figure 3g ). the trends of the immune response to covid-19 are summarized in figure 3h . most markers of inflammation, the immune response, and liver damage presented in patients with a fatal course of covid-19 so far seem mostly on par with those seen in patients with a moderate form of the disease, suggesting a weaker response to the infection compared to severely ill patients, which resulted in the patients' deaths. other key characteristics of patients with a fatal course of the disease seen in our study were a mineral disbalance, particularly hypocalcemia, and renal insufficiency, with elevated serum urea and creatinine ( figure 4a ). although elevated urea and creatinine levels were also present in some moderately and severely ill patients, these tended to normalize eventually ( figure 4b ). although serum il-6 was not particularly high in fatally ill patients ( figure 1a) , there was a substantial production of il-6 in the lungs, which was driven by interstitially positioned leukocytes ( figure 4c ). pneumonia and acute respiratory distress syndrome were accompanied by numerous thrombi (figure 4d ), along with high plasma d-dimers. the trends of calcemia and markers of kidney failure in covid-19 are summarized in figure 4e . as demonstrated above, common trends arose when studying the immune response against the sars-cov-2 virus and the different facets of its pathogenicity against humans, as summarized in the trend graphs of figures 1-4 . to characterize the complexity of the differences between patients with an efficient, well-coordinated response to the infection, and therefore, only a mild course of the disease, and patients with a fatal course of covid-19, we constructed correlation matrices of selected laboratory parameters ( figure 5) . in patients with a mild course of covid-19 ( figure 5a ), we found a cluster of positively intercorrelated hematological parameters, such as the overall leukocyte count, neutrophils, and immature neutrophils, but also, interestingly, lymphocytes and t cells. markers of inflammation, such as crp, procalcitonin, il-6, and sil2r, positively correlated with the humoral immune responseserum igg, iga, igm, and specific anti-sars-cov-2 antibodies-clearly showing a well-orchestrated immune response of both the innate and humoral adaptive immunity. in contrast, patients with fatal covid-19 ( figure 5b ) displayed a negative correlation between leukocytes and lymphocytes, and their inflammatory markers increased with markers of organ failure (liver enzymes, amylase, ggt, urea, and creatinine) and cytotoxic cellular immunity (activated cd38+ hla-dr+ cd8 t cells) instead. interestingly, while sil2r is a marker of inflammation, it showed the opposite trend compared to crp, procalcitonin, il-6, or ferritin, which may be driven by its unique elevation in patients with a fatal course of the disease, as seen in figure 1a . covid-19 is a multifaceted disease with a striking stratification of the severity spectrum. as a contribution to the current knowledge pool, our report describes a representative cohort of covid-19 patients hospitalized during the pandemic in a large czech hospital. the distribution of mild, moderate, severe, and fatal courses of the disease aligns with previously described cohorts [3, 7, 22] . similar to others, we observed a correlation between a set of inflammatory markers, crp, procalcitonin, ferritin, and serum il-6, and additionally note that fatal cases failed to mount the corresponding elevation of these parameters, suggesting either the exhaustion or suppression of these key inflammatory components. instead, patients with a fatal course of the disease showed high sil2r and d-dimers. although unspecific, as a marker of t cell activation, sil2r has been shown to identify patients with multi-organ sarcoidosis [35] in a similar fashion to our patients with fatal covid-19. the elevation of d-dimers accompanies a hypercoagulation state that manifests as macroand microvascular thrombotic complications in severe covid-19 patients [12, 17, 36] , and has been implied as an independent marker of increased mortality [4, 32] . indeed, here we show venous thrombi in the lungs of a deceased covid-19 patient. furthermore, by directly demonstrating the presence of il-6-producing cells and cd8+ t cells in the lungs, we document a cellular inflammation-related mechanism of lung damage beyond the systemic cytokine storm. abnormalities in the white blood count, i.e., lymphopenia with marked neutrophilia, are now well-established features of severe covid-19 that we can confirm in our cohort [20, 24] . additionally, we describe a marked shift toward immature granulocyte forms, which became more pronounced with increasing severity of the disease, and a stark decline in both the mature neutrophil and their precursor counts was found in the fatal courses. the expansion of developing neutrophils in patients with severe covid-19 was recently identified through single-cell rna sequencing [37] and their reduction may imply a primary failure to efficiently recruit these innate immune responders. moreover, the severe and fatal cases displayed profound t cell, and particularly cd8 t cell, depression, but an unusual presence of activated cd38+ hla-dr+ cd8 t cells. this reflects the observations that t cells express one of the sars-cov-2 receptors cd147 [15] , rendering the t cells susceptible to viral entry, and that the infection is associated with a reduction of the naive cd8 t cell percentage [38] . additionally, activated t cells are more permissive to viral entry and replication [39] . the lymphopenia observed in covid-19 may, in part, arise as a result of il-2 signaling inhibition due to the increased soluble il-2 receptor seen in ours and other cohorts [40] . taken together, t lymphocyte damage is likely an important aspect of clinical deterioration in covid19 . hepatopathy has been reported in 16-53% of symptomatic patients with covid-19 [6, 41] . although severe liver dysfunction has been described, the liver injury appears to be mild and transient in the majority of patients, with the median transaminase level remaining lower than twice the upper reference [34, 42] , which corresponds well with our mild cohort. the elevation of ggt, which is a marker of cholangiocyte injury, has only rarely been reported in covid-19 so far [34, 43] . interestingly, in our severe, but not fatal, subgroup, we observed an excessive increase of ggt that was strikingly disproportionate to the increase of alt and ast. the progression to severe disease has previously been associated with predominantly hepatic (elevated alt and ast) or mixed hepatic and biliary (elevated ggt and alp) types of liver injury [43] . in our severe cohort, biliary or mixed biliary and hepatic damage was found in the majority of patients. therefore, we suggest that in covid-19-related hepatopathy with biliary injury, the predominant elevation of ggt may represent a new independent negative prognostic marker. although the hepatopathy and cholestasis present in our cohort of patients may be, at least in part, of hypoxemic or drug-induced origin, the permissiveness of hepatocytes and cholangiocytes to sars-cov-2 entry has also been documented [11, 44] . therefore, a direct viral-induced injury to these cells is feasible. to the best of our knowledge, no direct evidence for pro-inflammatory cytokine involvement in the hepatopathy displayed in patients with covid-19 has been reported. the infiltration of il-6-producing cells into liver sinusoids and the interstitium may accelerate the production of other markers of inflammation. however, their relative scarcity and the lack of infiltrating cd8 t cells suggests that immune cells, unlike in the lungs, are not the main drivers of pathology in covid-19 liver disease, despite the correlation between activated cd8 t cells and serum ggt levels. overwhelming evidence thus points to the multi-organ affinity of the virus, which also extends to the kidneys [16] . indeed, our finding of elevated markers of kidney damage in patients with a more severe course of the disease echoes the data from china, where high creatinine and acute kidney injury were risk factors for in-hospital death [45, 46] . however, the observed renal pathophysiology is likely multifactorial, involving hypoxemic, hypovolemic, thrombotic, and medication-induced insults. a comprehensive mapping of markers of the immune and metabolic response in our cohort illustratively documented its uncoordinated orchestration, which was highlighted in the comparison of mild and fatal cases. while systems biology approaches may help to decipher the pathophysiology of covid-19, especially due to its multi-organ affinity, limitations imposed by heterogeneous cohorts, temporal changes in examined parameters, and interindividual variability due to comorbidities and medication should be kept in mind. these are indeed the main limitations of ours and most other published studies on covid-19. in summary, we demonstrated the complexity of immune and metabolic disturbances in covid-19 patients. our experiments contribute to the current understanding of the nature of sars-cov-2-driven immunopathology and tissue injury, particularly the systemic inflammation, lymphopenia with t cell activation, and organ infiltration. we observed that severe covid-19-related hepatopathy may be associated with a marked biliary lesion, which was hallmarked by a stark elevation of ggt, and suggest that this enzyme may represent an additional negative prognostic marker. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/9/3000/s1. figure s1 : healthy control biopsy. world health organization coronavirus disease (covid-19) pandemic clinical features of patients infected with 2019 novel coronavirus in 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 course and outcomes of critically ill patients with sars-cov-2 pneumonia in wuhan, china: a single-centered, retrospective, observational study clinical characteristics of 113 deceased patients with coronavirus disease 2019: retrospective study structure analysis of the receptor binding of 2019-ncov functional assessment of cell entry and receptor usage for sars-cov-2 and other lineage b betacoronaviruses cell entry mechanisms of sars-cov-2 specific ace2 expression in cholangiocytes may cause liver damage after 2019-ncov infection endothelial cell infection and endotheliitis in covid-19 high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa single-cell rna-seq data analysis on the receptor ace2 expression reveals the potential risk of different human organs vulnerable to 2019-ncov infection. front distribution of ace2, cd147, cd26 and other sars-cov-2 associated molecules in tissues and immune cells in health and in asthma, copd, obesity, hypertension, and covid-19 risk factors multiorgan and renal tropism of sars-cov-2 pulmonary vascular endothelialitis, thrombosis, and angiogenesis in covid-19 pulmonary post-mortem findings in a series of covid-19 cases from northern italy: a two-centre descriptive study immune dysfunction leads to mortality and organ injury in patients with covid-19 in china: insights from ers-covid-19 study dysregulation of immune response in patients with covid-19 in wuhan covid-19: immunopathology and its implications for therapy correlation analysis between disease severity and inflammation-related parameters in patients with covid-19 pneumonia immune phenotyping based on neutrophil-to-lymphocyte ratio and igg predicts disease severity and outcome for patients with covid-19 pathological findings of covid-19 associated with acute respiratory distress syndrome elevated exhaustion levels and reduced functional diversity of t cells in peripheral blood may predict severe progression in covid-19 patients functional exhaustion of antiviral lymphocytes in covid-19 patients predictive symptoms and comorbidities for severe covid-19 and intensive care unit admission: a systematic review and meta-analysis risk factors for severity and mortality in adult covid-19 inpatients in wuhan laboratory characteristics of patients infected with the novel sars-cov-2 virus risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in spice: exploration and analysis of post-cytometric complex multivariate datasets liver impairment in covid-19 patients: a retrospective analysis of 115 cases from a single centre in wuhan city serum soluble interleukin-2 receptor level is more sensitive than angiotensin-converting enzyme or lysozyme for diagnosis of sarcoidosis and may be a marker of multiple organ involvement incidence of thrombotic complications in critically ill icu patients with covid-19 a single-cell atlas of the peripheral immune response in patients with severe covid-19 flow cytometry identifies risk factors and dynamic changes in patients with covid-19 restrictions to hiv-1 replication in resting cd4 + t lymphocytes potential contribution of increased soluble il-2r to lymphopenia in covid-19 patients liver injury in covid-19: management and challenges covid-19 and the liver: little cause for concern covid-19: abnormal liver function tests a human pluripotent stem cell-based platform to study sars-cov-2 tropism and model virus infection in human cells and organoids 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 we would like to thank the biotechnological company exbio (prague, czech republic) for providing the prefabricated lymphocyte phenotyping kits free of charge as part of the global anti-covid-19 initiative. we would also like to thank all the patients included in this study, their attending physicians, and hospital staff for their hard work during the covid-19 pandemic. the authors declare no conflict of interest. key: cord-011426-jn29kica authors: portero de la cruz, silvia; cebrino, jesús; herruzo, javier; vaquero-abellán, manuel title: a multicenter study into burnout, perceived stress, job satisfaction, coping strategies, and general health among emergency department nursing staff date: 2020-04-02 journal: j clin med doi: 10.3390/jcm9041007 sha: doc_id: 11426 cord_uid: jn29kica burnout is a major problem among nurses working in emergency departments and is closely related to a high turnover of personnel, nursing errors, and patient dissatisfaction. the aims of this study were to estimate burnout, perceived stress, job satisfaction, coping and general health levels experienced by nurses working in emergency departments in spain and to analyze the relationships between sociodemographic, occupational, and psychological variables and the occurrence of burnout syndrome among these professionals. a cross-sectional study was conducted in four emergency departments in andalusia (spain) from march to december 2016. the study sample was composed of n = 171 nurses. an ad hoc questionnaire was prepared to collect sociodemographic and work data, and the maslach burnout inventory, the perceived stress scale, the font–roja questionnaire, the brief cope orientation to problem experience and the general health questionnaire were used. the prevalence of high burnout was 8.19%. the levels of perceived stress and job satisfaction were moderate. the most frequent clinical manifestations were social dysfunction and somatic symptoms, and problem-focused coping was the strategy most used by nurses. lack of physical exercise, gender, years worked at an emergency department, anxiety, social dysfunction, and avoidance coping were significant predictors of the dimensions of burnout. burnout is a psychological syndrome emerging as a prolonged response to chronic interpersonal stressors at work. the three key dimensions of this response are: (i) emotional exhaustion (ee), or a loss of enthusiasm in one's work; (ii) depersonalization (dp), or an impersonal response to patients; and (iii) personal accomplishment (pa), or a feeling of carrying out one's job successfully [1, 2] . burnout has been included in the 11 th revision of the international classification of diseases as an occupational phenomenon [3] affecting a broad spectrum of workers [4] . healthcare professionals are more likely to develop burnout [5] , and nurses in particular are among the major risk groups [6] . studies have reported high values in this respect for nurses [7, 8] . however, the various clinical contexts differently affect the nurses' susceptibility to burnout [9] [10] [11] . it is estimated that 26% of nursing professionals working in emergency departments (eds) suffer from burnout, defined as a state of depletion of resources of an employee as a result of negative perception per year and a reference population of 461,078 inhabitants; ed 3 has 48,000 visits per year and a reference population of 62,234 inhabitants; ed 4 has 18,000 visits per year and a reference population of 24,287 inhabitants. in order to assess the suitability of the study population, the required sample size was calculated using epidat version 3.1 (general directorate of public health, galicia, spain). we considered a 95% confidence level, an absolute precision of 3%, and a prevalence of burnout among ed nursing staff of 26% [12] . with these data, the estimated minimum sample size was 169 subjects. 212 cover letters with the questionnaires were sent in sealed envelopes to all the nurses in the four eds selected. this number comprised the total number of nurses employed by the four eds at the start of the study which complied with the inclusion criteria, which included all the active nurses during data collection who had worked at the ed for at least one year. the exclusion criteria were nurses on sick leave or unpaid leave during data collection. in the end, 171 questionnaires were completed (80.66% response rate). the data were collected from march to december 2016. the study data were compiled for the following sociodemographic, occupational, and psychological variables: sociodemographic variables, including sex (male, female), age (years), marital status (single, married, separated/divorced, widowed), daily physical exercise (yes, no), and daily tobacco use (yes, no). the occupational variables included type of employment contract (permanent, indefinite, part-time), time of service at the ed (years), and work experience (years). the psychological variables were burnout, perceived stress, job satisfaction, coping strategies, and general health. burnout syndrome was measured using the maslach burnout syndrome (mbi) [37] adapted for the spanish population [38] . this instrument contains 22 items scored on a seven-point likert response scale ranging from 0 (never) to 6 (every day). the mbi result is presented with reference to three dimensions: ee (nine items), dp (five items), and pa (eight items). the dimensions were categorized into low, average, and high levels considering the cut-off points established previously in the literature [39, 40] : ee: low: 0-18, medium 19-26, high: ≥ 27; dp: low: 0-5, moderate: 6-9, high: ≥ 10; and pa: low: 0-33, moderate: 34-39, high: ≥ 40. low scores for ee and dp and high ones for pa indicate the absence of burnout. the rest of the cases are indicative of burnout (high level of burnout was defined by high scores for ee and dp and low ones for pa, and moderate level of burnout was determined by the rest of the cases). the following reliability coefficients (α) for the mbi scales were calculated: ee (α = 0.90), dp (α = 0.60), and pa (α = 0.81). perceived stress was measured using the perceived stress scale [41] adapted for the spanish population [42] . the main characteristic of perceived stress is that the response of an individual is not based exclusively on the characteristics of the stimulus, but is greatly influenced by personal and contextual factors [42] . this tool evaluates the degree to which individuals believe their life has been unpredictable, uncontrollable, and overloaded over the previous month. the assessed items are general in nature rather than focusing on specific events or experiences, and it contains 14 items scored on a five-point likert response scale ranging from 0 (never) to 4 (very often). the overall perceived stress is obtained by adding the scores of the 14 items. the results range from 0 to 56 points. perceived stress increases with higher scores. a score between 20-22 points is considered a level of perceived stress within the normal range [42] . the internal consistency value measured in terms of the cronbach's alpha for the perceived stress scale was 0.73. the questionnaire used to assess job satisfaction was the font-roja questionnaire [43] . it consists of 24 items and explores 9 dimensions that determine a professional's level of satisfaction: job satisfaction, work-related tension, professional competence, job pressure, professional promotion, interpersonal relationship with their superiors, interpersonal relationship with coworkers, extrinsic characteristics of status, and job monotony. each item is valued using a likert scale, with values ranging from 1 (totally disagree) to 5 (totally agree). the overall job satisfaction is obtained by the addition of the scores of the 24 responses and ranges from 24 to 120 points. the higher the score, the greater the job satisfaction. in this study, the cronbach's alpha coefficient was 0.83 for the overall job satisfaction. the spanish version [44] of the brief cope [45] was used to evaluate how individuals cope with stressful situations. coping is defined as "constantly changing cognitive and behavioral efforts to manage specific external and/or internal demands that are appraised as taxing or exceeding the resources of the person" [30] . this questionnaire is made up of 28 items on a four-point likert scale from 0 (i absolutely never do this) to 3 (i do this often). the items are grouped into 14 subscales measuring 3 strategies: problem-focused coping (active coping, planning, and search for instrumental support), emotion-focused coping (search for emotional support, positive reinterpretation, negation, acceptance, religion, and humor), and avoidance coping (self-distraction, venting, behavior disconnection, substance use, self-blame). a higher score indicates a higher use of the coping strategy. cronbach's alpha coefficients for the 3 coping strategies were: α = 0.83 (problem-focused coping), α = 0.85 (emotion-focused coping), and α = 0.90 (avoidance coping). health status was measured using the general health questionnaire (ghq) [46] validated for the spanish population [47] . this tool contains 28 items in 4 subscales referring to somatic symptoms (7 items), anxiety (7 items), social dysfunction (7 items), and depression (7 items). answers follow a 4-point likert scale, ranging from 0 (less than usual) to 3 (much more than usual). the total score for each scale ranged from 0 to 21 points. the total score of the ghq ranged from 0 to 84 points. a higher score is related to worse health status. the following reliability coefficients (α) for the total score of the ghq and for the scales were calculated: α = 0.88 (total ghq), α = 0.85 (somatic symptoms), α = 0.81 (anxiety), α = 0.85 (social dysfunction), and α = 0.90 (depression). data research is available as supplementary file. the study was approved by the clinical research ethics committee (approval number 249, reference 3050). a cover letter explaining the voluntary and confidential nature of the study was delivered to all ed healthcare personnel. a descriptive analysis was performed using the means and the standard deviations for the quantitative variables, and frequencies and percentages for the categorical variables. the kolmogorov-smirnov test was used to check the normality of the variables. student's t-, mann-whitney, analysis of variance and kruskal-wallis tests were used to analyze the relationships between the sociodemographic, occupational, and psychological characteristics and the mbi dimensions. correlations between the quantitative variables were tested using the pearson correlation and the spearman's rho tests. three univariate linear regressions were created in order to assess the relationship between the sociodemographic, occupational, and psychological characteristics and each mbi dimension. those variables that showed a statistically significant relationship with each of the considered dimensions (p < 0.05) were subsequently included in a multivariate linear regression model. in this way, 3 elimination multiple linear regression models were created for each mbi dimension (ee, dp, pa). for purposes of the multivariate analysis, the variables were reorganized as follows: marital status (married, not married) and type of employment contract (permanent, non-permanent). all the results were considered statistically significant with the p-value < 0.05. the statistical analyses were performed with statistical package g-stat v.2.0 (glaxosmithkline s.a., madrid, spain). a total of 171 nurses participated in the study. the participants' mean age was 47.85 (8.11) years, and 73.10% were women. of the group, 60.23% were single, 48.54% did daily physical exercise, and 67.25% had a permanent contract. other sociodemographic and work characteristics are shown in table 1 . as regards the levels of burnout (figure 1) , ed nurses had a higher prevalence of low levels of ee (59.65%) and high levels of dp (43.27%) and pa (53.22%). the prevalence of high level of burnout was 8.19%. as shown in table 2 , the average perceived stress and job satisfaction scores among all the workers were found to be 21.30 (5.94) and 67.19 (6.98) points, respectively. higher scores of ee were positively correlated with anxiety (p = 0.001) and social dysfunction (p = 0.002). a significant negative correlation was also found between perceived stress and job satisfaction (p = 0.0004). burnout variables ee and dp and the subscales corresponding to the ghq showed positive relationships with avoidance coping (p < 0.01 and p < 0.001, respectively). problem-focused coping was negatively correlated with depression (p = 0.003) and social dysfunction (p = 0.0002). it should also be noted that a significant positive correlation was found between emotion-focused coping and depression (p = 0.002). the differences between the average burnout (ee, dp, and pa) scores were evaluated according to the participants' sociodemographic data and occupational characteristics. accordingly, those who did not take part in daily physical exercise had higher mean dp (p = 0.005) scores. there were negative significant relationships between pa and age (p = 0.03), time of service at the ed (p = 0.03), and work experience (p = 0.02) ( table 3) . table 4 shows the multivariate linear regression models obtained for each of the mbi dimensions. the results indicate that the use of avoidance coping (p = 0.03), anxiety (p = 0.02), social dysfunction (p = 0.02), and being female (p = 0.01) were statistically significant predictors of ee. dp was determined by the absence of daily physical exercise (p = 0.006), being female (p = 0.01), and the use of avoidance coping (p = 0.03). pa seems to be influenced by the years worked at eds (p = 0.03). in this study, 21.05% and 43.27% of the participants had high levels of ee and dp, respectively. 26.31% had low pa. among the nursing staff, the prevalence of each of the 3 dimensions of burnout according to the mbi range was as follows: high level of ee (20-44%), high level of dp (23-51%), and low level of pa (15-44%) [11] . regarding the prevalence of burnout, the study carried out in ed nurses showed that 3.40% suffered from high levels of burnout [48] . this prevalence is more than half than that obtained in our study. the participants' perceived stress score was within the normal range, similar to the results of mirhagi and sarabien [49] . however, lower scores have been reported in the literature. hutchinson et al. [50] found that the average score of perceived stress among ed medical personnel was 15.53 points, and wong et al. [51] reported 12.30 points. these variations may be due to the use of the 10-item version of the perceived stress scale. although the perceived stress score obtained was not high, we consider that the level of perceived stress is in fact higher among ed nursing professionals due to lack of personnel, work overload, shift work, role ambiguity, lack of autonomy, rapid technological changes, and increased pressure in decision-making [52, 53] . despite the fact that the impact of working in an ed on the level of stress and burnout among nurses has now been established [54] [55] [56] , less is known about its impact on job satisfaction. in the present study, it was found that the level of job satisfaction among the participants was moderate, which is consistent with another study [28] . in the field of health, most of the studies present similar results: medium-high level of job satisfaction in medical staff [57] and lower levels among nursing personnel [58, 59] . in eds, nurses show a higher degree of dissatisfaction than nurses working in other specialties, due mainly to understaffing and poor professional status [60] . in addition, we found a negative correlation between job satisfaction and perceived stress, which matched results from other studies [61, 62] . in the current study, somatic symptoms and social dysfunction were the most frequent clinical manifestations among ed nurses. this is consistent with the results of another study carried out among emergency and intensive nursing staff [63] . the use of adaptive coping styles produces a positive effect on physical and psychological well-being, management of stress, and overall performance among healthcare professionals [64] , which is related to an improvement in the quality of care, greater patient safety, and a fall in health service costs [65] . this is congruent with our results that showed that the use of problem-focused coping reduced both social dysfunction and anxiety and depressive symptomatology. we found that the most commonly used coping strategy was problem-focused coping and the least common was avoidance coping, as in similar studies [18, 64, [66] [67] [68] . the use of avoidance coping may be explained by the low level of personal suffering due to the high turnover rate occurring in eds [56] . in the multivariate analysis, ee and dp were determined positively by the use of avoidance coping, which was consistent with the findings of other authors [69, 70] . it should be noted that some authors have long argued that the dp dimension is in fact a coping style [71, 72] . in addition, this type of coping was positively related to somatic symptoms, anxiety, social dysfunction, and depression. these results were similar to those of yates et al. [73] . nevertheless, avoidance coping may be the best option for ed personnel when an event occurs in order to avoid emotional involvement [74] . we found that ee and dp were influenced positively by being a female. however, this result should be viewed with caution due to the sample of our study consisting predominantly of women. while previous studies noted that gender is an important variable in ee and that women experience more burnout than men [75, 76] , there are other studies which suggest that the burnout is not associated with gender in eds [77, 78] . the significantly higher ee scores in women may be due to the social role played by women and their effort to strike a better work-life balance [79] . in addition, work-family conflicts are considered important risk factors in the development of burnout among women [80] . regarding the age of ed nurses, gökçen et al. [81] determined that this was positively related to ee. on the other hand, schooley et al. [75] also found the same relationships and a significant positive relationship with dp. lloyd et al. [82] showed that with age, the level of dp decreased, while the level of pa increased in ed physicians. in the present study, a significant negative relationship was found between age and the pa level, which is due to the fact that, over time, daily work with people tends to lead to feelings of personal inadequacy and low professional self-esteem as a result of the lack of concern for the problems of others and the loss of empathy [81] . in this study, no relationship was found between marital status and the dimensions of burnout. in this, the findings from the literature are again unclear and contradictory. some authors suggest that burnout is associated with people who have no partner [83] , while others argue otherwise [78] and find no relationship between these variables [75] . these disagreements highlight the importance of exploring the role of marital status in the workplace. the role of lifestyles in ed healthcare professionals' burnout levels needs to be studied extensively [79] . furthermore, no relationship was found between smoking and the dimensions of burnout. this result is similar to that obtained in ed physicians [84] . dp, in the multivariate analysis, was determined by the absence of daily physical exercise. likewise, goldberg et al. [85] reported that low levels of physical exercise were a predictor of burnout in ed personnel. it has been suggested that regular physical exercise facilitates psychological detachment from work and increases people's self-efficacy. as a result, ed nurses may feel more able to cope with their work duties and may find the tasks less demanding, which reduces the risk of burnout. in addition, regular physical exercise may result in the body recovering faster after exposure to stress and may induce changes in several neurotransmitters and neuromodulators, leading to a better mood and increased energy, thus reducing the risk of burnout [86] . as regards job characteristics, the correlational analysis showed an inverse relationship between work experience and pa. in the multivariate analysis, pa was influenced negatively by the years worked in eds. working in an ed involves dealing with unexpected situations, patients who have life-threatening pathologies, and more frequent attacks or assaults than other specialized medical units, which may produce lack of assertive skills in nursing professionals and, as a result, low pa [11, 87] . however, here, too, there are conflicting findings. while popa et al. [88] found no relationship between years worked in eds and the level of burnout, other studies have found a significant positive relationship evident in two periods, corresponding to workers in the first two years of their professional career and those with over ten years of experience. in these stages, the relationship with burnout is lower [85, 89] . no differences were found between the type of employment contract and the dimensions of burnout, which was not consistent with the results from garcia et al. [90] , who revealed that ed staff with permanent contracts had a lower level of dp than those with part-time contracts. our study has certain limitations. first of all, because of the cross-sectional study design, it is not possible to establish any cause-effect relationships. secondly, findings may not necessarily be representative, as a convenience sample was used. thirdly, the study was carried out only in the region of andalusia, which may limit the generalization of the results. in further studies, it would be interesting to consider using a wider geographical range, and to use longitudinal research methods and randomized sampling. it is vital for health services to be aware of the relationships between burnout, perceived stress, job satisfaction, coping strategies, and general health. since ed nursing professionals provide a valuable service to the community, the levels of these factors should be taken into account, as they have an important impact on patients, as well as on the general population. understanding the influence that work characteristics have on burnout is crucial to inform policy and practice in designing suitable interventions to prevent illnesses and improve motivation among ed nurses. high burnout affects 8.19% nurses working in the eds of four hospitals in the region of andalusia. perceived stress is within the normal range and job satisfaction level is moderate. problem-focused coping is the most commonly used strategy, and somatic symptoms and social dysfunction are the most frequently experienced clinical manifestations. the absence of physical exercise, gender, years worked in eds, anxiety, social dysfunction, and avoidance coping are the main predictors of burnout. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/4/1007/s1, file s1. research data. understanding the burnout experience: recent research and its implications for psychiatry job burnout international classification of diseases 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among canadian emergency physicians burnout and satisfaction with work-life balance among us physicians relative to the general us population sense of meaning as a predictor of burnout in emergency physicians in israel: a national survey burnout and its correlates in emergency physicians: four years' experience with a wellness booth systematic review of the association between physical activity and burnout assertiveness and its relationship to emotional problems and burnout in healthcare workers occupational burnout levels in emergency medicine-a stage 2 nationwide study and analysis aspectos epidemiológicos del síndrome de burnout en personal sanitario the relationship between psychosocial job stress and burnout in emergency departments: an exploratory study this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank the managers of the participating hospitals for their help in data collection, and the professionals who collaborated in the study. the authors declare no conflict of interest. key: cord-294910-gnc04ax1 authors: nogueira, paulo jorge; de araújo nobre, miguel; costa, andreia; ribeiro, ruy m.; furtado, cristina; bacelar nicolau, leonor; camarinha, catarina; luís, márcia; abrantes, ricardo; vaz carneiro, antónio title: the role of health preconditions on covid-19 deaths in portugal: evidence from surveillance data of the first 20293 infection cases date: 2020-07-24 journal: j clin med doi: 10.3390/jcm9082368 sha: doc_id: 294910 cord_uid: gnc04ax1 background: it is essential to study the effect of potential co-factors on the risk of death in patients infected by covid-19. the identification of risk factors is important to allow more efficient public health and health services strategic interventions with a significant impact on deaths by covid-19. this study aimed to identify factors associated with covid-19 deaths in portugal. methods: a national dataset with the first 20,293 patients infected with covid-19 between 1 january and 21 april 2020 was analyzed. the primary outcome measure was mortality by covid-19, measured (registered and confirmed) by medical doctors serving as health delegates on the daily death registry. a logistic regression model using a generalized linear model was used for estimating odds ratio (or) with 95% confidence intervals (95% ci) for each potential risk indicator. results: a total of 502 infected patients died of covid-19. the risk factors for increased odds of death by covid-19 were: sex (male: or = 1.47, ref = female), age ((56–60) years, or = 6.01; (61–65) years, or = 10.5; (66–70) years, or = 20.4; (71–75) years, or = 34; (76–80) years, or = 50.9; (81–85) years, or = 70.7; (86–90) years, or = 83.2; (91–95) years, or = 91.8; (96–104) years, or = 140.2, ref = (0–55)), cardiac disease (or = 2.86), kidney disorder (or = 2.95), and neuromuscular disorder (or = 1.58), while condition (none (absence of precondition); or = 0.49) was associated with a reduced chance of dying after adjusting for other variables of interest. conclusions: besides age and sex, preconditions justify the risk difference in mortality by covid-19. system for epidemiological surveillance), which is the national database for mandatory diseases and public health problems notifications, managed by dgs, that provided the data fully anonymized. the research team filled the publicly available form and submitted an analysis project. the project was approved by an ethics committee (number 165/2020). the provided data concern information between 1 january and 21 april 2020 on infected cases and deaths by covid-19. a confirmed case was based on a positive polymerase chain reaction test. the primary outcome measure was mortality by covid-19, measured (registered and confirmed) by medical doctors serving as health delegates on the daily death registry. the dataset received on 27 april, encompasses confirmed deaths up to 21 april. potential deaths from cases registered later in the database (in particular between early april and 27 april) are not confirmed and accounted for in the dataset. as referenced above, the data were registered in sinave, an electronic system for epidemiological surveillance of mandatory diseases that includes laboratory notification. sars-cov-2 cases are registered by doctors in sinave allowing the electronic communication with local, regional, and national health authorities [11] . the sars-cov-2 case definition used is based on the world health organization case definition. the sinave allows epidemiological surveillance and epidemiological data registration including laboratory notification, ongoing treatment, hospitalization, existing health preconditions (morbidity), and death registration. individualized health precondition indicator variables were constructed from the two variables provided in the database ("precondition" and "other precondition"). the data retrieved include individuals' demographic characteristics (age, sex, region), covid-19 disease information (death, recovery, still in treatment, hospitalization, intensive care, respiratory support), and preconditions (asthma, cancer, cardiac disease, hematological disorder, diabetes, hiv and other immune deficiency, kidney disorder, liver disorder, neuromuscular disorder, other precondition and none (absence of precondition)). the data on intensive care and respiratory support include a number of individuals with "unknown" status that we considered to not have had these interventions if they were never hospitalized. this work considers as primary outcome mortality among those with a positive molecular test for covid-19 on record. descriptive statistics, such as absolute and relative frequencies, mean, standard deviations, and medians were used to summarize univariate characteristics. bivariable analyses were performed between the outcome variable (death) and variables potentially associated using the x 2 test or the fisher exact test. further bivariate analysis was performed to evaluate the difference in distribution between preconditions and sex, hospitalization, and intensive care. we analyzed regional differences based on the second level of the nomenclature of territorial units for statistical purposes (nuts ii) classification for portugal. logistic regression models were performed using a generalized linear model with binomial error distribution and logit link function to estimate the crude and adjusted odds ratio (or) with 95% confidence intervals (95% ci) for each potential risk factor. the model performance was assessed using the area under the curve (auc) and corresponding 95% confidence intervals (95% ci). the significance level was set at 5%. statistical analysis was performed using r software version 3.6.3 (r foundation for statistical computing, vienna, austria). the available information comprises all 20,293 individuals reported as infected with sars-cov-2 in portugal between 1 january and 21 april 2020. there were 11,903 female (58.7%) and 8390 male cases (41.3%). the average age (standard deviation) of the individuals was 52.1 years (21.3 years), with a majority between 18 and 55 years of age (57.2%, minimum: 0 years; maximum: 104 years). the majority of cases (60%) occurred in the north region (table 1) . considering the geographical region, the distribution of infection rates per 100,000 inhabitants was the following: 341.8 in the north region (12, in terms of health preconditions, the most common observed condition was diabetes (5.1%), followed by neuromuscular (3.4%) and lung disorders (3.4%). there are 502 deaths registered in the database representing an overall lethality of 2.5% of all infected cases at that point (note that cases registered later in the database may not have their final outcome yet). among the deceased, the mean age was 81.5 ± 10.5 and median 84 years, while in the group that recovered or was still in treatment, mean age was 51.3 ± 21.0 years. from the available information, at least 2973 (14.7%) infected cases were hospitalized (the data include 1623 cases with unknown hospitalization status), at least 261 (1.3%) infected cases were subjected to intensive care, and at least 86 (0.4%) had respiratory support (oxygen or ventilator). mortality within the hospitalized cases was 11.1% (330), within those subjected to intensive care 10.3% (27) . at this point, no deaths were registered among those submitted to respiratory support (table 1) . lethality was higher in men (2.97%) than in women (2.13%). lethality increased with age reaching 9.5% in infected cases aged 75 to 85 and 12.61% in infected cases older than 85 years old. considering the geographical region, the highest lethality was observed in the centre region with 3.44% (97 deaths), followed by the north region with 2.61% (319 deaths) and lisbon metropolitan area with 1.74% (74 deaths). most of the preconditions were significantly associated, in bivariate analyses, with the death outcome (lethality), with increased lethality in those carrying the precondition. the highest lethality was observed among those infected with a prior history of cardiac and kidney disorders (table 1) . the results of the analyses between preconditions and sex, hospitalization, and intensive care are depicted in table 2 . most preconditions were significantly more prevalent in men (except for asthma and none). hospitalization of infected cases was associated, albeit weakly, with all preconditions. cardiac disease, kidney, neuromuscular, and hematological disorders had higher chances of hospitalization. most preconditions were associated with the use of intensive care, with cardiac disease, kidney disease, hiv/other immune deficiency, lung disease, and diabetes with higher chances of being submitted to intensive care. the univariable and multivariable logistic regression analysis for the outcome "death" in infected patients with covid-19 is shown in table 3 and figure 1 . table 3 registers three multivariable models: model 1, a model only including patients without preconditions; model 2, for each individual precondition adjusted for age and sex; model 3, the full model. considering the full multivariable model, males exhibited a 47% increase in mortality compared to female patients. concerning age, an overall "j" shape along age was defined by the given parameters (odds ratios). after adjusting for the remaining variables, a less defined "j" shape results. this still means that lethality risk increases with the infected cases' age after adjustment. considering the preconditions, the majority were bivariately associated with mortality. after adjustment, three preconditions (cardiac disease, kidney disease, neuromuscular disorder, and none (absence of precondition)) remained significantly associated. the presence of cardiac disease and kidney disease in the infected cases doubled the chance of mortality by covid-19. the absence of preconditions (none) showed a protective effect, reducing the chance of mortality by covid-19. complementary calculations (multivariable models) were performed with the objective of evaluating the data robustness and model consistency (table 4) . first, we analyzed a model excluding data from the last 3 weeks so that cases with insufficient follow-up were excluded. next, we analyzed a model including only the hospitalized cases, assuming that these cases would have more accurate recording of comorbidities. as shown in table 4 , the results of these two reduced models are consistent with the model for the full dataset in table 3 . the full model performance for predicting covid-19 mortality is illustrated in figure 2 , where an excellent discrimination capacity was observed (auc (95% ci): 0.909 (0.900;0.919)). 1.05 (0.14;5.00) =0.960 0.57 (0.12;2.04) =0.426 * full model with cases up to 31 march (n = 11103 individuals; n = 328 deaths); ** model of hospitalized cases (n = 2973 individuals; n = 230 deaths). the full model performance for predicting covid-19 mortality is illustrated in figure 2 , where an excellent discrimination capacity was observed (auc (95% ci): 0.909 (0.900;0.919)). our study evaluating a national database of more than 20,000 infected individuals constitutes one of the largest population studies on covid-19 to date. a total of 14.7% of patients needed hospitalization, associated with a case fatality rate (cfr) of 11.1%, while 1.3% were admitted to the intensive care unit (icu) with an associated cfr of 10.3%. these figures were somewhat lower when compared to previous systematic reviews, where a 20.3-29.3% and 6.8-13.7% of icu admission and cfr were reported, respectively [12, 13] . this difference may be related with a different stage of the epidemic in portugal at the time of data analysis, and differences in the timing of non-pharmaceutical actions (schools and medical faculties closures and lockdown declared one and two weeks after the first covid-19 case, respectively) [14] , as the majority of studies included in the systematic reviews were from china. moreover, it is important to consider the variations of proportions that were found in other individual studies. this study represents one of the first attempts to understand the lethality of covid-19 in portugal from infected cases, proposing risk factors based on multivariable analysis. the risk factors for lethality by covid-19 were sex (male), advanced age, kidney disorder, cardiac disease and neuromuscular disorder, while the absence of a precondition was associated with a reduced chance of mortality after adjusting for other variables of interest. the principal risk factors for lethality by covid-19 reported in the present study are supported by the literature. the majority of studies report male patients, older patients, and patients with preconditions at an increased risk of infection and mortality irrespective of the region of the globe [12, 15] . a recent meta-analysis of thirteen studies registered male individuals (or = 1.76), age over 65 years old (or = 6.06), smoking habits (or = 2.51), and preconditions including cardiovascular disease (or = 5.19), diabetes (or = 3.68), respiratory disease (or = 3.68), and hypertension (or = 2.72) as significantly higher in critical/mortal patients compared to non-critical patients [15] . men registered a 48% increased risk of death by covid-19 in our study, a result previously observed [9] . we registered an increased prevalence of preconditions in men when compared to women (all preconditions except asthma); while the absence of our study evaluating a national database of more than 20,000 infected individuals constitutes one of the largest population studies on covid-19 to date. a total of 14.7% of patients needed hospitalization, associated with a case fatality rate (cfr) of 11.1%, while 1.3% were admitted to the intensive care unit (icu) with an associated cfr of 10.3%. these figures were somewhat lower when compared to previous systematic reviews, where a 20.3-29.3% and 6.8-13.7% of icu admission and cfr were reported, respectively [12, 13] . this difference may be related with a different stage of the epidemic in portugal at the time of data analysis, and differences in the timing of non-pharmaceutical actions (schools and medical faculties closures and lockdown declared one and two weeks after the first covid-19 case, respectively) [14] , as the majority of studies included in the systematic reviews were from china. moreover, it is important to consider the variations of proportions that were found in other individual studies. this study represents one of the first attempts to understand the lethality of covid-19 in portugal from infected cases, proposing risk factors based on multivariable analysis. the risk factors for lethality by covid-19 were sex (male), advanced age, kidney disorder, cardiac disease and neuromuscular disorder, while the absence of a precondition was associated with a reduced chance of mortality after adjusting for other variables of interest. the principal risk factors for lethality by covid-19 reported in the present study are supported by the literature. the majority of studies report male patients, older patients, and patients with preconditions at an increased risk of infection and mortality irrespective of the region of the globe [12, 15] . a recent meta-analysis of thirteen studies registered male individuals (or = 1.76), age over 65 years old (or = 6.06), smoking habits (or = 2.51), and preconditions including cardiovascular disease (or = 5.19), diabetes (or = 3.68), respiratory disease (or = 3.68), and hypertension (or = 2.72) as significantly higher in critical/mortal patients compared to non-critical patients [15] . men registered a 48% increased risk of death by covid-19 in our study, a result previously observed [9] . we registered an increased prevalence of preconditions in men when compared to women (all preconditions except asthma); while the absence of preconditions was 30% more likely in female individuals. moreover, the fact that women could have stronger responses than men in many infectious pathogens [16] , the likelihood of women to search for health care services more than men [17] , and in the adoption of hygiene practices [18] may further complement the explanation of this result. age was an important predictor for mortality in our study, with lethality-adjusted ors increasing after 55 years of age. moreover, there was a thirty-year difference in the average age between fatal (average age of 81 years) and non-fatal cases (average age of 51 years). age was a predictor for mortality in patients both with and without preconditions, a result supported by a previous multicenter cohort study of 191 individuals where increased odds of in-hospital death were associated with older age (odds ratio 1.10, 95% ci 1.03-1.17, per year increase [19] ). these results are partially explained by the increased burden of pre-conditions in older age groups, where dramatic increases for the prevalence of preconditions (particularly for cardiac disease, kidney disorder, and neuromuscular disorder) were registered in older age groups when compared to individuals of less than 55 years of age. nevertheless, age remained a risk factor for death in our study even in patients without preconditions, as would be expected given that advanced age is a risk factor for death even in the absence of covid-19 infection. indeed, it is possible that the increase in risk of death is more due to aging than to covid-19 infection proper. still, the link between older individuals and the likelihood to develop severe and critical cases of covid-19 has been made before, either due to immunosenescence, malnutrition, or ignoring more easily the early symptoms and consequently missing the best time to seek medical advice [20, 21] . furthermore, several studies registered an important association between increased age and covid-19 severity/fatality [22] [23] [24] [25] [26] . the vulnerability of elderly individuals is illustrated in recent studies. a retrospective observational study investigating mortality in hospitalized patients with covid-19 registered the great vulnerability of patients residing in retirement homes, with older age independently associated with mortality when adjusted for other variables of interest [22] . on the opposite residence condition, the shape of covid-19 vulnerability was estimated based on a random infection of 10% of the population living in private households (excluding individuals living in retirement homes) of 81 countries [27] . in this study, it was estimated that national age and coresidence patterns can alter the vulnerability of a country to covid-19 outbreaks, with direct effects dependent on a country's age structure and indirect effects dependent on the size and age structure of a country's households [27] . the specific comorbidities that emerged as risk factors for mortality in our study (cardiac disease, kidney disorder, and neuromuscular disorder) should be interpreted considering the covid-19 physiopathology. in our study, the prevalence of kidney disease on admission in patients with covid-19 was high and associated with clinical stage decline. the highest in-hospital mortality rate (24.4%) and chances of mortality (2.43-fold increase) were registered for individuals with kidney disease precondition. other studies in patients with kidney disorders recorded comparable results. a meta-analysis of eleven covid-19 studies registered an association between acute kidney injury and a higher risk of mortality of almost 16-fold (or = 15.93), with creatinine levels significantly higher in non-survivors compared to survivors [28] . nevertheless, the meta-analysis reported a high heterogeneity and a difficulty in adjusting for confounders [28] . pathophysiological mechanisms may be involved. considering that co2 is an independent determinant of ph adjusted by alveolar ventilation, the disturbance of the acid-base regulation (through the interplay of bicarbonate buffer and respiratory and renal systems) may induce acid-base imbalance and in this way may pose a life-threatening situation [29] . a second hypothesis consists in the direct kidney infection by sars-cov-2, which recognizes the human angiotensin-converting enzyme 2 as a cellular receptor that allows it to infect different host cells, a mechanism previously expressed by sars-cov virus [30] . this mechanism could explain the particular importance of acute kidney injury during hospitalization, considering the exhibited conditions of proteinuria; hematuria; and elevated levels of either serum creatinine, blood urea nitrogen, or both, rendering a significant increase of in-hospital mortality between 1+ and 5.5-fold [31] . patients with cardiovascular disorder exhibited a nearly 3-fold increase in the chance of dying from covid-19. cardiovascular disease has been consistently reported as one of the main risk factors for covid-19 mortality. two recent meta-analysis reported an odds ratio (95% ci) of 5.19 (3.25; 8.29) (1) and a risk ratio (95% ci) of 2.25 (1.53; 3.29) ) for mortality in cardiovascular disease patients [32] . our results are in concordance with the systematic review by pranata et al. [32] and lower than the systematic review of zheng et al. [15] . a possible reason may be the small study effect on the estimates from zheng et al. [15] , considering the majority of the 13 studies included were of smaller sample size compared to both our study and pranata et al. [32] . nevertheless, the direction of the estimate towards risk is clear. the pathophysiological mechanisms behind this association may be multiple: from severe infection with sars-cov-2, precipitating myocardial infarction, myocarditis, heart failure, and arrhythmias as well as an acute respiratory distress syndrome and renal failure [33, 34] ; through the evolution along with multiorgan failure directly due to sars-cov-2-infected endothelial cells and resulting endothelitis [33] ; to the potential impacts of therapies considering the likely increase in the number of ace 2 receptors and the corresponding increase in the susceptibility [33] . furthermore, the link between pneumonia and cardiovascular complications should be accounted for: recent studies explore/registered myocardial injury during sars-cov-2, secondary to type 2 myocardial infarction, a consequence of increased oxygen demand or reduced oxygen supply during respiratory failure [34] [35] [36] . in this scenario, cytokines microvascular activation can cause not only myocardial injury but also harm other systems involved in covid-19 infections, including the kidneys [37] . consequently, the relation between cardiovascular disease, kidney disease and diabetes should not be ruled out [33, 38] . patients with neuromuscular disorders registered a 41% increase in lethality. the pathophysiological mechanisms related to this association could be: (i) the fact that patients with this precondition are under the use of immunosuppressive therapies and therefore more likely to increase the severity of covid-19 infection [39] ; and (ii) risk of exacerbation of myasthenia gravis and qt prolongation in patients with pre-existing cardiac involvement secondary to the treatment with hydroxychloroquine and azithromycin [40] . however, given that the present database did not provide the patient-specific pharmacological therapies, this question remains open. diabetes, previously registered as a significant risk factor for covid-19 mortality [3] , was not significant in our study when adjusted for other variables of interest. a potential reason for this result might be related to the level of glycemic control. a recent study evaluating the impact of blood glucose control and outcomes of covid-19 in pre-existing type 2 diabetes noted that when adjusting the model for well-controlled blood glucose, a marked lower mortality was registered compared to individuals with poorly controlled blood glucose [41] . data from a national study in portugal with 1688 individuals reported about 82% of portuguese diabetics were pharmacologically medicated and that 60.7% were controlled [42] . however, since we were not able to retrieve the level of glycemic control in diabetic individuals in our study, this question remains open. the strengths of this study include being a population-based study, the large sample size, and the origin of the data. sinave is the electronic platform for notification and cases monitoring of mandatory communicable diseases, allowing the analysis and evaluation of emergent situations, particularly large-scale epidemic outbreaks and pandemics, such as a covid-19. this system allows for the electronic articulation of doctors (who notify cases of illness), health authorities (responsible for epidemiology at local, regional and national levels), and laboratories (cases notification and cases confirmation). sinave allows real-time notification, admitting the implementation of control measures to control and limit the spread of disease and the occurrence of additional cases. the sinave database is relevant for the present covid-19 study by its quality and extensibility since it is based on the information registered by the medical doctor who notified the case. furthermore, it contains all the notified cases up to the extraction of the data due to the interoperability characteristics between the sinave computer application and clinical process computer applications. the limitations of the present study include the absence of potentially important data from the database, missing data, underreporting of mild cases, the impossibility of accounting for the temporal sequence of events, and under-reporting of preconditions. the limitations are presented and discussed in detail below, including the potential bias and corresponding direction. the database did not include reported symptoms and laboratory test results. the existence of unknown values in the data for some outcomes, together with the reporting of preconditions in the medical record, may lead to an underestimation of some risk indicators. it is likely that some of the preconditions were under-reported both in quantity and importance: a particular example is cerebrovascular disease, considered one of the comorbidities with significant impact in covid-19 prognostic [7] , and conspicuously absent from our database. the temporal sequence of events was not taken into account (time elapsed between the onset of symptoms and hospital admission, or time between hospital admission and death), which may imply an underestimation of preconditions. finally, because the patients' clinical observation is still ongoing, many individuals have not reached clinical endpoints (recovery or death). the authors performed complementary calculations to evaluate data robustness and model consistency: the results of these complementary estimations (one model excluding cases from the last 3 weeks and the other model on hospitalized cases) show reasonable consistency with the full multivariable model. a further limitation is related to the data that concern only the initial phase of the pandemic in portugal up to 21 april. the pandemic is still ongoing, registering on june 25 about 40,866 infected individuals and 1555 deaths [43] . nevertheless, no update of the data was made available up to the moment, and therefore the hypothesis of a difference in mortality between the initial and actual phase remains open. the results of the present study registered potential different pathophysiological mechanisms for covid-19 mortality, suggesting the need for a team approach between different medical specialties in order to maximize the probabilities of recovery for covid-19 patients. future research with larger data sets should include the study of effect and impact of preconditions with individuals reaching clinical endpoints to gain a better understanding of risk factors, as well as the economic and health impacts of covid-19. based on the results, lethality by covid-19 in portuguese infected individuals was significantly associated with demographics (males; advanced age) and the preconditions cardiac disease, kidney disease, and neuromuscular disorder. the present study successfully modeled the condition to assess the prognosis of each patient with high precision. being one of the first studies in europe not only to identify the main preconditions associated with covid-19 lethality but also 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covid-19 and pre-existing type 2 diabetes diabetes na população portuguesa: uma análise do estudo e_cor novo coronavirus covid-19 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would like to thank the dgs for providing the database. the authors would further like to acknowledge all health professionals that made these data analyses possible while taking care of all individuals infected during the covid-19 pandemic. the authors also acknowledge the computational support provided by infraestrutura nacional de computação distribuída (incd) (national infrastructure for distributed computation) institution funded by the fundação para a ciência e tecnologia (fct) and feder under project 01/saict/2016 nº 022153. the authors declare no conflict of interest. j. clin. med. 2020, 9, 2368 key: cord-281039-a7q5nzwn authors: rodilla, enrique; saura, alberto; jiménez, iratxe; mendizábal, andrea; pineda-cantero, araceli; lorenzo-hernández, elizabeth; fidalgo-montero, maria del pilar; lópez-cuervo, joaquín fernandez; gil-sánchez, ricardo; rabadán-pejenaute, elisa; abella-vázquez, lucy; giner-galvañ, vicente; solís-marquínez, marta nataya; boixeda, ramon; de la peña-fernández, andrés; carrasco-sánchez, francisco javier; gonzález-moraleja, julio; torres-peña, josé david; guisado-espartero, maría esther; escobar-sevilla, joaquín; guzmán-garcía, marcos; martín-escalante, maría dolores; martínez-gonzález, ángel luis; casas-rojo, josé manuel; gómez-huelgas, ricardo title: association of hypertension with all-cause mortality among hospitalized patients with covid-19 date: 2020-09-28 journal: j clin med doi: 10.3390/jcm9103136 sha: doc_id: 281039 cord_uid: a7q5nzwn it is unclear to which extent the higher mortality associated with hypertension in the coronavirus disease (covid-19) is due to its increased prevalence among older patients or to specific mechanisms. cross-sectional, observational, retrospective multicenter study, analyzing 12226 patients who required hospital admission in 150 spanish centers included in the nationwide semi-covid-19 network. we compared the clinical characteristics of survivors versus non-survivors. the mean age of the study population was 67.5 ± 16.1 years, 42.6% were women. overall, 2630 (21.5%) subjects died. the most common comorbidity was hypertension (50.9%) followed by diabetes (19.1%), and atrial fibrillation (11.2%). multivariate analysis showed that after adjusting for gender (males, or: 1.5, p = 0.0001), age tertiles (second and third tertiles, or: 2.0 and 4.7, p = 0.0001), and charlson comorbidity index scores (second and third tertiles, or: 4.7 and 8.1, p = 0.0001), hypertension was significantly predictive of all-cause mortality when this comorbidity was treated with angiotensin-converting enzyme inhibitors (aceis) (or: 1.6, p = 0.002) or other than renin-angiotensin-aldosterone blockers (or: 1.3, p = 0.001) or angiotensin ii receptor blockers (arbs) (or: 1.2, p = 0.035). the preexisting condition of hypertension had an independent prognostic value for all-cause mortality in patients with covid-19 who required hospitalization. arbs showed a lower risk of lethality in hypertensive patients than other antihypertensive drugs. the novel coronavirus disease is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). worldwide, as of 14 august 2020, nearly 21 million (20, 960, 424) people had been diagnosed with covid-19 and 760,371 had died [1] . one characteristic of this recent covid-19 epidemic, described in the first reports coming out of china and italy, was the observation that older patients with cardiovascular diseases (cvd) seemed to be highly represented, suggesting that there was a higher risk for worse outcomes of covid-19 in this population [2, 3] . accordingly, hypertension (ht), which represents the single most important risk factor for cvd [4] , has been repeatedly proposed as an independent prognostic factor of severe covid-19 and has been included in clinical risk scores to predict the occurrence of critical illness in hospitalized patients with covid-19 [5] . several plausible arguments support the hypothesis of a causal association between ht and covid-19. first, microvascular inflammation plays an important role in both the pathogenesis of ht and covid-19, as illustrated by the high cytokine levels found in both ht and covid-19 [6, 7] . second, angiotensin-converting enzyme 2 (ace2) plays a pivotal role as a binding receptor for the cellular penetration of sars-cov-2 [8] . it is widely distributed on the respiratory epithelium as well as in the heart, kidney, and blood vessels [9] . angiotensin-converting enzyme inhibitors (aceis) and angiotensin ii receptor blockers (arbs)-the most frequent antihypertensive drugs used in ht treatment [10] -have been linked in both animal models and in humans with up-regulation of ace2 [11] , thereby enhancing the ability of sars-cov-2 to infect cells and reducing the physiological degradation of angiotensin ii. it has been claimed that the activation of this ace/angiotensin/angiotensin ii type-1 receptor (at1r) axis [12] not only enhances susceptibility to but also the severity of sars-cov-2 infection [13] . the alternative hypothesis postulates that cvd, including ht, are simply confounding factors for the genuine association between older age and covid-19 [14, 15] . more evidence is still needed to support the idea that the association between ht and other cvd with covid-19 is fully independent of age. in fact, prevalence of ht increases with advancing age, reaching a prevalence of >60% in people aged >60 years [16] . it is also well-known that age is a powerful risk factor for ht and other cvd. furthermore, the causal role of age in explaining coronary heart disease and stroke increases in parallel with age [17] . treatment of ht with renin-angiotensin-aldosterone system (raas) inhibitors might have a beneficial effect on covid-19 patients. according to the ace2/angiotensin 1-7/mas receptor axis theory, aceis and arbs would contribute to counteracting the pro-inflammatory role of elevated angiotensin ii levels as a result of decreased ace2 activity [18] . the extent to which aceis and arbs might have similar or different mechanisms of actions in covid-19 patients is not known, but some authors predict a beneficial effect of arbs compared to aceis [19] , as angiotensin ii represents the final product of the raas, whose pro-inflammatory effects should be avoided. therefore, many scientific societies have published recommendations to continue antihypertensive treatment with aceis and arbs in covid-19 patients [20, 21] . interventional studies are now underway to test the anticipated benefit of adding aceis/arbs to covid-19 treatment, even in normotensive patients. the covid-19 pandemic has hit spain with unexpected severity; the country ranks fifth on the list of deaths per million inhabitants. the semi-covid-19 network was created by the spanish society of internal medicine (semi) to establish a nationwide, observational registry of patents who have been diagnosed with covid-19. it includes epidemiological, laboratory, treatment, and outcome data [22] . the main objective of this study is to analyze whether ht represents an independent risk factor for death as a hard endpoint in patients hospitalized with sars-cov-2 in spain. more specifically, it seeks to examine the effect previous treatment with aceis/arbs may have on these patients. additionally, the association between ht and aceis/arbs with intensive care unit (icu) admission and/or assisted ventilation was analyzed. the semi-covid-19 registry is an ongoing nationwide, multicenter, observational, retrospective cohort registry. information on the registry and data collection have been described elsewhere [22] . in summary, a total of 150 hospitals from the 17 regions that comprise spain participated in the registry, thus assuring a representative sample of the entirety of the country. inclusion criteria were age ≥18 years and first admission to a hospital in spain with diagnosis of covid-19 confirmed microbiologically by reverse transcription polymerase chain reaction (rt-pcr) testing of a nasopharyngeal sample, as per the recommendations of the world health organization [23] . the exclusion criteria included subsequent admissions of the same patient and denial or withdrawal of informed consent. admission and treatment of patients took place at the discretion of the attending physicians based on their clinical judgment, local protocols, and the updated recommendations of the spanish ministry of health. a total of 13121 consecutive patients were recruited from 1 march 2020 to 24 june 2020, when the last patient entered this study. patients were 18 to 106 years of age. the processing of personal data strictly complied with spanish law 14/2007, of july 3, on biomedical research; regulation (eu) 2016/679 of the european parliament and of the council of 27 april 2016 on the protection of natural persons with regard to the processing of personal data and on the free movement of such data, and repealing directive 95/46/ec (general data protection regulation); and spanish organic law 3/2018, of 5 december, on the protection of personal data and the guarantee of digital rights. the semi-covid-19 registry has been approved by the provincial research ethics committee of málaga (spain), following the recommendation of the spanish agency of medicines and medical products (aemps, for its initials in spanish). all patients gave their informed consent. when there were biosafety concerns and/or when the patient had already been discharged, verbal informed consent was requested and noted on the medical record. the conduct and reporting of the study were performed according to the strobe statement guidelines. an online electronic data capture system (dcs) was developed on behalf of semi. after receiving training, at least one physician belonging to each hospital's internal medicine department was responsible for acquiring and inputting the requested medical information into the dcs. this work was performed on a voluntary basis without remuneration. in order to ensure the highest possible quality of data collection, a database manager (jmcr) was designated and data verification procedures were implemented. the study's scientific steering committee and an independent external agency performed database monitoring. data analysis and logistics coordination were also carried out by independent external agencies. alphanumeric sequences of characters based on identification codes were used to pseudoanonymize dissociated patient identifiable data so that the dcs did not contain any direct identifiers. a secure server hosts the database platform and all information is fully encrypted through a valid transport layer security (tls) certificate. approximately 300 variables were retrospectively collected under various headings: (1) inclusion criteria; (2) epidemiological data; (3) rt-pcr and serology data; (4) personal medical and medication history, including antihypertensive treatment categorized as aceis, arbs, or other; (5) symptoms and physical examination findings at admission; (6) laboratory (blood gases, metabolic panel, complete blood count, and coagulation) and diagnostic imaging tests; (7) additional data at seven days after admission or at admission to the icu; (8) pharmacological treatment and ventilator support during hospitalization; (9) complications during hospitalization; and (10) progress after discharge and/or 30 days from diagnosis. the raw and age-adjusted charlson comorbidity index (cci) score was calculated from the data collected [24] . a complete list of variables collected can be found in the source paper [22] . all-cause mortality during hospitalization versus hospital discharge was the primary endpoint. secondary endpoints such as invasive or non-invasive ventilation and icu admission were also explored. time of follow-up was the period from admission to discharge or death. mortality is expressed as the case fatality rate (cfr). the data this study is based on are available from the corresponding author upon reasonable request. continuous variables were tested for normal distribution using the kolmogorov-smirnov test. results are shown as means (standard deviation, sd) or medians (25th to 75th percentile) for continuous variables and numbers (%) for categorical variables. to compare baseline demographic data and clinical characteristics among the different groups, we used analysis of variance (anova) or the kruskal-wallis test for continuous variables. differences in proportion were analyzed using the chi-square test. ht was categorized as absent or present; when present, it was further categorized into three groups according to treatment received: (a) non-aceis/arbs, (b) aceis, and (c) arbs. the association between these four categories (normotension, non-aceis/arbs, aceis, and arbs and death was analyzed using kaplan-meier survival curves; the log-rank test was calculated from baseline to time of death according to the ht groups. we used a multivariate logistic regression with all-cause mortality as the dependent variable to evaluate the role of normotension, previous treatment with non-aceis/arbs, aceis, and arbs and other comorbidities as predictor variables with a 95% confidence interval (ci). variables with p < 0.1 on the univariate analysis were included. sensitivity analysis was carried out through a second logistic regression with the composite secondary endpoint as dependent variable. all statistical analyses were performed using spss software (version 26.0, chicago, il, usa). a two-sided p value < 0.05 was considered statistically significant. the semi-covid-19 registry collected data from 13,121 records. of these, 895 were missing information. in the end, 12,226 (93.2%) participants were included in the study. a subject inclusion flow chart can be seen in figure 1 . demographic and baseline clinical features are listed in table 1 . subjects' mean age was 67.5 ± 16.1 years and 42.6% were women. the ethnicity of our study population was mostly white european origin (90.1%), followed by latin american origin (8.2%). the data presented only represent patients who were hospitalized and discharged: 73.1% returned home, 5.9% continued their recovery in non-hospital healthcare institutions, and 21.0% died. concerning information about terminal complications of covid19 patients with fatal outcome, the most prevalent cause of death was adult respiratory distress syndrome (78.7%), followed at a considerable distance by acute renal failure (36.2%), multiorgan failure (27.0%), secondary bacterial pneumonia (22.1%), sepsis (20.5%), shock (16.2%), heart failure (hf, 14.8%), and cardiac arrhythmia (9.4%). disseminated intravascular coagulation (3.3%), myocarditis (2.4%), acute coronary disease (2.3%), pulmonary embolism (1.8%), and stroke (1.7%) were also present, but far more rare. this proportion of deaths is in line with official data from the spanish ministry of health as of 29 may 2020 (20,534 deaths out of 99,808 hospitalized patients, 20.6%) [25] . mean time of hospitalization was 11.3 days (±10.3), ranging from 1 to 107 days. the demographic and baseline clinical features of our population can be observed in table 1 . among our subjects, ht was the most frequent comorbidity (50.9%), far ahead of diabetes mellitus (19.1%), atrial fibrillation (11.2%), chronic heart disease (chd (8.0%), stroke (7.7%), hf (7.1%), chronic obstructive pulmonary disease (copd) (7.0%), and chronic kidney disease (ckd) (6.0%). when stratifying our population by survival/non-survival, age was markedly higher in the non-survivor group. likewise, all of the chronic comorbidities listed in table 1 showed a significantly higher prevalence in the non-survivor group. the demographic and baseline clinical features of our population can be observed in table 1 . among our subjects, ht was the most frequent comorbidity (50.9%), far ahead of diabetes mellitus (19.1%), atrial fibrillation (11.2%), chronic heart disease (chd (8.0%), stroke (7.7%), hf (7.1%), chronic obstructive pulmonary disease (copd) (7.0%), and chronic kidney disease (ckd) (6.0%). when stratifying our population by survival/non-survival, age was markedly higher in the non-survivor group. likewise, all of the chronic comorbidities listed in table 1 showed a significantly higher prevalence in the non-survivor group. to further characterize the association between ht and outcomes, we compared normotensive patients with hypertensive patients from all three hypertension categories. we observed a highly significant increase in the mortality rate of hypertensive versus normotensive subjects as well as worse outcomes in the non-acei/arb group versus the acei and arb groups. interestingly, we also saw better outcomes among subjects in the arb group compared to the acei group ( figure 2) . to further characterize the association between ht and outcomes, we compared normotensive patients with hypertensive patients from all three hypertension categories. we observed a highly significant increase in the mortality rate of hypertensive versus normotensive subjects as well as worse outcomes in the non-acei/arb group versus the acei and arb groups. interestingly, we also saw better outcomes among subjects in the arb group compared to the acei group ( figure 2 ). kaplan-meier survival curves ( figure 3 ) according to blood pressure status confirm a clear increase in all-cause mortality in hypertensive patients in the non-acei/arb group and the acei group compared to normotensive patients (log-rank p < 0001) from the very beginning of the observation period. of note, the arb group initially matches the curve of the former two groups, but there is a clear separation at around the third week of hospitalization, with figures then approaching the curve for normotensive patients. remarkably, the maintenance of arbs during the first two weeks and thereafter was very similar (47.0% vs. 44.3%, respectively). kaplan-meier survival curves ( figure 3 ) according to blood pressure status confirm a clear increase in all-cause mortality in hypertensive patients in the non-acei/arb group and the acei group compared to normotensive patients (log-rank p < 0001) from the very beginning of the observation period. of note, the arb group initially matches the curve of the former two groups, but there is a clear separation at around the third week of hospitalization, with figures then approaching the curve for normotensive patients. remarkably, the maintenance of arbs during the first two weeks and thereafter was very similar (47.0% vs. 44.3%, respectively). table 2 shows differences between normotensive and hypertensive patients according to the treatment groups. the figures indicate a similar distribution of comorbidities across the three treatment groups. when analyzing the acei and arb groups, it is noteworthy that not only age, but also the charlson comorbidity index scores were almost identical, pointing to a homogeneous distribution of comorbidities in both groups. there was a slightly higher percentage of males in the acei group (60.9% vs. 57.6%, p = 0.03). table 2 shows differences between normotensive and hypertensive patients according to the treatment groups. the figures indicate a similar distribution of comorbidities across the three treatment groups. when analyzing the acei and arb groups, it is noteworthy that not only age, but also the charlson comorbidity index scores were almost identical, pointing to a homogeneous distribution of comorbidities in both groups. there was a slightly higher percentage of males in the acei group (60.9% vs. 57.6%, p = 0.03). using all covariates with a significant association (p < 0.1) with all-cause mortality as the dependent variable, we performed a multivariate stepwise logistic regression analysis adjusting for age and gender (table 3) . we also included in-hospital use of aceis/arbs to control for discontinuation of these drugs as confusion factors. in fact, only 44.9% of patients previously on aceis treatment and 46.4% of those on arbs before hospitalization, maintained these antihypertensive drugs during the active phase of the disease. the two main factors that were independently predictive of death were the charlson comorbidity index score and age. male gender, atrial fibrillation, and hf remained significant determinants, but diabetes, copd, and peripheral arterial disease did not. ckd was borderline significant. of particular interest is the observation that compared to normotensive subjects, treated ht was significantly associated with increased all-cause mortality, independently of previous antihypertensive treatment. on the contrary, in-hospital treatment with aceis/arbs exerted an independent and significant protective effect for fatal outcomes. to increase the sensitivity of our study, we also analyzed a composite endpoint consisting of death, need for ventilatory support, and admission to the icu. table s1 (supplementary material) shows the results of the multivariate logistic regression for the composite endpoint outcome as the dependent variable. an analysis confirms the independent predictive value of age, charlson comorbidity index score, sex, and antihypertensive treatment. on the other hand, hf, ckd, and atrial fibrillation were shown to have borderline significance with similar ors. the independent protective effect of in-hospital use of aceis/arbs was confirmed. to the best of our knowledge, this observational, cross-sectional, multicenter study constitutes the largest analysis of hospitalized, treated, and discharged covid-19 patients worldwide. data from 12,226 participants from 150 hospitals throughout the nation were collected, achieving a representative sample of the pandemic in spain. our analysis was strictly limited to patients who required in-hospital treatment. focusing on the comorbidities associated with covid-19 severity and using in-hospital all-cause mortality as a hard endpoint, the following conclusions can be drawn. first, a previous diagnosis of ht increased the risk of all-cause death in covid-19 patients who required hospitalization on the order of approximately 20% and independently of age and other cardiovascular comorbidities, such as hf and atrial fibrillation. in line with the vast majority of studies, our results support the observation that covid-19 patients are generally older and more fragile than the general population [2, 26] , as confirmed by the use of different scores reflecting concurrent chronic diseases [5] . in our study population, we observed that the higher the corrected charlson comorbidity index score was, the higher all-cause mortality was. however, the specific association between ht and covid-19 remains controversial. some studies have linked presence of ht to worse outcomes in covid-19 [27] , whereas others consider ht to simply be a potential confounding factor for the real, causal relationship between age, cardiovascular disease, and increased mortality due to covid-19 [15, 28] . in a recent cross-sectional, observational, multicenter study, iaccarino et al. [15] analyzed the comorbidities of 1591 covid-19 patients in italy. they found that ht was the most frequent preexisting condition (73.4%). nevertheless, after adjusting for all other clinical conditions, only age, diabetes mellitus, chronic obstructive pulmonary disease, and chronic kidney disease-not ht-showed prognostic value for death from covid-19. although the work by iaccarino et al. and our study share many similarities, such as the median age of survivors and non-survivors, the prevalence of baseline conditions, multivariate adjustment for all of them, and all-cause mortality as the primary endpoint, a fundamental difference was that up to 72.7% of the italian study's patients were still in the active phase of the covid-19 disease. at that stage of the disease, the outcome should be at least considered uncertain in relation to a hard endpoint. in contrast, all of our patients had either been discharged or had died, therefore offering a definite result in terms of the principal outcome variable. further arguments that could explain conflicting results between the studies include the fact that up to 7% of patients in the italian study received outpatient treatment, as hospital admission was not an inclusion criterion. this could reflect a different degree of disease severity in those patients. second, previous treatment with aceis/arbs in hypertensive patients was not associated with a higher risk of all-cause mortality in hypertensive hospitalized covid-19 patients compared to other antihypertensive drugs. two recent papers addressing the relationship between previous treatment with aceis/arbs and covid-19 seem to confirm our observation; they found no increase in severity of covid-19 in the group of patients treated with aceis/arbs compared with other drugs for cvd [14, 29] . it is important to underline that use of aceis/arbs in both studies was not synonymous with ht, as the prevalence of ht in the group of patients treated with aceis/arbs ranged between 58% and 71%. in other words, other diseases in which aceis/arbs are also indicated as a baseline therapy were included in the analysis in these studies and may act as confounding factors of the benefit or harm of aceis/arbs from the perspective of their role as treatment before hospital admission. in contrast, our study addressed the use of aceis/arbs exclusively for ht and before admission. the results of both studies should therefore not be interpreted as arguments against the association between ht and covid-19, but as evidence that previous treatment of cvd with aceis/arbs does not relate to severity of or susceptibility to covid-19 per se. third, the lowest risk of all-cause mortality in previously treated hypertensive covid-19 patients was observed in the group of arbs. furthermore, patients previously treated with arbs showed a tendency to become protective two weeks after admission. the fact that the so-called "cytokine storm" syndrome generally develops after the second week of covid-19 infection [30] , might explain the delay in the possible beneficial action of arbs. most studies include aceis/arbs in a single group, as they share a common pathway. nevertheless, angiotensin ii has been shown to increase in covid-19 and arbs act on the final step of the raas system, precisely blocking the at1-receptor for angiotensin ii. it has therefore been claimed that for this reason, arbs might be superior to aceis in improving covid-19 prognosis [19] . as shown in this study, mortality increased in patients prevented from continuing their previous treatment with aceis/arbs during their hospital stay. therefore, a careful evaluation of medications used in hypertensive patients diagnosed with covid 19 is mandatory. it is of capital importance to emphasize the scope of our study on ht treatment before hospital admission. in line with the evidence published to date, our study does not examine in-hospital management of covid-19 patients as the main objective. although the underlying mechanisms might be equally related to the ace2/angiotensin 1-7/mas receptor axis, additional studies are necessary to evaluate confounding factors, especially the incidence of cardiovascular complications and in-hospital treatment with aceis/arbs that may alter the extent of the association of ht and its previous treatment with all-cause mortality. our data about terminal complications contribute to understand the pathophysiological mechanisms of fatal outcomes in hospitalized covid19 patients. death was overwhelmingly caused by adult respiratory distress syndrome, suggesting that the cytokine storm may play a major role, although other pathways affecting especially the kidneys and the cardiovascular systems are also definitely involved. the strengths of our study include the large number of participants, the use of a hard endpoint for analysis, and previous experience in handling databases by a scientific society. nevertheless, its cross-sectional design, the high proportion of patients of white ethnicity, the unknown real spread of covid-19 in outpatients, and the strict inclusion of covid-19 patients requiring hospital admission do not allow for our results to be extrapolated to the general population. further studies, using data from death certificates, should be carried out to further explore the association between hypertension and all-cause mortality at population level. improving medical certification of cause of death across countries with an analysis protocol for uniform minimum data reporting is necessary for addressing the growing burden of the covid19 pandemic [31] . hypertension is associated with a higher risk for all-cause mortality independently of other comorbidities, sex, and age. previous treatment with aceis/arbs, compared to other antihypertensive drugs, does not alter outcomes in hypertensive patients. compared to other antihypertensive drugs, hypertensive patients previously treated with angiotensin ii receptor blockers (arbs) had the lowest risk for all-cause mortality. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/10/3136/s1, table s1 : association with the composite endpoint. univariate analysis and adjusted multivariate logistic regression model. ckd: chronic kidney disease; hf: heart failure. clinical characteristics of coronavirus disease 2019 in china baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region global atlas on cardiovascular disease prevention and control; world health organization development and validation of a clinical risk score to predict the occurrence of critical illness in hospitalized patients with covid-19 immune mechanisms of hypertension nadph oxidases and oxidase crosstalk in cardiovascular diseases: novel therapeutic targets sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor ace2: from vasopeptidase to sars virus receptor esc/esh guidelines for the management of arterial hypertension: the task force for the management of arterial hypertension of the european society of cardiology (esc) and the european society of hypertension (esh) urinary angiotensin-converting enzyme 2 in hypertensive patients may be increased by olmesartan, an angiotensin ii receptor blocker the ace2/angiotensin-(1-7)/mas axis of the renin-angiotensin system: focus on angiotensin are patients with hypertension and diabetes mellitus at increased risk for covid-19 infection? renin-angiotensin-aldosterone system blockers and the risk of covid-19 age and multimorbidity predict death among covid-19 patients: results of the sars-ras study of the italian society of hypertension prevalence, awareness, treatment, and control of hypertension in rural and urban communities in high-, middle-, and low-income countries impact of aging on the strength of cardiovascular risk factors: a longitudinal study over 40 years a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury angiotensin receptor blockers and covid-19 addresses concerns re: using raas antagonists in covid-19 european society of cardiology. position statement of theesc council on hypertension on ace-inhibitors and angiotensin receptor blockers características clínicas de los pacientes hospitalizados con covid-19 en españa: resultados del registro semi-covid-19 clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance a new method of classifying prognostic comorbidity in longitudinal studies: development and validation análisis de los casos de covid-19 notificados a la renave hasta el 10 de mayo en españa a 29 de mayo de 2020. equipo covid-19. renave. cne. cnm (isciii) clinical course and risk factors for mortality of adult inpatients with covid-19 in wu-han, china: a retrospective cohort study comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis arterial hypertension and the risk of severity and mortality of covid-19 association of angiotensin-converting enzyme inhibitor or angiotensin receptor blocker use with covid-19 diagnosis and mortality hlh across speciality collaboration, uk. covid-19: consider cytokine storm syndromes and immunosuppression medical certification of cause of death for covid 19 we gratefully acknowledge all the investigators who participate in the semi-covid-19 registry. we also thank the semi-covid-19 registry coordinating center, s&h medical science service, for their quality control data, logistic and administrative support. the authors declare that there are no conflicts of interest. the authors declare no conflict of interest. j. clin. med. 2020, 9, 3136 key: cord-273567-8fp3a9h8 authors: zipprich, hannah m.; teschner, ulrike; witte, otto w.; schönenberg, aline; prell, tino title: knowledge, attitudes, practices, and burden during the covid-19 pandemic in people with parkinson’s disease in germany date: 2020-05-29 journal: j clin med doi: 10.3390/jcm9061643 sha: doc_id: 273567 cord_uid: 8fp3a9h8 background: adherence to measures that have been adopted during the covid-19 pandemic is crucial to control the spread of the coronavirus. methods: semi-structured telephone interviews were performed with 99 patients with parkinson’s disease (pd) and 21 controls to explore knowledge, attitudes, practices, and burden in order to elucidate nonadherence to preventive measures. results: the majority of patients understood the preventive measures and felt sufficiently informed. analysis of qualitative answers, however, showed that about 30% of patients had an insufficient level of knowledge, which was not associated with educational level, cognitive disorders, or depression. changes in behaviour were reported by 73 patients (99% performed at least one specific preventive behavior, and 86.9% have reduced social contacts and stayed home). a closer analysis of qualitative answers showed that 27.3% of patients continued to meet relatives face-to-face almost daily. anxiety and worries about the current situation were reported by 58.6% of patients; 31.3% complained about a decrease in their mobility since the beginning of the restrictions, mainly because of worsening of pd and because regular therapies (e.g., physiotherapy) were canceled. conclusions: about 30% of pd patients are nonadherent to preventive measures. use of simple dichotomous questions overestimates adherence to preventive measures in patients with pd. severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a novel virus causing covid-19 infection, has led to a deadly pandemic. this virus has poorer outcomes and higher mortality rates in older adults and those with comorbidities or chronic diseases such as parkinson's disease [1] [2] [3] . sars-cov-2 appeared in early december 2019 in the city of wuhan, hubei, china. since then, local and national governments have taken unprecedented measures in response to the outbreak of sars-cov-2-induced coronavirus disease in 2019 (covid-19), including quarantining infected individuals and their family members, canceling public transportation, exit controls, travel restrictions, contact restrictions, curfews, school closures, and requiring people to wear mouth and nose masks [4, 5] . these measures may have several short-term as well as long-term adverse consequences for people with parkinson´s disease, such as worsening of motor function and stress-related psychiatric symptoms such as anxiety and depressive mood [3] . however, for successful containment of the spread of the virus, it is essential that people with parkinson´s disease follow the measures. three factors, among others, are decisive for adherence to these measures: the knowledge of the population, their attitudes, and practical implementation of the recommendations [6] . the lessons learned from the sars outbreak in 2003 suggest that knowledge and attitudes toward infectious diseases influence the degree of emotional response in the population. above all, panic can further complicate attempts to prevent the spread of the disease [7] . in order to facilitate the management of the covid-19 outbreak there is an urgent need to understand public awareness of covid-19 and the reasons for nonadherence to measures at this critical moment. perception of risks is important for human decision making. regarding behavior, emotions such as fear or the feeling of being threatened also play a role. so far, one study has investigated the knowledge, attitudes, and practices (kap) of chinese people with respect to covid-19 [8] . in that study, the majority of the mostly female and well-educated respondents was well informed about covid-19 and followed the guidelines. however, their average age was 33.0 years, and the results are not transferable to people with parkinson´s disease. a recent study using telephone interviews of 100 people with parkinson´s disease suggested that most patients and caregivers were well informed and were coping well with the pandemic [9] . however, multiple-choice or dichotomous questions such as those in the recent study by prasad et al. cannot adequately reflect kap [9] . moreover, one cannot make valid conclusions about the true rates of adherence to preventive measures, and one has to take into account the sociodemographic circumstances of the patients. for this purpose, qualitative methods are necessary. there are no comprehensive data on kap about covid-19 in patients with parkinson´s disease. describing kap about covid-19 in patients with parkinson´s disease may help to improve adherence to preventive measures. this cross-sectional survey was conducted from april 2 to 17, 2020. patients with parkinson's disease who were enrolled in the neurogeradh study (drks00016774) between august 2019 and february 2020 were interviewed by telephone. the neurogeradh study is a longitudinal observational study of adherence in patients with neurological disorders. this study was approved by the local ethics committee (approval number 5290-10/17) of the jena university hospital, and all patients provided written informed consent. the semi-structured questionnaire consisted of 22 questions to assess the patient's current situation and adherence to the ongoing regulations, with four of these questions examining the patient's knowledge of preventive measures (questions 4, 5, 6, and 7), three capturing their attitude toward the virus (questions 8, 9, and 16) , and six exploring practices and behavioral changes regarding covid-19 (questions 10, 11, 12, 13, 14, and 15 ). in addition, seven questions were included to evaluate the burden and physical and emotional strain felt by the patients due to the ongoing restrictions (questions 16 to 22). finally, information was obtained on current restrictions, contact with covid-19 patients, and experience with quarantine. the questionnaire is given in the supplementary table s1 ). knowledge of covid-19 was measured by questions 7 (knowing the correct function of the robert koch institute) and 9 (knowing the correct aims of measures and current restrictions). patients were grouped according to their knowledge as having good (knows both robert koch institute and aims of measures), moderate (knows one), or poor (knows neither) knowledge. the robert koch institute is an independent german federal authority for infectious diseases. as a public health care institution, it focuses on the health of the entire population and is the government's central scientific institution in the field of biomedicine in germany. in terms of covid-19 the robert koch institute is continuously monitoring the situation, evaluating all available information, estimating the risk for the population in germany and providing health professionals with recommendations. its tasks are, among others, the identification, surveillance and prevention of infectious diseases, monitoring and analyzing long-term public health trends in germany, performing epidemiological and medical analyses, providing a scientific basis for health-related political decision-making, and informing and advising political decision-makers, the scientific sector and the general public (https://www.rki.de). for each patient, the following parameters were extracted from the medical records: age, gender, marital status, level of education (high: german abitur or university; low: german realschule or general certificate of secondary education, german hauptschule, or no school), and employment status. information about cognitive state (montreal cognitive assessment (moca)) [10] , depressive mood (beck's depression inventory ii (bdi)), motor function (movement disorder society-sponsored revision of the unified parkinson's disease rating scale iii (mds-updrs iii)) [11] , presence of non-motor symptoms (revised non-motor symptoms questionnaire (nms-q)) [12, 13] , and adherence to medication (stendal adherence with medication score; sams) was extracted from the medical records. the sams includes 18 questions forming a cumulative scale (0-72) in which 0 indicates complete adherence and 72 complete nonadherence [14, 15] . the sams is available online (cc by nc 3.0 license; https://data.mendeley.com/datasets/ny2krr3vgg/1) [16] . because of the study design, data on these clinical parameters were obtained from two to six months before the interviews were performed. one hundred patients with parkinson´s disease were screened for the study. one potential subject could not be reached by telephone (we made three attempts to call the patients). in addition, 25 elderly patients without parkinson´s disease were randomly selected from the database as controls; 21 of these patients could be interviewed, and 4 were not reached (sociodemographic characteristics are given in table 1 ). statistical analysis was performed with the statistical software spss 25.0 (spss inc., chicago, il, usa). data were analyzed by descriptive statistics: means, standard deviations, medians, interquartile ranges, frequencies, and percentages. data were checked for normality by the shapiro-wilk test. for comparison of groups, the cohort was split into patients <70 and ≥70 years of age. correlation between different clinical variables was tested with spearman correlation for non-normally distributed data. group comparisons were performed by analysis of variance or the kruskal-wallis test with bonferroni correction and the chi-square test. p-values <0.05 were considered to indicate statistical significance. the final sample included 35 women (35.4%) and 64 men (64.6%) with parkinson's disease, with a median age of 72 years (iqr, 12 years). the median mds-updrs iii score was 26 (iqr, 18), and the mean nms-q score was 10.5 (sd, 4.2). most patients were married, receiving a pension, and had completed middle or high school. the majority (55.6%) lived in larger cities (20,000 to 250,000 inhabitants), and 14.1% lived in villages (<2000 inhabitants). none of the patients were infected with coronavirus. only two patients reported having had contact with a covid-19-positive person and were in quarantine. most patients reported that they were well or sufficiently informed about covid-19. thirty-two (32.3%) reported that they were very well informed, 38 (38.4%) that they were well informed, 16 (16.2%) that they were sufficiently informed, and 5 (5.1%) that they were poorly informed (3 missing). of note, five patients (5.1%) reported that they received too much information about the virus. most subjects (patients with parkinson's disease and controls) (94.9%) received their information from television news ( figure 1 ). j. clin. med. 2020, 9, 1643 4 of 11 wilk test. for comparison of groups, the cohort was split into patients <70 and ≥70 years of age. correlation between different clinical variables was tested with spearman correlation for nonnormally distributed data. group comparisons were performed by analysis of variance or the kruskal-wallis test with bonferroni correction and the chi-square test. p-values <0.05 were considered to indicate statistical significance. the final sample included 35 women (35.4%) and 64 men (64.6%) with parkinson's disease, with a median age of 72 years (iqr, 12 years). the median mds-updrs iii score was 26 (iqr, 18), and the mean nms-q score was 10.5 (sd, 4.2). most patients were married, receiving a pension, and had completed middle or high school. the majority (55.6%) lived in larger cities (20,000 to 250,000 inhabitants), and 14.1% lived in villages (<2000 inhabitants). none of the patients were infected with coronavirus. only two patients reported having had contact with a covid-19positive person and were in quarantine. most patients reported that they were well or sufficiently informed about covid-19. thirtytwo (32.3%) reported that they were very well informed, 38 (38.4%) that they were well informed, 16 (16.2%) that they were sufficiently informed, and 5 (5.1%) that they were poorly informed (3 missing). of note, five patients (5.1%) reported that they received too much information about the virus. most subjects (patients with parkinson's disease and controls) (94.9%) received their information from television news ( figure 1 ). figure 1 . actively reported sources of information (%). active reporting means that patients were not given a choice of answers but were free to choose their own. almost half of the patients (47.5%) actively sought additional information on the coronavirus pandemic, mainly via the internet or through acquaintances in the medical profession. among patients not seeking additional information, 23 patients (46.0%) reported that they already had enough information, 12 (24.0%) that they did not have the ability to obtain additional information (e.g., no internet access), and 7 (14%) that they were not interested. ten percent of patients stated that they did not want to get carried away by too much information. seventy-seven patients (77.8%) almost half of the patients (47.5%) actively sought additional information on the coronavirus pandemic, mainly via the internet or through acquaintances in the medical profession. among patients not seeking additional information, 23 patients (46.0%) reported that they already had enough information, 12 (24.0%) that they did not have the ability to obtain additional information (e.g., no internet access), and 7 (14%) that they were not interested. ten percent of patients stated that they did not want to get carried away by too much information. seventy-seven patients (77.8%) reported that they knew about the robert koch institute; however, only 66 patients (66.7%) could describe its function correctly. most patients (94.9%) stated that the virus was dangerous (in general and/or to them personally). their most common reason (in approximately one third of patients) was that they saw themselves as patients at risk. between 13.8% and 17.0% attributed danger to the deadly course of the disease, the lack of any specific cure, and the lack of knowledge about the virus, followed by other reasons, such as the high rate of infection. most patients (94.9%) felt that current restrictions on everyday life (social distancing, etc.) were necessary (94.9%). most patients (94.9%) felt that these measures to contain the virus were sufficient, and 4.0% felt that they were excessive. ninety-five percent stated that they understood why the measures were necessary, and 70% gave the correct reasons for the preventive measures (e.g., reducing the distribution of the virus or "flattening the curve"). almost two thirds of the patients (61.6%) were recommended to stay at home more often by their family members. seventy-two patients (72.7%) reported that they had changed their behavior since the appearance of the virus; only 4.0% reported taking no measures to protect themselves. the most common actively reported preventive behaviors are shown in figure 2 . active reporting means that patients were not given a choice of answers but were free to choose their own. j. clin. med. 2020, 9, 1643 5 of 11 reported that they knew about the robert koch institute; however, only 66 patients (66.7%) could describe its function correctly. most patients (94.9%) stated that the virus was dangerous (in general and/or to them personally). their most common reason (in approximately one third of patients) was that they saw themselves as patients at risk. between 13.8% and 17.0% attributed danger to the deadly course of the disease, the lack of any specific cure, and the lack of knowledge about the virus, followed by other reasons, such as the high rate of infection. most patients (94.9%) felt that current restrictions on everyday life (social distancing, etc.) were necessary (94.9%). most patients (94.9%) felt that these measures to contain the virus were sufficient, and 4.0% felt that they were excessive. ninety-five percent stated that they understood why the measures were necessary, and 70% gave the correct reasons for the preventive measures (e.g., reducing the distribution of the virus or "flattening the curve"). almost two thirds of the patients (61.6%) were recommended to stay at home more often by their family members. seventy-two patients (72.7%) reported that they had changed their behavior since the appearance of the virus; only 4.0% reported taking no measures to protect themselves. the most common actively reported preventive behaviors are shown in figure 2 . active reporting means that patients were not given a choice of answers but were free to choose their own. others include increased use of disinfectants (1), separation of towels (1), avoid public transport (1), and additional vaccination (1). active reporting means that patients were not given a choice of answers but were free to choose their own. when asked "have you reduced social contacts?," 86.9% of patients stated that they had reduced their contacts with relatives and friends and stayed at home more. among patients who had not reduced their contacts, most stated that they had only a few contacts already and/or that they had to stay home anyway, mostly due to their parkinson's disease. when asked about contacts with their relatives and friends, 77 patients (77.8%) reported that they had contact with their families at least several times a week, and 40 patients (40.4%) reported that they had a similar amount of contact with their friends. thirty-nine patients (39.4%) saw their family members face-to-face, 42 (42.4%) only talked to them by telephone, and 16 (16.2%) used more up-to-date means of communication, such as video telephony and smartphone messenger services. others include increased use of disinfectants (1), separation of towels (1), avoid public transport (1), and additional vaccination (1). active reporting means that patients were not given a choice of answers but were free to choose their own. when asked "have you reduced social contacts?", 86.9% of patients stated that they had reduced their contacts with relatives and friends and stayed at home more. among patients who had not reduced their contacts, most stated that they had only a few contacts already and/or that they had to stay home anyway, mostly due to their parkinson's disease. when asked about contacts with their relatives and friends, 77 patients (77.8%) reported that they had contact with their families at least several times a week, and 40 patients (40.4%) reported that they had a similar amount of contact with their friends. thirty-nine patients (39.4%) saw their family members face-to-face, 42 (42.4%) only talked to them by telephone, and 16 (16.2%) used more up-to-date means of communication, such as video telephony and smartphone messenger services. the majority of patients (61.6%) kept in touch with friends by telephone. among the 73 patients with grandchildren (73.7%), 61 patients (83.6%) did not have contact with their grandchildren at present (including 8 who did not have contact with them before the coronavirus). the discontinuation of contact was mainly proposed by the parents of the grandchildren, the patients themselves, or both (86.8%). thirty-six (73.6%) of those who did not have contact with their grandchildren stated that they suffered from this. anxiety and worries about the present situation were reported by 58 patients (58.6%). their greatest fear was infection with the coronavirus. in addition, the general uncertainty, the economic and social developments, and the possible loss of other medical care worried the patients. restrictions on everyday life due to the coronavirus were reported by 54 patients (54.5%). as physical burdens, lack of outdoor activities (10 patients [10.1%]), worsening of their parkinson's disease (19 patients [19.2%]), and further illness or injury were mentioned. as mental stresses, 22 patients (22.2%) mentioned the lack of social contacts and the ability to go outside, 10 (10.1%) were afraid of becoming infected, and 32 (32.3%) were burdened by other fears and worries. on the physical level, 31 patients (31.3%) complained about a decrease in their mobility since the beginning of the restrictions, mainly because of worsening of their parkinson's disease and a lack of treatment. consistent with this, many prescribed therapies had been canceled (63.2% of physiotherapy, 57.5% of occupational therapy, 62.5 of speech therapy). thirty-four patients (34.3%) also reported that they had to refrain from sports activities (such as sports therapy, but also walking outside, or sports groups). however, 28 of these 34 patients (82.4%) found alternative forms of activity by doing their exercises at home or going outside. older patients with parkinson's disease had lower moca scores and higher mds-updrs iii scores than younger patients, indicating poorer cognitive function and greater motor impairment (table 1) . older and younger patients did not differ in bdi, sams, or nms-q scores ( table 1) . the items for kap and burden with respect to covid-19 did not differ between younger and older patients ( table 2) . compared with control subjects, more patients with parkinson's disease (younger and older) reported having actively sought information (question 6). in comparison to the controls, more patients with parkinson's disease (younger, older and entire group with parkinson's disease) believed that the virus was dangerous (question 8). the other items for kap and burden with respect to covid-19 did not differ between patients with parkinson's disease and controls. most items of kap and burden did not differ between male and female patients; however, female patients more frequently perceived the current situation as threatening (p = 0.014, chi-square test). values in the same row and sub-table where the sub-script (a, b) is not identical differ at p < 0.05. in sub-tables with an expected cell frequency <5 the exact test (monte carlo) was used. knowledge and attitudes are crucial elements of adherent behavior. we observed some discrepancies between answers to simple yes/no questions and to detailed qualitative questions, indicating that around 30% of patients were nonadherent. although 86 patients (89.6%) felt that they were sufficiently to very well informed about preventive measures and covid-19, 25 of these patients (29.1%) were not able to correctly describe the function of the robert koch institute. this is surprising, because the robert koch institute was omnipresent in the media during the time of the study. it is germany's national public health institute in the field of surveillance, control, and prevention of diseases, comparable to the us centers for disease control and prevention. although 77 patients (77.8%) reported that they knew what the robert koch institute was, the qualitative question showed that only 67 (67.7%) were able to correctly name one of its functions. thirty patients (29.7%) were not able to name a correct aim of the current restrictions, and this was also associated with limited knowledge about the robert koch institute (p = 0.04, chi-square test). fourteen patients (13.6%) did not know what the robert koch institute was nor why the measures were taken. missing knowledge about the robert koch institute or the aim of the measures was not associated with educational level (p = 0.08, p = 0.133, respectively), moca score (p = 0.13, p = 0.38, respectively), bdi score (p = 0.81, p = 0.62, respectively), or mds-updrs iii score (p = 0.14, p = 0.57, respectively). patients with limited knowledge were older (mean age, 74.4 years; sd = 7.2) than patients with good knowledge (mean age, 70.0 years; sd = 8.5) (p = 0.03). patients who felt that they were very well informed about covid-19 were more likely to search actively for further information (p = 0.03) than patients who felt that they were not sufficiently informed about covid-19; in contrast, none of the five patients who felt that they were poorly informed about covid-19 actively searched for information. ninety-five percent of patients believed that the virus was dangerous, mainly because they regarded themselves as being at higher risk. ninety-five percent of patients also perceived the current restrictions on everyday life to be necessary or sufficient. approximately 60% perceived the current situation as threatening. perceiving the situation as threatening was not associated with regarding the virus as dangerous (p = 0.58), actively searching for further information (p = 0.20), general changes in behavior (p = 0.09), or reducing social contact (p = 0.30). seventy-two patients (72.7%) reported that they had changed their behavior (question 10), and 99% reported at least one specific preventive behavior such as washing hands or social distancing. eighty-six patients (86.9%) reported that they had reduced social contacts and stayed at home. however, 27 (27.3%) of the patients (who did not live together with their families in one house) had personal contact with their relatives several times a week. patients who did not reduce personal contact with relatives were significantly more likely to have only moderate or poor knowledge of covid-19 (p = 0.003). of note, six patients (6.1%) who had good knowledge were nonadherent and did not reduce their social contacts. the sams score was weakly correlated with the number of preventive practices performed by the patients (r = −0.19, p = 0.037). this means that adherent patients (lower sams) took more measures to protect themselves and others from the virus. in this study, the kap of patients with and without parkinson's disease was analyzed by means of semi-structured telephone interviews to estimate the extent of nonadherence to preventive measures. the majority of patients with parkinson's disease understood the containment measures in the context of the coronavirus pandemic and felt sufficiently informed. most patients informed themselves via television news, which is in line with the study by prasad et al. [9] . in addition, younger patients tended to inform themselves via the internet, and older patients got their information from the radio or from family members. a closer analysis shows, however, that only about 30% of patients could name the correct function of the robert koch institute, although it had been present daily in the most diverse media since the outbreak of the coronavirus pandemic in germany. moreover, 30% of patients could not explain why specific measures (such as frequent handwashing) were necessary. we therefore conclude that about 30% of the patients had an insufficient level of knowledge, although all patients indicated that they obtained information from various media. a low level of knowledge was not associated with a lower educational level, cognitive disorders, or depression. with regard to attitudes, the picture is more homogeneous. almost all patients stated that they considered the virus to be dangerous and that the preventive measures were reasonable. however, this attitude did not seem to be transformed into corresponding adherent behavior. on the basis of the simple yes/no questions (e.g., "have you reduced contact?", "have you changed your behavior?"), the majority of patients seemed to be adherent and to adhere to the instructions. this was also the case in the recent study by prasad et al. [9] in which all patients reported following "any preventive measure" against covid-19 and all patients reported performing social distancing. however, if one adds the qualitative questions, as in our study, the reported high level of adherence is put into perspective. although almost all of our patients claimed to have reduced contact, a closer analysis of the qualitative answers shows that a considerable proportion of the patients continued to meet relatives in person almost daily. this shows that the use of a simple dichotomous question ("have you reduced social contact?") overestimates adherence to this preventive measure. such simply structured queries are rather unsuitable for patients with parkinson's disease. on the other hand, it is alarming that 27% of patients did not adhere to the important preventive measure of contact reduction. in general, patients with good drug adherence (according to the sams) also seemed to practise more preventive measures (e.g., handwashing, mouth and nose protection) than did nonadherent patients. however, adherence to the measure of contact reduction was also related to knowledge. patients who did not reduce personal contact with relatives had significantly less knowledge. on the other hand, there were also patients who did not reduce social contact against their better knowledge. this means that both intentional and unintentional nonadherence play a role. in terms of burden, anxiety and worries about the current situation were reported by 58.6% of patients, consisting of fear of becoming infected as well as worries about future economic and social development. in the study by prasad et al., only 11% of patients reported worsening of parkinson's disease symptoms following the onset of the covid-19 pandemic [9] . in contrast, in our study, 31.3% of the patients complained about a decrease in their mobility since the beginning of the restrictions, mainly due to worsening of their parkinson's disease and cancelation of their regular therapies (e.g., physiotherapy). the discrepancy between these studies may be partly explained by differences in the degrees of preventive measures in germany and india and the younger age of the patients in the study by prasad et al. within the framework of this telephone survey, we did not want to overburden the patients with too many questionnaires. however, it would be of great interest in further studies to investigate how anxiety, depression and psychotic symptoms appear in patients with parkinson's disease before and after covid-19. for this purpose, disease-specific questionnaires would have to be used. the crucial question is, what measures can improve kap and adherence in elderly people? this study cannot provide a conclusive answer to this question. some preliminary conclusions can nevertheless be drawn. first of all, it is crucial to know how (i.e., on which channels) we reach older people in the first place. the most important sources of information are news (television, radio, newspaper) and relatives. direct communication via social media is therefore not very effective. social media would only reach older people indirectly at most if younger relatives were informed via these channels. despite the fact that almost everyone stated that they knew something about the pandemic (some even found the wealth of information too great), it became apparent that the purpose of the measures was not always understood through the existing communication channels. perhaps it would make more sense at this point to communicate the measures in a form that allows interaction between the communication partners, e.g., via telephone consultation or personal contact (with appropriate preventive measures). the present study has several limitations. the patients were recruited from a specialized neurological hospital, and the interviews were performed in a short time interval during the ongoing pandemic. this limits the generalizability of the results. on the other hand, it is important to keep in mind that simple dichotomous questions cannot provide a valid estimate of true adherence to preventive measures. because of the restrictions, it was not possible to provide current clinical characteristics of the cohort by personal assessments. the clinical data provided, such as mds-updrs iii scores, were obtained between two and six months before the interviews were performed and can therefore only provide an estimate of the characteristics of the cohort. nevertheless, we decided to provide these clinical data, because we assume that dramatic changes in mds-updrs iii scores did not occur during a few months for the whole cohort. moreover, we think that this may help to make the cohort comparable to other parkinson's disease cohorts. it remains important to note that the results for some items (e.g., how do patients receive information?; what preventive measures do they perform?) are based on actively reported answers. we did not provide a selection of answers (multiple-choice) in order to avoid answers according to social desirability and in order to get an impression of what was truly prominent in the minds of the interviewed patients. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/6/1643/s1, table s1 : the questionnaire. covid-19 and older adults: what we know novel coronavirus infection (covid-19) in humans: a scoping review and meta-analysis the impact of the covid-19 pandemic on parkinson's disease: hidden sorrows and emerging opportunities interventions to mitigate early spread of sars-cov-2 in singapore: a modelling study the effect of control strategies to reduce social mixing on outcomes of the covid-19 epidemic in wuhan, china: a modelling study the impact of knowledge and attitudes on adherence to tuberculosis treatment: a case-control study in a moroccan region fear and stigma: the epidemic within the sars outbreak knowledge, attitudes, and practices towards covid-19 among chinese residents during the rapid rise period of the covid-19 outbreak: a quick online cross-sectional survey parkinson's disease and covid-19: perceptions and implications in patients and caregivers the montreal cognitive assessment, moca: a brief screening tool for mild cognitive impairment movement disorder society-sponsored revision of the unified parkinson's disease rating scale (mds-updrs): process, format, and clinimetric testing plan international multicenter pilot study of the first comprehensive self-completed nonmotor symptoms questionnaire for parkinson's disease: the nmsquest study validation of the non-motor symptoms questionnaire (nms-quest) comparison of anonymous versus nonanonymous responses to a medication adherence questionnaire in patients with parkinson's disease clusters of non-adherences to medication in neurological patients data on adherence to medication in neurological patients using the german stendal adherence to medication score (sams). data brief this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we thank lena sand, dorothea berges, and eric winter for assistance in data acquisition. the authors report no conflict of interest. key: cord-254148-wc762p6v authors: prell, tino; siebecker, frank; lorrain, michael; eggers, carsten; lorenzl, stefan; klucken, jochen; warnecke, tobias; buhmann, carsten; tönges, lars; ehret, reinhard; wellach, ingmar; wolz, martin title: recommendations for standards of network care for patients with parkinson’s disease in germany date: 2020-05-13 journal: j clin med doi: 10.3390/jcm9051455 sha: doc_id: 254148 cord_uid: wc762p6v although our understanding of parkinson’s disease (pd) has improved and effective treatments are available, caring for people with pd remains a challenge. the large heterogeneity in terms of motor symptoms, nonmotor symptoms, and disease progression makes tailored individual therapy and individual timing of treatment necessary. on the other hand, only limited resources are available for a growing number of patients, and the high quality of treatment cannot be guaranteed across the board. at this point, networks can help to make better use of resources and improve care. the working group pd networks and integrated care, part of the german parkinson society, is entrusted to convene clinicians, therapists, nurses, researchers, and patients to promote the development of pd networks. this article summarizes the work carried out by the working group pd networks and integrated care in the development of standards of network care for patients with pd in germany. of evidence-based treatment guidelines, the selection of motivated practitioners, regular training, commitment to compliance with the guidelines, patient-centered treatment, and transparent outcome quality [9, 12] . the core element of patient care within a network should be the implementation of a standardized treatment pathway. this defines the best possible sequence of treatment steps on the basis of guidelines and medical expertise. if possible, all patients within a network should be treated with specialized neurologists, registered neurologists, and gps working in a collaborative manner. there should be flowing boundaries to allow equal, individual care concepts based on medical necessity. in our opinion, the establishment of such a treatment pathway must be individually oriented in each network to the corresponding network structures, regional characteristics, and resources available in each case, making simple transferability between different networks impossible. nevertheless, core elements will certainly be available in different networks. this review, therefore, provides an overview of evidence-based recommendations for the network care of patients with pd within the framework of a multisectoral, multiprofessional setting. the authors met in cologne in 2019 for a roundtable discussion and to organize the foundation of the working group pd networks and integrated care, part of the deutsche gesellschaft für parkinson und bewegungsstörungen (dpg). the working group has the following aims: synchronization of supply networks in germany, development of minimum standards, development of joint research projects, further development of nursing staff qualifications, and development of qualification standards for therapy groups. the dpg working group pursues the goal of improving patient care in close cooperation with other physicians, therapists, and patient support groups. the roundtable discussion was sponsored by dpg (travel costs). following introductions and stated aims, various points of interest (existing german network structures and aims, communication strategies, standards of network care, etc.) were discussed. no formal votes were taken at the meeting. the discussions identified general points of agreement. to give recommendations for a standard of network care, one has to acknowledge the existing care paths for patients with pd in germany. typically, the initial symptoms are not identified as parkinsonism or pd-specific symptoms by the patients themselves. instead, they usually approach their gp with motor or even more important and frequent nonmotor complaints (e.g., obstipation, pain and depression). occasionally, physiotherapists treating back pain or degenerative joint symptoms realize that these are the first signs of motor symptoms (e.g., rigidity) related to pd and that the patient should be referred for a pd diagnostic workup. patients who live in rural areas are more likely to have their gp identify symptoms as being related to pd (13) . however, the gp model in germany is not as rigid as in other countries, and many citizens not only have a primary care physician but can also consult specialists (e.g., internal medicine, orthopaedists, etc.), depending on their prior health care contacts and requirements throughout their lifetime. parkinsonism refers to a clinical presentation characterized by the presence of bradykinesia plus rest tremor or rigidity [13] . bradykinesia is a generalized slowing of movements and repetitive motion fatigue. it may present as hypomimia ("masked face"), hypophonia, worsening of fine-motor tasks, micrographia, difficulty turning in bed, or reduced arm swing with side difference. additionally, distinct changes of gait and balance, like short steps, shuffling gait, and uncertainty when turning around are common. rigidity is the resistance that can be assessed clinically by passively flexing and extending a patient's limb. typically, the patient complains about stiffness and pain, which often manifests as shoulder or back pain. while kinetic and postural tremors may occur, the rest tremor is the most common type of tremor in early pd. patients with these clinical signs should be referred to a neurologist for further diagnosis, because, in germany, the time between the appearance of the first symptoms and the diagnosis is significantly longer when patients with these symptoms see their gp [5] . patients with symptoms that may be related to pd should be asked about pd-typical nonmotor signs such as sensory symptoms (loss of smell, pain), depressed mood, rapid eye movement-sleep behavior disturbance, periodic limb movement disorder, or constipation, which frequently occur years before motor signs are realized. thus, one recommendation for standard of care in the initial phase of the disease course is physician awareness of the first signs of pd (which could be achieved with better information and secondary prevention standards in the network) and early referral of patients to a movement disorder specialist (which could be achieved by specific disease management programmes). the movement disorder specialist should be a neurologist with many years of experience caring for patients with pd. in germany, there are specific recommendations for patient referral in this context that can guide decisions in the outpatient setting [6] . in 2015, the official international parkinson and movement disorder society (mds) clinical diagnostic criteria for pd were proposed [13] . the benchmark for these criteria is an expert clinical diagnosis. however, the criteria can be easily applied by clinicians with less expertise in pd diagnosis [14] . in the mds criteria, motor symptoms remain the core feature of the disease, defined as bradykinesia plus rest tremor or rigidity (explicit instructions for defining these symptoms are given). after consideration of absolute exclusion criteria (which rule out pd), red flags, and supportive criteria, the diagnosis of clinically established pd or probable pd can be made, or pd can be ruled out. besides anamnesis, clinical assessment with cerebral imaging (cranial mr imaging [mri] is preferred) should be performed to exclude symptomatic causes of pd symptoms. in case of divergent clinical and mri-based diagnoses, the clinical assessment should take precedence. however, a differentiated approach is required for the numerous subsequent apparatus and drug tests. the evidence shows that levodopa and apomorphine tests are not as meaningful as standard levodopa therapy in differentiating between established pd and atypical parkinson's syndrome. a negative test does not rule out a response to longer-lasting levodopa treatment. this suggests that levodopa and apomorphine tests should not be routinely used in differential diagnosis but may be valuable in specific clinical situations [15] . a reduction in olfactory capacity is a sensitive but not specific indicator of pd. therefore, standardized olfactory tests are only recommended in combination with other diagnostic procedures for the diagnosis of pd. striatal dopamine active transporter-single photon emission computed tomography (spect) imaging should be used early in the course of the disease to detect a nigrostriatal deficit in clinically unexplained parkinsonism or tremor syndrome [15] . in contrast, the postsynaptic (i 123-iodobenzamide) spect should not be used for the differential diagnosis of established parkinson's syndrome (syn. idiopathic parkinson syndrome) to differentiate atypical neurodegenerative disease variants. the myocardial 123 mibg-spect can be used to distinguish multiple system atrophy from pd [15] . in addition, functional brain imaging with positron emission tomography is a valuable diagnostic tool for the differential diagnosis of idiopathic parkinson syndrom and atypical parkinsonism [16, 17] . for clinical neurological confirmation of the diagnosis and therapy control, the patient should be examined after 3 months, and thereafter according to clinical need but at least once a year [15]. because even proven experts have to revise the diagnosis of an ips during the course of the disease, the diagnosis should be reviewed at regular intervals. with the rising availability of electronic patient records, another recommendation is that a standard set of information should be generated and stored in the record of each patient with pd after the results of the first diagnostic tests. this information should be available for the patient and his or her health care provider team. referral to a movement disorder specialist is important to improve the accuracy of diagnosis, for case selection and to provide guidance in terms of specialized device-aided therapies, namely, dbs, levodopa/carbidopa intestinal gel (lcig) and apomorphine. consultation from the medical staff of a specialized center may improve motor function and the quality of life in patients in advanced pd stages [18, 19] . patients with the following constellations and symptoms should be referred to a movement disorder specialist even if the disease duration is <4 years [20] [21] [22] : referral to a movement disorder specialist should also be considered for the patient to have access to the most innovative treatment and clinical research options. a substantial number of patients are highly interested in contributing to research, the opportunities for which are typically limited to regional neurologists. recommendations for these patient referrals in the outpatient setting in germany have been proposed [6] . some health care insurance systems reimburse treatment of patients with pd in specialized units. a well established and frequently used multiprofessional inpatient treatment concept in germany is pd multimodal complex treatment (pd-mct). prerequisites for patients taking part in mct are documented physician diagnosis of pd, a constant anti-parkinsonian drug titration, and the application of activating therapies (at least 7.5 h/week). it involves physicians, physiotherapists, occupational therapists, speech therapists, and other specialists for the optimization of pd treatment [3] and usually lasts 7 to 21 days. this therapy programme has been shown to be effective, with a reduction of motor symptoms and nms [23, 24] . richter et al. [3] performed an analysis of 55,141 inpatients with pd who were integrated into this mct from 2010-2016. they found that a large majority of patients with pd need to leave their residence county for an inpatient stay in a specialized pd unit. this limited access to multimodal therapy programmes means that patients sometimes have to travel long distances to receive specialized therapy [3] . there are no generally valid definitions of which patients should be treated within the complex programme and which should not. in view of the heterogeneity, it is difficult to make binding statements about this. a prerequisite should be that the motor or nmss can no longer be satisfactorily treated by outpatient therapy. another prerequisite should be that patients are dealing with limitations in their activities of daily life and have a reduced quality of life. this can be the case, for example, with side effects under oral therapy, motor deterioration, or the high burden of nms. other typical indications for inpatient treatment would be the discontinuation of dbs or the initiation and optimization of therapy with lcig or apomorphine. however, as the disease progresses and progressive limitations in mobility and cognition are observed, the benefits of inpatient treatment must be weighed against the increasing risk of delirium. overall, clinical experience shows a substantial benefit of pd-mct for a large number of patients. the preselection process could ideally be managed by network structures and players. additionally, the positive effect achieved by intense medical and nonmedical intervention should be maintained after release by immediate intensified ambulatory intervention and home-training concepts in order for the patients to benefit from the positive experience. this would be an important incentive for the patient to take part in pd-mct. for patients with pd who need to adapt to complex medication schemes, drug pumps, or dbs devices, a classical outpatient or inpatient setting is not appropriate to sufficiently address clinical problems, while in a neurologist's office or even in a movement center, outpatient clinic time and staff capacities are limited and the results of changes in medication or stimulation of the dbs device can only be monitored in the next (often late) consultation. an in-house stay is associated with an artificial environment that does not reflect the individual's everyday life demands and is less suited for patients with dementia who often cannot cope with an altered environment. furthermore, many patients with pd decline hospitalization for personal reasons such as job issues or having to care for other family members. for these patients, at the border between inpatient and outpatient care and the need for sophisticated treatment strategies, the new comprehensive, individual, and interdisciplinary concept of a pd day clinic has proven to be effective [25] . in the meantime, in germany, several university clinics with a pd focus have established this or a similar pd day clinic concept to close the gap in pd care that have been found to be a transnational issue [26] [27] [28] . the concepts and standards of qualified pd day clinics have been certified recently by the tüv and the german parkinson patient society [29] . in general, a neurologist should be responsible for long-term medical care of patients with pd, and movement disorder specialists should be involved when there is a special issue. however, for various reasons, this is not always possible. neurologists may not be available in rural areas, and even for patients in nursing homes, access to specialized neurological treatment is often limited. this is an important issue, because the number of patients in long-term care facilities will rise sharply in the coming decades [30] . for patients with pd, the interaction between the gps and neurologists is essential. pd networks can make a decisive contribution to ensuring high-quality care of these patient groups. medical treatment is not the only option to control the motor symptoms and nms during the course of the disease. other nonmedical treatment options from other specialists are frequently necessary to improve functional status, performance of daily activities, and quality of life. these specialists include, among others, physiotherapists, occupational therapists, speech therapists, pd nurse specialists, and social workers [31] . specific recommendations for physiotherapists, physicians, and patients with pd were published in the european physiotherapy guidelines for parkinson's disease [32] . health professionals must have sufficient pd-specific knowledge and expertise [33] . physiotherapy has a positive impact on functional activities involving gait, transfers, and balance [32, 34] . the occupational therapist focuses on enabling performance and engagement in meaningful activities [35] . home-based, individualized occupational therapy can improve the self-perceived performance of daily activities in patients with pd [36] . timely referral to physiotherapy, and occupational therapy is recommended because difficulties in daily activities can occur in every disease stage. given the high prevalence of dysphagia and dysarthria during the course of the disease [37] , speech-language therapy, including swallowing techniques, is frequently necessary for patients with pd. a collaborative approach between these disciplines should focus on complementary and different aspects. therapists have to be aware of each other's expertise, and effective and timely communication is essential [35] . pd networks are promising tools to share information about diagnostic results, current treatment goals, and plans. in addition, there are many different nonphysician pd specialists for inpatient and outpatient care, such as pd nurse specialists or parkinson assistants (passs). their different roles and functions are described in another paper in this issue. depending on the location (inpatient or outpatient), the focus of their tasks can be different. these specialists are often familiar with aspects of case management; medication adherence; provision of information, education, psychosocial support, and coping skills; and caregiver support [38] . patients with pd should have 1) regular access to clinical monitoring and adjustment of medication in consultation with the treating physician; 2) regular contact with caregivers, including home visits, as appropriate; and 3) access to reliable sources of information on clinical and social issues affecting patients with pd and their caregivers/families. these functions could be provided by pd nurse specialists or a pass. the positive therapeutical effects of pd nurse specialists are currently evaluated for their health economic impact [39] . in particular, patients with advanced pd may benefit early from palliative care. doctors and nursing staff can provide information about the final phase so that the family can take advantage of adequate care options. palliative care should be aligned with patient priorities and complement other treatments. therefore, advanced care planning might also increase knowledge about end of life issues. generally, it should start early in the course of the disease. it can be started when particular symptoms occur (pain, dyspnoea, dysphagia, and aspiration) or at the very end of life [40, 41] . besides general markers of advanced disease (frequent infections and hospitalizations, malnutrition, etc.), the palliative performance scale can be used to measure the functional status of a patient and to determine the eligibility for enrolment in a palliative care programme [41, 42] . dysphagia with symptomatic aspiration might be taken as a clear indicator when palliative care should begin, because it also involves a discussion about life-prolonging therapies such as tube feeding. figure 1 provides an overview of common players and structures in a local supply network. information on clinical and social issues affecting patients with pd and their caregivers/families. these functions could be provided by pd nurse specialists or a pass. the positive therapeutical effects of pd nurse specialists are currently evaluated for their health economic impact [39] . in particular, patients with advanced pd may benefit early from palliative care. doctors and nursing staff can provide information about the final phase so that the family can take advantage of adequate care options. palliative care should be aligned with patient priorities and complement other treatments. therefore, advanced care planning might also increase knowledge about end of life issues. generally, it should start early in the course of the disease. it can be started when particular symptoms occur (pain, dyspnoea, dysphagia, and aspiration) or at the very end of life [40, 41] . besides general markers of advanced disease (frequent infections and hospitalizations, malnutrition, etc.), the palliative performance scale can be used to measure the functional status of a patient and to determine the eligibility for enrolment in a palliative care programme [41, 42] . dysphagia with symptomatic aspiration might be taken as a clear indicator when palliative care should begin, because it also involves a discussion about life-prolonging therapies such as tube feeding. figure 1 provides an overview of common players and structures in a local supply network. the therapist network (outpatient) directly surrounding the patient and his or her environment is not only linked to the patient, but therapists are also linked to each other. this results in mutual inter-relationships and a flow of information between all professional groups involved (not only between the directly neighbouring ones). a supraregional supply network in the form of clinics and centers is connected to this ''micro-network''. here, exchange and cooperation results. different stationary and semistationary care options are offered and supplemented with, for example, telemedical services (e.g., medical video observation and sensor-based motion analysis). the therapist network (outpatient) directly surrounding the patient and his or her environment is not only linked to the patient, but therapists are also linked to each other. this results in mutual inter-relationships and a flow of information between all professional groups involved (not only between the directly neighbouring ones). a supraregional supply network in the form of clinics and centers is connected to this ''micro-network". here, exchange and cooperation results. different stationary and semistationary care options are offered and supplemented with, for example, telemedical services (e.g., medical video observation and sensor-based motion analysis). self-management means having knowledge, skills, and confidence to manage daily tasks when living with a chronic disorder such as pd. it includes the concepts of self-management tasks (medical, role, and emotional management) and self-management skills (problem solving, decision-making, resource utilization, the formation of a patient-provider partnership, action-planning, and self-tailoring) [43] . patients with pd should be able to monitor progress and problems and to set, communicate, and harmonize their individual therapeutic goals with all members of the health care provider team. in addition, required information for the individual aspects of the disease symptoms, treatments, and side effects/risks should be tailored to the patient requirements and transferred adequately to the patient. health care providers involved in the care of patients with pd can positively influence self-management skills with distinct approaches that mainly focus on education and support. self-management in pd may, therefore, contribute to slower disease progression, reduced complications, and lowered costs [44] . however, self-management support interventions for patients with pd vary in content, structure, and intensity, and little is known about which existing self-management support programmes are most effective. as indicated by a recent overview of self-management support programmes for patients with pd, clinicians should ensure that the key components of education, goal setting, and guided problem solving are included. moreover, adding these skills to the rehabilitation process and including caregivers and peer support systems seems promising [44] . as mentioned above, pd requires close interaction between different care partners in order to provide the best possible care for the patient. rural location, nursing home residence, and the presence of physical or cognitive impairment are common reasons for limited access to specialized pd health care [45] . a pd network can improve access to specialized health care and manage the distribution of resources, tasks, and responsibilities. by doing so, pd networks can help to avoid unnecessary hospitalization and reduce costs [8] . different methods exist to bring pd-specific knowledge and care to the patients in a pd network structure. in this context, telemedicine has shown promising effects for the management of pd. this includes synchronous methods (videoconferencing) and asynchronous methods (e.g., e-mail, smartphone assessments, remote monitoring, and wearable devices) [10, 11, 46] . telemedicine has the potential to allow pd-specific efficient care to be delivered to more patients and more regularly than a traditional model of care [47] . from the patient's view, telemedicine has the advantages of access to specialists, convenience, and time savings [48] . at present, it is applied in several clinical settings due to sanctions imposed for infection prophylaxis in the current sars-cov-2 pandemic, and it is seen to be a suitable tool with which to give advice and treat patients with pd. it also can be used to support outpatient palliative care teams with special neurological knowledge when the patient chooses to die at home [49] . since 2019, the remuneration of video consultation hours has been based on the insured, basic, or consultation flat rate in germany. nevertheless, telemedicine is still limited by patients' limited access to high-speed internet and usability issues (especially in elderly patients) [46] . nevertheless, with the new digital health act (''digitale-versorgung-gesetz" (dvg)), reimbursement for video-based home telemedicine support has begun in germany, and now, home telemedicine needs to be integrated into pd health care workflows. the german health care system is struggling with the issues of separation of care sectors (e.g., outpatient vs. inpatient care) and considerable differences in the provision of care in urban and rural areas. in order to optimize the specialized care of patients with pd in germany, the current care structures must be changed. this can be achieved by establishing pd networks, which act as a link between outpatient and inpatient treatment as well as between patients, caregivers, gps, nonspecialized neurologists, movement disorder specialists, and other therapists. this is a promising way to ensure that a stage-appropriate and patient-specific therapy for pd can be initiated promptly and maintained permanently in accordance with the current guidelines. additionally, new e-health processes might overcome current barriers and limited access to specialized health care and provide both patients and health care professionals with the potential for future seamless care, a strong interaction between health care partners, and involvement of patients and caregivers. interestingly, many patients with pd are using digital media tools and smartphones and thus have access to digital technology [50] . furthermore, the recently released digital health act (dvg) will enable patient-centered technologies as digital health care applications for better support of trans-sectoral pd health care. especially against the background that some studies have found only limited benefits from specialized network structures, it is very important to provide scientific support for the formation of networks in germany. these studies from england or the netherlands that focus on very limited aspects (e.g., pd nurses, physiotherapy) are only transferable to germany to a limited extent [33, 51] . decision makers, planners, and managers need evidence-based policy options and information on the scope of networks [52] . the dpg working group networks and integrated care is therefore an opportunity to provide a framework for various forms of networks, to facilitate the exchange of experience, and to provide scientific support for the various structures and networks with their regional characteristics. the epidemiology of parkinson's disease: risk factors and prevention nonmotor symptoms in parkinson's disease dynamics of parkinson's disease multimodal complex treatment in germany from 2010-2016: patient characteristics, access to 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committee. international parkinson and movement disorder society evidence-based medicine review: update on treatments for the motor symptoms of parkinson's disease physical therapy and occupational therapy in parkinson's disease otip study group. efficacy of occupational therapy for patients with parkinson's disease: a randomized controlled trial dysphagia in parkinson's disease a guideline for parkinson's disease nurse specialists, with recommendations for clinical practice the cost-effectiveness of specialized nursing interventions for people with parkinson's disease: the nice-pd study protocol for a randomized controlled clinical trial specialist palliative care for parkinson's disease palliative care for parkinson's disease using the palliative performance scale to provide meaningful survival estimates self-management education: history, definition, outcomes, and mechanisms self-management support programs for persons with parkinson's disease: an integrative review patientenperspektive auf die versorgungssituation im krankheitsbild morbus parkinson in deutschland-eine querschnittserhebung the promise of telemedicine for chronic neurological disorders: the example of parkinson's disease the promise of telemedicine for movement disorders: an interdisciplinary approach patient views on telemedicine for parkinson disease telemedicine in palliative care: implementation of new technologies to overcome structural challenges in the care of neurological patients the use of digital technology and media in german parkinson's disease patients effects of community based nurses specialising in parkinson's disease on health outcome and costs: randomized controlled trial this article is an open access article distributed under the terms and conditions of the creative commons attribution acknowledgments: travel costs for experts meeting by dpg. tino prell has received bmbf research grant, and honoraria for presentations/lectures abbvie gmbh, ucb pharma gmbh, desitin gmbh, licher mt gmbh, and bayer ag deutschland. frank siebecker reports no conflict of interest. michael lorrain has received honoraria and compensation for consultancy and lecturing from abbvie, afi, bayer, bial, biogen, desitin, merck, nordrheinische akademie, teva, ucb, and zambon. carsten eggers received payments as a consultant for abbvie inc. ce received honoraria as a speaker from abbvie inc., daiichi sankyo inc., bayer vital inc. ce received payments as a consultant for abbvie inc. and philyra inc. stefan lorenzl reports no conflict of interest. jochen klucken reports institutional research grants from bavarian research foundation; emerging field initiative, fau, eit-health, eit-digital, eu (h2020), german research foundation (dfg), and bmbf, and industry-sponsored institutional iits and grants from teva gmbh, licher mt gmbh, astrum it gmbh, and alpha-telemed ag. he is coemployed by the university hospital erlangen, germany, fraunhofer institute for integrated circuits e.v., germany, and the medical valley digital health application center gmbh, bamberg, germany. he works on advisory boards in the field of healthcare technologies and digital health of different associations of medical professionals, industries, and political authorities. he holds shares of portabiles healthcare technologies gmbh, portabiles gmbh, alpha-telemed ag, and received compensation and honoraria from serving on scientific advisory boards for lichermt gmbh, abbvie gmbh, ucb pharma gmbh; he has lectured at ucb pharma gmbh, teva pharma gmbh, licher mt gmbh, desitin gmbh, abbvie gmbh, solvay pharmaceuticals, bial deutschland gmbh; celgene gmbh, lundbeck-foundation. dr. klucken has a patent related to gait assessments pending. tobias warnecke has received honoraria from abbvie (lecture fees, consultant). abbvie acts as coinitiator of the parkinsonnetwork muensterland+ (pnm+) and is cocontractor of the university hospital of muenster. carsten buhmann has received fees as speaker and/or advisor from abbvie, bial, desitin, grünenthal, licher, novartis, tad pharma, ucb, and zambon. lars tönges has received travel funding and/or speaker honoraria from abbvie, bayer, bial, desitin, ge, ucb, and zambon, and consulted for abbvie, bayer, bial, desitin, ucb, and zambon, in the last 3 years. reinhard ehret reports no conflict of interests. ingmar wellach has received honoraria an compensation for consultancy and lecturing from abbvie gmbh, ucb pharma gmbh, desitin gmbh, bial deutschland gmbh, zambon deutschland gmbh, fagron gmbh & co. kg, grünenthal gmbh, and bayer ag deutschland. martin wolz has received honoraria for presentations/lectures from zambon, valeant, desitin, teva, ucb pharma, abbvie, bial, licher, and daiichi sankyo. key: cord-333959-8ermzrmr authors: gao, zan; lee, jung eun; mcdonough, daniel j.; albers, callie title: virtual reality exercise as a coping strategy for health and wellness promotion in older adults during the covid-19 pandemic date: 2020-06-25 journal: j clin med doi: 10.3390/jcm9061986 sha: doc_id: 333959 cord_uid: 8ermzrmr the december 2019 covid-19 outbreak in china has led to worldwide quarantine, as recommended by local governments and the world health organization. particularly affected are older adults (i.e., those aged ≥ 65 years) who are at elevated risk for various adverse health outcomes, including declines in motor ability and physical activity (pa) participation, increased obesity, impaired cognition, and various psychological disorders. thus, given the secular increases in the older adult population, novel and effective intervention strategies are necessary to improve physical activity behaviors and health in this population. virtual reality (vr)-integrated exercise is a promising intervention strategy, which has been utilized in healthcare fields like stroke rehabilitation and psychotherapy. therefore, the purpose of this editorial is to synthesize recent research examining the efficacy and effectiveness of vr exercise in the promotion of favorable health outcomes among the older adults. results indicate the application of vr exercise to facilitate improved physical outcomes (e.g., enhanced motor ability, reduced obesity), cognition and psychological outcomes. vr exercise has also been observed to be an effective intervention strategy for fall prevention in this population. future research should employ more rigorous research designs to allow for a more robust quantitative synthesis of the effect of vr exercise on the preceding outcomes to elucidate which type(s) of vr-based pa interventions are most effective in promoting improved health outcomes among older adults. findings from this study will better inform the development of technology-savvy pa programs for wellness promotion in older adults who practice social distancing and exercise from home under the unprecedented global health crisis. the december 2019 novel coronavirus outbreak in china has infected more than 9.97 million people and resulted in over 480,000 deaths worldwide [1] , which has led to global quarantine as recommended by local governments and the world health organization. indeed, quarantine can help mitigate individuals' exposure to covid-19 and, therefore, minimize the risk of contracting the virus. however, the quarantine orders have created many national challenges that have had profound impacts on financial, physical, psychological, and emotional health among people of all ages [2, 3] . particularly affected are older adults (i.e., those 65 years and older) who are more likely to suffer from serious covid-19 illness. in fact, 8 out of 10 deaths reported in the u.s. have been in older adults [4] . this may be partially attributed to compromised immune systems with age, making it harder to fight off coronavirus diseases and infection. in the past 30 years, researchers have found regular physical activity (pa) participation to have beneficial effects on older adults' health and wellbeing [5, 6] . from 2020 to 2030, the number of older adults in the u.s. is expected to increase from 56 million to 74 million, which amounts to one in five americans [7] . this generation has higher rates of chronic disease and disability compared to any other generation [8] , and studies have shown that the four most common poor health conditions seen in older adults are decreased motor ability, increased obesity, impaired cognition, and psychological disorders, which lead to a lower quality of life [9, 10] . for example, an inactive lifestyle, along with a natural decline in physiological markers with age, contributes to a loss in muscle strength and balance [9] , and, through deterioration of motor abilities, older adults' risk for falls and fractures increases [8] . furthermore, the prevalence of obesity in older adults puts them not only at higher risk for developing cardiovascular diseases but also acquiring a disability and remaining physically impaired [10] . cognitive impairment is a health concern that makes it difficult for older adults to live independently and also places them at a higher risk for falls [9] . for instance, it has been shown that older adults with cognitive impairment are twice as likely to have a fall compared to older adults without impaired cognition [9] . lastly, depression, anxiety disorder, and dementia are the most prevalent psychological problems in older adults [10] . it is dismaying that 21% of older adults report experiencing symptoms of anxiety that contribute to significant distress, lower quality of life, and a higher chance of having depression [11] . thus, it is important to develop and implement effective intervention strategies that can prevent or reverse these adverse health outcomes in order to improve older adults' quality of life. given that many are experiencing stressful life challenges under the covid-19 pandemic crisis, it is imperative to develop innovative and effective pa intervention programs that reduce stress and promote health and wellbeing in older adults [12, 13] . one innovative intervention strategy that has shown promise in the healthcare field is virtual reality (vr)-based pa interventions [14] . however, reviews investigating the effectiveness of vr in the promotion of better health outcomes in older adults are scarce [15] . therefore, the purpose of this editorial was to determine the efficacy and effectiveness of vr exercise in aiding healthy older adults to have increased motor ability, reduced obesity, improved cognition, and better psychological outcomes. as known, vr is a new and engaging technology that has received limited research with regards to health promotion in older adults. the findings of this paper may provide healthcare practitioners and researchers with valuable information on the utility of vr that they could apply in community and home settings under challenging circumstances. one intervention strategy which has shown promise for promoting healthy aging among older adults is vr-integrated exercise [16, 17] . vr exercise is a novel and innovative technology, which immerses individuals in a computer-generated, multi-sensory, three-dimensional world wherein they interact with the virtual environment using either a headset and/or exercise equipment [18] [19] [20] . vr technology can be dichotomized by immersion (i.e., immersive and non-immersive). immersive vr typically requires the use of a head-mounted display (e.g., oculus rift, menlo park, ca, usa) or an entire room display which encloses the user (e.g., cave automatic virtual environment (cave)) [21] . non-immersive vr, on the other hand, offers users a computer-generated world which typically uses a desktop or projector [18] . examples of non-immersive vr include the nintendo wii switch and the xbox 360 kinect, which are often more cost-effective and better for use in the home setting compared to immersive vr equipment [22] [23] [24] [25] [26] [27] [28] . vr technology is currently used in a variety of health field areas, such as psychotherapy and stroke rehabilitation [14] , and has been shown to be effective in improving balance and overall health and promoting weight loss in older adults [9, 14, 16, 29] . for instance, vr has been implemented within therapeutic programs for phobias related to height and public speaking, in which patients were immersed into an environment where they progressively worked on their fears [30, 31] . furthermore, vr exercise has been successfully used within rehabilitation settings for motor learning following a stroke, which led to patients' increased brain plasticity [17, 32] . vr has also been shown to be effective in exercise promotion, which led to multiple health benefits, including reduced obesity and anxiety, as well as improved cognition [9, 16, 29] . additionally, studies have suggested that vr consisting of cognitive behavioral treatment could aid in weight loss and alleviation of psychological disorders [11, 33] . along with all these health benefits, vr also presents itself as a potential candidate for promoting leisure activity. participants who were immersed into nature via vr while using a traditional exercise bike reported that it was much more enjoyable than traditional exercise biking alone [34] . the application of vr has been shown to have positive benefits on older adults' physical and mental health; however, these findings are still limited. therefore, more innovative and technology-savvy interventions need to be employed to help control obesity rates and poor health concerns in this population. due to aging, older adults naturally exhibit decreased motor ability, including compromised coordination, balance, muscular strength, and speed [22] . in general, vr exercise has demonstrated positive effects on the preceding components of older adults' motor ability by engaging older adults' motor skills and promoting sensorimotor learning and cortical plasticity to improve their motor ability. for example, a home-based vr intervention, which used an xbox 360 gaming console and your shape fitness evolved software and consisted of tai chi and yoga exercise programs, indicated positive effects of vr exercise on older adults' motor ability outcomes, such as hip muscle strength and balance control [22] . furthermore, significantly improved muscle strength as assessed by hand grip dynamometry and an arm curl test and improved balance measured by a postural sway test were evident in another study that implemented a three-dimensional vr kayak program [9] . while these two studies had muscular strength as an outcome measure which significantly improved, one looked at hip strength and the other used grip strength. due to this difference, the effect of vr on targeted muscle strength is inconclusive, and more research is needed in the future. rehabilitation methods (e.g., therapeutic exercise) have been employed extensively with the aim of improving older adults' motor ability. however, current rehabilitation methods with this aim often fail to account for the characteristics and needs of patients and, consequently, the patients often do not see rehabilitative success in the real world [35] . findings suggest that the learning of new skills and activation of brain plasticity are enriched when a patient is placed in an appropriate environment that resembles real life [35] . for example, in a recent study that employed an immersive vr intervention (the cave), the scenario placed participants in an apple orchard, where they had to reach out as quickly as possible to grab the virtual apple then place it in the basket to score points [35] . the results demonstrated a gradual increase in scores and improved postural stability, which is an important component of motor ability. overall, existing vr exercise programs were all shown to significantly improve older adults' motor ability through increased balance. with improved balance control, older adults can achieve better health outcomes, such as reduced falls. however, research examining the effect of vr exercise on strengthening the larger musculature (e.g., hips, arms) is needed to determine if it is an effective intervention strategy for improving motor ability in older adults. studies show that over one third of older adults are obese, and the prevalence is steadily increasing [36] . this calls for effective and innovative intervention strategies to manage and prevent obesity in older adults. while vr exercise's utility for weight loss and control is relatively new, it is well established that technology-based interventions targeting weight loss are scalable and cost-effective [37] . for example, manzoni and colleagues [33] and thomas and bond [37] examined the efficacy of vr-integrated cognitive-behavioral treatment (cbt) for reducing obesity among older adults. cbt is a type of psychotherapy commonly used to help treat eating disorders, which aims to change individuals' thinking patterns using a goal-oriented approach [37] , whereas vr-integrated cbt aims to teach problem-solving techniques and reduce body weight and problematic eating. manzoni and colleagues [33] utilized the neurovr open space software to station participants in real-world environments where they had to handle situations of daily living, such as working out at a gym, shopping at the grocery store, or dining at a restaurant. the researchers observed at one-year follow up that the vr group displayed consistent weight loss maintenance, whereas the control group gained back most of their lost weight. additionally, thomas and bond [25] conducted research using a vr-based behavioral weight loss program (second life virtual world), in which participants learned to navigate difficult situations. although the sample size in this study was small, the results suggest that vr may be more beneficial for long-term weight loss compared to traditional, face-to-face treatments. beyond vr-integrated cbt's implications for weight loss, vr may also encourage weight loss indirectly through the promotion of pa. wii fit, for example, is readily accessible, affordable, and motivating for older adults and has shown promise for promoting pa and weight loss in this population. for example, one study observed wii fit sports to increase daily energy expenditure and time spent in moderate to vigorous pa in older adults at risk for obesity [38] . although no significant correlations could be made due to the small sample size, the findings showed modest weight loss and enjoyment among participants while they engaged in the exercise, which may be promising for long-term adherence [38] . while vr-integrated cbt studies [33, 37] have reported chronic effects on weight loss as compared to controls, the preceding wii fit study [38] primarily targeted participants' attitudes toward pa, which indicated vr exercise to be more a more engaging form of exercise compared to traditional exercise. overall, findings suggest vr-integrated cbt is effective for assisting older adults in weight loss maintenance for months after the cessation of the intervention programs [33, 37] . further, wii fit sports games increased participants' pa levels and pa-related enjoyment following an 8-week program. notably, however, given the small sample size and short intervention length, these findings warrant further empirical support [38] . vr-based exercise interventions like cbt and wii fit exercise programs are highly accessible, cost-effective, and motivating strategies, which show promise for obesity reduction in older adults. however, further research addressing the preceding research gaps are needed. declines in cognitive ability is a part of normal aging and may eventually develop into cognitive disorders [11] . vr has shown promise for improving cognitive functions, such as executive function, visuospatial processing, and memory [9] . specifically, vr interventions like immersive memory training and a three-dimensional kayaking exercise program significantly improved older adults' shortand long-term memory [9, 39] . further, another study observed a 6-week vr kayaking program to significantly improve cognitive older adults' cognitive functioning, including executive functions, conceptual thinking, concentration, attention, visuoconstructive skills, working memory, mathematical calculations, language, and orientation [9] . results indicated these cognitive domains to significantly improve from pre-to post-intervention only in the vr experimental group. another study indicated that vr exercise may also be a promising tool for improving cognitive functioning using vr memory training [39] . in this study, participants in the vr group used a head-mounted display and a joystick to maneuver along city paths within the immersive vr environment and were then asked to memorize and recall those paths. findings from the neuropsychological tests showed significant improvements in overall cognitive functioning and verbal memory. notably, only small, non-significant improvements in executive functioning and visuospatial processing were observed. this may be attributable to some of the tests requiring drawing pictures, and not all participants may have had the natural drawing abilities needed to adequately perform on these tests. although these two studies [9, 39] targeted similar cognitive domains, such as executive function, memory, and visuoconstruction/visuospatial skills, as health outcomes, the two differed in terms of the level of improvement in such outcomes. possible explanations for these differences include different samples and intervention components and inconsistency in the employed cognitive domain tests. therefore, more research with consistent intervention components and testing is warranted to determine if vr exercise truly facilitates significant improvements in these cognitive functions. however, memory was observed to significantly improve in both studies. in sum, vr exercise shows promise for improving cognitive functioning and memory in older adults as well as other cognitive outcomes, but further research is warranted to confirm this. with an increase in cognitive function and ability, older adults will experience improved mental health outcomes and exhibit a lower risk for falls. findings indicate that over 21% of older adults experience anxiety symptoms [11] . the use of vr exercise has shown promise for decreasing anxiety and depression in older adults, which may translate to improved overall mental health outcomes in this population [29] . this preliminary review identifies and examines five eligible studies, which reported that vr exercise programs can relieve feelings of anxiety and depression and increase enjoyment and daily energy levels [29] . for example, one study had 54 older female participants undergo either a group-based exercise program or a vr-based tai chi exercise program. the investigators observed the vr exercise group to report significantly greater decreases in anxiety and depression compared to the traditional exercise program. on the other hand, one study not included in the review utilized the geriatric depression scale and observed no significant differences in these outcomes following a vr-based, wii fit balance intervention [40] . there is also the possibility of using vr with cbt to decrease anxiety in older adults. in another preliminary review, which examined three meta-analyses to determine the potential of vr-enhanced cbt in treating anxiety disorders in older adults [11] , the authors revealed that the number of cbt randomized controlled trials in older adults was half that of studies on younger adults and none have been designed to explore vr-enhanced cbt for adults 65 and older. since vr-enhanced cbt has been successful in treating anxiety disorders in younger adults, grenier et al. [11] proposed a pilot study that investigates the efficacy of an 8-week cbt program which integrates vr. the treatment will teach participants how to cope with the triggers and episodes of anxiety. in sum, vr has been purported as a promising tool for facilitating better mental health outcomes in older adults when combined with cbt and for its ability to relieve feelings of anxiety and depression. however, more supporting empirical evidence is needed in this field of inquiry, considering that only one empirical study and two preliminary reviews were identified. both the wii fit and vr-based tai chi studies used anxiety and depression as the mental health outcomes. however, compared to controls, only the vr-based tai chi pa program prompted significant improvements in feelings of anxiety and depression. conversely, the wii fit program, compared to the control group, observed some improvement in feelings of anxiety and depression, though statistical significance was not reached for either outcome. differences in outcomes between studies may be explained by differences in modality, duration, intensity, and/or frequency of the exercise programs. thus, more research is needed to discern the effectiveness of vr exercise in the promotion of improved psychological outcomes in older adults, such as depression and anxiety. approximately 30% of older adults experience at least one fall each year, and those that have a fallen are at increased risk of falling again [41] . older adults who have a history of falls tend to have significantly lower muscle strength in their hip musculature [22] . pa has been shown to improve muscular strength and balance, and, therefore, reduce the risk of falls among the elderly [42] . research has identified two main types of vr-based exercises that are related to older adults' reduced fall rates: vr-based treadmill exercises and wii fit exercises. to date, two studies have examined the effects of vr-based treadmill exercise, both of which found significant decreases in the incidence of falls in the vr training group compared with a traditional treadmill exercise group [43, 44] . with regard to wii fit exercise, studies suggested that both immersive and non-immersive wii fit exercise can decrease older adults' risk for falls by improving their motor functioning, such as by improving their center of balance [14, 45] . chiarovano et al. [45] used immersive vr (oculus rift dk2 vr headset) in conjunction with the wii fit balance board and the balancerite application, while other researchers [14] used a non-immersive nintendo wii fit exercise wherein participants played ski slalom, table tile , and balance bubble. findings suggested that, through having older adults perform the dual task of working on postural stability as well as respond to powerful visual stimuli, older adults increased their capacity for attention demands and decreased their risk of falls. these findings support the effectiveness of vr exercise interventions in reducing fall rates and improving balance in older adults. thus, the use of vr exercise training can be a more effective fall prevention tool compared to treadmill exercise training alone through increased balance and speed and the teaching of reactive strategies. it has been reported that age-related cognitive declines increase older adults' risk for falls, which are a major contributor to morbidity and mortality rates in this population [43] . for instance, 60-80% of older adults who have cognitive impairments report at least one a year. these falls often occur due to compromised executive functioning and, therefore, navigation, causing them to trip over obstacles and basic objects [43] . therefore, improving cognition is of paramount importance for reducing the risk of falls and improving quality of life in older adults. one study [43] that examined the use of vr treadmill exercise as an intervention strategy to reduce falls also targeted cognitive functioning. in detail, the vr simulation was composed of real-life situations and challenges, such as obstacles and distractions, in order to enhance older adults' cognitive functioning (i.e., executive function and attention) while walking. executive functioning and attention play a major role in obstacle clearance and are, therefore, essential in the prevention of falls. the findings from this study indicate that treadmill training concurrent with vr exercise is more effective than treadmill exercise alone for improving cognitive functioning and, therefore, reducing falls among older adults. in addition, findings from two other studies [22, 45] showed that both immersive and non-immersive vr treadmill exercise and the wii balance board were effective for reducing rates of falls in older adults by lessening the severity of falls and teaching more effective fall prevention strategies. fear of falling in older adults entails an intense fear of standing or walking. the prevalence of this phenomenon is 24-55% in older adults and up to 92% in older adults who have experienced at least one fall [46] . serious consequences come with fear of falling, including decreased social interactions, physical injury, reduced quality of life, and accidental death, which further supports the need for effective exercise-based therapeutic interventions. current available treatments include traditional exercise interventions and protectors worn at the hip. however, these methods have only shown minimal effects and do not consider the psychological aspect of the fear of falling [46] . that said, vr exercise has shown promise for addressing the fear of falling in older adults. for instance, levy and colleagues [46] examined the effect of immersive vr games in participants who reported having a fear of falling, such as fighting off enemies by moving their hands and washing a window with foam. a questionnaire regarding the activities of daily life (e.g., getting out of bed, putting on clothes) demonstrated significant improvements in older adults' fear of falling after the vr exercise intervention compared to a control group. these findings showed promise for the utility of vr-based exercise interventions for successfully reducing older adults' fear of falling and, thus, improving their motor ability and overall quality of life. noteworthy is the fact that this study had a small sample size and, therefore, more research is needed to further support these findings. vr is a promising tool for effective treatment in the rehabilitation setting. by implementing non-immersive vr on the treadmill or immersing a patient into a realistic environment, such as a city or park setting with a head-mounted display or within the cave, physical and occupational therapy sessions can be enhanced, subsequently increasing the chance of successful adaptation to the real world [35] . participants also found that exercising on a stationary bike with vr that immersed them into nature was significantly more enjoyable than traditional biking without vr [34, 47] . since vr was found to be an engaging activity for older adults, this could lead to better adherence to a rehabilitation program, which in turn may lead to better health outcomes in patients. vr exercise interventions also include home-based interventions, such as vr-based tai chi and yoga programs [22] . the use of at-home rehabilitation techniques would lead to more effective rehabilitation, as older adults can receive real-time feedback from home by using vr during times in which they are not at the clinic. this may be especially important during the covid-19 pandemic, as older adults may wish to remain quarantined in their homes given their increased risk of contracting the virus. home-based vr exercise interventions can also help relieve stress from healthcare services with the surge of baby boomers reaching older age. this reduction in overscheduling for physical and occupational therapists may allow them to provide better care during their sessions. further, during in-person appointments, vr exercise can be supplemented to increase patients' exercise motivation and enjoyment. though some studies support vr exercise's effectiveness in promoting better health outcomes among older adults, they are not without limitations. for example, older adults' success in using vr-integrated exercise may be limited by perceptual, mental, and physical declines that naturally come with age [10] . thus, these individuals may be discouraged from participating in vr exercise interventions and may negatively impact retention rates in such studies. second, many of the included studies had small sample sizes (≤30 participants), which may have affected the external validity of the findings. additionally, the implemented vr exercise interventions varied greatly, in that immersive and non-immersive vr-integrated exercise equipment and vr-enhanced cbt, among other intervention strategies, were used across studies. this renders it difficult to confidently conclude that all vr exercise modalities and programs can facilitate better health outcomes in older adults. as such, we recommend more research be conducted in this area of inquiry to better discern which vr interventions are the most effective among older adults. future studies should address the research design issue observed in most studies by increasing sample sizes [48] . more research focusing on the mental health problems seen in older adults is also needed. in addition, there is a need for more research investigating the effectiveness of vr exercise programs on older adults' weight loss, as vr exercise has only recently been applied as a means for weight control. in addition, examining the motivation to maintain or increase pa participation [49] during leisure time among older adults using vr at homes or community centers is warranted. finally, as stated above, health professionals need to determine which specific types of vr are most effective for improving health outcomes in healthy older adults. this may include determining factors, such as modality, intensity, duration, and frequency, as well as vr exercise setting(s) most suitable for older adults. the purpose of this paper was to explore the potential of using vr exercise as a coping strategy for health and wellness promotion in older adults during the covid-19 pandemic. vr is an emerging technology that is a valuable tool for healthy aging in older adults. empirical studies support that vr leads to improvements, although not always significant, in the four most common health concerns seen in older adults: decreased motor ability, increased obesity, impaired cognition, and various psychological disorders. across studies, findings demonstrate that vr exercise interventions lead to significant improvements in older adults' balance and memory, which contribute to a lower risk for falls. given the secular increases in the older adult population, healthcare services must be equipped to meet their specific health needs. indeed, chronic disease and disability prevalence in this generation of older adults can be compared to any other generation and vr is purported to be a valuable intervention tool and strategy in rehabilitation and/or home settings in this population. integrating vr into physical and occupational therapy may serve to minimize stress in clinicians and patients by allowing patients to engage in vr-based rehabilitation from home. further, compared to traditional exercise intervention strategies, vr exercise has been shown to be more effective in leading to more significant and faster recoveries. this may be partially attributed to vr's engaging nature, making it well tolerated by older adults. additionally, vr exercise interventions may have multiple health benefits pertaining to older adults' motor ability, obesity status, cognition, and psychological outcomes. however, much more research is needed to investigate this novel treatment strategy among older adults. it is especially imperative for health professionals to deliver exercise programs remotely due to social distancing under covid-19 and for possible future pandemic crises. author contributions: z.g. conceived the study and drafted the manuscript; j.e.l., d.j.m. and c.a. helped draft the manuscript. all authors agree with the order of presentation of the 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pasqualato, sebastiano; mapelli, marina; facciotti, federica title: persistence of anti-sars-cov-2 antibodies in non-hospitalized covid-19 convalescent health care workers date: 2020-10-01 journal: j clin med doi: 10.3390/jcm9103188 sha: doc_id: 318018 cord_uid: ybdkp398 although antibody response to sars-cov-2 can be detected early during the infection, several outstanding questions remain to be addressed regarding the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response. an elisa assay has been developed by expressing and purifying the recombinant sars-cov-2 spike receptor binding domain (rbd), soluble ectodomain (spike), and full length nucleocapsid protein (n). sera from healthcare workers affected by non-severe covid-19 were longitudinally collected over four weeks, and compared to sera from patients hospitalized in intensive care units (icu) and sars-cov-2-negative subjects for the presence of igm, igg and iga antibodies as well as soluble pro-inflammatory mediators in the sera. non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles as compared to patients in intensive care units (icu), irrespective of the antibodies tested. noteworthy, in non-severe covid-19 infections, antibody titers against rbd and spike, but not against the n protein, as well as pro-inflammatory cytokines decreased within a month after viral clearance. thus, rapid decline in antibody titers and in pro-inflammatory cytokines may be a common feature of non-severe sars-cov-2 infection, suggesting that antibody-mediated protection against re-infection with sars-cov-2 is of short duration. these results suggest caution in using serological testing to estimate the prevalence of sars-cov-2 infection in the general population. the coronavirus disease-19 (covid-19) is a respiratory illness caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a novel beta-coronavirus firstly described in wuhan city, china, on december 2019 [1] . sars-cov-2 spreading has been declared pandemic in mid-march 2020 by who [2] . at present, the virus has infected more than 33 million people worldwide with an associated case mortality rate of 1% to 15%, depending on the country [3] . covid-19 is associated with a broad range of mild-to-severe symptoms, potentially leading to hospitalization in intensive care units (icu) for the most severe cases. the respiratory tract is initially involved with possible development of severe interstitial pneumonia [4, 5] , albeit the gastrointestinal tract can also significantly participate in disease pathogenesis as a consequence of the expression of the ace2 receptor, that mediates sars-cov-2 viral entry [6] , on both alveolar and enteric epithelial cells [7] . infected subjects manifest a complex clinical pattern appearing as early as two days post exposure and lasting several weeks [1] . infection with sars-cov-2 induces a prompt activation of the immune system, finalized to the clearance of infected cells [8] . innate and adaptive immune cells accumulate at the site of infection, where production of cytokines and inflammatory mediators may result in patient recovery or, in case of ineffective viral clearance, in hyperactivation of the immune system and development of severe complications, such as acute respiratory distress syndrome ards [4, 9] . overexpression of pro-inflammatory cytokines (i.e., il-1 beta, il-2, il-6, il-17, tnfα etc.) and impairment of humoral immunity have been described in patients with the most severe form of the disease [5] . antibodies against sars-cov-2 proteins are produced as a consequence of the activation of the humoral arm of the immune system. virus-specific igm antibodies are secreted as first class of immunoglobulins, followed by the more specific igg [10] . among the latter, those specific for the viral spike receptor binding domain (rbd) when expressed at higher titer manifest direct neutralizing activity towards the viral entry into cells, as they prevent effective engagement of surface ace2 receptors by the spike protein [11, 12] . the iga response against sars-cov-2 has been shown to be rapid and persistent [13, 14] and is associated with mucosal responses, including respiratory and gastrointestinal responses. serological testing is a valuable tool to monitor viral spreading throughout the population [15] . furthermore, serological assays allow the identification of past infection in individuals with viral rna levels undetectable by rt-pcr for epidemiological purposes [16] . various commercial and in-house assays that utilize distinct viral antigens and detect different antibody classes are currently available. however, sars-cov-2 serological tests available on the market do not always allow systematic simultaneous detection of a wide antibody spectrum for several antigens in a reliable manner, and this may hamper a proper population testing for clinical or epidemiological purposes [17] . conversely, serological enzyme-linked immunosorbent assays (elisa) to detect immunoglobulins raised against the viral spike soluble ectodomain (spike) and its highly immunogenic receptor binding domain (rbd), or against the nucleocapsid protein (n), provide promising results in terms of accuracy and reproducibility [11] . recently, these elisa assays have been used to show that neutralizing antibodies (nab) against different viral antigens may decline after 20-30 days post symptoms onset, and that the magnitude of nab response may be associated with disease severity in covid-19 patients [18] . in order to measure the presence and variation of antibody responses against different viral proteins, we set up and validated an in-house direct elisa assay based on three distinct sars-cov-2 viral antigens, i.e., eukaryotically-expressed rbd and spike and bacterially-expressed nucleocapsid protein. using this assay, we simultaneously measured igm, igg and iga anti-viral antibodies titers in the sera of covid-19 patients, as well as levels of pro-inflammatory cytokines. in addition, we longitudinally collected the sera of 16 convalescent healthcare workers who tested positive for sars-cov-2 by nasopharyngeal (nf) swabs, and were symptomatic but not hospitalized. our data show that humoral immune responses against sars-cov-2 correlated with disease severity in terms of both antibody titers, persistence over time and serum levels of pro-inflammatory cytokines. notably, 90% of covid-19 mildly symptomatic patients halved their anti-rbd igg titers after 4 weeks from viral negativization, thus confirming the short lifespan of humoral immune responses against sars-cov-2. health care workers of two different covid hospitals in milan (n = 16) with documented covid-19 infection (by nf swab), not hospitalized but with manifested covid-19 symptoms (supplementary table s2 ) were monitored for seroconversion by igm, igg and iga serum levels at two time points after viral clearance between april and june 2020. the study has been conducted in accordance with the standards of good clinical practice, with the ethical principles deriving from the helsinki declaration and the current legislation on observational studies. clearance from the ethical committee has been obtained (ieo1271). additional study populations were icu hospitalized severe covid-19 patients (n = 24) and (n = 58) covid-19 negative subjects whose sera were collected between april and june 2020. in total, 436 pre-covid subjects enrolled in ieo studies between 2009 and 2015 were used to calculate the roc curves for the assays. the exclusion criterion was, for all subjects involved in the study, the inability to provide informed consent. the inclusion criteria were, for those not hospitalized with covid-19, (i) being health care workers (medical doctors, practitioners, post-graduate students, nurses), potentially exposed to sars-cov-2 between february and june 2020, (ii) documented sars-cov-2 infection by nf swab, (iii) not being hospitalized for covid-19; for those hospitalized with covid-19: (i) documented sars-cov-2 infection by nf swab, (ii) being admitted in the icu between february and june 2020 for covid-19; for negative controls: (i) sera being collected before 2019. the recombinant spike sars-cov-2 glycoprotein receptor binding domain (rbd) and the soluble full-length trimeric ectodomain have been produced in mammalian hek293f cells as glycosylated proteins by transient transfection with pcaggs vectors generated in prof. krammer's laboratory [11] . the constructs were synthesized using the genomic sequence of the isolated virus, wuhan-hi-1 released in january 2020, and contain codons optimized for expression in mammalian cells. briefly, hek293f cells were seeded at a final concentration of 0.5 million/ml in freestyle medium (thermo fisher scientific, milano, italy), incubated at 37 • c, 5% co 2 at 120 rpm o/n in an eppendorf new brunswick s41i incubator. the day after hek293f cells were transfected using 1 µg of dna per 1 × 10 6 cells and a dna: pei max ratio of 1:5 in optimem medium. four hours post-transfection, the medium was supplemented with peptone primatone rl (merck) to a final concentration of 0.6% w/v. cells were then incubated for 6 days, checking cell viability daily if needed (a mortality higher than 30% is indicative of a toxic protein). for protein purification, the culture supernatant was transferred to conical centrifuge tubes, cleared by centrifugation at 1000×·g for 15 min and filtered with 0.22 µm stericup filters. the filtered medium addition with 1:10 volume of 570 mm nah 2 po 4 ph 8.0, 300 mm nacl and loaded on a hisprep fast flow 16/60 column (ge-healthcare) equilibrated in 57 mm nah 2 po 4 , 300 mm nacl. his-tagged protein was eluted with step gradients of 50-100-150-235 mm imidazole. peak fractions were pooled, dialyzed overnight against pbs and concentrated to 0.4 mg/ml (spike soluble) or 1.0 mg/ml (rbd) in 10 kda-mwco amicon filter units. retrieved proteins were quantified, flash frozen in liquid nitrogen in aliquots and stored at −80 • c. his-tagged sars-cov-2 full length n-protein plasmid (kind gift of david d. ho, md, columbia university, new york, ny, usa) was transformed in e.coli bl21 plyss cells. protein expression was induced with 0.5 mm iptg and carried on at 18 • c overnight. cells were harvested by centrifugation in lysis buffer (25 mm tris-hcl ph 8.0, 500 mm nacl, 1 mm dtt, 5% glycerol, 10 mm imidazole, with calbiochem protease inhibitor cocktail iii). all following steps were carried out at 4 • c or using ice-cold buffers. cells were lysed by sonication; lysate was cleared by centrifugation at 20,000× g for 40 min, then pei (ph 7.5-final concentration 0.02%) was dropwise added, under stirring, and lysate was then further cleared by centrifugation at 20,000× g for 40 min. next, 5 ml ni-nta beads per liter of culture, pre-equilibrated in lysis buffer, were added to the cleared lysate and protein binding was continued for 1 h in gentle agitation at 4 • c. beads were washed with at least 20 column volumes of 25 mm tris-hcl ph 7.4, 500 mm nacl, 1 mm dtt, 5% glycerol, 50 mm imidazole and his-tagged protein was eluted with 4 column volumes of 25 mm tris-hcl ph 7.4, 200 mm nacl, 1 mm dtt, 5% glycerol, 250 mm imidazole. the eluted fractions containing protein were diluted with heparin buffer (25 mm tris-hcl ph 7.4, 1 mm dtt) to reach final a nacl concentration of 0.1 m and were subsequently loaded onto a hi-trap heparin hp column (ge healthcare) equilibrated in 25 mm tris-hcl ph, 50 mm nacl, 1 mm dtt (buffer a). a linear gradient reaching 100% buffer b (25 mm tris-hcl ph, 1 m nacl, 1 mm dtt) in 30 column volumes was applied and fractions containing his-tagged n-protein were pooled, concentrated and loaded onto a superdex 200 16/60 size exclusion chromatography. fractions containing n-protein were pooled. a 4 l culture yielded 12.5 ml of 1.12 mg/ml pure n-protein, which was flash frozen in liquid nitrogen in aliquots and stored at stored at −80 • c. the elisa assay to detect immunoglobulins (ig) uses fragments of the sars-cov-2 spike glycoprotein (s-protein) and the nucleocapsid (n) as antigens based on the protocol published in [11, 19] . after binding of the proteins to a nunc maxisorp elisa plate, and blocking aspecific bindings with pbs-bsa 3%, patients' sera to be analyzed were applied to the plate to allow antibody binding at a final dilution of 1:200, revealed with secondary anti-human-igg (bd, clone g18-145), igm (merck, polyclonal code a6907), iga (biolegend, poly24110) antibody conjugated to hrp. samples are read on a glomax reader at 450 nm. this elisa test is not intended for commercial use and is currently under evaluation at the italy's ministry of health (aut.min.rich. 15.05.2020) for emergency use approval. the assay has been validated with a cohort of n = 56 covid-19 subjects (severe, moderate and mild disease) and n = 463 (subjects collected in pre-covid era (between 2012 and 2015)). roc curves have been implemented to determine the sensitivity and specificity of the assay (supplementary figure s1 ). quantification of soluble biomarkers was performed in sera of patients collected immediately after virus clearance (2 consecutive negative nf swabs) and one month post virus clearance using a luminex immunoassay (human cytokine/chemokine/gf procartaplex 45plex, thermo fisher) with map technology according to manufacturer's protocol. samples were acquired on a luminex 200sd and analyzed with xponent software 4.2. the sera of healthy subjects (n = 20) collected between april and june 2020 as well as icu covid-19 patients (n = 24) were used as control groups. the categorical variables were described as absolute frequency and percentage. the continuous variables with normal distribution were described as median ± standard deviation (sd), whereas the continuous variables without normal distribution were given as median and range. normality of continuous variables was checked with d'agostino-pearson omnibus normality test. the mann-whitney test or student's t-test for continuous variables, and the chi-square or fisher's exact tests for categorical variables, were used to associate clinical variables with the result of sars-cov-2 serological test (positive or negative). the p values lower than 0.05, two-tailed, will be considered statistically significant. graphpad prism software was used for all statistical analyses. to evaluate the antibody response of individuals infected by sars-cov-2, elisa assays were developed in-house by producing and purifying recombinant rbd, spike and nucleocapsid proteins of the sars-cov-2 virus following the protocols published in [19] ( figure 1a) . the performances of these elisa assays were assessed for the different viral antigens and classes of antibodies by determining roc curves using (i) a cohort of 56 sera from covid-19 patients collected between april and june 2020 and tested positive for nasopharyngeal swabs, and (ii) 436 pre-covid-19 sera, collected between 2012 and 2015 (supplementary table s1 and figure s1 ). anti-sars-cov-2 igg showed the highest specificity and sensitivity, irrespective of the antigen used (supplementary figure s1a,b) . anti-rbd igg showed a specificity and sensitivity of 97% and 95%, respectively, while the assay performed with the spike ectodomain reached values of 98.5% and 77% and the one with the n protein values of 91% and 95% (supplementary table s1 and figure s1 ). these performances are in line with those published for both in-house and commercial assays approved for emergency use by the fda [20, 21] . the performance of iga detection was high for the rbd assay (91.5% specificity and 95% sensitivity), while it was slightly lower for the n protein (85% and 69%) and for the spike (73% and 71%). the performance of the igm assay was comparatively lower for all the viral proteins tested (supplementary figure s1a,b) . the validated elisa assays were then used to systematically test the antibody titers of different classes of sars-cov-2 specific antibodies in sera from the following groups of patients: (i) 24 severe covid-19 patients admitted to icus; (ii) 16 health care workers from two hospitals in milan, exposed to the virus between february and march 2020 and confirmed positive to sars-cov-2 rna by rt-qpcr on nasopharyngeal swabs. fifty-eight sars-cov-2-negative subjects collected between april and june 2020 were used as negative controls (supplementary table s1 ). sera of the 16 health care workers were collected in the convalescence phase of the disease after two consecutively negative nasopharyngeal swab tests. time between the first detection of the virus and the first negative swab ranged from 14 to 35 days from onset of symptoms to disappearance of viral rna (supplementary table s2 ). these subjects all manifested clinical symptoms strongly related to sars-cov-2 infection, including fever, ageusia, anosmia, fatigue, myalgia, diarrhea, coryza and cough [5] . two of them manifested a more severe disease course with episodes of dyspnea. none of the patients required hospitalization and they all recovered from the disease (supplementary table s2 ). non-hospitalized covid-19 subjects manifested a lower antibody titer as compared to severe icu patients for all the tested antibody classes and viral antigens ( figure 1b-d) . this finding is in accordance to what published for asymptomatic [22] and paucisymptomatic [14] patients whose antibody titers were detected using commercial elisa or chemiluminescence assays against either the spike or the n-protein. when comparing the presence of the different classes of antibodies, all the covid-19 positive subjects resulted positive for the presence of igg antibodies against all the viral antigens tested ( figure 1e ). interestingly, a few of them were igm negative or with an antibody concentration close to the detection limit of the spike and rbd assay, as compared to the n protein. the observation that all of them instead showed n-specific igm antibodies may be a genuine persistence of anti-n protein igm or the consequence of a lower specificity of the n assay, possibly reflecting the high conservation of the n proteins among beta-coronaviruses other than sars-cov-2 [23] . interestingly, 25% of the non-hospitalized covid-19 patients did not develop rbd-specific iga, and only 1 out of 4 developed n-specific iga antibodies, a percentage that was instead above 80% for the hospitalized ones ( figure 1e ). since severe covid-19 is associated with a strong release of pro-inflammatory cytokines [8] , the sera from covid-19 patients were analyzed for the presence of pro-and anti-inflammatory cytokines, chemokines and growth factors by multidimensional analysis (figure 2, supplementary figures s2 and s3 and supplementary table s3 ). icu patients, whose sera were collected in the acute phase of the disease, showed a sustained production of pro-inflammatory mediators, among which il-6, il-17a, il-12p70, il-1beta, il-4, il-5 and il-13, all associated with the "cytokine storm" observed in very severe covid-19 patients, were the most abundantly detected (figure 2a ). on the contrary, even in the early convalescent phase, those cytokines were undetectable in the sera of non-hospitalized covid-19 patients (figure 2a) . interestingly, pro-inflammatory cytokines-such as ifn-gamma, tnf, il-23, il-15, il-21 and ip-10/cxcl-10-were detected both in the sera of severe icu hospitalized and of non-hospitalized covid-19 patients ( figure 2b ). to note, chemokines involved in the recruitment in inflamed tissues of both monocytes and t cells like mcp1/ccl2, rantes/ccl5, mip1alpha/ccl3 and eotaxin/ccl11 ( figure 2c ) were present at comparable concentrations in severe icu hospitalized and in non-hospitalized patients, indicating active recruitment of immune cell populations also in milder forms of covid-19. in order to evaluate the kinetics of antibody titers in convalescent non-hospitalized covid-19 patients, serum ig levels were measured at different time points, i.e., two days (t1) and one month (t2) after the first negative nf swab ( figure 3a) . interestingly, within a month after negativization of the viral rna, rbd-and spike-specific antibody titers halved in the sera of the vast majority of convalescent covid-19 patients ( figure 3b ,c). when tested against the rbd, 14/16, 13/16 and 14/16 patients showed a decrease in the antibody title ranging from 30% to 90% in their viral-specific igm, igg and iga antibodies classes ( figure 3b,e) . similarly, 8/16, 11/16 and 13/16 patients showed a decrease of at least 50% of their spike igm, igg and iga antibody titers ( figure 3c ,e). in both cases antibodies titers were still above the od detection threshold. on the contrary, antibodies against the viral nucleocapsid protein did not show a significant decrease at the second time point of evaluation ( figure 3d,e) . interestingly, similarly to the antibody titers, the presence of proinflammatory mediators in the sera of convalescent patients also decreased over time and became almost undetectable one month after a negative pcr for viral rna, a finding that mirrors the successful control of the infection and the consequent switch off of the immune response ( figure 3f, supplementary figure s4 ). during the last months many key aspects of the immune response to sars-cov-2 have been elucidated. however, given the complexity and diversity of the clinical manifestation of covid-19 disease, several outstanding questions remain still to be addressed. here we show that humoral immune responses against sars-cov-2 correlated with disease severity in terms of both antibody titers, persistence over time and serum levels of pro-inflammatory mediators. moreover, we showed that the vast majority of covid-19 mildly symptomatic patients analyzed in the study halved their anti-rbd antibody titers after 4 weeks from viral negativization, thus confirming the short lifespan of humoral immune responses against sars-cov-2. humoral immune response against sars-cov-2 proteins leads the production of antibodies against the portions of the viral proteins [10] [11] [12] . in this sense, serological tests, based on the search of specific anti-sars-cov-2 antibodies, represent a useful tool aimed at identifying patients who contracted the infection and, consequently, comparing the clinical course and eventual complications between the general population and population at risk, such as health care workers [15] . importantly, measurable variations in the humoral response might account for a re-activation of the immune system as a consequence of viral re-exposure, both in healthcare workers and in the general population. serological monitoring of antibody levels can thus provide information on the actual circulation of the virus, which can be used by decision makers to adapt safety and restriction measures according to the real presence of the virus within the population. nonetheless, the specificity and sensitivity of the different assays greatly vary among kits taking into consideration the different techniques implemented (elisa, clia, lateral flow) and the antigens used (spike ectodomain, s1-s2 of the spike, spike rbd, nucleocapsid). thus, only highly sensitive tests can detect with high accuracy whether people, including mildly symptomatic or asymptomatic subjects, have specific anti-sars-cov-2 antibodies present in their blood. the test utilized in this study is a robust elisa assay imported from the laboratory of prof. krammer at mount sinai, that has been approved for emergency use by the fda [11, 19] . we reproduced its excellent performance in our lab, that allowed us to detect a broad range of antibody levels, spanning form those measured in the blood of severe hospitalized patients and not hospitalized mild covid-19+ individuals. the elisa assay has been validated with a cohort of more than 500 positive and negative subjects, giving rise to extremely high performance values. specificity and sensitivity of the elisa assays were high for anti-rbd igg and iga (92-97%) and slightly lower for igm and the spike and n proteins (70-85%). these performances are in line with those published for both in-house and commercial assays [20, 21] . for this reason, this test is also being currently evaluated by the italy's istituto superiore di sanita' (iss) for its emergency use approval. one additional key strength of this assay as compared to other types of serological assays is its flexibility, i.e., the possibility to simultaneously assess different classes of antibodies against a broad panel of sars-cov-2 antigens within the same assay. thus, this elisa assay gave us a comprehensive understanding of the magnitude and persistence of antibody titer against different viral proteins and their correlation with the strength of the immune response, as measured by the serum levels of pro-inflammatory mediators. the presence of few false positives among the covid-negative population tested with the viral nucleocapsid protein as compared to the rbd might be a consequence of a mistakenly detection of anti-n antibodies previously raised against common cold coronaviruses which cross-react with the sars-cov-2 nucleocapsid [23] . the nucleocapsid protein is the more conserved protein among different coronaviruses. it is possible to speculate that antibodies produced against previous common cold coronaviruses (and cross-reacting with the sars-cov-2 antigens) might still be present in the sera at high levels, and therefore be detectable. as a consequence, when analyzed longitudinally, we observed that only the antibodies specific to sars-cov-2 decline while those aspecific and possibly reacting to previous coronaviruses remain detectable at the same levels over time. a similar observation was recently published by a large longitudinal study [24] . moreover, a recent paper evaluated the persistence of anti-n specific antibodies raised against four different common cold coronaviruses in a cohort of hiv+ individuals followed longitudinally for more than 10 years [25] . the study confirmed that n-specific antibodies undergo fluctuations in their detection levels as a consequence of seasonal re-infections with a kinetic of 6-12 months. interestingly, the authors reported that 2 out of 10 patients (20% of the individuals enrolled in the study) showed cross-reactive antibodies against the viral n-proteins of the four viruses, and in one of them these cross-reactive antibodies persisted over the years. the duration of circulating igg antibodies is still unclear and might depend on several factors, including the type and extent of immune response elicited upon the encounter with the virus [17] . in this study, non-hospitalized subjects showed lower antibody titers and blood pro-inflammatory cytokine profiles compared to patients in intensive care units (icu), irrespective of the antibodies tested. this finding is in accordance to what published for asymptomatic [22] and paucisymptomatic [14] patients whose antibody titers were detected using commercial elisa or chemiluminescence assays against either the spike or the n-protein. anti-rbd iga antibodies manifested a similar kinetic compared to that of igg. iga response against sars-cov-2 has been reported to be rapid and persistent [18, 19] and possibly associated with mucosal immune response in the gut and lungs. notably, iga production has been associated with disease severity, suggesting that iga production might occur locally at the mucosal sites, possibly correlating with the viral load, the duration of the viral exposure and the virus entry route [13, 26] . consistently, a recent communication [14] confirmed that the highest levels of igg and iga antibodies against the spike s1 domain, encompassing the n-terminal half of the protein with the rbd, were associated with severe disease [13, 14] . severe hospitalized covid-19 patients overexpressed pro-inflammatory cytokines (i.e., il-1 beta, il-2, il-6, il-17, tnfα). in one of the very first reports of the clinical course of covid-19 patients, as early as march 2020, serum increase in interleukin (il)-2, il-7, gmcsf, ip-10, mcp 1, mip1-α, and tnf-α was associated to disease severity [5] . elevated il-6 levels were detected in hospitalized patients and have been associated with icu admission, respiratory failure, and poor prognosis in several studies [5, 27, 28] . presently, conflicting results regarding il-1b and il-4 have been reported [29] [30] [31] . the elevation of pro-inflammatory cytokines, albeit being widely described in covid-19 patients, does not seem presently to have prognostic value, because they do not always differentiate moderate cases from severe cases [32] . levels of il-6 at first assessment might predict respiratory failure [33] , other publications with longitudinal analyses demonstrated that il-6 increases fairly late during the disease's course, consequently compromising its prognostic value at earlier stages [34] . moreover, serum concentrations of kl-6, a molecule elevated in serum of patients with interstitial lung diseases (ilds), such as idiopathic pulmonary fibrosis and hypersensitivity pneumonitis, was recently proposed to be capable of differentiating between severe and mild covid-19 patients, being mainly produced by damaged or regenerating alveolar type ii pneumocytes [35, 36] . conversely, ip-10, mcp-3, and il-1ra were capable of differentiating between severe and mild covid-19 patients [32] . interestingly, mip 1 alpha, il8 and eotaxin, similarly to the results published by long et al. [22] , were expressed to a greater extent by healthy subjects compared to covid-19 patients. human mip 1 alpha and eotaxin were reported to be potent inhibitors of m-tropic hiv-1 infection, and were therefore considered as potential hiv-1 inhibitors [37] . a similar protective mechanism of action might be envisaged in sars-cov-2 infection. we also observed that during non-severe covid-19 infections, pro-inflammatory cytokines are produced and correlate with the severity of the disease. similarly to anti-sars-cov-2 antibodies, pro-inflammatory mediators also decreased within a month after viral clearance, as expected upon the resolution of the disease. overall, we suggest that the decline in antibody titer and pro-inflammatory cytokines is a common characteristic of sars-cov-2 infection. this study therefore has important implications for the use of serological testing for the monitoring of infection outbreaks against re-infection with sars-cov-2. our results indicate that the detection of antibodies with serological assays for epidemiological and monitoring purposes in non-hospitalized seroconverted covid-19+ subjects, who most likely represent the majority of people who encountered the virus, is only highly reliable within a limited window of time after viral clearance. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/10/3188/s1, figure s1 : roc curves, figure s2 : cytokine levels in sera of covid-19 patients, figure s3 : sera growth factors concentration, figure s4 : not significant longitudinal variation of serum cytokines and chemokines in non-hospitalized covid-19 patients table s1 : patients' clinical characteristics table s2: covid-19 non-hospitalized patients clinical symptoms table s3 . luminex analytes. funding: this research was funded by a generous contribution from giuseppe caprotti and the fondazione guido venosta and partially supported by the italian ministry of health with ricerca corrente and 5 × 1000 funds; we thank the enthusiastic support of francesco niutta and nicolo' fontana-rava. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. a new coronavirus associated with human respiratory disease in china johns hopkins coronavirus resource center clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in clinical features of patients infected with 2019 novel coronavirus in angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target evidence for gastrointestinal infection of sars-cov-2 the trinity of covid-19: immunity, inflammation and intervention risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease antibody responses to sars-cov-2 in patients with covid-19 a serological assay to detect sars-cov-2 seroconversion in humans analysis of a sars-cov-2-infected individual reveals development of potent neutralizing antibodies with limited somatic mutation iga-ab response to spike glycoprotein of sars-cov-2 in patients with covid-19: a longitudinal study spectrum of innate and adaptive immune response to sars cov 2 infection across asymptomatic, mild and severe cases meta-analysis of diagnostic performance of serological tests for sars-cov-2 antibodies up to 25 temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study antibody tests in detecting sars-cov-2 infection: a meta-analysis longitudinal evaluation and decline of antibody responses in sars-cov2 infection sars-cov-2 seroconversion in humans: a detailed protocol for a serological assay, antigen production, and test setup medicaldevices/emergency-situations-medical-devices/eua-authorized-serology-test-performance (accessed on covid-19 in vitro diagnostic devices and test methods database clinical and immunological assessment of asymptomatic sars-cov-2 infections crystal structure of sars-cov-2 nucleocapsid protein rna binding domain reveals potential unique drug targeting sites humoral immune response to sars-cov-2 in iceland seasonal coronavirus protective immunity is short-lasting distinct early iga profile may determine severity of covid-19 symptoms: an immunological case series detectable serum sars-cov-2 viral load (rnaaemia) is closely correlated with drastically elevated interleukin 6 (il-6) level in critically ill covid-19 patients the role of interleukin-6 in monitoring severe case of coronavirus disease 2019 virologic and clinical characteristics for prognosis of severe covid-19: a retrospective observational study in wuhan correlation analysis between disease severity and inflammation-related parameters in patients with covid-19 pneumonia immune cell profiling of covid-19 patients in the recovery stage by single-cell sequencing plasma ip-10 and mcp-3 levels are highly associated with disease severity and predict the progression of covid-19 elevated levels of interleukin-6 and crp predict the need for mechanical ventilation in covid-19 clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study serum kl-6 concentrations as a novel biomarker of severe covid-19 peripheral lymphocyte subset monitoring in covid19 patients: a prospective italian real-life case series identification of rantes, mip-1α, and mip-1β as the major hiv-suppressive factors produced by cd8+t cells the authors declare no conflict of interest. key: cord-315696-43wmazxa authors: marinaki, smaragdi; tsiakas, stathis; korogiannou, maria; grigorakos, konstantinos; papalois, vassilios; boletis, ioannis title: a systematic review of covid-19 infection in kidney transplant recipients: a universal effort to preserve patients’ lives and allografts date: 2020-09-16 journal: j clin med doi: 10.3390/jcm9092986 sha: doc_id: 315696 cord_uid: 43wmazxa the coronavirus disease 2019 (covid-19) pandemic has posed a significant challenge to physicians and healthcare systems worldwide. evidence about kidney transplant (ktx) recipients is still limited. a systematic literature review was performed. we included 63 articles published from 1 january until 7 july 2020, reporting on 420 adult ktx recipients with confirmed covid-19. the mean age of patients was 55 ± 15 years. there was a male predominance (67%). the majority (74%) were deceased donor recipients, and 23% were recently transplanted (<1 year). most patients (88%) had at least one comorbidity, 29% had two, and 18% three. ninety-three percent of cases were hospitalized. among them, 30% were admitted to the intensive care unit, 45% developed acute respiratory distress syndrome, and 44% had acute kidney injury with 23% needing renal replacement therapy. from the hospitalized patients a total of 22% died, 59% were discharged, and 19% were still in hospital at the time of publication. immunosuppression was reduced in 27%, discontinued in 31%, and remained unchanged in 5%. hydroxychloroquine was administered to 78% of patients, antibiotics to 73%, and antivirals to 30% while 25% received corticosteroid boluses, 28% received anti-interleukin agents, and 8% were given immunoglobulin. the main finding of our analysis was that the incidence of covid-19 among kidney transplant patients is not particularly high, but when they do get infected, this is related to significant morbidity and mortality. as the first wave of the coronavirus disease 2019 (covid-19) pandemic is continuing with different effects in different countries, our knowledge about disease features and outcomes of this novel coronavirus in the general population has grown substantially [1] . kidney transplant (ktx) recipients have been recently classified by the center for disease control and prevention (cdc) as a high-risk group for severe covid-19 [2] . emerging evidence suggests 10-fold higher rates of early case fatality rate (cfr) in transplanted patients compared to that in the general population (gp) [3, 4] , due to the immunocompromised status resulting in impaired immunological response to pathogens [5] and to the almost universal presence of comorbidities [6, 7] . the body of literature regarding covid-19 infection in kidney transplantation is growing every day; however, it comprises mostly case reports, small case series, and small cohorts. there is a broad variation in studies among different countries across the globe. the reported ktx recipients are heterogeneous in terms of race, ethnicity, time from transplantation, and baseline status at the time of covid-19 infection. in this systematic review, we analyzed the data of all studies reporting on adult ktx recipients with confirmed covid-19. we focused on the following: (1) transplant characteristics and patient's baseline status at the time of covid-19 infection, (2) major outcomes of covid-19 infection, and (3) therapeutic interventions including modifications of immunosuppression and investigational agents used for covid-19. a systematic search of the literature published from 1 january to 7 july 2020, including pubmed, web of science, scopus, and the cochrane library was conducted. the search terms incorporated were: "covid 19" or "sars cov2" or "coronavirus" or "2019-ncov" or "sars-cov-2" or "sars-cov" and "renal" or "kidney" and "transplant" or "transplantation" and "recipient" or "patient", using boolean operators, wildcards, and special characters as described in supplementary item s1. authors k.g. and s.m. independently reviewed the titles and abstracts for inclusion. this systematic review was conducted in accordance with the preferred reported items for systematic reviews and meta-analyses (prisma 2009) [8] , and the flow diagram is depicted in figure 1 . the prisma checklist is shown in supplementary item s2. we included the following types of articles: case reports, case series, case-control studies, cohort studies, and correspondence articles. we did not include other systematic reviews, editorials, and conference abstracts. since almost all studies were observational with small numbers of patients, we included all that were in accordance with our inclusion and exclusion criteria. according to the p.i.c.o model for clinical questions in systematic reviews, our intended patient population (p) comprised all adult (over 18 years old) solid organ transplant recipients who had undergone either kidney transplantation only or multiorgan-including as per definition kidney-transplantation with covid-19 from 1 january until 7 july 2020. the intervention-exposure (i) was covid-19 infection confirmed by nucleic acid amplification technique (nat). we did not have a comparison (c) group. all major adverse outcomes (o) of covid-19 infection, i.e., hospitalization, intensive care unit (icu) admission, mechanical ventilation (mv), acute kidney injury (aki), acute respiratory syndrome (ards), and death, were recorded as were recovery and discharge. we collected and analyzed the following parameters: last name of the first author, city or region, country, sample size, infection rate, hospitalization, duration of hospitalization (days), ards, aki, icu admission and duration of stay in icu, type of mv (invasive and non-invasive), discharge, recovery, death, and case fatality rate (cfr). we included the following types of articles: case reports, case series, case-control studies, cohort studies, and correspondence articles. we did not include other systematic reviews, editorials, and conference abstracts. since almost all studies were observational with small numbers of patients, we included all that were in accordance with our inclusion and exclusion criteria. according to the p.i.c.o model for clinical questions in systematic reviews, our intended patient population (p) comprised all adult (over 18 years old) solid organ transplant recipients who had undergone either kidney transplantation only or multiorgan-including as per definition kidney-transplantation with covid-19 from 1 january until 7 july 2020. the intervention-exposure (i) was covid-19 infection confirmed by nucleic acid amplification technique (nat). we did not have a comparison (c) group. all major adverse outcomes (o) of covid-19 infection, i.e., hospitalization, intensive care unit (icu) admission, mechanical ventilation (mv), acute kidney injury (aki), acute respiratory syndrome (ards), and death, were recorded as were recovery and discharge. patients' demographics and baseline characteristics included: age; gender; type of donor: living, deceased, donor after brain death (dbd), donor after cardiac death (dcd); multiple organ transplantation including, as per definition, kidney transplantation; recipient of first, second, or third kidney transplant; time since transplantation. serum creatinine levels and, if available, estimated glomerular filtration rate (egfr) were recorded at three time points: baseline at admission, peak during hospitalization, and at discharge. comorbidities recorded were arterial hypertension (htn); diabetes mellitus (dm); cardiovascular disease (cvd); malignancy (solid tumors and hematologic malignancies); obesity (ob); chronic obstructive pulmonary disease (copd); and chronic viral infections including human immunodeficiency virus (hiv), hepatitis b virus (hbv), and hepatitis c virus (hcv). baseline immunosuppressive agents and regimens were also recorded, as were modifications in immunosuppression (is) consisting of discontinuation, reduction, or switch from one agent to another. induction therapy consisting of anti-interleukin-2 (anti-il-2) agent basiliximab or antithymocyte globulin (atg) were recorded in those transplanted for less than one year. covid-19-targeted therapies recorded included: antivirals, hydroxychloroquine (hcq), antimicrobial agents, corticosteroid boluses, anti-interleukin-6 (anti-il-6) monoclonal antibodies, interferon (ifn), immunoglobulin, colchicine, and the anti-chemokine-receptor-type 5 (ccr5) inhibitor leronlimab. we used the microsoft office version 2019 platform to extract, collect, and analyze data. individual participant data (ipd) were used for all patients reported separately. aggregated data were also used when information about individual patients with kidney transplantation and confirmed covid-19 infection was not available. continuous variables were reported as mean ± sd and/or median. categorical variables were reported as count and percentage. our initial search retrieved a total of 328 articles. an additional 6 records were identified through manual screening. after the removal of duplicates, using endnote online as citation manager, the remaining 175 studies were screened by title and abstract. subsequently, 90 articles were removed, based on relevance and inclusion and exclusion criteria. full-length text was assessed for eligibility in 85 articles. another 22 studies were excluded due to duplicate or mixed population, aggregated or missing data, and articles in a language other than english. finally, 63 articles reporting on 420 ktx recipients with confirmed covid-19 infection were included. there were 28 case reports, 14 case series, 1 case-control study, 1 cohort study, and 19 correspondence articles from a total of 18 countries (table 1; table 2 ). for identification of the risk of bias, authors s.m. and k.g. independently performed a quality assessment of 63 studies. in case of disagreement, the problem was solved by discussion. the quality of case series included was assessed using the joanna briggs institute's (jbi) critical appraisal checklist for case series, which consists of 9 quality items. studies with up to 4 positive responses were considered to have low quality, while those with 5 to 9 positive responses had high quality. the study quality of case reports was assessed with the jbi checklist for case reports. the range of the jbi scale is between 0 and 8, with a score of 0-3 denoting low quality and 4-8 denoting high quality. accordingly, all studies were included in the analysis. the risk of bias assessment is shown in supplementary items s3 and s4. the mean and median age of the 169 patients for whom ipd were available were 55 ± 15 years and 55 years (range 21-80) respectively. the reported median age from available aggregated data ranged from 57 to 60 years. there was a male predominance of 67%. the majority of ktx recipients (74%) had received a deceased donor transplant (dbd in 95% of cases), while 26% had been transplanted from a living donor. in 156 cases, the donor source was not reported. seven out of 420 patients (2%) were multiple organ transplant recipients: 2 received liver and kidney, 2 received heart and kidney, and 3 received pancreas and kidney, respectively. from the total cohort, only 2% patients had undergone subsequent kidney transplantations. median time from ktx to covid-19 infection was 6.5 years (range 0-33) while 23% of the patients were transplanted for less than one year. the majority of patients (81%) suffered from htn, while only 12% had no comorbidities. the second most frequent associated medical condition was dm (36%), followed by cvd (21%), ob (15%), copd (5%), malignancy (4%), and chronic viral infection (2%). out of 162 patients with comorbidities, 41% had one, 29% had two, and 18% suffered from three comorbidities. baseline renal function was relatively well-preserved with a median serum creatinine of 1.47 mg/dl in 91 patients. baseline egfr was assessed in few studies (16 out of 63) with a mean of 40 ± 23 ml/min/1.73 m 2 . median peak serum creatinine during hospitalization was 2.2 mg/dl (range 0.62-10.94) and returned to 1.4 mg/dl at discharge. the patients' clinical features and outcome as well as management strategies are depicted in table 3 . infection rate in our study ranged from 0.27% to 1.67% and was calculated in those studies where the number of the total cohort of ktx recipients was available. from the total cohort, 93% of patients were hospitalized. median duration of hospitalization was 16 days (range 1-100). from 391 hospitalized patients, 118 (30%) were admitted to the icu. in 32 out of 118 patients, the median duration of the icu stay was 8.5 days (range 1-34). non-invasive mechanical ventilation (niv) was applied to 7% and invasive mechanical ventilation (imv) to 23% of patients. ards was reported in (175/391) 45% of patients. a substantial proportion of patients, (150/345) 44% developed aki, with need for renal replacement therapy (rrt) reported in 23%. death was recorded in (93/420) 22% of patients. most patients, (232/391) 59% were discharged; 29 patients remained hospitalized when the studies were published, 14 of whom were still in the icu. case fatality rates in the case series including more than ten patients ranged from 6% up to 67%. the most frequently applied immunosuppressive regimen at baseline consisted of a calcineurin inhibitor (cni), an antimetabolite, and corticosteroids (cs) in 73% of patients. in total, 64% (147/230) of patients were receiving tacrolimus (tac), 10% (18/176) cyclosporine (csa), 68% (217/319) mycophenolic acid (mpa), 14% (26/184) everolimus, 4% (9/211) azathioprine (aza), and a minority (<2%) of patients other agents such as belatacept, mizoribine, or leflunomide. overall, is was reduced in 27% (97/357), discontinued in 31% (66/212), and remained unchanged in 5% (14/275) of patients. the most frequently discontinued drug was the antimetabolite in 91% (227/250) of patients. calcineurin inhibitors were reduced in 32% (65/204) and discontinued in 58% (118/204) of patients. switch from tac or mammalian target of rapamycin inhibitor (mtori) to csa occurred in 7% (24/358) of patients. the mtori was reduced in 7% (2/27) and discontinued in 67% (18/27) of cases. the main agents used for covid-19 infection were antivirals, antibiotics, hydroxychloroquine (hcq), anti-il monoclonal antibodies, and steroid boluses. in total, 30% (123/414) of patients received antivirals. the most frequently used antiviral was lopinavir/ ritonavir, administered to 76% (94/123) of those. other antivirals included darunavir/ritonavir (13%), ritonavir-darunavir/lopinavir (4%), oseltamivir or arbidol (11%), umifenovir (7%), and darunavir/cobicistat (2%). hydroxychloroquine was administered to 78% (320/409) of patients. the majority of patients (73%, 290/399) received antibiotics: azithromycin was administered to 53% (155/290) and other broad-spectrum antibiotics to 17% (50/290) of cases. corticosteroid (cs) boluses or dexamethasone were used in 25% (83/331) of patients. anti-il agents were introduced in 28% (59/213) of patients with more severe illness; tocilizumab was the preferred agent in 56 of 59 patients. less frequently used agents were immunoglobulin in 8%, colchicine in 0.5%, interferon in 0.5%, and leronlimab in 1.4% of patients. we separately investigated three patient groups: recent transplanted recipients, elderly patients, and those who died. outcomes are depicted in table 4 . table 4 . demographic and baseline characteristics, treatment, and clinical outcomes of specific subgroups of kidney transplant recipients with covid-19 infection. age (years, mean ± sd) (n = 29) 53.9 ± 13.9 gender (male) 18 in this review, we focused on the baseline status of the patients at the time of acquiring the infection, on the major clinical outcomes, as well as on the therapeutic interventions. emerging evidence suggests that kidney transplant recipients are not at particularly high risk of acquiring the infection. infection rates in our review range from 0.27% to 1.67%, with the highest rate of 5% reported in a study from spain [56] , in a cohort of elderly (>65 years) recipients. of note, the infection rate among younger recipients in the same cohort was at 0.8%. however, infection rates depend greatly on the number of tested individuals; therefore, it is impossible to draw definite conclusions. most agree that presenting symptoms are similar to those of non-transplanted patients with fever (85%), dry cough (70%), myalgia (60%), and dyspnea (57%) being the most frequently reported symptoms [6, 69] . in a substantial number of transplanted patients, mild and/or atypical initial presentation with less fever and dyspnea and predominantly gastrointestinal symptoms has been reported [3, 20, 64, 68, 70] , suggesting need for increased vigilance. illness severity at presentation among ktx recipients may vary significantly, similar to the case in the general population. however, acute respiratory decompensation and rapid clinical deterioration have been described in hospitalized as well as outpatient ktx recipients at an average of 7-10 days after disease onset. [3, 7, 53, 61, 71] . though the management of ktx recipients with mild symptoms as outpatients may be a reasonable option, given the lack of prognostic indicators for eventual deterioration and current evidence about acute decompensation, rapid testing and early hospitalization is advisable. the initial suggestion that an immunocompromised status would hypothetically limit a striking cytokine release and lead to a milder disease course [72] has been confuted by current evidence. on the contrary, kidney tx recipients acquiring covid-19 infection are at high risk of developing severe disease due in fact to their immunocompromised status. the presence of at least one comorbidity is an almost universal finding in transplanted patients. in non-transplanted individuals, comorbidities have been associated with adverse covid-19 outcomes [73] . the mean age of the ktx recipients was 55 ± 15 years, and they were predominantly male. regarding transplantation parameters, most patients were deceased donor recipients. time from transplantation to covid-19 infection varied greatly: median 6.5, range 0-33 years. only 23% were recently transplanted. renal function at baseline was relatively well-preserved: mean serum creatinine was 1.68 ± 0.77 mg/dl and mean egfr 39.7 ± 23 ml/min/1.73 m 2 . our analysis confirmed the almost universal presence of at least one comorbidity in the ktx population: htn was the most prevalent in 81% of patients, followed by dm in 36%, cvd in 21%, obesity in 15%, and copd in 5%. remarkably, a substantial proportion (29%) of patients suffered from two and another 18% from three comorbidities. hypertension, diabetes, cvd, and copd have been identified early during the covid-19 outbreak as risk factors for adverse outcomes [74] . african-american race as well as obesity have been recently associated with more severe covid-19 [74, 75] . all major adverse clinical outcomes related to covid-19 including death were more prevalent in ktx recipients [76] . we found a hospitalization rate of 93%. this may include a selection bias, since indeed most studies reported those patients who had the most severe disease course and were hospitalized. we also found prolonged hospital stay (median 16 days) and a high icu admission rate of 30% among them. a total of 30% of ktx recipients needed mechanical ventilation: 23% invasive (imv) and another 7% required non-invasive mechanical ventilation (niv) support, while 45% of ktx developed ards. we found a high rate of aki in 44% of ktx recipients, compared to 29% in critically ill covid-19-infected patients of the gp and to 15% in covid-infected patients in general [77] . a substantial proportion of those developing aki in our study (23%) needed rrt. aki was reversible in most cases who recovered. regarding the etiology of aki, direct damage of the proximal tubular epithelial cells by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been reported early during the outbreak [78] but could not be confirmed later on. aki as a result of renal damage due to uncontrolled cytokine storm seems to be currently the most prevalent theory [79] . furthermore, in a kidney transplant recipient, is reduction or withdrawal may lead to acute allograft rejection. no renal biopsy was reported in any of the 420 analyzed recipients. in the absence of biopsy confirmation and given the fact that aki resolved in most of them who recovered, the most plausible explanation is the occurrence of aki in the setting of multiorgan failure in patients with a sole functioning kidney and preexisting chronic kidney disease (ckd). the overall death rate in our analysis was 22%. in 13 case series including more than 10 patients, cfr ranged from 6% up to 67%. the highest cfr was reported by nair (u.s., 33%), crespo (spain, 50%), abrishami (iran, 67%), akalin (u.s., 28%), and bossini (italy, 28%) [3, 6, 50, 53, 56] . nair [6] and akalin [3] reported on 10 and 36 ktx recipients from ny city with cfrs of 33% and 28%, respectively. the small numbers of patients, the racial diversity, and the fact that the reports come from the epicenter of the pandemic in the u.s., are all factors that may have contributed to the high cfr in this series. the study by crespo et al. [56] reports a cfr of 50% in a selected cohort of 16 transplanted patients with confirmed covid-19 infection who were all above 65 years. older age is a known risk factor for adverse outcomes in patients with covid-19. further risk factors in this cohort included frailty, obesity, and underlying heart disease. abrishami et al. [50] from iran, reports a cfr of 67% in a cohort of predominantly young patients with well-preserved renal function and few comorbidities. there is no obvious cause for this inexplicably high cfr in this study. in a recent study, bossini and alberici [53] analyzed 53 ktx recipients from italy, 45 of whom were hospitalized. the cfr was 33% in hospitalized patients and 28% in the entire cohort also including outpatients. at this point, it is important to underline the high rates of adverse outcomes associated with covid-19 infection in ktx recipients. compared to the outcomes of influenza in solid organ transplant (sot) recipients, as described in a review by mombelli et al. [80] , all adverse outcomes were higher in those with covid-19 infection: 93% vs. 70% hospitalization rate and 30% vs. 16% icu admission, respectively. the most striking difference was in early cfr: 22% vs. 3-8%. managing is in a ktx recipient in the context of severe infection is a complex approach. since immunological response to infections is reduced in immunocompromised hosts, there is rationale to reduce and even to temporarily withdraw is in case of severe covid-19 disease. one has to balance the benefit of at least partially "restoring" the immune response in order to save the patient's life against the risk of losing the graft due to acute rejection. in our analysis, there was a high rate of is reduction or withdrawal. total is was reduced in 27% and completely withdrawn, with the exception of steroids, in another 31% of patients. the antimetabolites should be discontinued first because of their effect on inhibiting t-cell function and proliferation [81] . we found a rate of antimetabolite discontinuation of 91%. though mtor-inhibitors have antiviral potential [82] , they have been associated with various types of lung injury [83] and should preferentially also be withdrawn. in our study, the rate of mtori reduction was 7% and the rate of withdrawal 67%. regarding cnis, the most common approach is to minimize doses, which has proven efficacy in severe viral or opportunistic infections [5] . cnis were withdrawn in 58% and reduced in 32% of cases in our study. specifically, for cyclosporine, there are in vitro studies demonstrating that csa suppresses viral replication through the inhibition of cyclophilin and this effect could be also demonstrated for the virus sars-cov-2. based on this theoretical benefit and with the fear of completely withdrawing is, another approach is to switch the tacrolimus or mtori-based regimen to csa, which occurred in 7% of cases in our study. as for corticosteroids, since all practices including withdrawal, reintroduction, dose reduction, maintenance or increase, switch from oral to intravenous, or administration of boluses had been applied, we recorded only patients who had taken boluses as discussed in the next section. the main pharmacological interventions for the treatment of covid-19 infection included antivirals in 30% of patients, broad-spectrum antibiotics in 73%, hydroxychloroquine in 78%, tocilizumab in 26%, steroid boluses in 25%, and less frequently other anti-il agents (clazakizumab, anakinra), colchicine, immunoglobulin, interferon, and the anti-ccr5, leronlimab. from the 30% of ktx patients who received antivirals, the majority, 76% received the combination of protease inhibitors lopinavir/ritonavir. they interact with cnis by dramatically increasing their levels and prolonging half-lives; if used concomitantly with cnis, drastic dose reduction and prolonged dosing intervals of the cni are mandatory, otherwise patients will be exposed to prolonged overimmunosuppression, with detrimental effects in critically ill individuals [84] . the safest approach is to completely withdraw cnis if they are co-administered. moreover, they induce qt prolongation which, especially in combination with hcq or azithromycin, is additive and may lead to severe arrhythmia [85] . remdesivir is a nucleotide analogue initially developed to treat ebola virus [86] . it has not been used until the outbreak of the covid-19 pandemic but has shown efficacy in the gp [87] . regarding hydroxychloroquine, early reports suggested a role in reducing the viral load [88] . since it is cheap and easily available, it has been applied broadly. in total, 78% of ktx recipients received hcq. current data do not further support the use of lopinavir/ritonavir and hcq in hospitalized patients with covid-19 infection. on july 4, the world health organization (who) announced the discontinuation of the two treatment arms (hcq/lop-riton) of the solidarity trial based on results of the interim analysis that showed no effect in terms of reducing mortality [89] . corticosteroid boluses have been used in 25% of ktx recipients. since they increase viral replication, they are not desirable at the first phase of covid-19 infection. in critically ill patients, they have immunomodulatory effects [90] . steroid boluses are recommended for patients with ards; furthermore, the recovery trial has shown benefit of high doses of dexamethasone in patients under mechanical ventilation [91] . the most reasonable approach is to maintain the lowest possible cs doses in the first phase of the infection and to administer boluses in those who develop severe illness. since severe covid-19 infection has been associated with a cytokine storm [92] , there is rationale for the use of anti-il agents. monoclonal antibodies that inhibit cytokines were used in 28% of ktx recipients in our study. the most commonly used was the il-6 receptor antagonist tocilizumab in 26% of them. less frequently applied agents included clazakizumab, anakinra, and leronlimab. unfortunately, due to the small numbers of patients in the studies investigated, the different time points during the disease course at which therapies were applied, and the different time points at which studies have been published, it is impossible to draw conclusions about the impact of therapeutic interventions on outcomes. three patient groups, i.e., recent transplanted recipients, elderly patients, and those who died, were analyzed separately. a total of 23% with available ipd were recent ktx recipients. they did not differ from the total cohort in means of baseline characteristics, with the exception of better renal function. in terms of outcomes, though nearly half of them had received atg, they had lower rates of aki (38% vs. 44%), ards (31% vs. 45%), and death (15% vs. 22%) compared to those in the entire cohort. this finding further confirms preliminary data suggesting at least not worse outcomes in this subgroup of ktx recipients. patients older than 65 analyzed, comprised 34% of those for whom information was available. only 4% had no comorbidity vs. 12% in the total cohort, while baseline creatinine was higher, at 1.8 mg/dl. a substantial proportion (17%) of them were on mtori-based is at baseline. the icu admission rate was 19% vs. 30% in the total population, indicating either healthcare resource unavailability or decision not to intervene due to frailty and comorbidities. the death rate was higher at 32% vs. 22%. those who died (93 out of 420) were older: 27 out of 55 were over 65 years and predominantly male (61%), and they had worse baseline renal function. all adverse outcome rates were strikingly higher: icu admission at 58%, 62% had need for invasive mechanical ventilation support, 81% developed ards, and 58% had aki. in terms of therapeutic intervention, they had higher rates of intravenous cs (43%) but not of tocilizumab (20%) administration. the major limitation of our analysis was that the included articles were case reports and case series, which are subject to selection and publication bias. thus, it is uncertain whether the results of our systematic review can be extrapolated to the general ktx-recipient population. there were a limited number of patients from all over the globe with a broad diversity in terms of race, ethnicity, and country of origin, as well as transplant and clinical characteristics. besides patients' heterogeneity, there is also variation in outcomes, disease course, and management of transplanted patients described, according to the time of publication of the study. moreover, therapeutic interventions varied among countries depending on local policies, ethical issues, and healthcare resource availability conjointly with the "total covid-disease burden" of the specific country. thus, we could perform only descriptive statistics, and no conclusion could be drawn about the impact of therapeutic interventions on outcomes. in view of the absence of a commercially available vaccine in the near future and given the fact that covid-19 is our new reality, especially for vulnerable patient groups such as ktx recipients, large registry data and targeted studies assessing the impact of therapeutic strategies are urgently awaited. in conclusion, the main finding in our analysis is the high rate of all major adverse outcomes of covid-19 infection in hospitalized ktx recipients. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/9/2986/s1, supplementary item s1: search strategy, supplementary item s2: prisma checklist, supplementary item s3: study quality assessment-jbi critical appraisal checklist for case series, supplementary item s4: study quality assessment-jbi critical appraisal checklist for case reports. author contributions: s.m. performed the design of the study, conducted the main literature search, wrote the manuscript, and supervised the work. k.g. contributed to the study design, conducted the main literature search, and participated in the writing of the "methods" section. m.k. and s.t. collected and analyzed data, did the statistics, and wrote the tables. i.b. and v.p. have reviewed and approved the final version of the manuscript. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. understanding of covid-19 based on current evidence people with underlying medical conditions, immunocompromised state (weakened immune system) from solid organ transplant covid-19 and kidney transplantation covid-19 in solid organ transplant recipients: initial report from the us epicenter infection in solid-organ transplant recipients covid-19 in kidney transplant recipients a single center observational study of the clinical characteristics and short-term outcome of 20 kidney transplant patients admitted for sars-cov2 pneumonia preferred reporting items for systematic reviews and meta-analyses: the 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transform study a. mtor inhibitors and renal allograft: yin and yang antiretroviral and immunosuppressive drug-drug interactions in human immunodeficiency virus-infected liver and kidney transplant recipients chronic hydroxychloroquine use associated with qt prolongation and refractory ventricular arrhythmia therapeutic efficacy of the small molecule gs-5734 against ebola virus in rhesus monkeys remdesivir for the treatment of covid-19-preliminary report hydroxychloroquine and azithromycin as a treatment of covid-19: results of an open-label non-randomized clinical trial hydroxychloroquine and lopinavir/ritonavir treatment arms for covid-19 on the use of corticosteroids for 2019-ncov pneumonia dexamethasone in hospitalized patients with covid-19-preliminary report this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this review is dedicated to all transplanted patients who have lost their lives due to covid-19 worldwide. the authors declare no conflict of interest. key: cord-011965-n0re0u5u authors: bibbò, stefano; settanni, carlo romano; porcari, serena; bocchino, enrico; ianiro, gianluca; cammarota, giovanni; gasbarrini, antonio title: fecal microbiota transplantation: screening and selection to choose the optimal donor date: 2020-06-05 journal: j clin med doi: 10.3390/jcm9061757 sha: doc_id: 11965 cord_uid: n0re0u5u in the past decade, fecal microbiota transplantation (fmt) has rapidly spread worldwide in clinical practice as a highly effective treatment option against recurrent clostridioides difficile infection. moreover, new evidence also supports a role for fmt in other conditions, such as inflammatory bowel disease, functional gastrointestinal disorders, or metabolic disorders. recently, some studies have identified specific microbial characteristics associated with clinical improvement after fmt, in different disorders, paving the way for a microbiota-based precision medicine approach. moreover, donor screening has become increasingly more complex over years, along with standardization of fmt and the increasing number of stool banks. in this narrative review, we discuss most recent evidence on the screening and selection of the stool donor, with reference to recent studies that have identified specific microbiological features for clinical conditions such as clostridioides difficile infection, irritable bowel syndrome, inflammatory bowel disease, and metabolic disorders. in the last decade, multiple studies have expanded knowledge in the field of gut microbiota, including pathogenesis, diagnosis, and therapeutics [1] . to date, the therapeutic modulation of the intestinal microbiota is performed with traditional approaches such as antibiotics and probiotics, or increasingly through fecal microbiota transplantation (fmt), which is defined as the transfer of fecal material from a healthy donor into the gastrointestinal tract of a recipient [2] . fecal material has been used in medicine since almost two thousand years. the first description of the use of fecal material for medical purposes dates back to about 1700 years ago; traditional chinese medicine in particular had perceived the potential role of this biological material and used it for several clinical indications such as gastrointestinal, nervous system, skin, and gynecological diseases [3] . in western countries, the first description of ancestral fmt dates to the 17th century, when fabricius acquapendente reported the transplantation of feces for the cure of animals unable to ruminate [4] . more recently, anecdotal use has been reported during the second world war. german soldiers residing in north africa suffered from recurrent episodes of diarrhea that they treated by eating camel stool, being inspired from the local practice of the bedouins [5] . western medicine began to study the potential role of fmt only in the second half of the 20th century. firstly in 1958, ben eiseman reported the successfully treatment of four patients with pseudomembranous colitis using fecal enemas [6] , and over 20 year later, schwan et al. reported new evidence supporting the efficacy of fmt in c. difficile infection (cdi) [7] . in the following years, several other reports came out, and a growing body of evidence showed the efficacy of fmt in the treatment of recurrent cdi, and, furthermore, the feasibility of fmt was gradually suggested for other clinical indications. the first randomized controlled trial that investigated the role of fmt for recurrent cdi was published by van nood et al. in 2013. they reported that a single infusion of fecal material by nasoduodenal route was superior to standard therapy with vancomycin [8] . in further years, other routes of administration were successfully tested in clinical trials, demonstrating the efficacy of fmt by lower route through colonoscopy [9] or upper administration with capsule [10] . therefore, the growing interest of the scientific community towards fmt has meant that a large amount of data has been published in the last decade; for this reason, a panel of european experts met in rome in 2017 to release the first evidence-based consensus report for the use of fmt in clinical practice [11] . over the years, further issues have emerged, which are still not clarified to date. in particular, in view of the growing number of patients who could benefit from fmt, it is necessary to identify innovative ways to storing fecal material to be used if necessary. indeed, in the early experiences, fmt was performed only with fresh material from occasional healthy donors, but this approach is not feasible for large-scale use of fmt. to solve this problem, the possibility to create structures to bank the feces after manipulation was suggested, and this approach is supported by the evidence of the effectiveness of fmt performed with frozen material [12] . in consideration of the increasing interest of the scientific community on this topic, a panel of international experts met in rome in 2019 to define the general guidelines for the creation of stool banks [13] . despite these efforts, many problems remain to be solved. above all, the identification of the optimal donor is a fundamental clinical issue of rising relevance. indeed, the increasing number of clinical indications suggests the need to identify the ideal donor for each disease or patient that cannot be treated indiscriminately with the same fecal biomass. the fascinating idea of identifying the "perfect" intestinal microbiota has motivated the scientific community for at least one century-ever since in the early 20 century elie metchnikoff suggested the role of intestinal bacteria in the development of many pathological conditions and health in the homeostasis of the microbial species [14] , generating the concepts of "eubiosis" and "dysbiosis," which for years were considered only fascinating hypotheses without strong scientific bases. however, in recent years, the molecular techniques of genomic sequencing have allowed to understand the link between gut microbiota and several diseases [15] , giving evidence to this old intuition. in particular, we refer to "eubiosis" as considering a status characterized by a preponderance of potentially beneficial species, while "dysbiosis" is a condition characterized by the loss of homeostasis and by the proliferation of microbial species considered potentially pathogenic and, moreover, favor a "milieu" triggering the hyper-inflammatory state [16] . to date, an increasing number of studies confirm these hypotheses, in particular the reduced diversity of gut microbiota, simply defined as the variety and abundance of species in a defined microbial ecosystem [17, 18] , which is known to characterize several chronic diseases compared to a control group [19, 20] . therefore, in this narrative review, we report the most recent evidences on the screening and selection of the stool donor, with special efforts to describe findings that may lead to the optimal donor in several disease looking for an "optimal microbiota" to be transplanted (cdi, inflammatory bowel disease (ibd), irritable bowel syndrome (ibs), and other emerging pathological conditions). to date, the only recommendation for fmt in clinical practice is the treatment of recurrent cdi, although a large number of emerging indications are being experienced in several studies [21] . cdi is a burdensome clinical issue and represent the most relevant cause of antibiotic-associated diarrhea; its incidence has evolved in recent years and the risk of recurrence after standard antibiotic therapy has widely increased [22, 23] . the standard treatment for the first occurrence of cdi is still represented by antibiotic therapy, mainly with metronidazole or vancomycin [24] . however, the clinical success rate of antibiotics in the recurrence of cdi is dramatically decreased, consequently, more effective therapies have been proposed, including fmt [25, 26] . the clinical success of fmt, in contrast to the loss of efficacy of standard antibiotic therapy, could be explained by understanding the mechanism of action. in fact, fmt is a restorative treatment of gut microbiota alterations, unlike antibiotics, which is a disruptive treatment; accordingly, the administration of fmt results in a prompt and sustained normalization of microbial community structure and then metabolic activity of gut microbiota [27] . indeed, cdi develops only in subjects with disruption of gut microbiota [28] ; supporting this idea, it was demonstrated that the feces of patients with recurrent cdi have a higher relative abundance of several bacterial family as enterobacteriaceae, veillonellaceae, and lactobacillaceae, and lower relative abundance of ruminococcaceae, bacteroidaceae, and lachnospiraceae [29] . furthermore, fmt recipients have shown changes in microbial profiles and shifts in the gut microbiota composition towards a profile similar to that of the healthy donor; this finding is obtained in a few days and is observed for at least six months [30] . to date, several systematic review and meta-analyses have shown an overall cure rate of fmt of up to 90% in preventing further cdi recurrence [31, 32] . moreover, a recent meta-analysis has shown that both the upper and the lower route are effective, with a slight advantage of colonoscopy over other techniques [32] . based on these positive evidences, scientific societies have included fmt among the recommended treatment for recurrent cdi. already in 2014, fmt was strongly recommended in recurrent cdi by the european society of clinical microbiology and infectious disease (escmid) [33] , while the american college of gastroenterology (acg) stated that fmt can be considered after the third recurrence [34] ; more recently, the infectious disease society of america (idsa) confirmed the indication in the treatment of recurrent cdi with fmt [24] . alongside well-established indications such as cdi, several studies have found emerging clinical conditions for which fmt may represent a promising alternative to standard therapies. most evidence comes from inflammatory bowel disease (ibd) studies. several alterations of gut microbiota has been proposed as factors contributing to the development of the aberrant immunological response in ibd [35] , but it is still unclear if the perturbations of microbiota are the cause or consequence of the mucosal inflammation associated to ibd [36] . in particular, ulcerative colitis (uc) is the most suitable ibd model for the study of fmt, considering the characteristics of inflammation of the mucosa and the established role of the microbiota in pathogenesis [37] . to date, a little number of clinical trials have reported promising results, but several concerns suggest to better investigate this potential clinical application [38] . according to a cochrane systematic review of four clinical trials, the overall remission rate at week 8 was 37% (52/140 uc patients) in patients receiving fmt, compared with 18% (24/137 patients) in those receiving placebo; additionally, clinical response and endoscopic remission improved in patients treated with fmt [39] . however, several factors appear to influence the clinical response in uc patients, as the condition during the manipulation of the feces or the donor selection. for instance, anaerobic conditions during the manipulation of stool were associated with better performance considering clinical remission or steroid free response [40] . donor selection might be a relevant factor considering that a study reported higher success rates with one particular donor compared with other donors [41] . furthermore, an emerging relevant indication for fmt was represented by the flare of uc associated with concurrent c. difficile over infection. a recent clinical trial, including patients affected by uc or crohn disease with recurrent cdi, reported that fmt has a curative effect on the recurrence of cdi, but has no apparent beneficial effect on the ibd course [42] . gut microbiota disturbance was also involved in other gastrointestinal diseases such as irritable bowel syndrome (ibs). a systematic reviews with meta-analysis showed that fmt may be beneficial in ibs [43] , but this finding is limited by the small number of patients included and by the relevant differences in the design of the studies. in particular, ibs is triggered by multiple factors, and furthermore, is a heterogeneous condition that may require a selection of the donor in each case. for instance, el-salhy et al. have recently reported that fmt administered through gastroscope was highly effective in ibs if a well-defined donor was chosen with a normal disbyosis index and favorable specific microbial signature [44] . furthermore, metabolic and hepatic diseases are also considered emerging indications for fmt. there is great interest towards the modulation of the gut microbiota in metabolic syndrome, as two studies reported promising results in improving peripheral insulin sensitivity [45, 46] . unfortunately, the improvement of metabolic profile was not maintained in the long term, and a recent systematic review including three studies reported the absence of significant benefits from fmt in metabolic syndrome [47] . thus, further studies to clarify the feasibility of this approach in metabolic disorders are needed. furthermore, fmt was able to reverse encephalopathy derived from disturbed gut-brain axis in patients with liver chronic disease, two clinical studies shown promising results in this field of application [48, 49] . fmt was also proposed in the treatment of several other clinical conditions, but evidence is limited and results were reported by small studies; thus, the application is limited to clinical studies and selected cases. for instance, fmt was reported as effective in the decolonization of patients carrier of multi-drug resistant organism [50] , in reducing symptoms in autism spectrum disorders [51] , or in reliving symptoms and increasing progression free survival in graft versus host disease after hematopoietic stem cell transplant [52] . donor selection represents a fundamental challenge in view of the implementation of fmt programs worldwide. to date, there is a broad debate regarding the preference of donor selection, whether the stool donor should be known to the patient or whether it is preferable to use feces from unrelated donor. moreover, in the case of non-related donor, fecal material could be banked at dedicated structures that provide support to the hospital that will perform fmt [53] . in particular, the ideal stool donor should be a healthy volunteer, without risk factors for infectious or other chronic diseases, and who is willing to "donate" frequently if needed. unfortunately, although the conditions do not seem too selective, it is not always easy to identify an adequate number of donors to meet the needs of the fmt program. indeed, data from large stool bank suggest high rates of donor drop out due to high commitment required [54] ; furthermore, physicians often give up fmt because of the complexity and costs of screening [55] . consequently, to solve these problems, it would be appropriate to implement the undirected donor selection program. hence, the related donors should be only limited in cases of patient preference. indeed, undirected donors reduced the likelihood of confidentiality concerns, and then, they are essential for the implementation of stool banking in consideration of easy availability, traceability, and reduction of screening expenses [56] . the screening of potential donors consist in two key landmarks, the preliminary interview and the laboratory testing [13] . a preliminary interview is usually performed by a structured questionnaire that investigated several risk factors to minimize the risk of transferring infections or adverse gut microbiota profile. in particular, the medical interview screen potential donors inquiring about the use of drugs that can alter gut microbiota, known history or risk behaviors for infectious disease, and for disorders potentially associated with the disruption of gut microbiota. the schedule of questions reported in this review (table 1) includes the most frequently investigated features in leading fmt centers. obviously, this draft of interview is not mandatory, but can be adapted to the socio-cultural context of potential donors. for example, it would be advisable to carefully investigate the eating habits of potential donors from country where the consumption of raw meat and fish is widespread, thereby increasing the risk of transmission of enteric pathogens, or who eat exotic animals that are potential carriers of unknown pathogens; or seasonal habits that increase the risk to get infected with intestinal pathogens (e.g., summer holidays and risk of sea food of poor quality). these examples allow to understand how the aim of the interview is to early intercept potential risks of pathogen transmission; thus, each center should adapt the medical interview to its socio-cultural context to make it more efficient. the optimal donor correspond at young individual (preferably < 50 years, as suggested by a panel of experts [13] taking into account that increasing age has been associated with altered gut microbiota composition [57] ; moreover, aged microbiota could have a negative effect contributing to the inflammatory state of the recipient [58] ), although is important to exclude candidates with personal history of malignancies or autoimmune disease [13] . moreover, there are concerns regarding the exclusion of healthcare workers considering the supposed increased risk of colonization by antibiotic-resistant bacteria; however, available data suggest a low prevalence in this population [59] . potential donors who have a permissive medical history must undergo to blood and fecal examination to exclude infective disease transmittable trough fecal transfer [13] . the tests may change between the various protocols, but there are some mandatory examinations ( table 2 ). in fact, blood testing should include complete blood cell count, liver enzyme, creatinine, and c-reactive protein to check overall clinical condition, serology for hepatitis virus, and human immunodeficiency virus (hiv). furthermore, blood tests can be considered in case of anomalies of the first round of laboratory tests, endemic spread of some pathogens, emergence of new pathogens or selected cases of recipients (e.g., immunosuppressed). in particular, there is debate about the usefulness of serology for ebv and cmv, as the high prevalence of prior exposure among adult individuals weakens the diagnostic power of this approach, limiting the clinical utility to igm cmv in donors dedicated to immunosuppressed recipients. of course, it is not appropriate to exclude subjects with prior exposure to ebv or cmv from the donation because of the unlikely risk of transmission, unless clinical or laboratory suspicion of reactivation. finally, the candidates could be considered for testing the serology for nematodes, based on social and geographical features and tests availability [13] . stool testing should include common enteric pathogens, clostridium difficile, fecal parasites, and helicobacter pylori antigen (this last exam only for upper route of fmt delivery). enteric pathogens, which must also be investigated in asymptomatic subjects, should be detected with conventional methods (culture, microscopy, or antigen test) and/or with molecular diagnosis (pcr-based panels) that have shown a high specificity and sensitivity compared to conventional methods in rapid detection of pathogens [60] . furthermore, it is mandatory to test all fecal samples for antibiotic-resistant bacteria (including meticillin-resistant staphylococcus aureus (mrsa), vancomycin-resistant enterococci (vre), extended-spectrum β-lactamase-producing enterobacteriaceae, and carbapenem-resistant enterobacteriaceae/carbapenemase-producing enterobacteriaceae), considering the burden of the gastrointestinal carriage in asymptomatic subjects [61, 62] and the reporting of some serious adverse events associated to sepsis after fmt [13] . nowadays, due to the emerging covid-19 pandemic, a panel of international experts has suggested to include in the tests for sars-cov-2 a thorough nasopharyngeal swab and/or rna detection in stool [63] . finally, if all blood and fecal tests are negative, the candidate is accepted to become a stool donor. especially in the fecal bank program, the donor should be available to donate on many occasions over time. for this reason, it is advisable to repeat the screening tests every 8-12 weeks and administer a short questionnaire on the same day of the donation to check for any recent-onset harmful events. in this paragraph we have reported the general rules to select and to screen potential donor for fmt, mainly to treat cdi that is cured by the restorative effect of fecal transfer on gut microbiota. however, for other clinical indications, which find their rationale in the modification of metabolic and inflammatory pathways mediated by gut microbiota, it would be appropriate to identify a specific donor for each case. this issue will be discussed later. the correct recruitment of healthy donors is essential for a standardized and safe fmt procedure [11, 13] . fmt is considered a safe procedure; however, mild adverse effects attributable to fmt are reported in about one third of the recipients, such as self-limiting abdominal discomfort or changes of bowel habits, and unfortunately, about 2-6% of patients experienced serious adverse events, such as infection, relapse of pre-existing disease, or death [64] . moreover, the difficulty of selecting the appropriate candidates is increasing due to emerging concerns, as the possibility of transmission of putative procarcinogenic bacteria [65] or the potential risk of serious life threatening infections with multi-drug resistant organisms after fmt [66] . moreover, recent evidences showed that the efficacy of fmt in recurrent cdi treatment, in clinical trials and in other healthcare settings seems to be linked to different variables, such as the delivery methods of fecal infusate, the bowel preparation, the number of infusion, the disease severity, and in particular to the microbial diversity and composition of the transplanted stools [32, 44, 67] . since the idea that the success rate of fmt could be related to the gut microbiota or other features of the donor, the term "super-donors" has been introduced to indicate the ideal individuals whose stools could ensure a better outcome for recipients compared to others fecal donations [68] . therefore, assuming that dysbiosis-related disorders have been associated to different imbalanced microbial signatures [15] , in order to restore the eubiosis, it is reasonable to assume that reaching the correct donor-recipient match with targeted fmt based on specific microbial disturbances might be the key to improve fmt response. accumulating evidence strengthens this hypothesis, leading to discard the concept of "one stool fits all" and to search an optimal donor [68] , as in other organ transplantation procedures [69] . the research of the ideal donor in recurrent cdi is obviously a widely debated topic of study. for example, one study identified the optimal donor among nine healthy vegetarian or vegan candidates, selecting the candidate who had a balanced bacteroidetes/firmicutes ratio, the highest alpha diversity among screened individuals, and high butyrate concentration. after 10 weeks from a single or multiple fmt, none of the 10 patients experienced cdi recurrence [70] . of interest, the gut virome may also play a role in cdi treatment [71] . indeed, enteric virome alterations marked by an increase in the abundance of caudovirales, together with a decreased caudovirales diversity, richness, and evenness, have been reported in patients with cdi. moreover, cd eradication was associated with the colonization of a higher abundance of donor-derived caudovirales contigs detected during follow up. these findings could possibly explain why bacterial fecal filtrate infusion resulted in effective treatment of cdi [72] , and shifted the attention on the importance of the bacteriophages and on the potential role of selecting donors on the basis of their gut virome. finally, some authors reported that selecting specific enteric bacterial strains with bacterial cultures from healthy donors to prepare a stool substitute blend might be a winning strategy to cure recurrent and antibiotic-resistant c. difficile colitis [73, 74] . however, it is likely that the relevant impact on fmt success in cdi depends on the transfer of a complete fecal microbiome rather than specific bacterial strains; moreover, the promising results reported by the study that transfer the fecal filtrate alone suggest a predominant role for bacteriophages rather than for the specific relative abundance pattern of the gut microbiota of donor, shifting the central role from bacteria to viruses in the therapeutic challenge of fmt in cdi; however, these data are still preliminary and need to be confirmed by further studies. many studies analyzed the microbial profile of donors and tried to relate it with clinical and laboratory outcomes in patients with ibd. clinical outcomes and immunological changes after fmt in patients with ibd were significantly related to the variations of several specific strains in recipients of fecal microbiota [75] . for instance, intensive fmt in uc patients were associated with negative outcomes in case of abundance of fusobacterium spp and sutterella spp in recipients' fecal microbiota after the fmt [76] . furthermore, a study that involved refractory uc patients reported that pre-treatment with antibiotic plus repeated fmts using fecal material from donor with a high bacterial richness and high relative abundance of akkermansia muciniphila, unclassified ruminococcaceae, and ruminococcus spp. was more likely to induce remission compared to antibiotics alone [67] . as also described in other studies [41, 77, 78] , it is plausible that choosing donors based on their taxonomic composition, in particular low or high abundance of specific strains, might reflect the possibility for future trials in ibd. for this purpose, methods aimed at preventing an inflammatory response of the recipient's intestinal immune system by selecting compatible donors on their microbial profiles are under study [79] . furthermore, the gut virome could represent a potential marker for fmt response in uc patients. in particular, results from a small case series reported that fmt responders already presented, before undergoing to fmt, a significantly lower eukaryotic viral richness than non-responders. moreover, the richness of donor virome was not associated with the fmt outcome, as instead proposed for bacteria [80] . several preclinical and clinical studies supported the rationale for donor selection based on gut microbial profile in other disorders associated to gut dysbiosis. indeed, in the field of anti-cancer treatment, it has been reported that microbiota can influence chemotherapy response [81] . preclinical studies found a clinical improvement in mouse models of melanoma on anti-pd-1 therapy that received fmt from donors with a melanoma "responder-like" microbial signature (with high alpha diversity and abundance of ruminococcaceae, faecalibacterium, bifidobacterium longum, collinsella aerofaciens, and enterococcus faecium) when compared to mice that received "non responder-like" microbiome (characterized by low microbial diversity and high relative abundance of bacteroidales) [82, 83] . nevertheless, trials on humans, testing the effect of fmt in increasing the response to cancer therapies, are still in progress [84] . recently, a randomized placebo-controlled trial of fmt in ibs reported that the abundance of streptococcus, dorea, lactobacillus, and ruminococcaceae spp in the donor microbiota was associated with efficacy in relieving ibs symptoms [44] . interestingly, a small open-label clinical trial evaluated the impact of prolonged fmt with antibiotic pre-treatment in children with autism; authors reported a decrease of gastrointestinal symptoms and an improvement of behavior, together with specific genera increase in recipients (bifidobacterium, prevotella, and desulfovibrio). conversely, prevotella, and desulfovibrio were more represented in recipients after fmt than in the donor samples, suggesting that unknown factors changed the intestinal ecosystem, making it more hospitable to these strains [85] . within the context of metabolic diseases, the effect of allogenic fmt post-roux-en-y gastric bypass donors was compared with metabolic syndrome donors on glucose metabolism and other parameters in treatment-naïve patients with metabolic syndrome. the authors assessed a decrease of insulin sensitivity in recipients who received fmt from donors with metabolic syndrome compared with using post-surgical donors. moreover, they identified several microbial otus possibly predictive of metabolic response, suggesting a microbiota-related transmissible mechanism of insulin resistance [86] . similarly, another study reported a significant increase in insulin sensitivity, together with altered microbiota composition, in patients with metabolic syndrome who received allogenic fmt from lean donors compared to those who underwent autologous fmt [46] . to date, these results appear promising but partially controversial; thus, findings need to be confirmed with stronger evidence and by standardized clinical trial. further research is needed to identify the favorable microbial signature of donor or other ideal features in disease-specific settings. in this review, the stool donor screening process has been described, and recent evidence has been reported that try to identify the optimal donor for each clinical condition ( figure s1 ). to date, the clinical characteristics of the donor are well defined; in particular, they are recommended to be a healthy volunteer with a balanced lifestyle, without chronic diseases or family history of metabolic diseases or cancer, and defined laboratory exams must certify the current absence of disease. however, identification of the ideal donor through the microbiological typing of the stool is currently not suitable. first of all, understanding the role of the intestinal microbiota in each chronic disease is an indispensable condition before hypothesizing a personalized approach through fmt. in fact, while the restorative mechanism of fmt in recurrent cdi is now understood, many aspects still need to be understood regarding the treatment of other chronic conditions. interesting evidence has been reported regarding dysbiosis in ibd or in other chronic conditions, but the contrasting results reported in clinical trials of fmt could be justified by the choice of unsuitable donors. the identification of the microbiological characteristics of the ideal donor for each disease appears to be an achievable goal but still far from being accomplished due to the lack of clinical studies. the current evidence is still limited and insufficient for explaining and resolving the complexity of the interaction between the intestinal barrier and its role in gut-related chronic diseases. however, further studies need to be designed to confirm the encouraging results that have been reported in recent years. in particular, it will be necessary to type the fecal microbiota of the donor and the recipient, and to understand how environmental factors, such as diet, or individual features may benefit (or not) the clinical response to fmt. understanding the microbial characteristics of the optimal donor, in particular if they are modifiable through lifestyle changes or pharmacological measures, could increase the therapeutic potential of fmt. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/6/1757/s1, figure s1 : optimal stool donor to treat specific disorders. author contributions: conceptualization, s.b. and a.g.; data curation, s.b., c.r.s. and s.p.; writing-original draft preparation, s.b. and c.r.s.; writing-review and editing, s.b., c.r.s., s.p., e.b., g.i., g.c. and a.g. all authors have read and agreed to the published version of the manuscript. funding: this research received no external funding. the authors declare no conflict of interest. the gut microbiota and host health: a new clinical frontier fecal microbiota transplantation: past, present and future perspectives fecal medicines used in traditional medical system of china: a systematic review of their names, original species, traditional 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conditions of the creative commons attribution (cc by) license key: cord-289219-qjxdggz3 authors: sebio-garcía, raquel title: pulmonary rehabilitation: time for an upgrade date: 2020-08-25 journal: j clin med doi: 10.3390/jcm9092742 sha: doc_id: 289219 cord_uid: qjxdggz3 pulmonary rehabilitation is a notoriously known but highly underused intervention aimed to restore or improve functional capacity, symptom management and health-related quality of life among patients with chronic respiratory diseases. since early 1980s, pulmonary rehabilitation has been acknowledged as a comprehensive intervention with hundreds of studies being performed over the past thirty years demonstrating its benefits on multiple outcomes; nevertheless, there are still multiple unresolved challenges, and new ones are currently emerging, with the covid-19 outbreak now in the spotlight. in this editorial, these issues are summarized and discussed, while presenting some of the latest findings in research and clinical practice, with the ultimate goal of raising awareness of the future of pulmonary rehabilitation in the post covid-19 era. pulmonary rehabilitation (pr) is a well-established, widely known intervention that needs little introduction among the research community especially for those working with chronic respiratory diseases. pr stems from a comprehensive evaluation of the patient aimed to design an individually-tailored, multi-component intervention to optimise symptom control, pulmonary function, exercise capacity and health-related quality of life [1] . as a multi-component intervention, pr consists of different elements including but not limited to exercise training (both endurance and resistance), breathing exercises, smoking cessation, education as well as psychological and nutritional support, among others [2] . the frequency, intensity and method of delivery of these components might vary between individuals depending on their characteristics and specific needs but the ultimate focus should be always improving patient's life and achieving behavioural change. the effectiveness of pr on different outcomes such as exercise capacity, muscle function, dyspnoea and symptom control, is quite robust, so it is currently recommended in the management of different chronic respiratory conditions, especially for patients with chronic obstructive pulmonary disease (copd). in 2001, the global initiative for obstructive lung disease (gold) endorsed pr as a standard of care for people with copd. furthermore, pr is the most cost-effective intervention along with smoking cessation for patients with copd [3] . currently, there is also evidence that pr might improve prognosis of the disease by reducing exacerbations, readmissions and potentially mortality [4, 5] . however, despite the bulk of evidence showing the benefits of pr and its acknowledgement as a core therapy in patients with chronic respiratory diseases, there are still unresolved issues and knowledge gaps that need our attention. one of the core difficulties that pr faces is its lack of and limited access to programmes for a large population of patients worldwide. in a study conducted in the us, only 2.7% of patients were referred to a pulmonary rehabilitation programme within 12 months of a copd exacerbation [6] . reasons for this underutilization are several but commonly identified issues include insufficient funds, lack of awareness/referral of patients, inadequate allocation of healthcare resources, lack of specialised healthcare professionals and/or adequate training opportunities [7] . in 2015 the american thoracic society and the european respiratory society developed a policy statement in which they addressed these limitations and provided different strategies to improve implementation of pr [7] . in addition to these policies which largely depend on governments and funding bodies, new research has been recently conducted to increase delivery and uptake of pulmonary rehabilitation. for instance, marques and colleagues [8] have designed a real-world non-randomised controlled study, where they plan to engage primary healthcare centres where programmes are not available, by training healthcare professionals in the basics components of pr. this programme has the benefit of bringing pr closer to the community of the patients, thus reducing commuting times to hospital, and therefore improving both access and adherence. home-based pr has also been studied as a feasible option to increase the delivery of pr especially among patients who live far from their hospital of reference, and/or have problems commuting because of their physical limitations, or cannot rely on their caregivers to travel. several studies have shown that home-based pr can be as effective as supervised face-to-face sessions to improve exercise capacity and health-related quality of life in patients with copd [9] , while others have found no effects [10] . choosing the most appropriate setting according to patients' characteristics (stage of the disease, transportation options, stable or unstable, degree of disability) and goals (maintenance, improvement, etc.) is definitely an important gap in the current literature, which should be tackled to improve the uptake of pr. another unanswered problematic that has been extensively investigated over the past years is how to maintain the benefits achieved during the programme in the long term. it is commonly acknowledged that if no maintenance strategy is provided, the benefits achieved during pr are likely to disappear after 6 to 12 months [1] . among the potential ways to extend the benefits of pr the use of telemedicine and medical technology has gained quite popularity. in a recent study published by jimenez et al. [11] , the addition of a mhealth application with a patient-educator interaction following a pulmonary rehabilitation programme of 12 weeks resulted in an increase in adherence to respiratory physiotherapy treatment compared to a control group. technology-supported exercise interventions, with the addition of a monitoring device, such as a pedometer or a fitness tracker, have shown to be effective in improving physical activity when a target is set (for example, 10.000 steps per day) [12] . however, the success of these devices in improving or maintaining physical activity and/or exercise capacity are likely to be subject to the support of a behavioural change intervention. therefore, a multi-disciplinary team should be monitoring and supporting the use of this technology instead of just giving it to the patient and "let him be". undoubtedly, the biggest challenge that pr is facing today and will continue to do so in the future is the covid-19 outbreak, which has had a tremendous impact on the healthcare systems around the world and has dramatically affected not only those diagnosed by the disease but also those vulnerable or at higher risk. as of august 12th, more than 20 million people have been diagnosed with the disease, and more than 740.000 have died worldwide. people surviving the disease, especially those that have been admitted to the icu, are at risk of developing long-term complications and sequelae, such as pulmonary fibrosis, persistent dyspnoea, impaired pulmonary function, decreased functional and exercise capacity, as well as neurological and cognitive impairments. according to a study conducted in italy, at the time of being discharged from the hospital the one-minute sit-to-stand was below the 2.5th percentile of the reference values in 33% of the patients. in addition, the barthel index which measures participation and limitation in activities of daily living (adls) was found to be poor in almost half the patients (64% were dependent for bathing, 24 for dressing/undressing, 35% for toilet use, 35% were immobile and 30% were wheelchair dependent, while 17.5% were still bedridden) [13] . as time goes by since the beginning of the pandemic, more studies are being published highlighting the long-term effects of the disease and the potential role of rehabilitation. in a recent cross-sectional study conducted among patients who had been hospitalised for covid-19, more than half of patients were still experiencing fatigue, breathlessness, and decreased health-related quality of life 48 days after discharge [14] . this situation comes as no surprise as evidence from the 2003 sars as well as icu survivors show that these patients are at a high risk of developing post-intensive care syndrome (pics) which is characterised by the presence of cognitive, psychiatric and physical impairments. there's an urgent need to provide an adequate response to the demands that this population is facing with the on top difficulty of adapting traditional face-to-face rehabilitation to other delivery platforms as we continue under the social distancing premises. the covid-19 should work as a "wake-up call" for governments and healthcare systems to implement telehealth solutions including tele-rehabilitation and remote monitoring of patients in preparation for future waves with potential new periods of isolation which will compromise again the ability to deliver pr interventions at the hospital or in the community. in addition to those who are already undertaking pr, rehabilitation services will also have to provide safe alternatives for the screening, inclusion and monitoring of new patients (both covid-19 and non-covid19) in this context. to this matter, experts on the field have recently proposed what kind of test should be used in a remote environment based on their safety and appropriateness. the short physical performance battery test (sppb), the sit-to-stand test, the stair climbing test and the timed up and go test have thus been recommended both in those recovering from covid-19 and also in other patients with respiratory conditions who are vulnerable and at risk [15] . overall, there's enough evidence to support the use of tele-rehabilitation as a safe, effective alternative to traditional pr not only as a short-term solution to the current situation but also to alleviate the burden on rehabilitation services and healthcare systems and increase reach to pr to more patients in need [16] . in summary, this is definitely an exciting year to contribute to the body of knowledge in pulmonary rehabilitation and embrace the challenges that it brings. we highly encourage researchers to submit your latest manuscript to this special issue in the journal of clinical medicine, a top open-access journal in its category and a reference for researchers in the different fields of medicine. statement: key concepts and advances in pulmonary rehabilitation time to say farewell to therapeutic nihilism pulmonary rehabilitation following exacerbations of chronic obstructive pulmonary disease lower mortality after early supervised pulmonary rehabilitation following copd-exacerbations: a systematic review and meta-analysis participation in pulmonary rehabilitation after hospitalization for chronic obstructive pulmonary disease among medicare beneficiaries policy statement: enhancing implementation, use, and delivery of pulmonary rehabilitation improving access to community-based pulmonary rehabilitation: 3r protocol for real-world settings with cost-benefit analysis feasibility and effectiveness of a home-based exercise training program before lung resection surgery the impact of home-based pulmonary rehabilitation on people with mild chronic obstructive pulmonary disease: a randomised controlled trial development and preliminary evaluation of the effects of an mhealth web-based platform (happyair) on adherence to a maintenance program after pulmonary rehabilitation in patients with chronic obstructive pulmonary disease: randomized controlled trial (preprint) using pedometers to increase physical activity and improve health: a systematic review low physical functioning and impaired performance of activities of daily life in covid-19 patients who survived the hospitalisation post-discharge symptoms and rehabilitation needs in survivors of covid-19 infection: a cross-sectional evaluation home-based and remote exercise testing in chronic respiratory disease, during the covid-19 pandemic and beyond: a rapid review pulmonary telerehabilitation: an international call for action this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license funding: this research received no external funding. the author declares no conflict of interest. key: cord-013457-rqon1adg authors: de cannière, hélène; smeets, christophe j. p.; schoutteten, melanie; varon, carolina; morales tellez, john f.; van hoof, chris; van huffel, sabine; groenendaal, willemijn; vandervoort, pieter title: short-term exercise progression of cardiovascular patients throughout cardiac rehabilitation: an observational study date: 2020-09-29 journal: j clin med doi: 10.3390/jcm9103160 sha: doc_id: 13457 cord_uid: rqon1adg cardiac rehabilitation (cr) is a highly recommended secondary prevention measure for patients with diagnosed cardiovascular disease. unfortunately, participation rates are low due to enrollment and adherence issues. as such, new cr delivery strategies are of interest, as to improve overall cr delivery. the goal of the study was to obtain a better understanding of the short-term progression of functional capacity throughout multidisciplinary cr, measured as the change in walking distance between baseline six-minute walking test (6mwt) and four consecutive follow-up tests. one-hundred-and-twenty-nine patients diagnosed with cardiovascular disease participated in the study, of which 89 patients who completed the whole study protocol were included in the statistical analysis. a one-way repeated measures anova was conducted to determine whether there was a significant change in mean 6mwt distance (6mwd) throughout cr. a three-way-mixed anova was performed to determine the influence of categorical variables on the progression in 6mwd between groups. significant differences in mean 6mwd between consecutive measurements were observed. two subgroups were identified based on the change in distance between baseline and end-of-study. patients who increased most showed a linear progression. in the other group progression leveled off halfway through rehabilitation. moreover, the improvement during the initial phase of cr seemed to be indicative for overall progression. the current study adds to the understanding of the short-term progression in exercise capacity of patients diagnosed with cardiovascular disease throughout a cr program. the results are not only of interest for cr in general, but could be particularly relevant in the setting of home-based cr. cardiac rehabilitation (cr) is an evidence-based intervention that uses a multidisciplinary approach to improve secondary prevention outcomes in cardiovascular patients [1] [2] [3] [4] [5] . the clinical effectiveness of conventional center-based cr is well established and the beneficial effects of cr on mortality and hospital readmissions have been extensively discussed [6] [7] [8] [9] [10] [11] . although a large and increasing number of cardiovascular patients need cr, many will not participate due to issues such as limited referral, enrolment problems or suboptimal completion rates [12] . participation rates are especially low for women, elderly patients, and socially disadvantaged groups [1, 13] . new cr delivery strategies are needed to improve enrollment, adherence, and completion in eligible patients. however, the heterogeneity in cr programs implemented across europe makes it difficult to properly study and interpret the effects of the intervention. moreover, to be able to accurately measure quality of care improvement, a cr standardization process is needed. the european association of preventive cardiology (eapc) accreditation program focusses on developing minimum standards for the evaluation of quality of a cr program, including characterization of eligible patients, ideal timing of cr and defining necessary components of cr [14] . these standardization procedures are also wanted within home-based cr. a potential new approach to overcome some of the current enrollment barriers is by moving cr to an in-home setting [14] [15] [16] . several studies and meta-analyses have shown that home-based cr and center-based cr have similar effects on clinical primary outcomes, including total mortality, exercise capacity, and health-related quality of life. however, these analyses also report the heterogeneity of cr programs among studies as a major limitation, further emphasizing the need for standardization procedures [17] [18] [19] . furthermore, parameters of care, defined by standardization, can contribute to the personalization of treatment strategies, optimizing the patients' needs and preferences. in addition, wearable telemonitoring solutions support patients in following personalized treatment recommendations (i.e., exercise training, diet, medication . . . ), both in home-and center-based cr programs [15] . although the majority of studies on home-and center-based cr programs report data on changes in exercise capacity measured at baseline and on completion of the intervention limited information is available on the short-term progression in exercise capacity throughout the cr [20] [21] [22] . the six-minute walking test (6mwt) is one of the outcome measures used to report progression [23] . the 6mwt is often used as a testing modality in clinical practice due to its simplicity, safety, low-cost and ease-to-administer [23] [24] [25] . in addition, the 6mwt is reflective for activities of daily living and can thus be easily translated to a standardized activity in an in-home environment. consequently, the 6mwt is not only suitable for studying short-term changes in exercise capacity, but it could also be used to report changes in exercise capacity at home [26, 27] . however, the 6mwt is characterized by some limitations. compared to the cardiopulmonary exercise test (cpet), which provides a global assessment of functional capacity, it is impossible to determine the causes or mechanisms of exercise limitation or dyspnea on exertion. however, despite these differences, good correlations have been shown between both tests [25, 28] . in this study, we hypothesized that monitoring progression in detail during the course of a standardized rehabilitation program could provide more insight into the actual improvement in exercise capacity. to our knowledge, the present research is the first to study the effects of exercise training at short-time intervals in the course of a standardized cr program. future studies could potentially use this information to optimize short-term guidance and treatment strategies, namely in home-based cr programs. one hundred and twenty-nine patients diagnosed with cardiovascular disease, participating in a structured ambulatory multidisciplinary cr program in a single tertiary care center (ziekenhuis oost-limburg, genk, belgium), were included. criteria for eligibility were being 18-years-old or older, providing informed consent, hf with reduced ejection fraction (ef), hf with preserved ejection fraction and patients referred to cr after myocardial infarction, coronary artery bypass grafting, percutaneous intervention or other, with a left ventricular ef less than or equal to 55%, measured during the baseline echocardiography measurement. the diagnosis (i.e., hf) were considered as according to the electronic patient records and as described by the patient's physician. a cut-off value of 55% in terms of ef was chosen based on the values that are reported by lang et al. values below 55% are considered to be abnormal. this experience-based partition value allows to achieve meaningful clinical categorization of the severity of abnormality [29] . exclusion criteria were orthopedic or neurological limitations. the study complied with the declaration of helsinki (fortaleza, brazil, 2013) and the local institutional ethical committee (cme zol genk) approved the study protocol. on average, one out of ten eligible patients declined participation. written informed consent was obtained from all participating subjects. after a cardiovascular-related hospitalization or consultation, patients were referred to the multidisciplinary cr program, according to standard of care in our center. baseline cardiopulmonary exercise testing (cpet) on a stationary bicycle and echocardiography measurements (philips medical systems, ie33, andover, ma, usa) were performed prior to program enrollment. for cpet, a ramp protocol starting at 50 w and increasing 30 w every 2 min was used. patients on beta-blocker therapy were asked to discontinue beta-blocker treatment prior to testing in order for their heart rate to be able to reach the age-predicted value. the modified simpson's biplane method was used to calculate ef. moreover, clinical assessment was undertaken during cpet measurements at the beginning and end of the program. enrolled patients had to follow 45-sessions of supervised ambulatory rehabilitation at a frequency of three one-hour-sessions a week. the training protocol consisted of both aerobic and resistive exercises. seventy percent to 85% of the maximal heart rate (hr) was chosen as target hr during aerobic training. aerobic training consisted of in total 30-40 min of aerobic exercise on bicycle, handbike, treadmill and/or stepper. resistive training, added halfway into the rehabilitation program, was performed at 50-80% of one repetition maximum. resistive training consisted of three times fifteen repetitions on both the leg and arm press. the supervising physiotherapist increased training intensity based on individual performance every two weeks. additionally, when deemed necessary or upon patient request, dietary sessions, psychological support, and social consultations were included in the multidisciplinary program. a cpet was repeated and at the end of the rehabilitation program, together with an echocardiographic assessment. adherence to the rehabilitation program was evaluated by measuring attendance at the exercise sessions. demographic and clinical data (baseline cpet, new york heart association (nyha)-class, medical therapy, baseline echocardiography, reason for referral, and medical history) were collected from the electronic medical record. health-related quality of life (hrqol) assessments were carried out using the short form-36 (sf-36) questionnaire and the minnesota living with heart failure questionnaire (mlhfq) at baseline and end-of-study, both validated in cr patients. a 6mwt was taken at baseline (start of rehabilitation program) and was repeated four times (every three weeks), for five times in total. the 6mwt was performed according to a standardized protocol [24] . the test was done in a 45-m-corridor and standardized encouraging sentences were repeated by the researcher every minute throughout the test. the distance traveled after completing the 6mwt was determined. in response to exercise training, a change in walking distance of 30-50 m is considered clinically relevant [24] . both the patient's self-assessment of wellbeing on a scale of one to ten and the nyha classification were determined before. hemodynamic parameters, including hr, spo 2 and blood pressure were assessed before and after the test. the borg score of perceived exertion for dyspnea and fatigue was also recorded before and upon completion of the test. the baseline characteristics between patients who completed all five 6mwts and patients who failed to finish the study protocol were compared. this study focused on the progression of functional capacity throughout a standardized rehabilitation program. the functional improvement was measured as the change in distance between baseline 6mwt and every consecutive follow-up test. continuous variables were expressed as mean (standard deviation) if normally distributed or as median (interquartile range) if non-normally distributed and dichotomous data were expressed as n (%). normality was assessed by the shapiro-wilk statistic. a one-way repeated measures anova was conducted to determine whether there were statistically significant differences in mean 6mwt distance (6mwd) over the period of a three-month rehabilitation program. when significant, a post hoc bonferroni adjustment investigated the pairwise comparison of the different measurements. the friedman test was used as a non-parametric alternative to assess differences in ordinal variables over time. a paired-samples t-test was used to determine whether there was a statistically significant difference between cpet at the start and at the end of the rehabilitation program. continuous variables were compared between groups with the student's t-test or mann-whitney u test as appropriate. a chi-square test was used to compare categorical variables between groups. a three-way-mixed anova was performed to determine the influence of categorical variables on the progression in 6mwd over the period of a three-month rehabilitation program between groups. patients unable to complete the cr program, due to health-related problems, lack of motivation, work commitment, or family commitment, failed to complete all five 6mwts and were therefore excluded from analysis. only patients who completed the five 6mwt measurements were included in the statistical analysis. the statistical significance was always set at a 2-tailed probability level of <0.05. statistics were performed using spss version 24 (ibm, chicago, il, usa). one-hundred and twenty-nine patients, enrolled in the multidisciplinary cr program, consented to participate in the study. sixty-nine percent of the patients (n = 89) completed all five 6mwts. patients unable to complete the cr program, due to health-related problems, lack of motivation, work commitment, or family commitment, failed to complete all five 6mwts and were therefore excluded from analysis when investigating progression throughout the complete cr ( figure a1 ). clinical and baseline characteristics of the patients are summarized in table 1 . mean age was 63 years and 71.3% of patients were male. twenty-two percent were actively smoking during the rehabilitation program. baseline median ef was 45% for the total population and 42% suffered prior myocardial infarction. twenty-eight percent of patients were classified as nyha i, 46.5% as nyha ii, and 25.6% as nyha iii. fifty-one percent of subjects were on ace-i therapy, 75.2% were also on beta-blocker therapy, 51.9% were taking diuretics, and 72% were on statins therapy. the mean baseline peak vo 2 was 16.21 ml kg-1min-1, while 6mwd at baseline was 468 m. reasons of referral were post myocardial infarction (24.8%), hf (29.5%), coronary artery bypass grafting (cabg) (13.2%), percutaneous coronary intervention (11.6%), other (20.9% (table a1) ). there was no statistical significant difference in baseline physical performance, i.e., peak vo 2 and 6mwd, between referral groups. one-hundred-and-two patients were referred to cr after hospitalization (79%). the time-delay after hospitalization until the start of the program was on average 21 (18) days. table a2 shows the baseline characteristics for the patients who completed the five 6mwts (n = 89) and the patients who failed to finish the study protocol (n = 40). forty-three percent of patients who dropped-out early were referred to cr for hf, compared to 24% in the group who completed the study protocol. the distance walked during the baseline 6mwt was significantly lower for the patients who did not complete the study protocol (484 vs. 431 m). moreover, peak vo 2 values and peak power were significantly lower (17.00 vs. 14.06 ml/kg/min and 114 vs. 96 watts) for the drop-out group. overall compliance rate to the exercise program was 91.9%, with respect to a total of 45 sessions that needed to be followed for the 89 patients who completed the five 6mwts. while exercise adherence on a three-weekly basis (with respect to 3 sessions per week, 9 in total), in between the consecutive 6mwts, changed respectively from 86.3% to 86.6%, to 82.8% and to 83.5%. for the 89 patients who completed the whole study protocol, mean peak vo 2 increased significantly with 3.93 (95% ci, 2.60 to 5.32) ml kg −1 min −1 between baseline 16.18 (5.03) ml kg −1 min −1 and end-of-study 20.14 (5.24) ml kg −1 min −1 (t(38) = 5.86, p ≤ 0.001, d = 0.94). an improvement in hrqol was seen after completing cr. more specifically, a significant difference was seen for both mlhfq and sf-36 questionnaires when comparing baseline with end-of-study measurements. a significant decrease of 14.13 (95% ci, 9.54 to 18.72, t(75) = 6.13, p ≤ 0.001, d = 0.70) in total mlhfq score and a significant increase for almost all sf-36 subclasses, except for the general health subclass, was noted ( table 2 ). no significant change in blood pressure between baseline and end-of-study measurements was seen (127/75 (19/11) vs. 128/74 (17/12)). the short-term change in exercise capacity at different time intervals throughout the cr program was studied by means of the 6mwt. a total of 89 patients completed the whole study protocol and performed all five 6mwts (table 3) . based on the one-way repeated measures anova, mean distance differed significantly across consecutive time points during rehabilitation, f (2.357, 207.421) = 164.828, p ≤ 0.001, partial η 2 = 0.652. post hoc analysis revealed significant differences in 6mwd for each pair of consecutive time points, except for the mean distance between the second and third follow-up measurement. respectively, an increase of 48.5 (95% ci, 36.0 to 60.9, p ≤ 0.001,) m was seen between baseline and first follow-up, 30.8 (95% ci, 20.5 to 41.1, p ≤ 0.001) m from first to second follow-up, 6.4 (95% ci, −2.5 to 15.3, p = 0.413) m from second to third follow-up and 15 (95% ci, 7.3 to 22.8, p ≤ 0.001,) m from third follow-up to end-of-study measurement. the distance tended to increase throughout the exercise program ( figure 1 ). however, towards the end of the study, the slope decreases. end-of-study measurement. the distance tended to increase throughout the exercise program ( figure 1 ). however, towards the end of the study, the slope decreases. fluctuations in hr measured before and after the 6mwt (hrbefore and hrafter) and changes in nyha classification during rehabilitation can provide additional information on exercise capacity in patients diagnosed with cardiovascular disease [30, 31] . hrbefore altered across the consecutive time points during rehabilitation, f (4, 19945) = 2.950, p ≤ 0.021, partial η 2 = 0.038. a significant decrease of 4 bpm from baseline to end-of-study was observed (95% ci, 0 to 8, p = 0.024). although no significant difference could be determined between pairs of mean hrafter, one-way repeated measures anova showed a significant increase in hrafter during the exercise program (f (4, 292) = 3.747, p ≤ 0.001, partial η 2 = 0.049). friedman testing showed a significant decrease in nyha classification at the different time points, χ 2 (4) = 36.314, p ≤ 0.001 (baseline: 1.93 (0.70), first: 1.74 (0.70), second: 1.64 (0.66), third: 1.56 (0.68), end-of-study: 1.48 (0.67)). next, to determine whether the decrease in progression during the second half of the cr program was characteristic for all patients, the increase in 6mwd was investigated on a patient level. each individual patient was characterized by a unique progression pattern throughout the cr program, representing the complexity of this study population. therefore, a median split for the increase in distance walked throughout the rehabilitation program, was performed. two groups with an equal number of patients were created based on an improvement in 6mwd of more or less than 90 m (median value). two progression patterns emerged (figure 2 ). fluctuations in hr measured before and after the 6mwt (hr before and hr after ) and changes in nyha classification during rehabilitation can provide additional information on exercise capacity in patients diagnosed with cardiovascular disease [30, 31] . hr before altered across the consecutive time points during rehabilitation, f (4, 19945) = 2.950, p ≤ 0.021, partial η 2 = 0.038. a significant decrease of 4 bpm from baseline to end-of-study was observed (95% ci, 0 to 8, p = 0.024). although no significant difference could be determined between pairs of mean hr after , one-way repeated measures anova showed a significant increase in hr after during the exercise program (f (4, 292) = 3.747, p ≤ 0.001, partial η 2 = 0.049). friedman testing showed a significant decrease in nyha classification at the different time points, χ 2 (4) = 36.314, p ≤ 0.001 (baseline: 1.93 (0.70), first: 1.74 (0.70), second: 1.64 (0.66), third: 1.56 (0.68), end-of-study: 1.48 (0.67)). next, to determine whether the decrease in progression during the second half of the cr program was characteristic for all patients, the increase in 6mwd was investigated on a patient level. each individual patient was characterized by a unique progression pattern throughout the cr program, representing the complexity of this study population. therefore, a median split for the increase in distance walked throughout the rehabilitation program, was performed. two groups with an equal number of patients were created based on an improvement in 6mwd of more or less than 90 m (median value). two progression patterns emerged (figure 2 ). (27) m between third follow-up and end-of-study an increase of 35 ± 34 m was seen for the linear progression group between the second follow-up measurement and endof-study. this increase differed significantly from the 8 ± 25 m change of the less than 90 meters' group (95% ci, 14.3 to 39.4, p ≤ 0.001). there were no statistically significant differences between both groups with respect to the demographics and baseline characteristics, except for diabetes (11 (24.4%) in less than 90 m group vs. 1 (2.3%) in more than 90 m group). moreover, no difference in peak vo2 and respiratory exchange ratio (rer) were observed (17.2 (5.3) ml/kg/min with a rer of 1.1 (0.1) for the less than 90 m group vs. 16.8 (4.9) ml/kg/min with a rer of 1.1 (0.1) for the more than 90 m group). the reasons of referral were equally divided between both groups. no significant difference in compliance rate was observed between both groups. however, a significant difference between both groups was seen in distance increased between baseline and the initial follow-up measurement. patients who increased more than 90 m throughout rehabilitation, increased more than 70 m from baseline to first follow-up. the patients who improved less than 90 m during cr, increased less than 30 m between baseline and first follow-up measurement (mean difference of 45.02 m, 95% ci, 30.64 to 59.42, p ≤ 0.001). the current study adds to the knowledge on the short-term progression in exercise capacity of patients following a multidisciplinary cr program. an increase in 6mwd between consecutive 6mwts throughout the cr was seen, which confirmed the beneficial effects of exercise training. our study population increased on average 101 m in 6mwd when comparing baseline and endof-study measurements. this is a large increase when compared to the outcome of two meta-analysis investigating the effects of cr in a hf population, where an increase of 41 m and 50 m, respectively, was noted [32, 33] . on the other hand, a larger improvement was seen in a study investigating the increase in 6mwd after cr in a population with a recent history of acute coronary syndrome [34] . although only a limited amount of data is available in current literature, making it difficult to define a clinically significant increase in 6mwd, it is clear that it differs among and between patient populations. moreover, it is dependent on patient characteristics, emphasizing the need to interpret improvement in 6mwd on an individual patient level. additionally, there was an increase of 4 bpm there were no statistically significant differences between both groups with respect to the demographics and baseline characteristics, except for diabetes (11 (24.4%) in less than 90 m group vs. 1 (2.3%) in more than 90 m group). moreover, no difference in peak vo 2 and respiratory exchange ratio (rer) were observed (17.2 (5.3) ml/kg/min with a rer of 1.1 (0.1) for the less than 90 m group vs. 16.8 (4.9) ml/kg/min with a rer of 1.1 (0.1) for the more than 90 m group). the reasons of referral were equally divided between both groups. no significant difference in compliance rate was observed between both groups. however, a significant difference between both groups was seen in distance increased between baseline and the initial follow-up measurement. patients who increased more than 90 m throughout rehabilitation, increased more than 70 m from baseline to first follow-up. the patients who improved less than 90 m during cr, increased less than 30 m between baseline and first follow-up measurement (mean difference of 45.02 m, 95% ci, 30.64 to 59.42, p ≤ 0.001). the current study adds to the knowledge on the short-term progression in exercise capacity of patients following a multidisciplinary cr program. an increase in 6mwd between consecutive 6mwts throughout the cr was seen, which confirmed the beneficial effects of exercise training. our study population increased on average 101 m in 6mwd when comparing baseline and end-of-study measurements. this is a large increase when compared to the outcome of two meta-analysis investigating the effects of cr in a hf population, where an increase of 41 m and 50 m, respectively, was noted [32, 33] . on the other hand, a larger improvement was seen in a study investigating the increase in 6mwd after cr in a population with a recent history of acute coronary syndrome [34] . although only a limited amount of data is available in current literature, making it difficult to define a clinically significant increase in 6mwd, it is clear that it differs among and between patient populations. moreover, it is dependent on patient characteristics, emphasizing the need to interpret improvement in 6mwd on an individual patient level. additionally, there was an increase of 4 bpm in hr measured immediately after the 6mwt when comparing baseline with end-of-study measurements. these hr measurements are collected at specific and discrete moments in time. the absence of continuous hr monitoring makes it difficult to determine the cause of this increase. it is possible that patients are more motivated and try harder, causing their hr to increase. however, together with the decrease in the resting hr and the increase in peak vo 2 , it can be stated that the difference in hr is most likely caused by an improvement in fitness level [35] . an improvement in hrqol was seen after completing cr, except for the general health status subclass. these results indicate that patients do not evaluate their personal health differently at the end of cr when compared to the start of cr. however, they do evaluate their personal health differently when compared to healthy subjects. in other words, they do not consider themselves as healthy individuals and feel compromised by the disease. although both their mental and physical status improve, cr patients still feel different when compared to healthy subjects after following a cr program. there is limited evidence available on studying this subtopic of the sf-36 questionnaire in this specific patient population. in most research, focus is only put on the average increase in mental and physical health with respect to sf-36 results, no subtopics are discussed. during the second half of rehabilitation the increase in 6mwd levelled-off. the 6mwt is an outcome measure representative for exercise tolerance, limited by several cardiovascular related and non-cardiovascular related factors, which could all influence the decrease in progression. firstly, an increase in ef can be present throughout the cr program [36] . it is possible that similar to the 6mwd, a decrease in ef improvement is seen, explaining the stagnation of progression. however, perreti et al. investigated the relationship between functional improvement (evaluated by a 6mwt) and the improvement in ef after cr in a population with a recent history of acute coronary syndrome. although patients with a lower ef showed a larger increase in distance walked after cr compared to patients with a higher ef (168 m vs. 194 m), no significant correlation was found [34] . another possible explanation is that the 6mwt is a submaximal exercise test limited by the patients' maximal walking speed, thereby limiting the distance they can cover in six minutes [37] . moreover, osteoarticular pathology or skeletal muscle malfunctioning are determinants of exercise capacity and could influence the rate of improvement. non-cardiovascular related factors are equally important to consider when studying the decrease in progression. patient motivation, effort, and willingness to perform the exercise could influence the outcome of a 6mwt [38] . in our study, although not statistically significant, a decreasing trend in compliance rate was seen throughout the program, which could potentially explain the reduced improvement. there are several other known non-cardiovascular factors that can potentially influence the response of patients to cr, which can however often be related to failure to participate [39] . however, in our heterogeneous study population it is difficult to define these (non-) cardiovascular factors that determine the decrease in progression during the second half of cr. next, the differences between the patients who completed all five 6mwts and the patients who failed to finish the study protocol were investigated. notably, almost half of the patients who failed to complete the study protocol, were patients who were referred to cr for hf. the high number of hf patients in the drop-out group can be a possible explanation for the differences observed during baseline cpet testing. patients from the drop-out group are characterized by a lower exercise capacity. ritchey et al. reported similar results showing low participation rates for hf patients [40] . these high drop-out rates for hf referral patients can be explained by the fact that these patients tend to be older and sicker, making them less motivated to finish the rehabilitation program. an additional explanation can be found in the fact that non-procedure related secondary events as a reason for referral, are often characterized by low participation rates. the secondary events are typical the motivation behind referring hf patients to cr [40] . to investigate whether the decrease in progression was characteristic for all patients, the increase in 6mwd was investigated on a patient level. a unique progression pattern for each individual patient was seen throughout the cr program, representing the complexity of this study population. therefore, patients were divided into two groups by means of a median split based on the increase in distance walked throughout the rehabilitation program. two different progression patterns emerged. the group who increased more than 90 m showed a near-linear improvement, while the other group's progression levelled off during the second half of the rehabilitation program. limited improvement can be associated with female gender and body mass index [41] . however, there were no differences in baseline characteristics between both groups except for the prevalence of diabetes. previous research groups showed that diabetes patients benefitted less from cr when compared to patients who did not have diabetes, which can be explained by a dysregulation of cardiovascular and metabolic functions [42] [43] [44] . the lack of improvement in exercise capacity can potentially be explained by genetic factors but also by a decline in motivation or cardiac related barriers causing a decrease in adherence. therefore, a personalized approach consisting of exercise training programs adjusted to individual needs is recommended in these patients [45] . a higher prevalence of diabetes in the group who improved less than 90 m could (at least partially) explain the difference in progression between both groups. although it is difficult to determine at baseline which patients will benefit more, a distinction can be made early on in cr. an important difference between the two progression pattern groups is the improvement in 6mwd obtained during the initial three weeks of cr. patients increasing on average more than 60 m between the initial two measurements, will improve more throughout cr, while patients improving less than 30 m will progress less. these results showcase the potential importance of short-term follow-up, which could allow a fast optimization of exercise management, possibly improving outcome for specific patient subgroups. future studies should investigate whether similar progression patterns emerge in both center-based (including with larger patient groups) and in home-based cr programs and whether this short-term information on progression can be used to optimize outcomes by improving exercise capacity and motivation. the results from this observational study can be used as a framework for future studies to compare the short-term progression in functional capacity between center-based and home-based rehabilitation studies. the authors of this study believe that continuous monitoring of various physiological parameters throughout a 6mwt at the start of cr could provide even more information needed to fine-tune exercise management in a home-based rehabilitation program. previous research demonstrated that additional parameters optimize the interpretation of the results of a 6mwt. heart rate recovery, measured immediately after exercise, can function as a powerful prognosticator in cardiovascular disease. heart rate recovery was shown to be a significant predictor of adverse events and strong predictor of survival [46] . furthermore, information extracted from accelerometer data can be used to reliable evaluate exercise performance, allowing routine assessment in an in-home setting [27] . step frequency and activity counts, both parameters extracted from accelerometer data have been proven to be correlated to the 6mwd. moreover, the activity counts can be used to assess walking speed in a hf population [27] . our research group showed that wearable sensor technology can be used to characterize response to cr by measuring heart rate parameters and digital biomarkers, derived from continuous measurements. these digital biomarkers allow for in-depth insights into the cardiac response of patients during a standardized activity [47] . therefore, wearable and mobile technologies, capable of continuously monitoring patients following cr during exercise, could give more insight in the actual progression on patient level throughout rehabilitation. these technologies are not able to provide the same clinical insights as compared to the cpet measurements. the ability of deriving respiratory variables, i.e., oxygen consumption, that would enable insights into the pathogenic mechanisms that lead to exercise limitations (dyspnea and fatigue sensation) are restricted. however, they can expand the capacity of the 6mwt to assess and monitor patients at home by monitoring activity levels, distance walked, and hr measured during exercise, e.g., device-generated data showed a similar effect on hf related events compared to 6mwd alone [48] . this study should be interpreted in the light of some limitations. first, the learning effect of the 6mwt should be taken into account when interpreting the change in distance between baseline and first follow-up measurement [26] . although a larger increase was seen between the initial two measurements, the increase persists during the following 6mwts ensuring an improvement in functional capacity. other limitations to the 6mwt include the difficulty to determine the causes or mechanisms of exercise limitations, compared to the cpet. moreover, the performance of the patient is motivation dependent [25] . secondly, some limitations need to be considered on the exercise modalities used to assess functional capacity. during the 6mwt patients can potentially reach an exercise capacity level that makes the 6mwt unsuitable to follow up progression as maximal walking speed can limit their performance [37, 49] . the low number of patients who performed a cpet halfway into the rehabilitation program is an important limitation, as it would be of interest to compare these data with the outcome of the 6mwts. the low number of patients, missing information on marital and educational status, the heterogeneous character of the population, the lack of information on biochemical parameters, and the imbalance between male and female patients are all limitations of the study that need to be taken into account when interpreting the results. in addition, the absolute number of patients screened for study participation and their characteristics have not been described. another limitation to the study is that changes in medication during the study period were not accounted for. however, it should be mentioned that the 6mwt can be used for the assessment of changes in symptoms, but there are still some hinderances concerning its full scope in terms of assessment of pharmacological interventions [50] . furthermore, the lack of comparison with a group undergoing home-based cr makes it difficult to implement these results in home-based rehabilitation. however, these interpretations are exploratory and future studies with a matched patient group undergoing home-based cr should be considered. the differences between the patient groups who completed the study protocol and those who did not should be taken into account as almost a third of the patients initially enrolled in the study did not complete the protocol. especially when considering the present data as a framework for future studies, it is important to emphasize that these results only apply to a subset of patients which were less likely to be referred due to hf and which presented a higher functional capacity at baseline. the results of this study should be investigated in a larger patient group. the current study investigated the effect of cr on the progression of exercise capacity in patients diagnosed with cardiovascular disease within short-time spans. the main findings of the study were: (1) after six weeks of initial progression in exercise capacity, a flattening of improvement is reached; (2) two different patterns of progression emerge when analyzing the data on a more personal level. the group who progressed most, showed a near linear improvement in exercise capacity throughout cr. the improvement of patients who responded less to exercise training, leveled of half-way into rehabilitation. future studies could use this information to optimize short-term guidance and treatment strategies, namely in home-based cr programs. 1s53616n) . svh acknowledges the financial support of imec. svh's research received funding from the flemish government (ai research program). svh, jfmt and cv's research is supported by agentschap innoveren en ondernemen (vlaio) (150466), osa+. the funding organizations did not make a 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for patients with type 2 diabetes and cardiovascular disease: what to pursue and how to do it. a position paper of the european association of preventive cardiology (eapc) heart rate recovery after the 6 min walk test rather than distance ambulated is a powerful prognostic indicator in heart failure with reduced and preserved ejection fraction: a comparison with cardiopulmonary exercise testing using biosensors and digital biomarkers to assess response to cardiac rehabilitation: observational study six-minute walk test for assessing physical functional capacity in chronic heart failure 6-min walking test: a useful tool in the management of heart failure patients six minute corridor walk test as an outcome measure for the assessment of treatment in randomized, blinded intervention trials of chronic heart failure: a systematic review this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this research is part of the limburg clinical research center (lcrc) uhasselt-zol-jessa, supported by the foundation limburg sterk merk, province of limburg, flemish government, hasselt university, jessa hospital and ziekenhuis oost-limburg. we would like to thank the engineers from holst center/imec the netherlands for their technical support. we thank the physiotherapists of the cardiac rehabilitation center, ziekenhuis oost-limburg, for their support and guidance during the study. we furthermore thank all the participants of this study. key: cord-252687-7084pfqm authors: szelenberger, rafal; saluk-bijak, joanna; bijak, michal title: ischemic stroke among the symptoms caused by the covid-19 infection date: 2020-08-19 journal: j clin med doi: 10.3390/jcm9092688 sha: doc_id: 252687 cord_uid: 7084pfqm the 2019 global pandemic of coronavirus disease 2019 (covid-19) caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) has been declared a public health emergency of international concern by the world health organization (who). the who recognized the spread of covid-19 as a pandemic on 11 march 2020. based on statistics from 10 august 2020, more than 20.2 million cases of covid-19 have been reported resulting in more than 738,000 deaths. this completely new coronavirus has spread worldwide in a short period, causing economic crises and healthcare system failures worldwide. initially, it was thought that the main health threat was associated with respiratory system failures, but since then, sars-cov-2 has been linked to a broad spectrum of symptoms indicating neurological manifestations, including ischemic stroke. current knowledge about sars-cov-2 and its complications is very limited because of its rapidly evolving character. however, further research is undoubtedly necessary to understand the causes of neurological abnormalities, including acute cerebrovascular disease. the viral infection is inextricably associated with the activation of the immune system and the release of pro-inflammatory factors, that can stimulate the host organism to defend itself. however, the body’s immune response is a double-edged sword that on one hand, destroys the virus but also disrupts the homeostasis leading to serious complications, including thrombosis. numerous studies have linked coagulopathies with covid-19, however, there is great uncertainty regarding it functions on the molecular level. in this review, a detailed insight into the biological processes associated with ischemic stroke in covid-19 patients and suggest a possible explanation for this phenomenon is provided. coronavirus disease 2019 (covid-19) is a new infectious disease caused by the newly identified severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which was classified by the world health organization (who) as a pandemic on 11 march 2020. belonging to the orthocoronavirinae subfamily in the coronaviridae family, sars-cov-2 is the seventh member of all coronaviruses with the ability to infect humans [1] . as regards its origin, there are few theories, the most probable one being that sars-cov-2 has a natural, zoonotic origin. it is closely related to bat coronaviruses, pangolin coronaviruses and sars-cov. the first diagnostic reports of an unusual respiratory disease appeared in december 2019 in the city of wuhan (hubei province), china and were linked to a cluster of wet markets processing bat meat and their guano [2] . however, new reports from china suggest that a 55-year-old person from hubei may have been the first person infected on 17 november 2019. isolation and genome sequencing of the new virus led to the discovery of a new pathogen that primarily caused "pneumonia of an unknown etiology" [1] . however, current knowledge about this virus is very limited and is mostly derived from previous coronaviruses. longitudinal serological and immunological studies are necessary to assess the efficiency of an immune response to sars-cov-2 [3] . initially, sars-cov-2 was thought to cause fever, dyspnea, cough and fatigue via infection of the host's respiratory system [2] . however, the ongoing scientific effort in order to profiling of covid-19 patients revealed that patients exhibit a broader range of atypical symptoms affecting the severity and disease progression, including headache, nasal congestion, diarrhea, loss of taste or smell, rash and conjunctivitis [4] . furthermore, there is the onset of a wide range of symptoms, the presence of comorbidities and response to existing therapies failure, which may result in mild pneumonia that quickly develops into the acute respiratory syndrome sepsis, and even to multi-organ dysfunction within a short period of time [5] . the vast majority of sars-cov-2 infections are asymptomatic at the time of testing. however, most of infected people developed symptoms later, which enhance the virus transmission. furthermore, the presence of flu-like symptoms with a prolonged viral incubation period may result in wrong diagnosis or the disease not being detected at all. not isolated and infected individuals are a vector for the rapid spread and advanced migration of sars-cov-2. an estimation of the basic reproduction number (r0) for covid19 in january showed that it may be about 5.7 (with a 95% confidence interval of 3.8 to 8.9). the low mortality threshold facilitates a host-to-host transfer, increasing the number of cases exponentially [6] . according to the 10 august 2020 who data, more than 20.2 million cases of covid19 have been reported resulting in more than 738,000 deaths. this number will continue to grow unless an effective treatment or vaccine is developed. the appearing threat associated with covid-19 pandemic is related to the virus ability to induce microvascular, venous and arterial thrombosis, thus exacerbating the functionality of organs. many clinical studies have shown an association between sars-cov-2 infection and hypercoagulability diagnosed on the basis of abnormal coagulation parameters, including activated partial thromboplastin time, prothrombin time, fibrinogen, d-dimer and c-reactive protein level. furthermore, studies showed that ischemic events, including venous thromboembolism, were present in 25-49% of patients with severe viral infection. statistics proved that patients with thrombotic complications have 5-fold augmented mortality. what is more, autopsy series on covid-19 non-survivors found not only macrovascular complications, but also microvascular thrombosis. small thrombi were found in over 80% samples of pulmonary vasculature. several groups reported also augmented rates of ischemic stroke in covid-19 patients admitted to hospital [7] . all those evidence indicate that sars-cov-2 may contribute to a number of vascular disorders, indicating the necessity for detailed patients diagnoses in order to avoid further complications that significantly reduce life quality. in this review, the potential mechanism and the effect of the sars-cov-2 viral infection on the development of ischemic stroke in covid-19 patients were carefully studied. thrombosis is a pathological process associated with the blood clots formation in circulatory system. thrombosis may occur within the venous and arterial system and contribute to various medical complications, including stroke, myocardial infarction or pulmonary embolism [8] . as mentioned above, many studies confirm the presence of thrombosis in patients diagnosed with covid-19. although studies do not implicate sars-cov-2 to have procoagulant effect itself, scientists more likely assess covid-19 coagulopathy with profound inflammatory response [9] . spreading the viral infection can contribute to the formation of many inflammatory foci in the human body in various places. the proliferation of the virus in the lungs causes diffuse interstitial and alveolar inflammatory exudation, which leads to edema and gas exchange disorders, resulting in hypoxia in the central nervous system (cns). thus increasing oxygen-free metabolism in the brain cells mitochondria [10, 11] . what is more, rapidly progressing inflammation, activation of the coagulation system and an imbalance between pro-and anti-coagulant properties may lead to the formation of disseminated intravascular coagulation (dic) syndrome. moreover, a systematic disorder characterized by a widespread activation of the hemostatic system leading to excessive blood clot formation in small vessels with simultaneous, massive consumption of blood platelets and coagulation factors, resulting in hemorrhagic complications are observed [12, 13] . the presence of dic was confirmed by the tang et al. study, in which most non-survivor covid-19 patients' (71.4%) blood tests showed prolonged prothrombin time and an increased d-dimer levels, which indicated the state after activation of the plasma coagulation system [14] . data from many studies showed a significant decrease in the platelet count, increased fibrinogen and d-dimer levels and prolonged prothrombin time, which was associated with severe covid-19 infections. thus indicating excessive activity of the coagulation system and the risk of dic development [10, [15] [16] [17] . ranucci et al. besides the augmented level of fibrinogen and d-dimer levels, also presented a significant increase of il6 and antithrombin levels, prolonged coagulation indicator-activated partial thromboplastin time (aptt) and elevated parameters of blood viscoelasticity [18] . coagulation changes were also proven by magro et al. in lung histopathological analysis and skin biopsies, which showed generalized microvascular thrombotic disorder [19] . furthermore, in a study conducted by carsana et al. a pulmonary autopsy showed that small arterial vessel fibrin thrombus was observed in 86.8% of examined, non-survived patients [20] . stroke is a medical condition caused by a deficit of blood flow in the brain causing neurological dysfunctions [21] . global epidemiologic reports ranked stroke as the second death cause globally, with a mortality rate of approximately 5.5 million per year. stroke survivors are at high risk of chronic disability leading to loss of their independence, work capacity, employment and material resources [22] . a sudden loss of neurological function is caused by infarction or cerebral vessels hemorrhage, the spinal cord or retina. clinically, patients mostly experienced unilateral weakness, ataxia, altered speech, numbness and/or visual loss. however, atypical symptoms like amnesia, dysphagia, dysarthria, anosognosia, headache and confusion may occur simultaneously [23] . the term "stroke" is not commonly used in clinical practice, because of its various etiology. the most common and generally diagnosed subtype of stroke is ischemic stroke, which constitutes 88% of all diagnosed cases. this subtype of stroke is caused by a partial or complete blockage of blood flow in the brain, which results in cerebral ischemia. a reduction in blood circulation to 16 ml/100 g of the brain tissue per minute may cause irreversible tissue damage within one hour. moreover, full occlusion and the absence of blood flow leads to the death of brain cells within 4 to 10 minutes [21] . most commonly, ischemia is caused by local vessel injury as an effect of atherosclerosis. the formation of plaque in the vessel lumen begins with damaged endothelium, ongoing inflammation and activation of the coagulation system. along with the increased severity of pathological processes, plaque forms become thicker and fibrous. in the final step, a clot that forms may partially or completely limit the blood flow in the vessels, or break free, forming an embolus, which is able to travel through vessels and block the blood flow further on [21] . a cerebral hemorrhage is the next subtype of stroke, caused by the rupture of a cerebral vessel, resulting in extravasation of blood within the brain [21] . generally, hemorrhagic stroke is a complication of hypertension, cerebral amyloid angiopathy, anticoagulation therapy and/or vascular structural lesions [23] . symptoms may vary between patients, depending on the anatomical site of the hemorrhage [21] . the major risk factors for the stroke development are: modifiable and include hypertension, atrial fibrillation and atrial cardiopathy, dyslipidemia, obesity, lack of physical activity, diet, untreated co-morbidities and inflammation, alcohol consumption and smoking. mostly, they contribute to the elevation of blood pressure and the progression of atherosclerosis. health improvement associated with the elimination of behavioral and medical risk factors can significantly reduce the risk of stroke. however, non-modifiable risk factors including age, sex, genetics and ethnicity can also increase the chance of stroke development [24] . identification of a stroke syndrome is usually easy to recognize because of visible neurologic deficits. however, symptoms differ among various regions of the brain and types of stroke. therefore, neuroimaging is a gold standard method for all stroke diagnostics. the vast majority of strokes may be recognized using fast acronym, which means facial droop, arm droop, speech disturbances and time. computed tomography (ct) is the first examination that can with almost 100% certainty confirm stroke and in over 95% accuracy assess the type of stroke. however, small-volume ischemia may not be detected in ct because of insufficient resolution. for higher resolution, magnetic resonance imagining (mri) is recommended. for all acute stroke syndromes, ct angiography is recommended due to the identification of ischemic area. the determination of occlusion and evaluation of extracranial vertebral and carotid, aortic arch and proximal great vessels is necessary for further management. although patients with acute coronary syndromes have helpful diagnostic biomarkers (i.e., serum troponin, electrocardiography), for stroke patients those tests are not available [25] . despite the available clinical studies evaluating the potential role of hemostatis biomarkers (i.e., von willebrand factor (vwf), p-selectin, fibrinogen, thrombomodulin, tissue factor, d-dimer, etc.) in ischemic stroke patients, the value of studied biomarkers is still unproven and requires further investigation [26] . ischemic stroke is a dynamic process that persists for more than 24 h. an ischemic cascade is activated rapidly after lack of blood flow in the brain, resulting in an ionic imbalance, excitotoxicity, blood-brain barrier dysfunction, generation of nitrosative and oxidative stress and inflammation ( figure 1 ). shortages in glucose and oxygen delivery, caused by the ischemic event, force the human body to use alternative biochemical pathways and substrates like glycogen, fatty acids or lactate. however, lack of oxygen leads to the reduction of adenosine triphosphate (atp) (inducing glycolytic metabolism), accumulation of lactate and protons and diminishment in intracellular ph. dysfunction in the activity of the electron transport chain in mitochondria causes a further reduction in atp concentration and disturbances in the functioning of ionic pumps. a loss of potassium ion concentration and an increase in sodium, chloride and calcium ion concentration leads to the depolarization of the cell membrane of astrocytes and neurons and to the secretion of neurotransmitters causing excitotoxicity [27] . during the excitotoxicity process, neuronal cells are exposed to a high amount of glutamate. the augmented concentration of glutamate may occur after neuronal depolarization, which is excessively released after neuronal depolarization. increased exposition of brain tissue to glutamate induces neuronal death, mitochondria failure and apoptosis. an influx of calcium ions causes degeneration of organelles and disrupts the integrity of cellular membrane [28] . removal of excess calcium ions is possible through atp-dependent mitochondria activity. however, this involves the production of reactive oxygen species (ros), thus inducing the peroxidation of lipids, activation of proteases, disruption of cell membrane integrity, dysfunction of mitochondria, stimulation of microglia and production of cytotoxic factors. during shortages of oxygen and glucose, mitochondria switches to anaerobic atp production, resulting in the formation of lactic acid and hydrogen ions, which provide a substrate for the conversion of superoxide anion into hydrogen peroxide or hydroxyl radical. along with nitrogen oxides, oxidative and nitrosative stress increase, thus enhancing brain tissue damage. ongoing ischemia and associated pathological processes cause necrotic cell death [27] , which induces the release of damaged-associated molecular patterns (damps), endogenous biomolecules responsible for the activation of the innate immune system from dead cells [29] . ischemic stroke also triggers the inflammation of the brain tissue as a result of oxidative and nitrosative stress and the formation of free radicals, hypoxia or necrotic cell death [27] . the inflammatory response to ischemia causes the rapid activation of microglial cells, which induce the infiltration of circulating inflammatory cells. ischemic cell damage generates and releases pro-inflammatory mediators and ros, thus promoting transendothelial migration of circulating leukocytes and inducing the expression of adhesion molecules in endothelial brain cells. within hours and days, mobilized leukocytes release chemokines, cytokines and ros, which enhance the inflammatory response in brain tissue [30] . circulating monocytes activated by cytokine storm and chemotactic factors roll from the central axis to the peripheral marginal bloodstream and bind with the endothelium surface. the rapidly repeating and overlapping processes of cytokine releasement, monocyte migration and its binding with endothelium cause excessive cell accumulation. trapped monocytes undergo a transformation process into macrophages, which intensively internalize and accumulate lipids, thus transforming into foam cells [31] . oxidized low-density lipoproteins inhibit a tethered macrophages chemotaxis, thus preventing them from leaving the endothelium and amplifying the accumulation [32] . the leukocytes sequential migration causes lymphocytopenia, which contributes to the increased risk of infection via immunodepression [28] . the ongoing pathological state results in the expression of pro-inflammatory genes and the augmented production of pro-inflammatory factors via the nf-κb pathway. intra-and extracellular signaling pathways trigger the interaction among brain tissue, endothelial cells, immune cells and hemostatic cells, thus stimulating the release of cytotoxic molecules like matrix metalloproteinases (mmps), which initially, causes the disruption of blood-brain barrier (bbb) permeability, nitric oxide, which constitute an independent source of reactive nitrogen species and damps, which enhance the cells mobilization and migration. disruption of bbb permits the infiltration of leukocytes, neurotoxic substances, cytokines, chemokines and pathogens to enter the brain tissue, exaggerating the infarct zone and resulting in the microvascular occlusion [27, 33] . figure 1 . the brain ischemia pathway. brain ischemia causes shortages in the oxygen supply, brain tissue necrosis and release of cytokines and chemokines that cause an inflammatory response. lack of oxygen causes the dysregulation of mitochondria and induces the anaerobic production of adenosine triphosphate (atp), which generates the reactive oxygen species (ros). disorders in the concentration of ions cause excitotoxicity, which results in cell damage and brain tissue necrosis. necrotic cells release damaged-associated molecular patterns (damps), which induce the activation of microglia, resulting in a massive release of cytokines and chemokines. pro-inflammatory factors mobilize leukocytes to migrate into the infarct zone enhancing the release of inflammatory response molecules. cerebral endothelium is stimulated to express the adhesion molecules on its surface and accumulate the cells, narrowing the vessel lumen and elevating the formation of atherosclerotic plaque. the ongoing mobilization of leukocytes results in the immunodeficiency caused by lymphocytopenia, thus increasing the risk of infection, which complicates the stroke by increasing the activation of the immune system and its interaction with endothelial and neural cells. neuronal damage caused by brain injury may be monitored by some brain markers including s100b protein and neuron-specific enolase (nse). s100b belongs to the ca 2+ binding protein family and is responsible for intracellular level of ca 2+ ions regulation. the concentration of s100b in cerebrospinal fluid and plasma is correlated with brain damage and disease severity. serum s100b levels are 40-fold decreased in comparison to cerebrospinal fluid level, however, serum protein is significantly easier and less invasive to collect and measure. several studies concluded that serum s100b level shows strong correlation with the volume of infarct and the size of neurological deficit [34] . nse is an isoenzyme of the enolase found in neuron's cytoplasm and is considered as neuronal damage biomarker. nse is present in peripheral blood serum in negligible concentration and its level rise during cell death. the study conducted by bharosay et al. has shown that nse serum level increases significantly due to cerebrovascular stroke (p < 0.001) and is correlated with score and disability degree [35] . both neuronal damage biomarkers have a potential to be use in the determination of the reason of brain damage (injury caused by sars-cov-2, or injury caused by stroke). however, there are currently no studies that describe this association. the contribution of viral infection in atherogenesis has been discussed for many years. studies showed that viral infection can be associated with endothelial dysfunction, the progression of atherosclerosis and future cardiovascular mortality. pathogens residing in the vascular wall induce the response of the immune system and the endothelium dysfunction, promoting the inhibition of vasodilatation, elevating the expression of pro-inflammatory factors and reactive oxygen species (ros), as well as contributing to the rupturing of plaque caused by mmp activity. unfavorable features of an ongoing pathological state of viral infection devastate the host organism and may contribute to severe complications of the initial pneumonia [32] . the formation of blood clots in the cerebral vessel as a complication of sars-cov-2 infection, has been reported in a significant number of research articles. in a study conducted by mao et al. of the 214 patients diagnosed with covid-19 who enrolled for their study, 78 had neurological disorders categorized into three categories: cns, which included headache, dizziness, impaired consciousness, ischemic stroke and cerebral hemorrhage; skeletal muscular injury defined as pain muscle or augmented level of serum creatine kinase (higher than 200 u/i); and peripheral nervous system (pns), which included smell, taste or vision impairment, and/or nerve pain. cns symptoms were the most relevant among all the neurological manifestations in patients. of 5 patients with diagnosed ischemic stroke, only one survived. the authors showed that patients with cns symptoms had lower platelet counts, lower lymphocyte levels and augmented blood urea nitrogen levels compared to patients without cns symptoms. what is more, patients with severe infections had augmented d-dimer levels [10] . similar results were conducted by beyrouti et al. where the clinical characteristics of six patients were presented. the first patient, a 64-year-old man diagnosed with covid-19 and exhibiting symptoms like cough, fever, breathlessness, myalgia and poor appetite was admitted to the intensive care unit due to respiratory failure. during hospitalization, the patient developed mild left upper limb weakness and incoordination. magnetic resonance imaging (mri) showed acute left posterior inferior cerebellar artery territory infarct with petechial hemorrhage and intradural left vertebral artery occlusion. moreover, the patient had markedly elevated d-dimer levels (>80,000 µg/l). the patient's deteriorating health revealed a bilateral pulmonary embolism and acute bilateral incoordination, high homonymous hemianopia and extensive acute posterior cerebral artery territory infarction diagnosed with mri. the second patient was a 53-year-old woman with valvular atrial fibrillation and confirmed covid-19 with cough, dyspnea, acute confusion, incoordination and drowsiness. a computed tomography (ct) scan showed acute large left cerebellar and right parieto-occipital infarcts. at the time of the stroke, there was an onset of symptoms: the patient had augmented d-dimer levels (7750 µg/l) and a prolonged prothrombin time with an international normalized ratio (inr) of 3.6. cardiorespiratory deterioration and disease severity contributed to the patient's decease. the third patient, an 85-year-old man diagnosed with covid-19 and risk factors like hypertension, atrial fibrillation and ischemic heart disease, developed a left posterior cerebral artery occlusion and infarction confirmed with a ct scan. the d-dimer levels were also highly increased (16,100 µg/l). the fourth patient, a 61-year-old man admitted to the hospital with hypertension, a high body mass index and previous stroke history at the time of the medical interview, had acute right striatal infarct detected by a brain mri, and markedly elevated d-dimer levels (27, 190 µg/l). during hospitalization, the patient developed respiratory symptoms with a pulmonary embolus confirmed with ct angiogram and was diagnosed with covid-19. the fifth patient, an 83-year-old man diagnosed with covid-19, diabetes, hypertension, smoking and alcohol consumption and ischemic heart disease, developed a thrombotic occlusion of proximal m2 branch of the right middle cerebral artery and infarct in the right insula. similarly to all patients, the d-dimer levels were augmented (19,450 µg/l). the final and sixth patient, a 70-year-old man with common covid-19 symptoms, was admitted to the hospital with dysphasia and right hemiparesis. an mri brain test confirmed bilateral p2 segment stenosis, thrombus in the basilar artery and multiple acute infarcts in the left pons, right thalamus, right cerebellar hemisphere and right occipital lobe. the d-dimer levels were 1080 µg/l and measured after intravenous thrombolysis. based on their observations, the authors suggest that ischemic stroke is a complication of covid-19, and may have distinct characteristics. however, the mechanisms of this disorder are not yet understood [16] . oxley et al. published a case report study, in which five patients younger than 50 years of age, diagnosed with covid-19, developed a large-vessel stroke. the first and second patients were a 33-year-old female and 37-year-old man, respectively, displayed no risk factors for stroke in their medical records. the female patient had mild covid-19 symptoms like cough, headache and chills. medical tests showed a partial infarction of the right middle cerebral artery with a partially occlusive thrombus in the right carotid artery at the cervical bifurcation, hemiplegia on the left side, dysarthria, sensory deficit, homonymous hemianopia and facial droop. the male patient, recently exposed to a sars-cov-2 infected family member, showed no symptoms of covid-19. however, medical tests confirmed ischemia in a left middle cerebral artery, and stroke symptoms such as sensory deficit, dysarthria, hemiplegia on the right side, reduced consciousness and dysphasia. other patients, a 39-year-old man, a 44-year-old man and a 49-year-old man were diagnosed with ischemia in a right posterior cerebral artery, left middle cerebral artery and right middle cerebral artery respectively. patients had a burden of medical records with risk factors like hypertension, hyperlipidemia, diabetes or previous mild stroke, with various covid-19 symptoms (from none symptoms to lethargy). the authors suggest that vascular endothelial dysfunction and coagulopathy are a complication of the ongoing covid-19 disease. furthermore, before the world pandemic, the same hospital in the same 2-week period admitted 0.73 patients on average, in comparison to five admitted patients during the pandemic [36] . the large disproportion in the number of patients admitted suggests that neurological manifestations, including ischemic stroke, are very serious complications of the ongoing sars-cov-2 virus infection and differential diagnoses should be implemented to hospitals to avoid delays in the diagnosis of concomitant complications. furthermore, the above-mentioned studies showed that patients with severe infections manifested neurologic symptoms more often. merkler 8%) were admitted because of cerebrovascular infarction. d-dimer levels and c-reactive protein were significantly augmented (with median values of 3913 ng/ml and 1011 ng/ml, respectively). the development of stroke with unknown etiology may be related with hypercoagulability caused by sars-cov-2 infection [40] . in the study performed by qin et al. the clinical characteristics and outcomes of covid-19 patients with and without history of stroke were evaluated. authors showed that patients with a history of stroke presented more comorbidities, more coagulation disorders and more aggressive inflammatory response. moreover, those patients had poorer outcomes and higher risk of severe events. patients with history of stroke had elevated number of neutrophils and interleukin 6 level, which may induce the cytokine storm and augmented, harmful immune system response. however, more severe course of the disease in patients with stroke history may not be associated with viral infection, but with enhanced risk factors and poorer health condition [41] . genetically, sars-cov-2 shows about 79% similarity with sars-cov, and about 50% similarity with the middle-eastern respiratory syndrome coronavirus (mers-cov). studies showed that this new coronavirus enters the human cells by binding to the angiotensin-converting enzyme 2 (ace2), such as sars-cov [42, 43] . the parallels between those two viruses are very important in laboratory diagnostics, medical treatment, spreading prevention and clinical characteristics, because since its discovery, the virus has proved itself to be extremely harmful and highly contagious. however, very few studies have yielded any conclusive explanations regarding the virus properties. ace2 is mainly expressed in the human airway epithelia, lung parenchyma, kidney cells, heart, testis, vascular endothelial cells, intestinal epithelial cells and brain [44] . hamming et al. carried out research based on immunohistochemistry testing on 15 different human tissue organs, localized ace2 in endothelial cells from arteries and veins in all the studied samples, including the brain [45] . these studies, published in 2004 and 2005, demonstrated that sars-cov was found in the brain samples of infected patients. interestingly, virus particles were found mostly in the neurons [46] [47] [48] . in order to find the means of virus neuroinvasion, netland et al. performed a study on transgenic mice, infected intranasally with sars-cov, and confirmed viral antigen distribution in the brain. thus suggesting that the virus can enter the brain via the olfactory nerve [49] . there are currently no similar studies that could confirm sars-cov-2 brain infection through the olfactory system, however, the similarity between these viruses may suggest that this new coronavirus may invade the brain in the same way. ace2 is a part of the renin-angiotensin system (ras), which is very important in the cardiovascular functions regulation, through the degradation of angiotensin ii to angiotensin [1] [2] [3] [4] [5] [6] [7] . experimental studies have shown that angiotensin ii induces myocardial hypertrophy, interstitial fibrosis, endothelial dysfunction, hypertension, vasoconstriction, oxidative stress, coagulation and enhances inflammation. the opposite role was shown in the case of angiotensin, which provides anti-inflammatory properties, thus reducing inflammation, fibrosis, migration and infiltration of cells. sars-cov-2 binding with the ace2 receptor leads to its down-regulation, increasing harmful and pathological state development in the host organism [50] . in the cns, angiotensin ii increases blood pressure and releases vasopressin. moreover, in ace2 knockout mice models, gene deletion was correlated with the augmented level of superoxides [51] . the severity and mortality of covid-19 are correlated with the body's immune response. in a study conducted by chen et al. most patients diagnosed with covid-19 had fewer lymphocytes and more c-reactive protein. furthermore, 52% of enrolled patients had an increased level of serum interleukin 6 (il6) [52] . huang et al. conducted a study, in which patients with severe infections, admitted to the intensive care unit, had elevated levels of plasma pro-inflammatory cytokines like il2, il10, il7, granulocyte colony-stimulating factor (gscf), interferon γ-induced protein (ip10), monocyte chemoattractant protein-1 (mcp1), macrophage inflammatory protein-1-α (mip1a) and tumor necrosis factor α (tnfα). what is more, the concentration of platelet-derived growth factor (pdgf), vascular endothelial growth factor (vegf), il1β, il8, il9 and interferon γ (ifn-γ) were elevated in all diagnosed patients [53] . in order to better understand all molecular processes ongoing during viral infection, the molecular mechanisms occurring in various cell types were described, maintaining the events chronology during the infection. a series of processes causing harmful body response to a viral infection leading to thromboembolic complications, begin with the endothelial cells. ace2 receptor located in endothelium allows the virus to connect and enter in the cells [44] . although the adhesion of leukocytes and blood platelets to endothelium is normally prevented, localized pro-inflammatory mediators (cytokines and chemokines), clotting cascade factors, growth factors and nitric oxide effect the reduced barrier integrity [54] . furthermore, experimental studies showed that tnf and il1β, which are released from endothelial cells during viral infection, are able to activate endothelial cells via nfκb pathway, which finally induces the new genes expression associated with the inflammatory response, i.e., adhesion molecules like vascular cell adhesion protein 1 (vcam-1) and intracellular adhesion molecule 1 (icam-1) [55] . furthermore, il1 and tnf have an ability to increase tissue factor (tf) and plasminogen activator inhibitor, increase the endothelium adhesivity for leukocytes and stimulate the secretion of pdgf. these effects tip the balance between pro-and anti-coagulant properties towards intravascular coagulation [56] . ongoing inflammation in the endothelium causes changes in vascular permeability and leads to the cells death [54] . release of damps from injured endothelial cells induces the migration of immune system cells, whose task is to eliminate the pathogen [29] . a very important role in the viral infection response is played by neutrophils, which are the first cell population that migrates to the damaged area. to eliminate the threat, neutrophils are equipped with various biological features, including chemokines, ros and proteases (i.e., mpps). however, all the invasive and aggressive mechanisms responsible for the pathogens elimination also work efficiently with host cells, which can cause damage to the inflamed tissue [29] . mobilization of macrophages, leukocytes and neutrophils (which constitute an innate immunity system) at the site of infection involves a massive release of cytokines and chemokines in damaged tissue. in the case of the brain tissue, mainly tnfα, il1β and il6 were found to be associated with ischemic stroke [48, 57] . cytokines and chemokines, which are released activate endothelium cell adhesion molecules that capture macrophages, leukocytes and neutrophils. the other pro-inflammatory molecule, ifn-γ, may increase the immune response by augmented infiltration of monocytes and lymphocyte into the damaged vessel. thus elevating the level of surface adhesion molecules and chemokines [58] . released il2 possesses an ability to induce t-cell proliferation (which constitute an adaptive immune system) and regulates their development, function and survival, and induces the differentiation of t-helper cells [59] . t-cell development is regulated also by released il7, which shown the properties to stimulate the recruitment and adhesion of macrophages and monocytes to endothelial cells, and upregulated mcp1 in the endothelium, which is responsible for the antiviral immune response, and the migration and infiltration of monocytes and t-cells [60, 61] . chemokines have a similar effect to mip1a and ip10 [62, 63] . migration of neutrophils and activation of mast cells are mainly provided by releasing il8 and il9, respectively [64, 65] . what is more, il9 may contribute to the augmented production of other pro-inflammatory cytokines in airways, resulting in its hyperresponsiveness [65] . to increase the immune response, gcsf stimulates the generation of the granulocytes, mainly neutrophils, and their release into the bloodstream. however, gcsf has also shown to inhibit the production of tnf and il8 in monocytes, macrophages and neutrophils, and to induce the expression of il10, anti-inflammatory cytokine, which enables the reduction of interaction between monocytes and endothelial cells resulting in decreased adhesiveness [66, 67] . strengthening the inflammation as a result of the body's response to infection is caused by the rapid production of il6, which is released by microglial, leukocytes, endothelial cells and astrocytes, and is responsible for the stimulation of production of c-reactive protein and fibrinogen. thus increasing the risk of a thrombotic event. furthermore, il6 may accelerate the migration of leukocytes as well as the production of adhesion molecules and chemokines. studies showed that il6 is associated with neurovascular dysfunction, neurodegeneration and inflammation of peripheral nerves [28] . as a result of cell death, released damps activate astro-and microglia, thus amplifying the mobilization of immune response cells [29] . the accumulation of immune cells in the vascular wall in response to the viral infection, especially among patients with ischemic risk factors, induces endothelial dysfunction, migration and proliferation of cells, activation of coagulation cascade and production of fibrous plaques. tf, which is activated by cytokines is the key initiator that triggers the coagulation cascade. blood platelets are the smallest nucleated blood morphotic elements, which are responsible for the maintaining a hemostasis process. in addition to that, platelets are the only cytoplasmic fragments of megakaryocytes, they are equipped with large number of receptors and biologically active compounds that interact with vascular microenvironment. under physiological conditions, platelets freely circulate in bloodstream without interacting with endothelium. this property is ensured by a glycoproteins layer and proteoglycans present between endothelium and blood, known as the glycocalyx [68] . however, inflammatory mediators released during viral infection, such as tnfα and lipopolysaccharide (lps), can cause degradation of the glycocalyx, thus regulating the permeability of endothelium. the injured endothelium expose tf, which triggers the coagulation cascade. firstly, tf binds with serine protease factor viia, which further activates factor x and factor ix, resulting in thrombin generation in the final [69] . the positive thrombin feedback brings a blood platelet activation. activated platelets change their shape in order to expose their adhesion receptors and to release granular content, pro-inflammatory cytokines and chemokines, and other activators (i.e., adp, vwf, thromboxane a2) that enhance thrombus formation [68] . simultaneously, during the progression of the coagulation cascade, factor xiia cleaves plasma prekallikrein to form the active serine protease plasma kallikrein that generates bradykinin. its binding to endothelium resulting in the induction of glial activation, enhancing an inflammation and neuronal death, which in turn enhances the secretion of damps [70] . activated blood platelets interact with leukocytes via glycoprotein p-selectin platelets and its ligand (p-selectin glycoprotein ligand-1; psgl-1) on leukocytes, and support their migration to inflamed endothelium [68] . adhesive molecules on endothelium (e-selectin, p-selectin, vcam-1) trap the rolling leukocytes [71] . during the accumulation of immune and hemostatic cells, thrombin generates the insoluble fibrin from fibrinogen [69] . furthermore, this effect is enhanced by cytokines that stimulate the plasminogen activator inhibitor-1 (pai-1), that reduces the fibrinolysis efficiency and effectivity [56] . the ongoing recruitment of platelets and successive infiltration of leukocytes, neutrophils and macrophages cause thickening of plaque that blocks the blood flow, resulting in the ischemic event ( figure 2) [21, 70] . severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infects human cells via the angiotensin-converting enzyme 2 (ace2) receptor. neutrophils migrate to the infected area in order to eliminate the pathogen. release of biologically active compounds (i.e., chemokines, reactive oxygen species-ros) stimulates the inflammation, causing mobilization of other immune system cells. dead cells from the injured zone release damaged-associated molecular patterns (damps), which activate microglia, inducing the migration of macrophages, leukocytes and neutrophils. the ongoing endothelial dysfunction activates the coagulation cascade via tissue factor (tf). generated thrombin stimulates blood platelets activation and shape change. thus exposing adhesion receptors and secreting granular content, enhancing inflammation and coagulation. activated platelets interact with leukocytes and support their migration to the damaged area. the interaction among endothelium, immune system and hemostatic cells enhances the ischemia and inflammation, simultaneously reducing the fibrinolysis effectivity and efficiency. pharmacological treatment of stroke patients must be matched with the stroke type. that is why a detailed medical interview and examination have to be performed before drug supplementation. ischemic stroke patients mostly received thrombolytic therapy. clinical trials showed that recombinant tissue plasminogen activator (rt-pa, also named alteplase) administered in maximal 4.5 h after onset of symptoms, significantly reduce hypoxia and improve patients outcomes [72] . according to the early management of patients with ischemic stroke guidelines, the combination of rt-pa with antiplatelet medicaments (excluding heparin, thrombin inhibitors, factor xa inhibitors and gpiib/iiia inhibitors) is recommended because of their beneficial character [73] . due to the national institutes of health guidelines, hospitalized adult patients should be administered with venous thromboembolic events (vte) prophylaxis, if hematologic and coagulation parameters indicate the possibility of thrombotic complications, or patients are at high risk of thromboembolic event. what is more, patients receiving antiplatelet and anticoagulant therapies before covid-19 diagnosis should continue the treatment. however, available data are insufficient to recommend the use of thrombolytics and anticoagulant drugs. in the case of sars-cov-2 infection, there is no antiviral agent for covid-19, however, several medicaments, including remdesivir, chloroquine, lopinavir, rotinavir and other hiv protease inhibitors, are evaluated as a potential antiviral drug (table 1) . administration and selection of anticoagulant or antiplatelet drug for covid-19 patients should be always considered to potential drug-drug interactions. for this reason, the university of liverpool collated a list of drug interactions for medical personnel [74] . table 1 . potential antiviral drugs under evaluation for the treatment of coronavirus disease 2019 (covid-19) [74] . intravenous prodrug responsible for inhibiting viral replication via binding to the viral rna polymerase. antimalarial drug, which inhibits the fusion of virus with host cell membranes. in vitro studies showed that both drugs may block the viral transport from endosomes to endolysosomes, thus regulating the releasement of viral genome. chloroquine has an ability to inhibits glycosylation of ace2 receptor, thus interfering the viral linkage. lopinavir/ritonavir lopinavir/ritonavir inhibits the activity of proteases responsible for replication of sars-cov-2. numerous studies showed that covid-19 may cause thromboembolic complications, which lead to many vascular disorders, including ischemic stroke. the rapidly growing number of case-reports demonstrates the need for more detailed medical examination of patients, especially those with severe infections. oxygen and nutrient shortages caused by a viral infection, along with the release of cytokines and chemokines, migration and influx of immune defense cells, their interactions with endothelium and accumulation in the damaged area, activation of the coagulation system and generation of thrombus result in many thromboembolic 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responses to arterial injury il-9: function, sources, and detection the multifaceted effects of granulocyte colony-stimulating factor in immunomodulation and potential roles in intestinal immune homeostasis effects of interleukin-10 on monocyte/endothelial cell adhesion and mmp-9/timp-1 secretion interaction between platelets and endothelium: from pathophysiology to new therapeutic options continuing education course #2: current understanding of hemostasis thromboinflammation in stroke brain damage the role of platelets in the recruitment of leukocytes during vascular disease pharmacological interventions and rehabilitation approach for enhancing brain self-repair and stroke recovery guidelines for the early management of patients with acute ischemic stroke: a guideline for healthcare professionals from the covid-19) treatment guidelines. national institutes of health this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-320882-cr0ccsnp authors: li volti, giovanni; caruso, massimo; polosa, riccardo title: smoking and sars-cov-2 disease (covid-19): dangerous liaisons or confusing relationships? date: 2020-05-02 journal: j clin med doi: 10.3390/jcm9051321 sha: doc_id: 320882 cord_uid: cr0ccsnp we read with great interest the article by brake sj and colleagues [...]. keywords: covid-19; sars-cov-2; smoking; angiotensin-converting enzyme-2 we read with great interest the article by brake sj and colleagues [1] investigating the relationship between smoking and angiotensin-converting enzyme-2 (ace-2) and the potential implication for covid-19. the authors present findings linking ace-2 expression to smoking in a variety of experimental models together with observations of their own; immunohistochemistry data showing an increased expression of ace-2 in a series of biopsies from a group of current smokers with chronic obstructive pulmonary disease when compared to a control group. the authors then venture into reporting existing chinese case reports to support their hypothesis that smoking could increase the risk of covid-19 via upregulation of ace-2 expression, a known cellular entry gateway for sars-cov-2 [2] . however, there are a number of problems with their hypothesis. first, the virus spike protein responsible for ace-2 binding requires its counterpart to be localized on the plasma membrane in order to be subsequently internalized [3, 4] . therefore, the mere total protein or gene expression is not conclusive to suggest a possible increased virus infection risk. second, it is known that ace-2 expression is down regulated on plasma membranes following sars-cov-2 infection because of successive internalization of ace-2-virus complex [5] . third, simple ace-2 expression on plasma membranes may be not a conclusive element in order to establish a potential risk factor for virus infection. in fact, once the spike protein is bound to ace-2, the cell is required to trigger a complex series of biochemical (i.e., activation of specific protease) and molecular signals in order to internalize the virus [3] . in addition, the interplay between covid-19 and the renin-angiotensin-aldosterone system is complex [6] . the view that overexpression of ace2 is detrimental does not take into account more recent evidence that up-regulation of ace2 may in fact be protective against disease severity [7] . experimental data suggest that infection with sars-cov and sars-cov-2 leads to down-regulation of ace2, and this downregulation is harmful due to uncontrolled ace and angiotensin ii activity [2, 7] . it has been observed that decreased ace2 availability contributes to lung injury and ards development [8, 9] . therefore, higher ace2 expression, while seemingly paradoxical, may protect against acute lung injury caused by covid-19 [10] . to the best of our knowledge, there are no experimental or clinical evidence establishing the potential impact of smoking on the above-described complex mechanisms, some of which remain still elusive. consistently, several recent clinical and demographical evidence further support the idea that the impact of smoking and risk of sars-cov-2 infection is still an open question and a matter of debate. in a recent systematic review of 13 chinese studies, smoking is vastly protective for hospitalized covid-19 and similar findings have been now noted in the us [11] . the centers for disease control and prevention (cdc) [12] report an unusually low prevalence of current smoking among covid-19 cases (1.3%) compared to the population smoking prevalence in the us (16.5%) [13] . a cross-sectional analysis of 4103 laboratory-confirmed covid-19 patients treated at academic hospitals in new york city demonstrated again a low smoking prevalence (5.2%) [14] . consistent with the findings of farsalinos et al. [11] and cdc [12] , the multivariate analysis performed by the new york researchers showed a significant protective effect against hospitalization for current and former tobacco use (or = 0.71, 95% ci 0.57-0.87 p = 0.001). moreover, smoking was not a risk factor for critical disease or death. finally, the authors stated that electronic cigarettes and "heat-not-burn" devices are not "safer" than cigarettes since they are still tobacco products producing vapor or smoke and therefore, similarly could cause infectious lung damage as we see with traditional cigarettes. such statements are highly inaccurate; uk and us health authorities have stated that combustion free tobacco products are less harmful than combustible cigarettes [15, 16] . last but not least, to date, no data or research on vaping and covid-19 is available. the assertions made by the authors on vaping and covid-19 are pure speculation. the complex interaction between smoking and raas/ace-2 poses multiple challenges for the researcher, the clinician and the covid-19 patient. the jury is still out, and the relationship between smoking and covid-19 should be carefully investigated. funding: this research received no external funding. in relation to his work in the area of tobacco control and respiratory diseases, riccardo polosa has received lecture fees and research funding from pfizer, inc., glaxosmithkline plc, cv therapeutics, neurosearch a/s, sandoz, msd, boehringer ingelheim, novartis, duska therapeutics, and forest laboratories. he has also served as a consultant for pfizer, inc., global health alliance for treatment of tobacco dependence, cv therapeutics, neurosearch a/s, boehringer ingelheim, duska therapeutics, forest laboratories, ecita (electronic cigarette industry trade association, in the uk), and health diplomat (consulting company that delivers solutions to global health problems with special emphasis on harm minimization). lecture fees from a number of european ec industry and trade associations (including fédération interprofessionnelle de la vape in france and federazione italiana esercenti svapo elettronico in italy) were directly donated to vaper advocacy no-profit organizations. he is currently head of the european technical committee for standardization on "requirements and test methods for emissions of electronic cigarettes" (cen/tc 437; wg4). he is also founder of the center of excellence for the acceleration of harm reduction at the university of catania (coehar), which has received a grant from the foundation for a smoke free world to support 8 independent investigator-initiated research projects on tobacco harm reduction, and scientific advisor for liaf, lega italiana anti fumo (italian acronym for italian anti-smoking league). giovanni li volti is full professor of biochemistry at the university of catania and the new director from 2020 of the coehar mentioned above. massimo caruso has no conflicts of interest to declare. smoking upregulates angiotensin-converting enzyme-2 receptor: a potential adhesion site for novel coronavirus sars-cov-2 (covid-19) angiotensin-converting enzyme 2 (ace2) as a sars-cov-2 receptor: molecular mechanisms and potential therapeutic target sars-cov-2 cell entry depends on ace2 and tmprss2 and is blocked by a clinically proven protease inhibitor ace2 receptor expression and severe acute respiratory syndrome coronavirus infection depend on differentiation of human airway epithelia differential downregulation of ace2 by the spike proteins of severe acute respiratory syndrome coronavirus and human coronavirus nl63 renin-angiotensin-aldosterone system inhibitors in patients with covid-19 a crucial role of angiotensin converting enzyme 2 (ace2) in sars coronavirus-induced lung injury replication-dependent downregulation of cellular angiotensin-converting enzyme 2 protein expression by human coronavirus nl63 angiotensin-converting enzyme 2 protects from severe acute lung failure angiotensin receptor blockers as tentative sars-cov-2 therapeutics smoking, vaping and hospitalization for covid-19 preliminary estimates of the prevalence of selected underlying health conditions among patients with coronavirus disease tobacco product use and cessation indicators among adults factors associated with hospitalization and critical illness among 4103 patients with covid-19 disease board on population health and public health practice; committee on the review of the health effects of electronic nicotine delivery systems. public health consequences of e-cigarettes key: cord-280111-6hiuzkvz authors: maspero, cinzia; abate, andrea; cavagnetto, davide; el morsi, mohamed; fama, andrea; farronato, marco title: available technologies, applications and benefits of teleorthodontics. a literature review and possible applications during the covid-19 pandemic date: 2020-06-17 journal: j clin med doi: 10.3390/jcm9061891 sha: doc_id: 280111 cord_uid: 6hiuzkvz background: covid-2019 spread rapidly throughout the world from china. this infection is highly contagiousness, has a high morbidity, and is capable of evolving into a potentially lethal form of interstitial pneumonia. numerous countries shut-down various activities that were considered “not essential.” dental treatment was in this category and, at the time of writing, only non-deferrable emergencies are still allowed in many countries. therefore, follow-up visits of ongoing active therapies (e.g., orthodontic treatment) must be handled taking special precautions. this literature review aims at reducing in-office appointments by providing an overview of the technologies available and their reliability in the long-distance monitoring of patients, i.e., teledentistry. methods: a literature review was made according to preferred reporting items for systematic reviews and meta-analyses protocols (prisma-p) guidelines. randomized clinical trials, cross sectional, observational, and case-control studies were evaluated with the mixed methods appraisal tool for quality assessment and study limitations. results: a primary search found 80 articles, 69/80 were excluded as non-relevant on the basis of: the abstract, title, study design, bias, and/or lack of relevance. twelve articles were included in the qualitative analysis. conclusions: teleorthodontics can manage most emergencies, reassuring and following patients remotely. the aim set by dental teleassistance was met as it reduced patients’ office visits whilst maintaining regular monitoring, without compromising the results. although our preliminary findings should be further investigated to objectively evaluate the efficacy, cost-effectiveness, and long-term results, we are confident that teleassistance in orthodontics will have a role to play in the near future. a new type of coronavirus initially named novel coronavirus pneumonia (ncp) and later renamed new corona virus 2019 (2019-ncov or covid-19) spread rapidly from china to the world from december 2019. it is the seventh coronavirus known to spillover to humans [1] . this viral infection is of great concern due to its high contagiousness and morbidity, as well as its ability to evolve into a potentially lethal form of interstitial pneumonia and its possible evolution into a potentially lethal form of interstitial pneumonia [2] . preventive hygiene measures such as social distancing, quarantine, and isolation have been taken to limit its diffusion in most countries to different extent [3] . on 30 january 2020, the world health organization (who) stated that covid-19 constituted a public health emergency of international relevance [4] . the national health committee keeps receiving an ever-increasing number of confirmed, suspected, and fatal cases reported from all over the world. to date, they are still carrying out world surveillance. there was an estimated human-to-human healthcare-related transmission of about 41% at the beginning of the outbreak [5] . many health care workers got and still are getting infected [5] . government and healthcare services have put on their thinking hats to re-organize triage services in an attempt to reduce nosocomial infection by covid 19 [5] . this task is particularly arduous as transmission is mainly through droplets and numerous subjects may be asymptomatic and/or in the incubation period. dental clinics belong to a high-risk category as infection can be facilitated during dental maneuvers that generate droplets, including restorative procedures, professional hygiene sessions, etc., or whilst patients are in the waiting room [6] . therefore, strict and effective hygiene protocols for infection control are urgently needed for dental practices to reduce dental practitioners' and patients' risk to get infected. the use of appropriate personal protective equipment (ppe) is pivotal in avoiding cross infection during clinical practice between patients and healthcare workers and the adoption of adequate decontamination measures can help to reduce the risks. although it has also currently been suggested that dental clinics limit their practice only to not deferrable emergencies, this is not always possible. some ongoing treatment such as orthodontic therapies and/or critical situations, like conditions that must be identified in the early stages and treated immediately to avoid more serious outcomes, require timely follow-up appointments. indeed, continuous monitoring by the orthodontist is a must in orthodontic treatment so as to evaluate the efficacy and/or any undesirable effects [7, 8] . however, some periodic visits are not strictly necessary and others could be delayed by instructing the patient how to make simple changes to the appliance, for example by indicating which teeth to put the intraoral elastics on or how many activations to perform on the central screw of a rapid palatal expander. at the time of writing, despite huge investments and research efforts, the current pandemic is still under investigation as are the best preventive measures to be adopted in individual fields. however, we are of the opinion that avoiding unnecessary follow-up appointments whilst maintaining the monitoring of treatment outcomes and current health status would be of great interest and importance for healthcare providers. recently, an innovative approach has been proposed in the medical field. although it was originally developed to provide healthcare services in remote areas, it may well be of use in managing healthcare services in this unprecedented emergency situation, i.e., telemedicine. the world health organization (who) defines telemedicine as the use of telecommunications and virtual technologies to provide healthcare outside of traditional healthcare facilities [9] . in more detail, telemedicine is a set of technologies, especially information and communication technologies (ict), specifically aimed at providing healthcare services from a distance to lessen the need for contact between the patient and the healthcare provider [10, 11] secure communication of medical information, notes, sounds, pictures, or any other form of data necessary are required to prevent, or to diagnose pathologies, and therefore, to treat and to monitor patients [9] . moreover, telemedicine is not only able to facilitate communication and interaction between the healthcare provider and the patient, but also between the providers themselves. indeed, it can, to a certain extent, remove geographical and temporal barriers, bridging gaps in the dishomogeneous distribution of the healthcare offer. therefore, it can provide care for more people, enabling them to benefit from healthcare services, especially those who live in remote areas and/or have poorly developed healthcare facilities. it can simplify online transmission of diagnostic tests and reduce waiting lists for consultations through an enhanced organization of appointments [12] [13] [14] . this makes these technologies a great resource in optimizing and reducing in-office visits and does not compromise necessary check-ups. treatment progress and efficacy can be monitored in this time of social distancing, which will most likely be prolonged into the year to come as the international scientific community has declared that a definitive cure and/or vaccine is not yet available as research is still ongoing. nowadays, telemedicine is becoming more and more widespread in the fields of oncology, cardiology, pediatrics, psychiatry, psychology, radiology, pneumology, dermatology, neurology, orthopedics, ophthalmology, and dentistry [15] . although teleassistance in dentistry is far from new, it seems that its advantages in orthodontics have not yet been fully explored and is used on a limited scale. indeed, there are some reviews on teledentistry in general but none on teleorthodontics as most articles about teleorthodontics are relatively new. as no reviews have yet been carried out on the efficacy of teleassistance in orthodontics as a way to manage patients at a distance, we would like to report on the evidence available as to the possibility of implementing new technologies in teleassistance, generally known by teleorthodontics to help during the covid-19 pandemic to remotely monitor patients' conditions. this topic is far from new, however, few studies have been reported, there is need for exploratory research for a better understanding. given the above, a non-systematic literature review was performed [16] . the electronic literature was searched using the following databases: medline, pubmed, embase, cochrane library, ebm reviews, web of science, ovid, and google scholar. the search was mainly based on five terms, i.e., teledentistry, teleorthodontics, virtual assistance, tele assistance, and telemedicine. embase and pubmed were searched respectively using also the terms embase subject headings (emtree) and medical subject heading (mesh). the endnote software reference manager (version x7 × 9.21, thomson reuters, released september 2014, toronto, on, canada) was used/adopted to store/archive and view/analyze retrieved references studies. the research refers to the preferred reporting items for systematic reviews and meta-analyses (prisma-p) 2015 [17, 18] . grey literature was also searched, but no data met the inclusion criteria. a hand search for relevant studies in the selected bibliography was also performed. studies involving new or already existing devices and software for teleassistance in orthodontics were included. service provided, type of intervention, clinical outcomes, efficacy and efficiency of assessed methods, and possible time saving compared to traditional methods were evaluated. the following study designs were included: observational studies, longitudinal studies, prospective studies, case-control studies, systematic and narrative reviews, and clinical trials. given the state of technology and its rapid evolution, the search was limited to papers published over the previous 15 years. studies in a language other than english or on application areas unrelated to orthodontics were excluded. articles with a poor methodology description lacking at least two of the following were excluded: study design, sample size, hardware utilized, software installed. letters to the editor, short communications, and all other publications not subjected to the peer review process were also excluded. considering the variety of study designs in the articles included, mixed methods appraisal tool (mmat) was used for quality assessment [17] . the score of each article was calculated by dividing the criteria that were considered satisfied by 4 (25% by 1 criterion, 100% if all 4 criteria were considered satisfied). the use of this system is compatible with a literature review that analyzes different research methodologies, as reported by whittemore [18] . two of the authors of this study (a.f and m.e.m) read the titles of the retrieved articles independently to ensure they met the eligibility criteria. if in doubt, the abstracts were read and the same method was applied. a final selection was then made by an independent evaluation of the full text of aforementioned papers before inclusion. any disagreement between the assessors was resolved by their discussing the full texts. the studies selected according to eligibility criteria are reported in the evidence table (table 1 ). one reviewer (a.f) extracted data from the full-texts and the other (m.e.m), independently verified the extracted data. data extraction included: journal and year of publication, study design, clinical outcomes, and the conclusions of the research. the description of the included studies is reported in table 1 . teleassistance in dentistry is a new and powerful tool that makes for effective communication between the care provider and patient and between the providers themselves. the study stated that these technologies can be of significant help in treating orthodontic emergencies. mmat score 75%. the sample is not representative of the target population. observations taken from this pilot study through the assessment of treatment planning and comparison between patients' opinion about traditional dental examinations (face to face) and teleorthodontics showed that the treatment planning was influenced by the diagnosis of the observer. however, the consultation system satisfied both the clinician and the patient. the study concluded that in-office assessment of a rapid maxillary expander can be successfully substituted with teleorthodontics (dm software). mmat score 50%. this paper presents a small sample size due to several dropouts. the sample was selected from a pool of patients attending a single hospital and, therefore, the conclusions cannot be extended to a private setting. teleorthodontics is a useful tool to manage emergencies, and monitor patients at a distance using whatsapp. mmat score 50%. the study is based on personal experience. no patients were analyzed, no results were evidenced due to the differences in the design of the selected studies, understandable given the heterogeneity of what was taken into account, it was not deemed fit to apply common methods for evaluation of the risk of bias. there was a low or absent overall risk of bias as to data description but high risk of bias for the efficacy analysis of such a technology. all considered papers had high or unknown selection bias and reference standards. moreover, as this is a novel topic, to the best of our knowledge, no validated protocol has yet been reported. due to the heterogeneity of study designs and the technological tools assessed, it was not possible to carry out a meta-analysis of the data. therefore, a thematic investigation was made, targeting the main topics that were analyzed in the selected papers. no other limitations appear to be present, as the review was carried out according to the prisma-p guidelines. initially 129 articles were found. the primary search retrieved 80 articles, net of elimination of duplicates (n = 49). a total of 31 articles were then deemed irrelevant after screening the abstract, the title, or the study design and were therefore excluded. twenty records were screened from the database and another 8 articles were excluded due to bias. twelve studies were read in extenso and were included in the qualitative analysis. the summary of the studies that met the inclusion criteria is shown in table 1 . the prisma flow chart reports the search methodology ( figure 1 ). five studies assessed the benefits of teleassistance in orthodontics for the management of patients at a distance. they all stated that teleorthodontics has the potential to provide significant and determinant help even if further investigation is deemed necessary [19] [20] [21] [22] [23] . five papers evaluated the efficacy and reliability of orthodontic teleassistance in the diagnosis treatment and follow-up of patients [24] [25] [26] [27] [28] . five studies assessed the benefits of teleassistance in orthodontics for the management of patients at a distance. they all stated that teleorthodontics has the potential to provide significant and determinant help even if further investigation is deemed necessary [19] [20] [21] [22] [23] . five papers evaluated the efficacy and reliability of orthodontic teleassistance in the diagnosis treatment and follow-up of patients [24] [25] [26] [27] [28] . one study endorsed the use of teleorthodontics for remote patient management [29] . one study evaluated which of the available it technologies would allegedly be used in the near future for remote patient management [30] . all the included studies agreed on the advantages of introducing teleorthodontics into clinical practice. taking into consideration the 11 included studies, one was not analyzed by mmat, as this system is not suitable for non-empirical studies such as reviews and theoretical papers [20] . a total of 11 studies were analyzed by mmat, nine of them were quantitative, two qualitative, and no studies used mixed methods. all the papers that were included were rated equal to or above 50% (average score 79.5%) according to the mixed method assessment tool and were therefore included. table 1 presents a detailed summary of each of the 11 studies included. one study endorsed the use of teleorthodontics for remote patient management [29] . one study evaluated which of the available it technologies would allegedly be used in the near future for remote patient management [30] . all the included studies agreed on the advantages of introducing teleorthodontics into clinical practice. taking into consideration the 11 included studies, one was not analyzed by mmat, as this system is not suitable for non-empirical studies such as reviews and theoretical papers [20] . a total of 11 studies were analyzed by mmat, nine of them were quantitative, two qualitative, and no studies used mixed methods. all the papers that were included were rated equal to or above 50% (average score 79.5%) according to the mixed method assessment tool and were therefore included. table 1 presents a detailed summary of each of the 11 studies included. currently, available technologies that can be used in teleorthodontics are: high-speed internet connection, digital videos and photographs, smartphones, and websites. a review by costa et al. [30] . emphasized that peer-to-peer communication services (msn, skype, etc.) can be helpful in patient management but that they are not sufficiently reliable by themselves, since they are products of big companies, they may be subjected to unpredictable changes. the authors of the aforementioned review thus recommend using websites instead, as they are easier to use and require no installation. in order to minimize problems involving safety, the same authors recommend using anti-virus and/or firewalls and adopt only sites with valid digital certification and end-to-end data encryption [31] . whatsapp messenger seems to be the most widely used communication tool according to available literature [29] . maintaining periodic virtual contacts, while it is impossible to do otherwise, is a valuable tool to build and maintain a positive patient-clinician relationship and a valuable therapeutic allegiance [29] . digital technology in imaging and impression taking, that is now commonplace in most dental practices, is a powerful tool for the orthodontist to access, analyze and, if need be, communicate with patients, colleagues, and/or dental technicians. the widespread diffusion of smart phones among doctors and patients led to the development of a new option. [32, 33] indeed, an application for smartphones that allows remote monitoring of orthodontic patients using an algorithm of artificial intelligence, has recently been developed. this application is called dental monitoring tm (dm) [34] . its purpose is to provide a precise record of the patient's occlusion with the integrated phone camera. dm was designed to carry out orthodontic follow-up at a distance. it tracks tooth movement through a 3d reconstruction of an intraoral movie taken with the smartphone camera and specific cheek retractors. the patients themselves make a video that is processed into a scan by dm tm . therefore, orthodontists can perform real-time monitoring of treatment outcomes anywhere and anytime. this smartphone application (android, ios) was originally designed to provide access to orthodontic treatment for people living in places with limited access, to improve comfort and fruibility of the service for people who have busy schedules or travel frequently for work. similarly, patients who are on orthodontic treatment during the covid 19 pandemic period can benefit tremendously from remote monitoring, avoiding unnecessary follow-up appointments. patient monitoring through this simple software may also improve treatment efficacy by avoiding late detections of problems such as debonded brackets, broken ligatures, non-tracking aligners, and are therefore able to solve the problem in the early stage [22] . a review on the benefits of teledentistry published in 2018, which considered only papers with high quality assessment scores, stated that not only is teledentistry potentially an effective tool for patient management, but it also has a positive economic impact on the dental profession. this review also pointed out that there is a rapid increase in the number of publications as to the efficacy of teledentistry, especially in oral medicine, periodontics, pediatric dentistry, and orthodontics [20] . however, due to the lack of conclusive evidence and the different methods (outcomes, assessment methods, main goal, etc.) they adopted, the findings cannot be generalized. other papers evaluate teleorthodontics as a means of performing initial examinations and report that there was no disagreement between in-office assessment and remote assessment through clinical photographs as to diagnosis and treatment planning [11, 21] . they demonstrated that teleorthodontics reduced costs and provided treatment access to a wider range of persons able to benefit from specialist treatment at a distance, without compromising the quality of care [11, 35, 36] . a study by favero et al. reported how new technologies applied to orthodontics allowed for remote management of several common orthodontic questions that would have otherwise necessitated in-office treatment: e.g., ligature displacement, discomfort from the appliance, cheek irritation [21] . a preliminary study by hansa et al. [22] . evaluated whether the use of remote monitoring, carried out with dm tm software, is able to reduce the number of in-office visits compared to the traditional appointment management. the same study used a questionnaire to assessed patients' attitude towards the use of a remote monitoring software during treatment the patients who had remote monitoring had fewer in-office appointments: 1.68 in average during the 7-month follow-up taken into consideration. this means that over a 2-year treatment period, an average of 5.8 in-office appointments could be avoided by the use of dm software. most patients classified the application as user-friendly (easy or very easy) (86%) and (84%) thought it was useful for their treatment. the questionnaire revealed that most patients using dm had the sensation of enhanced communication with their dentist and better convenience. however, this study [22] provides only preliminary results and, as do other studies, suggests that if the whole treatment period were to be considered instead of just 7 months, more precise information on the effects of teleorthodontics could be obtained. some studies analyzed the benefits of teleassistance in orthodontics and reported their utility in periodic check-ups for those in retention to make an early identification of problems and immediately book in-office appointments, thus maintaining a good doctor-patient relationship and a good level of surveillance over finished cases, without taking up the dentists' and patients' time unnecessarily [26] . several studies have described teleorthodontics as an effective tool that allows the orthodontist to maintain treatment control in situations where the patient cannot go to the clinic [37] . these results are in agreement with data reported by berndt et al. [19] . the authors provided evidence of the viability of teleorthodontics during interceptive treatment. other studies have shown that the use of new patient monitoring technologies has enabled dental professionals to enhance the quality of treatment provided to their patients, as reported by mandall and stephens [23, 38] . these authors stated that teledentistry is an effective way to identify appropriate referrals and that teledentistry may well increase treatment efficacy [20] . dunbar et al. compared the reproducibility of treatment planning performed on digital records, clinical examinations, and standard records. the paper also considered patients' opinion of in-office visits and teleassistance [25] . it showed that that 50% of the observers were influenced by the type of records used to decide which treatment was more appropriate. the agreement between doctors was higher on standard records than on digital ones. the authors of this study concluded that it is possible to save money, time, and avoid the need to go to the dental office for a consultation [25] . bradley et al. also made favorable comments about this system [28] . the attitudes toward teleassistance in orthodontics, and in general, dentistry by respective dental care professionals, was investigated in several studies which confirmed it was as an effective alternative to in-office visits for several routine procedures and to make consultations more accessible to dentists and patients [23, 38] . mandall concluded that teleassistance in dentistry is a reliable tool, enabling the screening of new patients and therefore, that it was of substantial help in lowering incorrect referral rates and reduced the waiting list for fist consultations [23] . morris et al. [24] stated that three dimensional impressions taken with dental monitoring software do not differ greatly to those taken with an intraoral scanner (iteroelement, align technology, santa clara, ca, usa). there was a clinically insignificant mean difference of 0.02 mm between the digital models generated with dental monitoring and intraoral scans, suggesting and therefore judging it clinically insignificant [24] . heather et al. [26] evaluated the reliability and accuracy of dm. they assessed intercanine and intermolar distances during rapid maxillary expansion (rme) on dm scans and on digital models taken at in-office follow-up appointments. the paper reported a slightly higher margin of error for dm scans compared to digital model at the molar level. however, in-office and dm measurements differed by less than 0.5 mm. therefore, the author of this study concluded that, as long as dm scans are of acceptable quality, they can be reliable in the formulation of clinical decisions. the reliability of dm in the evaluation of rapid palatal expansion treatment, compliance and satisfaction were also studied by kuriakose et al. [27] , who was in agreement with the aforementioned claims. that is, dm was able to make a remote assessment of the condition of posterior crossbite. no significant difference was noted in intermolar width between dm, digital model, or intraoral examination. on the basis of these data, it seems that in-office control of maxillary expansion can be substituted by teleassistance with dm software [27] . bernd et al. [19] stated that facial orthopedic treatments can be delivered by sufficiently trained general dentists through remote supervision of an orthodontist using teleassistance technology. this may well make a significant improvement in conditions of malocclusion in children, who for various reasons, cannot be treated in-office by an orthodontist. even if most patients treated with phase i orthodontics usually require a phase ii treatment cycle, malocclusion is far less complex and is, therefore, easily managed. the ever more powerful capacity of modern computers has led to a continuous development of innovative technologies. indeed, currently, various branches of medicine and dentistry are benefiting from the advances provided by new technologies for the diagnosis and treatment of several pathologies. teledentistry is the part of telemedicine that deals with the application of ict to dental care. moreover, teledentistry and dental video phoning allow colleagues to readily exchange information. it can also become a cutting-edge screening system able to reduce patients' waiting time for specialist advice. as long as it is correctly set up, it is capable of improving service and working conditions and may even reduce costs [21] . teleorthodontics generally refers to any orthodontic care delivered through information technology. a common and relevant example could be that of colleagues being able to discuss the digital records of clinical cases over the internet and to exchange advice and share experience. the first studies on teleorthodontics date back to the early 2000s [23, 38] . a remarkable example that yielded promising results was a paper investigating the possibility to deliver orthodontic treatments through the remote real-time supervision of an orthodontic specialist for general dentists so as to reach patients with limited access to orthodontic care [19, 38] . another useful application is remote retention check-up by sending images rather than physically going to the dentist [22] . however, most likely, we shall have to wait for yet another decade before teleorthodontics becomes a viable option as technological and cultural obstacles still have to be overcome. in recent years, the number of patients who wish to undergo orthodontic treatment requiring fewer in-office visits, while at the same time allowing the specialist to maintain control over the progress of their treatment, has grown. teleorthodontics as a mean to further reduce unnecessary journeys to the orthodontic practice while maintaining control over treatment is allegedly one of the main reasons teleorthodontics has gained ground over the past few years. the development of clear aligners and lingual custom prescription brackets with robotic multi-wires has significantly reduced chair-side time and in-office visits [39, 40] . as a rule, aligners or wires are changed during in-office visits at pre-established appointments that have been made on the basis of personal experience and common knowledge of an approximated time span for the wire to have exhausted its biological efficacy. however, a one size fits all approach is not always ideal, as average values do not take into account a patient's individual biological response. teleorthodontics allows for tailor-made scheduled in-office visits though remote monitoring, promoting a more productive workflow. these procedures are capable of reducing chair time and improving patient convenience. as reported in the results of our review, it appears that the most promising technology for teleassistance in orthodontics is dental monitoring tm (dm). this is based on three integrated platforms: a smartphone application that takes the patient through correct record taking; a software that adopts an algorithm that quantifies individual tooth movements (less than 0.5 • for mesiodistal angulation and faciolingual inclination, and rotation) and an internet-based interface where the dentist can check patients' updates as soon as they are uploaded and interact with the patient. alerts can be set at certain thresholds to receive warnings should an emergency condition arise, e.g., debonded brackets, gingival issues depending on poor hygiene, non-tracking aligners, and so forth and/or for specific treatment objectives. all images recorded by the software are available on the clinician's platform. physicians can take advantage of in-office photos (baseline and interim photos) as reference to better understand changes. the software allows four possible monitoring levels, i.e., the number of photos per period of time. routine pre-treatment monitoring requires one picture every couple of months. the monitoring of active treatment requires one or two photos per week (for aligners and the other therapies respectively). the monitoring of the retaining phase has a more complex picture timing scheme: once weekly for a month, then once monthly for six months, followed by once every couple of months. the last possibility is known as dm go live, which is for use in aligner therapy, where pictures are taken once weekly and the patient is informed whether to keep the same aligner or to proceed to the next one. although expectations are promising, teleassistance in orthodontics could have some limitations. forwarding scans each 7 days may become a nuisance and frustrating for the patient as they may sometimes need to be taken again. moreover, the reduction in the time spent visiting the patient in person may deteriorate the patient-doctor relationship. therefore, consent and education are needed for the patient to begin a similar path in order to build a positive and strong therapeutic allegiance [22] . during the pandemic outbreak, orthodontists had to significantly reduce, and in certain cases, suspend follow-up visits of patients currently under active treatments. we can therefore say that the use of applications for monitoring orthodontic therapy could be an effective solution to continue to keep deferrable orthodontic patients under control during the closure of dental practice due to covid 19 and to reduce unnecessary in-office appointments. the italian society of orthodontics (sido) has recently published the recommended guidelines on the management of orthodontic patients during the covid-19 outbreak. orthodontic emergencies are unpredictable issues caused by orthodontic appliances that provoke pain or discomfort, thus requiring urgent dental care [29] . orthodontic emergencies should be faced using a stepwise approach. the first recommended approach should be virtual assistance through photographic documentation or a video call. it is important to perform a preliminary triage to distinguish situations that require in-office treatment rather than those that are remotely manageable. unlike other dental questions, orthodontic problems like traumatic injuries of teeth and periodontal structures, abscesses, etc., have a lower degree of severity and often do not necessitate in-office care to be solved. the most common orthodontic emergencies are related to the detachment of one or a few brackets and acute stinging of the lips and the oral mucosa caused by orthodontic wire or scraping brackets. many of these problems can be readily solved at home with less stress for patients' families, saving time for both patients and dentists alike. since they are not true emergencies, they can, more often than not, be easily resolved by providing the patient with simple instructions during a video call or with typed messages after photographic documentation of the problem, describing the intraoral condition. it has been suggested that dental caregivers become familiar with the potential social networks and modern web-based communication platforms have, thanks to the possibility of making a precise evaluation of indications and contraindications [41, 42] . patients should continue ongoing therapies, but should also be video checked periodically. dental professionals and their team must select the number of eligible patients and organize this procedure [29] . in all other cases, it is advisable to contact each individual patient in therapy actively in order to give specific indications and it is recommended to make telephone appointments with patients 4-6 weeks apart to carry out a further check-up or fix an appointment in the studio, if strictly necessary [29] . patients must be reassured and periodically checked, in particular if they have discomfort or problems related to their orthodontic appliance. it is important to emphasize that not only must emergencies be managed by the orthodontist, but also all other patients with both mobile and fixed appliances. teleorthodontics relies on information technology and telecommunications and allows for various types of orthodontic follow-up visits at a distance. therefore, it is of fundamental importance to be able to avail oneself of teleorthodontics for the constant monitoring of all patients should the orthodontic practice be shut-down and/or visits significantly limited. the professional, thanks to special devices, can check a patient's real situation remotely and compare it with the digital setups previously made, especially in the case of treatments with lingual orthodontics and aligners [26] . based on the revised articles, new information technology improved the management of orthodontic patients, and in numerous cases, allowed for their remote management. teleorthodontics has the potential to improve patient management and reduce treatment costs. this review does have some limitations. one such limitation is the fact that the studies included had only a fair scoring in mmat. moreover, although most papers reported a positive attitude towards teleorthodontics, a publication bias may be present since all papers reported one or more positive outcomes for accuracy and/or efficacy and some papers are technical reports or pilot studies. only a limited number of papers made a controlled comparison of teleassistance in orthodontics with traditional methods. many included studies focused on the evaluation of efficacy rather than the effectiveness of teleorthodontics. it would be of great importance to evaluate the appropriateness of teleassistance in orthodontics by assessing clinical outcomes and costs/template details the sections that can be used in a manuscript. this review found a growing number of studies sustaining the efficacy of teleassistance in orthodontics. the advent of a large number of technological innovations over the past few years in dental and in orthodontic practices has allowed for substantial improvement. the covid-19 pandemic will surely have long-term effects on patient management as it seems unlikely there will be any definitive treatment or vaccines available in the near future. this condition will require a different organization of dental appointments for several months to come and remote patient management could be efficiently carried out using instant messaging platforms to deliver healthcare consultancies. we believe that teleassistance in orthodontics should be considered a welcome resource, as it is able to successfully manage many dental emergencies, to reassure and follow patients at a distance without exposing them and/or dental practitioners to unnecessary risks. moreover, most case issues can be promptly solved without the patients coming to the orthodontist office by communicating with photos and/or videos, saving both the patient's and clinician's valuable time. the aim of teleorthodontics is fulfilled by reducing unnecessary follow-up visits while maintaining regular monitoring, thus not jeopardizing expected results. the potential of teleorthodontics is virtually endless; remote consultations could be carried out across the globe without the obstacles of distances or of scheduling appointments. this kind of approach could be of great help in the management of all dentofacial orthopedic removable appliances and of orthodontic treatments that need little in-office maintenance, such as some clear aligner therapies. even though in-office visits are still required for many dental and orthodontic procedures, teleorthodontics opens up open new horizons in the treatment and follow-up of many patients. nevertheless, currently, most studies report only pilot studies and evaluate short-term results of teleassistance in orthodontics. therefore, there is limited evidence and the study designs differ, as do the interventions and endpoints assessed in the papers included, meaning that our findings cannot be strictly generalized. however, although we are of the opinion that further studies with higher levels of evidence are needed to objectively evaluate efficacy, cost-effectiveness, and long-term results, we are confident that teleassistance in orthodontics will have a role to play in the future. technological advances have substantially changed dentistry. in orthodontics, software like dental monitoring, which is capable of providing web-based platforms for sharing health data [43] between patients and doctors, may allow orthodontists to closely monitor their patients' status, reducing in-office visits and delivering more patient-centered treatment. the aim of teleassistance in orthodontics is to reduce unnecessary in-office visits and improving monitoring and early treatment of problems that may jeopardize the desired final outcome. teleassistance in orthodontics may allow for remote consultations that can be carried out wherever without the need for the patient to be anywhere near the office. the downside of teleorthodontics is the reduced time to develop and maintain a positive relationship between doctor and patient [44, 45] . dunbar [25] reported in a feasibility study that 70% of patients thought that in-office consultation was extremely important, and most patients preferred it to teleorthodontics assistance. another important consideration are the points of law regarding patient confidentiality that may be in danger because of digital communication of sensible data over the internet [46] . the quality of the doctor-patient relationship is particularly important should the treatment have complications or if outcomes are deemed unacceptable. malpractice lawsuits may increase if patients feel they are not receiving treatment of a satisfying quality. moreover, teleassistance in orthodontics and patients' confidentiality issues are more complicated in orthodontics because patients are often minors. therefore, the teleorthodontics we are implementing during the covid 19 pandemic should not be seen in the future as a new treatment option for patients looking for cheaper and aesthetic alternatives to traditional orthodontics, as dental and orthodontic care should not be reduced to a simple question of "commodity" [47] . as already reported by hansa et al. [22] , orthodontists are still a little doubtful about treatment at a distance because of the possibility of limiting their patient base and due to the risk patients may face. it is the authors' opinion that, in the future, teleassistance in orthodontics will be helpful to maintain high standards of care while reducing unnecessary in-office visits in order to improve rather than reduce the quality of the service already provided by conventional orthodontics. a pneumonia outbreak associated with a new coronavirus of probable bat origin a novel coronavirus from patients with pneumonia in china the novel coronavirus originating in wuhan, china: challenges for global health governance china coronavirus: who declares international emergency as death toll exceeds 200 clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in wuhan, china dentistry and coronavirus (covid-19)-moral decision-making treatment of dental dilacerations comparison of the dental and skeletal effects of two different rapid palatal expansion appliances for the correction of the maxillary asymmetric transverse discrepancies opportunities and developments in member states: report on the second global survey on ehealth 2009 telemedicine: a new horizon in public health in india applications of teledentistry: a literature review and update telemedicina linee di indirizzo nazionali addressing health disparities in rural communities using telehealth telemedicine: a guide to assessing telecommunications for health care chapter 8: executive summary prática clínica baseada em evidências na área da saúde proposal: a mixed methods appraisal tool for systematic mixed studies reviews the integrative review: updated methodology using teledentistry to provide interceptive orthodontic services to disadvantaged children a systematic review of the research evidence for the benefits of teledentistry communication through telemedicine: home teleassistance in orthodontics tele-orthodontics": concept, scope and applications teledentistry for screening new patient orthodontic referrals. part 1: a randomised controlled trial accuracy of dental monitoring 3d digital dental models using photograph and video mode the influence of using digital diagnostic information on orthodontic treatment planning-a pilot study accuracy of a smartphone-based orthodontic treatment-monitoring application: a pilot study the assessment of rapid palatal expansion using a remote monitoring software profiling the interest of general dental practitioners in west yorkshire in using teledentistry to obtain advice from orthodontic consultants management of orthodontic emergencies during 2019-ncov teleortodontia: ferramenta de auxílio à prática clínica e à educação continuada implementation and evaluation of a low-cost telemedicine station in the remote ecuadorian rainforest the world of orthodontic apps. apos trends orthod up in the air: orthodontic technology unplugged! apos trends orthod dental care: virtual consultations and remote monitoring a systematic review of clinical outcomes, utilization and costs remote patient monitoring via non-invasive digital technologies: a systematic review do-it-yourself orthodontics, and remote treatment monitoring attitudes of uk consultants to teledentistry as a means of providing orthodontic advice to dental practitioners and their patients effectiveness and efficiency of a cad/cam orthodontic bracket system efficacy of clear aligners in controlling orthodontic tooth movement: a systematic review considerazioni sull'uso di whatsapp nella comunicazione e relazione medico-paziente whatsapp in clinical practice: a literature review transverse dental and dental arch depth dimensions in the mixed dentition in a skeletal sample from the 14th to the 19th century and norwegian children and norwegian sami children of today everything in its place! " apos trends orthod showing you care: an empathetic approach to doctor-patient communication being as good as your dumbest competitor? this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors thank barbara wade, contract professor at the university of torino, for her linguistic advice. the authors declare no conflict of interest. key: cord-318211-hhp84ygq authors: ticconi, carlo; pietropolli, adalgisa; specchia, monia; nicastri, elena; chiaramonte, carlo; piccione, emilio; scambia, giovanni; di simone, nicoletta title: pregnancy-related complications in women with recurrent pregnancy loss: a prospective cohort study date: 2020-09-01 journal: j clin med doi: 10.3390/jcm9092833 sha: doc_id: 318211 cord_uid: hhp84ygq the aim of this prospective cohort study was to determine whether women with recurrent pregnancy loss (rpl) have an increased risk of pregnancy complications compared to normal pregnant women. a total of 1092 singleton pregnancies were followed, 431 in women with rpl and 661 in normal healthy women. the prevalence of the following complications was observed: threatened miscarriage, miscarriage, cervical insufficiency, chromosomal/genetic abnormalities, fetal anomalies, oligohydramnios, polyhydramnios, fetal growth restriction, intrauterine fetal death, gestational diabetes mellitus (gdm), preeclampsia, placenta previa, abruptio placentae, pregnancy-related liver disorders, and preterm premature rupture of the membranes. the odds ratio and 95% ci for each pregnancy complication considered were determined by comparing women with rpl and normal healthy women. women with rpl had an overall rate of pregnancy complications higher than normal women (or = 4.37; 95% ci: 3.353–5.714; p < 0.0001). their risk was increased for nearly all the conditions considered. they also had an increased risk of multiple concomitant pregnancy complications (or = 4.64; 95% ci: 3.10–6.94, p < 0.0001). considering only women with rpl, women with ≥3 losses had a higher risk of pregnancy complications than women with two losses (or = 1.269; 95% ci: 1.112–2.386, p < 0.02). no differences were found in the overall risk of pregnancy complications according to the type, explained or unexplained, of rpl. women with secondary rpl had an increased risk of gdm than women with primary rpl. pregnancy in women with rpl should be considered at high risk. recurrent pregnancy loss (rpl), defined as the spontaneous loss of two or more pregnancies (according to the american society for reproductive medicine [1] ) or the loss of two or more pregnancies before the 24th week of gestation (according to the european society of human reproduction and embryology [2]), presents several still incompletely defined aspects. among these is the outcome of the successive pregnancy in women with a history of rpl. indeed, there is considerable discrepancy between the reported birth rates and the rates of gestational complications of the successive pregnancy in women with rpl. the likelihood of a live birth in the successive pregnancy in untreated women with rpl has been reported to range 42-86% after three miscarriages and decreases with increasing the number of pregnancy losses, reaching only 23-51% after ≥5 losses [3] . this observation suggests that the number of miscarriages-a likely indicator of the gravity of the condition-is a major determinant of the reproductive success of women with rpl; in fact, it has been reported that the live birth rates in the successive pregnancy in women with two consecutive losses is around 75% [4, 5] . on the other hand, several studies and reviews investigating the outcome of the successive pregnancy in women with rpl found that it was burdened by many obstetric and perinatal complications which occurred more frequently than in normal control women without rpl [6] [7] [8] [9] , even though this finding has not been observed in all studies [10] . therefore, it is difficult for the clinicians to ensure a clear counseling, in terms of prognosis, to women with rpl about the subsequent pregnancy once the diagnostic workup has been completed. whereas the outcome of the subsequent pregnancy in women with rpl in terms of live birth, labor, and perinatal complications is well established [3, 6, 8] , less information is available on the obstetric risks that can occur in these women during their pregnancy before labor. moreover, limited information is available concerning the gestational complications in women with rpl with regard to their specific features (primary/secondary or explained/unexplained rpl). the present study was carried out to investigate the outcome of the first pregnancy in women with rpl after their referral compared with healthy pregnant women without rpl, with specific application to the gestational complications and to the particular characteristics of these women. further information on this issue could allow a more comprehensive counseling of women with rpl and could help to better clarify whether these women actually need a more thorough monitoring throughout their pregnancy. this prospective, observational, study has been carried out to investigate the occurrence rates of major gestational complications in a cohort of women with rpl compared to normal healthy women without rpl followed during their first subsequent pregnancy after referral. the study subjects were enrolled from 1 january 2017 to 31 january 2020. overall 1782 women were initially enrolled in the study. they attended as outpatients the gynecology and obstetrics unit of the policlinico tor vergata university hospital or the università cattolica of the sacred hearth at the policlinico gemelli hospital of rome, italy. in both hospitals, the women with rpl were followed at the rpl units, whereas the control women were followed throughout their pregnancy in the low-risk obstetric clinics. the study subjects were divided into two groups: group 1, rpl (n = 1030): non-pregnant women with rpl, enrolled at their first visit carried out to investigate the possible causes/risk factors of the rpl. the women of this group who entered in pregnancy were followed during their gestation. group 2, control (n = 752): pregnant normal women with low risk pregnancy who had at least one uncomplicated pregnancy at term with any previous pregnancy loss. they were followed throughout their gestation. all the control women have had at least two pregnancies at term without any losses and were selected consecutively. of the initial population of enrolled women, 139 women (13.5%) with rpl were lost at follow-up without any information on whether they get pregnant. of the remaining 891 women followed-up, 511 (57.3%) become pregnant; of these women, 70 (13.7%) were lost at the successive follow-up while pregnant, whereas 431 (84.3%) were followed-up during their pregnancy. ninety-one women (12.1%) of the control group were lost at follow-up during their pregnancy, while 661 women (87.9%) were followed up. therefore, the final number of pregnant women included in the study was 1092. these numbers have been reported for clarity in figure 1 . j. clin. med. 2020, 9, x for peer review 3 of 12 up, 511 (57.3%) become pregnant; of these women, 70 (13.7%) were lost at the successive follow-up while pregnant, whereas 431 (84.3%) were followed-up during their pregnancy. ninety-one women (12.1%) of the control group were lost at follow-up during their pregnancy, while 661 women (87.9%) were followed up. therefore, the final number of pregnant women included in the study was 1092. these numbers have been reported for clarity in figure 1 . the final number of 1092 pregnant women to be included was calculated by taking into account the following conditions: (a) the null hypothesis (cases and controls have the same pregnancy complications) is refused if the difference between the means of the cases and of the controls (size effect) is >13.8% of the jointed variance of the two distributions; (b) the verification test is two-tailed student's t-test in which α = 0.05 and β = 0.10, with a power = 90%. with the above conditions, the overall number of women to be included (cases and controls) is no less than 1092. the women with rpl were investigated according to a standardized diagnostic protocol already reported in detail [11, 12] which included the collection of general and obstetrical history, gynecologic examination with a pelvic ultrasound scan, karyotype of both partners, hysteroscopy, hormonal profile, autoantibodies panel, metabolic evaluation, and screening for coagulation and thrombophilic disorders. the diagnostic workup was aimed to identify defined and probable causes of rpl. the women with rpl with treatable causes (medical and/or surgical) were treated according to the the final number of 1092 pregnant women to be included was calculated by taking into account the following conditions: (a) the null hypothesis (cases and controls have the same pregnancy complications) is refused if the difference between the means of the cases and of the controls (size effect) is ≥13.8% of the jointed variance of the two distributions; (b) the verification test is two-tailed student's t-test in which α = 0.05 and β = 0.10, with a power = 90%. with the above conditions, the overall number of women to be included (cases and controls) is no less than 1092. the women with rpl were investigated according to a standardized diagnostic protocol already reported in detail [11, 12] which included the collection of general and obstetrical history, gynecologic examination with a pelvic ultrasound scan, karyotype of both partners, hysteroscopy, hormonal profile, autoantibodies panel, metabolic evaluation, and screening for coagulation and thrombophilic disorders. the diagnostic workup was aimed to identify defined and probable causes of rpl. the women with rpl with treatable causes (medical and/or surgical) were treated according to the european society of human reproduction and embryology (eshre) guidelines [2] . treatments in women who were enrolled before the publication of these guidelines were changed accordingly. the women in the control group were followed throughout the pregnancy until term in the low-risk pregnancy unit according to the standardized protocol used in our units, which complies with the national institute for health and clinical excellence (nice) clinical guidelines [13] . the present study was carried out in accordance with the declaration of helsinki, modified tokyo 2004, and was approved by the institutional review board (irb) of policlinico tor vergata university hospital (protocol number: 230/19). all women gave their informed written consent to the study. rpl was defined according to the eshre 2017 guidelines [2]. rpl was defined as unexplained when no definite cause could be found at the end of the diagnostic workup. primary rpl was defined as the absence of previous pregnancy at term or beyond the 24 weeks of gestation; secondary rpl was defined as the presence of two or more consecutive losses occurring in women with a previous child or whose previous pregnancy reached the 24 weeks of gestational age. all the women of both groups with pre-existing diabetes and hypertension before the onset of pregnancy were excluded, in order to avoid confounding factors as much as possible, since the objective of the study was to investigate with specific attention the pregnancy-related complications in relation to rpl. women with multiple pregnancies were also excluded from the study. the following additional inclusion criteria were followed for control women: absence of any pre-existing medical conditions, no previous gynecologic surgery, no assumption of drugs before pregnancy. the definitions of the pregnancy complications of interest for the present study are reported in table 1 . table 1 . definitions of pregnancy complications. threatened miscarriage an abnormal vaginal bleeding and abdominal pain occurring before the 24 weeks in an otherwise ongoing pregnancy miscarriage a spontaneous pl occurring before the 24 weeks gestation cervical insufficiency cl < 25 mm by transvaginal ultrasound, or cervical changes detected on physical examination before 24 weeks of gestation [14] chromosomal abnormalities any detected alteration of the fetal karyotype and/or dna fetal anomalies any structural/morphological abnormality detected by ultrasound oligohydramnios afv < 5% for ga, or afi < 5 cm or mdp < 2 cm [15] polyhydramnios a deepest vertical pocket of >8 cm or an afi > 24 cm [16] fetal growth restriction fetus with an uefw 5-10th percentile for ga, calculated using the igc according to snijders and nicolaides [17] intrauterine fetal death fetal death at 24 weeks gestation or late gdm gdm was defined following who criteria [18] preeclampsia preeclampsia was defined according to acog 2013 [19] placenta previa/low-lying placenta defined according to the criteria of rcog [20] placental abruptio the premature separation of the placenta before delivery [21] pregnancy-related liver disorders all the collected data of interest for the present study were reported in a preconceived template. a computerized database available for the successive analyses was then constructed. any collected information was anonymized and de-identified prior to analysis. data are presented as means ± standard deviation (sd) or percentages or proportions, or odds ratios (or) and 95% confidence intervals (ci) as appropriate. statistical analysis was carried out by using student's t-test and chi-square test. bravais-pearson coefficient was determined to analyze correlations. the software used was the statistical software spss release 23 (ibm ® , armonk, ny, usa). the effect of age and bmi for each complication is counted as percentages of the total population of women (cases and controls); the data were elaborated by using the method of analysis of the averages; by using this approach, the "effect size" expressed in percentages maintains the same dimensional magnitude of the original data. this statistical elaboration has been applied separately for the two above factors in relation to each complication; the results have been analyzed by student's under the usual hypotheses of normality and homogeneity of the corresponding distributions. significance was set at p < 0.05. overall 1092 singleton pregnancies were followed (431 in women with rpl and 661 in normal healthy controls). the major clinical characteristics of the study women are reported in table 2 . ethnicity distribution was not different between the two study groups (chi-square test = 4.41, p = 0.29, not significant difference). no difference was found between the rates of women with two (48.5%) or ≥3 (51.5%) losses in the population of women with rpl (chi-square test = 0.205, not significant). conversely, significant differences were found in the rates of women with primary vs. secondary rpl (chi-square test = 28.72, p < 0.001), as well as in the rates of women with explained vs. unexplained rpl (chi-square test = 11.35, p < 0.001). no differences were found between the two groups in the rates of pregnant women lost at follow-up (figure 1 ; chi-square test = 0.78, not significant). the mean length of follow-up was similar in the two groups (table 2) . table 2 . general characteristics of study women. no maternal deaths were observed in the overall study population. live births were 371/431 (86%) in women with rpl and 643/661 (97.2%) in women of control group. women with rpl during their subsequent pregnancy after referral, had a significantly increased risk of not having a live birth compared with control women (or = 5.77, 95% ci: 3.359-9.933, p < 0.0001). women with rpl also had a significantly higher overall rate of pregnancy complications (231/431, (53.6%) than control women (138/661, 20.9%): or = 4.37, 95% ci: 3.353-5.714; p < 0.0001). the rates by specific complications are reported in table 3 . the factorial analysis (reported in detail in supplemental tables s1 and s2) has been carried out to ascertain the effect of age and bmi, that were higher in rpl than in control women; it revealed that both age and bmi had a significant effect on the distribution of nearly all of the above considered complications. this effect was particularly relevant in: (a) the case of age for spontaneous miscarriage; and (b) the case of bmi, for spontaneous miscarriage, chromosomal abnormalities, fetal growth restriction, gestational diabetes mellitus, and preeclampsia. table 3 . pregnancy complication rates in women with rpl and control women. the factorial analysis of the effect size of age and bmi is reported as ∆% of the whole study population. women with rpl (%) the fetal chromosomal abnormalities detected were: trisomy 21; trisomy 22; monosomy 45, x0; trisomy 47, xxy; autosomal triploidy. the fetal anomalies detected during prenatal ultrasonography were: clinodactyly, pre-axial polydactyly, hydrops fetalis, interventricular septal defect, tricuspidal insufficiency, micrognathia, trigonocephaly, femoral heterometry, liver calcifications, cystic hygroma, bilateral pyelectasis, alterations of head circumference, persistence of the right umbilical vein. the detail of the pregnancy-related liver disorders is reported as supplementary material (supplementary table s3 ). the effect of the number of previous losses on the risk for each specific pregnancy complication was also investigated and the results have been reported in table 4 . a "gravity-response" effect was clearly evident in the case of spontaneous miscarriage, cervical insufficiency, chromosomal abnormalities and preterm prom. the number of women who had more than one pregnancy complication was higher in the rpl group (93/431, 21.57%) than in the control group (37/661, 5.59%; or = 4.64, 95% ci: 3.10-6.94, p < 0.0001). the detailed rates of concomitant pregnancy complications in study women are reported in table 5 . two hundred and nine women with rpl had two previous pregnancy losses and 222 had three or more previous losses. when the women with rpl were stratified in two major groups according to the number of previous losses (two and ≥3) the pregnancy complication rate in the women with ≥3 losses (132/222, 59.45%) was higher than that of women with two losses (99/209, 47.36%; or = 1.269; 95% ci: 1.112-2.386, p < 0.02). the detailed rates of pregnancy complications by the number of previous losses are shown in supplementary table s4 . the pregnancy complications in women with rpl were then stratified by the main diagnostic categories, i.e., explained and unexplained. one hundred and thirty-seven (52.89%) out of the 259 women who had an explained rpl and 94 (54.65%) out of the 172 women who had an unexplained rpl had a pregnancy complication, respectively. this overall difference was not significant (or = 0.931; 95% ci: 0.632-1.371; p = 0.72). however, when the two populations of women with rpl were analyzed by specific type of complications, women with unexplained rpl had an increased risk to develop preeclampsia (or = 2.35, 95% ci: 1.26-4.38, p < 0.05) and pregnancy-related liver disorders (or = 2.34, 95% ci: 1.12-4.89, p < 0.05). these findings are illustrated in detail in supplementary table s5 . in women with explained rpl the following causes were detected: anatomic causes, 21.7%; endocrine causes, 15%; thrombophilias (hereditary and acquired), 6%; immunologic causes, 11.7%; parental chromosomal disorders, 1.8%; and environmental and health behaviors causes, 3.9%. finally, the pregnancy complications in women with rpl were stratified by the other main diagnostic categories, i.e., primary and secondary. in our study, 284 women had primary rpl and 147 women had secondary rpl. women with secondary rpl had a higher rate of pregnancy complications (93/147, 63.26%) than women with primary rpl (138/284, 48.59%; or = 1.822, 95% ci: 1.211-2.470, p < 0.005). however, no differences between women with primary and secondary rpl were found for any of the specific complications considered, with the exception of gestational diabetes mellitus that was more frequent in women with secondary rpl (or = 0.40; 95% ci: 0.21-0.77, p < 0.01). these findings are shown in detail in supplementary table s6 . the results of the present study show that women with rpl during their first gestation after the completion of the diagnostic workup had a significantly higher rate of several pregnancy complications than normal healthy women without rpl. nearly all the pregnancy complications considered in our study occurred more frequently in women with rpl and that women with rpl had an increased risk to have multiple pregnancy complications than control women. to our knowledge, this aspect has been scarcely explored in women with rpl. these observations suggest that pregnancy in women with rpl could be considered high-risk in its entirety and support the general concept that these women could have a wide reproductive disorder not limited to early pregnancy establishment and maintenance; rather, it can be extended also to late gestation, once the implantation of the embryo and its initial development have been successfully established. however, this hypothesis could be in contrast with the high rate (86%) of live births observed in our study. there are several potential explanations for this. it is known that the final outcome of the successive pregnancy in women with rpl in terms of live births could be considered, all in all, satisfactory, particularly in those women with a limited number of previous losses [5, 22, 23] . this is the case of our study, in which the majority of women studied (332/431, 77.03%) had two or three previous losses, while the women with ≥4 previous losses, those at highest risk of an unfavorable outcome, were 99/431, (22.97%), i.e., less than one quarter of the overall population of women with rpl). it is also possible that the high rates of live births in our study women, albeit are within or near the high range reported in previous studies [4, 5, 22] , are linked to complications of moderate severity; moreover, our live birth rates could be to some extent overestimated, since a part of our cohort of pregnant women (13.7% of women with rpl and 12.1% of control women) was lost at follow-up and this is a limitation of the present study. the rate of spontaneous miscarriage in women with rpl was much higher than that found in control women. the risk of miscarriage increased by increasing the number of previous losses. the higher risk of fetal anomalies in women with rpl is in substantial accordance with the observations of previous studied carried out on this issue [24, 25] , even though other more recent studies could not find this association [8, 26] . however, the postnatal genetic follow-up of the newborns was incomplete in our series and this limitation does not allows to draw firm conclusions on this issue. in our study, women with rpl had an increased risk of fetal growth restriction and intrauterine fetal demise compared to control women. these findings are in accordance, although to a variable extent, with many of the studied previously carried out [6, 7, 24, 27] . however, several other studies could not demonstrate the above associations with rpl [26, 28] . similar considerations can be made for gestational diabetes mellitus, preeclampsia, placenta previa and abruptio placentae, conditions for which the association was either found to variable extent or not found at all [5] [6] [7] [8] [9] [10] 24, [26] [27] [28] [29] [30] . pregnancy-related liver disorders were found to be more frequent in women with rpl than in control women. this finding is in accordance with what has been observed by cozzolino et al. [31] . in the present study there are additional major findings related to the specific population of women with rpl. (1) by increasing the number of previous losses, the rates of women who became pregnant decreased, however the rates of women with pregnancy complications have the tendency to increase. since the number of previous losses is considered an indicator of the severity of the rpl condition [3, 32] , it is possible that the biological factors underlying multiple pregnancy losses can continue to act by impairing the successive pregnancies, even though they have the strength to evolve towards advanced gestational ages. further research is needed to check this hypothesis. a clear effect of the gravity of rpl condition, in terms of number of previous losses, has been shown in the case of selected pregnancy complications (table 4 ). (2) when the women with rpl were stratified according to the two major diagnostic categories, explained and unexplained, the rates of overall pregnancy complications were similar. however, the analysis carried out by specific complication revealed that the risk of preeclampsia and abruptio placentae was higher in women with unexplained rpl. a possible, plausible explanation for this finding-taken into account that the above conditions are linked since preeclampsia is a known major risk factor for abruptio placentae-is that in some or several women with unexplained rpl a disorder in the placentation could occur. (3) when the women with rpl were stratified according to the other two major diagnostic categories, primary and secondary rpl, the rates of overall pregnancy complications were similar. however, the analysis carried out by specific complication revealed that the risk of gdm was higher in women with secondary than in women with primary rpl. a possible explanation for this finding could be that women with secondary rpl have been more exposed than women with primary rpl to the well-known diabetogenic effect of pregnancy that is exerted mainly in the second half of pregnancy, making them more susceptible for gdm in a successive pregnancy. this possibility is also supported by recent observation showing the association between high numbers of pregnancies and the increased prevalence of gdm [33] . on the basis of all the above consideration, it is clear that assessing the outcome of the first pregnancy after referral with the aim to establish a clear prognosis is highly problematic [3] . in fact, it is very difficult to make comparisons and fully explain the differences in the specific pregnancy complications observed between the studies, including the present one. this can be due to multiple reasons, including the heterogeneity of the studies with regard to the study design (retrospective/prospective), the inclusion/exclusion criteria, the different specific complications taken into account and their clear definitions, the stratification of women with rpl in specific subgroups, the potential impact of different therapeutic managements; in several studies a control group is lacking [4, 5, 27, 29] . on the other hand, the major limitations of our study are the incomplete follow-up of the initially included women, particularly the pregnant ones, and the limited number of women with multiple pregnancy losses, i.e., ≥4. another limitation of the present study is that the intrapartum complications of pregnancy, as well as the neonatal complications, have not been reported because they were not included in the design of the present study, whose aim was to gain and report as much information as possible on the prepartum outcome of the investigated subjects. finally, there is evidence suggesting that women with rpl are at increased risk of long-term cardiovascular complications [34] , so that recently figo had published guidelines regarding long-term follow up on these women in order to decrease this risk [35] . we believe that this issue is worth to be investigated in depth. the results of the present study show that women with rpl have an increased risk to develop pregnancy-related complications during the first gestation after their referral; their pregnancy should be considered at high-risk and deserves special attention and care, even though caution is needed before drawing firm conclusions on this relevant issue, as it has recently reported [34] . clearly, further investigation is needed to fully clarify still many aspects of this important issue. 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impact of consecutive versus non-consecutive losses? spontaneous abortion and subsequent adverse birth outcomes worldwide collaborative observational study and meta-analysis on allogenic leukocyte immunotherapy for recurrent spontaneous abortion the japan environment, children's study (jecs) group; adverse pregnancy and perinatal outcome in patients with recurrent pregnancy loss: multiple imputation analyses with propensity score adjustment applied to a large-scale birth cohort of the japan environment and children's study outcome of pregnancies progressing beyond 28 weeks gestation in women with a history of recurrent miscarriage obstetric outcome in women after multiple spontaneous abortions risk factors and outcomes of recurrent pregnancy loss in japan obstetric outcome after recurrent pregnancy loss ongoing pregnancies in patients with unexplained recurrent pregnancy loss: adverse obstetric outcomes investigation protocol for recurrent pregnancy loss higher numbers of pregnancies associated with an increased prevalence of gestational diabetes mellitus: results from the healthy baby cohort study recurrent pregnancy loss: a risk factor for long-term maternal atherosclerotic morbidity? international federation of gynecology and obstetrics) postpregnancy initiative: long-term maternal implications of pregnancy complications-follow-up considerations this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license acknowledgments: this article has been written during the so far hardest time of the covid-19 outbreak in italy. our thanks to all our colleagues who died in fighting the infection. the authors declare no conflict of interest. the following are available online at http://www.mdpi.com/2077-0383/9/9/2833/s1, supplementary key: cord-252902-qtfx49qp authors: scott, jodie; oxlad, melissa; dodd, jodie; szabo, claudia; deussen, andrea; turnbull, deborah title: creating healthy change in the preconception period for women with overweight or obesity: a qualitative study using the information–motivation–behavioural skills model date: 2020-10-19 journal: j clin med doi: 10.3390/jcm9103351 sha: doc_id: 252902 cord_uid: qtfx49qp worldwide, half of women begin a pregnancy with overweight or obesity, which increases the risk of pregnancy and birth complications and adversely affects the lifelong health of the offspring. in order for metabolic changes to influence the gestational environment, research suggests that weight loss should take place before conception. this study aimed to understand women’s emotional and social contexts, knowledge, motivations, skills and self-efficacy in making healthy change. semi-structured interviews conducted with twenty-three women with overweight or obesity, informed by the information–motivation–behavioural skills (imb) model, were analysed using reflexive thematic analysis. information-related themes identified included poor health risk knowledge, healthy food decisions and health versus convenience. the motivation themes comprised taking responsibility, flexible options, social expectations, interpersonal challenges and accountability. behavioural skills entailed themes such as the mental battle, time management, self-care and inspiration. an environmental factor was identified in affordability—limiting access to healthier alternatives. women wanted simple, flexible options that considered family commitments, time and budgetary constraints. unprompted, several mentioned the importance of psychological support in managing setbacks, stress and maintaining motivation. strategies for enhancing self-efficacy and motivational support are required to enable longstanding health behaviour change. findings will inform intervention mapping development of an ehealth solution for women preconception. worldwide, half of all women of childbearing age have overweight (body mass index (bmi) ≥ 25.0 to 29.9 kg/m 2 ) or obesity (bmi ≥ 30 kg/m 2 ) [1] . this figure is significant as retrospective, case-control and cohort studies have found that women who enter pregnancy with obesity are at higher risk of gestational diabetes mellitus (gdm) [2] and pre-eclampsia [2, 3] -both associated with long-term morbidities [4] . a review of reviews on the risks of maternal obesity also found these women are more traditional weight-loss interventions across all life stages have included advice and strategies to adopt healthy behaviours via diet and/or physical activity. those adopting an interdisciplinary approach using behavioural strategies and psychological techniques, and specifically targeting the preconception period are almost non-existent. there are also significant barriers to behaviour change regarding healthy lifestyle. within the target group of women for this study, there is low health literacy, and they are often from low-income families. in a large stratified study on the socioeconomic differences in health behaviours, those with lower socioeconomic status (ses) also displayed less health consciousness (thinking about ways to keep healthy), stronger beliefs about the role of chance on their health and lower rates of thinking about the future [26] . a qualitative study focused on improving health in women of childbearing age identified that dietary knowledge, cooking skills and the time and cost of preparing healthy food were significant barriers to adopting a healthier diet [23] . a lack of support from partners and family members was also cited as a significant barrier to healthy change, likewise, finding the time and inclination to exercise. while preconception care largely focusses on women's health and care, fathers are increasingly becoming involved in pregnancy planning and lifestyle changes in efforts to conceive. however, little is known about partners' attitudes and roles in supporting positive preconception health behaviours [27] . a high bmi category is associated with a range of co-morbidities, and poorer general health may affect motivation and perceived ability to adhere to healthy lifestyle recommendations [26] . behavioural programs have the potential for substantial weight loss, yet significant problems remain due to program attrition and poor maintenance of healthy habits-with authors citing the lack of innovation as a factor in this area [28] . significant gaps in knowledge exist, with further research exploring women's perspectives required to inform effective preconception health promotion strategies [29] . this study aims to develop an understanding of preconception health awareness, potential barriers to adopting a healthier lifestyle, motivations, current behaviours and the practical skills required to change behaviour, for women with overweight or obesity. we sought to understand experiences from the women's perspectives [30] consistent with the aims of the study in fostering empathy with their desires, needs and challenges. the intent is to understand the women's emotional and social contexts, to inform which behaviour change techniques and intervention components are likely to be most engaging. this qualitative study explored the perceptions and experiences of women related to healthy lifestyle change and weight management before conception. the information-motivation-behavioural skills (imb) model [31] addresses some of the existing attitudes, beliefs and values that may impact behaviour [32] and was used as the theoretical model for the interview topics. the model, which asserts that when individuals are well-informed, motivated to act, and hold the necessary behavioural skills, they will likely initiate and maintain a health behaviour [33] , is highly applicable to obesity management [32, 33] . the imb model is based on a critical review and integration of relevant constructs in social and health psychology theories and seeks to address limitations to these [33] . the constructs are supported in the literature to improve healthy lifestyle behaviours and have been tested and used successfully in obesity prevention [34] [35] [36] and in improving dietary and physical activity behaviours [37, 38] . this model was chosen for its simplicity of structure and the fact that the constructs can be easily translated into intervention components. the imb model's elicitation-intervention-evaluation approach to the promotion of health behaviour, begins with seeking to identify this cohort's existing weight management knowledge, motivation and behavioural skills assets and deficits [33] . individual determinants of behavioural change were explored: information, including behaviour-related information, knowledge about the impact of obesity, but also heuristics that permit automatic decision-making; motivation, comprising personal motivation (beliefs about intervention outcome and attitudes towards obesity prevention behaviours) and social motivation (including perceived social support for engaging in that behaviour); and behavioural skills (individual skills and self-efficacy) [33] . qualitative investigation enables a deep and thorough understanding of the topic, yielding rich data [39] , and is recommended to develop effective interventions [40] . the relevant literature informed the development of the interview schedule, with probes allowing for exploration of topics driven by participants. under the belief that knowledge is socially situated [41] , demographic information was also gathered, allowing the researchers to reflect on the relationship between the results and the sample [42] . the present research, drawing on the problem-solving principles of design thinking [43] , uses a human-centred approach that holds the emotional, functional and motivational needs of the user at the centre of the development process [44] . this study represents the first phase of this process in empathic engagement-with these findings being used to inform the development of an ehealth solution using the intervention mapping approach. interview participants were a sample of women who have participated previously in diet and lifestyle intervention studies (the limit [45] and/or grow [46] randomised trials) at the women's and children's hospital-a high-risk specialty hospital with approximately 5000 deliveries annually-and who had given their consent to be contacted about future research. a purposeful sampling frame was adopted, with eligibility limited to those of reproductive age (15-49 years) [47] , who were above the healthy weight range (bmi > 25 kg/m 2 ) and identified that they would like to lose weight. intention to become pregnant was not a prerequisite. women were contacted via telephone, with the purpose and methods of the study explained, and eligibility confirmed. those who expressed an interest in the research were emailed an information sheet and consent form, with interview times confirmed via email or telephone. the primary researcher (js) conducted twenty-three interviews during september 2019. eight participants attended face-to-face interviews at the university of adelaide robinson research institute, with the remaining 15 conducted via telephone. the interviews lasted 20-72 min (mdn = 33). written consent was obtained from those attending a face-to-face interview, with verbal consent gained from those participating via telephone. the interviews followed after the consent process. the interviewer was a psychology researcher (js) trained in qualitative methods and interviewing skills, with knowledge of pre-pregnancy health and wellbeing, and no previous connection with any participants. a pilot interview was conducted with an eligible woman to determine the level of comprehension and natural flow of the intended questions, with this data excluded from the dataset as the interviewee was known to the researcher. after several interviews, some questions were revised for greater understanding, with others modified to broaden the scope for response. no further changes were made after the seventh interview. this process of revision is accepted as best practice within qualitative interviewing [48] . each interview commenced with broader questions about lifestyle to build rapport. more sensitive and descriptive questions were asked later when participants were more comfortable. the interview schedule closed with questions that empowered the women to give their opinions and advice on the broader issue. questions were formulated to address the imb model constructs yet allowed scope for participants to speak freely outside of these topics. indicative topics included awareness of maternal and neonatal health risks, previous experience of weight loss, lifestyle and social factors, motivations, challenges and self-efficacy. a sample of questions is provided in table 1 . participants who attended a face-to-face interview received a 20 aud gift card as reimbursement for travel costs. after the interview, all women were provided with information on the maternal and neonatal health risks associated with overweight or obesity in pregnancy. all interviews were digitally recorded and transcribed verbatim by the first author. data analysis software nvivo 12 ® [49] was used to store and manage the transcripts, with each participant given a pseudonym and identifying information removed from the transcripts. despite recurring patterns indicating that code saturation was reached after 12 participants, additional women were interviewed to provide meaning saturation-thereby identifying further insights and the nuances of issues required to understand this complex topic [50] . it is believed that depth requires further data, especially for codes that are conceptual in nature [51] . the analysis followed the six-phase process of reflexive thematic analysis set out in braun and clarke (2019) [39] : familiarisation, generating codes, constructing themes, revising themes, defining themes and producing the report. this recursive process begins with repeatedly reading the data in an immersive manner to gain a sense of the whole. the analysis took both a deductive and inductive approach [52] . while guided by the imb model and obesity literature, the coding process ensured that concepts falling outside of this were also captured. key concepts relating to the research aims were coded, then sorted into meaningful clusters that were assessed for applicability to the imb model constructs. two additional members of the research team (mo, dt) co-coded several transcripts to ensure rigour and transparency of interpretation, with the researchers working collaboratively to ensure the codes and candidate themes fit both the evidence, and the constructs of the imb model. the themes were then defined and named, with compelling extracts selected to illustrate the findings. perspectives that differed from the dominant beliefs were not excluded, with counter-instances considered to add rigour [53] . the study was conducted in accordance with the declaration of helsinki, with the research approved by the women's and children health network (hrec/19/wchn/108) and the university of adelaide human research ethics committee. this research was reported as per the consolidated criteria for reporting qualitative studies (coreq) [54]-a 32-item checklist across three domains governing reflexivity, study design and analysis. each author approached the research from their respective positions and biases. while the primary researcher (js) who conducted the interviews, is not above the healthy weight range, she has an understanding of preconception health. the research team also comprised: a clinical and health psychologist (mo) with extensive experience in preconception care and health literacy; an obstetrician and academic researcher (jd) with extensive experience in maternity care; a digital technology specialist (cs) with an interest in its practical application within the health domain; a clinical trials manager (ad) within reproductive health and preventive medicine; and an academic researcher (dt) with a specialist interest in health psychology and maternity care. all authors are women with children of their own. a triangulated review process was used, with codes and candidate themes discussed by three researchers (js, mo, dt). an audit trail was maintained throughout data collection and analysis [41, 54] , and participant confidentiality ensured by assigning pseudonyms. participants were able to review their transcripts to verify accuracy [41] and were sent a summary of the findings [54] . due to the sensitive nature of the topic, and participants not providing consent for sharing of their data beyond the current research, data will not be made publicly-available. a total of 23 women, aged between 23 and 48 years (m = 34.78, sd = 7.33) participated. each had between one and four children, with two women intending to conceive in the next 12 months and three unsure on pregnancy intentions. participants self-reported their weight and height-most considered themselves above the healthy weight range (n = 22), with bmi categories ranging between overweight (26.1 kg/m 2 ) and obese class iii (56.3 kg/m 2 ). all women expressed a desire to lose weight, with amounts ranging between five and 60 kg. demographic characteristics of the participants can be seen in table 2 . the themes identified in the data reflect the individual perspectives of participants concerning healthy lifestyle changes. these comprise four overarching themes: the imb model constructs (information, motivation, behavioural skills) and one issue beyond the scope of these (environmental factors) that encompass the diverse experiences of these women. within these, several themes were derived from the data, depicted in figure 1 . the themes identified in the data reflect the individual perspectives of participants concerning healthy lifestyle changes. these comprise four overarching themes: the imb model constructs (information, motivation, behavioural skills) and one issue beyond the scope of these (environmental factors) that encompass the diverse experiences of these women. within these, several themes were derived from the data, depicted in figure 1 . information-related themes included poor health risk knowledge, making healthy food decisions and health versus convenience. motivation-related themes-divided into personal and social motivation-included concepts such as taking responsibility, flexible options, interpersonal challenges and accountability. behavioural skills themes covered concepts such as the mental battle, time management, self-care and inspiration. the women conveyed their feelings and attitudes as they discussed previous experiences, their social environments, and aspects they considered presented barriers to adopting a healthy lifestyle. themes are presented in greater detail below. knowledge of the potential maternal and neonatal health risks associated with overweight or obesity before pregnancy was alarmingly low. while many participants had some knowledge of the risks to their health, most commonly gestational diabetes and pre-eclampsia, others expressed this knowledge in more general terms. of those who knew some of the risks, several disclosed they had experienced these in a previous pregnancy. information-related themes included poor health risk knowledge, making healthy food decisions and health versus convenience. motivation-related themes-divided into personal and social motivation-included concepts such as taking responsibility, flexible options, interpersonal challenges and accountability. behavioural skills themes covered concepts such as the mental battle, time management, self-care and inspiration. the women conveyed their feelings and attitudes as they discussed previous experiences, their social environments, and aspects they considered presented barriers to adopting a healthy lifestyle. themes are presented in greater detail below. knowledge of the potential maternal and neonatal health risks associated with overweight or obesity before pregnancy was alarmingly low. while many participants had some knowledge of the risks to their health, most commonly gestational diabetes and pre-eclampsia, others expressed this knowledge in more general terms. of those who knew some of the risks, several disclosed they had experienced these in a previous pregnancy. knowledge of neonatal health risks and outcomes was much less evident, with only half the participants able to recall any at all. of these, the majority were unable to describe them in detail or they were based on false information. while several women knew the risk of high birthweight, only two participants mentioned that the child may be at risk of having weight issues over their life course. however, some women did express informed knowledge of longer-term health risks for their child. "yeah well, stillbirth, um, high birth weight, um, i might be pulling this out of thin air, but i believe it puts your child at higher risk of having diabetes themselves." (amber, obese class ii) even those women who knew some of the risks to their health or their baby's health were unaware of the genetic traits they may pass on to their child, by entering a pregnancy with overweight or obesity. in many instances, participants only followed a healthy lifestyle after conception-discussing the more immediate effects of the foods they ate during the pregnancy and how this may impact the baby. " . . . you want what's best for your kids, and that starts from the second you see those two pink lines." (annie, obese class iii) when asked about the benefits of losing weight before pregnancy, some women expressed that they may fall pregnant more easily, and many conceded the healthier they were, the easier the pregnancy and birth would be, with less strain on their body and fewer complications. some requested information be freely available on the benefits, for themselves and their baby, of being a healthy weight before conception. many of the women had inadequate information on nutrition to make autonomous healthy food decisions for themselves or their families. while no participants were currently on an intensive weight loss program, some foods they reported consuming were carbohydrate-laden or high in sugar or fat-suggesting poor nutritional knowledge and self-monitoring of eating habits. for some, knowledge was based on misinformation such as considering some high sugar or "light" version of foods as healthy. " . . . i suppose an example is when i had gallstones and i wasn't supposed to be eating fat, i ate kabana [high-fat cured sausage], not realising that it was full of fat, and i ended up in the hospital with a gall bladder attack. it was just a . . . you know, a lack of information i suppose, i just didn't know." (erin, overweight) portion control, snacking, willpower and knowledge of consumption norms were among the greatest food challenges-even for those who considered their current diet to be healthy. positive health knowledge and behaviours were also displayed, with some reporting a good understanding of nutrition to aid weight loss. several women recognised the value in clean eating and shopping the perimeter of the supermarket to avoid heavily processed foods. "don't do fad diets, just eat clean. your food is 80% of it. if you've got your food under control, then . . . other things will fall into place." (elizabeth, overweight) while over half of the participants read the ingredients list and/or nutritional information panels, there appeared to be confusion around interpreting these. many appeared overwhelmed by the volume of nutrition advice available and whether it could be trusted. "there's a whole confusing world out there when it comes to diet" (mary, obese class iii) several women requested information being more freely available on healthy substitutions for unhealthy ingredients or dishes, plus alternatives to processed foods. participants emphasised the complexity of their lives, with busy schedules a contributing factor in choosing convenience meals over healthy ones. many women spoke of being too exhausted to cook and often relied heavily on processed foods or take-away, while half of the women reported cooking from scratch most of the time. physical and emotional exhaustion, general busyness and a lack of forward planning were cited as reasons for making poorer choices. when asked about their primary motivators for losing weight, some of the women expressed aesthetic motivators over health ones, in wanting to feel good about themselves and more confident. "almost entirely aesthetic. like i don't like the way i look in photos. and i understand the health risks, you know, the higher, higher incidence of diabetes, heart disease, all of the fat diseases, but that's not my main motivator if i'm honest." (amber, obese class ii) a powerful motivator for nearly all participants was role modelling healthy behaviours to their children, so they can also make good choices. several women did not want their children to focus on weight or dieting; instead, they wanted to set an example with positive health talk. within this, a couple of women conceded that the food they fed their children was much healthier than what they ate themselves. several women expressed their primary motivation was to improve their overall health, with some conceding that co-morbidities they suffered were an added incentive to lose weight. "just to feel healthier in myself. also to kick my depression a bit." (lucinda, obese class iii) most women cited several reasons for wanting to lose weight-even those with aesthetic motivators also wanted to provide a good example to others and see their children grow up. other reasons cited were to improve self-esteem, have more energy, feel proud next to their partner or to simply be a normal bmi. some women looked to the future, in wanting a long healthy life for their children, or more self-sufficient as they age. several expressed motivators related to the health of their potential baby or having an easier pregnancy. most women held positive beliefs about how important it was for them to manage their weight. it was widely recognised that they needed a "lifestyle" rather than a "diet"-having had negative experiences of weight loss programs in the past. many women attributed their excess weight to unhealthy behavioural patterns and felt a responsibility, whether trying to conceive or not, for managing their weight and health. " . . . trying to conceive, or have already had children and trying to conceive again, you know, you've got another body to look after, like it's not just you any more" (sasha, obese class ii) many women felt a sense of personal autonomy in choosing to improve their health-recognising that their lifestyle choices were modifiable and to have a healthy life, they had to take stock of their habits and change their mindset. "i don't blame my kids or my husband and i don't think that i don't have enough time." (mary, obese class iii) many were ready to change their lifestyle but perceived a barrier in making that first step-not knowing where to start in a task that seemed overwhelming. beliefs about other parts of women's lives often affected their motivation to begin a program of change. some women conceded they had no reason to eat unhealthily and were ignoring satiety cues or lacking in motivation. all participants had previous experience with trying to lose weight. these included meal replacement shakes, the ketogenic (or other low carbohydrate) diet, duromine or other diet pills, the "healthy mummy" app, intermittent fasting, weight watchers and counting calories. most had tried at least two of these methods. although some women had experienced success, many failed to lose weight or found the programs too restrictive, costly, or rigid to maintain with other responsibilities. women required options for both healthy eating and exercise that were flexible enough to accommodate family commitments. "i like taking the ideas from it, but i need the flexibility, with the family . . . for what's best to eat for us." (chloe, overweight) women talked about establishing a new routine that became an automatic habit. several women noted that they wanted a program that they could use long term to manage their weight. some noted that small steps and achievable goals would make women more motivated to succeed, along with devising a program that was simple to follow. social motivation many women reported feeling that societal norms dictated how they should look and feel and felt judged when they did not fit the "social" mould. some felt they were perceived differently due to their weight and did not want to shame their children or partners. . ] there's always going to be that voice in the back of your head that there is someone judging you, that doesn't even know you." (annie, obese class iii) a handful of participants could not relate to some of the advice or services they had accessed in the past, believing they target women who were already fit and a healthy weight, and found this de-motivating. despite experiencing hostility from others, or feeling out of place in fitness classes, some women were still able to push forward to improve their health. several women noted their partners were a barrier to eating more healthily, despite how supportive they may be in other ways. either they prepared unhealthy foods or ate unhealthy food in front of them. more than half of the women reported that their children were fussy eaters-which provided an added barrier to motivation, in that mothers preferred to cook food they knew their children would eat, at the expense of their own health. some struggled to strike a balance between healthy foods and the nutritional needs and preferences of their children, so as not to have to cook separate meals. "i've got two kids that are fussy eaters, to try and provide foods that are easy and carb-loaded for them [ . . . ] so that's where i've had issues with putting on weight, because trying to motivate the kids to eat and put on a bit of weight, you have to eat what they're eating. so, it's just trying to get that balance." (ella, obese class ii) some had difficulty convincing others of the need for healthy eating and felt this attitude sabotaged any efforts to lose weight. many women reported ongoing encouragement as holding power to maintain motivationregardless of whether they saw results-citing it as a significant reason not to abandon a healthy lifestyle program. "just nice words of encouragement, like you know 'you're doing a good job, and well done on you know, getting through the day, and you know, reaching your goal of however many steps' or 'it's ok you didn't get there today, but, you know, you're still doing good'." (olivia, overweight) women often find motivation wanes when "life gets in the way" and wanted gentle encouragement to keep on track after a setback. methods were highly individualized-while some wanted strategies that challenge on an individual level with personal messages, others found a group setting or competitive environment more motivating in that they saw others push themselves. the most powerful motivational support for women was seeing results-several had abandoned previous weight loss attempts due to not seeing results quickly enough. overwhelmingly, women felt they needed to be held accountable-some preferred a real person rather than goals set in an app, feeling they may be more likely to move the goalposts or lose motivation if they could not reach them. "i can fool myself pretty easily and make excuses for why i haven't done what i've done. it's different when you've got to explain to someone else your pathetic reasons for not having done something." (amber, obese class ii) several participants saw value in family members also committing to improving their health habits, to create an environment more conducive to success. some saw reciprocal benefits in encouraging others-such as peer interaction within an online support forum-and valued the camaraderie with those on the same journey. behavioural skills unprompted, several women mentioned psychological support in managing setbacks, stress and maintaining motivation-knowing the difficulties that can be encountered. " . . . mental health is a huge thing when you're trying to lose weight. [ . . . ] [it] can be really demoralising, especially if you're not achieving those goals. or if people around you are achieving them, and you're not." (elizabeth, overweight) weight management was viewed as much more complicated than just calorie input versus output. one participant noted that for most, obesity is about more than just food, and she would value being able to talk to someone about other issues that may impact behaviours. some women talked about their mental barriers and expressed the need to change their thinking around old habits. "you've really got to sort of train yourself mentally as well, to . . . um, eat better and to exercise more. it's kind of a mental battle as well." (olivia, overweight) participants overwhelmingly had stressful lives, with factors such as juggling work and caring for children, financial difficulties, children with complex health issues, or managing a hectic schedule contributing to this. for some, exercise was noted as being particularly beneficial for stress, with several women requesting stress management or mindfulness approaches within an intervention. some also valued a holistic approach incorporating increasing self-esteem too, with the belief it would help maintain good health decisions. most women recognised time as one of the biggest barriers to a healthier lifestyle. prioritising their health over other responsibilities proved difficult and contributed to the abandonment of previous weight loss attempts. some participants recognised that exercising would be easy if they were able to manage their time better. "i feel like it's just managing my time better isn't it? just getting up, even like a little bit earlier, i can do it in the morning . . . " (alex, obese class ii) some felt that time spent cooking from scratch or exercising meant that other priorities in their lives piled up and left them further behind. several women conceded that they do not really have an excuse not to eat well or exercise, but probably do not make the best use of their time, with "perceived busyness" often an excuse. "i think if someone can help you plan your whole day, so you can fit it in." (mary, obese class iii) exercise proved more of a challenge than healthy eating, and women wanted ideas for how to integrate physical activity into their lifestyle with small changes. women often felt a trade-off between spending time with their children and exercising, with the unpredictable nature of family life a barrier to maintaining physical activity and healthy eating regimes. many women also recognised other benefits, beyond weight management, in eating well and exercising. participants noted these self-care behaviours were rewarded with improvements in sleep, energy, clearer skin, mental clarity, less digestive issues, and some felt it gave them more motivation in other areas of their lives. overwhelmingly, women assigned greater importance to eating well than physical activity. "when i eat well, i feel well if that makes sense." (erin, overweight) some noted it took longer to notice the health benefits of physical activity, yet several women placed higher importance on exercise than healthy food, for the additional psychological benefits. "for me it's got the stress relief component, it's my 'me' time, it feels good, i feel good afterwards, i tend to be . . . i find the flow on effects, so when i exercise that's when i do tend to be less likely to go to straight the cupboard because i'm still feeling motivated." (chloe, overweight) it was also mentioned that, as mothers, they often prioritised everything else before their health. some found value in exercise as it allows them to focus on themselves, rather than just taking care of their families. many women lacked skills such as nutrition planning and meal preparation. participants indicated they often found healthy food boring or repetitive and wanted ideas to make meals more interesting and attractive to themselves and their families. " . . . knowing how to make foods interesting so you're not eating the same old things continuously [ . . . ] how to make vegetables more interesting [ . . . ] without adding massive amounts of calories to them." (lucinda, obese class iii) around half the women regularly cooked meals from scratch with fresh produce. while not always intentional with their meal planning, a handful of women noted they were skilled in creating basic meals, based on ingredients at hand and their personal preferences. some participants noted that what they needed was inspiration to implement changes. " . . . oh gosh, i need more inspiration than information" (amber, obese class ii) several women expressed a desire for basic meal plans and simple, quick recipes that were within their skillset, using easily accessible, cost-effective ingredients. women knew their old, unhealthy patterns of eating, and highly restricted regimes were not sustainable in helping them establish healthy patterns for weight loss. many wanted a more structured plan with interesting ideas for both nutrition and exercise. 3.3.14. theme: believing in myself to change my lifestyle several women noted they felt confident to make healthy changes to their diet and exercise levels, but motivation held them back. many placed caveats on their ability-contingent upon managing their time better, managing exhaustion, or finding adequate motivation. for some, previous successful experience of weight loss gave them confidence that they could make the changes required. "reasonably confident. i know i can do it. i know i can, because i've done that with little things, like the soft drink as i said. so i know i could do it." (erin, overweight) women often reported better results with changing their diet than exercising and expressed more confidence in being able to modify their diet. several cited the 80/20 rule-believing that weight loss is 80% diet and 20% exercise. given the correct information, many felt confident about easily integrating healthy foods into their daily life. however, a handful of women recognised that their lack of cooking skills might hinder their progress. some felt it would be easy to include physical exercise in their daily life, but only after attending to other responsibilities. environmental factors several women considered healthy food to be more expensive than convenience foods, and some cited this as a reason for their poor diet at times. they noted that the foods on special offer were the "cheat" foods, not the healthier options, and this influenced their purchase choices. "so it's more about finding the alternate to those expensive things [ . . . ] because it's very expensive starting a diet and that can sometimes put a lot of people off, because budget-wise you just can't fit it in. and i find that's why people go for the easy foods, because it is cheaper." (april, overweight) especially for families with limited income, cost encouraged reliance on cheaper fat-, sugar-or carbohydrate-laden foods. frozen or packaged food was sometimes bought in favour of fresh, as it could be relied upon if money was tight. leaner cuts of meat were also seen as less affordable. " . . . that makes it a bit harder sometimes, especially if we're like, tight on money and the only thing that we can whip up is like sausages and packet pasta and stuff like that." (carla, obese class iii) some were unable to continue with previous weight management programs due to food costs. physical exercise often presented a cost barrier too, with several noting the cost of gym memberships, boot camp and exercise clothing suitable for overweight women. this qualitative investigation examined the experiences and beliefs of women with overweight or obesity related to managing their weight before pregnancy. in general, the participants displayed poor health literacy on the impact of entering pregnancy with overweight or obesity. while some women were aware of the risks to their health, few recognised the potential risks to their future baby. this finding corresponds with previous research noting a poor understanding of neonatal outcomes for pregnant women with overweight or obesity [55] . while preconception care and counselling may include advice about smoking, alcohol intake, nutritional supplementation and immunisations, less attention has been paid to diet and lifestyle advice for women with overweight or obesity [56] . the topic holds such sensitivity that it is often not raised by doctors during consultations. previous systematic review research has found that the beliefs and attitudes of partners, peers and family exert a powerful influence on women's health behaviours that may undermine the advice of health professionals [57] . the fact that many women reported interpersonal challenges suggests that partners need greater involvement in the process of preconception counselling and creating and supporting a healthy lifestyle. pregnancy is considered a powerful "teachable moment" [58] for weight control and health behaviour change, which enhances the perceived value of nutrition and exercise. while the focus of the current study has been on efforts women can undertake in the preconception period to maximise outcomes for their child, pregnancy is often a joint endeavour and partners are becoming increasingly involved in preconception planning. it is evident that partners can help or hinder women in their efforts to make lifestyle changes. it is crucial that any future intervention consider the impact of partners including ways to foster social support by making lifestyle or behavioural changes themselves, and to reduce social pressure by lifting the stigma around weight. this would provide a supportive environment to initiate healthy change within the whole family. interestingly, many of the women conceded they were motivated more by aesthetics and social expectation than health considerations. this finding reiterates previous research that found motivations beyond health, such as self-image, may be more engaging for some women [59] , and enable those not yet considering pregnancy to be captured by the public health messages. while it has been reported that between 56% [60] and 70% [29] of pregnancies are planned, it is thought that women with overweight or obesity are more likely to have unplanned pregnancies [61] and therefore are less able to optimise their health before conception. thus, interventions may need to target not just those intending to conceive, but all women of childbearing age. for many women in the study, the prime consideration was being healthy during the pregnancy, rather than in preparation for conception. greater understanding of the health implications of weight status may provide an added motivation to improve lifestyle. while the current study is concerned with behaviour change on an individual level, interactions with surrounding influences-family, community, plus social, environmental and policy contexts-exert a powerful force on health behaviours, as noted by many of the women in this study and previous research [12] . a two-pronged approach is required-empowering women with the tools to change the way they respond to the environment, and also changing their environment, where possible, to make healthier choices easier to make. the sugar tax, intended to create shifts in consumer behaviour, has reduced sugar consumption in the united kingdom and mexico [62] , with higher effects for lower-income households. while food taxes and subsidies have not yet been implemented in australia [63] , it stresses the importance of giving women evidence-informed guidance they can trust, but also individual support to counteract obesogenic environments [64] . unsurprisingly, nutrition knowledge was relatively poor amongst participants, with many consuming processed foods high in sugar or fat. it is known that unconscious and instinctual processes can prompt poorer eating practices, with people relying on heuristic cues to make food decisions which often lead them to choose larger and less nutritious options [65] . heuristics are mental shortcuts-in this instance, made in response to contextual food cues and are thought to reduce the cognitive depletion associated with making health decisions [65] -especially salient in a population with lower health literacy. many women had requested simple solutions, rather than complex calorie-counting and food logging. these findings emphasise the need for interventions to respond to the preference for heuristic processing, to help people make better choices with regard to food, portion control, even leisure activities, as suggested in a previous study [65] . previous experience of dieting practices meant that many of the women were wary of highly restrictive eating regimes, which often affected adherence and success. this result corresponds with previous research on barriers to following a mediterranean style diet for women of childbearing age [23] , with women perceiving the term "diet" to have negative connotations. the language used will be critical to the success of any future intervention, a sentiment shared by previous research on public perceptions of the terms used to improve eating habits [66] . this information highlights the need to frame the intervention with clear communication and a positive focus, along with making guidelines more flexible to accommodate busy lifestyles and families, as noted by previous research [67] . accessibility was also problematic, with the perception that healthy food was more expensive-a finding common to other studies [23, 29] . this finding points to the need for education on better food choices and substitutions-being mindful of budgetary constraints. women had stressed the importance of social support, encouragement and accountability, suggesting the need for an inclusive community to be created, where women feel empowered and supported to reach their goals-as noted in previous research on mutual-help groups [68] . avenues for peer support have never before been so important, with social barriers such as those imposed by the covid-19 pandemic, along with caring responsibilities for other children, giving women less opportunity to interact with peers and health professionals for encouragement. many participants also recognised the benefits of a healthier lifestyle beyond weight loss itself. a holistic intervention therefore presents an opportunity to promote factors such as stress reduction, increased self-esteem, improved mental health or role modelling. several women in the study expressed a need for psychological support along their weight loss journey. a technology-delivered motivational interviewing approach [69] could provide tactics to increase self-efficacy through small steps and achievable goals, elicit change talk and garner family support. a regime of establishing and recognising partial success in changing behaviour, plus strategies to manage setbacks, may promote greater motivation to continue. mindfulness-based interventions may have particular relevance for obesity with a meta-analysis suggesting benefit for improving both psychological health and eating behaviours [70] . likewise, open trial and case series evidence suggests that cognitive behavioural therapy (cbt) [71] and acceptance and commitment therapy (act) [28] can provide useful adjuncts to other behavioural techniques for weight management. however, few studies exist that assess these techniques when delivered via digital health intervention. an ehealth intervention offers a solution that is low cost, high reach, with the potential for personalisation and the use of adaptive and agile design to improve efficacy. this study presents an opportunity to understand the behaviour change techniques and digital functionality to which women may best respond. in-depth qualitative research is crucial in understanding the personal experiences and the contexts within which potential intervention users live, and to help tailor interventions to specific life stages. this person-based approach complements the theory-and evidence-based approaches to intervention development [72] . an intervention mapping approach will be guided by these interviews, the literature on obesity, behaviour change and psychological techniques. it is hoped that themes and subthemes derived from this study, having been identified by the women as important, will directly inform modules to be delivered in the intervention. the intent is to develop an intervention that the women want to engage with, using strategies that address some of the existing barriers to change and help them to create a sustainable healthy lifestyle. to date, there has been little qualitative research conducted into the experiences and beliefs of women regarding weight management before pregnancy-within an australian context. to the authors knowledge, there have been none that use the imb determinants of behaviour within this specific target group. this cohort displayed a diverse profile across age, family circumstance, bmi and socioeconomic factors. credibility was added through the multivocality of the participants, with the authors aware of the empathic understanding required to let this wide range of insights emerge. both the interviews and analysis were conducted with rigour, adhering to best-practice guidelines for qualitative research [41, 54] . the research was not without limitations. the women interviewed were not necessarily intending pregnancy (n = 2 intending pregnancy in next 12 months, n = 3 unsure, n = 18 not planning pregnancy in the next 12 months). therefore, motivations may be different from those in the preconception phase. however, it is thought that similar health and role-modelling sentiments would stand for those intending pregnancy, in wanting the best outcomes for their children. the women were recruited from a pool who had previously participated in studies concerning gestational weight gain, so may have prior knowledge of managing their weight through pregnancy in a supported manner. in addition, some women reflected on previous pregnancies in their responses, discussing lived experience of the health risks associated with their weight status. moreover, the cohort was not culturally diverse, in that recruitment was limited to those who spoke english. the cultural implications of dietary and social habits need to be considered and, therefore, future research with diverse populations is recommended. the preconception period is now acknowledged as a critical window in which to intervene for preventing obesity in pregnancy, with significant potential benefits-both health and economic. however, obesity is a complex and challenging issue, with multiple genetic, social, environmental and behavioural influences. promoting meaningful change in this group requires a multifactorial approach, involving a complex interaction between the necessary determinants of behaviour-information, motivation, and behavioural skills. this elicitation study, the first step in the imb approach to health behaviour change, provided insights about the beliefs and psychosocial contexts of women in this particular population. important factors for consideration include psychological support, flexibility, enhancing 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weight management intervention peer support groups for weight loss technology-delivered adaptations of motivational interviewing for health-related behaviors: a systematic review of the current research mindfulness-based interventions for adults who are overweight or obese: a meta-analysis of physical and psychological health outcomes the effectiveness of cognitive behavioral therapy with mindfulness and an internet intervention for obesity: a case series the person-based approach to intervention development: application to digital health-related behavior change interventions the authors would like to sincerely thank the women who participated for their generosity in sharing their thoughts and experiences with us. we would also like to thank the coordinating team who supported recruitment in this study: deussen a, kannieappan l. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-286298-pn9nwl64 authors: helmy, yosra a.; fawzy, mohamed; elaswad, ahmed; sobieh, ahmed; kenney, scott p.; shehata, awad a. title: the covid-19 pandemic: a comprehensive review of taxonomy, genetics, epidemiology, diagnosis, treatment, and control date: 2020-04-24 journal: j clin med doi: 10.3390/jcm9041225 sha: doc_id: 286298 cord_uid: pn9nwl64 a pneumonia outbreak with unknown etiology was reported in wuhan, hubei province, china, in december 2019, associated with the huanan seafood wholesale market. the causative agent of the outbreak was identified by the who as the severe acute respiratory syndrome coronavirus-2 (sars-cov-2), producing the disease named coronavirus disease-2019 (covid-19). the virus is closely related (96.3%) to bat coronavirus ratg13, based on phylogenetic analysis. human-to-human transmission has been confirmed even from asymptomatic carriers. the virus has spread to at least 200 countries, and more than 1,700,000 confirmed cases and 111,600 deaths have been recorded, with massive global increases in the number of cases daily. therefore, the who has declared covid-19 a pandemic. the disease is characterized by fever, dry cough, and chest pain with pneumonia in severe cases. in the beginning, the world public health authorities tried to eradicate the disease in china through quarantine but are now transitioning to prevention strategies worldwide to delay its spread. to date, there are no available vaccines or specific therapeutic drugs to treat the virus. there are many knowledge gaps about the newly emerged sars-cov-2, leading to misinformation. therefore, in this review, we provide recent information about the covid-19 pandemic. this review also provides insights for the control of pathogenic infections in humans such as sars-cov-2 infection and future spillovers. coronaviruses are enveloped, single-strand rna viruses that can infect a wide range of hosts including avian, wild, domestic mammalian species, and humans. coronaviruses are well known for their ability to mutate rapidly, alter tissue tropism, cross the species barrier, and adapt to different coronaviruses are enveloped, icosahedral symmetric particles, approximately 80-220 nm in diameter containing a non-segmented, single-strand, positive-sense rna genome of about 26-32 kb in size [34] . coronaviruses (covs) are one of the largest groups of viruses that belong to the order nidovirales, suborder cornidovirineae, and family coronaviridae. coronaviridae is classified into two subfamilies, namely, letovirinae and orthocoronavirinae. letovirinae includes the alphaletovirus genus, while orthocoronaviridae is further classified on the basis of phylogenetic analysis and genome structure into four genera: alphacoronavirus (αcov), betacoronavirus (βcov), gammacoronavirus (γcov), and deltacoronavirus (δcov), which contain 17, 12, 2, and 7 unique species, respectively (ictv 2018). the most recent classification of the coronaviridae is shown in table 1 . corona in latin means crown, and this name was attributed to the virus due to the presence of spike projections from the virus envelope that give it the shape of a crown under the electron microscope; nido means nest and refers to the ability of the viruses of this order to make a nested set of subgenomic mrna [25, 35] . coronaviruses infect a wide range of wild and domestic animals; αand βcovs infect mammals, while γand δcovs primarily infect birds (table 1) . a human coronavirus (hcov) was first isolated in 1960 from hospitalized patients who suffered from common cold symptoms and was named b814 [36] . so far, the seven different hcovs that infect humans are 229e, nl63, which belong to α covs, and hku1, oc43, sars, mers, sars-cov-2, which belong to βcovs. in 2002-2003, a pandemic caused by sars-cov (lineage b βcov) originated in china [37] . in the middle east, mers-cov (lineage c βcov) emerged in 2012 [27] . in 2019, a newly emerged sars-cov-2, closely related to bat sars-related covs, was clustered with lineage b βcov. chan et al. [13] demonstrated that sars-cov-2 represents a distinct lineage in the subgenus sarbecovirus (previously, lineage 2b of βcov) [38] . additionally, other coronaviruses have caused pandemic diseases in domestic and wild mammals and birds, leading to high mortality rates and severe economic losses. these viruses include ibv in chickens [40] , beluga whale coronavirus sw1 (bwcov-sw1) [41] , bat coronaviruses cdphe15 and hku10 (ictv 2018), porcine epidemic diarrhea virus (pedv), tgev, and sudden acute diarrhea syndrome (sads-cov) [42] . since the emergence of sars-cov-2 in wuhan city, china, in december 2019, many laboratories have been working on sequencing the genome of the causative agent. as of 14 april 2020, there are a total of 7655 complete genomes from 67 countries in the global initiative on sharing all influenza data (gisaid) database [43] (table 2) . a reference genome is now available in the ncbi genome database (29,903 nucleotide, reference sequence: nc_045512.3) [44] . to date, there are a total of 875 sequences including one refseq sequence and 768 complete genomes at ncbi. sars-cov-2 is a monopartite, single-stranded, and positive-sense rna virus with a genome size of 29,903 nucleotides, making it the second-largest known rna genome. the virus genome consists of two untranslated regions (utrs) at the 5 and 3 ends and 11 open reading frames (orfs) that encode 27 proteins (table 3) . the first orf (orf1/ab) constitutes about two-thirds of the virus genome, encoding 16 non-structural proteins (nsps), while the remaining third of the genome encodes 4 structural proteins and at least 6 accessory proteins. the structural proteins are spike glycoprotein (s), matrix protein (m), envelope protein (e), and nucleocapsid protein (n), while the accessory proteins are orf3a, orf6, orf7a, orf7b, orf8, and orf10, as shown in figure 1 [2, 13, 38, 45] . the 5′utr and 3′utr of sars cov-2 are comprised of 265 and 229 nucleotides, respectively. orf1ab is 21,290 nucleotides and encodes either replicase proteins pp1a of 4405 amino acids (aa) (nsp1-nsp11) or pp1ab of 7096 aa (nsp1-nsp16), according to ribosomal frameshift. of these proteins, (1) nsp1 suppresses the antiviral host response, (2) nsp3 is a papain-like protease, (3) nsp5 is a 3clpro (3c-like protease domain), (4) nsp7 makes a complex with nsp8 to form a primase, (5) nsp9 is responsible for rna/dna binding activity, (6) nsp12 is an rna-dependent rna polymerase (rdrp), (7) nsp13 is confirmed as a helicase, (8) nsp14 is a 3′-5′ exonuclease (exon), 9) nsp15 is a poly(u)-specific endoribonuclease (xendou). the remaining nsps are involved in transcription and replication of the viral genome [13, 46, 47] . single-stranded rna viruses exhibit a faster biological mutation rate due to the lack of proofreading activity of viral rna polymerases [48] ; however, unlike other mutation-prone rna viruses, with the exception of the arenaviridae family, covs do have limited proofreading capabilities, with the nsp14 protein allowing for the enhanced genome size of cov family members [49] . recombination is another mechanism of evolution in coronaviruses [50] . a high recombination frequency was demonstrated in murine hepatitis virus during mixed infection, where the majority of viruses recovered after three passages were recombinants [51] . recombination was also reported for mers-cov and sars-cov. seven putative recombination regions were detected in orf1ab and s protein between sars-cov and six other coronaviruses by in silico analysis of their genomes [52] . similarly, bioinformatic analysis of mers-cov genomic data revealed 28 recombinant sequences from humans and camels [53] . recombination in sars-cov-2 is not yet clearly understood. initial studies suggested that it may have occurred in the course of sars-cov-2 evolution [9] , while other researchers excluded the possibility of recombination based on a full genome evolutionary analysis investigating putative recombination events [11] . to better understand the evolution of sars-cov-2, we performed a phylogenetic analysis of 45 representative coronaviruses from 18 countries including sars-cov, sars-cov-2, hcov, bat sars . the other third of sars cov-2 includes four genes (in green) that encode four structural proteins (s, m, e, n), and six accessory genes (in blue) that encode six accessory proteins (orf3a, orf6, orf7a, orf7b, orf8, and orf10). the 5 utr and 3 utr of sars cov-2 are comprised of 265 and 229 nucleotides, respectively. orf1ab is 21,290 nucleotides and encodes either replicase proteins pp1a of 4405 amino acids (aa) (nsp1-nsp11) or pp1ab of 7096 aa (nsp1-nsp16), according to ribosomal frameshift. of these proteins, (1) nsp1 suppresses the antiviral host response, (2) nsp3 is a papain-like protease, (3) nsp5 is a 3clpro (3c-like protease domain), (4) nsp7 makes a complex with nsp8 to form a primase, (5) nsp9 is responsible for rna/dna binding activity, (6) nsp12 is an rna-dependent rna polymerase (rdrp), (7) nsp13 is confirmed as a helicase, (8) nsp14 is a 3 -5 exonuclease (exon), 9) nsp15 is a poly(u)-specific endoribonuclease (xendou). the remaining nsps are involved in transcription and replication of the viral genome [13, 46, 47] . single-stranded rna viruses exhibit a faster biological mutation rate due to the lack of proofreading activity of viral rna polymerases [48] ; however, unlike other mutation-prone rna viruses, with the exception of the arenaviridae family, covs do have limited proofreading capabilities, with the nsp14 protein allowing for the enhanced genome size of cov family members [49] . recombination is another mechanism of evolution in coronaviruses [50] . a high recombination frequency was demonstrated in murine hepatitis virus during mixed infection, where the majority of viruses recovered after three passages were recombinants [51] . recombination was also reported for mers-cov and sars-cov. seven putative recombination regions were detected in orf1ab and s protein between sars-cov and six other coronaviruses by in silico analysis of their genomes [52] . similarly, bioinformatic analysis of mers-cov genomic data revealed 28 recombinant sequences from humans and camels [53] . recombination in sars-cov-2 is not yet clearly understood. initial studies suggested that it may have occurred in the course of sars-cov-2 evolution [9] , while other researchers excluded the possibility of recombination based on a full genome evolutionary analysis investigating putative recombination events [11] . to better understand the evolution of sars-cov-2, we performed a phylogenetic analysis of 45 representative coronaviruses from 18 countries including sars-cov, sars-cov-2, hcov, bat sars cov, bat sars-like cov, and mers-cov. the viral genomes were obtained from the gisaid and ncbi databases. multiple sequence alignment was performed using kalign 3 [54] . a phylogenetic tree was constructed based on whole-genome sequences (coding sequences of all genes) in iq-tree, using the maximum likelihood method, ultrafast bootstrap approximation, and modelfinder [55, 56] . the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. the bootstrap values were determined by 1,000 replicates. the tree was visualized in mega x [57] ( figure 2 ). cov, bat sars-like cov, and mers-cov. the viral genomes were obtained from the gisaid and ncbi databases. multiple sequence alignment was performed using kalign 3 [54] . a phylogenetic tree was constructed based on whole-genome sequences (coding sequences of all genes) in iq-tree, using the maximum likelihood method, ultrafast bootstrap approximation, and modelfinder [55, 56] . the tree was drawn to scale, with branch lengths measured in the number of substitutions per site. the bootstrap values were determined by 1,000 replicates. the tree was visualized in mega x [57] ( figure 2 ). in the analysis we performed, all sars-cov-2 samples from the 18 countries clustered together and were close to bat sars or sars-like coronaviruses, with wuhan bat cov ratg13 being the closest virus. in addition, mers-cov and human cov hku1 were very distant from sars-cov-2 ( figure 2 ). within mers-cov samples, the south china mers-nl13892 clustered separately from other mers-covs. the two bat sars-like covs (bat-sl-covzc45 and bat-sl-covzxc21) were the second closest viruses from bats to sars-cov-2 ( figure 2 ). all sars-covs from china, canada, england, and the us were in a single cluster. the tree was constructed in iq-tree using the maximum likelihood method, modelfinder, and ultrafast bootstrap approximation (1000 replicates). the tree is drawn to scale, with branch lengths (numbers below the branches) measured in the number of substitutions per site. branch lengths less than 0.3 are not shown. numbers above the branches represent the percentage of replicate trees in which the associated viruses clustered together in the bootstrap test. the tree is rooted with two human coronavirus species from the genus alphacoronavirus as an outgroup (hcov-229e and hcov-nl63). in the analysis we performed, all sars-cov-2 samples from the 18 countries clustered together and were close to bat sars or sars-like coronaviruses, with wuhan bat cov ratg13 being the closest virus. in addition, mers-cov and human cov hku1 were very distant from sars-cov-2 ( figure 2 ). within mers-cov samples, the south china mers-nl13892 clustered separately from other mers-covs. the two bat sars-like covs (bat-sl-covzc45 and bat-sl-covzxc21) were the second closest viruses from bats to sars-cov-2 ( figure 2 ). all sars-covs from china, canada, england, and the us were in a single cluster. zhou et al. [9] conducted a phylogenetic analysis of sars-cov-2 against previously identified coronaviruses based on their whole-genome sequences, main structural protein genes, and non-structural protein genes. sars-cov-2 clustering was different depending on whether the whole genome or specific genes were used in the analysis. for example, sars-cov-2 clustered with the members of the subgenus sarbecovirus including the sars-cov (79.5% identical) that caused the global pandemic in 2003 and other bat sars-like viruses (96% identical at the whole-genome level), but the topological position within the sarbecoviruses changed when individual genes (orf1ab, s, e, m, and n) were used for clustering [2, 9] . based on the whole-genome sequence alignment, sars-cov-2 shares 89% identity with bat sars-like covzxc21, 82% with sars-cov, and 96.3% with bat cov ratg13 [11, 13] . alignment of the predicted protein sequences of sars-cov-2 to those of sars-cov or sars-like coronaviruses revealed a total of 380 amino acid substitutions between these viruses [2] . these amino acid substitutions were distributed as follows: 348 mutations in nonstructural proteins (orf1ab, 3a, 3b, 7a, 7b, 9b, and orf14), 27 in s protein, and 5 in n protein. no amino acid substitutions were detected in e or m proteins, indicating that e and m proteins are highly conserved among these viruses. it has been reported that sars-cov-2 uses the same cellular receptor, hace2, as sars-cov to gain entry into the cell [9, 58, 59] . the analysis of the receptor-binding domains (rbd) of sars-cov and sars-cov-2 s protein revealed similar binding affinities [60] . wu et al. [2] found a total of 27 amino acid substitutions in the s protein but not in the receptor-binding motif (rbm) that directly interacts with hace2, which may affect host tropism. these 27 substituted residues were distributed as follows: 17 in the s1 subunit [6 in the rbd and 6 in the subdomain (sd)] and 10 in the s2 subunit. wan et al. [58] reported similarity in the spike protein rbd, including rbm, of both sars-cov and sars-cov-2, in addition to the presence of several residues in sars-cov-2 rbm that favor the interaction with human ace2. these results agree with the genomic analysis of sars-cov-2, according to which the s2 subunit of the spike protein shares 99% identity with those of two bat sars-like covs (sl-covzxc21 and zc45) and of human sars-cov [13] . while the sars-cov-2 s2 subunit was conserved, the s1 subunit shares an overall 70% identity with those of bat and human sars-cov. the rbd core domain of s1 is highly conserved, with most of the amino acid differences located in the external subdomain that is responsible for the direct interaction with host receptors [13] . investigators have also reported the presence of a polybasic cleavage site and predicted o-linked glycans that are unique to sars-cov-2 s protein. differences in sars-cov-2 s protein and the high contagious nature of this virus suggest that sars-cov-2 has evolved via natural selection for binding to human ace2 receptor [61] . orf3b also differs in sars-cov-2. orf3b deletion mutations in sars-cov do not affect viral replication in vitro [62] . orf3b may play a role in viral pathogenicity in addition to its inhibitory effects on interferon (ifn) expression and signaling [63, 64] . recently, a novel short putative protein was identified in orf3b of sars-cov-2 [13] ; however, the function of this novel protein is still not known. sars-cov-2 orf8 is closer to those of bat sars cov zxc21 and zc45 and distant from that of human sars-cov [13] . the assessment of genetic diversity among 86 complete or semi-complete genomes of sars-cov-2 viruses revealed three deletions in the genome of isolates from japan, usa, and australia in addition to many other substitution mutations. the deletion mutations were in the orf1ab gene (3-nucleotide and 24-nucleotide deletion) and at the 3 end of the genome (10-nucleotide deletion). of the 93 substitution mutations, 42 changed the amino acid sequence of structural and non-structural proteins [65] . the 3and 24-nucleotide deletions in orf1ab are expected to reduce the protein sequence by 1 and 8 amino acid residues, respectively, without changing the reading frame, but the functional effects have yet to be investigated. the alignment of sars-cov-2 reference s protein gene against all sars-cov-2 sequenced genomes from china, usa, japan, australia, and taiwan revealed 99.97-100% identity, with 100% query coverage (also confirmed by our phylogenetic analysis, figure 2 ), while the identity and coverage for sars-cov s protein gene were 74.5% and 91%, respectively. also, the s protein gene from bat sars and sars-like coronavirus isolates shared 76.5-83% identity with that of sars-cov-2. this agrees with previous conclusions regarding the evolutionary analysis of sars-cov-2 [11, 44] . in the phylogenetic analysis we performed, sars-cov-2 viruses were in the same cluster regardless of the geographic region ( figure 2 ). these results strongly suggest the possibility of a recent common ancestor for all sars-cov-2 or the transmission of the same virus strain across countries. the outbreak of covid-19 originated from wuhan city, hubei province, in china. fifty-five percent of the infected cases before 1 january 2020 were linked to the huanan seafood wholesale market. however, the first human-to-human case of sars-cov-2 infection reported on 1 december 2019 did not have any exposure to this market [66, 67] . in mid-january 2020, sars-cov-2 spread to other provinces of china due to the spring festival travel season. sars-cov-2 was transmitted from china to other countries via international travelers. on 13 january 2020, the first case of sars-cov-2 infection was confirmed outside china in thailand, and on 16 january 2020 the first infected case was confirmed in japan. these cases were also linked to the huanan seafood wholesale market. by 25 january 2020, the number of confirmed cases had risen to 2062, including 2,016 in china, thailand, hong kong, macau, australia, malaysia, singapore, france, japan, south korea, taiwan, the us, vietnam, nepal, and sweden. on 30 january 2020, china reported a sharp rise in the number of infected cases, with the presence of infection in more than 18 countries. therefore, who declared the sars-cov-2 outbreak to be a public health emergency of international concern [68] . as of 16 march 2020, more than 150 countries and territories have been affected, with major outbreaks in central china, south korea, italy, iran, france, and germany [69] . there were 167,511 confirmed cases of sars-cov-2 infections, with 6606 deaths and about 8% estimated mortality rate. more than 73% of these cases have been reported in mainland china [69] . at this time, the number of global cases has shown a drastic increase within a short time, confirmed cases and deaths in china have not increased too much, while confirmed cases and deaths in other countries have drastically increased ( table 4 ). the number of confirmed cases increased from 2798 to 17,391 in one week (between 27 january and 3 february), and the number of infected countries doubled (from 12 to 24). due to the rapid increase of the number of infected cases and infected countries, the who declared sars-cov-2 a pandemic on 11 march 2020 and on 13 march 2020, the who declared europe to be the new center of the pandemic due to the massive increase of confirmed cases there [70] . on 15 ,889, were in italy). one week later (13 april 2020), the number of confirmed cases of sars-cov-2 increased 1.7 times (up to 524,514 confirmed cases), and the number of deaths increased 2.5 times (up to 20,444 deaths) in the usa alone. the number of confirmed cases, deaths, and infected countries are shown in table 4 . the origins of more than 75% of coronavirus infections are considered zoonotic, i.e., animals are the main source of the outbreaks. for example, sars-cov was transmitted from palm civets to humans, and mers-cov from dromedary camels to humans. bats are currently considered a reservoir for all human coronaviruses, as mentioned above [5, 71] . many coronaviruses are circulating in animals but have not yet infected humans. the type of animal that sars-cov-2 originated from is still unclear. at the beginning of the outbreak in wuhan, china, many patients were linked to the huanan seafood wholesale market, suggesting animal-to-person spread. after retrospectively studying case reports, the number of patients that did not have exposure to animal markets has risen, indicating person-to-person spread was also occurring at that time [66] . sars-cov-2 is closely related to bat coronaviruses and sars-cov [8] . a group of researchers reported early in the outbreak that the novel sars-cov-2 has the highest similarity of codon usage bias with snakes [72, 73] ; however, this method to determine initial host origins is dubious. interestingly, researchers also reported one amino acid difference in the receptor-binding domain of the s protein of pangolin-cov compared to that of sars-cov-2, suggesting that pangolins might play a role as an intermediate host (xiao et al., data currently under review). another group of researchers reported that the virus originated from bats based on the genome sequence of sars-cov-2, which is 96% identical to bat coronavirus ratg13. there were speculations that sars-cov-2 is a laboratory-engineered cov and leaked directly from a laboratory in wuhan where a bat cov (ratg13) was recently reported. however, there is no evidence to support this allegation [74] . recently, a group of researchers found that sars-cov-2 replicates poorly in dogs, pigs, chickens, and ducks but efficiently in ferrets and cats [75] . scientists are still trying to find the main source of the disease outbreak and identify the definitive intermediate hosts. both established (sars-cov, mers-cov) and novel (sars-cov-2) coronaviruses were reported to spread from an infected person to a non-infected person through direct or indirect contact. sars-cov-2 infection was reported to be transmitted directly from person to person like most respiratory viruses via close contact with an infected person or through respiratory droplets (aerosol) produced when an infected person coughs or sneezes. these droplets can be inhaled to reach the lung. the virus can be indirectly transmitted via touching a surface or an object that was previously contaminated with the virus and then touching the face, eyes, or mouth [76] and possibly via the fecal-oral route [77, 78] . asymptomatic carriers (during the incubation period of the virus) and patients after recovery from the acute form of the disease are also considered a potential source of virus transmission to healthy persons [10, 12] . interestingly, human coronaviruses are able to survive on steel, metal, wood, aluminum, paper, glass, plastic, ceramic, disposable gowns, and surgical gloves for 2-9 days. high temperature (≥30 • c) can reduce the persistence period, while low temperature (4 • c) increases the persistence time up to 28 days [79] . transmission of the virus vertically from mother to fetus or via breast milk has not been confirmed yet [80] . the transmission cycle of coronavirus among animals and humans is shown in figure 3 . both established (sars-cov, mers-cov) and novel (sars-cov-2) coronaviruses were reported to spread from an infected person to a non-infected person through direct or indirect contact. sars-cov-2 infection was reported to be transmitted directly from person to person like most respiratory viruses via close contact with an infected person or through respiratory droplets (aerosol) produced when an infected person coughs or sneezes. these droplets can be inhaled to reach the lung. the virus can be indirectly transmitted via touching a surface or an object that was previously contaminated with the virus and then touching the face, eyes, or mouth [76] and possibly via the fecal-oral route [77, 78] . asymptomatic carriers (during the incubation period of the virus) and patients after recovery from the acute form of the disease are also considered a potential source of virus transmission to healthy persons [10, 12] . interestingly, human coronaviruses are able to survive on steel, metal, wood, aluminum, paper, glass, plastic, ceramic, disposable gowns, and surgical gloves for 2-9 days. high temperature (≥30 °c) can reduce the persistence period, while low temperature (4 °c) increases the persistence time up to 28 days [79] . transmission of the virus vertically from mother to fetus or via breast milk has not been confirmed yet [80] . the transmission cycle of coronavirus among animals and humans is shown in figure 3 . there are many factors that affect sars-cov-2 transmission and spread. these factors include, but are not limited to: (1) travel to or contact with individuals who have recently visited wuhan, china, or other places experiencing an outbreak; (2) close contact with persons who are diagnosed positive for the disease, such as healthcare workers caring for patients with sars-cov-2; (3) contact with droplets and secretions (produced by sneezing or coughing) from an infected person and eating or handling wild animals native to china such as bats. additionally, the risk of infection is higher for the elderly and for patients suffering from pre-existing illnesses such as cardiovascular disease, hypertension, diabetes, and chronic respiratory disease [66] . the reported fatality rate based on age is 14.8% for people ˃80 years of age, 8% for people between 70 and 79 years, 3.6% for people between 60 and 69 years, 1.3% for people between 50 and 59 years, 0.4% for people between 40 and 49 years, there are many factors that affect sars-cov-2 transmission and spread. these factors include, but are not limited to: (1) travel to or contact with individuals who have recently visited wuhan, china, or other places experiencing an outbreak; (2) close contact with persons who are diagnosed positive for the disease, such as healthcare workers caring for patients with sars-cov-2; (3) contact with droplets and secretions (produced by sneezing or coughing) from an infected person and eating or handling wild animals native to china such as bats. additionally, the risk of infection is higher for the elderly and for patients suffering from pre-existing illnesses such as cardiovascular disease, hypertension, diabetes, and chronic respiratory disease [66] . the reported fatality rate based on age is 14.8% for people >80 years of age, 8% for people between 70 and 79 years, 3.6% for people between 60 and 69 years, 1.3% for people between 50 and 59 years, 0.4% for people between 40 and 49 years, 0.2% for people between 10 and 39 years; no fatalities have been reported for children under 10 years of age. notably, the fatality rate is higher in males (2.8%) than in females (1.7%) [81, 82] . the estimated incubation period of the novel coronavirus ranges from 2 to 14 days. however, some cases had an incubation period of 21, 24, or 27 days [83] . the complete clinical picture of sars-cov-2 is still unclear. the disease begins with flu-like symptoms that include fever, fatigue, dry cough, sore throat, shortness of breath, headache, chest tightness, chest pain, and muscle pain. some of sars-cov-2 patients have runny nose, nausea, vomiting, and diarrhea [84] . people can be infected without showing symptoms, which allows the virus to spread more effectively from person to person. complications can occur due to covid-19 leading to severe infections, such as pneumonia (infection of the lungs), kidney failure, and death [8] . the mild phase of the disease can last up to 2 weeks, while severe or critical disease lasts approximately 3 to 6 weeks (this analysis was conducted on 55,924 confirmed cases). additionally, the time from the disease onset to the development of severe disease is one week, while the time from the onset of symptoms to death ranges from 2 to 8 weeks [82] . based on the data analysis of 72,314 confirmed cases of sars-cov-2 in wuhan city, china, by 11 february, 80.9% of the cases were mild with flu-like symptoms, and patients recovered at home, 13.8% were severe with pneumonia and shortness of breath, 4.7% were critical with respiratory failure and septic shock resulting in organs failure, and approximately 2% of the cases were fatal [85] . another study was conducted on 99 hospitalized patients, and symptoms were classified as follow: fever (83%), cough (82%), shortness of breath (31%), muscle ache (11%), confusion (9%), headache (8%), sore throat (5%), runny nose (4%), chest pain (2%), diarrhea (2%), and nausea and vomiting (1%) [8] . the rapid diagnosis of sars-cov-2 infection is the cornerstone of disease control. it depends on several criteria including case history, clinical symptoms, serology, molecular diagnosis, and computed tomography (ct) imaging. on 2 march 2020, who published interim guidance for laboratory testing of suspected human cases, with precautions for specimen collection, packing, shipment, and amplification of nucleic acid to detect viral genes (n, e, s, and rdrp) [82] . sars-cov-2 uses the same cell entry receptor, hace2, as sars-cov. therefore, oral swabs, bronchoalveolar lavage fluid (balf), blood, as well as anal swabs are the best samples used for virus diagnosis [86] . a proper diagnosis depends primarily on the factors described below. the strict monitoring of case history in clinically suspicious patients is considered the first step in the early diagnosis of sars-cov-2 infection. clinically suspicious patients are those who suffer from fever and lower respiratory tract infection symptoms (for details, see the clinical characteristics section) and reside within or have traveled to endemic regions or had close contact with a confirmed or suspected case. additionally, sars-cov-2 can be transmitted by symptomatic and asymptomatic patients especially to the high-risk group mentioned above (for details, see the risk assessment section) [13] . the blood profiles of patients suffering from sars-cov-2 infection revealed the following: (1) increased c-reactive protein and erythrocytes, (2) increased myohemoglobin, liver enzymes, and muscle enzymes, with a high level of d-dimer in severe cases, and (3) normal or decreased white blood cell counts and lymphocytes in the early stage of the disease, with advanced lymphocytopenia in severe cases [13] . in icu patients, high levels of plasma granulocyte colony-stimulating factor (gcsf), ip10, il2, il7, il10, tnf-α, and mip1a were reported [38] . electron microscope examination of sars-cov-2 revealed the typical coronavirus morphology. further, sars-cov-2 was successfully isolated from human respiratory epithelial cells or balf samples of infected patients using huh7 cells and vero e6 cells. the isolated strain was confirmed by immunofluorescent antibody techniques using the cross-reactive nucleoprotein (np) antibody. serum neutralization tests (snt) using vero e6 cells were conducted to confirm the neutralization activity in igg-positive viral samples [87] . igm and igg elisa detection kits using bat sarsr-cov rp3 np were developed with no cross-reaction against human coronaviruses except sarsr-cov [80] . using these serological tools, viral antibody titers were increased in sars-cov-2-infected patients [38] . the procedures of elisa for the determination of sars-cov-2 igg were described before [86] . nucleic acid detection is the main, fastest, and most sensitive test for the diagnosis of sars-cov-2 infection. recently, two nested rt-pcr and two real-time rt-pcr assays have been developed with successful detection of the first 25 positive cases of infection in japan [88] . three real-time rt-pcr techniques have been designed based on the e, rdrp, and n genes [89] . also, scientists established molecular detection tools for sars-cov-2 based on the s gene [86] . chest x-ray examination in the early stage of the disease shows interstitial changes and multiple small plaque shadows. chest ct scans play an important role in the diagnosis of acute respiratory disease syndrome (ards) and pneumonia as well as in the early detection of lung parenchymal abnormalities in patients at risk and provide an impression of secondary infection (figure 4 ). electron microscope examination of sars-cov-2 revealed the typical coronavirus morphology. further, sars-cov-2 was successfully isolated from human respiratory epithelial cells or balf samples of infected patients using huh7 cells and vero e6 cells. the isolated strain was confirmed by immunofluorescent antibody techniques using the cross-reactive nucleoprotein (np) antibody. serum neutralization tests (snt) using vero e6 cells were conducted to confirm the neutralization activity in igg-positive viral samples [87] . igm and igg elisa detection kits using bat sarsr-cov rp3 np were developed with no cross-reaction against human coronaviruses except sarsr-cov [80] . using these serological tools, viral antibody titers were increased in sars-cov-2-infected patients [38] . the procedures of elisa for the determination of sars-cov-2 igg were described before [86] . nucleic acid detection is the main, fastest, and most sensitive test for the diagnosis of sars-cov-2 infection. recently, two nested rt-pcr and two real-time rt-pcr assays have been developed with successful detection of the first 25 positive cases of infection in japan [88] . three real-time rt-pcr techniques have been designed based on the e, rdrp, and n genes [89] . also, scientists established molecular detection tools for sars-cov-2 based on the s gene [86] . chest x-ray examination in the early stage of the disease shows interstitial changes and multiple small plaque shadows. chest ct scans play an important role in the diagnosis of acute respiratory disease syndrome (ards) and pneumonia as well as in the early detection of lung parenchymal abnormalities in patients at risk and provide an impression of secondary infection ( figure 4 ). assessing these lungs parenchymal abnormalities conveys disease severity to clinicians. using artificial intelligence models in the future may be useful in mass screening, to allow risk prioritization and help to minimize turnaround time [90] . pan et al. [91] conducted a retrospective study to elaborate the time course of lung changes during recovery from infection. they described assessing these lungs parenchymal abnormalities conveys disease severity to clinicians. using artificial intelligence models in the future may be useful in mass screening, to allow risk prioritization and help to minimize turnaround time [90] . pan et al. [91] conducted a retrospective study to elaborate the time course of lung changes during recovery from infection. they described findings using international standard nomenclatures such as ground-glass opacity (ggo), consolidation, and crazy paving patterns. they established a semi-quantitative scoring system of 5 grades to quantify the degree of involvement based on an area ranging from 0% to >75%. the total score ranged from 0 to 25 (max), and involvement was subpleural, random, or diffuse. they found that in early stages (0-4 days after the onset of symptoms), ggo was the main finding in lower lung lobes; in progressive stages (5-8 days), the progression of lung disease involved three patterns of ground-glass, consolidation, and crazy paving, while in peak stages (9-13 days) , dense consolidation became the prevalent feature; in absorption stages (>14 days), ggo was detected with no crazy paving and resolution of consolidations [91] . more than 75% of sars-cov-2-affected patients suffered from bilateral lung involvement, and 71% have multilobe involvement. ct examinations of 21 patients showed 29% consolidation and 86% ggo in the chest [8, 9, 92, 93] . another study examined 51 cases by ct and reported that 77% showed pure ggo, 75% exhibited ggo with reticular and/or interlobular septal thickness, 59% had ggo with consolidation, while 55% revealed pure consolidation. bilateral lung involvement was reported in 86% of cases; in 80% of the cases the posterior part of the lung was involved, while in 86%, the periphery was involved [94] . in january 2020, the who issued guidance for the clinical management of sars when sars-cov-2 infection was suspected. in this guidance, the start of emergency treatments, immediate implementation of prevention and control strategies, early supportive therapy and prevention of sars-cov-2 complications were described in detail [15] . so far, there are no approved specific antiviral drugs for sars-cov-2 infection. therefore, preventive measures and inactivation of the virus are essential to stop and control the spread of the disease. human coronaviruses can be inactivated using 0.5% hydrogen peroxide, 62-71% ethanol, 0.1% sodium hypochlorite, 0.7-1% formaldehyde, 2% glutaraldehyde, or 0.23% povidone iodine within 1 minute. other disinfectants such as 0.02% chlorhexidine digluconate, 0.55% orthophtalaldehyde, or 0.05-0.2% benzalkonium chloride are less effective [79] . in light of the urgent clinical demand, many drugs are approved to be used for clinical trials against sars-cov-2 infection, such as lopinavir/ritonavir, arbidol, interferon-alpha, favipiravir, chloroquine phosphate, darunavir/cobicistat, oseltamivir, and methylprednisolone. the most used antiviral drugs [95] are summarized in table 5 . generally, coronaviruses are not sensitive to current antiviral drugs, and high concentrations of drugs effective on these viruses cannot be used in vivo. therefore, combinations of different therapies have been used for the treatment of coronavirus infections [96] . some drug combinations that could be successful for the treatment of sars-cov-2 patients are lopinavir and ritonavir [97, 98] , lopinavir/ritonavir plus arbidol [99] , and ribavirin and interferon [100, 101] . the use of anti-inflammatory drugs such as glucocorticoids, il-6 antagonist, janus kinase inhibitors (jak), and choloroquine/hydrocholoroquine in sars-cov-2 patients is a dilemma, especially in patients suffering from an impaired immune system. balancing the risk-benefit ratio is a critical issue. corticosteroids may delay the elimination of the virus and increase the risk of secondary infection. in addition, drugs targeting pro-inflammatory cytokines can only inhibit specific inflammatory factors and thus may not be very effective in curbing the cytokine storm (excessive and uncontrolled release of pro-inflammatory cytokines). moreover, some anti-inflammatory drugs such as jak block inf-α production, which is important in fighting the virus [102] . additionally, fecal transplantation was approved for clinical trials as a therapeutic option for sars-cov-2-related pneumonia based on the promising results obtained from fecal microbiota transplantation in patients suffering from antibiotic-associated diarrhea, active ulcerative colitis, and other viral infections [103] [104] [105] [106] . recently, it was found that intestinal microbiota-derived ifn in lung stroma confers protection against viral diseases such as avian influenza and respiratory syncytial virus [103] . moreover, based on historical records of the effect of antiviral herbs on sars and influenza h1n1, chinese herbal formulas could be an alternative approach for the prevention of sars-cov-2 in a high-risk population [107] , if no scientifically based therapeutics are available. it was found that sambucus formasana nakai exhibited a strong antiviral effect against human coronavirus nl63 [108]. increases ph in host cell lysosomes and negatively influences virus-receptor binding, as well as interferes with the glycosylation of cellular receptors of sars-cov -exhibited a promising antiviral effect against sars-cov-2 in vitro -improved covid-19-pneumonia patients and shortened the course of the disease [114] remdesivir a monophosphoramidate of adenosine prodrug that incorporates into nascent viral rna chains causing pre-mature termination -used against a wide range of rna viruses such as filoviridae, paramyxoviridae, pneumoviridae, and coronaviridae; used successfully in covid-19 treatment in the united states and showed no adverse events [115] darunavir and cobicistat inhibit 3 c-like protease (3clpro). -used for the treatment of mers-cov in experimental animals -used for the treatment of hiv-1 patients [116] to date, there is no vaccine to prevent sars-cov-2 infection, and trials for vaccine development are in the preliminary stages of research. several vaccine candidates such as live attenuated, adenovirus-vectored, recombinant protein, and nucleic acid (dna and mrna) vaccines are in the pipeline [123] . the epidemiology of sars-cov-2 is still unclear, and data availability is limited. therefore, it is imperative to follow preventive measures and safety precautions issued by health authorities to limit exposure to the virus and to reduce further spread. general hygienic measures should be implemented, such as (1) washing hands often with soap and water or an alcohol-based hand sanitizer, (2) cough or sneeze etiquette, recommending covering of the mouth, (3) avoiding touching eyes, nose, and mouth if the hands are not clean, (4) avoiding close contact with sick persons, (5) avoiding sharing dishes, glasses, bedding, and other household items with sick people, (6) cleaning and disinfection of surfaces that are often touched, and (7) staying home from work, school, and public areas when feeling sick. the transmission route of sars-cov-2 is probably not only through cough, respiratory droplets, and/or contaminated surfaces [13, 124] , but also through fecal-oral transmission [78] . therefore, strict hygienic measures should be followed, especially in dense cities or agricultural spaces [125] . since the sars-cov-2 spread is primarily driven by travel, screening of travelers who arrive at airports from pandemic areas for possible sars-cov-2 infection and entry-screening procedures are necessary. also, general hygienic precautions during travel are highly recommended. travelers who suffered from acute respiratory infection should be tested and reported to the respective public health authorities [73] . in addition, people should be motivated to notify and report about travel history and close contacts in case of sars-cov-2 infection. asymptomatic carriers (during the incubation period) and patients after recovery from the acute form are also considered potential sources of the virus [12, 13] . strict hygienic measures should be implemented to avoid virus transmission to healthcare workers and other contacts, i.e., placement of sars-cov-2 suspected or confirmed patients in single-person rooms and wearing personal protection equipment (ppe) such as masks, goggles, and protective gowns. because early diagnosis and detection of asymptomatic carriers of sars-cov-2 are successful factors for the treatment and prevention of transmission, health authorities should designate laboratories to implement tests for a rapid and accurate diagnosis [126] . the control of coronaviruses is based on biosecurity regarding animals as well as on shifts in food habits, including discouraging the consumption of bushmeat and of animal products without appropriate cooking [127] . ban of wet marketplaces where live or dead animals are handled should be implemented. surveillance among people who have contact with wildlife and improvement of biosecurity regarding wildlife trade are urgently needed to prevent the next pandemic outbreak [128] . the epidemiology of sars-cov-2 is still unclear. many unresolved questions related to sars-cov-2 epidemiology and pathogenicity pose great challenges for researchers. these unresolved questions include: what is the origin of sars-cov-2? what is the intermediate host that transmitted the virus from bats? why does the virus cause severe disease and mortality in the elderly or those with co-morbidities, while it is milder in children? are aerosol, saliva, feces, urine, and foodborne the only routes of transmission? what are the other unknown routes of transmission? control of the sars-cov-2 outbreak and future epidemics requires global efforts among medical and veterinary clinicians, diagnosticians, epidemiologists, public health experts, vaccinologists, pharmaceutical industries, economists, and governments to implement a one-health approach [128, 129] . these measures must include: (1) writing policies and supporting funds required for the implementation of one health, prevention, and control measures, (2) hiring well-trained and professional personnel, (3) performing rapid and accurate diagnosis and treatment of infected persons, (4) developing and providing vaccines for virus control in humans, (5) conducting surveillance among wildlife for the identification and characterization of possible reservoirs and surveillance among people who are in contact with wildlife to identify risk factors in human behaviors and living environment, (6) improving hygienic measures, (7) assessing the social and economic impacts of covid-19 on the population, (8) utilizing veterinary experience in the disinfection of premises and gatherings under the supervision of health authorities to decrease outbreaks in humans, (9) providing antiviral drugs for the treatment of the disease in humans, and (10) increasing public health awareness about the virus and its transmission. the sars-cov-2 outbreak started in wuhan city, china, in december 2019. it is now a global pandemic, with 1,773,084 confirmed cases, 111,652 deaths, and 467,074 recoveries (as of 13 april 2020). the virus has the potential for rapid and extensive spread between people and countries. there are a lot of misleading information and knowledge gaps on the newly emerged sars-cov-2. therefore, we reviewed the latest updates about different aspects including epidemiology, source of infection, transmission dynamics, zoonotic potential, virus characteristics, and discovery of novel strategies for disease control to avoid spillover of infection in the future. bats play an important role in the transmission of the infection to humans. coronaviruses are genetically diverse and have a high tendency towards frequent genetic mutations and gene recombination, which increases the risk of interspecies transmission. information about the incubation period can help in establishing an effective quarantine for asymptomatic carriers, thus preventing the virus spread. from our perspectives and based on the currently available information 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future pandemics similar to the 2019-ncov outbreak the one health approach is necessary for the control of rift valley fever infections in egypt: a comprehensive review this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we gratefully acknowledge the authors, originating, and submitting laboratories of the sequences from the gisaid's epicov™ database, on which part of the phylogenetic tree construction is based. the authors declare no conflict of interest. key: cord-352668-qjlqsb2k authors: cabello, francisco; sánchez, froilán; farré, josep m.; montejo, angel l. title: consensus on recommendations for safe sexual activity during the covid-19 coronavirus pandemic date: 2020-07-20 journal: j clin med doi: 10.3390/jcm9072297 sha: doc_id: 352668 cord_uid: qjlqsb2k sexual activity offers numerous advantages for physical and mental health but maintains inherent risks in a pandemic situation, such as the current one caused by sars-cov-2. a group of experts from the spanish association of sexuality and mental health (aesexsame) has reached a consensus on recommendations to maintain lower-risk sexual activity, depending on one’s clinical and partner situations, based on the current knowledge of sars-cov-2. different situations are included in the recommendations: a sexual partner passing quarantine without any symptoms, a sexual partner that has not passed quarantine, a sexual partner with some suspicious symptoms of covid-19, a positive sexual partner with covid-19, a pregnant sexual partner, a health professional partner in contact with covid-19 patients, and people without a sexual partner. the main recommendations include returning to engaging in safe sex after quarantine is over (28 days based on the duration one can carry sars-cov-2, or 33 days for those who are >60 years old) and all parties are asymptomatic. in all other cases (for those under quarantine, those with some clinical symptoms, health professionals in contact with covid-19 patients, and during pregnancy), abstaining from coital/oral/anal sex, substituting it with masturbatory or virtual sexual activity to provide maximum protection from the contagion, and increasing the benefits inherent to sexual activity are recommended. for persons without a partner, not initiating sexual activity with a sporadic partner is strongly recommended. sexuality is one of the aspects of personality in which the degree of intimacy and privacy is great. asking patients about their sex life often arouses misgivings and feelings of shame and/or guilt [1] . however, scientific evidence shows that successful sexuality benefits males and females physically and emotionally to, having a favourable impact on their quality of life. there is evidence that sexual activity has advantages for humans, including increasing our longevity [2] [3] [4] and improving our immune system, among others [5, 6] . additionally, successful sexual activity increases psychic wellbeing by improving mood, even in depressed and high-anxiety patients [7, 8] , falling asleep [9] ; stress [10] ; relaxation [11] ; physical form and providing a younger body image [12] thereby contributing to the prevention of post-traumatic stress and anxiety disorders [13] . sexual experience regularises the menstrual cycle [14] , relieves dysmenorrhea and reduces the risk of endometriosis [15] . sexual dysfunctions can cause some interpersonal conflicts by deteriorating either self-esteem or partner relationships [16] . additionally, it may constitute an early sign of some organic pathology such as cardiovascular [17] , neurological or endocrine diseases. there is also some evidence that sexual inactivity correlates with an increased frequency of cancer, need for major surgery, worsening mental health and the increase of cognitive decline and cardiovascular disease risk factors such as diabetes, hypertension and hypercholesterolemia [18] . the effects of sars-cov-2 on human sexual and reproductive function, including whether the virus passes the blood-testis and ovary barriers and whether there is any effect on sexual hormone production, are still unknown [19] . additionally, some studies are currently seeking to identify similar impacts across the different populations impacted by hiv [20] . in the current sars-cov-2 pandemic situation, sexual activity during quarantine could be a relevant aid in reducing the onset of post-traumatic stress and anxiety disorders that were experienced in other previous pandemic confinements. in canada, during the outbreak of severe acute respiratory syndrome (sars) in 2003, a high level of acute stress was observed among health workers [21] . ebola virus confinement in africa also increased suicides [22] and gender-based violence [23] . during the australian equine fever quarantine, a high level of anxiety was observed in 34% of those confined compared to 12% in those not confined [24] . in china, some of the health workers quarantined during the sars epidemic-maintained symptoms of post-traumatic stress disorder three years later [25] . sexual satisfaction is a good predictor of global life satisfaction in young people and older adults [26] . in a large survey on sexual health in spain [27] , people were interviewed about their motivation for sexual intercourse, and the vast majority pointed out that the main reason was either the search for emotional intimacy or to satisfy the need to love and be loved. additionally, sexuality, as a basic aspect of mental health, is a current topic of interest for clinicians and researchers [28] . there is some literature indicating the potential benefits of increased sexual activity during periods of forced isolation indicating that those who maintain frequent in-person, but not remote, social and sexual connections have better mental health outcomes [29] . given the psychologically negative repercussions of previous quarantines and the preventive benefits of healthy sexuality, it is reasonable to maintain one's safe sexual frequency. however, sexual intercourse requires close physical contact, and sars-cov-2 is very easily transmitted with this level of closeness [30] . physical contact entails high viral exposure. when sharing a home with a covid-19-positive person, the virus has been detected in 63.2% of room air samples and 66.7% of corridor air samples [31] . other known coronaviruses do not appear to be sexually transmitted, but sars-cov-2 has been found in bodily fluids such as the saliva, mucus, and faeces of infected people, albeit slightly less in urine (6.9%). some recent studies have reported the virus to be present in the testicular seminal duct [32, 33] compromising the safety of sexual intercourse by persistence for at least 2 weeks postinfection in urine, faeces and nasopharynx secretions. considering that 80% of those infected have mild symptoms or are asymptomatic, it is advisable to take some precautions at least during quarantine. the use of condoms and noncoital behaviour that does not involve direct contact with semen is highly recommended [34, 35] . the virus was very recently found in the vaginal discharge of an infected 65-year-old female even while she was receiving oral lopinavir/ritonavir plus remdesevir. after two previous negative results, the vaginal swab tested positive via a real time reverse transcriptase-polymerase chain reaction on days 7 and 20 from symptom onset [36] . this new finding raises the possibility that sexual intercourse could be an additional direct vector of infection, adding to the recent evidence of a likely faecal-oral transmission vector [37] , or indirectly by exposure of the rectal mucosa to saliva [38] . additionally, patients can persistently test positive on rectal swabs even after negative results from nasopharyngeal testing [39] . thus sexual, transmission may be possible despite apparent clinical recovery. using real-time reverse transcription polymerase chain reaction to routinely test for sars-cov-2 in faeces was recently recommended [37] . patients' sexual habits should be routinely investigated in order to avoid direct sexual practices if infected with covid-19. physicians should always address these questions in epidemiologic surveys on transmission routes in order to determine effective strategies to control infection. information about changes in sexual habits in the isolated population and in those infected by the virus is scarce so far. an increase in both, sexual desire and the frequency of sexual intercourse during the current covid-19 pandemic, compared to the previous 6-12-month period, has recently been shown, although the quality of sex decreased significantly [40] . however, another recent study showed that during the covid-19 outbreak, the frequency of sexual activity in china decreased significantly in men and women, accompanied by a lowering of risky sexual behaviours [41] . the main objective is to avoid contagion by covid-19 and, at the same time, maintain, as far as possible, active sexuality, given the multiple advantages that healthy sexuality brings according to scientific evidence. due to the ease of contagion and the lack of information about the possible transmission of sars-cov-2, a group of experts from the spanish association for sexuality and mental health, covering the fields of sexology, psychiatry, psychology and medicine reached a consensus. the multidisciplinary panel included four experts in the fields of family medicine, sexology, epidemiology, psychology and psychiatry. a bibliographic search was performed in the medline, scopus, psycinfo and web of science databases without time limits. after searching the information sources, two reviewers independently preselected potentially relevant references using the keywords; sexual * and coronavirus or covid-19 or pandemic (645 refs). after preselection the search was refined, duplicates were removed, and 83 refs were found. after reading the complete articles, those that would ultimately form part of the review were selected (38) . based on the current knowledge of the scientific literature, and considering the absence of either clinical guidelines or recommendations in this regard, the authors have developed a consensus on some specific recommendations to maintain safe sexual activity and to prevent the transmission of covid-19. the authors carried out three preliminary drafts until a full consensus of the final text was reached. in order to avoid the risk of contagion, the main recommendation is that tongue kissing and oral-sex relationships should be avoided. as indicated by recent recommendations from the new york department of health, "you are your safest sexual partner" [42] . thus, during the pandemic, and until the end of quarantine, it is a good time to devote oneself to autoerotic growth, which means, improving sexual health, and therefore mood, by training to optimise sexual response. under confinement with a sexual partner (not always a household partner), including the full diversity of partner types (homo, hetero, bisexual, nonheteronormative couples with polyamory or those who maintain living apart), there are several possibilities we can recommend to improve safe sexual activities by avoiding the risk of contagion to the greatest extent. to clarify the concept of "safe quarantine", our recommendation includes avoiding contact with high-risk populations during quarantine and avoiding restarting sex when in contact with a confirmed or highly suspected case. we contemplate two main scenarios: (a) partners living in the same household and (b) partner/s not living in the same household/starting a new relationship or polyamory. a. partner/s living in the same household. a sexual partner after passing complete asymptomatic quarantine. a sexual partner during quarantine. a sexual partner with suspicious symptoms of covid-19. a sexual partner that is positive for covid-19 5. a pregnant sexual partner. a sexual partner in contact with covid-19 patients. b. partner/s not living in the same household/starting a new relationship or polyamory. current information indicates that the average incubation period for covid-19 is 5.1 days (95% ci 4.5-5.8 days) and that 97.5% of cases developed symptoms in 11.5 days (95% ci: 8.2-15.6 days). the incubation period before the onset of fever is 5.7 days, and 98% of those infected have a fever in the first 12.5 days. only 1% develop symptoms after 14 days; there is a small but real possibility of symptomatology at 28 days after infection [43] . the recommendation is that after 28 days of confinement without symptoms and without external contact, sexual intercourse can be carried out according to the usual habits of the couple, but while increasing hygiene before and after sexual activity. this would be a good time to enhance sexual creativity by increasing communication about one's preferences and desirable experiences, possibly by sharing fantasies and testing new scenarios, engaging in erotic readings, or viewing erotic films. before starting sexual activity, proper handwashing becomes essential to avoid virus transmission through fomites [44] and by touching the t-zones (mouth, nose and eyes) of the sexual partner's face [45] . it is also advisable to recommend strategies to avoid self-contact with one's own t-zones, as it happens without realisation at an average of 23 times per hour [46] . in this scenario, we must take into account that the severity of covid-19 depends on several factors, including viral load. it is possible that one member of the couple could be contaminated but asymptomatic as seen in a sample of 157 cases in singapore where 6.4% of presymptomatic transmission was identified [47] . in this case, sexual intercourse could be a major risk of contagion. the recommendation, before 28 days, is that one can practice safe sex using penetrative positions from behind, avoiding kissing, oral and anal sex, and by always using condoms. if a partner shows any suspicious symptoms, such as fever (which may be intermittent), cough, diarrhoea, severe and unexplained tiredness, sore throat, anosmia, hypogeusia, or other symptoms associated with covid-19, he or she may be in the incubation period or suffering from a mild form of the disease. the recommendation would be to avoid direct sexual activity. as a substitute, performing self-stimulation (masturbation while simultaneously keeping the safety distance of approximately 2 m), narrating erotic fantasies, sharing visualisations of erotic scenes, or using erotic board games might be suggested. if a sexual partner has tested positive for sars-cov-2, having sex is a possible source of contagion, so it is advisable to isolate in separate rooms. however, it is advisable to continue to practice some kind of sexual activity while avoiding physical contact, as recommended previously. one can, for example, use telematic applications to maintain erotic activities. it should be considered that people with active covid-19 infections may not want to engage in any sexual activity (even remote/virtual connections,) depending on the severity of their symptoms and personal preferences. once symptoms disappear, there is a high probability of contagion, as the virus persists in nasal secretions for an average of 9.5 days and in stool from 11 to 16 days (20 days if treated with corticosteroids) [48] . the duration for carrying sars-cov-2 in covid-19 (between 16-28 days) can increase up to 33 days in those over 60 years old, as well as in very severe cases [49] . waiting for this period of time before having coital sex and maintaining the recommended precautions for confined stable sex partners without passing quarantine are recommended. it is currently unknown whether pregnant individuals are more likely to get sick with covid-19, and there is still little information on the severity of the disease [50, 51] . other coronaviruses and viral respiratory infections, such as the flu, may carry a higher risk of developing complications or serious illnesses in pregnant women. therefore, since this is a population at risk, precautions should be increased [52] . pregnant individuals do not appear to be at greater risk than the general population, except for the presence of other associated factors, such as preeclampsia, hypertension, diabetes, and uterine atony. these individuals should follow the same preventative measures as those indicated for the general population. fetal transmission is unlikely as no viruses have been reported in either the umbilical cord, amniotic fluid, breast milk, or neonatal pharynx smears. however, in addition to the usual records, fetal ultrasounds and cardio recordings are recommended. in the case of maternal infection, there is a risk of postpartum transmission, for which screening tests will be needed [53, 54] . however, a recent joint review, including 37 infected pregnant women and 38 newborns in iran/china/usa aged 23-40 years-old, a placental transmission during the pregnancy was observed, while keeping normal amniotic fluid, vaginal discharge and milk secretion. no teratogenic effects were shown. however, the risk of infection with breast milk, cough or other vectors was high for 5-7 days after birth, so it is advisable to avoid breastfeeding if the mother is infected [55] . the recommendation is to use the same identified sexual strategies for stable sexual partners confined without passing quarantine, thus avoiding kissing, oral sex, and anal penetration, and using "a tergo" and posterior positions. the health workers, caregivers, and professionals in contact with infected people in hospitals, residential centres, or similar areas could spend a great deal of time exposed to the virus, possibly without adequate protection. thus, there is a high risk of accumulating a large viral load. the recommendation would be to follow the same strategy used for contaminated people (i.e., virtual sex). under no circumstances should sex be performed in vivo with a new partner unless there is the certainty that the partner has been immunised against the virus. for those who do not have a partner and for very erotophilic people, nonheteronormative couples with polyamory or those who maintain living apart, the recommendation is abstinence from sexual intercourse until the incubation time passes without symptoms after starting the coexistence. additionally, it might be worth using sexting or virtual sex. some swingers clubs organise erotic encounters through platforms in order to have virtual group sex. the measures that are generally taken to contain sars-cov-2 have undoubtedly decreased the professional activities of sex workers, and fewer casual sexual encounters in this context may affect individual risks for hiv infection and other stis [56] . in order to minimise the risk of infection when sporadic sexual intercourse occurs, condoms, dental dams or similar protection methods should be applied. in summary, we recommend maintaining an adequate level of eroticism and sexual activity, both in confinement and throughout the pandemic. this, along with other factors such as physical activity, social distancing or smoking cessation, will allow the minimisation of some part of its negative effects. however, it is necessary to meet the standards established by international organisations for the prevention of covid-19 [57] , given that sexual relations, regardless of sexual orientation-heterosexual, homosexual or bisexual-can facilitate its spread. a summary of the recommendations can be seen in table 1 , considering the previous different scenarios contemplated. our recommendations are comparable to those made by institutions such as new york citi health [53] , but so far, no specific recommendations have been published on safe sexual practices to avoid the transmission of the virus by other sources of trusted information such as the centers for disease control and prevention (cdc) or the world health organization. there is certainty that the vast majority of the global population will survive this pandemic, but associated stressors, such as uncertainty about the end of the pandemic, fear of contagion, and economic and social impairment, facilitate a weakening of the immune system [58, 59] . as there is evidence that sexuality promotes the immune system and physical and mental health, it is advisable to maintain an acceptable degree of sexual intercourse. there is some evidence that healthy sexual activity improves the immune system and serves as a more preventive factor against sars-cov-2. on the other hand, healthy, safe and frequent sexual activity might be attenuating (even if there is no evidence yet) the negative psychological effects associated with the infection. unfortunately, at this moment, there are no vaccines available for sars-cov-2 and we do not yet know how durable immunity is after an infection. currently, the epidemic is still spreading, and there are no effective means to prevent the infection. as most vaccines are under design and preparation, there is currently no possibility of having "certainty" around immunisation status [60] . additionally, sars-cov-2 may cause neurologic and psychiatric effects such as delirium in some patients in the acute stage and depression, anxiety and post-traumatic stress disorder in the long term [61] . therefore, the protective benefits of sexual activity and the maintenance of a suitable couple relationship could be beneficial to avoid the psychiatric and psychological deterioration that is secondary to the pandemic. on the other hand, it is very relevant not to stigmatise healthcare workers and consider them contaminated people. however, if the risk is high due to being in permanent contact with covid-19 patients, precautions should be taken to avoid infections due to the existence of frequent asymptomatic cases. in this sense, pregnant women should not be stigmatised either, although additional precautions are necessary to avoid possible consequences after infection in the mother and the fetus. undoubtedly, the pandemic has implied some big changes in the sexual behaviour of special groups such as sex workers, where it is difficult to issue recommendations because it is their only livelihood in low-income countries. the recommendation of sexual abstinence in this group is aimed at promoting changes to sex work due to increased potential exposure to infection and various health concerns, however, they could be very difficult to apply. innovative strategies launched by the leaders of sex workers and health workers, implementing new health protection guidelines and clinical spaces, are especially instructive [62] . additionally, other vulnerable populations such as men who have sex with men (msm), require special attention. a recent survey in the usa on sexual habits in msm during the pandemic showed that half had fewer sex partners and most had no change in condom access or use with increased difficulties in accessing hiv testing, prevention and treatment services. in addition to our recommendations to reduce the risk of sexual contact, it is necessary to apply other strategies such as telehealth and mailed testing and prevention supplies to avoid increased hiv incidence among msm during the covid-19 pandemic [63] . the risk of unwanted pregnancies is a consequence of the covid-19 pandemic [64] , because sexual and reproductive health services have remained closed during the pandemic. fortunately, in some countries, remote resources have allowed them to continue their preventive function by prescribing contraceptive methods, but unfortunately, in many others it has not been possible. we consider both, sexual and reproductive healthcare to be a priority, guaranteeing access to contraception as well as adequate information on sexual and reproductive health. some sexual intercourse-related scenarios may not be covered by these recommendations, and additionally, there is a lack of full information on virus behaviour and its impact on the sex life of infected patients and asymptomatic transmitters. however, keeping sex life healthy and safe is one of the biggest challenges for health prescribers and for the general population in this pandemic. furthermore, the peculiarities of sexual minorities during the pandemic are not yet known so these recommendations may not be widespread. there is not enough information about how the treatments received by infected patients affect their sex life or whether there are modifications to desire, arousal or orgasm compared to the previous stage. although many uncertainties still remain, the study of the sex life of this population could clear up scarce information about how the virus affects both the sexual and reproductive life. however, the benefits of maintaining a healthy sex life, harnessing its qualities and preventing contagion should be a priority in all sexual activity scenarios. these recommendations do not cover all minority sexual behaviours due to the great heterogeneity of human sexuality. however, they include most diversity of sexual activity regardless of orientation: heterosexual, homosexual or bisexual. our proposals for confinement include maintaining a security period of 28 days, before the couple can restart their regular sexual practices, since 1% of persons affected by covid-19 develop symptoms after an incubation period of 14 days. therefore, although we know that this could be a difficult recommendation for many couples to accept, we consider it relevant to safeguard security as much as possible. another relevant limitation is related to some still unknown facts, such as the duration of the immunity in people who have overcome the disease. this would be crucial to avoid reinfections when patients are supposedly immunised but do not respect the preventive measures implemented. further research is needed to obtain more precise data on the pathophysiology, prevention and treatment of covid-19, as well as to better understand the duration and level of immunity that overcoming the disease confers. with more scientific evidence, these are transcendental factors when recommending actions, that could avoid contagion through sexual activity. progress in the understanding of these aspects, through well-designed observational, case-control and experimental studies, will allow us to modify and enrich our current recommendations. staying at home and complying with strict confinement standards is the most active way to fight sars-cov-2. strengthening the immune system along with other factors such as increased positive interpersonal relationships and rewarding occupational activities, will help us fight the negative effects of the pandemic. this must be done without forgetting to reserve time for erotic activity that fosters creativity and communication. 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2020-09-12 journal: j clin med doi: 10.3390/jcm9092953 sha: doc_id: 267933 cord_uid: rg1yus8g background: treatment of cholangitis complicating choledocholithiasis includes biliary sphincterotomy and stone extraction. in certain cases of elderly comorbid patients with high risk for definitive endoscopic treatment, biliary stenting is the only measure for relieving biliary obstruction. aim: we aimed to report the safety of retained biliary stone. methods: a multi-center, retrospective case-control study conducted at two israeli medical centers from january 2013 to december 2018 including all patients 18 years of age or older who underwent ercp and biliary stent insertion for the treatment of acute cholangitis due to choledocholithiasis. results: three-hundred and eight patients were identified. eighty-three patients had retained long-term biliary stents of more than 6 months (group a) from insertion compared to 225 patients whose biliary stents were removed within a 6-month period (group b). the mean follow-up in group a was 66.1± 16.3 vs. 11.1 ± 2.7 weeks in group b. overall complications during the follow-up were similar between groups a and b (6% vs. 4.9%, or 1.24, chi square 0.69). similarly, the rate of each complication alone was not different when comparing group a to group b (3.6%, 1.2% and 1.2% vs. 2.7%, 0.44% and 1.8%) for cholangitis, stent related pancreatitis and biliary colic, respectively (chi square 0.85). even after 12 months, the rates of overall complications and each complication alone were not higher compared to less than 12 months (chi square 0.72 and 0.8, respectively). conclusion: endoscopic biliary stenting for cholangitis complicating choledocholithiasis is safe for the long-term period without increase in stent related complications. endoscopic retrograde cholangiopancreatography (ercp) is considered an essential procedure for the treatment of numerous pancreatic and biliary tract conditions [1] , mainly used for the management of choledocholithiasis complications [2, 3] . given its diagnostic and therapeutic potential coupled with its acceptable complications risk profile, ercp has largely replaced surgical exploration of the common bile duct (cbd). ercp has been reported to be successful in 80-95% of cases [4, 5] . the therapeutic interventions used to treat choledocholithiasis include papillotomy, basket and or balloon extraction and in cases of large solid stones, lithotripsy is used. in patients with septic cholangitis, most endoscopists prefer to perform endoscopic drainage via stenting without performing papillotomy [6] . moreover, endoscopic insertion of biliary endoprosthesis has been proposed as an alternative treatment for elderly patients or those with high surgical risks [7, 8] . stent related complications are divided into early and late, the former including cholangitis, pancreatitis, perforation and bleeding and the latter including stent occlusion, migration, cholangitis and cholecystitis [9] . it is advocated that plastic stents should be removed or replaced within 3-6 months after insertion [10] [11] [12] , while metallic stents are recommended to be replaced after 10 months to minimize the risk of stent occlusion and cholangitis [13] . several studies have shown the beneficial effect of short-and long-term biliary stenting for irretrievable cbd stones in high-risk elderly patients [7, 8, 14] . however, only few studies have reported long term complications; stent migration and cholangitis were the main complications with prolonged stenting duration [15] . previous studies have reported cholangitis to be the most common long-term complication of biliary stent in the elderly approaching 40% of patients, and 16% of patients died because of biliary-related causes [16] . given the paucity of data regarding retained biliary stents beyond the recommended period of extraction or replacement, we aimed to report our experience regarding the long-term safety and complications of retained biliary stents in elderly patients hospitalized with cholangitis complicating choledocholithiasis and treated by biliary stenting. we performed a multi-center, retrospective case-control study conducted at two israeli medical centers (galilee medical center and sha'arei zedek medical center from january 2013 to december 2018). inclusion criteria for the study endpoints included: patients 18 years of age or older who underwent ercp and biliary stent insertion for the treatment of acute cholangitis complicating choledocholithiasis. exclusion criteria included patients with cholangitis secondary to biliary disease other than choledocholithiasis such as malignancy and biliary stricture, patients with primary sclerosing cholangitis. all medical records of eligible patients were reviewed and the following parameters were collected: demographic data (age, gender), clinical data (aetiology of stent insertion), endoscopic parameters at the time of ercp performance (incomplete stone extraction and complication of ercp), period of follow-up, laboratory improvement after stent insertion) and follow up data on complications including cholangitis, stent related biliary pancreatitis, migration and biliary colic. cholangitis was defined as new-onset right upper quadrant pain accompanied by fever and jaundice. pancreatitis was defined as abdominal pain consistent with acute pancreatitis (acute onset of a persistent, severe, epigastric pain often radiating to the back) accompanied by elevated serum lipase or amylase activity at least three times greater than the upper normal limit and characteristic findings of acute pancreatitis on contrast-enhanced computed tomography or transabdominal ultrasonography [17] . biliary colic was defined as new-onset transient colicky right upper quadrant pain accompanied by normal or abnormal liver enzymes and normal serum bilirubin level. all patients' charts were extracted using icd-9 codes for choledocholithiasis and cholangitis. the primary endpoint was to compare the complications rate between all patients whose biliary stents were retained (patient refusal to remove or non-attendance to planned ercp session for stent removal or replacement) for more than 6 months as being confirmed by imaging studies performed for assessment of stent presence, and patients whose stent was removed or replaced within 6 months after the index ercp as recommended. secondary endpoints were to assess the complications rate at specific time intervals; for this purpose, we subdivided the study cohort into 2 comparison groups as follows: (0-12 months vs. >12 months and 0-18 months vs. >18 months). furthermore, we aimed to assess the complications rate according to the type of the stent (plastic vs. metallic). the study was approved by the institution's ethics committee (0034-16-szmc obtained on 21.3.2016). written informed consent was waived by the ethics committee due to the retrospective non-interventional nature of the study. in our study we used two types of biliary stents, mostly plastic stents (cotton-huibregtse stents 10 fr, 7 cm of cook medical) and metallic stents (10-mm, 6 cm wallflex of boston scientific). the inner tip of the stent was positioned in the cbd above the stone and its distal tip was positioned into the second part of the duodenum. stent exchange was performed only when recurrent episode of cholangitis was experienced. characteristics of participants are presented with descriptive statistics such as arithmetic means and standard deviation (sd) or range for continuous variables, or as frequencies (percentages) for categorical variables. the comparison of two independent groups was performed using student's t-test for continuous variables and the chi-square statistic for categorial variables. all tests applied were two-tailed. p value of 0.05 or less was considered to be statistically significant. normality test was performed and showed normal distribution. all analyses were carried out using the statistical analysis software (sas vs. 9.4 copyright (c) 2016 by sas institute inc., cary, nc, usa). a total of 3082 patients were identified from january 2013 to december 2018. of these we included 308 patients with choledocholithiasis who had ercp and biliary stent insertion. eighty-three patients who underwent urgent ercp for acute cholangitis complicating choledocholithiasis with biliary stent insertion mostly without papillotomy or with papillotomy when stone extraction was attempted, had retained long-term biliary stents of more than 6 months from insertion (group a) vs. 225 patients whose biliary stents were removed within 6 months following the index ercp (group b), ( figure 1 ). the average ages of group a and b were 83.5 ± 4.8 years and 61.3 ± 17.9 years respectively (p < 0.001). sixty-five percent and 63% of the patients were males in groups a and b respectively. none of the patients in group a had complete stone extraction, as 22.9% had incomplete stone extraction and 77.1% did not have their stone extracted. the causes of biliary stent insertion in group a were hemodynamic instability secondary to cholangitis (56 patients, 67.5%), technically difficult ercp due to the presence of duodenal diverticula (25 patients, 30.1%) and inability to complete the ercp due to sedation related complications (2 patients, 2.4%), while in group b, all patients had stent inserted as a preventive measure until performing cholecystectomy. the mean follow-up in group a was 66.1 ± 16.3 weeks (range 30-100 weeks) as compared to 11.1 ± 2.7 weeks (range 5-18) in group b. clinical and endoscopic characteristics are demonstrated in table 1 . tables 2 and 3 demonstrate the rate of overall complications and each complication alone (cholangitis, stent related pancreatitis and biliary colic). the rate of overall complications during the follow-up period at the primary endpoint were similar between groups a and b (6% vs. 4.9%, or 1.24, 95% ci 0.42-3.7, chi square 0.69). moreover, in the secondary endpoint analysis, comparing retained stents of <12 months vs. more than 12 months, again, there was no increase in the rate of all complication (5% vs. 6.1% respectively, or 1.23, 95% ci 0.38-3.96, chi square 0.72). even after 18 months, the rate of all complications was similar to those who had retained stents of less than 18 months (5.3% vs. 5.2% respectively, or 1.01, 95% ci 0.12-8.12, chi square 0.98). notably, the migration rate of biliary stent occurred in 5 patients (6%) in group a compared to 11 patients (4.9%) in group b (chi square 0.69). in spite of this, the complications rate was not higher in group a than in group b. there was no difference when we analyzed each complication alone. the rate of cholangitis, stent related pancreatitis and biliary colic were 3.6%, 1.2% and 1.2% vs. 2.7%, 0.44% and 1.8% in groups a and b respectively (chi square 0.85). moreover, in the secondary analysis, comparing retained stents of <12 months vs. more than 12 months, the rates of cholangitis, stent related pancreatitis and biliary colic were 2.8%, 0.4% and 1.65% vs. 3%, 1.5% and 1.5%, respectively (chi square 0.8). interestingly, the rate of each complication was not more common in patients with retained stents for more than 18 months (chi square 0.4) (tables 2 and 3 ). two-hundred eighty-eight patients had plastic stents as compared to 20 patients who had metallic stents. overall complications in the plastic stent group occurred in 15 patients (5.2%) as compared to 1 patient (5%) in the metallic group (or 0.95, 95% ci 0.12-7.64, chi square 0.97). similarly, the rate of each complication alone was not different between the plastic stent vs. the metallic stent (chi square 0.07). comparing patients according to stent type and stent retaining time, there was no difference between overall complications (or 1.1, 95% ci 0.34-3.62, chi square 0.8) and each complication alone (chi square 0.86) in patients with plastic stents of more than 6 months as compared to less than 6 months. similarly, patients with metallic stent of more than 6 months did not have either more overall complications (or 1.1, 95% ci 0.92-1.29, chi square 0.4) or more of each single complication alone (chi square 0.56) as compared to patients with metallic stent of less than 6 months (tables 4 and 5 ). our study demonstrated that approximately 10% of patients who underwent ercp due to cholangitis complicating choledocholithiasis in the long-term (over 6 months in the primary endpoint) and even over 12 and 18 months in the secondary endpoints, retained biliary stents (plastic and metallic) and were not associated with more overall complications or any specific complication including cholangitis, pancreatitis and biliary colic compared to stent duration of less than 6 months. actually, little is known about what happens when biliary stents are forgotten by patients for more than 6 months, despite several case reports of several dozens of patients reporting over 40% complications rate. the complications rates in our study were much lower (6% for overall complications and 3.6%, 1.2% and 1.2% for cholangitis, stent related pancreatitis and biliary colic, respectively) than previously reported in the literature. a study by de palma gd et al. of 49 patients with symptomatic choledocholithiasis and with irretrievable bile duct stone treated by endoscopic stenting for a median period of 39 months reported successful biliary drainage in all patients but found late complications in 40.8% of cases, with 3 cases of biliary-related death. they reached the conclusion that definitive biliary stenting for irretrievable stones should be relegated to highly selected cases [18] . similarly, bergman jj et al. reported 40% complications rate following stent insertion for symptomatic choledocholithiasis, mostly cholangitis when biliary stents (polyethelene 10 fr, 15 or 19 cm wedged in the intra-hepatic ducts) were inserted as permanent therapy in 58 patients for a median period of 36 months. they reached the same conclusion that permanent biliary stenting should preferably be restricted to patients unfit for elective treatment at a later stage and with a short life expectancy [16] . similarly, ang tl et al. reported the long-term outcome of plastic biliary stenting (7 fr or 10 fr) in 83 patients with symptomatic choledocholithiasis. in their report the complications rates for cholangitis, biliary colic, and pancreatitis were 71.4%, 3.6% and 3.6%, respectively. notably, most of the stents used were 7 fr in 79 patients, while only 4 patients had 10 fr stents [19] . on the other hand, a recent retrospective study comparing the outcome in 3 groups of patients with choledocholithiasis unfit for definitive endoscopic stone removal or surgery using plastic biliary stenting (7 fr, 7 cm), showed cholangitis rate of 2.9% and 8.6% in 6-and 12-month replacement groups, respectively, and 35.3% in the third group in whom stent replacement was carried out due to developing acute cholangitis with a median time for replacement of 16.3 months [20] . this study showed that the rate of acute cholangitis was low mainly in the 6 month replacement group, and increased as the stenting period increased, suggesting that 7 fr plastic biliary stenting should be replaced every 6 months [20] . in comparison with the previous study, slattery et al. showed a cholangitis rate in 7.4% with median stent patency of 11.8 months [21] . in our study, we showed a much lower complications rate even after a longer follow-up period. to the best of our knowledge, our study is the first to report these low complications rate probably due to the larger stent diameter and shorter length (10 fr, 7 cm) that we used. in fact, most of the studies cited above [19] [20] [21] used 7 fr stents, while one study reported the use of 10 fr stent (15 or 19 cm wedged in the intra-hepatic ducts) [16] . in all studies the rates of complications were higher compared to our study, suggesting that both the stent diameter and length probably has a potential role in the development of stent related complications in the long-term. notably, we did not find previous studies on the correlation between stent diameter and length with stent patency or complications, making our observation novel and necessitating further confirmatory studies. moreover, biliary stent migration rates have been reported to approach 4.9% and 5.9% for proximal (into the duct) and distal (out of the duct), respectively [22] . similarly, the migration rate in our study was 6% in group a and 4.9% in group b. however, it is important to address that stent migration did not predispose for more stent related complications. in our study, although more patients in group a had metallic stent compared to group b, after controlling for stent type, we found no difference in the rate of complications between the stents in the primary and the secondary endpoint analysis. moreover, studies recommend to replace or remove a metallic stent after approximately 10 months from insertion [13] ; in our study we found that stent duration of more than 12 months and even 18 months was not associated with more complications, again suggesting the long-term safety of metallic stenting. interestingly, most of the patients in group a (94%) did not perform cholecystectomy in the follow-up period, though 37.3% of them had gallbladder stones without increase in the rate of overall complications, suggesting that biliary stenting is effective in preventing further episodes of cholangitis and even cholecystitis in the setting of gallbladder stones. biliary stenting for choledocholithiasis has been used mainly as a temporary measure to restore bile flow in cases of stone impaction in the cbd until stabilizing the patient's condition followed by subsequent definitive endoscopic treatment or surgery [23] . with the rising age of our patient population, its associated comorbidities and prescribed medications including anticoagulants ant anti-aggregants, it has become nearly a daily dilemma which treatment suits the high-risk elderly comorbid patient. demonstrating the safety of long-term biliary stenting (probably 10 fr, 7 cm plastic stents or 10 mm, 6 cm metallic stents) in our study could provide an alternative safe and effective option for treating cholangitis complicating choledocholithiasis in this group of patients who are mostly elderly patients, adhering to one of the cardinal rules of practicing medicine "premium non nocere". this strategy also holds true for large, stiff, difficult-to-remove stones, where leaving a biliary stent seems to be an acceptable and safer option than insistence on removing an irretrievable stone, especially in high risk elderly comorbid patients [7, 8, 14] . remarkably, our study results may have special implications in the current covid-19 global pandemic period. sadly, in response to covid-19 pandemic most countries implemented lockdown as a major social distancing measure that naturally caused cancellation of elective gastrointestinal endoscopies as only emergent endoscopies were executed [24] . according to the recent position statement by the european society of gastrointestinal endoscopy (esge) regarding gi endoscopy units activity during covid; only patients presenting with obstructive jaundice or ascending cholangitis should undergo urgent ercp. conversely, patients who are scheduled for elective biliary stent replacement were listed in the high priority procedure group (a procedure to be done immediately or postponed for 12 weeks [25] . based on our results, postponing elective stent replacement is safe. our study findings may provide 'confidence' to clinician and patients to postpone elective biliary stent during the lockdown period. however, prospective multicenter international studies are required to further assess this concern. the main limitation of our study is its retrospective nature of data collection. on the other hand, to the best of our knowledge, this is the largest cohort of patients reported with retained biliary stents that were inserted for the indication of choledocholithiasis. in conclusion: we found that long term biliary stenting for cholangitis complicating choledocholithiasis was effective, feasible and safe without increase in the complications rates during a long-term period. factors associated with a low complication rate according to our study were larger stent diameter and probably shorter length. therefore, we suggest a prolonged stenting policy for elderly patients or patients with comorbidities who are poor candidates for further definitive endoscopic or surgical treatments. further multicenter, prospective studies with larger cohorts should be carried out to confirm our findings and better address this issue. the authors declare no conflict of interest. common bile duct ci confidence interval ercp endoscopic retrograde cholangiopancreatography or odds ratio sd 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stones endoscopy units and the coronavirus disease 2019 outbreak: a multicenter experience from italy esge and esgena position statement on gastrointestinal endoscopy and covid-19: an update on guidance during the post-lockdown phase and selected results from a membership survey key: cord-336257-f6yglaz8 authors: forte, giuseppe; favieri, francesca; tambelli, renata; casagrande, maria title: the enemy which sealed the world: effects of covid-19 diffusion on the psychological state of the italian population date: 2020-06-10 journal: j clin med doi: 10.3390/jcm9061802 sha: doc_id: 336257 cord_uid: f6yglaz8 background: starting from the first months of 2020, worldwide population has been facing the covid-19 pandemic. many nations, including italy, took extreme actions to reduce the diffusion of the virus, profoundly changing lifestyles. the italians have been faced with both the fear of contracting the infection and the consequences of enforcing social distancing. this study was aimed to understand the psychological impact of the covid-19 outbreak and the psychopathological outcomes related to the first phase of this emergency. methods: the study included 2291 respondents. an online survey collected information on socio-demographic variables, history of direct or indirect contact with covid-19, and additional information concerning the covid-19 emergency. moreover, psychopathological symptoms such as anxiety, mood alterations and post-traumatic symptomatology were assessed. results: the results revealed that respectively 31.38%, 37.19% and 27.72% of respondents reported levels of general psychopathological symptomatology, anxiety, and ptsd symptoms over the cut-off scores. furthermore, a significant worsening of mood has emerged. being a female or under the age of 50 years, having had direct contact with people infected by the covid-19, and experiencing uncertainty about the risk of contagion represent risk factors for psychological distress. conclusions: our findings indicate that the first weeks of the covid-19 pandemic appear to impact not only on physical health but also on psychological well-being. although these results need to be considered with caution being based on self-reported data collected at the beginning of this emergency, they should be used as a starting point for further studies aimed to develop interventions to minimize both the brief and long-term psychological consequences of the covid-19 pandemic. in december 2019, an outbreak of pneumonia associated with a new coronavirus (i.e., severe acute respiratory syndrome due to coronavirus 2 (sars-cov-2)) was reported in wuhan, china. in the following weeks, the infection attracted worldwide attention for its rapid and exponential diffusion across different countries around the world. on 12 february 2020, who named it coronavirus disease 2019 (covid-19) [1] . at the beginning of april 2020, covid-19 has infected more than one and a half million people, causing over 80,000 deaths in 204 countries [1] . this viral infection spread quickly, becoming unstoppable, and forcing the who to declare it a pandemic [1] . although the containment measures a web-based cross-sectional survey, implemented using the kobo toolbox platform and broadcasted through mainstream social-media (such as facebook, twitter, instagram, telegram), was used to collect data among the italian speaking population. in our opinion, this procedure represents the best data collection strategy in the present phase of forced social distancing, and it leads to reaching the largest number of people. the survey was carried out from 18 march 2020 to 31 march 2020. a brief presentation informed the participants about the aims of the study, and electronic informed consent was requested from each participant before starting the investigation. the survey took approximately 30 min to complete. when the participants' responses to the survey lasted less than 5 min or more than 60 min, data were excluded to ensure a standard quality of questionnaires. participation was entirely voluntary and free of charge. to guarantee anonymity, no personal data, which could allow the identification of participants, was collected. for the current research, being at least 18 years old was the only inclusion criterion employed. after a short demographic questionnaire, the participants answered questions that assessed knowledge and perceptions related to the spread of covid-19 and the government measures adopted to contain it. finally, italian versions of standardized questionnaires were administered to assess psychological dimensions. this study was conducted in accordance with the declaration of helsinki and was approved by the ethics committee of the department of dynamic and clinical psychology of the "sapienza" university of rome (protocol number: 0000266). participants could withdraw from the study at any time without providing any justification, and the data were not saved. only the questionnaire data that had a complete set of answers were considered. ninety-eight per cent of the total respondents (2291 out of 2332 people) who started the questionnaires completed the entire survey, and the related data were considered for statistical analyses. the main demographic characteristics of the sample are shown in table 1 . the authors assert that all procedures contributing to this work comply with the ethical standards of the relevant national and institutional committees on human experimentation and with the helsinki declaration of 1975, as revised in 2008. the first session of this questionnaire required information about gender, age, education and occupation, city, and region of origin. the second section aimed to evaluate personal knowledge about covid-19 diffusion, individual perception of the situation, and lifestyle changes related to government restrictions. the scl-90 [10] (italian version: 11) is a 90-items questionnaire aimed to assess psychological distress and symptomatology. the items are rated on a five-point likert scale, ranging from 'not at all' (0) to 'extremely' (4). ten primary symptom dimensions are measured: somatization, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, anger-hostility, phobic anxiety, paranoid ideation, psychoticism, and sleep disturbance. a global severity index provides measures of overall psychological distress. higher scores in each dimension indicate greater distress and psychopathological symptomatology. a cut-off score of 0.90 was selected to define higher psychopathological symptomatology, in line with previous studies on the general italian population [11, 12] . the internal consistency in the participants of the present study was α = 0.97. the stai measures state and trait anxiety [13] (italian version: 14). the questionnaire includes 40 items. twenty items refer to state anxiety (stai-s) and evaluate how participants feel about anxiety "right now, at this moment"; 20 items refer to trait anxiety (stai-t) and assess how people "generally feel" about anxiety. the items are rated on a four-point likert scale, ranging from 1 (not at all) to 4 (very much so). in both the state and trait anxiety scales, higher scores indicate greater anxiety levels. a cut-off point of 55 was used to define higher state anxiety, according to kvaal et al. [14] . although this study was interested in assessing state anxiety, trait anxiety was also measured to check whether the anxious state could be explained by a high anxious trait of the italian population. the internal consistency of stai in the sample of this study was adequate (α = 0.60). fifteen mood aspects (insecurity, helplessness, sadness, fear, anger, frustration, stress, anxiety, depression, boredom, serenity, happiness, preoccupation, tranquility, energy) both positive and negative were assessed to examine the emotional impact of the current situation. in these evaluations, the participant was required to refer to two different periods. the first was december, preceding the outbreak of the contagion (december 2019); the second period referred to the last week. the mood scales required a response on a 10-point likert scale [15] , from 0 (not at all) to 10 (very much). the use of mood scales has mainly been adopted to analyse the self-reported conditions of individual mood [16] [17] [18] . the items on the mood scales presented high internal consistency (α = 0.75). the ies-r is a self-report measure designed to assess ptsd symptomatology according to the diagnostic and statistical manual of mental disorders-fourth version (dsm-iv) criteria for ptsd. the questionnaire requires the indication of the magnitude of distress on specific dimensions (e.g., recurring dreams, feelings of anger and irritability) related to specific life events (i.e., the current covid-19 emergency) referring to the last seven days [19] (italian version: 20). the three subscales measure avoidance (the tendency to avoid thoughts or reminders about the incident), intrusion (difficulty in staying asleep, dissociative experiences similar to flashbacks), and hyperarousal (irritated feeling, angry, difficulty in sleep onset). the ies-r requires a response on a 5-point likert-scale, from 0 (not at all) to 4 (extremely). the score on an ies-r subscale is the mean of the scores of the items of that cluster. the ies-r also gives an overall score (ies-r total that is the sum of the scores of the three subscales). the cut-off of 33 was adopted to indicate a high risk of ptsd symptomatology [20, 21] . in the present sample, the ies-r presented high internal consistency (α = 0.95). descriptive analyses were conducted to describe demographic characteristics, and covid-19 related aspects in the italian population, considering the different italian territorial areas. student's t-test was performed to compare our data on anxiety, general psychological symptomatology, and ptsd symptomatology with data from the general italian population, reported by previous studies. specifically, our data on anxiety were compared with those reported by corno et al. [22] , scl-90 outcomes were compared with the data given by holi et al. [12] , and ptsd indices were compared with the results of ashbaugh et al. [23] . analyses of variance (anovas) were performed to explore the potential difference in the impact of covid-19 in the italian territorial areas. the differences between north italy, central italy, and south italy were reported for state and trait anxiety, psychopathological symptomatology (somatization, obsessive-compulsive, interpersonal sensitivity, depression, anxiety, anger-hostility, phobic anxiety, paranoid ideation, psychoticism, and sleep disturbance), and ptsd symptomatology (ies-r). furthermore, within-subjects anova designs were adopted to compare the respondents' self-reporting mood before and during the covid-19 emergency. logistic regressions were performed to explore the influence of demographic factors and experiences which were covid-19 related in determining risk for state anxiety (stai), psychopathological symptoms (scl-90), and ptsd symptomatology (ies-r). all data were analyzed using statistical package for social sciences (spss) version 24.0 and statistica 10.0 (statsoft.inc., tulsa, ok, usa). p-values of less than 0.05 were considered statistically significant. to better control the results for the multiple comparison analyses, the bonferroni correction was adopted; in these cases, an adjusted p-value of less than 0.01 was considered statistically significant. the characteristics of the respondents are shown in table 1 . two thousand two hundred ninety-one individuals completed the questionnaires, 580 (25.3%) were males, and 1708 (74.6%) were females; the mean age of the participants was 30.0 years (sd: 11.5 years; age range: 18-89). the most represented age range was 18-29 years (68.6%). most of the participants (1136; 49.6%) received a high school education and were students (1073; 46.8%) or employees (688; 30.0%). the respondents' current locations were sorted considering territorial area: north (23.6%), central (25.1%), and south (51.3%) of italy. most of the participants live in urban areas (937; 40.9%) with a number of inhabitants between 10,000 and 100,000. among all respondents, only 9 (0.4%) were infected by the covid-19, and 40 (1.7%) were sure that they had had close contacts with individuals suspected of covid-19 infection (see table 1 ). of the overall sample, 112 respondents (4.9%) and 177 (7.7%) respectively knew people dead and patients in intensive care units (icu) because of covid-19 infection. comparisons of state and trait anxiety, psychopathological symptomatology, and post-traumatic symptomatology during the covid-19 epidemic were made with data from the general population. the comparisons of psychological outcomes during the covid-19 epidemic in the italian population with data from the general population are presented in table 2 . considering scl-90 indices, depression (t = 6.14; p < 0.0001), anxiety (t = 7.83; p < 0.0001), anger-hostility (t = 1.89; p < 0.05), phobic anxiety (t = 9.71; p < 0.0001), psychoticism (t = 4.25; p < 0.0001), and global severity index (t = 4.18; p < 0.0001) significantly differ from holy's data [12] , indicating greater psychopathological symptomatology in our sample. considering stai indices, state anxiety appears to be higher in our sample compared to data reported by corno et al. [22] in an italian sample that considered the levels of anxiety separately in both males and females (males: t = 4.49; p < 0.0001; females: t = 9.64; p < 0.0001), while no significant differences were present considering trait anxiety. finally, ptsd related symptomatology assessed by the ies-r resulted higher in our sample compared to the data reported by ashbaugh et al. [23] (t = 2.41; p < 0.05) (see table 2 ). table 3 reports the differences in psychological outcomes, considering the three territorial areas of italy. considering psychopathological symptomatology assessed by the scl-90, significant differences were reported only in the sleep disturbance subscale (f 2,2288 = 4.55; p < 0.01; pη 2 = 0.004). people from north italy reported higher sleep disturbances compared to people from south italy (p < 0.003). however, no other significant differences were observed (see table 3 ). anovas on stai subscales did not highlight significant differences between individuals from north, central, and south italy. finally, considering ptsd, no significant differences were reported in ies-r subscales (see table 3 ). the results on the difference in subjective mood before and during the covid-19 epidemic are shown in table 4 and figure 1 . the analyses confirmed for all dimensions a perceived worsening of mood by the respondents. figure 2 shows the prevalence of psychopathological symptomatology, state of anxiety, and ptsd, stratified by gender, age, territorial areas, knowledge of people affected by covid-19, and loneliness in social distancing experience. table 5 ). sudden outbreak events always pose huge challenges to the countries where they occur, impacting not only on physical health but also on social and mental well-being. from this perspective, the covid-19 pandemic will have long-term consequences, influencing international and national public health policies. this study is part of a series of works aimed at investigating the characteristics and the psychological effects of the covid-19 pandemic and the restrictive measures adopted by the italian government during the early and more severe stages of the covid-19 outbreak [24, 25] . since the outbreak of the covid-19 epidemic, the italian government imposed a lockdown in north italy, expanding it nationwide following the exponential diffusion of the pandemic from the northern territorial areas to both the central and south areas. these severe limitations included the request for both people infected by the virus and healthy citizens to isolate themselves at home, prohibiting all other than indispensable activities, and making it mandatory to wear surgical masks to enter public places. our data were collected near the infection peak (between the end of march and the beginning of april 2020) [2] , and they provide an accurate snapshot of italians' perception of this emergency. this study delivers further information to add to the findings reported on the chinese population that was the first to be severely affected by 26] , indicating that the effects of this pandemic on the psychopathological conditions are similar in the italian and chinese populations. in both countries younger age, student status, female gender and direct contact with covid-19 infection are associated with a greater psychological impact of the emergency, involving many psychopathological dimensions (e.g., anxiety, distress, sleep disturbance) [5] [6] [7] [8] [9] 26] . one of the aims of the study was to analyse the psychological impact of the covid-19 outbreak in the different italian territorial areas. north italy was the first area in italy infected by the covid-19 and in which social distancing was imposed. it continues to have the highest prevalence of contagion and deaths, with a heavy burden on the public health system. accordingly, we expected an impact of these conditions on the psychological well-being and mental health of its inhabitants. however, although respondents from north italy reported more sleep disturbances and a relatively higher state of anxiety compared to those from central and south italy, no other differences were observed in psychopathological symptoms and ptsd risk [23] . these results would seem to underline that psychological status is not only influenced by the direct effects of a justifiable fear of contagion but also by the indirect consequences of the covid-19 outbreak such as the restrictive measures, that equally influenced people of all the italian regions, generating a similar psychological pattern. this assumption would be confirmed by the comparison of our results with data from the general italian population. the differences in the selection of the sample do not allow a generalizability of these results. most of the psychological symptoms assessed by the scl-90 subscales are significantly higher in our sample compared to data from the general population. only somatization and paranoid ideation resulted in being not significantly different from data on the general population. these last findings do not agree with recent data on the chinese population [27] , and they could appear incongruous because medical emergencies might induce higher somatization and intrusive and threatening thoughts. however, these results concord with those found during the sars epidemic [28] . the high prevalence of anxiety evidenced in our sample highlights that the covid-19 pandemic has increased alert levels and generated a high level of state anxiety in the population, confirming results of previous studies on sars, influenza a virus subtype h1n1 [29] [30] [31] , and covid-19 [6] [7] [8] . in our sample, 27.72% of the respondents presented ptsd symptomatology, and risk of ptsd higher than that reported in the general population, at least as regards the symptoms evaluated with the ies-r questionnaire [23] . this result should be interpreted with caution because it referred to the first weeks of the emergency when people could perceive the rapid spread of the virus and the extraordinary measures adopted by the government as sudden stressors, and it is known that sudden stressors affect the daily lives of individuals drastically. on the other hand, this first italian perception of the current situation would seem to give a photograph of the real impact of the covid-19 outbreak on mental health. another interesting result concerns the impact of the pandemic on mood. respondents perceived a significant change in their mood, with a sensitive decrease of positive mood (e.g., happiness, serenity) and a high increase of negative mood (e.g., sadness, preoccupation, boredom) after the covid-19 spread and the consequent social distancing measures. from a clinical point of view, this result could suggest a possible risk of mood disorders, such as depression, as long-term consequences of a pandemic [32] . however, it must be underlined that these data are not obtained prospectively, and the causal relationship cannot be confirmed. self-reported moods are subject to memory distortions and bias, and they should be taken with caution. overall, the results highlighted high levels of anxiety, psychopathological symptoms and ptsd symptoms in italian respondents during the first critical phase of the spread of the covid-19 pandemic and of the government measures taken to contain it. however, the results of the present study also suggested which people are most vulnerable to the psychological consequences of the covid-19 outbreak. this unexpected situation seems to have had a higher impact on females and people under 50 years. moreover, to have had direct contact with people infected by the virus, and to know people more or less severely infected by the covid-19 (i.e., people hospitalized in an intensive care unit or people dying as consequences of covid-19 infection) emerged as other relevant risk factors for psychological well-being. all these characteristics would make people more vulnerable to developing anxiety, psychopathological symptoms, and ptsd-related symptoms, confirming results observed in previous studies [8, 33] . these risk factors may depend on different aspects of the covid-19 pandemic. the high psychopathological risk related to direct experience with the covid-19 infection could depend on the fear of contagion, while being younger could be a risk factor due to the sense of constraint caused by social distancing and the other measures taken by the italian government [3]. our study reports that covid-19 infected 0.4% of the sample. this result is higher than the data on the general italian population (0.22%), updated on the 30 march 2020 [2] , but it indicates the high rate of healthy individuals in the sample. both this consideration and the data on risk factors would confirm that, even without real exposure to the covid-19 and an actual infection, fighting against an invisible enemy could affect mental health. uncertainty, fear about infection and social consequences of a pandemic could be triggers for psychopathological symptoms, and they should be considered in further studies. although some psychological characteristics are linked to medical conditions [34] [35] [36] [37] , psychological consequences of at-risk people are often overlooked during an epidemic emergency as reported for sars and h1n1 [29, 30, 33] . once again, the importance of not disregarding mental health and intervening during and after the pandemic emergency in the most affected psychological dimensions appear relevant in a long-term perspective. this study gives a picture of the psychological well-being of the italian population at the beginning of the covid-19 emergency. however, some limitations must be considered. despite the large sample size, it is not possible to overcome the limitation of a cross-sectional study, which does not allow us to determine a causal relationship between the variables. also, the use of an online survey presents other limitations. selection bias of participant recruitment is a consequence of this methodological choice. this bias is expressed by some characteristics of our sample, such as the higher number of respondents younger than 30 years, and the high number of females and people from south italy. another limit related to the online survey can be associated with convenience sampling that may have induced the collection of responses primarily from people who feel strongly about the considered issue. these limitations reduce the representativeness of our findings and may have influenced the results of the study. therefore, they must be considered. however, the adoption of an online survey was the best solution in this emergency in which social distancing measures limit data collection. in conclusion, a global response is desperately needed to prepare health systems to face the new challenge of the covid-19 outbreak. despite the underlined limitations, these preliminary findings, in line with the results of previous studies, evidenced that the diffusion of this pandemic can be related to anxiety, changes in mood, high psychopathological symptomatology, and could be associated with the development of ptsd. moreover, similarly to the results of other studies on the covid-19 pandemic, these findings should be considered preliminary, but they can be useful to predispose interventions aimed at 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received no specific grant from any funding agency, commercial or not-for-profit sectors. there is no funding support for this survey. we would like to thank gianluca pistore and all the people who helped in the data collection by sharing our survey on various social media. the authors declare no conflict of interest. key: cord-289034-yl3emjef authors: moro, loredana title: mitochondria at the crossroads of physiology and pathology date: 2020-06-24 journal: j clin med doi: 10.3390/jcm9061971 sha: doc_id: 289034 cord_uid: yl3emjef mitochondria play a crucial role in cell life and death by regulating bioenergetic and biosynthetic pathways. they are able to adapt rapidly to different microenvironmental stressors by accommodating the metabolic and biosynthetic needs of the cell. mounting evidence places mitochondrial dysfunction at the core of several diseases, notably in the context of pathologies of the cardiovascular and central nervous system. in addition, mutations in some mitochondrial proteins are bona fide cancer drivers. better understanding of the functions of these multifaceted organelles and their components may finetune our knowledge on the molecular bases of certain diseases and suggest new therapeutic avenues. mitochondria are semi-autonomous organelles with a double membrane system, namely the inner and the outer mitochondrial membrane that delimit the intermembrane space. the inner mitochondrial membrane demarcates the matrix, a viscous microenvironment that contains several enzymes catalyzing a plethora of anabolic and catabolic reactions. mitochondria contain their own genome, the mitochondrial dna (mtdna), a circular double-stranded dna molecule of 16,569 bp in humans, which encodes only 13 mitochondrial proteins belonging to the electron transport chain (etc), 22 transfer rnas and 2 ribosomal rnas needed to carry out the mitochondrial protein synthesis. all the other mitochondrial components are encoded by the nuclear genome. mitochondria are the energy powerhouses of the cell, being responsible for 90% of energy production in the form of atp by coupling the flux of electrons throughout the mitochondrial respiratory complexes i-iv with oxidative phosphorylation (oxphos). in brief, complete oxidation of nutrients through the tricarboxylic acid cycle (tca) within mitochondria produces reduced coenzymes (nadh, fadh2) that act as electron donors. the flux of electrons through the mitochondrial respiratory chain complexes produces an electrochemical gradient used by the mitochondrial respiratory complex v to generate atp. notably, the function of mitochondria in cell physiology goes beyond their role as energy producers and metabolic regulators. indeed, these multifaceted organelles play a pivotal role in the modulation of cell death pathways and intracellular signaling [1] . the etc is also the main cellular source of reactive oxygen species (ros), owing to an incomplete reduction of oxygen by complex i and complex iii. mitochondrial ros production can lead to oxidative damage to proteins, membranes and dna, thus impairing the ability of mitochondria to carry out their biosynthetic and catabolic reactions, including the tca cycle, heme synthesis, fatty acid oxidation, the urea cycle and amino acid metabolism [2] . mitochondrial oxidative damage can also promote permeabilization of the mitochondrial outer membrane (momp), resulting in release of intermembrane space proteins, such as cytochrome c, and activation of the mitochondrial apoptotic pathway. furthermore, mitochondrial ros production promotes the opening of the mitochondrial permeability transition pore (mptp), leading to permeabilization of the inner mitochondrial membrane to small molecules in pathological conditions, such as during ischaemia (loss of blood flow) and subsequent reperfusion [3] . two mitochondria quality control mechanisms are in place to meet the functional needs of any given cell under different physiological and pathological conditions: (a) mitochondrial biogenesis, fusion and fission [4] [5] [6] ; (b) mitophagy [7, 8] . the first mechanism is a balanced process that allows maintenance of the physiological mitochondrial homeostasis when cells face metabolic or microenvironmental stresses [9] . mitochondrial fission guarantees an adequate distribution of mitochondria in dividing cells. mitochondrial fusion allows complementation between dysfunctional mitochondria within the cell to maximize mitochondrial performance in response to stress. three gtpases, mitofusin 1 (mfn1), mfn2, and optic atrophy 1 (opa1), are primarily involved in the regulation of mitochondrial fusion. instead, mitochondrial fission is mainly controlled by the gtpase dynamin-related protein 1 (drp1) [10] . disruption of the balance between fusion and fission is associated with neurodegenerative diseases, such as parkinson's, and cancer [9, 10] . the second mechanism, mitophagy, is a specific form of autophagy that removes damaged mitochondria and reduces the mitochondrial mass upon microenvironmental stresses, such as hypoxia and nutrient starvation, promoting cell survival [11] . mitophagy dysregulation has been implicated in cancer development and progression [12] , neurodegeneration [13] and cardiovascular diseases [7] . mitochondrial dysfunction can lead to an array of diseases. depending on the nature of the defect leading to mitochondrial dysfunction, primary and secondary mitochondrial diseases can be distinguished. primary mitochondrial diseases develop as a consequence of germline mutations in mtdna and/or nuclear dna genes that encode proteins affecting mitochondrial functionality and energy production, including etc proteins and proteins involved in mtdna replication, such as polg. the first primary mitochondrial disease was described in 1962 [14] and involved a 35-year-old woman displaying excessive perspiration, polyphagia, polydipsia without polyuria, asthenia and decreased body weight, symptoms that started when she was seven years old. in addition, her basal metabolic rate was +172%, and she presented with creatinuria, myopathy and pathological cardiomyogram. she was diagnosed with a disorder of the enzymatic organization of the mitochondria. studies with mitochondria isolated from the skeletal muscle of this hypermetabolic patient revealed oxphos uncoupling [14] . since then, a range of primary mitochondrial diseases has been described (reviewed in [15] ). secondary mitochondrial defects can be caused by germline mutations in genes not involved in respiration/oxidative phosphorylation or can be acquired during the lifetime upon environmental insults. notably, environmental stress can induce mtdna alterations leading to mitochondrial dysfunction during aging, inflammatory response, etc. [16, 17] . from a pathological point of view, primary and secondary mitochondrial diseases can cause very similar symptoms, sometimes making diagnosis difficult. at the molecular level, mitochondrial dysfunction can affect the levels of key intracellular signaling regulators, such as ros and ca 2+ , that can be transmitted to the nucleus (mitochondria-to-nucleus signaling or retrograde signaling) resulting in changes in gene expression and modulation of a range of cellular functions [1, [18] [19] [20] . in addition, the release of mtdna and peptides from the mitochondrial matrix can activate an immune response that promotes a pro-inflammatory cascade [21] . mitochondrial metabolites can also act as signaling molecules and epigenetic modulators. in this context, citrate, an intermediate of the tca cycle, represents the major source of acetyl-coa for protein acetylation, a co-and post-translational modification that regulates protein levels and intracellular signaling in physiological and pathological conditions [22] . emerging data have also provided new evidences of connections between mitochondrial dynamics and physical contacts among mitochondria and the endoplasmic reticulum (er), known as mitochondrial-associated er membranes (mams), which can finetune the mechanisms of regulation of energy production, ca 2+ homeostasis, survival and apoptosis [23] . here, a synthetic overview of the role of mitochondria in specific physiopathological conditions is provided ( figure 1 ). cardiovascular diseases are a leading cause of death worldwide. this class of diseases comprises several pathologies, including ischemic heart disease, peripheral vascular disease, cardiac arrest, heart failure, cardiomyopathies, hypertension, atherosclerosis, and arrhythmia. mitochondria have been involved at various degrees in the pathological aspects of these diseases. notably, mitochondrial dysfunction of muscle cells represents a key event in the prognosis of peripheral arterial disease. reduced oxphos activity due to etc impairment increases ros levels and ca 2+ release from mitochondria, causing apoptosis [24] . however, if ros levels remain below a threshold, the cells activate a defense program involving production of antioxidants and increased mitochondrial biogenesis. these mechanisms, known as mitohormesis, can limit the damage caused by repeated cycles of ischemia-reperfusion in peripheral arterial disease [24] . pharmacological treatments that can improve mitohormesis might be a promising therapeutic approach for peripheral arterial disease and other cardiovascular diseases. disruption of mitophagy also exacerbates the development of cardiovascular diseases [7] . growing evidence indicates that the pharmacological targeting of the mitochondria with drugs/natural compounds able to modulate mitophagy can ameliorate cardiovascular disorders in patients and be cardioprotective [7, 25] . future studies that aim at a better understanding the pathogenesis of some cardiovascular diseases are crucial to develop mitochondria-targeting drugs in the clinic. inflammation is a complex, protective body response to infections and tissue damage. the inflammatory response signals the immune system to repair damaged tissue and defend against pathogens (viruses, bacteria, etc.) or other harmful stimuli through secretion of specific mediators. however, when inflammation persists, it may drive various diseases and tissue damage. mitochondrial-derived ros play a key role in the inflammatory response. notably, mitochondria are considered the main drivers of the nlrp3 (nod-, lrr-and pyrin domain-containing 3) inflammasome [26] [27] [28] [29] , representing a central hub that controls innate immunity and response to inflammation. among various inflammatory conditions, mitochondria are involved in the hyper-inflammatory response, also reported as cytokine storm, caused by the sars-cov-2 (covid-19) respiratory cardiovascular diseases are a leading cause of death worldwide. this class of diseases comprises several pathologies, including ischemic heart disease, peripheral vascular disease, cardiac arrest, heart failure, cardiomyopathies, hypertension, atherosclerosis, and arrhythmia. mitochondria have been involved at various degrees in the pathological aspects of these diseases. notably, mitochondrial dysfunction of muscle cells represents a key event in the prognosis of peripheral arterial disease. reduced oxphos activity due to etc impairment increases ros levels and ca 2+ release from mitochondria, causing apoptosis [24] . however, if ros levels remain below a threshold, the cells activate a defense program involving production of antioxidants and increased mitochondrial biogenesis. these mechanisms, known as mitohormesis, can limit the damage caused by repeated cycles of ischemia-reperfusion in peripheral arterial disease [24] . pharmacological treatments that can improve mitohormesis might be a promising therapeutic approach for peripheral arterial disease and other cardiovascular diseases. disruption of mitophagy also exacerbates the development of cardiovascular diseases [7] . growing evidence indicates that the pharmacological targeting of the mitochondria with drugs/natural compounds able to modulate mitophagy can ameliorate cardiovascular disorders in patients and be cardioprotective [7, 25] . future studies that aim at a better understanding the pathogenesis of some cardiovascular diseases are crucial to develop mitochondria-targeting drugs in the clinic. inflammation is a complex, protective body response to infections and tissue damage. the inflammatory response signals the immune system to repair damaged tissue and defend against pathogens (viruses, bacteria, etc.) or other harmful stimuli through secretion of specific mediators. however, when inflammation persists, it may drive various diseases and tissue damage. mitochondrial-derived ros play a key role in the inflammatory response. notably, mitochondria are considered the main drivers of the nlrp3 (nod-, lrr-and pyrin domain-containing 3) inflammasome [26] [27] [28] [29] , representing a central hub that controls innate immunity and response to inflammation. among various inflammatory conditions, mitochondria are involved in the hyper-inflammatory response, also reported as cytokine storm, caused by the sars-cov-2 (covid-19) respiratory infection ( [30] and references therein). when macrophages and other immune cells detect viruses, they start secreting cytokines and chemokines to communicate with other immune cells [31] . strikingly, wuhan's covid-19 patients with severe clinical symptoms requiring icu admission displayed higher levels of the cytokines/chemokines ccl2, tnf-α and cxcl10 compared to individuals with less severe symptoms [32] . the release of large quantities of pro-inflammatory cytokines and chemokines by overdriven immune effector cells sustains an aberrant systemic inflammatory response that results in the immune system attacking the body, which in turn causes the acute respiratory distress syndrome [33] . immune cells under a hyper-inflammatory state metabolically adapt to this stress condition by favoring aerobic glycolysis over oxphos for energy production. this metabolic rewiring allows macrophages to become more phagocytic and favors anabolic reactions for the synthesis and secretion of cytokines and chemokines in a vicious cycle ( [30] and references therein). side by side, many biosynthetic reactions occurring in mitochondria of hyper-activated macrophages are inhibited as a consequence of oxphos and tca cycle inhibition. melatonin's synthesis is among these reactions: acetyl-coa, a cofactor in the rate-limiting reaction for melatonin synthesis, lacks due to the tca cycle inhibition [30] . thus, melatonin cannot be synthetized. notably, melatonin is a potent anti-inflammatory and anti-oxidant and its administration to covid-19 patients has been recently proposed as potential adjuvant treatment strategy to reduce the severity of the covid-19 pandemic [34] [35] [36] . though clinical evidences are not yet available, several scientific data supports the potential utility of melatonin to attenuate the worst symptoms of covid-19 infection [37, 38] . mitochondrial dysfunction has long been recognized as a driver of the aging process. early studies have linked accumulation of mitochondrial dna mutations and the concomitant decline in etc and oxphos activity to aging [29, 39] . furthermore, genetic studies in mice support a causal relation between mtdna depletion and aging [40] . recent evidences have confirmed that healthy centenarians retain more "intact" mtdna copies than old people and frail centenarians [40] , suggesting that "healthy" mtdna is a hallmark of healthy aging. besides the mtdna status, activation of mitochondria-to-nucleus signaling pathways, particularly the mitochondrial unfolded protein response (upr mt ), has been implicated in aging. upr mt activation promotes transcription of several nuclear genes, such as those encoding antioxidant proteins and enzymes, which support survival, gain of the mitochondrial functionality and, thus, longevity and lifespan [41] . it should be noted that if a heteroplasmic mtdna pool is present, upr mt activation could exacerbate mitochondrial dysfunction as it may lead to accumulation of mutant mtdna [42] . alterations in the removal of damaged mitochondria through mitophagy have also been implicated in aging. mitophagy markedly decreases during aging in mammalian tissues and organs [43, 44] and this may be responsible for the known accumulation of damaged mitochondria in aging tissues. notably, genetic manipulations in c. elegans that increase mitophagy also extend the organismal lifespan [45] , strengthening the connection between altered mitophagy and aging. neurodegenerative diseases are characterized by changes in mitochondrial morphology and biochemical activity. alzheimer's (ad) and parkinson's (pd) disease are the most diffuse neurodegenerative illnesses among older adults. brain cells from ad and pd patients show reduced respiratory activity and mitochondrial biogenesis [46, 47] . a prominent pathological feature of ad is the impaired cerebral glucose metabolism, which is reduced by 45% in the early stages, preceding neurological impairment and atrophy, and further declines in the late stages of the disease [48] . the decrease in glucose metabolism is associated with reduced expression and activity of mitochondrial enzymes, including pyruvate dehydrogenase, isocitrate dehydrogenase and α-ketoglutarate dehydrogenase, three enzymes of the tca cycle [49] . in addition, reduced activity of the mitochondrial respiratory complexes i, ii, iii and iv has also been documented [46] . somatic mutations in the mitochondrial genome have been detected in postmortem brain tissue from ad patients, at levels higher than in healthy brains [50] . these mutations may not only affect the etc but also trigger other neuropathological consequences, such as increased ros production and oxidative stress in neurons and promotion of amyloidogenic processing of the amyloid precursor protein. mitophagy is also diminished in ad's neurons, and this may contribute to the etiopathogenesis of ad. indeed, mitophagy was able to prevent or reverse the cognitive impairment in several ad models [51] , confirming the critical involvement of mitochondria in ad. mutations in nuclear genes encoding mitochondrial proteins important for the proper function of mitochondria have been directly linked to pd. notably, mutations in proteins involved in mitochondrial quality control, such as pink1, parkin and lrrk2, are a frequent cause of monogenic pd [52] . loss or impaired functionality of these proteins results in mitochondrial fragmentation, dysregulation of calcium homeostasis and changes in mitochondria-endoplasmic reticulum contact sites (mercs). recently, mutations in miro1, a protein important for the regulation of the structure and function of mercs, have been causally linked to pd establishing that variants in the gene encoding for miro1 represent rare genetic risk factors for neurodegenerative diseases like pd ( [53] and references therein). although there is no doubt about the involvement of mitochondrial dysfunction in ad and pd, still more research is required to identify therapeutic targets that could improve mitochondrial activity and reduce oxidative stress in neurons in the early stages of these neurodegenerative diseases. future studies should be aimed at investigating the chronological sequence of molecular events involved in the pathogenesis of these diseases. further investigations are also needed to assess whether mitochondrial dysfunction represents a primary cause of ad or a consequence of other molecular/genetic events. mitochondrial dysfunction has been involved in different aspects of the pathogenesis of cancer, from the early steps of cancer development to cancer progression to a metastatic phenotype, and resistance to anti-cancer drugs [1, 19, 29] . in this context, mutations in three tca cycle enzymes, namely succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase, have been shown to play a causal role in carcinogenesis [54, 55] , thus providing compelling evidence for the involvement of mitochondrial metabolic alterations as cancer drivers. indeed, mutations in succinate dehydrogenase predispose to hereditary paragangliomas, pheochromocytomas, neuroblastomas, gastrointestinal tumors, renal cell cancers and thyroid tumors [54] . sporadic and hereditary mutations of fumarate hydratase trigger accumulation of an oncogenic metabolite, i.e., fumarate, that favors development of hereditary leiomyomatosis and renal cell carcinoma, ewing sarcoma and osteosarcoma, adrenocortical carcinoma, pheochromocytoma, glioma, neuroblastoma, paraganglioma, and ependymoma [55] . mutations in isocitrate dehydrogenase are only somatic and have been detected in about 20% of patients with acute myeloid leukemia or angioimmunoblastic t-cell lymphoma, and at lower frequencies in patients with thyroid, prostate, colorectal cancer and b-cell acute lymphoblastic leukemia [54, 56] . besides mutations in nuclear-encoded mitochondrial proteins, mutations in mtdna-encoded proteins have also been implicated in the pathogenesis of cancer. the spectrum of somatic mtdna mutations varies among different tissues, and increasing evidence shows that the load of mtdna mutations could have prognostic value. the majority of cancer-related mtdna mutations have been found in prostate cancer, with a total of more than 700 unique somatic mtdna mutations associated with this cancer [57] . there is increasing evidence that mtdna mutations/depletion may favor cancer progression to a metastatic and drug-resistant phenotype through increased production of ros and/or activation of a mitochondria-to-nucleus signaling that leads to expression of pro-metastatic and pro-survival nuclear genes [20, 29, [58] [59] [60] . although mtdna damage may not be the first driver of cancer progression, it is likely that it represents a "supporter" event that facilitates and accelerates different steps of the metastatic cascade, probably within a precise time window that remains to be identified. mitochondrial dysfunction is implicated in several pathological conditions, ranging from neurodegenerative and cardiovascular diseases, to aging, cancer and inflammation. each of these conditions shows a peculiar involvement of mitochondria. for example, up to 94% of pd patients show a defect in miro1 function, because this protein, located on the mitochondrial surface, fails to detach from depolarized mitochondria resulting in defective mitochondrial locomotion and clearance by mitophagy [61] . these new results suggest that miro1-based therapeutic strategies may provide new avenues to a personalized medicine for pd. the role of mitochondrial dysfunction in other diseases is still somehow controversial. in some cases, it may represent a driver event, like for mutations in the tca cycle enzymes succinate dehydrogenase, fumarate hydratase and isocitrate dehydrogenase that predispose to certain types of tumors. in other cases, a transient mitochondrial dysfunction may support a metabolic rewiring needed by the cells to adapt and survive to microenvironmental stressors. the author declares no conflict of interest. how mitochondria produce reactive oxygen species the role of the mitochondrial permeability transition pore in heart disease regulation of mitochondrial biogenesis mitochondrial quality control in cardiac cells: mechanisms and role in cardiac cell injury and disease intersection of mitochondrial fission and fusion machinery with apoptotic pathways: role of mcl-1 mitophagy in cardiovascular diseases mechanisms, pathophysiological roles, and analysis mitochondrial fission, fusion, and stress mitophagy and cancer parkin and mitophagy in cancer mitophagy in neurodegeneration and aging a case of severe hypermetabolism of nonthyroid origin with a defect in the maintenance of 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fumarate hydratase in cancer: a multifaceted tumour suppressor rescue of tca cycle dysfunction for cancer therapy mitochondrial genome variation and prostate cancer: a review of the mutational landscape and application to clinical management ros-generating mitochondrial dna mutations can regulate tumor cell metastasis mitochondrial dna depletion in prostate epithelial cells promotes anoikis resistance and invasion through activation of pi3k/akt2 mitochondrial dna depletion sensitizes cancer cells to parp inhibitors by translational and post-translational repression of brca2 miro1 marks parkinson's disease subset and miro1 reducer rescues neuron loss in parkinson's models this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-309876-l0xginsa authors: vena, antonio; berruti, marco; adessi, andrea; blumetti, pietro; brignole, michele; colognato, renato; gaggioli, germano; giacobbe, daniele roberto; bracci-laudiero, luisa; magnasco, laura; signori, alessio; taramasso, lucia; varelli, marco; vendola, nicoletta; ball, lorenzo; robba, chiara; battaglini, denise; brunetti, iole; pelosi, paolo; bassetti, matteo title: prevalence of antibodies to sars-cov-2 in italian adults and associated risk factors date: 2020-08-27 journal: j clin med doi: 10.3390/jcm9092780 sha: doc_id: 309876 cord_uid: l0xginsa we aimed to assess the prevalence of and factors associated with antisevere acute respiratory syndrome coronavirus-2 (sars-cov-2) positivity in a large population of adult volunteers from five administrative departments of the liguria and lombardia regions. a total of 3609 individuals were included in this analysis. participants were tested for anti-sars-cov-2 antibodies [immunoglobulin g (igg) and m (igm) class antibodies] at three private laboratories (istituto diganostico varelli, medical center, and casa della salute di genova). demographic data, occupational or private exposure to sars-cov-2-infected patients, and prior medical history consistent with sars-cov-2 infection were collected according to a preplanned analysis. the overall seroprevalence of anti-sars-cov-2 antibodies (igg and/or igm) was 11.0% [398/3609; confidence interval (ci) 10.0%–12.1%]. seroprevalence was higher in female inmates than in male inmates (12.5% vs. 9.2%, respectively, p = 0.002), with the highest rate observed among adults aged >55 years (13.2%). a generalized estimating equations model showed that the main risk factors associated with sars-cov-2 seroprevalence were the following: an occupational exposure to the virus [odd ratio (or) = 2.36; 95% ci 1.59–3.50, p = 0.001], being a long-term care facility resident (or = 4.53; 95% ci 3.19–6.45, p = 0.001), and reporting previous symptoms of influenza-like illness (or = 4.86; 95% ci 3.75–6.30, p = 0.001) or loss of sense of smell or taste (or = 41.00; 95% ci 18.94–88.71, p = 0.001). in conclusion, we found a high prevalence (11.0%) of sars-cov-2 infection that is significantly associated with residing in long-term care facilities or occupational exposure to the virus. these findings warrant further investigation into sars-cov-2 antibody prevalence among the italian population. in italy, the first case of pandemic severe acute respiratory syndrome coronavirus-2 (sars-cov-2) infection was reported on 20 february, 2020. since then, the number of cases increased rapidly in the north of the country, with the lombardia and liguria regions being heavily affected by the infection [1] . by the end of april 2020, approximately 85,000 laboratory confirmed cases -of sars-cov-2 infection were reported in this geographical area of the country [2] . however, these data included only a fraction of the real number of sars-cov-2 infections, since not all infected patients were symptomatic [3] [4] [5] , required hospitalizations, or provided specimens for laboratory testing. the extent to which surveillance data reflect the true burden of the disease can also be affected by changes in laboratory testing recommendation [1] . serology can represent a key element to overcoming these limits and to better understanding the infection statistics at a population level. the primary outcome of this study was to estimate the prevalence of sars-cov-2 antibodies. the secondary outcome was to evaluate possible factors associated with anti-sars-cov-2 positivity in a large population of individuals from five administrative departments of the liguria and lombardia regions. this was an observational study designed to evaluate the prevalence and factors associated with sars-cov-2 infections among voluntary, unpaid individuals tested for sars-cov-2 antibodies in three private institutions (istituto diagnostico varelli, medical center, and casa della salute di genova) during march and april 2020. these institutions altogether include approximately 5,784,974 inhabitants living in five administrative departments (milano, varese, pavia, genova and savona) of the liguria and lombardia regions. each laboratory process, about 500,000 samples per year, offers a comprehensive range of tests including clinical biochemistry, serology, and genetic analysis. we included non-hospitalized participants (aged > 18 years) who voluntarily tested for sars-cov-2 antibodies in an outpatient setting. after providing informed consent, a sample of venous blood was collected from each participant, all of whom also completed a questionnaire on potential risk factors for developing sars-cov-2 infection. recorded data included age, sex. and occupational or private exposure to sars-cov-2 infected patients. in addition, information regarding stays at a long-term care facilities or prior medical history consistent with sars-cov-2 infection (influenza-like illness defined according to who criteria [6] or loss of smell or taste) within the previous month, were also collected. the primary goal was to assess the prevalence of sars-cov-2 antibodies [either immunoglobulin m (igm) and g (igg)] positivity among the study population. the secondary goal was to investigate the association between positive tests and demographics (age and sex), occupational and private contact with sars-cov-2 infected patients, living in long-term care facilities, and prior symptoms consistent with sars-cov-2 infection. blood samples were analyzed for serological detection at each participating laboratory by trained staff, unaware of the clinical details of the tested patients. the first laboratory (istituto diagnostico varelli) used a chemiluminescent quantitative immunoassay detecting antibodies against nucleocapsid protein and spike protein (the maglumitm 2019) [7] . according to the manufacturer's recommendations, samples were considered positive above a threshold of 1.1 au/ml for igm and igg. this cut-off resulted in clinical sensitivities/specificities of 78.6%/97.5% and 91.2%/97.3% for igm and igg, respectively [7, 8] . the second laboratory (medical center) applied a rapid chromatographic immunoassay for the qualitative detection of igg and igm antibody against spike protein (realy tech ® 2019 ncov/covid-19 igg/igm rapid test device). the manufacturer's reported a clinical sensitivity of 92% for igm; 96% for igg; and a specificity of 100% for igm and igg. the third laboratory (casa della salute di genova) assessed anti-sars-cov-2 antibodies using a commercially available point-of-care lateral flow immunoassay (biosynex ® covid-19 bss, fribourg, switzerland) that can simultaneously detect igm and igg in human blood, with an overall sensitivity of 88.7% and specificity of 90.6% [9] . this qualitative test detected antibodies against nucleocapsid and spike proteins. all laboratories used internal procedures to validate the diagnostic performance of serological tests. in all cases, the results showed values of sensitivity and specificity consistent with those reported by each manufacturer. all statistics were analyzed using spss software. prevalence of anti-sars-cov-2 antibodies (igm or igg) was calculated and the exact binomial distribution was used to calculate 95% confidence intervals (cis). the association between positive sars-cov-2 antibodies and study variables was estimated in two steps. first, a general linear univariate analysis was performed using a chi-squared test. the second step used a generalized estimating equation (gee) model to consider laboratory provenience, with sars-cov-2 seropositivity used as a dependent variable. only differences with a p-values < 0.05 were considered statistically significant. the study protocol was approved by the ethics committee of liguria region (pi prof. matteo bassetti-n. cer liguria 381/2020-id 10770). between 1 march and 30 april 2020, 3609 individuals agreed to participate in the study. the mean number of screened individuals per administrative department was 721 (52-1430), representing 12 people per 100,000 inhabitants. the patients' demographics are outlined in table 1 . overall, 55.6% (2007/3609) were women and 44.4% were men (1602/3609). the median age was 51 years [interquartile range (iqr) 41-63], with the age group >55 years being most represented of the 3609 individuals included in the study population, 398 tested anti-sars-cov-2 positive [11.0% (ci 10.0%-12.1%)]. seroprevalence was higher among women vs. men (12.5% vs. 9.2%, p = 0.002) and varied with age. the rate was highest among adults aged >55 years (13.2%), followed by adults aged 18-35 years (11.9%). as for geographical distribution, the highest prevalence of anti-sars-cov-2 positivity was reported in the administrative departments of savona ( figure 1 ). of the 3609 individuals included in the study population, 398 tested anti-sars-cov-2 positive [11.0% (ci 10.0%-12.1%)]. seroprevalence was higher among women vs. men (12.5% vs. 9.2%, p = 0.002) and varied with age. the rate was highest among adults aged >55 years (13.2%), followed by adults aged 18-35 years (11.9%). as for geographical distribution, the highest prevalence of anti-sars-cov-2 positivity was reported in the administrative departments of savona (figure 1 ). table 2 shows estimated prevalence according to the three different laboratories. several factors showed an association with anti-sars-cov-2 antibodies positivity with univariable analysis ( table 3 ). the variables that showed a p-value < 0.10 were also included in the gee model ( table 4 ). the model showed that the main risk factors associated to sars-cov-2 seroprevalence were the following: occupational exposure to the virus (or = 2.36; 95% ci 1.59-3.50, p = 0.001), living in a long-term care facility (or = 4.53; 95% ci 3.19-6.45, p = 0.001), and reporting previous symptoms of influenza-like illness (or = 4.86; 95% ci 3.75-6.30, p = 0.001) or loss of sense of smell or taste (or = 41.00; 95% ci 18.94-88.71, p = 0.001). in the present observational study performed on a large sample of subject in northern italy, we found the following: (1) the overall seroprevalence of anti-sars-cov-2 antibodies (igg and/or igm) was 11.0%; (2) occupational exposure to the virus, long-term care facility residency, as well as previous symptoms of influenza-like illness or loss of sense of smell or taste were independently associated with anti-sars-cov-2 positivity. to the best of our knowledge, this is one of the first reports that attempts to describe the prevalence of coronavirus disease and to evaluate the potential circulation of sars-cov-2 in north italy. the findings of our study showed that in a definite geographical area of italy, approximately 630,000 people might have developed antibodies (11.0% of 5,784,974 inhabitants). this figure is significantly higher than the number of molecular-confirmed sars-cov-2 infections (~32,600 cases in the five administrative departments) reported by the protezione civile and the italian national institute of health as of 30 april 2020 [2]. the high observed seroprevalence is consistent with recent studies (table 5 ) performed in other heavily affected areas of europe: 9.7% in geneva, switzerland [10] and 10.0% in madrid, spain [11, 12] . table 5 . summary of articles published in the literature reporting data regarding prevalence of sars-cov-2 antibodies in the general population. petersen m.s. [13] faroe islands; nationwide study 1075 0.6% biggs h. [14] u.s.; two metropolitan atlanta counties 696 2.5% menachemi n. [15] u.s; indiana 3658 2.79% fischer b. [16] germany; three federal states 3186 0.91% pollan m. [11] spain; nationwide study 61,075 5.0% havers f. [17] u.s; 10 sites 16,025 from 1.0% (san francisco) to 6.9% (new york city) amorim filho l. [18] brazil; rio de janeiro 2857 4.0% percivalle e. [19] italy; lodi area 390 23.0% soriano v. [12] spain, madrid 674 13.8% stringhini s. [10] switzerland, geneve 2766 9.7% sood n. [20] u.s., los angeles 1702 4.3% living in a long-term care facility was the strongest predictors of sars-cov-2 infection and was reported by 21.6% of anti-sars-cov-2-positive participants (n = 86/398). this connection was not unexpected [21] [22] [23] , since long-term care facilities often have limited or no infection control programs [24, 25] and are usually congregative settings where elderly people have greater exposure to infected patients in the case of respiratory outbreaks [26] [27] [28] . therefore, our results emphasized the importance of implementing strategic bundles for infections prevention in long-term care facilities [29] . in this regard, educational interventions on healthcare providers' knowledge, as well as active surveillance of suspected cases and implementation of barrier precautions, were shown to play a vital role in limiting the spread of other respiratory outbreaks [26] [27] [28] . reporting an occupational exposure to the virus also emerged as an independent factor associated with sars-cov-2 infection and was reported by 8.7% of anti-sars-cov-2-positive participants (n = 35/398). however, approximately two-thirds of anti-sars-cov-2-positive participants did not report any apparent risk depicting the widespread circulation of the virus in the italian community, where it has become endemic. as for clinical symptoms, we found that the prevalence of sars-cov-2 antibodies depends on the type of clinical manifestation reported by the patient, being particularly high in people who reported loss of smell or taste [30, 31] . interestingly, 8.6% of participants (n = 277/3224) who did not report any symptoms presented antibodies positivity. this finding suggests that non-apparent infection is relatively common in a healthy, active population, thus supporting the hypothesis that, as is true for other coronavirus infections [32] , sars-cov-2 infection might also be asymptomatic or pauci-symptomatic and resolves spontaneously without any complications in many cases. in our opinion, the findings of our study could have several implications for pandemic management. because the real number of patients with sars-cov-2 infection is significantly higher than the pcr-confirmed cases, stringent lockdown strategies might possibly be re-implemented only when the intensive care units' capacities to handle emergencies are overwhelmed. since a large proportion of patients with sars-cov-2 infection are asymptomatic, contract tracing methods to limit the spread of the infection could be particularly challenging. thus, screening strategies beyond a symptoms-driven approach will be necessary for italy (e.g., use of mobile applications) to identify enough infected persons to reach sars-cov-2 elimination targets [33] ; our data could also be useful for vaccine design and implementation. there are several limitations that should be discussed. firstly, we do did have any information regarding previous sars-cov-2 molecular testing among those patients who tested positive. accordingly, we cannot provide valuable estimates of antibody prevalence in people positive and negative in pcr testing. secondly, we analyzed serum samples from patients who voluntarily decided to be tested. therefore, the clinical characteristics of the sample might differ from those of the general italian population. thirdly, geographical prevalence of anti-sars-cov-2 antibodies might have been influenced by the type of serological tests used. however, the diagnostic performances of each test are similar to each other; in addition, the highest percentage of infected patients in the liguria region agrees with recent evidence, suggesting the presence of anti-sars-cov-2 antibodies among blood donors from savona and genova since december 2019 (unpublished data reported by the ligurian regional health authority alisa). fourthly, all tests we used are non-fda approved and are yet to be validated. therefore, prevalence estimates could change once new information on the accuracy of tests are available. fifthly, the interpretation of the test is still under discussion, because even patients with confirmed sars-cov-2 infections have low or non-detectable antibodies titles several weeks after acute infection [34] . lastly, based on the specificities of testing kits, we cannot exclude that some participants had false positive results due to past or present infection with other viruses, including non-sars-cov-2 coronavirus strains [35] . in addition, antibody response may be impaired in elderly, immuno-compromised or immunosuppressed participants, and may produce false negative serology test results [36] . in conclusion, the results of the present study demonstrate that infection rates based on surveillance data considerably underestimated the infection rates during the sars-cov-2 virus pandemic in italy. the seroprevalence was much higher among people living in long-term care facilities or those with occupational exposure. in our opinion, these findings warrant further investigation into sars-cov-2 antibody prevalence among the italian population. outside the submitted work, m.b. (matteo bassetti) has participated in advisory boards and/or received speaker honoraria from achaogen, angelini, astellas, bayer, basilea, biomeérieux, cidara, gilead, menarini, msd, nabriva, paratek, pfizer, roche, melinta, shionogi, tetraphase, venatorx, and vifor and has received study grants from angelini, basilea, astellas, shionogi, cidara, melinta, gilead, pfizer, and msd. outside the submitted work, d.r.g. reports honoraria from stepstone pharma gmbh and unconditional grants from msd italia and correvio italia. the authors declare no conflict of interest. case-fatality rate and characteristics of patients dying in relation to covid-19 in italy the novel chinese coronavirus (2019-ncov) infections: challenges for fighting the storm clinical characteristics of asymptomatic and symptomatic patients with mild covid-19 estimating the asymptomatic proportion of coronavirus disease 2019 (covid-19) cases on board the diamond princess cruise ship working towards a simple case definition 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the united states seroprevalence of anti-sars-cov-2 among blood donors in rio de janeiro prevalence of sars-cov-2 specific neutralising antibodies in blood donors from the lodi red zone seroprevalence of sars-cov-2-specific antibodies among adults preventing the spread of covid-19 to nursing homes: experience from a singapore geriatric centre nursing homes with covid-19 cases epidemiology of covid-19 in a long-term care facility understanding infection prevention and control in nursing homes: a qualitative study infection prevention and control programs in us nursing homes: results of a national survey severe respiratory illness associated with human metapneumovirus in nursing home viral respiratory infections in a nursing home: a six-month prospective study outbreak of human metapneumovirus in a nursing home: a clinical perspective infections in nursing homes: epidemiology and prevention programs high prevalence of olfactory and taste disorder during sars-cov-2 infection in outpatients cov-2: olfaction, brain infection, and the urgent need for clinical samples allowing earlier virus detection asymptomatic coronavirus infection: mers-cov and sars-cov-2 (covid-19) sixty seconds on the contact tracing app neutralizing antibody responses to sars-cov-2 in a covid-19 recovered patient cohort and their implications the laboratory's role in combating covid-19 covid-19 serological tests: how well do they actually perform? diagnostics 2020 key: cord-315188-a9pvugjt authors: choi, min hyuk; ahn, hyunmin; ryu, han seok; kim, byung-jun; jang, joonyong; jung, moonki; kim, jinuoung; jeong, seok hoon title: clinical characteristics and disease progression in early-stage covid-19 patients in south korea date: 2020-06-23 journal: j clin med doi: 10.3390/jcm9061959 sha: doc_id: 315188 cord_uid: a9pvugjt a rapid increase in the number of patients with coronavirus disease 19 (covid-19) may overwhelm the available medical resources. we aimed to evaluate risk factors for disease severity in the early stages of covid-19. the cohort comprised 293 patients with covid-19 from 5 march 2020, to 18 march 2020. the korea centers for disease control and prevention (kcdc) classification system was used to triage patients. the clinical course was summarized, including the impact of drugs (angiotensin ii receptor blockers [arb], ibuprofen, and dipeptidyl peptidase-4 inhibitors [dpp4i]) and the therapeutic effect of lopinavir/ritonavir. after adjusting for confounding variables, prior history of drug use, including arb, ibuprofen, and dpp4i was not a risk factor associated with disease progression. patients treated with lopinavir/ritonavir had significantly shorter progression-free survival than those not receiving lopinavir/ritonavir. kcdc classification i clearly distinguished the improvement/stabilization group from the progression group of covid-19 patients (auc 0.817; 95% ci, 0.740–0.895). coronaviruses, enveloped viruses with a positive-sense single-stranded rna genome, comprise the family coronaviridae, order nidovirales and are widely distributed in birds, humans, and other mammals [1] . the novel coronavirus disease (covid-19) caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection emerged in wuhan, hubei, china, on 8 december 2019 [2] [3] [4] . the outbreak has spread worldwide, and the number of confirmed cases is growing rapidly [5] . most covid-19 patients have mild symptoms, such as fever and cough [5] , and have a favorable prognosis without specific treatment [4, 6, 7] . in severe cases, dyspnea and hypoxia may develop within one week after onset of the disease and may rapidly progress to acute respiratory distress syndrome (ards), acute respiratory failure, septic shock, metabolic acidosis, and coagulopathy [8] . the first case of covid-19 in south korea was a resident of wuhan, china, who entered incheon airport on 19 january 2020 [9] . on 17 february, the 31st covid-19 patient was confirmed to have participated in religious ceremonies in daegu city. subsequently, multiple covid-19 outbreaks occurred in south korea, including community-associated outbreaks in daegu city and healthcare-associated outbreaks in cheongdo, gyeongsangbuk-do province [10] . a rapid increase in number of patients with covid-19 can overwhelm the available medical resources, including intensive care units, negative pressure beds, and medical staff. therefore, early assessment of risk factors for disease progression and patient prognosis is critical to ensure that patients whose disease is more likely to increase in severity can receive proper treatment in a timely manner. thus, the korea centers for disease control and prevention (kcdc) have established a system to triage patients in public health centers, whereby mild cases are transferred to living treatment centers, mild to moderate cases to dedicated cohort hospitals, and severe cases to tertiary university hospitals [11] . large-scale diagnostic testing was performed to find hidden covid-19 cases in sub-populations that had a history of contact with confirmed cases. through these processes, south korean patients with asymptomatic infection or early symptoms of disease were identified, and early monitoring and treatment of patients with covid-19 were conducted. although many published studies have summarized the clinical features of covid-19 patients [4, 6, 7, [12] [13] [14] [15] , few have addressed the course of the disease in the early stages of symptom onset. therefore, the clinical characteristics, imaging features, and treatment outcomes of covid-19 patients before or immediately after onset of symptoms were investigated, with a particular focus on mild to moderate cases. we aimed to evaluate risk factors and kcdc classification models to predict disease progression in patients with early-stage covid-19. all consecutive patients with confirmed covid-19 admitted to the armed forces daegu hospital, daegu, south korea, from 5 march 2020 to 18 march 2020, were enrolled in this study. according to the arrangement established by the government, the hospital was designated as a covid-19-dedicated, 300-bed cohort hospital. the final follow-up date for this study was april 4, 2020. a total of 293 adult patients from daegu city was admitted to the hospital during the study period. all cases were confirmed as covid-19 using a real-time reverse transcription polymerase chain reaction (rt-pcr) (seegene inc., seoul, south korea, https://www.seegene.com) assay of nasal and oropharyngeal swabs [4] . this retrospective cohort study was approved by the institutional review board of the korean military medical association (seongnam-si, gyeonggi-do, south korea) (afmc-20015-irb-20-015). the following clinical data were collected using electronic medical records: age at diagnosis, sex, signs and symptoms, date of symptom onset, date of hospital admission, date of discharge or transfer, charlson comorbidity index [16] , eastern cooperative oncology group (ecog) performance status [17] , multilobular infiltration, hypo-lymphocytosis, bacterial coinfection, smoking history, hyper-tension and age (mulbsta) score [18] , pneumonia severity index [19] , confusion, urea, respiratory rate, blood pressure plus age ≥ 65 years (curb-65) [20] , respiratory support, and treatment agents administered before and during hospitalization. we also obtained radiologic findings. baseline non-contrast-enhanced chest computed tomography (ct) was completed for all patients to assess disease severity. to ensure the safety of medical staff by minimizing contact with patients, routine laboratory tests were not conducted in all patients but only for patients requiring clinical decisions. the triage algorithm and classification criteria according to the covid-19 response guidelines (version 7) developed by kcdc are presented in figure 1 . the criteria address the patient's mental state, age, history of underlying comorbidities, history of smoking, respiratory symptoms, and body temperature (bt). the "kcdc classification i" was applied if the patient's blood pressure (bp), pulse rate (pr), and respiratory rate (rr) could not be measured, and "kcdc classification ii" was applied if these parameters could be measured; thereafter, patients with covid-19 were classified into one of four groups (class i to iv). the living treatment center is a quarantine facility for mild or asymptomatic covid-19 patients who are unable to self-isolate at home. the patients were checked for vital signs twice a day and immediately transferred to hospitals if their symptoms worsened. if their symptoms resolved, the patient was tested according to the standards for lifting the quarantine. certain state-run facilities and accommodations are designated as living treatment centers and are supplied with medical staff, medical equipment (pulse oximetry device, thermometer, blood pressure monitor, cpr kit, chest x-ray radiograph, etc.), individual relief kits (underwear, toiletries, face masks, etc.), and hygiene kits (thermometer and medical supplies). the living treatment center is a quarantine facility for mild or asymptomatic covid-19 patients who are unable to self-isolate at home. the patients were checked for vital signs twice a day and immediately transferred to hospitals if their symptoms worsened. if their symptoms resolved, the patient was tested according to the standards for lifting the quarantine. certain state-run facilities and accommodations are designated as living treatment centers and are supplied with medical staff, medical equipment (pulse oximetry device, thermometer, blood pressure monitor, cpr kit, chest x-ray radiograph, etc.), individual relief kits (underwear, toiletries, face masks, etc.), and hygiene kits (thermometer and medical supplies). in previous studies of covid-19 patients [8, 12] , "mild cases" were defined as patients who experienced mild symptoms, with no manifestations of pneumonia on chest imaging. "moderate cases" referred to patients with uncontrolled fever despite administration of antipyretics and/or respiratory symptoms. severe cases of covid-19 were defined as patients with any of the following: respiratory distress, rr ≥ 30 breaths/min; mean oxygen saturation ≤ 93% at rest; arterial oxygen partial pressure/inspired oxygen fraction ≤ 300 mm hg. to quantify opacifications on pulmonary images, we applied the "ct score" as proposed in previous reports [21] [22] [23] . in brief, each pulmonary lobe was scored as 0 (none), 1 (diameter <1 cm), 2 (diameter 1 to 3 cm), 3 (diameter 3 cm to <50% of the lobe), or 4 (50% to 100% of the lobe) depending on lesion size and abnormal area. the overall score was calculated by summing all five lobar scores. depending on the course of the disease during hospitalization, patients were classified into either the progression or improvement/stabilization group. the progression group comprised mild or moderate cases that progressed to moderate or severe cases, while the improvement/stabilization group comprised mild cases that did not progress further. progression-free survival (pfs) was defined as the duration of time over which patients with covid-19 remained stable during their hospitalization. to adjust the outcomes of patients with covid-19 for potential confounding factors, we conducted a ps-matched case-control study. we selected 10 variables for adjustment using univariable analyses (table a1) : age, healthcare-associated infection, ecog performance status, asymptomatic on initial evaluation, bt at hospital admission, diastolic bp at hospital admission, pr at hospital admission, spo 2 at hospital admission, hypertension, and diabetes mellitus [24, 25] . we then performed ps-matched analyses by attempting to match cases and control patients (1:1 matching) using the nearest-neighbor-matching method. a match occurred when the difference in the logits of the ps was <0.2 times the standard deviation (sd) of the scores. we assessed all variables using the shapiro-wilk test to evaluate gaussian distributions. descriptive statistics are presented as median and interquartile range (iqr) for continuous and categorical variables. comparisons between groups were analyzed using the mann-whitney u test for continuous variables and fisher's exact test for categorical variables. pfs was analyzed using the kaplan-meier method, and differences between groups were qualified by log-rank testing. to obtain ors and hazard ratios (hrs), univariate regressions were performed using logistic and cox regression, respectively. all reported p values are two-tailed, and p values < 0.05 indicate statistical significance. we conducted statistical analyses using r statistical software (r studio, inc., https://www.r-project.org). the median days from the onset of symptoms to disease confirmation was 1 day (iqr, 0-6 days), and that from onset to hospital admission was 6 days (iqr, 0-12 days). the median duration of hospitalization was 18 days (iqr, 15-20 days), and hospitalized patients had a median duration of symptoms of 7 days (iqr, 0-15 days). as of april 4, 2020, 207 (70.6%) of 293 patients had been discharged, and 2 (0.7%) patients had been transferred due to symptom aggravation. the patients' discharge assessments were based on abatement of all symptoms, with two consecutive negative rt-pcr tests for covid-19. according to baseline chest ct imaging, 64 (21.8%) patients had findings consistent with bilateral pneumonia, and 56 (19.1%) patients had unilateral pneumonia. supplementary oxygen was required in 10 patients (3.4%). one hundred patients (34.1%) were administered antibiotics empirically: the treatment regimen was quinolone (84 patients [28. 7%]) or combination therapy with cefotaxime and doxycycline (14 patients [4.8%] ). in addition, 30 patients (10.2%) received lopinavir/ritonavir antiviral therapy, although it was withdrawn in 21 of these 30 patients (70.0%) due to side effects such as nausea and vomiting. thirty-six (12.3%) cases were classified as the progression group, and the remaining 257 (87.7%) cases were classified as the improvement/stabilization group. the progression group of covid-19 patients was significantly older than the improvement/stabilization group (49.5 vs. 27.0 years of age; p < 0.001). there were no statistically significant differences between sex and times from symptom onset to confirmation/ admission. the progression group included a greater proportion of cases of healthcare-associated infection than the improvement/stabilization group (p = 0.003). a greater proportion of patients in the progression group presented initial symptoms of fever, chest pain, dyspnea, myalgia or fatigue, chills, and diarrhea compared with patients in the improvement/stabilization group, while a greater proportion of patients in the improvement/stabilization group were asymptomatic. compared with the improvement/stabilization group, the progression group was more likely to have comorbidities such as hypertension (p = 0.003) and diabetes mellitus (p < 0.001). given the greater incidence of pre-existing conditions, a greater proportion of patients in the progression group had a history of drug use, including ibuprofen (p = 0.044), angiotensin ii receptor blockers (arb; p = 0.006), calcium channel blockers (ccb; p = 0.047), dipeptidyl peptidase-4 inhibitors (dpp4i; p < 0.001), metformin (p < 0.001), and/or statins (p = 0.006). initial symptoms (may be multiple) congestive heart failure 6 (2.0%) 2 (5.6%) 4 (1.6%) 0.338 prior history of drug use we conducted ps matching to adjust baseline demographics and clinical variables between the progression and improvement/stabilization groups, resulting in 36 matched pairs of patients. confounding variables were well balanced in the two groups, including all the 10 variables identified above in the methods section (table 2) . after ps matching, prior history of drug use, including ibuprofen, arb, dpp4i, was not statistically different between patients in the progression and improvement/stabilization groups. similarly, the effect of these drugs on patient prognosis did not differ significantly in subgroup analysis of patients with hypertension (table a2 ) or diabetes mellitus (table a3) . initial symptoms (may be multiple) to confirm that kcdc classifications were suitable for initial triage of patients with covid-19, the predictive values were compared to those of existing models using receiver operating characteristics analysis. as summarized in table 3 , all predictive values were significantly greater in the progression group than in the improvement/stabilization group (p < 0.001). kcdc classification i had the largest area under the curve (auc, 0.817; 95% ci, 0.740-0.895). after incorporating the ct score measured using baseline chest ct imaging into the kcdc classification i scheme, the auc was 0.846 (95% ci, 0.768-0.923), improving the predictive power. of the 293 patients with covid-19, 30 were treated with lopinavir/ritonavir (table a4) . patients chosen to receive lopinavir/ritonavir treatment were more likely to be in a higher risk group than patients who did not receive lopinavir/ritonavir treatment. after adjusting for confounding variables via ps matching, there were no significant differences between the groups for any of the 10 characteristics identified in the methods section above. however, even after matching, 18 of 30 (60.0%) patients who received lopinavir/ritonavir treatment showed disease progression, while 6 of 30 (20.0%) patients who did not receive lopinavir/ritonavir treatment experienced disease progression. patients treated in the lopinavir/ritonavir group had significantly shorter pfs than that in the group not receiving lopinavir/ritonavir both before and after ps matching, but there was no significant difference in the proportion of discharged patients between the two groups ( figure 2 and table a5 ). before and after propensity-score matching figure legend: patients treated with lopinavir/ritonavir group showed significantly lower progression-free survival than the without lopinavir/ritonavir group before and after propensity-score matching. but there was no statistical difference in discharge proportion between the two groups. in this cohort study, we reported the clinical characteristics of covid-19 patients and risk factors associated with disease progression, especially those associated with early stages of the disease. we also assessed the usefulness of the kcdc classification for initial patient triage. there are considerable differences between our study and previous studies of the course and severity of covid-19. in previous reports addressing chinese covid-19 patients, most of the patients were middle-aged and elderly, presented fever and/or cough, and chest ct indicated pneumonia in most patients [4, 6, 7, 12, 13, 26, 27] . in our study, the median age was 29 years, 33.1% of patients were asymptomatic at hospital admission, and pneumonia was evident in chest ct scans in only 41.3% of patients. in south korea, covid-19 patients were identified and classified at an early stage through use of large-scale diagnostic testing in accordance with national policy, which allowed both asymptomatic and symptomatic patients to receive inpatient treatment. the risk factors significantly associated with disease progression were older age, healthcareassociated infection, ecog performance status, presence of initial symptoms at the time of hospital admission, higher initial pr, lower initial spo2, hypertension, and diabetes, which were consistent with prior reports [4, 6, 12, 21, 23, 28] . in contrast, asymptomatic cases at the time of hospital admission had favorable outcomes. sars-cov-2 infects host cells by interacting with the angiotensin-converting enzyme (ace) 2 receptors [29] , which are expressed by epithelial cells in the lung, kidney, intestine, and blood vessels [30] . the high prevalence of ards and gastrointestinal symptoms such as diarrhea, nausea, and vomiting can be explained by this ace-2-receptor-mediated mechanism in covid-19 patients [31] . ace inhibitors (acei) and arbs that affect the renin-angiotensin-aldosterone system (raas) are commonly recommended for patients with hypertension [32] . in an animal model [33] and human studies [34, 35] , administration of acei and arbs has been shown to increase the number of ace2 receptors; ibuprofen and thiazolidinediones may also increase ace2 expression [36] . therefore, prior use of these drugs may be a risk factor for sars-cov 2 infection. similar to the ace2 receptor, human cd26 (also called dpp4) is also suggested as the potential binding site for covid-19 [37] . thus, dpp4i, which is widely used as a diabetes drug, may produce effects similar to arbs in covid-19 patients. however, given only small-scale clinical studies addressing acei/arb use and patient outcomes in hospital settings [28] have been completed to date, the impact of these drugs on covid-19 is controversial. moreover, vaduganathan et al. suggested that recombinant ace2 protein may restore balance to the raas and potentially prevent organ damage, and drugs acting on ace2 may benefit rather than harm covid-19 patients [38] . to analyze the impact of drugs acting on the ace2 receptor and human cd26 in covid-19 patients, we conducted a ps matched study. before matching, the proportion of patients reporting prior use of these drugs was significantly greater in the progression group; however, after adjusting for 10 confounding variables, including underlying comorbidities, there was no significant difference between patients with and without these medication histories. even after comparing the sums of three (ibuprofen, arbs, and thiazolidinediones) or four drugs (ibuprofen, arbs, thiazolidinediones, and dpp4i), no significant differences were found between the two groups. furthermore, in subgroup analysis of patients with hypertension and diabetes mellitus, the effect of these drugs on patient prognosis was not statistically significant. these results suggest that, in diseases such as hypertension and diabetes mellitus, the underlying pathophysiology associated with the raas affects the prognosis of covid-19 patients rather than the pharmacologic effects of the drugs used to control the disease. in the global covid-19 pandemic, the major challenge is the lack of medical resources. we evaluated use of the kcdc classifications to triage patients with covid-19 according to severity of the disease and to ensure they are treated at the appropriate medical institution. our results indicated that kcdc classification i had a good auc (0.817; 95% ci 73.98-89.46) and sensitivity, which suggested that this model is suitable for early screening of low-risk patients who are less likely to progress to severe disease. the use of the triage algorithm and kcdc classification for covid-19 patients saves medical resources, allowing more efficient treatment and management of patients. using the kcdc classification as a predictive model in the early stages of covid-19 outbreaks, more medical resources could be focused on patients with more severe disease, which may have underlain the relatively low cfr in south korea. the ct scores (auc > 0.7) for the covid-19 patients in this study clearly distinguished the progression group from the improvement/stabilization group, a finding which is consistent with previous reports [21] [22] [23] . the use of the kcdc classification i scheme with the ct score increased the auc and specificity of the predictive model. therefore, we suggest that triaging patients by applying these predictive models in accordance with the medical conditions and policies of each country may help manage patients in the covid-19 pandemic situation. in our clinical study, which comprised mostly mild to moderate cases, patients who received lopinavir/ritonavir treatment were not likely to experience a decrease in pfs; rather, the patients' symptoms may have been aggravated due to side effects of the antivirals. in a previous randomized controlled trial conducted in patients with severe covid-19, there was no treatment benefit of lopinavir/ritonavir: of 95 patients receiving lopinavir/ritonavir treatment, 48 (48.4%) had gastrointestinal side effects [39] , which is consistent with our data. the retrospective and single-center nature of our study may limit wider applicability of the results. due to the limited number of cases in the progression group, it was difficult to analyze risk factors for disease progression using multivariable-adjusted methods. thus, hidden bias and residual confounding factors might have influenced our results. another limitation of our study was that, to protect medical staff and minimize further spread of the disease in the hospital setting, routine laboratory tests were not conducted in all patients, and these data were not available for inclusion in the analyses. however, we tried to analyze risk factors for disease progression and treatment outcomes for covid-19 patients while minimizing selection bias using the ps matched study. after controlling for potential biases using ps matching analysis, drugs acting on the ace2 receptor and human cd26 were not risk factors for disease progression. we also demonstrated that the kcdc classification i was able to distinguish the improvement/stabilization group from the progression group of covid-19 patients, and the triage algorithm system saved medical resources, enabling efficient treatment and management of covid-19 patients in south korea. the authors declare no conflicts of interest. clinical virology outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle the continuing 2019-ncov epidemic threat of novel coronaviruses to global health-the latest 2019 novel coronavirus outbreak in wuhan clinical features of patients infected with 2019 novel coronavirus in 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medical literature between 1996 and 2003" by peter austin, statistics in medicine host susceptibility to severe covid-19 and establishment of a host risk score: findings of 487 cases outside wuhan clinical characteristics of coronavirus disease 2019 in china clinical characteristics and outcomes of 112 cardiovascular disease patients infected by 2019-ncov evolution of the novel coronavirus from the ongoing wuhan outbreak and modeling of its spike protein for risk of human transmission receptor recognition by the novel coronavirus from wuhan: an analysis based on decade-long structural studies of sars coronavirus covid-19 patients' clinical characteristics, discharge rate, and fatality rate of meta-analysis angiotensin converting enzyme inhibitors and angiotensin receptor blockers in the treatment of hypertension: should they be used together? effect of angiotensin-converting enzyme inhibition and angiotensin ii receptor blockers on cardiac angiotensin-converting enzyme 2 human intestine luminal ace2 and amino acid transporter expression increased by ace-inhibitors angiotensin receptor blockers as tentative sars-cov-2 therapeutics ibuprofen attenuates cardiac fibrosis in streptozotocin-induced diabetic rats emerging wuhan (covid-19) coronavirus: glycan shield and structure prediction of spike glycoprotein and its interaction with human cd26 renin-angiotensinaldosterone system inhibitors in patients with covid-19 a trial of lopinavir-ritonavir in adults hospitalized with severe covid-19 this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-322184-kgv9f58a authors: sohn, yujin; jeong, su jin; chung, won suk; hyun, jong hoon; baek, yae jee; cho, yunsuk; kim, jung ho; ahn, jin young; choi, jun yong; yeom, joon-sup title: assessing viral shedding and infectivity of asymptomatic or mildly symptomatic patients with covid-19 in a later phase date: 2020-09-10 journal: j clin med doi: 10.3390/jcm9092924 sha: doc_id: 322184 cord_uid: kgv9f58a background: the coronavirus disease (covid-19) pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has become a major global public health issue. sars-cov-2 infection is confirmed by the detection of viral rna using reverse transcription polymerase chain reaction (rt-pcr). prolonged viral shedding has been reported in patients with sars-cov-2 infection, but the presence of viral rna does not always correlate with infectivity. therefore, the present study aimed to confirm the presence of viable virus in asymptomatic or mildly symptomatic patients in the later phase of the disease, more than two weeks after diagnosis. method: asymptomatic or mildly symptomatic covid-19 patients who had been diagnosed with the disease at least two weeks previously and admitted to a community treatment center (ctc) from 15 march to 10 april 2020 were enrolled in this study. nasopharyngeal and salivary swab specimens were collected from each patient. using these specimens, rt-pcr assay and viral culture were performed. result: in total, 48 patients were enrolled in this study. there were no significant differences in baseline characteristics between the asymptomatic and mildly symptomatic patient groups. rt-pcr assay and viral culture of sars-cov-2 were performed using nasopharyngeal and salivary swabs. the results of rt-pcr performed using salivary swab specimens, in terms of cycle threshold (ct) values, were similar to those of rt-pcr using nasopharyngeal swab specimens. in addition, no viable virus could be cultured from swab specimens collected from the late-phase covid-19 patients with prolonged viral rna shedding. conclusions: in conclusion, our study suggests that even if viral shedding is sustained in asymptomatic or mildly symptomatic patients with later phase of covid-19, it can be expected that the transmission risk of the virus is low. in addition, saliva can be used as a reliable specimen for the diagnosis of sars-cov-2 infection. the coronavirus disease pandemic, caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2), has become a major global public health issue. at the time of writing, the massive viral outbreak has affected 216 countries, with more than 13 million people infected and 580,000 having died [1] . in south korea, the number of patients has increased rapidly since february 2020. for the proper management of covid-19 patients, asymptomatic or mildly symptomatic patients were admitted to community treatment centers (ctcs), which are non-medical facilities for isolation and monitoring. however, patients with prolonged viral rna detection after resolution of symptoms presented difficulties in terms of their safe discharge from isolation. for efficient distribution of medical resources, the revised who discharge guidelines recommend that symptomatic patients should be discharged 10 days after the onset of symptoms, following at least three additional days without symptoms, and that asymptomatic patients should be discharged 10 days after testing positive for sars-cov-2 [2] . diagnosis of covid-19 is achieved using a nasopharyngeal swab. at present, real-time reverse transcription polymerase chain reaction (rt-pcr) analysis of respiratory specimens is the gold standard test for detecting sars-cov-2 infection [3] . over the course of infection, viral rna has been identified in respiratory tract specimens 1-3 days prior to the onset of symptoms. the viral load is thought to persist for up to 7-10 days after the onset of symptoms in mild cases. in more severe cases, it tends to peak up to 11 days after the onset, followed by gradual decrease over time [4] . viral rna has been detected in various specimens of the human body, such as the upper and lower respiratory tract, blood, pharyngeal swabs, saliva, urine, and feces [5, 6] . prolonged viral shedding has been reported from nasopharyngeal swabs up to 63 days after the onset of symptoms among adult patients [7, 8] . however, viral shedding of sars-cov-2 does not always indicate infectivity, unless the virus is isolated and cultured from the specimens. the revised who release criteria do not require additional examination prior to discharge. therefore, there is a residual risk that the disease can still spread when these criteria are adhered to. accordingly, in late-phase covid-19 patients with prolonged viral rna detection, it is important to determine the risk of transmission. in this study, we attempted to confirm the presence of viable virus by performing rt-pcr assay and culture using salivary and nasopharyngeal swabs of asymptomatic or mildly symptomatic covid-19 patients who had been diagnosed with the disease and admitted to a ctc at least two weeks previously. asymptomatic or mildly symptomatic covid-19 patients who had been admitted to the ctc between march 15 and april 10, 2020 and had been diagnosed at least two weeks previously, were enrolled in the current study. asymptomatic or mildly symptomatic covid-19 patients were defined as those with an early warning score of <3 for sars-cov-2 infection [9] . patients referred to other hospitals due to worsening of symptoms during isolation were excluded. written informed consent was obtained from all study participants. nasopharyngeal and salivary swab specimens were obtained from each patient. ethical statement: this study was approved by the institutional review board (irb number: 4-2020-0133) of severance hospital (seoul, south korea) and informed consent was obtained. rt-pcr assays targeting three genes of sars-cov-2, the e (envelope protein), rdrp (rna-dependent rna polymerase), and n (nucleocapsid protein) genes, were performed using the allplex™ 2019-ncov assay (seegene inc., seoul, south korea) with nasopharyngeal and salivary swab specimens. patients with negative rt-pcr results were tested again by rt-pcr the following day; those with positive rt-pcr results were re-tested by rt-pcr after a week; and those with inconclusive rt-pcr results were re-tested by rt-pcr after three days. the inconclusive result refers to a case in which one or more, but not all genes included in the kit show an amplification curve after the cut-off when using a follow-up sample [10] . rt-pcr assay results were expressed as the cycle threshold (ct) value. ct values ≥ 40 were considered negative results. negative conversion was defined as two consecutive negative rt-pcr results at a 24 h interval. rebound ct value was defined as a negative (ct value ≥ 40) from the single rt-pcr assay and positive (ct value < 40) from the following rt-pcr result. the nasopharyngeal swab sample was placed in 2 ml of viral transport medium. saliva samples were collected in a sterile container and both specimens were stored frozen −70 • c. nasopharyngeal swab specimens were subjected to total nucleic acid extraction using a viral rna mini kit (qiagen, hilden, germany). after extraction, the total nucleic acid was recovered using 60 µl of elution buffer. for salivary swab specimens, the same amount of pbs was added, and following the vortex process, rna was extracted using the same process as for the nasopharyngeal swab samples. using 8 µl of rna, rt-pcr assay targeting the three genes of sars-cov-2 was performed using a seegene kit (allplex 2019-ncov assay kit, seegene, korea). when the upper respiratory specimens including the nasopharyngeal swab were examined using the allplex 2019-ncov assay, a positive percent agreement (ppa) was 100% (95% ci: 92.75~100%), and a negative percent agreement (npa) was 93.07% (95% ci: 85.76~96.93%) [11] . sars-cov-2 was cultured in a biosafety level 3 facility. vero e6 cells were used for isolating sars-cov-2. vero e6 cells were cultured in eagle's minimum essential medium (emem) supplemented with heat-inactivated fetal bovine serum (fbs; 10%). both nasopharyngeal and salivary swab specimens were used for virus isolation. vero e6 cells were seeded with 1 ml of emem at a density of 1.5 × 10 4 cells/well in culture tubes and incubated at 37 • c in a carbon dioxide incubator for 24 h until confluence for inoculation was achieved. the next day, each nasopharyngeal and salivary swab specimen was diluted at a ratio of 1:10 and inoculated into four wells containing emem (2% fbs, 1% p/s). cells in the media were fixed with 10% formaldehyde and stained with 1% crystal violet dye. the virus-induced cytopathic effect was examined daily for up to seven days [12, 13] . the analysis was performed on asymptomatic or mildly symptomatic covid-19 patients who had been diagnosed and admitted to a ctc at least two weeks previously. in total, 48 patients were enrolled in this study. the mean age of all patients was 32.62 ± 14.59 years, and 14 of the patients were male (29.2%). of all the patients, 11 (22.9%) were asymptomatic. baseline characteristics were similar between the symptomatic and asymptomatic patient groups. there was no statistically significant difference in terms of age, sex, comorbidities, and symptoms. the most common symptoms were myalgia (32.4%), fever (29.7%), and headache (24.3%); chest pain (2.7%) and vomiting (2.7%) were uncommon (table 1) . nasopharyngeal and salivary swab specimens were collected from the patients approximately 30.40 ± 5.71 days after initial diagnosis. the average ct value of patients on the day of culture was over 30 in both the symptomatic and asymptomatic groups. a total of 17 cases (35.4%) showed rebound ct values, which included 11 cases in the symptomatic group and 6 in the asymptomatic group (table 2) . rt-pcr assay was performed using saliva specimens to determine the effectiveness of saliva as a diagnostic tool. as shown in table 3 , in the case of patient 8, the saliva rt-pcr result was positive, although the rt-pcr result of the nasopharyngeal swab specimen was negative. additionally, in eight patients, the mean ct values of the nasopharyngeal and salivary swab specimens were 33.7 and 33.96, respectively, indicating that saliva swabs can serve as a reliable tool for diagnosing sars-cov-2 infection. cell culture was performed using nasopharyngeal and salivary swab specimens to confirm the isolation of viable virus. vero cells were inoculated with nasopharyngeal and salivary swab specimens and microplates were observed for the evidence of cytopathic effect. specimens were collected from patients at least 20 days after diagnosis, and we found no microplates showing any cytopathic effect with these specimens (figures 1 and 2) . 33.96, respectively, indicating that saliva swabs can serve as a reliable tool for diagnosing sars-cov-2 infection. cell culture was performed using nasopharyngeal and salivary swab specimens to confirm the isolation of viable virus. vero cells were inoculated with nasopharyngeal and salivary swab specimens and microplates were observed for the evidence of cytopathic effect. specimens were collected from patients at least 20 days after diagnosis, and we found no microplates showing any cytopathic effect with these specimens (figures 1 and 2) . in this study, we aimed to determine whether infectious viruses could be isolated using salivary and nasopharyngeal swab samples from patients with persistent positive rt-pcr results or rebound ct values more than two weeks after diagnosis. sars-cov-2 could not be cultured from the patient specimens. consequently, we surmised that there is no viral transmission risk in the later phase of sars-cov-2 infection. in addition, as indicated by the fact that the ct values derived from salivary swab rt-pcr were similar to those of nasopharyngeal swab rt-pcr, saliva was shown to be an effective diagnostic tool for the detection of sars-cov-2. sars-cov-2 infection is confirmed by detecting the presence of viral rna through molecular testing, usually by rt-pcr [14] . the presence of viral rna alone does not indicate that a patient is infectious or can transmit the virus to others. factors affecting the transmission risk include whether the patient has symptoms, such as cough, which can spread droplets, whether a virus is infectious, and environmental factors or the behavior of the infected patient. usually, 5 to 10 days after the initial sars-cov-2 infection, the patient begins to produce neutralizing antibodies. the binding of these neutralizing antibodies to the virus is expected to decrease the risk of viral transmission [4, 15] . in many studies, viral shedding detected by rt-pcr from respiratory specimens has been found to persist for more than 20 days and sometimes up to 63 days after the onset of symptoms, and it in this study, we aimed to determine whether infectious viruses could be isolated using salivary and nasopharyngeal swab samples from patients with persistent positive rt-pcr results or rebound ct values more than two weeks after diagnosis. sars-cov-2 could not be cultured from the patient specimens. consequently, we surmised that there is no viral transmission risk in the later phase of sars-cov-2 infection. in addition, as indicated by the fact that the ct values derived from salivary swab rt-pcr were similar to those of nasopharyngeal swab rt-pcr, saliva was shown to be an effective diagnostic tool for the detection of sars-cov-2. sars-cov-2 infection is confirmed by detecting the presence of viral rna through molecular testing, usually by rt-pcr [14] . the presence of viral rna alone does not indicate that a patient is infectious or can transmit the virus to others. factors affecting the transmission risk include whether the patient has symptoms, such as cough, which can spread droplets, whether a virus is infectious, and environmental factors or the behavior of the infected patient. usually, 5 to 10 days after the initial sars-cov-2 infection, the patient begins to produce neutralizing antibodies. the binding of these neutralizing antibodies to the virus is expected to decrease the risk of viral transmission [4, 15] . in many studies, viral shedding detected by rt-pcr from respiratory specimens has been found to persist for more than 20 days and sometimes up to 63 days after the onset of symptoms, and it appears to last beyond symptom resolution [6, 8, 16, 17] . it has also been proven that transmission occurs in asymptomatic patients. in a study conducted by zou et al., the viral loads of nasal and throat swabs were similar in symptomatic and permanently asymptomatic patients [18] . in addition, in the present study, the ct values of symptomatic and asymptomatic patients were not significantly different. this might be because in the late phase of infection the viral load is close to the detection limit, but there is evidence that viral shedding occurs in both symptomatic and asymptomatic patients after symptom improvement; however, the relationship between the detection of viral rna and infectivity is still unclear. rt-pcr results do not necessarily indicate the possibility of viral transmission and cannot distinguish between infectious and non-infectious virus [7, 15] . viral rna has been detected in the upper and lower respiratory tract, blood, pharyngeal swabs, saliva, urine, and feces, regardless of the severity of the disease [5, 16] . the virus has also been detected in water gargled by patients diagnosed with covid-19 [17] . we used saliva specimens for rt-pcr tests to confirm sars-cov-2 infection. it was confirmed that saliva showed a high concordance rate of 90% or more with the nasopharyngeal specimens in the detection of respiratory viruses including coronavirus [13, 19, 20] . a study by zhou et al. indicated that angiotensin-converting enzyme ii (ace2) is likely the cell receptor for sars-cov-2; it was also the receptor for sars-cov and hcov-nl63 [4] . according to a previous study, ace2 is presented on the epithelial cells of oral mucosa, suggesting that the oral cavity might be at high risk of sars-cov-2 infection [21] . these findings suggest that ace2-expressing cells may act as target cells and are therefore vulnerable to sars-cov-2 infection [22] . since many viruses, including sars-cov-2, can be detected in saliva, saliva is considered a major factor in the risk of transmission of viruses that can cause respiratory infections [21] . in our study, a viral load was detected in saliva, therefore, saliva can also be expected to serve as an effective diagnostic tool. viral rna tends to be detected for a longer period of time in more severe cases. according to several studies, viral rna has been detected in respiratory tract specimens 1-3 days before the onset of symptoms. viral load is thought to increase for up to 7-10 days after the onset of symptoms in mild cases. in more severe cases, it tends to peak up to 11 days after the onset, followed by gradual decrease over time [4, 23] . some studies have predicted that transmission risk is the highest at or around the time of symptom onset and during the first five days of disease [16] . viral culture studies using patient specimens to confirm the presence of infectious sars-cov-2 are still limited. in some studies, viable viruses have been isolated from viral cultures using respiratory samples collected during the early stages of the disease or at least within eight days after the onset of symptoms [4] . according to a study investigating the relationship between the ct value and culture positivity rate, all samples with ct values of 13-17 led to positive culture; however, the culture positivity rate decreased as ct values increased. cultures could not be obtained from specimens with ct values ≥ 34 [24] . in the present study, the mean ct value of specimens was approximately >30, and no virus was isolated in cultures using these samples. this study has several limitations. firstly, the sample size of this study was small and larger studies are needed to confirm the relationship between infectivity loss and rt-pcr results. secondly, the infectivity of individual cases and accuracy of our culture analysis may have individualized variations. furthermore, subculture is known to improve the sensitivity of culture assays, but subculture was not performed in this study. fourth, since serial saliva specimens were not available, serial viral load monitoring was not possible in this study. finally, changes in the dynamics of viral shedding could have occurred due to the treatments administered to patients. although viral rna can be detected in rt-pcr analyses even after the improvement of symptoms, the amount of viral rna gradually decreases over time, eventually reducing below the level at which viable virus can be isolated. therefore, based on the evidence that the virus is rarely detected in respiratory specimens after 10 days following the onset of symptoms, especially in mild or asymptomatic cases of sars-cov-2 infection, even if viral shedding is sustained in the later phase of covid-19, it can be expected that the transmission risk of the virus is low. accordingly, it seems safer to release patients from quarantine based on the revised who discharge criteria that require minimum isolation for 13 days, than to repeat rt-pcr assays. although the transmission risk is thought to be minimal in the later stages of covid-19, it cannot be completely ruled out. therefore, further investigations are warranted to understand the relationship between sars-cov-2 detection, viral culture, and transmission depending on the clinical course. coronavirus disease (covid-19) situation report-179 world health organization. criteria for releasing covid-19 patients from isolation saliva is a reliable tool to detect sars-cov-2 virological assessment of hospitalized patients with covid-2019 sars-cov-2 can be detected in urine, blood, anal swabs, and oropharyngeal swabs specimens temporal profiles of viral load in posterior oropharyngeal saliva samples and serum antibody responses during infection by sars-cov-2: an observational cohort study cov-2: the viral shedding vs infectivity dilemma clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study daegu medical association a brief telephone severity scoring system and therapeutic living centers solved acute hospital-bed shortage during the covid-19 outbreak in daegu covid-19 molecular testing in korea: practical essentials and answers from experts based on experiences of emergency use authorization assays in vitro diagnostics of coronavirus disease 2019: technologies and application isolation and characterization of sars-cov-2 from the first us covid-19 patient consistent detection of 2019 novel coronavirus in saliva localising an asset-based covid-19 response in ecuador temporal dynamics in viral shedding and transmissibility of covid-19 prolonged virus shedding even after seroconversion in a patient with covid-19 sars-cov-2 viral load in upper respiratory specimens of infected patients 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 saliva is a non-negligible factor in the spread of covid-19 high expression of ace2 receptor of 2019-ncov on the epithelial cells of oral mucosa asymptomatic cases in a family cluster with sars-cov-2 infection viral rna load as determined by cell culture as a management tool for discharge of sars-cov-2 patients from infectious disease wards key: cord-325014-n7mnhk2v authors: gujski, mariusz; jankowski, mateusz; pinkas, jarosław; wierzba, waldemar; samel-kowalik, piotr; zaczyński, artur; jędrusik, piotr; pańkowski, igor; juszczyk, grzegorz; rakocy, kamil; raciborski, filip title: prevalence of current and past sars-cov-2 infections among police employees in poland, june–july 2020 date: 2020-10-11 journal: j clin med doi: 10.3390/jcm9103245 sha: doc_id: 325014 cord_uid: n7mnhk2v background: coronavirus disease 2019 (covid-19) is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). we aimed to determine the prevalence of current and past sars-cov-2 infections among police employees. methods: this cross-sectional survey was undertaken among 5082 police employees from mazowieckie province, poland. rt-pcr testing for current sars-cov-2 infection and serological tests (elisa) for the presence of anti-sars-cov-2 igm+iga and igg antibodies were performed. results: all rt-pcr tests were negative. the anti-sars-cov-2 igm+iga index was positive (>8) in 8.9% of participants, including 11.2% women and 7.7% men (p < 0.001). equivocal igm+iga index (6–8) was found in 9.8% of participants, including 11.9% women and 8.7% men (p < 0.001). the igg index was positive (>6) in 4.3% and equivocal (4–6) in 13.2% of participants. a higher odds of positive igm+iga index was found in women vs. men (or: 1.742) and police officers vs. civilian employees (or: 1.411). participants aged ≥60 years had a higher odds of positive igg index vs. those aged 20–29 years (or: 3.309). daily vaping also increased the odds of positive igg index (or: 2.058). conclusions: the majority of polish police employees are seronegative for sars-cov-2 infection. vaping and older age (≥60 years) were associated with a higher risk of sars-cov-2 infection. coronavirus disease 2019 (covid-19) is caused by a novel strain of coronavirus, severe acute respiratory syndrome coronavirus 2 (sars-cov-2), which appeared in china in 2019 [1] and evolved into the current pandemic. although the definite laboratory diagnosis of sars-cov-2 infection is currently based on real-time reverse transcriptase-polymerase chain reaction (rt-pcr) testing, and rt-pcr is recommended for clinical testing in cases of suspected covid-19 disease [2], asymptomatic infected individuals (infection carriers) who do not come to medical attention may play an important role in transmitting infection within the population [3] . as the time window for a positive rt-pcr result is short, serological testing, which provides information about whether a person has been exposed to sars-cov-2, may be useful for epidemiological purposes to detect the overall burden of previous infection in a given community. currently, two types of serological assays are available for sars-cov-2 testing [2]. laboratory-based immunoassays, including enzyme-linked immunosorbent assays (elisas), chemiluminescentmicroparticle immunoassays, and immunometric assays, detect various classes of immunoglobulins (ig) against sars-cov-2, including igm, iga, and igg, can be qualitative or quantitative, and are generally performed using serum samples. rapid diagnostic tests are typically lateral-flow assays that can be used for point-of-care testing to detect anti-sars-cov-2 igg, igm, or viral antigens and are usually performed in fingerstick blood samples, although some may use saliva or other specimen types. the rationale for screening using tests detecting sars-cov-2-specific antibodies is that these antibodies develop regardless of symptoms and are present for several months after infection [4] . in addition, population data from spain and iceland indicate that a substantial proportion of infected persons, both asymptomatic and symptomatic, are never tested with rt-pcr in the acute phase [3, 4] . the data obtained during the current sars-cov-2 epidemic in poland indicate that more than 90% of infections within the workforce outbreaks, e.g., among miners, which involve younger and generally more healthy persons, were asymptomatic or oligosymptomatic [5] . poland is a country where the coronavirus epidemic arrived relatively late. the first laboratoryconfirmed covid-19 case was reported on 4 march 2020 [6] . as of 21 september, 78,330 laboratoryconfirmed covid-19 cases and 2282 related deaths were reported in poland [7] . the settings of sars-cov-2 transmission during the first 2 months of the epidemic were mostly hospitals and long-term care facilities, followed by outbreaks in workplaces, including coal mines, furniture factories, and meat-processing plants [8] . polish police officers are at an increased risk of acquiring sars-cov-2 infection due to their duties, including protection of public gatherings and daily verification (by direct visual contact) of compliance with mandatory quarantine rules. nationwide, the number of quarantined persons was >160,000 at the peak in early april 2020 [9] and currently (early september 2020) is about 70,000 [10] . the aim of this study was to determine the prevalence of current and past sars-cov-2 infections among police employees, a high-risk population due to their professional duties, during the covid-19 epidemic. this cross-sectional sars-cov-2 screening survey was carried out from 22 june to 8 july 2020 among police employees (police officers and civilian employees) from mazowieckie province in poland. random-cluster sampling was performed. out of 327 police units (including headquarters/police stations and departments), 170 were randomly selected. the smallest units (<5 people) were excluded from the sampling procedure. the cluster and stratified selection method was applied for sampling procedures to improve accuracy. all police employees from the randomly selected units were invited to participate in the study (8789 individuals from 170 units). due to refusal to participate and exclusion of the smallest units, the study was finally carried out in 122 police units. the questionnaire was completed by 5363 police employees, and biological samples (swab and blood) were effectively collected from 5082 of them. the exclusion criteria include refusal to participate, lack of a signed informed consent, hospitalization, quarantine, leave, and secondment to work in another police unit that was not selected for the survey. participants were invited via email or the police's internal communication system. after completing the questionnaire, each respondent received an individual id code for personal data protection. test samples were collected on the premises of the police units (in a dedicated room) on a day designated by the research team. samples were collected by a nurse or a paramedic equipped with personal protective equipment according to the applicable safety procedures. nasopharyngeal swab samples were collected by a nurse or a paramedic using transport sets specially designed for collecting clinical material for the diagnosis of sars-cov-2 infection. the diaplexq™ novel coronavirus (2019-ncov) detection kit (solgent co., ltd.; daejeon, korea) was used for the detection of sars-cov-2 rna by rt-pcr. virus identification (positive result) was based on the orf1ab and n target gene regions of sars-cov-2. rt-pcr testing was carried out in the national reference laboratory (national institute of public health-national institute of hygiene, warsaw, poland) by qualified clinical laboratory personnel specifically instructed and trained in rt-pcr techniques and in vitro diagnostic procedures. the testing procedure met the requirements of the who recommendations for covid-19 laboratory testing [11] . serum samples (up to 5 ml) were collected for the detection of anti-sars-cov-2 igm+iga and igg antibodies using indirect immunoenzyme assay (elisa). commercially available covid-19 elisa igg and igm+iga kits (vircell s.l., granada, spain) were used, targeting sars-cov-2-specific antigens, spike glycoprotein (s), and nucleocapsid protein (n). serological testing was carried out in the diagnostic laboratory of the central clinical hospital of the ministry of the interior and administration in warsaw by qualified clinical laboratory personnel. the testing procedure was performed according to the test manufacturer's instructions and met the spanish society for infectious diseases and clinical microbiology (seimc) recommendations. an in-house validation was carried out according to the validation protocol for users provided by the test manufacturer. according to the test manufacturer's guidelines, the results were presented in a semiquantitativemanner andthe antibody index was calculated using the following formula: antibody index = (sample optical densities/cutoff serum mean optical densities) × 10. samples with the anti-sars-cov-2 igm+iga index below 6 were considered negative, those with the index between 6 and 8 were considered indeterminate/equivocal, and those with the index above 8 were considered positive. samples with the anti-sars-cov-2 igg index below 4 were considered negative, those with the index between 4 and 6 were considered indeterminate/equivocal, and those with the index above 6 were considered positive. the study used an original 30-item questionnaire adapted to this particular group of police employees. in preparation of the questionnaire, we analyzed the previously published covid-19-oriented research, with special emphasis on the studies and reports published by the who [12] . the questionnaire was made available to the respondents via an internet platform. the computer-assisted web interview (cawi) method was applied. field control was enabled to avoid accidental missing data. the total number of police employees in the mazowieckie province was 17,400. the effective sample size of 5000 was assumed. due to estimated nonresponse rate of 35-45% (the beginning of the holiday season and the presidential elections being held in poland, which influenced the level of police unit involvement), a total of 8789 police employees from 170 police units were invited to take the survey. finally, 5082 individuals took part in both questionnaire and laboratory parts of the study. the questionnaire included several questions related to the personal characteristics, including age, size of the place of residence, living alone or with someone, and the presence of children in the respondent's home. the participants were also asked about self-declared health status (very good, good, fair, or poor), presence of chronic diseases (yes/no), ever and current (past 6 months) tobacco or e-cigarette use, international travel in the past 3 months, and the nature of their work. the type of employment was categorized as a police officer (uniformed and armed force) or a civilian employee (nonuniformed, work closely with uniformed officers, e.g., administrative support). based on the settings of official duties' performance, the following categories were designated: office work, fieldwork, and both office work and fieldwork. participants were also asked about the number of people with whom they had contact during the day, participation in the control of compliance with the quarantine rules, and participation in securing gatherings of more than 50 people. respondents were asked about the presence of 8 symptoms accompanying sars-cov-2 infections in the last 4 months (march-june 2020) (fever, cough, dyspnea/breathlessness, diarrhea, anorexia/lack of appetite, nausea or vomiting, loss of smell, and loss of taste). for the present analysis, only responses regarding the absence of symptoms within the given period were used. for logistic regression analysis, all analyzed variables were recoded into a series of dummy variables (0-1). the questionnaire data were supplemented with available epidemiological data on the number of registered cases and deaths per 10,000 residents in individual poviats (administrative regions) of the mazowieckie province as of 8 july 2020, obtained from the state sanitary inspection [13] . these data were incorporated into the model as continuous variables. the data were analyzed using spss version 26 (ibm, armonk, ny, usa, 2020), r version 4.0.2. (r foundation for statistical computing, vienna, austria, 2020), and h2o version 3.30.0.7 (apache license 2.0). the χ 2 test was used to assess the significance of differences in cross-tables. the associations between continuous variables (igm+iga and igg indexes) were measured by the pearson's linear correlation and the spearman's rank correlation. a logistic regression model was used to determine the strength of the effect of the analyzed factors on the risk of sars-cov-2 infection. machine learning techniques were used to improve the fit of the model-glm with the binominal function with ridge and lasso regularization (with cross-validation). samples were collected by qualified healthcare professionals (nurse or paramedic) in accordance with the standards set out in the ordinances of the polish minister of health. participation in the study was voluntary and free of charge. all participants gave written informed consent before participation in the study. the study protocol was reviewed and approved by the ethics review board at the medical university of warsaw, warsaw, poland (approval number: kb/87/2020). completed questionnaires were obtained from 5363 police employees (61%), and complete samples (nasopharyngeal swab and serum sample) were collected from 5082 police employees (33.5% females; response rate 57.8%). the mean age (sd) was 39.6 years (9.0) overall, 49.7 years (9.6) among women and 39.0 years (8.5) among men. among participants, 79.2% were police officers and 20.8% were civil employees. almost one-third (30.1%) of participants declared office-based work, 17.3% declared fieldwork, and 52.6% declared both office work and fieldwork. a quarter of the participants (25.9%) lived in rural areas, 44.8% lived in cities up to 500,000 inhabitants, and 29.3% lived in the city above 500,000 inhabitants (warsaw). the mean anti-sars-cov-2 igm+iga index was 4.4 ± 0.5 (range: 0.0-43.5). the mean anti-sars-cov-2 igg index was 3.1 ± 0.4 (range: 0.0-44.8) ( table 1) . the anti-sars-cov-2 igm+iga and igg indexes were linearly correlated at r = 0.209 (p < 0.001). using rank correlation, the coefficient rho = 0.355 was obtained (p < 0.001). of those with negative anti-sars-cov-2 igg index (<4), 7.6% had positive anti-sars-cov-2 igm+iga index (>8) and equivocal results were observed in 8.8%. of those with positive anti-sars-cov-2 igg index (>6), 18.0% had positive anti-sars-cov-2 igm+iga index (>8) and equivocal results were observed in 13.8% (table 2 ). the differences were statistically significant (p < 0.001). less than 1% of participants had both positive anti-sars-cov-2 igm+iga and igg indexes. there were no current sars-cov-2 infections among 5082 police employees in this study (all rt-pcr tests were negative). the anti-sars-cov-2 igm+iga index was positive (>8) in 8.9% of participants (95%ci: 8.1-9.7%) overall, in 11.2% (95%ci: 9.7-12.7%) of women, and in 7.7% (95%ci: 6.8-8.6%) of men (p < 0.001). an equivocal (6-8) anti-sars-cov-2 igm+iga index was found in 9.8% (95%ci: 9.0-10.6%) of participants, with a significant difference (p < 0.001) between women (11.9%; 95%ci: 10.4-13.5%) and men (8.7%; 95%ci: 7.8-9.7%) ( figure 1 ). the size of the place of residence also differentiated results in a statistically significant way (p <0.01). no other variable listed in figure 1 was significantly associated with the igm+iga results. overall, 4.3% participants (95%ci: 3.7-4.9%) were igg-seropositive (antibody index > 6). an equivocal (4-6) anti-sars-cov-2 igg index was found in 13.2% (95%ci: 12.3-14.1%) of participants. neither sex (p =0.155) nor other variables listed in figure 2 were significantly associated with the igg results ( figure 2 a logistic regression model predicting a positive anti-sars-cov-2 igm+iga index was developed (cox and snell r square at 0.015 andnagelkerke r square at 0.033). after including all variables listed in figures 1 and 2 along with the number of registered cases and deaths due to covid-19 (per 10,000 inhabitants), only 4 variables showed a correlation with a positive anti-sars-cov-2 igm+iga index. a higher odds of a positive anti-sars-cov-2 igm+iga index was observed among women compared to men (or: 1.742; 95%ci: 1.377-2.203), inhabitants of towns up to 20,000 residents and cities from 20,000 to 500,000 residents (or: 1.526; 95%ci: 1.099-2.119 and or: 1.657; 95%ci: 1.257-2.183, respectively) vs. those living in rural areas, and police officers compared to civilian employees(or: 1.411; 95%ci: 1.004-1.981) ( table 3) . (20-29 years) ; place of residence (rural); living alone; lack of children at home; civil employee; type of employment (fieldwork); average number of contacts per person perday (>100 person);verification of compliance with mandatory quarantine rules (no); protection of public gatherings > 50 persons (no); presence of chronic condition (no); tobacco smoking (no); e-cigarette use/vaping (no); heated tobacco use (no); foreign trip in the first half of 2020 (no). in a logistic regression model predicting a positive anti-sars-cov-2 igg index (cox & snell r square at 0.009, nagelkerke r square at 0.029), only 2 variables showed a correlation with a positive anti-sars-cov-2 igg index. compared to the age group 20-29 years, participants aged ≥60 years had higher odds of a positive anti-sars-cov-2 igg index (or: 3.309; 95%ci: 1.442-7.595). daily vaping (e-cigarette using) also increased the odds of a positive anti-sars-cov-2 igg index (or: 2.058; 95%ci: 1.097-3.861) ( table 3) . of the 217 igg-positive subjects, 56.7% (95%ci: 50.0-63.2%) did not notice any of the 8 most common covid-19 symptomsbetween march and end of june 2020, 18.0% (95%ci: 13.3-23.5%) reported 1 symptom, and 14.7% (95%ci: 10.5-19.9%) reported 2 symptoms. similar responses were obtained in those with negative (n = 4196) and equivocal (n = 669) anti-sars-cov-2 igg index (p = 0.954). the most common symptom was cough (27.4% of all respondents; 95% ci: 26.2-28.6%), but its rates did not differ significantly in relation to the igg result (p = 0.731). of the 8 symptoms, a significant correlation (p < 0.01) was found only for fever, which was reported by 17.1% (95%ci: 12.5-22.5%) of subjects with positive igg index, 12.4% (95%ci: 11.4-13.4%) of those with a negative igg index, and 9.0% (95%ci: 7.0-11.3%) of those with an equivocal igg index. no significant correlations were observed between the iga+igm result and the 8 analyzed covid-19 symptoms between march and end of june 2020, with the difference close to statistical significance only for cough (p = 0.052). our study is the first large cross-sectional sars-cov-2 screening survey performed among the personnel of the uniformed services in europe. in our study population, the anti-sars-cov-2 igm+iga index was positive in nearly 9% of participants, and igg index was positive in over 4% of participants, indicating a previous infection/exposure to sars-cov-2. both indexes were positive in <1% of participants. notably, all rt-pcr tests were negative, indicating no current sars-cov-2 infection, in all 5082 police employees in this study. the relatively low individual overlap between positive results of the igm+iga and igg indexes may be explained by the dynamics of various ig class formation. during the course of sars-cov-2, igm and/or iga are detected first, followed by a longer-lasting igg response. in most patients, seroconversion occurs between 7 and 14 days after the covid-19 diagnosis [14] . however, the speed and strength of individual immune response may be variable, e.g., depending on the viral burden upon exposure, and in some people, detectable antibodies may be not generated after infection due to such factors as an underlying immune disorder, immunosuppression, or other reasons. it is more difficult to explain why we did not have any positive rt-pcr test results despite a 9% rate of positive igm+iga results, presumably indicating early infection. due to the nature of police officers' activities, one may speculate whether our results might reflect a contact, possibly recurrent, with low viral burden that would be insufficient to generate true infection/virus replication (as detectable by rt-pcr) but enough to trigger antibody (iga/igm) production. studies show that while antibody responses may be undetectable or short-lived, memory t cell responses can persist for much longer and, indeed, sars-cov-2-specific t cells were detectable in antibody-seronegative-exposed family members and convalescent individuals with a history of asymptomatic or mild covid-19 [15] . obviously, it also possible that rt-pcr testing might have missed some active cases, and the antibody test used might have yielded some false positives. many of our igg-positive subjects reported some symptoms consistent with covid-19, although only fever was significantly more common in those with positive igg results compared to those without. there is a growing body of scientific data on nonrespiratorysymptoms of sars-cov-2 infection [16] [17] [18] . an analysis of covid-19 symptom profiles showed that gastrointestinal symptoms (diarrhea/lack of appetite), neurological symptoms (loss of smell and/or taste), as well as chills, myalgia, headache, and fatigue, were commonly reported by patients with covid-19 [16] . we can hypothesize that some igg-positive subjects may have observed nonrespiratorycovid-19 symptoms and therefore did not report them to a physician or sanitary inspection. although the sensitivity of rt-pcr is very high, its overall diagnostic accuracy depends on the quality of sampling (nasopharyngeal swab) and subsequent sample handling. antibody tests used might show cross-reactivity with other viruses, such as endemic coronavirus strains [19] . in a dutch summary of various elisa tests, the sensitivity of the vircell igg test at >14 days after illness onset was 97% in severe and 89% in mild/asymptomatic cases and the specificity was 94%. the sensitivity of the vircell igm+iga test at >14 days after illness onset was 97% in severe and 70% in mild/asymptomatic cases and specificity was 82% [20] . in a recent study, the vircell igg test had a sensitivity of 98% and specificity of 83% [21] . in another unpublished study, the sensitivity was much lower (65% for igg and 77% for igm+iga), whereasthe specificity was 96% for the vircell igg test and 83% for the vircell igm+iga test [22] . significant predictors of a positive igm+iga result included female gender, place of residence (town <20,000 inhabitants and city 20,000-500,000 inhabitants), and being a police officer (compared to civilian police employees). both positive and equivocal igm+iga results were significantly more common in women compared to men. although the clinical course of covid-19 is more severe in men [23] and the overall prevalence may be slightly higher in men, italian data indicate that among women aged 20-49 years, the prevalence was higher in women, and only after age 50, women outnumbered by men [24] . this has been explained by the fact that younger women are more represented in jobs (health, education, hotels, and restaurants) exposing them to a higher risk of contagion due to personal contacts. such an explanation seems less valid for police employees, but the higher rate in women may still be explained by gender differences in the nature of social contacts in general. regarding other predictors of positive igm+iga results, association with the local community size might be related to personal contacts with a larger number of people at risk of covid-19, either related or unrelated to police employees' professional activities, and being a police officer might involve a higher risk of exposure to covid-19 compared to civilian employees. however, these might also be chance findings, as other variables potentially reflecting increased exposure, such as the estimated number of persons contacted daily and involvement in the surveillance of quarantined individuals and protection of public gatherings, did not emerge as significant predictors of positive igm+iga and igg results. the two significant predictors of positive igg results were age ≥60 years and daily vaping. use of e-cigarettes, both alone and in combination with conventional cigarette smoking, has been associated with increased virulence and inflammatory potential of respiratory pathogens in general [25] and with covid-19 in particular [26] . however, the opposite results were also found for smoking. in a french cohort study, a lower proportion of participants with confirmed sars-cov-2 infection based on antibody detection was found in smokers compared to non-smokers [27] . regarding the association with age, a number of studies provided somewhat divergent results compared to our findings. in a dutch plasma donor study, antibodies against sars-cov-2 were detected significantly more often in younger people (18-30 years) , which was thought to be related to different social behaviors and higher exposure to the virus before social distancing was implemented [28] . similarly, the seroprevalence in those aged 20-49 years was significantly higher compared to those aged 50 years and older in geneva [29] . in healthy blood donors in milan, seroconversion to igg was noted more commonly among younger subjects, while seroconversion to igm was more common in older subjects [30] . in addition to positive igg and igm+iga results, we also obtained equivocal results of these tests in a significant proportion of participants. in our study population, an equivocal igm+iga index was found in nearly 10% of participants, and igg index was equivocal in 13% of participants. in general, repeated testing is recommended in case of an equivocal serological test result. in our study, testing was performed at a single time-point only, which constitutes a study limitation. an equivocal result indicates that antibodies were detected at a level close to the diagnostic threshold. equivocal results may represent an early infection, detection of decreasing antibody level after a past infection, cross-reactivity with other viruses, an underlying immune disorder, immunosuppression, or other reasons. our findings showed that 17.5% of participants had a positive or equivocal anti-sars-cov-2 igg index. based on this observation, we can hypothesize that due to the asymptomatic course of sars-cov-2 as well as due to the presence of nonrespiratory symptoms, the number of covid-19 cases in the polish population may be underestimated. testing strategies for sars-cov-2 should be regularly revised to include new scientific data on nonrespiratory symptoms of covid-19. regarding comparison of our findings to anti-sars-cov-2 seroprevalence estimates in the general population, no such data are available for poland but they are emerging for other european countries. the estimated seroprevalence was about 5% in spain (by point-of-care lateral-flow assay for anti-sars-cov-2 igm/igg and chemiluminescentmicroparticle immunoassay for igg) [3] and up to 11% in geneva (by another commercially available elisa igg test) [24] , both countries with a several-fold higher per capita covid-19 prevalence in the general population compared to poland. no large population studies have been published with the vircellcovid-19 elisa igg or igm+iga kits. this study has several limitations. the overall response rate was slightly below 58% (based on laboratory test sampling), which might have introduced some bias in terms of potential exposure to sars-cov-2, e.g., employees with more duties and responsibilities might have been less likely to participate. to determine seroprevalence, we relied on a single type of a serological test and a single kit manufacturer, although previous comparisons of the vircellcovid-19 elisa igg or igm+iga kits used do not indicate their inferior performance compared to other tests. the sensitivity and specificity of all currently available serological tests creates some potential for both false negatives and false positives, including cross-reactivity with other (corona) viruses, and false negatives are also possible with rt-pcr due to suboptimal nasopharyngeal swab technique or inadequate sample handling. the study protocol did not allow for retesting in individuals with an equivocal serological test result, which was dictated by feasibility issues and the intent to perform a single-time-point evaluation during a short period of up to 14-21 days. the majority of polish police employees are seronegative for sars-cov-2 infection. most sars-cov-2 infections were asymptomatic or oligosymptomatic, and fever was the only symptom more often reported by igg-positive subjects. e-cigarette use and older age (≥60 years) were associated with a higher risk of sars-cov-2 infection, which emphasizes the importance of quitting smoking to reduce the risk of infection. relatively high proportions of study subjects were igm+iga-positive with negative rt-pcr, or had equivocal igm+iga or igg indexes, an observation requiring further analyses. clinical features of patients infected with 2019 novel coronavirus in wuhan prevalence of sars-cov-2 in spain (ene-covid): a nationwide, population-based seroepidemiological study humoral immune response to sars-cov-2 in iceland 95% of infected are asymptomatic). available online epidemiological analysis of the first 1389 cases of covid-19 in poland: a preliminary report covid-19) as of 21 dynamics of the coronavirus disease 2019 outbreak in poland: an epidemiological analysis of the first 2 months of the epidemic daily up-date on covid-19, as of laboratory testing for coronavirus disease 2019 (covid-19) in suspected human cases survey tool and guidance. rapid simple, flexible behavioural insights on covid-19. available online chief sanitary inspectorate) immunology of covid-19: current state of the science robust t cell immunity in convalescent individuals with asymptomatic or mild covid-19 symptom profiles of a convenience sample of patients with covid-19-united states a study on the predictors of disease severity of covid-19 covid-19: specific and non-specific clinical manifestations and symptoms: the current state of knowledge clinical performance of sars-cov-2 igg antibody tests and potential protective immunity status of the validation of elisa and auto-analyser antibody tests for sars-cov-2 diagnostics: considerations for use performance evaluation of serological assays to determine the immunoglobulin status in sars-cov-2 infected patients evaluation of diagnostic accuracy of 10 serological assays for detection of sars-cov-2 antibodies gender differences in patients with covid-19: focus on severity and mortality. front epidemiological characteristics of covid-19 cases in italy and estimates of the reproductive numbers one month into the epidemic electronic cigarette vapour increases virulence and inflammatory potential of respiratory pathogens association between youth smoking, electronic cigarette use, and coronavirus disease 2019 cluster of covid-19 in northern france: a retrospective closed cohort study herd immunity is not a realistic exit strategy during a covid-19 outbreak seroprevalence of anti-sars-cov-2 igg antibodies in geneva, switzerland (serocov-pop): a population-based study sars-cov-2 seroprevalence trends in healthy blood donors during the covid-19 milan outbreak this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-315634-fkm6slkc authors: jouffroy, romain; kedzierewicz, romain; derkenne, clement; bertho, kilian; scannavino, marine; frattini, benoit; lemoine, frederic; jost, daniel; prunet, bertrand title: hypoxemia index associated with prehospital intubation in covid-19 patients date: 2020-09-20 journal: j clin med doi: 10.3390/jcm9093025 sha: doc_id: 315634 cord_uid: fkm6slkc background: there exists a need for prognostic tools for the early identification of covid-19 patients requiring prehospital intubation. here we investigated the association between a prehospital hypoxemia index (hi) and the need for intubation among covid-19 patients in the prehospital setting. methods: we retrospectively analyzed covid-19 patients initially cared for by a paris fire brigade advanced life support (als) team in the prehospital setting between 8th march and 18th april of 2020. we assessed the association between hi and prehospital intubation using receiver operating characteristic (roc) curve analysis and logistic regression model analysis after propensity score matching. results are expressed as odds ratio (or) and 95% confidence interval (ci). results: we analyzed 300 consecutive covid-19 patients (166 males (55%); mean age, 64 ± 18 years). among these patients, 45 (15%) were deceased on the scene, 34 (11%) had an active care restriction, and 18 (6%) were intubated in the prehospital setting. the mean hi value was 3.4 ± 1.9. hi was significantly associated with prehospital intubation (or, 0.24; 95% ci: 0.12–0.41, p < 10(−3)) with a corresponding area under curve (auc) of 0.91 (95% ci: 0.85–0.98). hi significantly differed between patients with and without prehospital intubation (1.0 ± 1.0 vs. 3.6 ± 1.8, respectively; p < 10(−3)). roc curve analysis defined the optimal hi threshold as 1.3. bivariate analysis revealed that hi <1.3 was significantly, positively associated with prehospital intubation (or, 38.38; 95% ci: 11.57–146.54; p < 10(−3)). multivariate logistic regression analysis demonstrated that prehospital intubation was significantly associated with hi (adjusted odds ratio (ora), 0.20; 95% ci: 0.06–0.45; p < 10(−3)) and hi <3 (ora, 51.08; 95% ci: 7.83–645.06; p < 10(−3)). after adjustment for confounders, the ora between hi <1.3 and prehospital intubation was 3.6 (95% ci: 1.95–5.08; p < 10(−3)). conclusion: an hi of <1.3 was associated with a 3-fold increase in prehospital intubation among covid-19 patients. hi may be a useful tool to facilitate decision-making regarding prehospital intubation of covid-19 patients initially cared for by a paris fire brigade als team. further prospective studies are needed to confirm these preliminary results. the first cases of covid-19 were described in asia in late 2019 [1] [2] [3] [4] , and on 11 march 2020, the world health organization declared this disease to be a worldwide pandemic [5] . although the overall mortality rate is low [6] [7] [8] [9] , to date, covid-19 has caused~200,000 deaths worldwide, half of which are in europe. covid-19 is caused by infection with sars-cov-2 [10] , and~25% of patients suffer a severe form of this disease [6] . the most severe form of covid-19 involves acute respiratory failure (arf) due to hypoxia and hypoxemia, which sometimes necessitates intubation and mechanical ventilation prior to hospital intensive care unit (icu) admission. we previously reported that dyspnea is the main symptom requiring prehospital treatment by the paris fire brigade prehospital emergency service [11] . one peculiarity of respiratory sars-cov-2 infection is the presentation of a low respiratory rate (rr) increase despite severe hypoxemia reflected by low pulse oximetry (spo 2 ) of variable depth depending on the stage of the disease, along with a lack of functional signs and respiratory distress signs [11] . this remarkable disconnect in rest between profound hypoxemia and proportional signs of respiratory distress was named "happy hypoxia" by dhont et al. [12] . the covid-19 pandemic poses a risk of an inadequate ratio between needs (patients with severe covid-19 arf requiring support ventilation) and resources (available medical ventilator devices). thus, it would be useful to develop a simple tool for early assessment of prehospital intubation requirement-for example, something similar to the shock index, which is calculated as the ratio between heart rate and systolic blood pressure [13] and serves as a simple clinical tool allowing early recognition of sepsis in the emergency department [14] . this tool could be useful to physicians for the decision-making process according to evidenced-based medicine knowledge without being polluted by an infodemic and the spread of fake news about covid-19 [15] . in the present study, we report the relationship between covid-19 patients intubated in the prehospital setting by a paris fire brigade advanced life support team and the hypoxemia index (hi), defined as the ratio between initial pulse oximetry and initial respiratory rate. as previously described [11] , the prehospital paris fire brigade emergency medical system is a two-tiered response system-comprising a basic life support (bls) tier served by 200 teams of 3-5 professional rescuers deployed from 77 stations and an advanced life support (als) tier served by 44 ambulance teams, each including an emergency physician, a nurse, and a driver [16] . emergency calls are assessed by a dispatch center operator, who may decide to send a bls and/or als team on the basis of the clinical history and symptoms reported by the patient or witness. once rescue teams have arrived on the scene, the emergency physician examines the patient, and then the patient can either be left on the scene, admitted to the emergency department (ed), or admitted directly to the intensive care unit (icu), depending on the level of criticality. here we performed a retrospective observational study that included patients who required intervention by a paris fire brigade team (bls and/or als) between 8th march and 18th april of 2020. no exclusion criteria were applied. this retrospective study was approved by the french society of anaesthesia and intensive care ethics committee on 7 april 2020 (ref: irb 00010254-2020-055). to minimize the bias in data abstraction [17] , data collection was performed by a single investigator using a previously established standardized abstraction template. from als prehospital medical reports, we retrieved the patients' demographic characteristics (age and gender), medical history (previous hypertension, cardiopathy, coronaropathy, chronic renal failure, chronic obstructive pulmonary disease, diabetes mellitus, obesity, stroke, immunosuppression, asthma, and active smoking), initial (i.e., at the first medical contact) prehospital vital sign values (systolic blood pressure, heart rate (hr), pulse oximetry (spo 2 ), respiratory rate (rr), temperature, and glasgow coma scale (gcs)), and record of administered prehospital treatments (oxygen modality and catecholamine type and dose). we also recorded the date of suspected contamination, the date of first symptoms, and the date of contact. the covid-19 diagnosis was established after transfer to the hospital, and prehospital diagnosis was based on a bundle of arguments including clinical signs and recent contact with a covid-19 patient. the hypoxemia index (hi) was calculated as the ratio between the initial spo 2 (%), i.e., spo 2 on room air, and the initial rr (breaths per minute): hi = spo2 rr . results are expressed as mean and standard deviation for quantitative parameters with a normal distribution, as median and interquartile range (q1-q3) for parameters with a non-gaussian distribution, and as absolute value and percentage for qualitative parameters. all analyses were performed using r 3.4.2 (http://www.r-project.org; the r foundation for statistical computing, vienna, austria). first, we performed bivariate analyses evaluating the relationship between covariates and prehospital intubation. second, we analyzed the prehospital hi level as a continuous variable and as a binary variable using the optimal threshold defined by receiver operating characteristic (roc) curve analysis, i.e., the optimal threshold with the highest sensitivity and the highest specificity associated with prehospital intubation, using the youden index [18] . to limit the impact of outliers, and to enable provision of more robust presentations, we obtained an adjusted average roc curve by averaging 10,000 bootstrapped samples (sampling with replacement). we compared the hi and spo 2 curves using the de long method [19] . third, we assessed the relationship between hi and prehospital intubation using logistic regression-including the following potential confounders: age, hypertension, cardiopathy, coronaropathy, chronic renal failure, chronic obstructive pulmonary disease, diabetes mellitus, obesity, immunosuppression, asthma, active smoking, systolic blood pressure, and hr-based on previous studies and physiopathological knowledge [6, 9, 11, 19, 20] . the results are expressed as adjusted odds ratio (ora) and 95% confidence interval (ci). fourth, to reduce the potential effect of confounders, we performed a propensity score analysis. we estimated the propensity score, i.e., the probability of hi lower than the optimal threshold, using logistic regression on the basis of the following potential confounders: age, hypertension, cardiopathy, coronaropathy, chronic renal failure, chronic obstructive pulmonary disease, diabetes mellitus, obesity, stroke, immunosuppression, asthma, active smoking, systolic blood pressure, and hr [21] . we used nearest-neighbor matching to match patients on the basis of the logit of the propensity score [22] , and then we assessed the balance of covariates based on absolute mean differences. after matching, the baseline characteristics included in the propensity score were compared between cases (prehospital intubation) and controls (no prehospital intubation) using paired tests to reduce the influence of sample size on p value, with a threshold of 10% considered acceptable [23] . to estimate the average treatment effect, the ora and 95% ci of prehospital intubation was evaluated for a value lower than the optimal threshold. between 8th march and 18th april of 2020, a total of 300 consecutive patients suffering from covid-19 were attended to by a prehospital paris fire brigade als team. the mean age was 64 ± 18 years, and 165 patients (55%) were male. a total of 45 patients (15%) were deceased on the scene, 30 (66%) of whom were male. the deceased patients were significantly older than the alive patients (70 ± 14 years vs. 63 ± 19 years, respectively; p = 0.018). among the patients alive on the scene, 34 (11% of all patients) had an active care restriction in the prehospital setting. these 34 patients included 20 males (59%) and were significantly older than the living patients without an active care restriction (81 ± 9 years vs. 62 ± 18 years, respectively; p < 10 −3 ). finally, 18 patients (6%) required prehospital intubation ( figure 1 ). years vs. 62 ± 18 years, respectively; p < 10 −3 ). finally, 18 patients (6%) required prehospital intubation ( figure 1 ). table 1 summarizes the population demographics and clinical characteristics, and table 2 summarizes the main prehospital functional symptoms. in the overall population, the initial rr was 28 ± 10 bpm and initial spo 2 was 89% (95% ci: 76-98%). patients with and without prehospital intubation showed significantly different initial values for rr (27 ± 10 bpm vs. 35 ± 11 bpm, respectively; p = 0.005) and spo 2 (90% (80-98%) vs. 45% (43-56%), respectively; p < 10 −3 ). figure 2 illustrates the relationship between initial rr and initial spo 2 in intubated and non-intubated patients. a total of 215 patients (72%) did not require prehospital intubation and instead received a median oxygen flow supplementation of 15 l/min (95% ci: 9-15 l/min). all prehospital intubations were performed by physicians wearing an ffp2 facial mask, glasses, and non-sterile gloves. eighteen patients (6%) needed prehospital intubation after anesthesia induction based on ketamine (n = 14, 78%) or etomidate (n = 4, 12%) and succinylcholine (n = 18, 100%). sedation was a total of 215 patients (72%) did not require prehospital intubation and instead received a median oxygen flow supplementation of 15 l/min (95% ci: 9-15 l/min). all prehospital intubations were performed by physicians wearing an ffp2 facial mask, glasses, and non-sterile gloves. eighteen patients (6%) needed prehospital intubation after anesthesia induction based on ketamine (n = 14, 78%) or etomidate (n = 4, 12%) and succinylcholine (n = 18, 100%). sedation was maintained by an association of midazolam and sufentanyl (n = 18, 100%). fifteen patients (83%) were paralyzed with atracurium. after intubation, their median rr was 15 bpm (12-20 bpm), median inspired fraction of oxygen was 100% (80-100%), median end-expiratory pressure was 12 cm h 2 o (5-15 cm h 2 o), and mean tidal volume was 442 ± 71 ml, corresponding to an indexed ideal body weight of 6-8 ml.kg −1 . hi was significantly associated with prehospital intubation (or, 0.24; 95% ci: 0.12-0.41; p < 10 −3 ), with a corresponding auc of 0.91 (0.85-0.98). the spo 2 roc curve is depicted in figure 3 , and the corresponding auc was 0.87 (0.77-0.98) without a significant difference from the hi auc (p = 0.41). the hi significantly differed (p <10 −3 ) between patients with and without prehospital intubation: 1.0 ± 1.0 vs. 3.6 ± 1.8, respectively. figure 4 illustrates the hi distribution among intubated and nonintubated patients, and figure 5 shows the roc curve for hi. the hi significantly differed (p < 10 −3 ) between patients with and without prehospital intubation: 1.0 ± 1.0 vs. 3.6 ± 1.8, respectively. figure 4 illustrates the hi distribution among intubated and non-intubated patients, and figure 5 shows the roc curve for hi. the hi optimal threshold, i.e., the threshold with the highest sensitivity and the highest specificity using the youden index, was 1.3. in bivariate analysis, an hi of <1.3 was significantly, positively associated with prehospital intubation (or, 38.38; 95% ci: 11.57-146.54; p < 10 −3 ) ( table 1) . multivariate logistic regression revealed that prehospital intubation was significantly associated with hi (ora, 0.20; 95% ci: 0.06-0.45; p < 10 −3 ) and with hi <1.3 (ora, 51.08; 95% ci: 7.83-645.06; p < 10 −3 ). figure 6 shows the absolute mean differences between subgroups after matching. the hi optimal threshold, i.e., the threshold with the highest sensitivity and the highest specificity using the youden index, was 1.3. in bivariate analysis, an hi of <1.3 was significantly, positively associated with prehospital intubation (or, 38.38; 95% ci: 11.57-146.54; p < 10 −3 ) (table 1) . multivariate logistic regression revealed that prehospital intubation was significantly associated with hi (ora, 0.20; 95% ci: 0.06-0.45; p < 10 −3 ) and with hi <1.3 (ora, 51.08; 95% ci: 7.83-645.06; p < 10 −3 ). figure 6 shows the absolute mean differences between subgroups after matching. after adjustment for confounders, hi of <1.3 was significantly associated with prehospital intubation (ora, 3.6; 95% ci: 1.95-5.08; p < 10 −3 ). after adjustment for confounders, hi of <1.3 was significantly associated with prehospital intubation (ora, 3.6; 95% ci: 1.95-5.08; p < 10 −3 ). in the present analysis of 300 covid-19 patients attended to by a prehospital paris fire brigade als team, we observed that hi was significantly associated with prehospital intubation. specifically, an hi of <1.3 was associated with a 3-fold increase in prehospital intubation. patients who required prehospital intubation were those with lower initial spo2 and higher initial rr values. the severe respiratory form of covid-19 appears as acute respiratory failure (arf) with a notable discrepancy between the increases of rr and spo2. as previously reported [11] , the respiratory form of covid-19 differs from other types of arf commonly encountered in the prehospital setting [24, 25] , with covid-19 patients commonly exhibiting a relatively low spo2 value but with no physical signs of acute respiratory distress (e.g., cyanosis and intercostal and substernal retractions). arf treatment is symptomatically treated because, despite many studies, no etiological treatments are yet available [26] [27] [28] . symptomatic arf treatment is based on oxygen therapyranging from low-flow or high-flow nasal cannula therapy [29] to invasive mechanical ventilation [30, 31] . in arf covid-19 disease, the optimal ventilatory mode remains controversial [30] [31] [32] due to the lack of scientific evidence and concerns about healthcare provider contamination through diabetes diabetes figure 6 . standardized mean deviation between cases and controls after matching. copd = chronic obstructive pulmonary disease, gcs = glasgow coma scale, sbp = systolic blood pressure, immunod = immunosuppression, smoke = active smoking. in the present analysis of 300 covid-19 patients attended to by a prehospital paris fire brigade als team, we observed that hi was significantly associated with prehospital intubation. specifically, an hi of <1.3 was associated with a 3-fold increase in prehospital intubation. patients who required prehospital intubation were those with lower initial spo 2 and higher initial rr values. the severe respiratory form of covid-19 appears as acute respiratory failure (arf) with a notable discrepancy between the increases of rr and spo 2 . as previously reported [11] , the respiratory form of covid-19 differs from other types of arf commonly encountered in the prehospital setting [24, 25] , with covid-19 patients commonly exhibiting a relatively low spo 2 value but with no physical signs of acute respiratory distress (e.g., cyanosis and intercostal and substernal retractions). arf treatment is symptomatically treated because, despite many studies, no etiological treatments are yet available [26] [27] [28] . symptomatic arf treatment is based on oxygen therapy-ranging from low-flow or high-flow nasal cannula therapy [29] to invasive mechanical ventilation [30, 31] . in arf covid-19 disease, the optimal ventilatory mode remains controversial [30] [31] [32] due to the lack of scientific evidence and concerns about healthcare provider contamination through sars-cov-2 aerosolization [33] [34] [35] [36] [37] . it is presently unclear how to choose the adequate ventilatory mode for each patient, with the goals being to avoid excessive intubation and to not delay necessary invasive mechanical ventilation. this is a great challenge to the prehospital management of arf during the covid-19 pandemic, due to the unfavorable ratio between needs and resources. early identification of patients at high risk of unfavorable respiratory evolution in the prehospital setting would help avoid both under-triage and over-triage [38, 39] . in covid-19 arf, clinical signs may not adequately indicate severity [11] ; thus, there is a need to explore other means of severity assessment. outside the hospital, hypoxemia depth may be assessed by arterial blood gas analysis, especially since covid-19 patients exhibit lower partial arterial oxygen pressure(pao2) than spo 2 [12] . however, during the covid-19 pandemic, there may be a shortage of blood gas analysis medical devices for prehospital emergency services. thus, severity assessment based on physical examination should also be available in order to counteract the previous limitations. this is why we have developed and tested the hypoxemia index to identify patients with a higher risk of requiring prehospital intubation. the present study has several limitations that restrict the generalization of our conclusions. first, it was a retrospective study with a restricted number of patients intubated in the prehospital setting. second, there is the possibility of bias from misclassification of covariates since data were collected from prehospital reports. third, the study design and the statistical analyses do not permit any conclusion regarding the causal link between hi and prehospital intubation; the results only indicate an association. fourth, as the relationship between rr and spo 2 was not linear [11] , we cannot conclude that the relationship between hi and prehospital intubation is linear; consequently, it may affect the interpretation of hi, especially since hi is markedly influenced by spo2. so far, hi should not be used, prior to a prospective validation, as a decisional trigger for prehospital intubation. fifth, data were collected by a single investigator, potentially compromising data accuracy [40] . sixth, no pediatric patients were included in our analyses, and thus our results may not be transposable to a pediatric population, particularly since sars-cov-2 does not affect children as it affects adults [41] . seventh, spo 2 is not the best tool to reflect pao2, which mostly triggered dyspnea. eighth, this study was performed in a single city within one prehospital emergency system. ninth, intubation of covid-19 patients remains challenging and under debate [40] , with risk for healthcare providers [42, 43] and patients [44] [45] [46] [47] . consequently, the decision-making for intubation may be delayed until arrival at a high-level care facility with an experienced team member [48, 49] . beyond these limitations, our results appear to indicate that hi may be a useful tool for distinguishing covid-19 patients with a higher risk of prehospital intubation in a prehospital setting. the presently described hypoxemia index is associated with the requirement for prehospital intubation in covid-19 patients cared for in the prehospital setting. this hypoxemia index may be useful for screening covid-19 patients for risk associated with prehospital intubation, but these results must be confirmed by prospective studies. acknowledgments: thanks to the paris fire brigade basic life support teams for their daily engagement during the pandemic. the authors declare no conflict of interest. potential for global spread of a novel coronavirus from pneumonia of unknown aetiology in wuhan, china: potential for international spread via commercial air travel the wuhan sars-cov-2-what's next for china outbreak of pneumonia of unknown etiology in wuhan, china: the mystery and the miracle world health organization. who director-general's opening remarks at the media briefing on covid-19-11 case-fatality rate and characteristics of patients dying in relation to covid-19 in italy case fatality rate 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 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of patients with covid-19: a prospective international multicentre cohort study aerosol generating procedures and risk of transmission of acute respiratory infections to healthcare workers: a systematic review official ers/ats clinical practice guidelines: noninvasive ventilation for acute respiratory failure guidelines for the management of tracheal intubation in critically ill adults cardiac arrest and mortality related to intubation procedure in critically ill adult patients: a multicenter cohort study covid-19: intubation kit, intubation team or intubation spots? consensus guidelines for managing the airway in patients with covid-19: guidelines from the difficult airway society, the association of anaesthetists the intensive care society, the faculty of intensive care medicine and the royal college of anaesthetists this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-331347-imexhlwn authors: anzai, asami; kobayashi, tetsuro; linton, natalie m.; kinoshita, ryo; hayashi, katsuma; suzuki, ayako; yang, yichi; jung, sung-mok; miyama, takeshi; akhmetzhanov, andrei r.; nishiura, hiroshi title: assessing the impact of reduced travel on exportation dynamics of novel coronavirus infection (covid-19) date: 2020-02-24 journal: j clin med doi: 10.3390/jcm9020601 sha: doc_id: 331347 cord_uid: imexhlwn the impact of the drastic reduction in travel volume within mainland china in january and february 2020 was quantified with respect to reports of novel coronavirus (covid-19) infections outside china. data on confirmed cases diagnosed outside china were analyzed using statistical models to estimate the impact of travel reduction on three epidemiological outcome measures: (i) the number of exported cases, (ii) the probability of a major epidemic, and (iii) the time delay to a major epidemic. from 28 january to 7 february 2020, we estimated that 226 exported cases (95% confidence interval: 86,449) were prevented, corresponding to a 70.4% reduction in incidence compared to the counterfactual scenario. the reduced probability of a major epidemic ranged from 7% to 20% in japan, which resulted in a median time delay to a major epidemic of two days. depending on the scenario, the estimated delay may be less than one day. as the delay is small, the decision to control travel volume through restrictions on freedom of movement should be balanced between the resulting estimated epidemiological impact and predicted economic fallout. by early february 2020, it was evident that the incidence of novel coronavirus infections (covid19) was growing exponentially [1] . accelerated by human migration, exported cases have been reported in various regions of the world, including europe, asia, north america, and oceania [2] . to minimize the rapid growth of cases via human-to-human transmission [3] [4] [5] , the government of china suspended all modes of transportation to and from wuhan on 23 january 2020-including vehicles, trains, and flights-expecting that the intervention would prevent further spread of the disease [6] . as of 12 february 2020, two additional cities outside of hubei province-wenzhou (zhejiang province) and shenzhen (guangdong province)-have been placed on complete lockdown (i.e., no cross-border movement to and from the closed city) to prevent further spatial spread of covid-19. to our knowledge, such drastic movement restrictions are a historical first. since wuhan was placed on lockdown, travel restriction and border control have been implemented by various countries, either as: (i) complete travel bans, (ii) travel restriction and quarantine-which allows for restriction of healthy individuals, (iii) entry screening for all incoming travelers, or some combination thereof. most countermeasures are in line with (ii) and (iii), aside from the three cities on complete lockdown, while some countries at high risk refused any entry from china (e.g., australia) or those from hubei and zhejiang provinces (e.g., japan). all travel arrangements including tourist tours outbound from china (to international destinations) organized by chinese travel agencies were cancelled, and all non-urgent travel with business purposes both inbound and outbound were greatly reduced. the effectiveness of quarantine (i.e., lockdown) measures to prevent the spread of an epidemic due to a novel infectious pathogen where no vaccine is available has often been a subject of debate [7] [8] [9] . under ordinary circumstances, border control efforts do not go beyond entry screening. however, during the epidemic of severe acute respiratory syndrome (sars) in 2002-2003, although entry screening at airports and other key locations was adopted, in most countries its effectiveness was estimated to be very limited due to the relatively long incubation period and low prevalence of sars, which resulted in extremely low positive predictive values at screening locations [10] [11] [12] [13] . in the ongoing covid-19 epidemic, many countries have accompanied regular entry screening with drastic changes in travel restrictions. although the effectiveness of entry screening is likely very limited as already shown elsewhere [14] , the epidemiological impact of the change in movement restrictions has yet to be explicitly evaluated. in this study, we quantify the impact of the drastic reduction in travel volume-resulting from movement restrictions-on the transmission dynamics outside china. we aim to estimate reductions in the number of exported cases, probability of an outbreak occurring outside china, and any time delay to a major epidemic that may be gained with these policies. we use the example of japan, the country in asia that receives the largest number of visitors from china, to calculate our estimates. an epidemiological dataset of confirmed cases with covid-19 infection diagnosed outside china was collected from government and news websites quoting official outbreak reports. for each case, the date of reporting and country of diagnosis were recorded. the data included only cases diagnosed outside china, but for whom infection may have occurred either in or outside china. the dataset is available as supplementary material (table s1 ). all cases were confirmed using reverse transcriptase polymerase chain reaction (rt-pcr) apart from two cases in australia that were clinically diagnosed. the endpoint for data collection was set at 6 february 2020. we considered the impact of reduced travel volumes on covid-19 transmission dynamics outside china. specifically, we quantified the impact on: (i) the number of exported cases, (ii) the probability of a major epidemic, and (iii) the time delay to a major epidemic. figure 1 shows the observed number of infections in and outside china. the first exported case in thailand was reported on 13 january 2020. assuming the epidemic start date is set at 1 december 2019 (day 0), the city of wuhan was put in lockdown from day 53 (or 23 january 2020). considering that the mean incubation period of covid-19 approximately is 5 days, the impact of reduced travel volumes would start to be interpretable from day 58 (28 january 2020). we used data from day 43 (13 january) onwards because the first case diagnosed outside china was reported on that day. data from day 43 (13 january) onwards because the first case diagnosed outside china was reported on that day. to estimate the reduced volume of exported cases, we employ a counterfactual model. if we let c(t) be the incidence of exported cases on day t, poisson regression was used to fit the following model through day 57: where is the initial value at t = 0 and r is the exponential growth rate of exported cases outside china. using the estimated parameters and their covariance matrix, we obtain the expected number of exported cases from day 58 onwards. supposing that h(t) is the observed number of cases on day t, the reduced travel volume of exported cases by day 67 is calculated as: . (2) we assumed that the distribution of the number of secondary cases generated by a single primary case follows a negative binomial distribution with the basic reproduction number r0, i.e., the average number of secondary cases generated by a single primary case, and the dispersion parameter k. the probability of extinction defined by the first generating moment [15] is then modeled as: . (3) r0 is estimated to range from 1.5 to 3.7, and here we adopt 1.5, 2.2, and 3.7 as plausible values for our calculations [16] [17] [18] . the value of k, a dispersion parameter, is assumed to be 0.54 as estimated elsewhere [17] . supposing that there are n untraced cases that were independently introduced, the probability of a major epidemic is: now we compare two scenarios: the observed data as influenced by the reduction in travel volume, and a counterfactual scenario in which travel volume reduction does not take place. the cumulative number of exported covid-19 cases observed in the former scenario is denoted m, to estimate the reduced volume of exported cases, we employ a counterfactual model. if we let c(t) be the incidence of exported cases on day t, poisson regression was used to fit the following model through day 57: where c 0 is the initial value at t = 0 and r is the exponential growth rate of exported cases outside china. using the estimated parameters and their covariance matrix, we obtain the expected number of exported cases from day 58 onwards. supposing that h(t) is the observed number of cases on day t, the reduced travel volume of exported cases by day 67 is calculated as: we assumed that the distribution of the number of secondary cases generated by a single primary case follows a negative binomial distribution with the basic reproduction number r 0 , i.e., the average number of secondary cases generated by a single primary case, and the dispersion parameter k. the probability of extinction π defined by the first generating moment [15] is then modeled as: r 0 is estimated to range from 1.5 to 3.7, and here we adopt 1.5, 2.2, and 3.7 as plausible values for our calculations [16] [17] [18] . the value of k, a dispersion parameter, is assumed to be 0.54 as estimated elsewhere [17] . supposing that there are n untraced cases that were independently introduced, the probability of a major epidemic is: now we compare two scenarios: the observed data as influenced by the reduction in travel volume, and a counterfactual scenario in which travel volume reduction does not take place. the cumulative number of exported covid-19 cases observed in the former scenario is denoted m, while m describes the number of cases in the counterfactual scenario. this leads to the following sums: and accordingly, the reduced probability of a major epidemic is calculated as: it should be noted that the probability of a major epidemic is evaluated at the country level, and only results for japan are presented here. whereas, the proposed method can equally handle the probability of a major epidemic for each importing country. for the computation, we first subtracted m, the integral of e(c(t)), by the integral of h(t), assuming that all cases h(t) were already traced, and then we multiplied the difference by 0.9, 0.7, or 0.5 if only 10%, 30%, or 50% of contacts were traced, respectively. for m, we accounted for three symptomatic cases that were regarded as locally acquired infections in reports and diagnosed between day 58 and day 67. assuming that the asymptomatic ratio was 50% [19] , we considered that in total there were m = 6 untraced cases including the diagnosed cases. lastly, we measured the time delay to a major epidemic gained from the reduction in travel volume using the hazard function of a major epidemic, λ(t), in the absence of travel volume changes. we model the probability of a major epidemic by time t in the absence of travel volume reduction as follows: in the presence of travel volume reduction, the hazard is reduced by the relative reduction factor in the probability of a major epidemic: here, we consider the median time to a major epidemic in (8) and (9). since an exponential growth of cases has been observed, we let the hazard be an exponential function. then, the integral of the hazard function holds the form: c · (exp(rt) − 1), where c is a constant (assumed to be one for the following calculation), and r is the exponential growth rate estimated at 0.14 per day [16] . the doubling time is then calculated as t d = ln(2)/r = 4.95 days. the difference in the median date between (8) and (9) is thus described as: (2) . all computations were conducted in jmp version 14.0 (sas institute, cary, north carolina). the confidence intervals were calculated using profile likelihood method. as figure 1 shows, a total of 242 cases were diagnosed and reported outside of china in 27 countries between day 43 (13 january) and day 67 (6 february 2020). of these, 140 cases were considered to have been infected in china and 102 cases were considered to have been infected outside china. the country with the highest number of exported cases infected inside china was thailand (n = 20), followed by singapore (n = 18), australia (n = 14), and japan (n = 12). among 242 cases, we specifically focused on 140 cases who traveled while movement restrictions were in place and were likely affected by said restrictions. figure 2 compares the observed and expected number of cases diagnosed outside china by date of report. the exponential growth of cases is consistent with the exponential growth of incidence in china, which qualitatively captures the observed pattern of incidence from day 43 to day 57. using the predicted curve, the expected number of cases between day 58 (28 january 2020) and day 67 was 321 cases (95% confidence interval [ci]: 181, 544). in the empirical observation, a total of 95 cases were diagnosed, including 8 cases in japan. that is, following the time that wuhan city was put in lockdown, we estimate that 226 cases (95% ci: 86, 449) were prevented from being exported across the world. this corresponds to a reduction in the number of exported cases of 70.4% during that time period. j. clin. med. 2020, 9, x for peer review 5 of 9 considered to have been infected in china and 102 cases were considered to have been infected outside china. the country with the highest number of exported cases infected inside china was thailand (n = 20), followed by singapore (n = 18), australia (n = 14), and japan (n = 12). among 242 cases, we specifically focused on 140 cases who traveled while movement restrictions were in place and were likely affected by said restrictions. figure 2 compares the observed and expected number of cases diagnosed outside china by date of report. the exponential growth of cases is consistent with the exponential growth of incidence in china, which qualitatively captures the observed pattern of incidence from day 43 to day 57. using the predicted curve, the expected number of cases between day 58 (28 january 2020) and day 67 was 321 cases (95% confidence interval [ci]: 181, 544). in the empirical observation, a total of 95 cases were diagnosed, including 8 cases in japan. that is, following the time that wuhan city was put in lockdown, we estimate that 226 cases (95% ci: 86, 449) were prevented from being exported across the world. this corresponds to a reduction in the number of exported cases of 70.4% during that time period. observed cases (dots) include those infected in china. an exponential growth curve was fitted to the observed data from 27 january 2020. the dashed lines represent the 95% confidence interval on and after 28 january 2020. as another measure of impact, we estimated the probability of a major epidemic. figure 3a shows the probability of a major epidemic with three different levels of transmissibility assuming an r0 of 1.5, 2.2, or 3.7, and three different levels of contact tracing resulting in a success rate of isolation of the traced contacts of 10%, 30%, or 50%. without the reduction in the travel volume, the probability of a major epidemic exceeded 90% in most scenarios. however, considering there have been six untraced cases in japan under travel restrictions, the probability of a major epidemic more broadly ranged from 56% to 98%. figure 3b shows the reduced probability of a major epidemic. assuming an r0 of 2.2, the absolute risk reduction was 7%, 12%, and 20%, respectively, for contact tracing levels leading to isolation at 10%, 30%, and 50%. observed and expected number of cases diagnosed outside china by date of report. observed cases (dots) include those infected in china. an exponential growth curve was fitted to the observed data from 27 january 2020. the dashed lines represent the 95% confidence interval on and after 28 january 2020. as another measure of impact, we estimated the probability of a major epidemic. figure 3a shows the probability of a major epidemic with three different levels of transmissibility assuming an r 0 of 1.5, 2.2, or 3.7, and three different levels of contact tracing resulting in a success rate of isolation of the traced contacts of 10%, 30%, or 50%. without the reduction in the travel volume, the probability of a major epidemic exceeded 90% in most scenarios. however, considering there have been six untraced cases in japan under travel restrictions, the probability of a major epidemic more broadly ranged from 56% to 98%. figure 3b shows the reduced probability of a major epidemic. assuming an r 0 of 2.2, the absolute risk reduction was 7%, 12%, and 20%, respectively, for contact tracing levels leading to isolation at 10%, 30%, and 50%. figure 3b describes the absolute reduction in risk of a major epidemic. the largest reduction was 37% when r0 = 1.5 and 50% of contacts were traced. the smallest reduction was 1% when r0 = 3.7 and 10% of contacts were traced. using those estimated relative reductions, the median time of delay gained by travel volume reduction is shown in figure 4 . the time delay of a major epidemic was less than one day when r0 is 2.2 and 3.7, and 1 to 2 days when r0 is 1.5. . delay in the time to a major epidemic gained by travel volume reduction. the median delay is shown for japan, using relative reduction in the probability of a major epidemic. the vertical axis represents the time delay to a major epidemic (in days), and the horizontal axis represents the proportion of contacts traced. each shaped dot represents different values of the basic reproduction number. the present study explicitly quantified the epidemiological impact of reduced travel volume to and from china on the transmission dynamics of covid-19 outside china using simple statistical the solid lines represent the probability of a major epidemic in the counterfactual scenario, i.e., based on the expected number of cases diagnosed in japan. dashed lines represent the probability of a major epidemic in the presence of travel volume reductions, calculated using the number of traced and untraced cases was 6 in total in japan from day 58 to day 67. contact tracing leading to isolation was assumed at three different levels: 10%, 30%, and 50%. (b) the vertical axis represents the reduced probability of a major epidemic due to travel volume reduction. the horizontal axis shows the proportion of cases traced, adopting the same scenarios as panel a. figure 3b describes the absolute reduction in risk of a major epidemic. the largest reduction was 37% when r 0 = 1.5 and 50% of contacts were traced. the smallest reduction was 1% when r 0 = 3.7 and 10% of contacts were traced. using those estimated relative reductions, the median time of delay gained by travel volume reduction is shown in figure 4 . the time delay of a major epidemic was less than one day when r 0 is 2.2 and 3.7, and 1 to 2 days when r 0 is 1.5. j. clin. med. 2020, 9, x for peer review 6 of 9 figure 3b describes the absolute reduction in risk of a major epidemic. the largest reduction was 37% when r0 = 1.5 and 50% of contacts were traced. the smallest reduction was 1% when r0 = 3.7 and 10% of contacts were traced. using those estimated relative reductions, the median time of delay gained by travel volume reduction is shown in figure 4 . the time delay of a major epidemic was less than one day when r0 is 2.2 and 3.7, and 1 to 2 days when r0 is 1.5. the present study explicitly quantified the epidemiological impact of reduced travel volume to and from china on the transmission dynamics of covid-19 outside china using simple statistical . delay in the time to a major epidemic gained by travel volume reduction. the median delay is shown for japan, using relative reduction in the probability of a major epidemic. the vertical axis represents the time delay to a major epidemic (in days), and the horizontal axis represents the proportion of contacts traced. each shaped dot represents different values of the basic reproduction number. the present study explicitly quantified the epidemiological impact of reduced travel volume to and from china on the transmission dynamics of covid-19 outside china using simple statistical models. the three epidemiological outcomes we measured on the example of japan were: (i) the number of exported cases, (ii) the probability of a major epidemic, and (iii) the time delay to a major epidemic. when the volume of exported cases outside china was considered to have been reduced by 70.4%, the probability of a major epidemic was estimated to be reduced by 7%-20% in japan, and a 2-day delay was gained in the estimated time to a major epidemic between day 58 and day 67. the reduced volume of exported cases was estimated to be as large as 226 cases outside china. our estimate is consistent with an assessment by chinazzi et al. [20] , which indicated that the exported cases would be reduced by 80% by the end of february. in addition to appropriately quantifying the impact on prevention of exported cases, we have estimated the median time delay to a major epidemic assuming plausible values of r 0 at 1.5, 2.2, and 3.7. with reduced probability of a major epidemic, the time delay to a major epidemic was estimated at a maximum of 2 days in japan and a minimum of less than 1 day. the estimated effect of the delay to a major epidemic outside china is smaller than what was anticipated for cities in china other than wuhan. tian et al. [21] estimated that the reduction in travel volume led to a 2.9-day delay in the spatial spread in china. to our knowledge, the present study is the first to have used simple stochastic process models to explicitly estimate the time delay to a major epidemic in japan that gained by the drastic reduction in travel volume in and outside china. although the covid-19 epidemic was declared a public health emergency of international concern (pheic) by the world health organization (who), the who specifically called upon member states to not restrict the freedom of movement of persons as a result of the epidemic [22] . however, member states did not adhere to this recommendation and have varyingly restricted the free movement of people from china [23] . such restrictions were most drastic in china, where some cities were put on complete lockdown [23] . these political decisions regarding movement restrictions must balance the expected epidemiological impact with predicted economic burden-the latter of which we did not examine. while securing a few days delay to epidemic spread in china would secure time for healthcare systems in chinese cities that have not yet been affected to prepare for the appearance of case-patients [21] , the impact of such a delay outside china is not substantial enough to accomplish meaningful prevention, such as the development, manufacturing, and distribution of a vaccine. in modern history, this epidemic is perhaps the first instance where a large city populated with more than 10 million people was placed on lockdown. while countermeasures to prevent epidemic spread require the sort of strong political decisions that resulted in strong movement restrictions, our study indicated that the delay to a major epidemic in countries other than china (using japan as an example) was unfortunately minimal. while the complete lockdown of wuhan, wenzhou, and shenzhen likely helped reduce case incidence outside of these cities, migration from other cities in china could still contribute to the spread of infection locally and internationally [24] [25] [26] . to quantify the epidemiological impact for the entire course of the epidemic more precisely, a more detailed analysis using dynamic datasets, e.g., airline passenger data, should be explored in the future. limitations of the present study must be discussed. first, the present study relied on the volume of cases diagnosed outside china and did not directly examine human migration data. second, we were unable to classify exported cases into those who acquired infection in hubei versus elsewhere in china. having this information may offer additional insight. third, several rough assumptions (e.g., a fixed time delay from illness onset to reporting at 5 days) were imposed, and the results presented here should be regarded as a preliminary assessment. in conclusion, the present study explored the impact of reduced travel volume to and from china on the transmission dynamics of covid-19 outside china, estimating that the time delay to a major epidemic was on the order of 2 days by 7 february 2020 for japan. a relatively short time period of gain is likely due to high contingency of the sars-cov-2, and also that the substantial fraction of infected individuals with milder symptoms are likely to escape the border control. our proposed approach was kept simple and will be applicable to other emerging epidemics in the future. world health organization. novel coronavirus-china world health organization. novel coronavirus (2019-ncov) situation report-21 transmission of 2019-ncov infection from an asymptomatic contact in germany a familial cluster of pneumonia associated with the 2019 novel coronavirus indicating person-to-person transmission: a study of a family cluster large-scale quarantine following biological terrorism in the united states: scientific examination, logistic and legal limits, and possible consequences quarantine for pandemic influenza control at the borders of small island nations is there a case for quarantine? perspectives from sars to ebola impact of quarantine on the 2003 sars outbreak: a retrospective modeling study entry screening for severe acute respiratory syndrome (sars) or influenza: policy evaluation border screening for sars in australia: what has been learnt? real time estimation of the risk of death from novel coronavirus (2019-ncov) infection: inference using exported cases stochastic epidemic models: a survey effectiveness of airport screening at detecting travellers infected with novel coronavirus (2019-ncov) pattern of early human-to-human transmission of wuhan early transmission dynamics in wuhan, china, of novel coronavirus-infected pneumonia estimation of the asymptomatic ratio of novel coronavirus (2019-ncov) infections among passengers on evacuation flights early evaluation of the wuhan city travel restrictions in response to the 2019 novel coronavirus outbreak emergency committee regarding the outbreak of novel coronavirus (2019-ncov) coronavirus: us bars foreigners who recently visited china nowcasting and forecasting the potential domestic and international spread of the 2019-ncov outbreak originating in wuhan, china: a modelling study estimation of the transmission risk of the 2019-ncov and its implication for public health interventions novel coronavirus outbreak in wuhan, china, 2020: intense surveillance is vital for preventing sustained transmission in new locations author contributions: h.n. conceived the study, a.a. and t.k. collected the data and analyzed the empirical data using models. all authors participated in the study design. a.a, t.k. and h.n. drafted the manuscript. all authors gave comments on the earlier versions of the manuscript. all authors have read and agreed to the published version of the manuscript. the authors declare no conflicts of interest. the following is available online at http://www.mdpi.com/2077-0383/9/2/601/s1, table s1 : number of confirmed cases by date of report. key: cord-287102-o19uwryp authors: amit, moran; sorkin, alex; chen, jacob; cohen, barak; karol, dana; tsur, avishai m; lev, shaul; rozenblat, tal; dvir, ayana; landau, geva; fridrich, lidar; glassberg, elon; kesari, shani; sviri, sigal; gelman, ram; miller, asaf; epstein, danny; ben-avi, ronny; matan, moshe; jakobson, daniel j.; bader, tarif; dahan, david; king, daniel a.; ben-ari, anat; soroksky, arie; bar, alon; fink, noam; singer, pierre; benov, avi title: clinical course and outcomes of severe covid-19: a national scale study date: 2020-07-18 journal: j clin med doi: 10.3390/jcm9072282 sha: doc_id: 287102 cord_uid: o19uwryp knowledge of the outcomes of critically ill patients is crucial for health and government officials who are planning how to address local outbreaks. the factors associated with outcomes of critically ill patients with coronavirus disease 2019 (covid-19) who required treatment in an intensive care unit (icu) are yet to be determined. methods: this was a retrospective registry-based case series of patients with laboratory-confirmed sars-cov-2 who were referred for icu admission and treated in the icus of the 13 participating centers in israel between 5 march and 27 april 2020. demographic and clinical data including clinical management were collected and subjected to a multivariable analysis; primary outcome was mortality. results: this study included 156 patients (median age = 72 years (range = 22–97 years)); 69% (108 of 156) were male. eighty-nine percent (139 of 156) of patients had at least one comorbidity. one hundred three patients (66%) required invasive mechanical ventilation. as of 8 may 2020, the median length of stay in the icu was 10 days (range = 0–37 days). the overall mortality rate was 56%; a multivariable regression model revealed that increasing age (or = 1.08 for each year of age, 95%ci = 1.03–1.13), the presence of sepsis (or = 1.08 for each year of age, 95%ci = 1.03–1.13), and a shorter icu stay(or = 0.90 for each day, 95% ci = 0.84–0.96) were independent prognostic factors. conclusions: in our case series, we found lower mortality rates than those in exhausted health systems. the results of our multivariable model suggest that further evaluation is needed of antiviral and antibacterial agents in the treatment of sepsis and secondary infection. coronavirus disease 2019 is caused by severe acute respiratory syndrome coronavirus 2 (sars-cov-2). this novel coronavirus was identified as the cause of a pandemic that originated in wuhan, china, in december 2019. as of 8 may 2020, more than 3.9 million people had been diagnosed with the disease in over 187 countries, with more than 272,000 deaths. [1] the magnitude of this pandemic has overwhelmed health care systems worldwide. hospitals have been overcrowded with covid-19 patients, and medical providers have been challenged by shortages of intensive care unit (icu) beds, ventilators, and essential medical personnel [2] [3] [4] . differences in resource availability among countries, an absence of data on covid-19's clinical course, and the rapid development of the spread of the pandemic, with insufficient follow-up time, have not allowed informed and balanced decision making. for instance, accurate data on clinical outcomes (death versus discharge or transfer from icu) of ventilated patients would allow better planning of ventilator distribution and use. in addition, data are lacking on the effectiveness of novel therapies, such as antivirals, and existing therapies, such as glucocorticoids and antibiotics. as of 8 may 2020, israel had 16,409 covid-19 cases, with 245 deaths. israel has designated over 2600 icu beds for severely ill covid-19 patients. however, the country has had no more than 181 severely ill patients at a time [5] . analyzing data from a non-overwhelmed health system of the developed world may shed light on the "natural history" of this disease. in addition, focusing on patients with established outcomes provides a better understanding of the role of different interventions. in this report, we comprehensively assessed the clinical characteristics, interventions, and outcomes of severely ill patients who were treated in israel. we also identified factors that were associated with mortality. this retrospective registry study was performed at the medical corps-israel defense forces, israel, which is the national coordinating center for the israel covid-19 icu registry. we enrolled all consecutive patients with laboratory-confirmed sars-cov-2 infection who were admitted to one of the icus among 13 participating hospitals between 5 march and 27 april 2020. because of the crisis status declared in israel and the nature of this retrospective chart review, with minimal-risk research using data collected for routine clinical practice, the israel ministry of health (jerusalem, israel) waived the need for an individual institutional ethics board and the need for informed consent from individual patients. according to the world health organization (who) guidelines [6] , laboratory confirmation of sars-cov-2 was defined as a positive result on a real-time reverse transcriptase-polymerase chain reaction assay of nasal and pharyngeal swabs. this guidance was implemented locally with the adjunct of using reverse transcriptase-polymerase chain reaction assay from lower respiratory tract aspirates. after being de-identified, patients' data were recorded daily on an online questionnaire-based electronic worksheet (surveymonkey) that was accessible online to registry associates. this study included critically ill patients admitted to icu. criteria for icu admission were either acute respiratory distress syndrome (ards), sepsis or acute organ failure [7, 8] . ards was defined as pao 2 /fio2a ≤ 300 mmhg with positive end-expiratory pressure (peep) or continuous positive airway pressure (cpap) ≥ 5 cm h 2 o or non-ventilated; if pao 2 was not available, spo2/fio2 ≤ 315. sepsis was defined as life-threatening organ dysfunction caused by a dysregulated host response to a suspected or proven infection and a sepsis-related sequential organ failure assessment (sofa) score of ≥2 points. acute organ dysfunction was defined as respiratory (hypoxemia defined by low pao 2 /fio2), coagulation (low platelets), liver (high bilirubin), cardiovascular (hypotension), central nervous system (low level of consciousness defined by glasgow coma scale), or kidneys (low urine output or high creatinine). patients who died prior to icu admission and patients without outcome data were excluded. clinical data reported in this study were collected within the first 24-120 h following icu discharge or death. the recorded data included the following: age, sex, medical comorbidities (i.e., smoking status, hypertension, diabetes, ischemic heart disease, chronic heart failure, cancer, chronic kidney disease, immunosuppression, cirrhosis, and dementia), medication history, vital signs, chest x-rays, laboratory studies on admission to the icu, anti-covid-19 pharmacological therapy in the icu (antimalarials, antivirals, anti-inflammatories, and plasma from recovered patients), respiratory support method (invasive or noninvasive mechanical ventilation and oxygen mask), renal replacement therapy, nutrition methods (enteral and total parenteral nutrition), the use of extracorporeal membrane oxygenation (ecmo), complications, and outcome. the number of patients who had died, been discharged, and been transferred to a lower level of care as of 5 may 2020, were recorded; icu length of stay was also determined. no statistical sample size calculation was performed a priori, and the sample size was equal to the number of patients treated during the study period. the primary outcome was patient status on discharge from the icu (i.e., dead vs. alive). continuous variables are presented as the median and interquartile range (iqr) with 95% confidence intervals (cis). categorical variables were expressed as the number of patients (percentage). differences in the distributions of patient characteristics by median age subgroups and the presence or absence of hypertension were reported using differences with 95% cis. the distribution of data over the age subgroups was based on the available data for that variable, and the other percentages were calculated using the available data for that subgroup. the mann-whitney rank sum test was used to compare nonparametric continuous variables. χ 2 or fisher exact test was used for categorical variables as appropriate. the first step was to study the correlation between death and each covariate via a univariable analysis; this was followed by a preliminary multivariable logistic regression model and a wald test. thus, covariates with a univariable p < 0.05 were included in a preliminary multivariable wald regression model. variables that remained statistically significant (p < 0.05) were included in the final multivariable model. all statistical tests were two-tailed, and statistical significance was defined as p < 0.05. analyses were performed using jmp pro 14.0. the analyses were not adjusted for multiple comparisons, and given the possibility of a type i error, the findings should be interpreted as exploratory and descriptive. from 5 march to 27 april 2020, 156 patients with suspected or confirmed covid-19 were hospitalized at one of the participating centers in israel. table 1 shows patients' demographic and clinical characteristics. positive sars-cov-2 status was confirmed prior to hospitalization in 97 patients (62%); in these patients, the median time from laboratory-confirmed sars-cov-2-19 to presentation was 4 days (range = 1 to 18 days). the remaining 59 patients had pending test results for sars-cov-2, and their positive sars-cov-2 status was confirmed during hospitalization. patients' median age was 72 years (iqr = 60-82 years; range = 22-97 years); 64 patients (41%) were aged 76 years and older, and 31 (20%) were younger than 55 years. overall, 69% (108 of 156) of patients were male, with a similar sex distribution among patients younger than 85 years. among patients older than 85 years, 66% (19 of 29) were female. there was no significant variance in age distribution (p = 0.775, leven's test) between centers; at one center, 95% of patients (20 of 21) were male, resulting in a significant variance in sex distribution between centers (p = 0.042, pearson test). eighty-nine percent (139 of 156) of patients had at least one comorbidity (table 1) . hypertension was the most common, affecting 85 (54%) patients, followed by diabetes (62 patients (40%)) and ischemic heart disease (33 patients (21%)). only 13 patients (8%) had a history of chronic obstructive pulmonary disease, eight of whom (5%) were treated for cancer and five of whom (3%) had immunosuppression (i.e., as a result of organ transplant or chronic treatment with systemic corticosteroids). only one (1.5%) patient older than 75 years presented without preexisting comorbidities; and 89% (57 of 64 patients) presented with multiple comorbidities. body mass index (bmi) data were available for 109 patients: 46% (50 of 109) of patients were overweight (i.e., bmi between 25 and 30) and 29% (32 of 109) were obese (i.e., body mass index, bmi > 30). supplementary table s1 presents patients' medication histories. table 2 presents patients' vital signs, chest x-rays, and laboratory findings on admission. acute organ failure was the most common critical illness-defining condition (103 of 156 patients (66%)), followed by ards (69 of 156 (44%)) and sepsis (40 of 156 (26%)) ( table 2 ). deterioration in inpatients who were initially not classified as critically ill occurred in 56% of patients (88 of 156); in these patients, the median time to deterioration and icu admission was 3 days (iqr = 2-6 days, range = 1-20 days). hydroxychloroquine and chloroquine were the most commonly used anti-covid-19 pharmacological agents administered in the icu (124 of 156 (79%)), followed by corticosteroids (22%) and antiviral agents (19%) ( table 3) . of note, fresh plasma from patients who had recovered from covid-19 was administered in seven patients (5%). table 3 presents the icu interventions and organ replacement therapies used. among 156 patients who were admitted to the icu, 103 (66%) required endotracheal intubation and mechanical ventilation, and 39 patients (25%) were treated with noninvasive ventilation. there were no differences in mechanical ventilation rates among different age groups (71% of patients younger than 55 years, 61% of patients older than 75 years, and 69% of patients between 55 and 75 years; p = 0.725, likelihood ratio); however, noninvasive ventilation was used more frequently in patients younger than 55 years (16 of 31(52%)) than in patients older than 75 years (6 of 64 (9%)) and patients between 55 and 75 years old (17 of 61 (28%)), p < 0.001, likelihood ratio). tracheostomies were placed in 14% (13 of 103) of ventilated patients. ecmo and renal replacement therapy were used in 4% (6 of 156) and 9% (14 of 156) of patients with acute respiratory and renal failure, respectively. seventy-four (47%) patients required tube feeding via a nasogastric tube, and 15 (10%) were fed with total parenteral nutrition. a secondary infection was diagnosed in 27 (17%) patients, and 21 (13%) developed sepsis. an acute kidney injury developed in 40 (27%) patients, and an acute cardiac injury developed in eight (5%) ( table 3) . as of 8 may 2020, the median (iqr) length of stay in the icu was 10 days (iqr = 5-17 days; range = 0-37 days). among patients who were intubated (n = 103), the median (iqr) ventilation time was 13 days (range = 7-19 days) compared to 7 days (range = 2-11 days) in non-intubated patients (p < 0.001, analysis of variance-anova). we also found a significant correlation between patients' age and length of stay: the median (iqr) length of stay in the icu was 14 days (6-21 days) in patients younger than 55 years and 4 days (2-11 days) (p = 0.004, correlation coefficient = −0.225) in patients older than 85 years. with a total of 87 deaths, the overall mortality rate was 56%; 25 (16%) patients were discharged home, and 44 (28%) experienced clinical improvement and were transferred to a lower level of care (i.e., rehabilitation or covid-19 internal medicine departments). we found a significant difference in the length of icu stay between patients who died (median = 7 days; iqr = 3-12 days) and those who experienced improvement and were discharged from the icu (median = 15 days; iqr = 9-21 days). a univariate analysis revealed that patients' age, sex, comorbidity status, sepsis, white blood cell count, antiviral therapy, antimalarial therapy, and length of icu stay were all statistically significant predictors of outcome (table 4 ). we included the significant variables in a multivariable regression model. this analysis revealed that older age (or = 1.08 for each year of age; 95% ci = 1.03-1.13), the presence of sepsis (or = 1.08 for each year of age; 95% ci = 1.03-1.13), and short length of icu stay (or = 0.90 for each day; 95% ci = 0.84-0.96) were the only independent prognostic factors. in this multicenter case series, we evaluated 156 critically ill patients who were admitted to icus in israel with laboratory-confirmed sars-cov-2 from 10 march to 5 may 2020. while the pandemic has been subsiding in some parts of the world, there is still a stable plateau in the western world and north america. covid-19 adversely impacts health systems, mostly due to major uncertainties regarding the outcomes of this disease. these uncertainties explain the aggressive responses of policy makers that have detrimentally affected societies and economies around the globe [9] . here, we focused on critically ill patients because (1) they are at the highest risk, (2) their clinical course and management are poorly defined, and (3) they demand the most resources and care [10] . most of the recently published data were collected during or near the peak of the outbreak, and careful evaluation revealed that outcome data were available for only a minority of patients and were not available for many hospitalized patients. we focused on survival data in our analysis of critically ill covid-19 patients. the israeli health system has not reached its maximal treatment capacity (supplementary figure s1) , which has allowed us to provide the best possible care, with minimal to no resource constraints for each patient. this unique report provides a clear understanding of the course of the disease at its extreme and sheds light on its clinical course in a non-overwhelmed health system. in israel, the disease course is unique as the health system was underutilized; and most, if not all patients received best possible care with minimal resource constraints. this, might also explain the relatively low mortality compared to reports from other regions. the patient population in our cohort was similar to those reported elsewhere in the world. the majority of patients were older men, and a large proportion presented with multiple comorbidities. most of our patients were admitted with ards and respiratory failure and required respiratory support, similar to the patients described in reports from china [3] . approximately two-thirds of the patients required invasive mechanical ventilation, mostly in older patients. despite these similarities, we found a difference in outcomes. the following mortality rates have been found for covid-19 patients in the icu for whom outcome data are available (i.e., excluding patients who were still being treated in the icu at the time of the report): 61% (lombardy, italy [2] ), 78% (new york city, ny, usa [4] ), 79% (wuhan, china [11] ), 57% (seattle, wa, usa [12] ), 85% (washington state, usa [13] ), and 67% (china [3] )) the mortality rate in our series was 56% at the time of data cut-off. there are several possible reasons why our rate was lower. first, use of the health system for covid-19 in israel never reached its maximal capacity, allowing longer icu stays. the median icu length of stay in our cohort was 10 days, with an even longer stay in patients who survived (median, 15 days). in the above-mentioned studies, the median length of icu stay ranged between 4.1 and 8 days [1, 2, 12, 13] . a longer icu stay allows patients to be weaned more slowly from the ventilator and allows longer follow-up to monitor response to novel therapies that, in turn, might affect patients' outcomes and prevent relapses and readmission or subsequent death. second, the lag between the outbreak in china, italy, and spain and israel allowed israel's health system to adjust and implement some of the lessons learned in regions that had been severely impacted by the virus; these included the need for an isolation regimen, personal protective equipment, and "capsules" that allow complete separation between providers who treat sars-cov-2-infected patients and those who do not. lastly, novel and advanced therapies, such as plasma derived from patients who have recovered from covid-19 and ecmo, were readily available for these patients. ecmo was performed in six patients in our study who were younger (median age = 48 years, iqr = 39-49 years) than the mean of the cohort, and only one patient treated with ecmo died. although the differences in survival rates between patients who were and were not treated with ecmo were statistically significant, we have refrained from making a conclusive statement about the therapeutic yield of ecmo; however, we recommend its consideration in younger critically ill patients. by evaluating patient and outcome data, we were able to assess the effects of various patient and disease factors on outcome. while multiple factors were associated with death, our multivariable regression model indicated that only older age, longer length of icu stay, and the presence of sepsis were independent predictors of outcome. similar to our data, some studies have reported the presence of comorbidities, such as hypertension, in severely ill patients [2, 3, 14] . hypertension and associated therapies (e.g., angiotensin-converting enzyme and angiotensin receptor blockers) have been found to be associated with mortality [15] . the results of our multivariable analysis suggest that these factors (i.e., comorbidities and related medication used) are associated with patients' age rather than with actual outcome; thus, the mortality rate is also associated with age. we were able to include novel therapies in our analysis, some of which were considered compassionate (e.g., remdesivir). as a retrospective cohort, we suspect that our study was not designed to evaluate the efficacy of different anti-covid19 therapies and was underpowered. our data support the prospective evaluation of antiviral and antimalarial agents in critically ill patients. our data showed no difference in the outcomes of patients treated with glucocorticoids. however, the results of a recent study suggested that glucocorticoids are associated with better clinical outcomes in patients with covid-19 and ards [16] . on the basis of the results of previous studies that investigated phylogenetically similar viruses (sars-cov-1 (2003) and middle east respiratory syndrome coronavirus (mers-cov)), we hypothesize that glucocorticoid treatment was associated with a higher subsequent plasma viral load, a longer viremia duration, and worse clinical outcomes in our cohort [17] [18] [19] [20] . this hypothesis is also in agreement with the better overall outcomes in patients who were treated longer in the icu. a large proportion of patients in this series presented shock that required vasopressor support. many of these patients presented sepsis or developed secondary infection and septic shock. unlike other reports that have demonstrated no bacterial or viral coinfection, we found the presence of sepsis in some patients; this finding suggests that, similar to seasonal influenza, covid-19 is associated with bacterial coinfection due to pathogens that colonize the nasopharynx, such as staphylococcus and streptococcus, in critically ill patients [21] . this might also explain the lack of efficacy of glucocorticoid treatment that might be hindered. of note, most of our patients were treated with antibiotics for over 24 hours. we recommend the prospective evaluation of the role of antimicrobial therapy in critically ill patients. this study has several limitations. first, it was a retrospective study. as such, some variables were not available for assessment. for example, computed tomography scans were not done routinely in all cases, and in the few cases when it was performed, it was done in a single time point. these considerations precluded the utilization of computed tomography (ct) scan as a marker for disease severity or prognosis in our cohort. however, the data were collected no longer than 5 days after outcomes were achieved for each patient. second, although our data are comprehensive and complete, we could not include all of the collected variables in the regression model because of our considerably underpowered number of events. that said, considering the population size in israel (nearly 9 million citizens), the reported numbers of cases and deaths reflect the relative numbers of events in other countries affected by covid-19. third, while the post-discharge follow-up was short, the follow-up time in the hospital was considerably long compared with the course of the disease, and more data were available with regard to the reported mortality data and length of stay data reported in other studies. in this nation-based registry study of critically ill patients with covid-19 who were admitted to icus in israel, the majority of patients were 55 years and older men, and a large proportion required mechanical ventilation. the overall mortality rate was 56%; increasing age, shorter icu stay (median, 7 days versus 15 days), and the presence of sepsis were independently associated with death. we found no association between coexisting conditions and outcome. our findings also highlight the importance of novel therapies, antibiotics use, and the availability of resources such as icu beds and ventilatory support in the treatment of patients with covid-19. these data will inform quality improvement efforts and counseling of high-risk covid-19 patients. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/7/2282/s1. figure s1 : covid-19 burden in israel. a, overall number of cases (orange) and deaths (blue). logarithmic scale. b, number of available icu beds dedicated to sars-cov-2-positive patients (orange) and number of icu beds occupied by critically ill covid-19 patients. clinical features of patients infected with 2019 novel coronavirus in baseline characteristics and outcomes of 1591 patients infected with sars-cov-2 admitted to icus of the lombardy region clinical characteristics of coronavirus disease 2019 in china presenting characteristics, comorbidities, and outcomes among 5700 patients hospitalized with covid-19 in the country & technical guidance-coronavirus disease (covid-19) who clinical management of severe acute respiratory infection when novel coronavirus (ncov) infection is suspected: interim guidance jpj surviving sepsis campaign-society of critical care medicine care of the critically ill and injured during pandemics and disasters task force for mass critical care. introduction and executive summary: care of the critically ill and injured during pandemics and disasters: chest consensus statement clinical course and risk factors for mortality of adult inpatients with covid-19 in wuhan, china: a retrospective cohort study covid-19 in critically ill patients in the seattle region-case series characteristics and outcomes of 21 critically ill patients with covid-19 in washington state clinical characteristics of 138 hospitalized patients with 2019 novel coronavirus-infected pneumonia in renin-angiotensin-aldosterone system inhibitors in patients with covid-19 risk factors associated with acute respiratory distress syndrome and death in patients with coronavirus disease clinical progression and viral load in a community outbreak of coronavirus-associated sars pneumonia: a prospective study effects of early corticosteroid treatment on plasma sars-associated coronavirus rna concentrations in adult patients corticosteroids as adjunctive therapy in the treatment of influenza corticosteroid therapy for critically ill patients with middle east respiratory syndrome bacterial coinfection in influenza we thank ann sutton of the department of scientific publications at the university of texas md anderson cancer center for editing the manuscript. the authors declare no conflicts of interest. key: cord-300991-ipy24zxp authors: khan, amira sayed; hichami, aziz; khan, naim akhtar title: obesity and covid-19: oro-naso-sensory perception date: 2020-07-08 journal: j clin med doi: 10.3390/jcm9072158 sha: doc_id: 300991 cord_uid: ipy24zxp through a recent upsurge of severe acute respiratory syndrome coronavirus-2 (sars-cov-2) pandemic, the clinical assessment of most of the coronavirus disease 19 (covid-19) patients clearly presents a health condition with the loss of oro-naso-sensory (ons) perception, responsible for the detection of flavor and savor. these changes include anosmia and dysgeusia. in some cases, these clinical manifestations appear even before the general flu-like symptoms, e.g., sore throat, thoracic oppression and fever. there is no direct report available on the loss of these chemical senses in obese covid-19 patients. interestingly, obesity has been shown to be associated with low ons cues. these alterations in obese subjects are due to obesity-induced altered expression of olfacto-taste receptors. besides, obesity may further aggravate the sars-cov-2 infection, as this pathology is associated with a high degree of inflammation/immunosuppression and reduced protection against viral infections. hence, obesity represents a great risk factor for sars-cov-2 infection, as it may hide the viral-associated altered ons symptoms, thus leading to a high mortality rate in these subjects. in the month of december 2019, there was an uprising of pneumonia, marked with respiratory distress, among the residents of wuhan district, located in the north-east of china [1] . the virus responsible for this health disaster was identified as severe acute respiratory syndrome coronavirus-2 (sars-cov-2) which belonged to the single-stranded enveloped rna viruses, and the disease was termed as coronavirus disease 2019 (covid-19) [2] . it is surprising that in the beginning of the pandemic, most of the covid-19 patients in wuhan (china) had some primary health problems, including obesity [1] . a recent cohort, conducted in 12 hospitals of the new york state on covid-19 patients, has proposed that there were 41% obese patients, admitted between march 1, 2020 and april 4, 2020 [3] . the incidence of obesity is increasing steadily in all the corners of the world, with 650 million clinically ill subjects requiring either a surgical or medical treatment [4] . the management of obesity has become a challenging task because this pathology is a favorable ground for several chronic diseases, including cardiovascular complications, type-2 diabetes mellitus, cancer, atherosclerosis, arthrosis and renal dysfunction, and respiratory tract infections (rti) in virus-affected patients [5] [6] [7] . the rti are the main physiological targets in covid-19 illness [1] . we would like to recall that during 2009 influenza pandemic, obesity was associated with reduced pulmonary immune defenses against the virus [8] . indeed, obese subjects were not only more prone to infection with the influenza (h1n1) the virus [8] . indeed, obese subjects were not only more prone to infection with the influenza (h1n1) virus, but also developed post-infection severity of illness [9] . an increase in adiposity has been shown to alter the integrity of respiratory epithelium, which might lead to dysfunctional airway fluxes [10] . due to high weight load with excessive pressure on belly and thorax, obesity will contribute to reduced pulmonary gas exchange capacities, such as forced expiratory volume (fev) and forced vital capacity (fvc) . the experiments conducted on mice have suggested that obesity is associated with high lung permeability [11] . epidemiological data confirm that there is an increased rate of pneumonia and rti in covid-19 obese patients [12] . in fact, the first report on rti in obese subjects was published by a french team wherein 47% of covid-19 patients were found to be obese with a high degree (nearly 90%) of artificial ventilation [13] . the marked inflammation leading to immunosuppression in obesity seems to favor viral infections [14] [15] [16] . sheridan et al. [17] observed that high body mass index (bmi) was associated with a high decline in influenza antibody titers and decreased cd8 + t-cell activation after 12 months postvaccination. as far as sars-cov-2 infection is concerned, tan et al. [18] assessed immunological alterations in covid-19 patients, wherein they noted an overall decline in cd4 + t-cells, cd8 + t-cells, b cells and natural killer (nk) cells. moreover, the number of immunosuppressive t-regulatory, treg (cd4 + cd25 + foxp3 + ) cells and concentrations of il-6, il-10, and c-reactive protein (crp) were upregulated in patients with severe covid-19 [18] , suggesting that sars-cov-2 infection may lead to "over-immunosuppression" in the case of obesity ( figure 1 ). the figure shows the immunosuppression in obese subjects. the adipose tissue of the obese is highly inflamed and, consequently, releases a number of cytokines, particularly il-6 and tnf-α. whose secretion is further potentiated by leptin. the lipopolysaccharide (lps)-triggered endotoxemia further aggravates inflammatory condition by inducing the release of il-6 and tnf-α from macrophages via tlr4 activation. obesity is also marked with high production of il-10, which decreases the function of dendritic cells. the prolonged inflammation will lead to immunosuppression that may favor the viral infection. severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has also been shown to induce immunosuppression. once installed, sars-cov-2 will aggravate the obesity-induced lung dysfunctions. (+) and (−) show, respectively, stimulatory and inhibitory actions. since dendritic cells (dcs) are the key players in the regulation of th1/th2 dichotomy and t-cell tolerance, their importance to trigger an anti-viral response has been considered primordial [19] . o'shea et al. [20] have demonstrated that obesity impacts the functions of these cells to trigger appropriate t-cell responses. this interesting report further showed that not only the number of circulating dcs were significantly lower in obese participants than lean subjects, but also in vitro activated-dcs from obese participants expressed less cd83 (a dcs maturation marker) and also produced, in high quantities, the il-10, an immunosuppressive cytokine [21] . the il-10, in turn, has the adipose tissue of the obese is highly inflamed and, consequently, releases a number of cytokines, particularly il-6 and tnf-α. whose secretion is further potentiated by leptin. the lipopolysaccharide (lps)-triggered endotoxemia further aggravates inflammatory condition by inducing the release of il-6 and tnf-α from macrophages via tlr4 activation. obesity is also marked with high production of il-10, which decreases the function of dendritic cells. the prolonged inflammation will lead to immunosuppression that may favor the viral infection. severe acute respiratory syndrome coronavirus-2 (sars-cov-2) has also been shown to induce immunosuppression. once installed, sars-cov-2 will aggravate the obesity-induced lung dysfunctions. (+) and (−) show, respectively, stimulatory and inhibitory actions. since dendritic cells (dcs) are the key players in the regulation of th1/th2 dichotomy and t-cell tolerance, their importance to trigger an anti-viral response has been considered primordial [19] . o'shea et al. [20] have demonstrated that obesity impacts the functions of these cells to trigger appropriate t-cell responses. this interesting report further showed that not only the number of circulating dcs were significantly lower in obese participants than lean subjects, but also in vitro activated-dcs from obese participants expressed less cd83 (a dcs maturation marker) and also produced, in high quantities, the il-10, an immunosuppressive cytokine [21] . the il-10, in turn, has been shown to inhibit the ability of dcs to stimulate cd4 + t-cells and to downregulate mhc-ii, cd86 (a co-stimulatory signal protein), and antigen presentation to cd4 + t-cells [21] . obesity is also marked with high concentrations of leptin, which is also known to trigger the production of il-6 and tnf-α from adipose tissues ( figure 1 ) and to increase the risk for viral infection. indeed, tnf-α administration in mice favors the induction of an experimental autoimmune disease [22] . the adipose tissue is the main source of circulating tnf-α in obesity, as its synthesis is increased by adipocytes in obese subjects and a weight-loss results in its low concentrations [23] . in obesity, leptin further decreases the secretion of adiponectin, an anti-inflammatory adipokine. in fact, the adipose tissue of obese subjects is an inflammatory "hot spot" that is also infiltrated by macrophages [24] . besides, obesity is also marked with a change in gut microbiota that leaks the entry of lipopolysaccharide (lps) into blood circulation. the lps is directly responsible for endotoxemia, so-called, "low grade inflammation", via toll-like receptor-4 (tlr-4), by inducing the production of il-1β, tnf-α and il-6 from macrophages and, at the same time, some of the adipocytes are also differentiated into "macrophage-like" cells [25] . finally, we can state that il-6 and tnf-α are the main players of inflammation in obesity ( figure 1 ). these two cytokines, along with il-1β via the nf-kb pathway, have been proposed to be the major cause of immunosuppression [26] as they induce accumulation and activation of myeloid-derived suppressor cells (mdscs) whose expansion interrupts the maturation of macrophages, dcs and granulocytes [27] . obesity is also associated with other immunosuppressive landmarks, such as low lymphocyte subset counts and their decreased polyclonal proliferation and oxidative burst activity of monocytes, increased thymic aging, and reduced t-cell repertoire diversity, which lead to increased risk for viral infections and rti both in experimental models and clinical studies [28] . luzi and radaelli [29] have proposed that there would be high viral shedding in obese subjects, thus increasing the probabilities of spreading the viral infection. it is also noteworthy that obesity, complicated by diabetes, may further aggravate the patient's health status. indeed, bello-chavolla et al. [30] have tried to establish a link between obesity and diabetic condition in sars-cov-2 infection. these investigators concluded that obesity might increase the lethality of covid-19 in diabetic subjects. diabetes, due to the deleterious role of hyperglycemia on immune responses, represents a risk factor for covid-19 infection in obesity [31, 32] . a french nationwide study, coronado (coronavirus sars-cov-2 and diabetes outcomes), has clearly shown the deleterious role of obesity in life-threatening outcomes in a large diabetic population with covid-19 [33, 34] . a perusal of above-mentioned studies clearly demonstrates that chronic inflammation, leading to immunosuppression, may contribute to decreased protection against viral infections in obese subjects [35] . it has been recently reported that a significant number of covid-19 patients suffer from a sudden loss of their senses of smell and taste, even in clinical conditions that are not marked with common viral symptoms such as fever, dry cough or thoracic oppression [36, 37] . a large number of covid-19 patients (from 60% to 80%) from iran have complained of a complete loss of their sense of smell or taste [38] . a multicentric european study conducted on covid-19 patients demonstrated that nearly 87% of patients reported olfacto-gustatory dysfunctions [39] . a recent meta-analysis on covid-19 patients, incorporating 10 research articles from 7 countries, has reported that nearly 52% and 43% of them had, respectively, gustatory and olfactory dysfunctions [40] . in france, gautier and ravussin [41] reported that there was a sudden appearance of anosmia and/or ageusia in a small number of covid-19 patients. similarly, almost two-thirds of covid-19 patients from germany also complained of anosmia [42] . in the usa, a survey was performed in the month of april 2020 on covid-19 patients, and 37.7% of participants complained of altered smell and taste perception [43] . interestingly, the changes in gusto-olfactory perception in covid-19 patients were more prevalent in home-quarantined subjects, independently of age and gender [44] . it is important to mention that sars-cov-2 does not generate clinically significant nasal congestion or rhinorrhea as seen in general nasal infections [45] [46] [47] [48] . does sars-cov-2 infect taste buds or nasal mucosal epithelia? a recent report, conducted in mice, has demonstrated that mouse sustentacular cells, involved in the transfer of odorant messages to olfactory neurons, express angiotensin converting enzyme 2 (ace2), which is a port of entry of sars-cov-2 ( figure 2 ) [49] . j. clin. med. 2020, 9, x for peer review 4 of 12 buds or nasal mucosal epithelia? a recent report, conducted in mice, has demonstrated that mouse sustentacular cells, involved in the transfer of odorant messages to olfactory neurons, express angiotensin converting enzyme 2 (ace2), which is a port of entry of sars-cov-2 ( figure 2 ) [49] . the duration and intensity of sars-cov-2-induced inflammation will also depend on pre-existing inflammation (like in obesity) and genetic or epigenetic backgrounds of the subjects. for simplification, we do not show the structure of the tongue papillae. we show a taste bud that is the unit of lingual gustatory papillae. during viral-induced inflammation, the oro-nasal epithelia will be infiltrated by macrophages that will release the pro-inflammatory cytokines such as il-6 and tnf-α that may aggravate the epithelial integrity and lead to clinical symptoms such as loss of oro-naso-sensory (ons) functions. beside the implication of ace2, the viral-induced generalized inflammation in covid-19 patients would also affect the integrity of the olfactory epithelium. chronic rhinosinusitis has been shown to trigger alterations in the olfactory mucosa, such as goblet cell hyperplasia, squamous metaplasia, and loss of supporting cells and olfactory neurons, associated with infiltration of proinflammatory immune cells [50] . we propose that sars-cov-2 might affect the integrity or regeneration/renewal of the olfactory epithelium, impacting the physiological function of olfactory sensory neurons (figure 2 ). hence, we can cite the example of sendai virus which has been shown to impair olfaction by reducing the regeneration of the olfactory epithelium and olfactory bulb in the mouse [51] . in in vitro experiments on murine olfactory neurons infected with this virus, the number of odorant-responsive cells were decreased. by using a plausible transgenic mouse model, lane et al. [52] have demonstrated that the induction of tnf-α expression triggered inflammation in the olfactory epithelium and the reversal of tnf-α expression restored the olfactory function in these animals, demonstrating that inflammation is an important factor involved in the loss of olfactory the duration and intensity of sars-cov-2-induced inflammation will also depend on pre-existing inflammation (like in obesity) and genetic or epigenetic backgrounds of the subjects. for simplification, we do not show the structure of the tongue papillae. we show a taste bud that is the unit of lingual gustatory papillae. during viral-induced inflammation, the oro-nasal epithelia will be infiltrated by macrophages that will release the pro-inflammatory cytokines such as il-6 and tnf-α that may aggravate the epithelial integrity and lead to clinical symptoms such as loss of oro-naso-sensory (ons) functions. beside the implication of ace2, the viral-induced generalized inflammation in covid-19 patients would also affect the integrity of the olfactory epithelium. chronic rhinosinusitis has been shown to trigger alterations in the olfactory mucosa, such as goblet cell hyperplasia, squamous metaplasia, and loss of supporting cells and olfactory neurons, associated with infiltration of pro-inflammatory immune cells [50] . we propose that sars-cov-2 might affect the integrity or regeneration/renewal of the olfactory epithelium, impacting the physiological function of olfactory sensory neurons ( figure 2 ). hence, we can cite the example of sendai virus which has been shown to impair olfaction by reducing the regeneration of the olfactory epithelium and olfactory bulb in the mouse [51] . in in vitro experiments on murine olfactory neurons infected with this virus, the number of odorant-responsive cells were decreased. by using a plausible transgenic mouse model, lane et al. [52] have demonstrated that the induction of tnf-α expression triggered inflammation in the olfactory epithelium and the reversal of tnf-α expression restored the olfactory function in these animals, demonstrating that inflammation is an important factor involved in the loss of olfactory sensory neurons and olfaction sensitivity. the olfactory mucosa is very sensitive to macrophage-secreted inflammatory cytokines, such as macrophage inflammatory protein-1α (mip-1a) and monocyte chemoattractant protein-1 (mcp-1), that may influence the renewal/regeneration of nasal epithelial cells [53] . as regards taste dysfunction, ace2 was highly expressed by tongue epithelial cells, but to a lesser extent by buccal and other tissues of the mouth cavity [54] . these observations suggest that the tongue is equipped with a sars-cov-2 entry route, but we do not know whether taste papillae and taste bud cells (tbcs) express the ace2 receptor. we would like to introduce toll-like receptors (tlrs) that act as receptors for viral rna, and are abundantly expressed on taste bud cells, particularly on type ii and type iii cells [55] . the activation of tlrs by the administration of exogenous ifn-γ led to inflammation in taste bud cells and, consequently, to cell death. the autoimmune pathologies in humans or experimental rodent models have clearly demonstrated that inflammation, associated with infiltration by il-6 and ifn-γ in gustatory epithelium, impacts taste perception [56] [57] [58] . moreover, administration of exogenous ifn-γ, via stat-1 signaling, induced apoptosis of taste bud cells [59] . these observations strongly support that oral taste papillae inflammation may contribute to low oro-sensory perception of sapid molecules. beside the peripheral mechanism, different brain areas might be involved in the loss of taste and smell in covid-19. there are several reports indicating that covid-19 patients also suffer from neurological complications, such as skeletal muscle injury, delirium and acute cerebrovascular disease [47] . chigr et al. [60] have proposed that this virus might accede to the olfactory cortex either by the nasopharyngeal cavity or directly by hematogenous spread. there is no direct report on the entry of sars-cov-2 into the brainstem; however, clinical features such as vomiting, nausea and loss of appetite suggest a perturbation in the dorsal vagal complex (dvc), which belongs to the medulla oblongata, the lowest region of the brainstem that controls several physiological functions, including food intake. in the dvc, the nucleus of tractus solitaris (nts) is known to regulate food intake, not only via the vagus nerve that connects the gut, but also via chorda tympani and glossopharyngeal nerves that connect directly to the gustatory taste papillae in the tongue [61] . ralli et al. [62] have proposed that sars-cov-2 could infect the olfactory receptors in the nasal epithelium, through which it may travel to the olfactory bulb and certain brain structures, such as the medulla oblongata. this hypothesis was based on the observations in animal experiments wherein intranasal administration of sars-cov, a strain similar to sars-cov-2, could enter the brain via the olfactory nerves and spread to the thalamus and brainstem [48] . sars-cov-2, in analogy to sars-cov34 and mers-cov13 infection in transgenic mice, might attain the brainstem [63] . indeed, using the murine model of hcov infection, it was shown that sars and oc43 were able to enter the olfactory bulb via the nasal route and reach the central nervous system (cns) [64] . moreover, ct scans and mri of covid-19 patients demonstrated "bilateral inflammatory obstruction of the olfactory clefts" [65] . though we do not have experimental animal data on sars-cov-2 entry, we can state that sars-cov-2 may enter the cns, using the olfactory pathway [63] , and exert its action via ace2 that has been detected in the central nervous system [66] . the question arises whether the loss of ons perception can be considered as an early marker of sars-cov-2 infection. we should be very cautious in this regard, as the methods that have been used for the assessment of ons defects are self-reported examinations. generally, the investigators employ either a 3-armed forced choice (3-afc) test or a comparison with 6-n-propylthiouracil (prop) tasting with and without sodium chloride for oral chemosensory perception, and for the detection of olfactory thresholds, rose smell and n-butanol are employed. by using these techniques, one can be sure about the decrease (or increase) in taste detection thresholds. however, in none of the reports on covid-19 patients, such tests were employed. why do all the covid-19 subjects not complain of the loss of smell? is there any genetic or epigenetic predisposition? before going into detail, we would like to emphasize that a reduced oro-sensory perception would trigger high consumption of palatable food, thus either leading to obesity or worsening this pathology [67, 68] , though we should not ignore the implication of the food addiction component, particularly for sweet food and those rich in fat [69, 70] . the studies conducted on healthy and obese participants suggested that the latter group exhibited lower sensitivity than the former for sweet and sour taste [71] . diet-induced obesity, by maintaining mice on a high-fat diet for ten weeks, resulted in low taste bud cell number and taste-evoked calcium signaling in obese mice [72] . similar observations have been reported for bitter and salt tastes in obese subjects [73] . as regards fat taste perception, there was a decreased perception of dietary fatty acids in obese rodents and human beings [61, 74, 75] . the decrease in taste sensitivity to different taste qualities might be due to partially functional taste receptors/sensors, caused by obesity-induced downregulation [75] , genetic polymorphism [75] [76] [77] [78] or epigenetic signatures [79] . the olfaction is not only important for the detection of sense of smell, but also to appreciate the palatability of a hedonic food, as the retro-nasal detection of flavors is brought about by nasal sensory epithelial cells [80, 81] . as regards the olfactory cue, there was a significant influence of bmi on olfactory thresholds, which were increased with increasing body weight in obese subjects [82, 83] . patel et al. [84] reported that high bmi was associated with subjective olfactory dysfunction in obese patients. by employing the olfactory threshold-discrimination-identification (tdi) test, pastor et al. [85] observed that olfactory discrimination power was lesser in obese subjects than control participants. like taste modalities, the genetic polymorphism of olfactory receptor genes [85, 86] or their hypermethylation [87] , also contributes to obesity. the decreased smell perception in obesity is a multicomponent phenomenon that involves not only nasal epithelial receptor activation, but also different brain areas, such as the limbic system, thalamus and piriform cortex, as well as amygdala, which project to the orbitofrontal cortex [88] . beside the afore-mentioned factors that bring about a decrease in ons, we should not forget to mention the role of cytokine-induced (generalized or tongue-specific) inflammation in obesity. the mouse taste bud cells have been shown to produce both tnf-α and il-10 in the microenvironment of taste papillae [89, 90] . in a plausible study, kaufman et al. [74] showed that an increase in tnf-α in the tongue of obese mice was associated with a significant reduction in taste bud and taste progenitor cells in tongue papillae. moreover, tnf-α null mice were protected from obesity-induced reduced number of taste bud cells, and administration of exogenous tnf-α brought back taste buds to degeneration [91] . the adipose-specific deletion of sel1l in mice maintained on a high-fat diet resulted in reduced adiposity and showed neither an increase in tnf-α concentrations nor any sign of taste bud cell atrophy. these observations clearly indicate that tnf-α released from hyperplasic/inflamed adipose tissue in obesity may trigger a loss in gustatory taste perception. moreover, lps-induced inflammation was also found to decrease the lifespan of mature taste bud cells [92] . as regards olfactory perception, inflammation and obesity, a link between apoptosis and inflammation has been recently reported in the olfactory mucosa of obese mice fed with a moderate high-fat diet, where a significant increase in activated caspase-3 was associated with a marked loss of olfactory sensory neurons and their axonal projections, paralleled with an increased expression of iba-1, suggesting an increase in proinflammatory cells [92] . hence, if the diet-induced obese mice are re-fed a normal diet and return to normal weight, the loss in olfactory perception is also reinstated. in vitro, tnf-α has been shown to induce cell death in olfactory epithelial explants [93] . in transgenic mice, the expression of tnf-α resulted in the loss of olfactory neurons and odor perception. as regards il-6, its concentrations were found elevated in the blood of patients suffering from hyposmia [94, 95] . a perusal of above-mentions observations clearly suggests that obesity is associated with the loss of ons, and inflammation in the oro-naso epithelia plays an important role in this phenomenon. figure 2 shows that sars-cov-2 infection will install (or aggravate) an inflammatory state both in the lingual and nasal epithelia. in the lingual taste buds, the virus-induced inflammation will attenuate the gustatory perception of different taste qualities, whereas in the olfactory sensory neurons, the virus-triggered inflammation may contribute to decreased olfactory perception of odorants. it is also possible that sars-cov-2, by penetrating the olfactory bulb, may enter the brainstem and modulate ons. why do all covid-19 patients not exhibit a change in ons perception? it is possible that the alteration in oro-olfactory epithelium functions might be secondary to viral infection, which may depend on genetic (or epigenetic) and other life-style-related build-up of the patients. nonetheless, we can infer that obese subjects are at high risk for sars-cov-2 infection as they already exhibit a low ons capacity for different taste modalities. hence, the existing gustatory and olfactory sensory deficiency, due to obesity, will mask sars-cov-2-induced diminished taste and smell sensation and, thus, may aggravate the patient's health. sars-cov-2 infection may further aggravate the ons functions; mask the obesity-induced inflammation, including loss of taste and smell; and render the obese subjects more vulnerable and prone to severe pathophysiological consequences such as rti, leading to death. by now, we have observational/self-reported studies, but data regarding the duration and the time of the onset and reversal of ons symptoms in this infection are lacking. we need a complete follow-up study of these patients as a function of time on the loss of ons. as mentioned previously, we also lack the proper set-up for the detection of olfactory and taste thresholds. we still do not know whether sars-cov-2 infection alters the taste bud renewal/turn-over and taste bud physiology either upstream or downstream of the detection of sapid molecules. it is too early to predict clearly that sars-cov-2-induced changes in ons might be due to its direct or indirect deleterious effects on brain regions such as the insula, caudal orbitofrontal and anterior cingulate cortex that control the integration of both taste and smell information [96] . while we have mentioned that tongue epithelium expresses ace2 receptors [54] , we still do not know which cell type (type i, ii or iii) expresses this receptor. this information will be important to correlate the loss of a particular taste modality as type ii cells express sweet, bitter and umami receptors; type i cells express salt receptors; and type iii cells are involved in sour sensing [74, 75] . the vistas in the eating behavioral physiology with regard to sars-cov-2 infection require more detailed investigations in covid-19 patients as gustatory and olfactory receptors are also expressed in other tissues such as those in the gut, which is the main site of the release of small peptides (such as cholecystokinin and peptide-yy) that control eating behavior via the vagus nerve [97] . similarly, the olfactory bulb also expresses receptors for a number of appetite-regulating hormones and peptides such as insulin, leptin, ghrelin and orexin [98] . it is now well established that the gut microbiome of obese subjects is shifted from bacteriodetes to fermecutes, a pro-inflammatory phylum, and the effects of this change on sars-cov-2 infection susceptibility should be explored in the future. does this viral infection promote a particular microbiome in the gut and ons epithelia? a recent report has outlined that there is a significant persistent alteration in the gut microbiome in covid-19 patients [99] . can the strategies to alter the intestinal microbiota decrease the severity of sars-cov-2 infection? we think that sars-cov-2 infection is much more dangerous than what is reported now and a significant amount of clinical information remains undiscovered. this study was supported by a grant from the french national research agency (anr-11-labx-0021-lipstic). the authors 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possible roles of caspase 1 (ice), caspase 2 (ich-1), and caspase 3 (cpp32) velicu, i. interleukin 6 in hyposmia experience-dependent neural integration of taste and smell in the human brain olfactory receptors in non-chemosensory tissues olfaction under metabolic influences alterations in gut microbiota of patients with covid-19 during time of hospitalization key: cord-292341-uo54ghf3 authors: cocconcelli, elisabetta; biondini, davide; giraudo, chiara; lococo, sara; bernardinello, nicol; fichera, giulia; barbiero, giulio; castelli, gioele; cavinato, silvia; ferrari, anna; saetta, marina; cattelan, annamaria; spagnolo, paolo; balestro, elisabetta title: clinical features and chest imaging as predictors of intensity of care in patients with covid-19 date: 2020-09-16 journal: j clin med doi: 10.3390/jcm9092990 sha: doc_id: 292341 cord_uid: uo54ghf3 coronavirus disease 2019 (covid-19) has rapidly become a global pandemic with lung disease representing the main cause of morbidity and mortality. conventional chest-x ray (cxr) and ultrasound (us) are valuable instruments to assess the extent of lung involvement. we investigated the relationship between cxr scores on admission and the level of medical care required in patients with covid-19. further, we assessed the cxr-us correlation to explore the role of ultrasound in monitoring the course of covid-19 pneumonia. clinical features and cxr scores were obtained at admission and correlated with the level of intensity of care required [high(himc) versus low-intensity medical care (limc)]. in a subgroup of patients, us findings were correlated with clinical and radiographic parameters. on hospital admission, cxr global score was higher in himcs compared to limc. smoking history, po(2) on admission, cardiovascular and oncologic diseases were independent predictors of himc. the us score was positively correlated with fio(2) while the correlation with cxr global score only trended towards significance. our study identifies clinical and radiographic features that strongly correlate with higher levels of medical care. the role of lung ultrasound in this setting remains undetermined and needs to be explored in larger prospective studies. since december 2019, when the first cases of coronavirus disease 2019 (covid19) were reported, the diffusion of the severe acute respiratory syndrome coronavirus type 2 (sars-cov-2) has rapidly spread from the hubei province in china to involve up to 213 states and territories to date, reaching pandemic proportions [1] . despite epidemiological reports showing that approximately half of the infected people are asymptomatic [1] , the spectrum of respiratory manifestations may range from mild symptoms, such as dry cough, fever, and fatigue, to acute respiratory distress syndrome (ards), requiring admission to intensive care unit (icu) and mechanical ventilation (mv). in this scenario, thoracic radiology plays a key role in early detection of lung involvement from covid-19. chest computed tomography (ct) is the technique with the highest sensitivity, but the risk of contamination and the need for a dedicated hospital organization makes ct hardly available in an emergency setting. portable chest x-ray (cxr) and ultrasonography (us) are quicker, safer and less expensive alternatives [2] . cxr is recommended as first level assessment by several scientific societies (american college of radiology, society of thoracic radiology) in the context of the sars-cov-2 pandemic [3] . predominant cxr features in patients with covid-19 include lower lobe, peripheral, bilateral ground glass opacities (ggo) or consolidations [2] , similar to other forms of viral pneumonias, such as the h1n1 strain [4] . yet, cxr could be normal in as many as 31% cases, peaking its sensibility in patients with advanced disease [5] [6] [7] . in the last three decades, lung us (lus) has become increasingly important in clinical practice, particularly in the assessment of patients with pneumonia, with sensitivity and specificity of 92% and 93%, respectively, especially when performed by experienced operators [8] . in the covid-19 pandemic, lus has been used in multiple centers as first radiological approach in patients with suspected pneumonia. the main ultrasound findings include multiple b-lines (separated or coalescent), peripheral consolidations and thickened pleural lines [9] , which however are nonspecific and found in a number of infectious and non-infectious diseases [10] . the use of lus and cxr in combination has the potential to facilitate the identifications of ards [11] . with this background, we investigated the relationship between cxr severity score on admission and the level of medical care required in patients with covid-19. further, we assessed the radiographic-ultrasound correlation with the aim to explore the value of ultrasound in monitoring the course of covid-19 pneumonia. in this longitudinal retrospective study, we identified a cohort of clinically well-characterized patients with sars-cov-2 infection referred to the university hospital of padova (division of infectious and tropical diseases, respiratory disease unit and intensive care unit) between march and may 2020. one hundred and two patients were included in the study (table 1 ) since the diagnosis of sars-cov-2 infection was made based on nasopharyngeal swab positivity. clinical and demographics data, and cxrs were obtained on admission. a subset of 25 patients (25/102, 24.5%), who were hospitalized in a low-intensity care setting, underwent a bedside lus and a cxr in the late phase of covid-19 pneumonia. the aim of performing lus and cxr in parallel was to explore the relation between these two procedures. the need for invasive/non-invasive ventilation or high-flow nasal cannula (hfnc), which required admission to icu or to the respiratory icu, was considered as high-intensity medical care (himc), while the need for low flow oxygen supplementation through nasal cannula or face mask, which required the setting of a general ward, was considered as low-intensity medical care (limc). the level of care could change over time based on patient's clinical conditions. for all patients, clinical data (demographics and comorbidities), gas exchange values (fio 2 , po 2 and po 2 /fio 2 ) were collected on admission (table 1) . we have categorized the five most frequent type of comorbidities: cardiovascular diseases (cvd), respiratory diseases, metabolic diseases, autoimmune diseases and oncologic diseases. among the metabolic comorbidities, we have considered diabetes mellitus, obesity and dyslipidemia (13%). oncologic history mentioned the different organs affected (i.e., lung, prostate, pancreas. breast, colon). this was a retrospective study on anonymized patient's data collected from electronic medical records. the study protocol complies to the ethical guidelines of the 1975 declaration of helsinki and, in agreement with national regulation on retrospective observational studies, it was notified and approved by the local ethics committee (n • 46430/03.08.2020) and the need for patient's informed consent was waived. we retrieved data on patients hospitalized for covid-19 between march and may 2020 at the university hospital of padova, one of the most affected areas in north-east of italy. we screened records of all patients admitted to our hospital with a diagnosis of sars-cov2 infection. for each patient, a single image plane cxr was available on hospital admission. two radiologists (c.g., g.b.) with more than ten years of experience in the thoracic field, who were blind to clinical data, scored the images independently using a semi-quantitative scale. this represented a modification of previously reported scoring systems that allowed to evaluate the extension of ground glass opacities (ggo) and consolidation (co) [6, 12, 13] . for each lung lobe, the two radiologists assessed the extent of ggo and co using the following scale: 0 (normal), 1 (up to 30% of the lobe involved), 2 (30% to 60% of the lobe involved), and 3 (more than 60% of the lobe involved). the sum of the scores for each lung lobe and a final value of ggo and co score for each patient was then calculated ( table 2 ). the cxr "global" score was calculated as the sum of the ggo and co scores of each patient, with a maximum score of 36. finally, each patient was classified as "normal", "ggo prevalent", "co prevalent", or "mixed" based on the prevalent cxr pattern [14] . table 2 . baseline radiological scores of the overall population hospitalized for sars-cov-2 related infection, and of the two subgroups categorized in low (limc) and high (himc) intensity medical care. a subset of 25 patients underwent bed-side lus. the examination was conducted with a portable mylab tm 25 gold ultrasound unit (esaote, genova, italy) and a dedicated ca631 convex transducer (range of frequency 1-8 mhz). we used low frequency and a single-focal modality at the pleural line. the depth was arranged on 6-7 cm and the harmonic-imaging system was deactivated. the lus score was calculated across 12 chest zones (six on each hemithorax) using a scale from 0 (normal pattern, a-lines or non-significant b-lines), 1 (significant b-lines ≥3 per rib space), and 2 (coalescent b-lines with or without small consolidations) to 3 (consolidation), as previously reported [15] . a final "us global score" was calculated for each patient with a maximum score of 36. categorical variables were described as absolute (n) and relative values (%), whereas continuous variables were described as median and range. to compare demographic data and baseline clinical characteristics between limc and himc groups, chi square test and fisher's exact test for categorical variables and mann-whitney u test for continuous variables were used, as appropriate. the correlation between cxr global score and po 2 , fio 2 , p/f ratio on admission was assessed for the entire study population and in the limc and himc groups using the nonparametric spearman's rank method. univariate logistic regression analysis, followed by a multivariate logistic regression, was performed to detect the strongest predictors of level of care. the covariates included in the final model were those that were significant in the univariate regression analyses. the correlation between lus global score and the corresponding cxr global score and fio 2 was calculated using the nonparametric spearman's rank method. all data were analyzed using spss software version 25.0 (us: ibm corp., new york, ny, usa). p-values < 0.05 were considered statistically significant. the graphs were obtained using the statistical package graphpad prism 7.0 (graphpad software, inc., la jolla, san diego, ca, usa). demographic and clinical characteristics at baseline (i.e., on hospital admission) are summarized in table 1 . most patients were male (73%) with a median age on admission of 68 years. seventy-one patients required limc during hospitalization and thirty-one himc. patients requiring himc (himcs) were mainly male (87 vs. 67%; p = 0.05) and older [74 vs. 63 (22-94) years; p = 0.03], with a higher body mass index (bmi) [31 (21-43) vs. 24 (16-31) kg/m 2 ; p = 0.02]. moreover, they had a heavier smoking history (10 (0-60) vs. 0 (0-60) pack/year (py); p = 0.01) and were mainly former smokers (61%). the most common presenting symptoms were fever (92%), cough (61%) and shortness of breath (34%), and with 5% of patients complaining of impaired sensory. the frequency of these symptoms did not differ between himcs and limcs. interestingly, although the time interval between onset of respiratory symptoms and admission to the emergency unit was similar, himcs showed a greater impairment of respiratory gas exchange with a lower po 2 on room air on admission (60 (21-90) vs. 90 (54-119) mmhg; p < 0.0001), greater fio 2 requirement at the time of admission (39 (21-100) vs. 21 (21-51) %; p < 0.001) and worse p/f (158 (33-429) vs. 429 (106-567); p < 0.0001) compared to limcs. in the overall population, cvds were the most frequent comorbidities (55%) that we observed. among the metabolic comorbidities, diabetes mellitus was the most prevalent (26%), followed by obesity (8%) and dyslipidemia (13%). hypothyroidism was the most frequent condition among the autoimmune diseases (3%). oncologic diseases (13%) were equally distributed among organs affected (i.e., lung, prostate, pancreas. breast, colon). himcs reported more comorbidities, in particular cardiovascular diseases (cvds) (80 vs. 49% of cases; p = 0.002), metabolic diseases (61 vs. 37%; p = 0.002) and oncologic diseases (22 vs. 8%; p = 0.05). furthermore, this patient group showed a higher frequency of bacterial co-infections (42 vs. 15%; p = 0.002) during hospitalization. finally, the hospitalization time was significantly longer for himcs compared to limcs [26 (7-119) vs. 8 (2-50) days; p < 0.0001], with 4 patients dying among himcs and only one among limcs (p = 0.01). on admission, himcs showed a more severe radiological impairment compared to limcs, with higher x-ray global score [8 (0-35) vs. 3 (0-22); p < 0.0001], ggo score (5 (0-15) vs. 1 (0-18); p < 0.001) and co score (0 (0-35) vs. 0 (0-10); p = 0.02), respectively. when considering the prevalent cxr pattern, only one patient among himcs had a normal cxr on admission compared to limcs (14; p = 0.003), with similar proportion of patients with "ggo prevalent" and "co prevalent" patterns in the himc and limc groups. in the overall study population, a positive correlation was observed between cxr global score and fio 2 on admission (r = 0.6, p < 0.0001). when stratified by level of care, the correlation between cxr global score and fio 2 on admission was confirmed in limcs (r = 0.51, p < 0.0001) but not in himc (figure 1a ). in the overall study population, we observed a negative correlation between cxr global score and po 2 on admission (r = −0.6, p < 0.0001). when stratified by level of care, the correlation between cxr global score and po 2 on admission was confirmed in limcs (r = −0.37; p = 0.02) but not in himcs (figure 1b) . finally, in the overall study population, we observed a negative correlation between cxr global score and p/f on admission (r = −0.6, p < 0.0001). when stratified by level of care, the correlation between cxr global score and p/f at admission was confirmed in both limcs (r = −0.40; p = 0.0003) and himcs (r =−0.37; p = 0.04) (figure 1c) . univariate logistic regression analysis of factors associated with level of care revealed that sex, age, smoking history, fio 2 , po 2 in room air at admission, bacterial co-infections developed during hospitalization, cvds, metabolic and oncologic diseases and chest x-ray global score had significant positive association with a higher level of care in the entire study population (table 3) . multivariate analysis performed using variables with statistical significance in univariate analysis revealed that smoking history (odds ratio 6.55; 95% ci: 1.15-52.09; p = 0.04), po 2 values are expressed as odds ratio (95% confidence interval). logistic regression analysis in relation to level of care was used to determine the relationship of clinical and radiological characteristics with higher level of care needed during hospitalization. a subset of 25 patients underwent a bed-side lus after a median time of 11 days from admission. in parallel, cxrs were performed in the same patients at the same time point. the median lus global score was 7 , whereas the median cxr global score was 9 (3-13). the lus global score positively correlated with the fio 2 requirement at the time of the us examination (r = 0.36; p = 0.03) ( figure 2) . conversely, the correlation between lus global score and cxr global score only trended towards statistical significance (r = 0.36, p = 0.07) ( figure 3) . finally, the lus global score positively correlated with the cxr co score (r = 0.38; p = 0.05) (figure 4) but not with the ggo score. j. clin. med. 2020, 9, x for peer review 7 of 12 univariate logistic regression analysis of factors associated with level of care revealed that sex, age, smoking history, fio2, po2 in room air at admission, bacterial co-infections developed during hospitalization, cvds, metabolic and oncologic diseases and chest x-ray global score had significant positive association with a higher level of care in the entire study population (table 3) . multivariate analysis performed using variables with statistical significance in univariate analysis revealed that smoking history (odds ratio 6.55; 95% ci: 1.15-52.09; p = 0.04), po2 (36.7, 3.64-681.4; p = 0.005), cvds (10.89, 1.44-112; p = 0.02), and oncologic diseases (17.13, 1.76-242.6; p = 0.02) were independent predictors of higher level of care in patients with sars-cov-2 infection. values are expressed as odds ratio (95% confidence interval). logistic regression analysis in relation to level of care was used to determine the relationship of clinical and radiological characteristics with higher level of care needed during hospitalization. a subset of 25 patients underwent a bed-side lus after a median time of 11 days from admission. in parallel, cxrs were performed in the same patients at the same time point. the median lus global score was 7 , whereas the median cxr global score was 9 (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) (13) . the lus global score positively correlated with the fio2 requirement at the time of the us examination (r = 0.36; p = 0.03) ( figure 2) . conversely, the correlation between lus global score and cxr global score only trended towards statistical significance (r = 0.36, p = 0.07) ( figure 3) . finally, the lus global score positively correlated with the cxr co score (r = 0.38; p = 0.05) (figure 4) but not with the ggo score. this is a retrospective analysis of clinical features and radiographic severity scores in patients with covid-19 and how these parameters on hospital admission correlate with different levels of medical care (i.e., himc vs. limc). a subgroup of patients also underwent lus, which was correlated with chest radiographs. our study revealed that patients with covid-19 who required a himc are mainly men, former smokers with a higher pack/year of smoking history, older and with a higher bmi compared to patients requiring limc. furthermore, the majority of them reported at least one comorbidity (i.e., cardiovascular, metabolic, or oncologic) and required on emergency room oxygen supplementation due to low alveolar oxygen partial pressure (pao2). moreover, using a multivariate analysis, we found that a heavier smoking history, po2 level on room air, and presence of cardiovascular or oncological disease on admission were independent predictors of the need of himc. our findings mirror those from previous studies indicating that older male patients with comorbidities are at higher risk of pulmonary infection and fatal consequences from covid-19 [16, 17] . in our study, we show that the number of pack-years was significantly higher in former smokers who required intensive care compared to those requiring limc. moreover, the proportion of former smokers was markedly increased among severe patients, whereas nonsmokers with covid-19 experienced a milder illness, which required low-flow oxygen supplementation. this is in line with other reports that explored the association between smoking and progression of covid-19 pneumonia [18] . notably, in our study, multivariate analysis revealed that smoking this is a retrospective analysis of clinical features and radiographic severity scores in patients with covid-19 and how these parameters on hospital admission correlate with different levels of medical care (i.e., himc vs. limc). a subgroup of patients also underwent lus, which was correlated with chest radiographs. our study revealed that patients with covid-19 who required a himc are mainly men, former smokers with a higher pack/year of smoking history, older and with a higher bmi compared to patients requiring limc. furthermore, the majority of them reported at least one comorbidity (i.e., cardiovascular, metabolic, or oncologic) and required on emergency room oxygen supplementation due to low alveolar oxygen partial pressure (pao2). moreover, using a multivariate analysis, we found that a heavier smoking history, po2 level on room air, and presence of cardiovascular or oncological disease on admission were independent predictors of the need of himc. our findings mirror those from previous studies indicating that older male patients with comorbidities are at higher risk of pulmonary infection and fatal consequences from covid-19 [16, 17] . in our study, we show that the number of pack-years was significantly higher in former smokers who required intensive care compared to those requiring limc. moreover, the proportion of former smokers was markedly increased among severe patients, whereas nonsmokers with covid-19 experienced a milder illness, which required low-flow oxygen supplementation. this is in line with other reports that explored the association between smoking and progression of covid-19 pneumonia [18] . notably, in our study, multivariate analysis revealed that smoking this is a retrospective analysis of clinical features and radiographic severity scores in patients with covid-19 and how these parameters on hospital admission correlate with different levels of medical care (i.e., himc vs. limc). a subgroup of patients also underwent lus, which was correlated with chest radiographs. our study revealed that patients with covid-19 who required a himc are mainly men, former smokers with a higher pack/year of smoking history, older and with a higher bmi compared to patients requiring limc. furthermore, the majority of them reported at least one comorbidity (i.e., cardiovascular, metabolic, or oncologic) and required on emergency room oxygen supplementation due to low alveolar oxygen partial pressure (pao 2 ). moreover, using a multivariate analysis, we found that a heavier smoking history, po 2 level on room air, and presence of cardiovascular or oncological disease on admission were independent predictors of the need of himc. our findings mirror those from previous studies indicating that older male patients with comorbidities are at higher risk of pulmonary infection and fatal consequences from covid-19 [16, 17] . in our study, we show that the number of pack-years was significantly higher in former smokers who required intensive care compared to those requiring limc. moreover, the proportion of former smokers was markedly increased among severe patients, whereas nonsmokers with covid-19 experienced a milder illness, which required low-flow oxygen supplementation. this is in line with other reports that explored the association between smoking and progression of covid-19 pneumonia [18] . notably, in our study, multivariate analysis revealed that smoking history was an independent risk factor for himc. we speculate that cigarette smoke upregulates the expression of angiotensin-converting enzyme 2 receptors, which in turn facilitate sars-cov-2 entry in the respiratory epithelium; this implies that smoking habit may represent a risk factor for developing severe illness even among former smokers. in other words, having quit smoking does not seem to prevent the risk of severe covid-19 pneumonia [19] . chronic respiratory disease, including, among others, chronic obstructive pulmonary disease (copd), carry a worse prognosis when associated with chronic conditions, such as cardiovascular diseases [20] [21] [22] . interestingly, in our cohort, concomitant cvds and neoplasms were independent risk factors for hospitalization in himc, with up to 80% of patients who required himc reporting an history of cvd (mainly arterial hypertension). a recent meta-analysis of 1576 patients concluded that hypertension, chronic respiratory disease and cvd are risk factors for severe covid-19 disease [23] . considering our study population, we observed that cvds are the most frequent comorbidities (55% of cases), 27% of patients suffered from diabetes mellitus, 13% showed blood tests positive for dyslipidemia, and 8% of our patients were obese. we, therefore, are in line with an italian nationwide observational study of covid-19 inpatients which reported a linear direct relationship between the number of comorbidities and the risk of death [24] . all these findings emphasize the importance of past medical history and comorbidities in the disease course of covid-19 patients, as they may predispose to worse outcome and higher intensity of care. ppo 2 level < 90 mmhg on admission to emergency room was an additional independent predictor of himc requirement. this is interesting, as the duration of symptoms (i.e., median of 4 days) did not differ between patients requiring himc and patients requiring limc. thirty-one subjects required subsequent admission to icu due to worsening of pneumonia and gas exchange. on admission, these patients displayed extensive radiological impairment in terms of both ggo score and consolidation. in the overall population gas exchange parameters correlate significantly with radiological scores but, interestingly enough, this correlation was mainly due to patients who remained in the limc group. indeed, in this group, radiological score correlated negatively with ppo 2 levels and positively with fio 2 reflecting exact correspondence between respiratory failure and radiologic impairment. conversely, among patients who subsequently required himc, cxr at baseline showed a variety of radiologic impairment, ranging from normal to highly abnormal however without a concurrent relation with gas abnormality. this result might arise attention to that patients who display discrepancies between gas exchange parameters and cxr. pevious reports on cxr findings in covid-19 patients focused on the distribution and type of lung abnormalities. wong and coauthors demonstrated that cxr at baseline has a sensitivity of 69% for a diagnosis of covid-19 pneumonia, corroborating the utility of cxr in the initial evaluation of subjects with suspected covid-19 pneumonia, thus obviating the need for ct [6] . toussie and colleagues have recently reported that initial cxr severity score is also an independent predictor of outcome in covid-19 patients [3] . we could not replicate this finding, but our study population was older than that studied by toussie et al. the prognostic role of cxr in covid-19 pneumonia therefore needs to be clarified in larger studies. lung ultrasound has been suggested as a potential diagnostic tool for covid-19 pneumonia given the predominant involvement of the lung periphery [7] ; lung ultrasound is a relatively simple technique that can be easily applied at patient bedside [25] . in our study, we investigated its role in the late phase of covid-19 pneumonia and its relation with cxr in a subgroup of patients hospitalized in a low-intensity care setting. we found a significant correlation between lus features and fio 2 level, suggesting these two parameters can be integrated into the evaluation of patients with covid-19 pneumonia. lus global score positively correlated with cxr consolidation score while the correlation with cxr global score only trended towards statistical significance. although only exploratory, these findings may anticipate further studies mainly focused on the utility of lus as a monitoring tool, possibly limiting the use of serial cxr, at least in the advanced phase of covid-19 pneumonia. in this regard, lus has been suggested as a potential substitute for cxr in the follow-up of various lung diseases in icu [26] , reducing the number of cxrs performed and relative medical costs without affecting patient outcome. of interest in a recent study by møller-sørensen and colleagues, the usefulness of bed-side lus in icu patients treated with extracorporeal membrane oxygenation (ecmo) was assessed during the covid-19 pandemic. authors used a three-zone score for each lung (anterior, posterior and lateral) with a maximum of 102 points for patient. lus score demonstrated a strong correlation with compliance during mechanical ventilation. moreover, a lower lus score advanced weaning capacity from ecmo [27] . soldati and colleagues have also suggested that lus can be useful in covid-19 pneumonia by identifying disease extension and specific patterns, as well as their evolution toward the consolidation phase [28] , thus providing further support to the role of lus in the follow-up of patients with covid-19 pneumonia. at present, however, the majority of studies performed during the covid-19 pandemic focused on ultrasonographic signs and disease patterns at presentation rather than overtime [29] [30] [31] [32] [33] . accordingly, the role of lus in monitoring the evolution of covid-19 pneumonia needs to be confirmed in larger studies. the results of our study should be interpreted in the light of important limitations. first, this is not a longitudinal study and we retrospectively collected all clinical and radiological data; therefore, the accuracy of the clinical information depends on medical records, which may introduce inaccuracies. however, every effort was made to limit this risk, even asking to the patients to fill all the missing data when possible. second, the study population was relatively small, particularly the subset of patients for whom lus data were available, although this was an exploratory analysis, and its findings should be viewed as such. clearly, these data need to be validated in larger, independent, prospectively collected populations of patients. in summary, our study identified clinical features that strongly predict the level of medical setting required by patients with covid-19 pneumonia (himc or limc). these findings allow the identification of patients at risk for severe disease and worse outcome already on hospital admission. the correlation of lus with clinical parameters and radiological score provides the basis for future studies on the utility of lus in the follow-up of patients with covid-19 pneumonia. conflicts of interest: p.s. has received personal fees and non-financial support from roche, boehringer-ingelheim, zambon, and ppm services. e.b. has received personal fees from roche and boehringer-ingelheim. m.s. has received research grants for the department (not personal) to her institution from takeda ltd., chiesi farmaceutici and laboratori guidotti spa. these funds were not used to support this project. coronavirus suppression of a sars-cov-2 outbreak in the italian municipality of vo' review of chest radiograph findings of covid-19 pneumonia and suggested reporting language clinical and chest radiography features determine patient outcomes in young and middle age adults with covid-19 presenting 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study (italico). front. cardiovasc the use of lung ultrasound images for the differential diagnosis of pulmonary and cardiac interstitial pathology could the use of bedside lung ultrasound reduce the number of chest x-rays in the intensive care unit? cardiovasc covid-19 assessment with bedside lung ultrasound in a population of intensive care patients treated with mechanical ventilation and ecmo is there a role for lung ultrasound during the covid -19 pandemic? sonographic signs and patterns of covid-19 pneumonia a preliminary study on the ultrasonic manifestations of peripulmonary lesions of non-critical novel coronavirus pneumonia (covid-19) diagnostic accuracy of lung ultrasonography combined with procalcitonin for the diagnosis of pneumonia: a pilot study can lung us help critical care clinicians in the early diagnosis of novel coronavirus (covid-19) pneumonia? findings of lung ultrasonography of novel corona virus pneumonia during the 2019-2020 epidemic this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-326834-eeldyj2u authors: graziani, desirée; soriano, joan b; del rio-bermudez, carlos; morena, diego; díaz, teresa; castillo, maría; alonso, miguel; ancochea, julio; lumbreras, sara; izquierdo, josé luis title: characteristics and prognosis of covid-19 in patients with copd date: 2020-10-12 journal: j clin med doi: 10.3390/jcm9103259 sha: doc_id: 326834 cord_uid: eeldyj2u patients with chronic obstructive pulmonary disease (copd) have a higher prevalence of coronary ischemia and other factors that put them at risk for covid-19-related complications. we aimed to explore the impact of covid-19 in a large population-based sample of patients with copd in castilla-la mancha, spain. we analyzed clinical data in electronic health records from 1 january to 10 may 2020 by using natural language processing through the savana manager(®) clinical platform. out of 31,633 copd patients, 793 had a diagnosis of covid-19. the proportion of patients with covid-19 in the copd population (2.51%; 95% ci 2.33–2.68) was significantly higher than in the general population aged >40 years (1.16%; 95% ci 1.14–1.18); p < 0.001. compared with copd-free individuals, copd patients with covid-19 showed significantly poorer disease prognosis, as evaluated by hospitalizations (31.1% vs. 39.8%: or 1.57; 95% ci 1.14–1.18) and mortality (3.4% vs. 9.3%: or 2.93; 95% ci 2.27–3.79). patients with copd and covid-19 were significantly older (75 vs. 66 years), predominantly male (83% vs. 17%), smoked more frequently, and had more comorbidities than their non-copd counterparts. pneumonia was the most common diagnosis among copd patients hospitalized due to covid-19 (59%); 19% of patients showed pulmonary infiltrates suggestive of pneumonia and heart failure. mortality in copd patients with covid-19 was associated with older age and prevalence of heart failure (p < 0.05). copd patients with covid-19 showed higher rates of hospitalization and mortality, mainly associated with pneumonia. this clinical profile is different from exacerbations caused by other respiratory viruses in the winter season. chronic obstructive pulmonary disease (copd) is one of the most prevalent chronic diseases, one of the main diagnoses in hospital admissions (especially during winter), and the fourth leading cause of death worldwide. importantly, one of the main factors underlying the negative impact of the disease in patients and health systems is copd exacerbation [1] . in turn, these exacerbations are primarily caused by respiratory viral infections (especially during epidemic periods), which have a direct effect on the symptomatology and favor bacterial superinfections [2, 3] . copd exacerbations worsen the prognosis of the disease by increasing mortality when associated with hospitalizations [4] . caused by coronavirus-2 (sars-cov-2), the clinical manifestation of covid-19 varies from mild to very severe symptoms and can lead to death in some patients [5, 6] . since the onset of the covid-19 pandemic, the severity of the disease has been associated with pre-existing comorbidities, namely cardiovascular diseases, diabetes mellitus, and hypertension. in contrast with the reported burden of influenza epidemics in copd patients, the impact of covid-19 in these patients seems to have been less evident; however, copd patients usually present a fragile situation and a lower respiratory functional reserve [7, 8] . covid-19 severity and mortality have also been associated with patient's age. although the virus can infect individuals of all ages, the majority of severe cases to date have been described in people older than 55 years and with significant comorbidities [9, 10] . the prevalence of copd also increases markedly with age, with most diagnoses occurring in patients aged 40 and older. indeed, patients over 40 years old frequently present with more than one chronic disease, especially in the endocrine-metabolic and cardiovascular spheres. several observational and case-control studies have confirmed a higher prevalence of cardiovascular diseases in copd patients than in the general population, possibly due to the coexistence of common risk factors or an associated pathogenic mechanism [11] . although there are large discrepancies in the studies that have evaluated the relationship between copd and cardiovascular disease, copd patients undoubtedly have a higher prevalence of coronary ischemia and other risk factors that may worsen the prognosis of covid-19 [12] . based on the above, it is crucial to characterize the evolution of sars-cov-2 infection in copd patients and identify the impact of copd and associated comorbidities in the patient's evolutionary course. the combination of real-world data (rwd) with big data analytics and artificial intelligence has the potential to increase our understanding of covid-19 in a timely manner. using such methods, this study aims to reuse the clinical information contained in the electronic health records (ehrs) of the population with copd and covid-19 to (a) describe the clinical characteristics of patients with copd and covid-19 and (b) to assess the influence of copd and related comorbidities and treatments in the prognosis of covid-19. this was a multicenter, non-interventional, retrospective study using free text data captured in the ehrs of patients diagnosed with copd and covid-19. the study period was 1 january-10 may 2020. we followed the strengthening the reporting of observational studies in epidemiology (strobe) guidelines for reporting observational studies [13] . clinical data from a total of 1,164,283 patients with available ehrs throughout the community of castilla la-mancha (spain) were collected from all available departments, including inpatient, outpatient, emergency room, and primary care. natural language processing (nlp) and artificial intelligence (ai) techniques were used to extract and analyze the information in ehrs. the software used (savana manager ® ) is a powerful multilingual, free-text analysis engine capable of interpreting the content in clinical records, regardless of the system in which they operate. the software can capture numerical values and physician's notes and translate them into usable variables, thus allowing the reuse of information included in large-scale collections of clinical records; therefore, the processed free-text information captured in ehrs is treated as big data. the methodology used to generate the study database has four distinct phases for data extraction and aggregation, namely (a) acquisition: the acquisition of data is the responsibility of the healthcare center, in close collaboration with the staff of savana information technology. following the general data protection regulation (gdpr) of the european union, data are extracted, anonymized, and transferred to savana; (b) integration: in this phase, data are integrated into the database; (c) nlp processing: using the ehread ® technology developed by savana, nlp techniques are implemented to analyze and extract the unstructured, free-text information written in millions of ehrs. the output of nlp processing is a synthetic patient database, as the software creates a patient database from scratch. this ensures that this information is protected and makes traceability to individual patients impossible; (d) validation: this process consists of a medical validation carried out by doctors and researchers. the terminology used by savana is based on multiple sources, such as snomed ct [14] . this terminology includes codes, concepts, synonyms, and definitions used in clinical documentation. it also includes symptoms, diagnoses, body structures, and substances. due to the novel methodological approach of this study, we complemented our clinical findings with an evaluation of savana's performance. this evaluation aims to verify the precision of the system by identifying records that contain mentions of copd, covid-19, and its related variables. the results of the annotations were used to generate the gold standard and to calculate savana's performance. the performance of the system is calculated in terms of the standard metrics for precision (p), recall (r), and its harmonic mean f-score [15] : precision = tp/(tp + fp). this parameter gives us an indicator of the precision of the information that the system retrieves. recall = tp/(tp + fn). this parameter gives us an indicator of the amount of information that the system retrieves. f-score = 2 × precision × recall/(precision + recall). this parameter provides us with a general performance indicator for information retrieval. in all cases, tp is the number of true positives (i.e., records retrieved successfully), fn is the number of false negatives (i.e., records incorrectly not retrieved), and fp is the number of false positives (i.e., records recovered incorrectly). the search terms for copd and covid-19 have been previously described [16, 17] . for the linguistic evaluation of the variable "copd", we obtained precision, recall, and f-scores of 0.926, 0.895, and 0.912, respectively; these metrics indicate that patients with copd were properly identified within the target population. ehread identified covid-19 with a precision of 0.99, a recall of 0.75, and an f-score of 0.93; again, these results indicate that within our population with copd, covid-19 cases were accurately identified. for all statistical analyses, spss software (v 25.0) was used. categorical variables are reported as absolute frequencies and percentages, while continuous variables are presented using mean and standard deviation. for the assessment of statistical significance of numerical variables, we used t-tests for independent samples or anovas. to measure the relative distribution of patients assigned to different categories of qualitative variables, we used chi 2 tests. in all cases, a p value for statistical significance was set at 0.05. the study was compliant with legal and regulatory requirements and the research practices described in the ich guide to good clinical practice, the declaration of helsinki in its latest edition, good pharmacoepidemiology practices, and local regulations. since this is a retrospective and observational study using anonymous patient data, informed consent does not apply to the present study. all actions were taken following the code of good data protection practices for big data projects of the european data protection authority and the european gdpr. the study has been approved by the ethics and research committee of the university hospital of guadalajara (spain). a total of 31,633 patients with a diagnosis of copd were attended by the health system of castilla la-mancha (spain) between 1 january 2019 and 10 may 2020. among these, 793 patients were diagnosed with covid-19. the patient flowchart is depicted in figure 1 . the percentage of patients diagnosed with covid-19 in the copd population (2.51%; 95% ci 2.33-2.68) was significantly higher than in the general population older than 40 years, (1.16%; 95% ci 1.14-1.18); p < 0.001. covid-19 diagnosis was confirmed by pcr in 335 (42%) of patients; in the remaining cases, diagnosis was based on rapid serological tests or clinical, radiological, and/or analytical evaluation, considering the reduced availability of pcr testing in the study area between march and may 2020. the demographic and clinical characteristics of covid-19 patients with and without copd are shown in table 1 . compared with copd-free patients with covid-19 older than 40 years, patients with both covid-19 and copd were older ((mean age ± sd) 75 ± 12 years vs. 66 ± 15 years (p < 0.001)) and predominantly male. furthermore, these patients showed a higher prevalence of comorbidities and a worse prognosis, as evaluated by hospitalizations (31.1 % vs. 39.8%: or 1.57; 95% ci 1.14-1.18) and mortality rate (3.4% vs. 9.3%: or 2.93; 95% ci 2.27-3.79) ( table 1) . the percentage of patients diagnosed with covid-19 in the copd population (2.51%; 95% ci 2.33-2.68) was significantly higher than in the general population older than 40 years, (1.16%; 95% ci 1.14-1.18); p < 0.001. covid-19 diagnosis was confirmed by pcr in 335 (42%) of patients; in the remaining cases, diagnosis was based on rapid serological tests or clinical, radiological, and/or analytical evaluation, considering the reduced availability of pcr testing in the study area between march and may 2020. the demographic and clinical characteristics of covid-19 patients with and without copd are shown in table 1 . compared with copd-free patients with covid-19 older than 40 years, patients with both covid-19 and copd were older ((mean age ± sd) 75 ± 12 years vs. 66 ± 15 years (p < 0.001)) and predominantly male. furthermore, these patients showed a higher prevalence of comorbidities and a worse prognosis, as evaluated by hospitalizations (31.1 % vs. 39.8%: or 1.57; 95% ci 1.14-1.18) and mortality rate (3.4% vs. 9.3%: or 2.93; 95% ci 2.27-3.79) ( table 1) . in castilla-la mancha, the covid-19 pandemic began in march 2020. at that time, there were hardly any other viral infections, including influenza. to assess the burden of covid-19 in patients with copd, we compared the clinical characteristics and outcomes of patients with copd and covid-19 during the study period with existing data from copd patients during the last two winter seasons. although patients' characteristics (including comorbidities) were similar in the two periods, covid-19 was associated with poorer prognosis in terms of hospitalization and mortality (table 2) . the main diagnosis of copd patients with covid-19 requiring hospital admission was pneumonia (59% of patients); 19% of hospitalized patients had pulmonary infiltrates, in turn suggestive of pneumonia and heart failure. in patients who died, the distribution of patients with pneumonia and heart failure were 72% and 28%, respectively, similar to non-copd patients with covid-19 who died (71% had pneumonia and 24 pulmonary infiltrates with different diagnosis, mainly heart failure or acute respiratory distress syndrome). in the copd population with covid-19, those who died were older (77 ± 11 years vs. 74 ± 11 years; p = 0.03) and had a higher incidence of heart failure than those who did not die from the disease; no other prognostic factor was identified (table 3) . table 3 . impact of comorbidities on the mortality of patients with copd. copd-covidregarding pharmacological treatment, most patients were under treatment with bronchodilators, namely beta 2 agonists and anticholinergics (table 4 ); a significantly greater use of both drugs was observed in those patients who died. on the other hand, the elevated rates of inhaled steroids use in both patient groups forbid further assessment of differences regarding the use of these agents and mortality. finally, we did not observe any differences in covid-19-related mortality rates based on use of cardiovascular drugs. table 4 . impact of treatments on the mortality of patients with copd. (1 january-10 may 2020) finally, in table 5 , our finding observed in the crude analysis of an association with death in covid-19 patients with copd is further confirmed. the increased mortality risk of covid-19 patients with copd versus those without copd is sustained in several multivariate analyses adjusted by covariates, and very consistently, sequentially with or and 95% ci of 1.70 (1.29-2.23) adjusted by sex and age (model 1); of 1.52 (1.15-2.00) when we also added the two most relevant comorbidities, that is heart failure and high blood pressure (model 2); and of 1.42 (1.07-1.88) when we added all single comorbidities in a full model (model 3). since the who declared the covid-19 outbreak a global pandemic, clinicians have aimed at determining the impact of the disease on patients with chronic diseases, especially of pulmonary and cardiovascular nature. although the frequency and severity of covid-19 has been associated with pre-existing comorbidities such as heart disease, arterial hypertension, and diabetes. surprisingly and "against all prognosis", however, healthcare data show that the incidence of covid-19 in copd patients has been relatively low [18] . this trend was already observed from the onset of the pandemic; a study that evaluated 1590 hospitalized patients with covid-19 in china revealed a low incidence in patients with copd, with a total of 24 cases [19] . however, copd was linked with a higher risk for poor disease outcome (composite endpoint including admission to an intensive care unit, invasive ventilation, or death), reflected by a hazard ratio (hr) of 2681 (95% ci 1424-5.0480), after adjusting for age and smoking. in this study, the comorbidity of copd as a risk factor was exceeded only by malignancy (hr 3.50, 95% ci 1.60 to 7.64) [19] . subsequently, a systematic review and meta-analysis showed that, although the prevalence of copd in covid-19 cases was low, sars-cov-2 infection was associated with high rates of severity and mortality in patients with copd [20] . in our study, we have confirmed that the impact of covid-19 in copd patients has been relatively limited. plausible underlying reasons for this include remission of the seasonal flu period, an absence of exposure to environmental factors due to isolation, the significant drop in contamination, and better control of the disease by complying with the treatments conscientiously "out of fear". our results, however, indicate that patients with copd are at a higher risk of sars-cov-2 infection doubling the infection rates observed in the general population over 40 years of age. this increased risk has also been described in a concise meta-analysis showing that copd is associated with a significant, five-fold increased risk of severe covid-19 infection; of note, this analysis is focused on the chinese population and there was substantial variability among the included studies [21] . although a greater risk associated with covid-19 in copd patients seems clear, it is difficult to accurately determine the extent to which copd itself or associated comorbidities affect the higher prevalence of covid-19 and its prognosis. in our study, copd patients were older and had more comorbidities. these factors could have been critical in the reported higher rates of admissions and mortality. although it is not possible to accurately assess the impact of comorbidities in copd [12] in a cross-sectional study, there is no doubt that copd patients are at higher risk for covid-19, showing a higher incidence of the disease and worse prognosis (as determined by higher hospitalization and mortality rates). our multivariate analyses presented in table 5 adds strength to our finding of an increased risk of death, ranging from 72% to 42%, in covid-19 patients with copd versus those without copd. please note that in the full model 3, some individual comorbidities were associated with nominal increases, but not statistically significant increased risks, such as stroke, ischemic heart disease, ischemic heart disease, and sleep apnoea, as well as smoking with an odds ratio (or) of 1.31 95% confidence interval (ci) (0.99-1.71), likely due to mathematical collinearity of these comorbidities in multimorbid patients. given the scarcity of pcr tests for sars-cov-2 at the onset of the covid-19 pandemic, regional protocols established the performance of multiple pcr tests for respiratory viruses in all patients who were hospitalized for respiratory symptoms. the results of these tests allowed us to confirm the near absence of other viral infections, including influenza, during the covid-19 pandemic. this allowed us to compare the differential impact of covid-19 in copd patients by comparing data during this period with data from the last two winter seasons. although both populations showed similar age and comorbidities, covid-19 caused a higher rate of hospitalizations and in-hospital mortality. most patients admitted for covid-19 presented pulmonary infiltrates compatible with sars-cov-2 pneumonia and, in some cases, with associated heart failure; this finding markedly differed from patients with copd exacerbation due to other viral causes. these data indicate that patients who were admitted into a hospital or died from covid-19 have different clinical profiles compared to those with winter viral exacerbations. thus, these differences must be taken into account so we can adapt to a scenario where both clinical profiles can coexist. as faust comments in a recent article [22] , many hospitals in areas hard-hit by covid-19 (as is the case in our study population) have had an unprecedented overload and demand for hospital resources during the crisis that have never been seen before, even during the worst flu season. although the interaction that may exist between sars-cov-2 and other viruses is unknown, the scenario can be further complicated by their simultaneous presentation. the treatment of patients with chronic diseases have been another highly discussed topic with regards to the newly identified pandemic. faced with the initial alarm regarding the deleterious potential of certain drugs such as ace inhibitors or arbs, our data do not confirm a negative impact of these drugs in patients with copd and related comorbidities that justify their use. these data are consistent with other series in the general population [23, 24] . in patients with copd and asthma, although there is still very little scientific evidence, the treatment with inhaled glucocorticoids (igc) could have a "protective effect" against covid-19 since they may decrease the expression of the ace2 receptor genes and the tmprss2 transmembrane protease genes, both key for the virus to enter cells and make copies of itself [25] . previous clinical data in patients with asthma support this hypothesis in our population [17] . however, since most copd patients were under treatment with igcs, it was not possible to evaluate the specific effect of igcs in our population. since the prescription of beta 2 agonists and anticholinergics is guided by symptoms, the observed greater use in patients who died may simply be related to increased severity of the disease. perhaps one of the most controversial topics around covid-19 is the association between smoking and the manifestation of the disease. although a protective effect of nicotine was initially suggested, several meta-analyses have confirmed that smoking increases the risk of severe covid-19 and mortality; these results are not consistent, however [20, 26] . as is the case with the present study (where we were not able to determine with precision whether smokers were still active tobacco users or the intensity of exposure) the exact duration of smoking was not reported in most studies included in these meta-analyses. both copd and tobacco smoke can up-regulate ace-2 expression in lower airways, which in part may explain the increased risk of severe covid-19 in this population [27] . the results presented here must be interpreted in light of some strengths and limitations. data were extracted from the public health system of castilla-la mancha, with a population of 2,030,807 inhabitants. specifically, we analyzed data from the sescam health system, which operates the savana manager ® clinical platform with available data since 2011. the information obtained from 1,164,283 patients with available ehrs available during the study period is verifiable and includes the clinical management of all patients without any type of bias. this differs from other databases, where limited reliability has generated controversy [23] . importantly, since we collected information from the entire population, reproducibility of the results does not apply. on the other hand, the results reported for some variables rely on the quality of the data captured in the clinical reports, which in many cases may not include all the clinical information for a given patient. since this is not a study based on a strict registry of variables, the information that was not adequately documented was excluded from further analyses [28, 29] . in this study, we included covid-19 cases both confirmed with pcr or serological tests and those exclusively diagnosed based on clinical criteria (i.e., symptoms, imaging, and laboratory results). however, it should be noted that pcr and other rapid laboratory tests for the detection of sars-cov-2 were not used routinely in spain during the onset of the pandemic. furthermore, this decision is supported by reports questioning the clinical validity and high sensitivity of symptom and image-based identification of patients with covid-19, especially in the early stages of the disease [30] [31] [32] . finally, diagnoses for viral infections analyzed in previous winter seasons were mostly based on clinical evaluations, without specifications regarding the type of virus in most cases. the data, which spans two annual periods, are nevertheless representative of copd exacerbations due to viral processes during the winter season. in conclusion, the results of this study confirm a higher incidence of covid-19 in copd patients and higher rates of hospital admissions and mortality, mainly associated with pneumonia. this clinical profile is different from that observed during winter exacerbations caused by other respiratory viruses. overall, our data support the implementation of specific plans in this population with close monitoring of copd patients in sceneries at risk for covid-19. global strategy for the diagnosis, management, and prevention of chronic obstructive pulmonary disease epidemiology of chronic obstructive pulmonary disease exacerbations acute exacerbation of copd. factors associated with poor outcome severe acute exacerbations and mortality in patients with chronic obstructive pulmonary disease coronavirus disease (covid-19) outbreak. available online early transmission dynamics in wuhan, china, of novel coronavirus-infected 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perspective. chronic. obstr comorbidity and its impact on 1590 patients with covid-19 in china: a nationwide analysis severity and mortality associated with copd and smoking in patients with covid-19: a rapid systematic review and meta-analysis chronic obstructive pulmonary disease is associated with severe coronavirus disease 2019 (covid-19) assessment of deaths from covid-19 and from seasonal influenza drug therapy, and mortality in covid-19 use of renin-angiotensin-aldosterone system inhibitors and risk of covid-19 requiring admission to hospital: a case-population study national heart, lung, and blood institute severe asthma research program-3 investigators. covid-19 related genes in sputum cells in asthma: relationship to demographic features and corticosteroids the impact of copd and smoking history on the severity of covid-19: a systemic review and meta-analysis ace-2 expression in the small airway epithelia of smokers and copd patients: implications for covid-19 a global information extraction and terminology expansion framework in the medical domain extracting information from the text of electronic medical records to improve case detection: a systematic review diagnosis of the coronavirus disease (covid-19): rrt-pcr or ct? detection of sars-cov-2 in different types of clinical specimens correlation of chest ct and rt-pcr testing in coronavirus disease 2019 (covid-19) in china: a report of 1014 cases this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors declare no conflict of interest. key: cord-327610-cm3vkpcn authors: fukuda, yosuke; akimoto, kaho; homma, tetsuya; baker, jonathan r; ito, kazuhiro; barnes, peter j; sagara, hironori title: virus-induced asthma exacerbations: sirt1 targeted approach date: 2020-08-13 journal: j clin med doi: 10.3390/jcm9082623 sha: doc_id: 327610 cord_uid: cm3vkpcn the prevalence of asthma has increased worldwide. asthma exacerbations triggered by upper respiratory tract viral infections remain a major clinical problem and account for hospital admissions and time lost from work. virus-induced asthma exacerbations cause airway inflammation, resulting in worsening asthma and deterioration in the patients’ quality of life, which may require systemic corticosteroid therapy. despite recent advances in understanding the cellular and molecular mechanisms underlying asthma exacerbations, current therapeutic modalities are inadequate for complete prevention and treatment of these episodes. the pathological role of cellular senescence, especially that involving the silent information regulator 2 homolog sirtuin (sirt) protein family, has recently been demonstrated in stable and exacerbated chronic respiratory disease states. this review discusses the role of sirt1 in the pathogenesis of bronchial asthma. it also discusses the role of sirt1 in inflammatory cells that play an important role in virus-induced asthma exacerbations. recent studies have hypothesized that sirt1 is one of major contributors to cellular senescence. sirt1 levels decrease in th2 and non-th2-related airway inflammation, indicating the role of sirt1 in several endotypes and phenotypes of asthma. moreover, several models have demonstrated relationships between viral infection and sirt1. therefore, targeting sirt1 is a novel strategy that may be effective for treating virus-induced asthma exacerbations in the future. asthma is the most common chronic respiratory disease, as over 300 million individuals suffer from asthma worldwide [1] . although the rate of asthma-related mortality has declined for decades due to the advancement in treatment strategies, the prevalence of asthma has gradually increased from 1990 to 2005, and death rates have plateaued in some countries with aging populations [2] . among all asthma patients, it is thought that about 5-10% of patients have severe refractory asthma. this is due to inaccurate inhalation techniques, poor treatment adherence, and inadequate management of comorbidities [3] . however, even when these factors are excluded, asthma is often still poorly controlled in the population. patients still have to grapple with various issues associated with the condition; asthma exacerbation is one of the challenges that requires a more effective solution. among multiple causes, infectious diseases are the most important cause of asthma exacerbations. various microorganisms, such as bacteria, fungi, and viruses, can cause acute exacerbations of asthma [4] [5] [6] , and viral infection is the most common trigger. human rhinovirus (hrv) is one of the major pathogens of virus-induced asthma exacerbations. in a review of 670 samples of hrv infection detected in the nasal mucosa of infants, hrv caused severe symptoms in winter, and hrv-a and hrv-c caused moderate to severe illnesses [7] . a previous study confirmed elevated expression of interferon (ifn) and type 2 cytokines analyzed from bronchosorption and nasosorption in asthma patients infected with hrv [8] . in the presence of hrv infection, it was suggested that airway infections of streptococcus pneumoniae and moraxella catarrhalis might increase the risk of experiencing the severity and symptoms of asthma exacerbations [9] . respiratory syncytial virus (rsv) is also an important pathogen for asthma exacerbations. rsv is known to infect almost all children by the age of 2 years [10] , and childhood rsv infection puts adults at risk of developing asthma [11] . in addition, the annual incidence of rsv infection is 3-7% in healthy older adults, and 7.2% of patients hospitalized for asthma have comorbid rsv infection [12] . thus, it is an essential pathogen for all generations. type 2 and non-type 2 airway inflammation are the two major immune phenotypes of asthma [13] . these phenotypes and endotypes are defined by a variety of factors, including genetic predisposition and environmental factors such as antigen exposure, inflammatory biomarkers, tight junctions in the airway epithelium, and viral infections [14] [15] [16] . in addition to the viral infections themselves, it has been suggested that viral infections can reduce the species, numbers, and diversity of microbiota, so-called dysbiosis [17] , indicating that asthma is a very complex disease. viral infection, which can affect patients with both phenotypes, induces the formation of a wide range of cytokines and chemokines in the airway [18] [19] [20] [21] [22] . eosinophils, t helper-2 (th2) lymphocytes, type 2 innate lymphoid cells (ilc2), th17 cells, and neutrophils are involved in epithelial chemokine production in virus-induced asthma exacerbation [18] [19] [20] [21] [22] (figure 1 ). inhaled corticosteroids (ics), which have been used widely for treating asthma over the past few decades, inhibit the expression of inflammatory cytokines in the airway during virus-induced asthma exacerbations, especially th2-airway inflammation [23] . although ics ameliorate asthma exacerbation by limiting neutrophilic and non-th2 inflammation [24] , it is also known that viral infection induces steroid resistance by inducing mainly neutrophilic airway inflammation [25] . virus-induced asthma exacerbation overburdens healthcare systems, and it elevates the rates of morbidity and mortality [26] . moreover, a few patients, described as severe, do not respond to current therapies, and few prophylactic strategies are available for treating such refractory cases. therefore, new therapeutic targets and approaches are the need of the hour. cellular senescence is characterized by irreversible cell-cycle arrest and release of inflammatory mediators known as the senescence-associated secreted phenotype (sasp), which can exert paracrine and autocrine effects on naïve cells [27] . the silent information regulator 2 homolog 1 (sirt1), which is a nicotinamide adenine dinucleotide (nad+)-dependent class iii deacetylase, is one of the essential proteins that regulates aging, metabolism, dna repair, immunity, and inflammation, and it protects against cellular senescence [28, 29] . in recent years, age-related diseases such as heart disease, neurological diseases, cancer, and diabetes have been found to be closely related to sirt1 [28] , and similarly, sirt1 has garnered considerable attention because of its role in the pathogenesis of asthma [30] [31] [32] . an sirt1-targeted treatment strategy may be effective in patients with virus-induced asthma exacerbation, who respond inadequately to existing therapies. in this review, we discuss the function of sirt1 in inflammatory cells that play a role in virus-induced asthma exacerbations and examine the possibility of using sirt1 as a target for treating virus-induced asthma exacerbations in the future. activation of sirt1 induces sasp in t cells through deacetylation of several transcription factors, such as p53, nf-κb, forkhead box o (foxo)3, pi3k, hif-1α, and pgc1α [32, 33] , regulating autophagy, dna repair, mitochondrial function, and cellular senescence [34] . it was suggested that the mechanism for this was an enhanced glycolysis in helper t cells, which might lead to immune dysfunction [35] . on the other hand, the details of b cells involved in the humoral immune response are not yet well known [36] . in 2003, a screen for mammalian sirt1 activators identified sirt1 activators, including resveratrol, piceatannol, and quercetin, called sirtuin activating compounds (stacs). among them, resveratrol was shown to be the most potent activator of sirt1 [37] . in a clinical trial of sirt1 activators in mild to moderate ulcerative colitis, srt2104, a sirt1 activator, was well tolerated [38] . adverse events of srt2104 was reported to include upper abdominal pain, fatigue, photophobia, diarrhea, and headache [38] . it is considered to be a relatively safe drug to use. on the other hand, selisistat (ex527), a sirt1 inhibitor, has been studied in healthy individuals and patients with huntington's disease, and has also been shown to be safe [39, 40] . the association between sirt inhibitors and respiratory illness is not well reported. based on these previous reports, we will discuss about the impact of sirt1 on virus-induced asthma exacerbations below. neutrophilic inflammation occurs in the airway during virus-induced asthma exacerbation. cxcl8 is a crucial cytokine of the neutrophilic airway inflammatory process, which is also involved in virus-induced asthma exacerbation [41] . hrv infection increases cxcl8 and il-1β levels in the nasal lavage fluid of patients with asthma [42] . smoking is an important factor in the worsening of the disease in asthmatic patients. stimulating human airway epithelial cells with hrv infection and cigarette smoking extract (cse) enhances cxcl8 expression [43] . increased production of cxcl8 is one of the mechanisms of corticosteroid resistance [44, 45] . the antimicrobial drug azithromycin may be effective in asthmatics with a predominance of cxcl-8 and other neutrophilic cytokines [46, 47] . in a clinical trial of children with rsv-infected bronchitis, azithromycin significantly reduced the expression of cxcl8 in nasal lavage and reduced respiratory symptoms one-year post-use compared to a placebo [46] . azithromycin may exert its effects by inducing ifn-β and ifn2/3, but the clinical benefits of using antimicrobials, including azithromycin, in patients with asthma are still unclear [48, 49] . mmp-9, a type of matrix metalloprotease, has been found to be increased during viral infections [50] . mmp-9 also causes airway remodeling through neutrophilic airway inflammation [50, 51] . airway remodeling is associated with decreased respiratory function and disease severity [52] . despite the fact that both cxcl8 and mmp-9 are important factors associated with neutrophilic airway inflammation, there are few therapeutic agents for these factors involved in neutrophilic airway inflammation. furthermore, it may lead to severe virus-induced asthma, and effective treatment strategies are needed. several reports suggest that sirt1 regulates neutrophilic airway inflammation related to cxcl8 [53] [54] [55] [56] . cse-induced cxcl8 elevation in mature mononuclear cells was attenuated by the overexpression of sirt1 in vitro [55] . these studies showed that activation of nf-κb signaling and deacetylation of foxo 3a protein as mechanisms by which sirt1 regulates neutrophilic airway inflammation [55] . a previous study confirmed that foxo3a expression was upregulated by rsv infection [57] . thus, sirt1 activators, including resveratrol, may be effective in targeting cxcl8-induced neutrophilic airway inflammation in virus-induced and steroid-resistant asthma exacerbations [58, 59] . interestingly, in basic experiments with macrophages isolated from bronchoalveolar lavages of copd patients, resveratrol inhibited the release of nearly all cytokines from alveolar macrophages. in contrast, dexamethasone, a type of systemic corticosteroid commonly used in the treatment of asthma exacerbations, only partially inhibited the release of cxcl8 [60] . these lines of evidence suggest that activation of sirt1 may lead to suppression of neutrophilic inflammation, possibly through suppression of cxcl8 and may be an effective therapeutic strategy, especially for steroid-resistant virus-induced asthma exacerbations. suzuki et al. investigated the relationship between viral infections and mmp-9 expression using human nasal epithelial cells [51] . notably, they found that the expression of mmp-9, which was enhanced by poly(i:c), was attenuated by resveratrol. furthermore, in the presence of the sirt1 inhibitor splitomicin, poly(i:c) significantly enhanced the expression of mmp-9 [51] . another study showed that the increased mmp-9 was attenuated by not only the sirt1 activator resveratrol, but also by the diabetes drug metformin in a mouse model exposed to uv light [61] . these results indicated that sirt1 activation could be a novel therapeutic strategy for virus-induced asthma exacerbations by regulating mmp-9 expression and suppressing airway neutrophilic inflammation and remodeling. however, additional studies will be necessary to determine whether mmp-9 is a good biomarker for sirt1-targeted therapy. eosinophils play an essential role in virus-induced asthma exacerbation. ccl5 and ccl11, which are chemokines associated with eosinophils, may be upregulated and recruit eosinophils when a virus infects the airway epithelium [62] . eosinophil cationic protein (ecp), an inflammatory mediator, is released by eosinophils and correlates with airway hyperreactivity [63] . in addition to these, cytokines such as interleukin (il)-4, il-5, and il-13 are thought to be involved in a complex. calhoun et al. investigated whether hrv could trigger an allergic response in the airway [64] since it is a significant viral pathogen that exacerbates asthma in adults and children [65] . bronchoalveolar lavage was performed for both healthy and allergic participants with or without hrv infection, and they concluded that eosinophil recruitment in the airway occurred during or after hrv infection in allergic participants, but not in healthy participants [65] . kato et al. studied childhood asthma and reported that serum il-5 and ecp levels were significantly higher in the virus-induced asthma group than those in the control group [66] . they also showed that the profile of those cytokines and chemokines differed with age [67] . fractional exhaled nitric oxide is a good indicator of eosinophilic airway inflammation [68] . bjerregaard and colleagues reported that feno in virus-induced asthma exacerbation was higher than in the follow-up period [69] . they demonstrated that patients with higher feno levels had significantly shorter time to arrive at asthma exacerbation than those patients with lower feno [69] . activation of toll-like receptor 3 (tlr3), a virus receptor within the airway epithelial cells, led to the induction of eosinophil-attracting chemokines (ccl11, eotaxin) in the cellular bases [18] [19] [20] [21] [22] . these findings suggested that eosinophilic airway inflammation is an important aspect of virus-induced asthma exacerbation. several studies have demonstrated the importance of sirt1 in eosinophilic airway inflammation. wang et al., using an ovalbumin-induced asthma mouse model, found that sirt1 was associated with eosinophilic airway inflammation [70] . while they reported that serum sirt1 levels were increased in ova-sensitized and challenged mice model, sirt1 levels were decreased in lung tissue, and more il-4, il-5, and il-13 in balf were found in the ovalbumin-induced asthma mouse model compared to the controls [70] . they also showed that respiratory function (forced expiratory volume in 1 s/forced vital capacity) was negatively correlated with serum sirt1 in asthmatic human samples [70] . based on these results, they believed that the elevated serum sirt1 levels were due to the release of sirt1 from the tissues following airway inflammation. in another study, sirt1 activator (sirt1720) treatment decreased the eosinophil count and il-5 and il-13 levels, but not il-4 levels in the bronchoalveolar fluid and lung tissue in the ovalbumin-induced asthma mouse model [71] . they also reported that sirt1 activation significantly inhibited inflammatory cell infiltration in the airways, but it did not significantly affect goblet cell hyperplasia, and they attributed this to the possibility that sirt1 activation might be inadequate to control airway inflammation. resveratrol, a known sirt1 activator, also attenuated il-5 and il-13 as well as eosinophil accumulation in ovalbumin-induced allergic rhinitis in mice [72] . this may be attributed to the differential effect of sirt1 on transcription factors, including gata3, which is a transcriptional factor that regulates th2 differentiation and the expression of the t2 cytokines il-4, il-5, and il-13 [73] . sirt1 is a key regulator of gata3 via its deacetylation, and in t-lymphocytes from patients with severe asthma, a decrease in sirt1 has been linked to increased expression of il-4 via increased gata3 activation [73] . these findings supported the fact that sirt1 activation might suppress eosinophilic inflammation in the airway during acute asthma exacerbations. controlling eosinophilic inflammation is a key approach for predicting and treating virus-induced asthma exacerbations [68, 74, 75] . anti-il-5 therapies, such as mepolizumab and benralizumab, are effective against eosinophilic airway inflammation and markedly reduce virus-induced asthma exacerbations [76, 77] . although these biologics produce marked effects in patients with refractory asthma, their efficacy was limited in patients with non-th2-asthma [78, 79] . activation of sirt1 may facilitate control of eosinophilic inflammation and refractory eosinophilic asthma. ige is an important therapeutic target for virus-induced asthma exacerbation, which is mainly produced by plasma cells. the position of omalizumab, which is a humanized anti-ige monoclonal antibody, has been confirmed as an important therapeutic agent [80] . the prose study revealed a lower asthma exacerbation frequency in the omalizumab group than that in the placebo group [81] . in this study, they conducted a subgroup analysis of patients with hrv infection and found a significant increase in ifn-α in the group of patients treated with omalizumab, which may be a protective mechanism for viral-induced asthma exacerbations [81] . another clinical study showed that omalizumab decreased the frequency and duration of hrv infection in patients with childhood asthma [82] . despite the efficacy of omalizumab in patients with asthma with viral infection, it was reported that some patients, especially geriatric patients, responded poorly to anti-ige. this observation may be attributed to immunosenescence, which includes impaired mucociliary clearance, changes within the inflammatory cells in the airway, and decreased antigen response [83] . hence, there is a demand for other treatment options besides anti-ige therapy for patients who are unresponsive to anti-ige therapy. lipid profiling is thought to be important for understanding viral infections [84, 85] . when the virus reacts with airway epithelium, mast cells are activated in an ige-dependent or independent manner, and degranulation occurs [86] . this results in the production of lipid mediators such as prostaglandin (pg) and cysteinyl leukotriene (cyslts), which induce an immediate response in the airways and other target organs [86] . currently, drugs targeting lipid mediators in asthma are mainly the cyslt 1 receptor antagonists pranlukast and montelukast [87, 88] . it is known that obesity, a factor in refractory asthma, results in steroid resistance due to decreased adipokines. although leukotriene receptor antagonists are useful in such patients [89] , they are still not well controlled. a better understanding of the pathogenesis of refractory asthma by further approaches to lipid mediators is an important issue. resveratrol, a polyphenol found in grapes, berries, red wine, and peanuts [90] , can activate sirt1 [91] . lee et al. examined the possible anti-inflammatory effects of resveratrol in an ovalbumin-induced asthma mouse model [92] . resveratrol significantly reduced total ige and ovalbumin-specific ige levels and increased serum igg2a, which is associated with th1 response, in serum [92] . moreover, it reduced airway hyperresponsiveness and mucus hypersecretion compared to a placebo [92] . yet another study found that sirt1 regulated the pathways, amp-activated protein kinase (ampk), and protein tyrosine phosphatase 1b. modulation of these pathways via resveratrol attenuated signals from the ige receptor, fcεri, and inhibited the release of lipid mediators, leukotriene c 4 (ltc 4 ) and pgd2, and the inflammatory cytokines, tumor necrosis factor (tnf)-α and il-6 [93] . in a mouse model of ova-induced allergic rhinitis, sirt1 administration reduced symptoms such as sneezing and nasal rubbing events, and it led to a significant reduction in serum ige [94] . it is known that when ige antibodies bind to the antigen, intracellular secretory granules are transported to the cell surface, and chemicals, such as histamine, contained in the granules are released. previous reports demonstrated that sirt1 inhibited degranulation [95] . the study noted that inhibition of degranulation by sirt1 may be mediated through inhibition of the response mechanisms of the phosphorylation of protein kinase c (pkc) isomer, pkcµ and pkcθ. these data suggested that sirt1 activation may ameliorate ige-mediated airway inflammation in viral-induced asthma exacerbations, whereas the detailed mechanism by which omalizumab blocks ige is unclear and requires further study. some studies on the association between sirt1 and lipid mediators have been reported. tan et al. demonstrated in basic experiments using eosinophils isolated from whole human blood that trans-resveratrol suppressed the expression of ltc 4 [96] . another study showed that resveratrol reduced the expression levels of ltc 4 and pgd in a mouse model of eosinophilic sinusitis [97] . these results might be attributed to inhibition of the phospholipasea2 (pla2) and lipoxygenase (lox) pathways, which play an important role in the arachidonic acid cascade [96, 97] . as mentioned above, sirt1 is closely related to metabolism. it was reported that fisetin and licochalcone, which are polyphenols, improve hepatic lipid metabolism via the sirt1/ampk pathway in a mouse model [98, 99] . in a clinical trial on asthma and diet, a healthier diet correlated with better asthma control [100] . these evidences suggest that drug treatment and diet modification may be one of the lipid mediator-mediated sirt targeted treatment strategies for virus-induced asthma exacerbations. ilcs are a novel type of lymphocyte, which have been recently identified as playing an important role in immune diseases. th2-type cytokines were initially found to be produced solely by th2 cells, but recent findings show that ilc2 cells, although less numerous than th2 cells, are more efficient in producing th2-type cytokines [101] . moreover, ilc2 has been shown to play an essential role in virus-induced asthma [102, 103] . studies have confirmed that the expression of the upstream cytokines il-25, il-33, and tslp that regulate ilc2 is enhanced in rhinovirus infections [104] [105] [106] [107] . current knowledge suggests that airway epithelial damage triggered by viral infections promotes the production of il-25, il-33, and thymic stromal lymphopoietin (tslp), which in turn leads to the activation of ilc2 and exacerbation of asthma [102] . we showed that the late addition of budesonide attenuates the increase in tslp caused by viral infection [19] . although tezepermab, a biologic that targets tslp for treatment, was reported to reduce the frequency of asthma exacerbations [108] , the role of ilcs2, including il25 and il-33, is not fully understood, and we need to elucidate the full mechanism of its production and develop new treatment options. little is currently known about the association between airway inflammation, sirt1, and ilc2. basic experiments using mouse models of allergic diseases demonstrated that resveratrol inhibited the expression of il-25, il-33, and tslp in airway epithelial cells [109] . resveratrol also reduces caspase-3, an indicator of apoptosis [109] . in basic experiments using a mouse model of hdm-induced asthma, resveratrol reduced cell apoptosis and suppressed the expression of the γh2ax gene, which is associated with dna damage [110] . activation of sirt1 suppresses epithelial damage, which may inhibit apoptosis and control viral-induced asthma exacerbations. leptin is one of the adipokines secreted by adipocytes and is involved in increased energy metabolism and appetite suppression via hypothalamic receptors [111] . an increase in sirt1 led to an increase in the sensitivity of leptin [111] . it was suggested that elevated leptin could cause exacerbation of allergic diseases via ilc2 [112, 113] . zeng et al. tested the relationship between leptin and ilc2 in patients with allergic rhinitis [113] . the results showed that leptin expression correlated with the percentage of ilc2 in peripheral blood mononuclear cells. mapk signaling and pi3k signaling were thought to be the possible pathways involved in this response [112, 113] . it was reported that sirt1 was inhibited by p38 mapk and pi3k signaling via micro-rna (mirna) 34-a and mirna570 expression, if the airway epithelial cells were subjected to oxidative stress [114, 115] . the possible mechanisms by which sirt1 regulates allergic airway inflammation through ilc2, such as cellular apoptosis and lipid metabolic pathways, as well as the presence of mirnas, require further investigation. th17 cells also play an important part in virus-induced asthma, mediated by the role of il-17 family cytokines [116] [117] [118] . in basic experiments in which an ova mouse model was infected with rsv, il-17a regulated the airway hyperreactivity [116] . niwa et al. demonstrated in vitro experiments using normal human bronchial epithelium that the increase in ifn-λ, which plays a protective role in viral infection, was attenuated by the presence of il-17a [118] . increased il-17 is thought to be one of the mechanisms of steroid resistance in asthmatic patients [118] , and il-17 may be a novel therapeutic target for patients with viral-induced asthma who are refractory to treatment. il-6 and transforming growth factor (tgf)-β are required for th17 differentiation. viral infection, including hrv infection, increases il-6 levels in the respiratory tract [119, 120] , which correlates with airway remodeling and the severity of asthma [121, 122] . at present, the anti-il-6 antibody tocilizumab, which is used to treat rheumatoid arthritis, is not indicated for asthma, but basic experiments suggested that il-6 may be an important marker of asthma [123, 124] . tgf-β is an essential factor affecting airway remodeling along with mmp-9, amphiregulin, vascular endothelial growth factor (vegf), and fibroblast growth factor (fgf) [125] . repeated rv infection in mice not sensitized to allergens activated tgf-β in lung tissue, but neutralizing tgf-β reduced airway smooth muscle thickening [125] . tgf-β-deficient mice showed an earlier increase in ifn-β in lung tissue compared to that in controls [126] . collectively, tgf-β was found to contribute strongly to remodeling during virus-induced asthma exacerbations. one therapeutic candidate targeting th17 cells is brodalumab, a monoclonal antibody against the il-17 receptor, which has failed to show efficacy in the clinical trial [127] . further elucidation of their mechanisms and development of therapeutic agents are required to control the pathogenesis of virus-induced asthma exacerbations. it was reported that loss of sirt1 further induced mrna expression of il-17 in an animal model of rsv infection [128] . according to their considerations, sirt1-deficient bone marrow dendritic cells elevated acetyl coa carboxylase 1 (acc1), which is associated with fatty acid synthesis, resulting in the activation of an abnormal metabolic process, which in turn preceded an excessive virus-induced immune response [128] . in other words, sirt1 may regulate the th17 immune response from virus-infected dendritic cells by regulating their metabolic pathways [128] . the relationship between sirt1 and il-17 has been well studied in other diseases. previous studies investigated the effect of sirt1 activation in patients with psoriasis [129, 130] . they found a significant histological improvement in the sirt activator group compared to that in the placebo group, which was attributed to the inhibition of il-17 and tnf-α [129] . moreover, resveratrol, a sirt1 activator, suppressed the expression of ccl6, a chemokine that is essential chemokine for the production of il-17 [130] . the involvement of th17 cells was demonstrated in patients with diabetic ophthalmopathy [130, 131] . other studies reported that the sirt1 activator might inhibit the elevation in serum il-17 levels, and regulation of il-17 through sirt1 probably leads to a reduction in the development of diabetic ophthalmopathy [132] . by applying the proven relationship between sirt1 and il-17 in these other diseases to viral-induced asthma, sirt1 could become a new therapeutic target in the future. sirt1 regulated il-6 expression in the ovalbumin-induced asthma mouse model [17, 133, 134] . these studies confirmed that the activation of the pi3k-akt pathway led to an increase in il-6, and this response was attenuated by sirt1 inhibitors [133, 134] . ichikawa et al. showed that a sirt1 activator suppressed il-6 and tnf-α production by splenocytes in ovalbumin-challenged mice [17] . these indicate that the akt-sirt1 signaling is a crucial pathway to the control of il-6. interestingly, another study reported that metformin, a pharmacotherapeutic agent used to treat diabetes, reduced il-6, il-17, il-1β, and tnf-α levels in a mouse model of acute respiratory distress syndrome [135] . one possibility was that the low expression of mirna138 might suppress the mitogen-activated protein kinase (mapk) pathway upstream of sirt1 [135] . moreover, metformin, like resveratrol, was reported to inhibit hif-1α expression, but through a different pathway [136] . in clinical practice, oral metformin reduced asthma-related hospitalizations and asthma exacerbations [137] . metformin may be one of the treatment options in patients with virus-induced asthma exacerbations by regulating th17 cells. the relationship between sirt1 and tgf-β has often been studied in idiopathic pulmonary fibrosis (ipf). zeng et al. used a bleomycin-induced mouse model to explore the role of sirt1 in pulmonary fibrosis [138] . they demonstrated that activation of sirt1 by resveratrol and srt1720 inhibited myofibroblast differentiation induced by tgf-β1. overexpression of sirt6, a member of the sirt family, reduced e-cadherin, a marker of emt, through the tgf-β pathway [139] . this result might involve p21, a protein that regulates cell cycle progression [140] . another study showed that resveratrol inhibits airway remodeling through transforming growth factor (tgf)-β1/smad signaling [141] . the relationship between sirt1 and tgf-β, which was identified in ipf, has potential applications in virus-induced asthma exacerbations, but the mechanisms of this relationship are still unclear. therefore, the findings of these studies suggest that future treatments against cellular senescence, especially sirt1, may regulate th17 airway inflammation. we believe that metformin is an attractive treatment option because it is already used in many patients with few adverse events, but further research is needed to determine whether it is useful in patients with virus-induced asthma exacerbations who do not have diabetes mellitus. protein acetylation plays a crucial role in host response to viral infection. histone deacetylases (hdac) are enzymes that define chromatin structure and are closely associated with chronic respiratory diseases [142] . adenovirus infection resulted in reduced activation of hdac in ova sensitization mice [143] . experiments using human blood samples also showed reduced activation of hdac in asthmatics compared to that in healthy subjects [144] . nf-e2-related factor2 (nrf2) is a transcription factor that has a protective effect on cells from oxidative stress caused by reactive oxygen species. rsv, which frequently causes asthma exacerbations, induces deacetylation of nrf2 [145] . in vitro models of rhinovirus infection showed that inhibition of s-nitrosoglutathione reductase was reported to increase sqstm1, an nrf2-dependent gene, and suppress viral growth, with an effect on airway hypersensitivity [146] . although these viral proteins are important factors in the pathogenesis of virus-induced asthma exacerbations, there remain unanswered questions concerning established treatment. theophylline, a drug for asthma and copd, activates hdac and exerts an anti-inflammatory effect. when the activity of hdac was investigated in lps-stimulated macrophages, hdacs were activated by the combination of theophylline compared to dexamethasone alone [147] . in a clinical trial examining the effects of low volume theophylline, the addition of theophylline to inhaled steroids improved respiratory function [148] . in patients with acute exacerbations of copd, theophylline also improved hdac activity during the stable phase [149] . interestingly, steroid resistance correlated with a decrease in sirt, and a combination of steroid, theophylline, curcumin, or resveratrol treatment resulted in an increase in sirt1 as well as an increase in glucocorticoid receptors [150] . doxophylline, which is considered to have fewer side effects than conventional theophylline preparations, improved the protein expression of sirt1, which was reduced by lps stimulation [151] . as a treatment option for targeting sirt1, theophylline is very effective for patients with inadequate response to steroids. sirt1 regulates the expression of various genes by deacetylating histones and transcription factors, such as nf-kb, stat1, and stat3 [152] [153] [154] [155] . sirt1 deacetylates induced nf-kb activation in monocytes [152] . sirt1 inhibits growth hormone-stimulated stat3 activation in mouse embryonic cells via the deacetylation of stat3 [153] . treatment with sirt1 agonists deacetylates stat3, which inhibits t-cell differentiation into th7 and th17 cells [154] . a recent study reported that the activation of sirt1 by viral infection further affected stat1 activation [155] . these studies indicated that transcription factors, including stat1 and stat3, may be potential biomarkers in virus-induced asthma with sirt1-targeted therapy. sirt1 also regulates antioxidant genes, which are important antiaging genes, via deacetylation of foxo3 and nrf2. resveratrol increased the expression of nrf2 in obese rat models and paraquat-induced lung injury mouse models [156, 157] . apios americana medikus is an edible tuberous legume native to eastern north america. chu and colleagues showed that apios americana medikus leaf extract increased the expression of nrf2 in mouse macrophages stimulated with lps [158] . at present, there is no specific treatment for nrf2, and further studies are needed. roflumilast, a selective inhibitor of phosphodiesterase 4 (pde4), was reported to reduce exacerbations and hospitalization rates as a treatment for copd [159] . roflumilast is highlighted by the fact that it increased the sirt1 expression along with nrf2 expression in copd patients [160] . in vitro, rsv infection increased the nrf2 expression and its increase was attenuated by roflumilast [161] . the proinflammatory cytokines il-6, il-8, and tnf-α were also reduced in a capacity-dependent manner. these results suggest that nrf2 may be a potential therapeutic target for viral asthma exacerbations. middle east respiratory syndrome coronavirus (mers-cov) pp1ab protein is potentially regulated by sirt1 [162] . sars-cov-2 might have a similar motif, but further study is required. sirt1 and other sirtuins were also reported to have antiviral roles against several dna and rna viruses, including hcmv, hsv-1, adenovirus, and influenza a [163] . although resveratrol was found to inhibit hrv replication in nasal epithelial cells [164] , it is unclear whether this is due to intercellular adhesion molecule-1 (icam-1) as the hrv receptor, or reduction or deacetylation on potential viral protein acetylation, as the acetylation of hrv or rsv proteins remains unclear. caspase 3 cleavage levels, indicators of apoptosis, are known to elevate in mers-cov infection [165] , and resveratrol was found to reduce caspase 3 cleavage levels with less cytotoxicity [166] . activation of sirt1 may reduce apoptosis through deacetylation of viral proteins. in another report using airway epithelial cells, kim and colleagues examined the relationship between cellular senescence and the replication efficiency of influenza virus [167] . senescent cells infected with influenza virus had reduced expression of ifn-β, which plays an essential role in the immune response compared to nonsenescent cells. they also examined whether sirt1, an essential factor in cellular senescence, affected viral replication. interestingly, sirt1-knockdown cells showed enhanced expression of proteins associated with influenza virus and reduced cell viability [167] . collectively, with further research, therapies targeting sirt1 may control asthma exacerbations through acetylation of the viral protein. we reviewed the cellular interactions between sirt1 and inflammatory cells involved in virus-induced asthma exacerbations (table 1 ). viral infection is a common health problem for children and adults with asthma. especially with the recent rampant covid-19 pandemic, treating and preventing viral infections is becoming an area of focus. while the role of various respiratory viruses in inducing the exacerbation of asthma is well established, the pathophysiological mechanisms underlying virus-induced asthma exacerbation and its treatment remain controversial. existing drugs such as ics and biologics are among the best treatment options for asthma exacerbation triggered by viral infection, but they may be partially ineffective due to unknown mechanisms. the current guidelines do not consider the potential for therapeutic agents related to sirt1. the difference in the role of sirt1 in asthma between adults and children is not clear. however, as mentioned above, sirt1-related signaling pathways are closely related to airway inflammation in asthma. a better understanding of the sirt1 mechanism may aid in the prevention and treatment of virus-induced asthma exacerbations ( figure 2 ). the development of therapies targeting sirt1 could be a boon for patients with virus-induced asthma. further research is needed to clarify the relationship between known drugs and sirt1 and to explore the development of new drugs related to sirt1. funding: this research received no external funding. regional, and national deaths, prevalence, disability-adjusted life years, and years lived with disability for chronic obstructive pulmonary disease and asthma, 1990-2015: a systematic analysis for the global burden of disease study global asthma prevalence in adults: findings from the cross-sectional world health survey asthma in the elderly: what we know and what we have yet to know beneficial effect of early intervention with garenoxacin for 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diet-induced obese mice protective effects of licochalcone a ameliorates obesity and non-alcoholic fatty liver disease via promotion of the sirt-1/ampk pathway in mice fed a high-fat diet associations between dietary scores with asthma symptoms and asthma control in adults pulmonary group 2 innate lymphoid cells: surprises and challenges respiratory viral infection, epithelial cytokines, and innate lymphoid cells in asthma exacerbations t cells and ilc2s are major effector cells in influenza-induced exacerbation of allergic airway inflammation in mice the innate cytokines il-25, il-33, and tslp cooperate in the induction of type 2 innate lymphoid cell exansion and mucous metaplasia in rhinovirus-infected immature mice il-33-dependent type 2 inflammation during rhinovirus-induced asthma exacerbations in vivo virus/allergen interactions in asthma thymic stromal lymphopoietin is induced by respiratory syncytial virus-infected airway epithelial cells and promotes a type 2 response to 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responses in airway epithelial cells resveratrol protects against oxidative stress by activating the keap-1/nrf2 antioxidant defense system in obese-asthmatic rats resveratrol protects mice from paraquat-induced lung injury: the important role of sirt1 and nrf2 antioxidant pathways flavonoids from apios americana medikus leaves protect raw264.7 cells against inflammation via inhibition of mapks, akt-mtor pathways, and nfr2 activation effect of roflumilast on exacerbations in patients with severe chronic obstructive pulmonary disease uncontrolled by combination therapy (react): a multicentre randomised controlled trial the phosphodiesterase-4 inhibitor roflumilast reverts proteolysis in skeletal muscle cells of patients with copd cachexia roflumilast inhibits respiratory syncytial virus infection in human differentiated bronchial epithelial cells identification of lysine acetylation sites on mers-cov replicase pp1ab sirtuins are evolutionarily conserved viral restriction factors resveratrol inhibits rhinovirus replication and expression of inflammatory mediators in nasal epithelia middle east respiratory syndrome coronavirus efficiently infects human primary t lymphocytes and activates the extrinsic and intrinsic apoptosis pathways effective inhibition of mers-cov infection by resveratrol enhanced viral replication by cellular replicative senescence this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license we give thanks to manami matsuda and teru haba for their generous support. we thank editage (www.editage.jp) for english language editing. the authors declare no conflict of interest. kazuhiro ito is an employee of pulmocide ltd. key: cord-306438-db2rqz4d authors: kalathiya, umesh; padariya, monikaben; mayordomo, marcos; lisowska, małgorzata; nicholson, judith; singh, ashita; baginski, maciej; fahraeus, robin; carragher, neil; ball, kathryn; haas, juergen; daniels, alison; hupp, ted r.; alfaro, javier antonio title: highly conserved homotrimer cavity formed by the sars-cov-2 spike glycoprotein: a novel binding site date: 2020-05-14 journal: j clin med doi: 10.3390/jcm9051473 sha: doc_id: 306438 cord_uid: db2rqz4d an important stage in severe acute respiratory syndrome coronavirus 2 (sars-cov-2) life cycle is the binding of the spike (s) protein to the angiotensin converting enzyme-2 (ace2) host cell receptor. therefore, to explore conserved features in spike protein dynamics and to identify potentially novel regions for drugging, we measured spike protein variability derived from 791 viral genomes and studied its properties by molecular dynamics (md) simulation. the findings indicated that s2 subunit (heptad-repeat 1 (hr1), central helix (ch), and connector domain (cd) domains) showed low variability, low fluctuations in md, and displayed a trimer cavity. by contrast, the receptor binding domain (rbd) domain, which is typically targeted in drug discovery programs, exhibits more sequence variability and flexibility. interpretations from md simulations suggest that the monomer form of spike protein is in constant motion showing transitions between an “up” and “down” state. in addition, the trimer cavity may function as a “bouncing spring” that may facilitate the homotrimer spike protein interactions with the ace2 receptor. the feasibility of the trimer cavity as a potential drug target was examined by structure based virtual screening. several hits were identified that have already been validated or suggested to inhibit the sars-cov-2 virus in published cell models. in particular, the data suggest an action mechanism for molecules including chitosan and macrolides such as the mtor (mammalian target of rapamycin) pathway inhibitor rapamycin. these findings identify a novel small molecule binding-site formed by the spike protein oligomer, that might assist in future drug discovery programs aimed at targeting the coronavirus (cov) family of viruses. the global pandemic developing from december 2019 by a strain of severe acute respiratory syndrome coronavirus 2 (sars-cov-2) can cause coronavirus disease 2019 disease. this emergent variant adds to the additional coronavirus strains that can infect humans including human coronavirus oc43 (hcov-oc43), human coronavirus hku1 (hcov-hku1), sars-cov, human coronavirus 229e (hcov-229e), human coronavirus nl63 (hcov-nl63), and human coronavirus nl63 (hcov-nl63) [1] [2] [3] [4] [5] [6] . coronaviruses (covs) are positive-sense, enveloped, single-stranded rna viruses that are classified taxonomically as a family coronavirdiae and order nidovirales [4] . there are four genera of covs, including αcov, βcov, δcov, and γcov; most δcovs and γcovs target avians, whilst αcovs and βcovs infect rodents and bats [1, 7, 8] . severe acute respiratory syndrome cov (sars-cov) outbreaks have also emerged previously creating an epidemic [2, 4, [9] [10] [11] [12] [13] . although the mortality of mers-cov, sars-cov, and sars-cov-2 is substantial, there are no preventative vaccines or drugs available to treat patients infected with the virus [9, 11, 12] . the current public health emergency of international concern (pheic) by the world health organization (who) has declared sars-cov-2 (covid-19; a novel βcov) as a pandemic threat. the data obtained from who (08/may/2020) suggest that the virus has caused 3,759,967 infections, 259,474 deaths, and it has affected over 200 countries. the open reading frame 1ab (orf1ab) of sars-cov-2 encodes for three proteins that are broadly recognized as drug targets, since they are key components for infections and disease progression: the sars-cov-2 protease [14, 15] , the rna-dependent rna polymerase (rdrp) [14, 16, 17] , and the sars-cov-2 spike (s) glycoprotein [15, [18] [19] [20] . the sars-cov-2 protease processes the polyproteins that are translated from the viral rna, and it has been heavily studied using small molecules inhibitors [15] . to penetrate the host, the sars-cov-2 makes use of homotrimeric class i glycosylated fusion spike protein [18, 21, 22] . fusion of the viral and host cell membranes is facilitated by the spike glycoprotein, which undergoes a significant conformational change upon fusion [18, 21, 22] . sars-cov-2 studies suggest [18, 23, 24] that the spike glycoprotein functions as a homotrimer. the recognition and subsequent fusion of the viral and cellular membranes are triggered by the s1 subunit of the spike protein, which binds the host cell receptor; angiotensin converting enzyme-2 (ace2) [16, [25] [26] [27] [28] [29] [30] [31] . several insights from structural biology are consistent with the role for this domain in affecting the infection rate of the virus. this host-virus interaction is mediated by the receptor binding domain (rbd) domain from s1 subunit of sars-cov-2 spike glycoprotein that forms a hinge-like conformation [18, 32] , i.e., "down" and "up" states that represents the host cell receptor-inaccessible and receptor-accessible [18] . this receptor-accessible "up" conformation exists in a highly fluctuating state [33] [34] [35] [36] . binding to the host target destabilizes the pre-fusion homotrimer, which sheds off the s1 subunit, and allows for the transition of the s2 subunit to a highly stable postfusion conformation [18] . interestingly, protein-mediated cell-cell fusion assays suggest that sars-cov-2 spike protein displays an elevated plasma membrane fusion capacity when compared to that of sars-cov [32, 37] . several studies have aimed to define the mechanism of binding of sars-cov-2 to the host cell receptor [38] . molecular dynamics simulations of the spike (rbd)-ace2 complex, over 10 ns indicated that spike(rbd)-ace2 binding free energy for sars-cov-2 is better than for the sars-cov [39] . similarly, other studies have shown that the sars-cov-2 spike protein has a better binding affinity to ace2 at two different "up" angles of the rbd domain than the sars-cov [40] . structural features at the spike-ace2 interface suggest that residues q493 and p499 from the spike rbd domain are responsible for maintaining protein-protein stability [41] . using a virtual high-throughput screening approach, small-molecules have been identified that can interact with the rbd domain of sars-cov-2 spike protein [42] . natural compounds present in curcuma sp., citrus sp., alpiniagalanga, and caesalpiniasappan could also target the rbd domain of the sars-cov-2 spike glycoprotein, the protease domain (pd) from ace2, and the sars-cov-2 protease [20] . a set of b cell and t cell epitopes derived from the spike and nucleocapsid proteins that map identically to sars-cov-2 proteins, were identified as potential vaccine candidates [23] . applying an integrative, antiviral drug repurposing methodology, the interplay between the cov-host interactome and drug targets in the human protein-protein interaction network have been defined [43] . bioinformatics methodologies were used to identify neutralizing antibodies that might interact with interfaces formed by the spike glycoprotein and the ace2 host cell receptor [24] . by targeting the rbd domain of the spike protein using docking experiments, kanishka et al. identified small molecule inhibitors [44] . in the majority of studies, the most common strategy is focused on targeting the interface formed by sars-cov-2 spike glycoprotein and the ace2 host cell receptor (i.e., spike(rbd)-ace2). currently, there are no robust drugs for wide-spread dissemination available against coronaviruses including; the sars-cov-2 virus. due to the relatively rapid spread in the current outbreak and the relatively high mortality rate (3.5%), more rapid development of new or repurposed antiviral drugs is of high value. although the majority of drug discovery programs target classically druggable enzymes encoded by the virus, such as the viral rna polymerase inhibited by remdesivir [13, 17, 45] , there is a paucity of information concerning the other regions of spike glycoprotein outwith the ace2-binding domains, especially the domains interacting with the viral membrane. the sars-cov-2 spike protein is a homotrimer composed of three monomers (chains a, b, and c; figure 1a ). each monomeric protein contains an n-terminal ace2 binding domain (receptor binding domain; rbd), a central helix/heptad repeat, and a c-terminal region that interacts with the plasma membrane [18] . homotrimer spike protein assembly from monomeric forms can be rate limiting in cells, suggesting a possible space for intervention on the viral life cycle [46] . our current study focuses on understanding the variability of the trimer spike glycoprotein in sars-cov-2 with respect to the genomes from other coronavirus strains, and identifying the changes in the molecular properties due to conformational flexibility in the spike protein. the analysis suggests that residues in the s2 subunit are less variable compared to the other regions. in addition, the molecular dynamics simulations (mds) identified that residues from the rbd domain obtained substantial flexibility which may be an obstacle in finding active hits. by contrast, residues in the s2 subunit (trimer cavity) showed the least flexibility representing a novel binding region for ligands. this information was used to identify potentially novel drug pockets or the active site regions specifically in the oligomeric sars-cov-2 spike glycoprotein. we performed md simulations on the monomeric and trimeric form of the sars-cov-2 spike glycoprotein, and developed a virtual screening using a food and drug administration (fda) approved chemical library. we identified and focused on an apparent cavity formed by three subunits (the homotrimer), that our simulations suggest can mediate dynamic movements that mimic a "bouncing spring" or a "sarrus linkage (converting a circular motion to a linear motion or vice versa)" when interacting with the ace2 host cell receptor. this motion might be important in the fusion of the virion and the host cell membrane. we hypothesized that such a cavity formed by three monomers or subunits of the spike protein (i.e., chains a, b, and c) might form an acceptor for small molecules, and we asked whether small molecules could be identified with a relatively high binding energy. we identified several known compounds with predicted binding energy of gbvi/wsa dg (generalized-born volume integral/weighted surface area) from −35 to −71 kcal/mol, some of which are already proposed for clinical trials including an mtor (mammalian target of rapamycin) pathway inhibitor, sirolimus (rapamycin; a macrolide type; nct03901001 not yet recruiting) [47] [48] [49] and ritonavir (open-label trial in hospitalized adults with severe covid-19) [48, [50] [51] [52] . a recent study that screened hundreds of approved molecules in a sars-cov-2 assay using artificial intelligence-enabled phenomic assays [53] , also identified sirolimus (rapamycin) as a promising candidate. in addition to the macrolides, one of the top hits we have identified, chitosan, has a recently reported derivative inhibiting sars-cov-2 coronavirus replication in cell lines [54, 55] . a previous study has also shown that the chitosan derivatives can interact with the spike protein and block its interaction with the host receptor [56] . our data suggest a mechanism whereby chitosan (and possibly its derivatives), as well as macrolide type molecules, might bind to a pocket formed by the spike protein trimer and provide a novel domain to focus on for future drug discovery projects. [18, 19] . (c) receptor binding domain (rbd) illustrating the "up" or "open" (pdb id. 6vsb [18] ) and "down" or "closed" (pdb id. 6vxx [31] ) conformation. a total of 791 viral genome sequences were downloaded from the global initiative on sharing all influenza data platform (gisaid) [57] , in order to define the evolutionary variability in different domains of the spike glycoprotein. only genomes with high coverage and complete sequences were selected. further filtering was applied to obtain complete sequences on the targeted domains which reduced the total number of strains to 768. total protein sequences were acquired from 3 frame translation using the transeq tool from emboss (european molecular biology open software suite) package (version 6.5.7) (european bioinformatics institute (embl-ebi), hinxton, cambridge, uk) [58] . the amino acid chains from the spike glycoprotein were aligned to the reference protein (pdb id. 6vsb [18, 59] ) using muscle [60] . variations in the amino acid or the residue changes were scanned on the entire spike protein sequence, along with two areas of interest in the multiple alignment file, focusing on a subset of the s2 subunit (hr1, ch, and cd domains) and the rbd domain ( figure 1 and tables s1-s3). the cryo-em (cryogenic electron microscopy) homotrimer structure of sars-cov-2 spike glycoprotein was retrieved from the protein data bank database (http://www.rcsb.org/pdb; pdb id. 6vsb; figure 1 ) [18, 59] . in addition, the missing amino acid (residues range: 67-78, 96-98, 143-155, 177-186, 247-260, 329-334, 444-448, 455-490, 501-502, 621-639, 673-686, 812-814, and 829-852) coordinates in the structure of sars-cov-2 spike glycoprotein were built using the swissmodel ( figure 1 ) [61] . molecular dynamics simulations were carried on the model systems as per the standardized pipelines [62] [63] [64] (detailed method explained in the supplementary materials; file s1). the gromacs 4.6.5 [65] program (gromacs; groningen machine for chemical simulations, university of groningen, groningen, the netherlands) was used to perform md calculations assigning the charmm27 forcefield [66] . we performed 100 ns molecular dynamics simulations on two systems: (i) the monomeric form and (ii) the homotrimer form of the spike protein. in our analysis of the md simulations, the dynamics of the monomeric form of the spike protein serves as control to the homotrimer, which is the functional unit. initially, the model systems were energy minimized, which provides a base-line model structure and resolves poorly-resolved conformations often found in crystal structures [13, 18, 62, [67] [68] [69] [70] . a simulation box of solvent atoms is then added to enhance simulation realism. following that, using the npt (number of particles (n), system pressure (p), and temperature (t); isobaric-isothermal) thermodynamic ensemble, equilibration of the systems was performed to adjust solvent molecules with counter ions in the simulation box [71] . these equilibrated systems were subsequently used to perform the final md production runs for 100 ns, and results were analyzed using gromacs [56] , biovia discovery studio (dassaultsystèmes, biovia corp., san diego, ca, usa), chimera, and visual molecular dynamics (vmd) tools [71] [72] [73] . structure-based virtual screening (sbvs) is an application of in silico methods that identify promising lead molecules from chemical libraries or databases. these methods are computational counterparts of experimental biological evaluation methods, such as high-throughput screening (hts). fda approved drug libraries were retrieved from target molecule corp. (targetmol; www.targetmol.com) and selleck chemicals (selleckchem; www.selleckchem.com) vendors. the sbvs against the sars-cov-2 spike glycoprotein was performed using the molecular operating environment (moe; chemical computing group inc., montreal, qc, canada) package [74, 75] . receptor-ligand binding or docking using the charmm27 forcefield [66] was evaluated using the gbvi/wsa ∆g scoring function [76] . the compounds showing best energies with the spike protein were selected for further analysis. gbvi/wsa ∆g is a forcefield based scoring function which determines the free energy of binding of the ligand from a given position [75] . in addition, we have also selected the compounds that showed comparatively stable interactions with the homotrimer spike protein. applying the "triangle matcher"placement method, receptor-ligand docking was performed defining the receptor as rigid and ligands as flexible [74, 75] . we were interested to define the evolutionary variance in the sars-cov-2 spike protein. understanding regions of high and low variance can identify domains that may be functionally conserved and potentially important to the virus life cycle, or those under positive evolutionary pressure whose selection might avoid the immune system. examining the variability of the spike protein in sars-cov-2 and its different domains, a total of 791 genome viral sequences were retrieved from gisaid [57] . a global view of the mutation space of the virus is presented in figure 2a , which represents the amino acid substitutions in bins of 10 aminoacids across the spike glycoprotein. these hotspots of variation are mostly confined to the ntd and the rbd domains (figures 1b and 2b) . we investigated the variability in the entire sequence of the spike protein, focusing on the regions that showed low-variability in the structure (figure 2 ). by investigating the variations in the residue changes across the entire spike protein sequence or all the regions of lower variability (figure 2a and table s1 ), the s2 subunit exhibited the lowest sequence variability (residue range: 816-1141; figures 1b and 2c) . moreover, previous studies have identified that the active site region for this spike protein is located in the rbd domain which interacts with the ace2 host cell receptor [18, 20, 26, [38] [39] [40] 42, 44] . comparing the variability of the rbd domain and s2 subunit domains, the rbd domain was shown to contain more mutations in its region compared to the s2 subunit (hr1, ch, and cd) domains (figure 2b ,c, and tables s1-s4). these data suggest that during mutation by natural selection, the viral-host "arms race" might operate more frequently on the rbd domain. by contrast, the s2 subunit conservation is suggestive of an important core function where mutations cannot be tolerated. these findings prompted our focus on the s2 subunit as an important region to investigate for identifying potentially novel druggable pockets. we next traced the dynamics of different domains in the spike protein using md simulations (figure 1b ). the simulated model systems of the spike protein in the monomer and the homotrimer forms were first processed to check the stability of the protein. stability of the simulated spike protein in both forms in the solvent environment was traced by rmsds (root mean square deviation), a time dependent change in the non-hydrogen atoms (figure 3a ). the rmsd plots (figure 3a) suggest that the trimer form of the spike protein is more stable compared to the monomer form. in addition, chain a in trimer has a higher rmsd (~2 å) compared to the other two chains which is a consequence of the fact that the "up" (or ace2-active) conformation [18] induces flexibility. since the monomer form has a higher rmsds compared to the trimer (figure 3a) , we performed independent triplicates (mds was repeated three times) of md simulation for the monomer form ( figure s1 ). the findings from these replicates indicate that the monomer form has a higher rmsd compared to the trimer spike protein (figure 3a and figure s1 ). the root mean square fluctuations (rmsf) were computed on the cα atoms of each residue from the spike protein, in order to trace their flexibility and thereby define the motions of different domains (figure 3b ). the rmsf findings in both forms (monomer and trimer) indicated that the amino acids in the rbd domain (residue range: 329-521) were highly fluctuating (figures 1b and 3b ). in addition, the triplicate md simulations of the monomer form, also suggests that the rbd domain has a higher rmsf in all three simulation replicates ( figure s1 ). these analyses correlate with previous studies [33] [34] [35] [36] 39] . particularly, amino acids ranging from 470-490, responsible for interacting with the ace2, that were highly fluctuating. furthermore, examining other regions of the spike protein suggests that the s2 subunit domains (residue range: 850-1141; hr1, ch, and cd) showed the least fluctuations within the entire protein sequence (figure 3b ). this correlates with the cryo-em studies performed on the spike protein; that the s2 subunit is more stable [18] compared to the rbd domain, and that this subunit is responsible for a highly stable postfusion conformation of the spike protein [18, 32] . from the perspective of designing drugs, the more stable or less flexible a region is within a protein, the more accurately we can trace a better hit molecule. in the case of the spike protein, the rmsf findings guided us towards focusing on the s2 subunit ( figure 3b) . moreover, by tracing the residues involved in the h-bond interactions between two monomers (i.e., chains a-b, a-c, or b-c) of the homotrimer, we observed that the rbd domain residues were also involved in intermolecular interactions with each other and with high occupancy (%). this suggests that intermolecular interactions between chains in the homotrimer might equilibrate the spike protein, and might stimulate conversion from a "down" to "up" conformation of the rbd domain that interacts with the ace2 receptor ( figure s2 and table s5 ). the structural dynamics over the time course for spike protein in the monomer and the homotrimer form was monitored during md simulations (figure 3c,d) . the monomeric spike protein in the solvent environment exhibited a movement from the "up" active state towards the "down" inactive state for the rbd domain (figure 1c, figure 3c , and movie s1). these correlate with the previous findings that the rbd domain can form two different conformations, i.e., "down" and "up" states, which represents the host cell receptor-inaccessible and receptor-accessible, respectively [33] [34] [35] [36] . the sars-cov-2 spike protein has a better binding affinity to the ace2 receptor at two different "up" angles of the rbd domain compared to the sars-cov [40] . figure 3c (and movie s1) describes the conformational change in other regions of the spike protein, when the rbd domain moves towards an "up" to "down" state in the monomeric form. 3d (and movie s2) represents the dynamics of the homotrimer spike protein, suggesting that the rbd domain of chain a opens more widely in its "up" state. domains hr1, ch, and cd close to the viral transmembrane exhibited the least movement (figure 3d ) during md simulations. in addition, exploration of the structural orientation of these s2 subunit domains (figure 3d and movie s2) suggests that they form a large pocket or cavity using three chains (or monomers) from a homotrimer spike protein. the slight movement observed in homotrimer during md simulation of this cavity (movies s1 and s2), and the structural orientation suggest that it could work as "bouncing spring" or "sarrus linkage". one may postulate that, when the spike protein interacts with the ace2 receptor, this "bouncing spring" or "sarrus linkage" movement may be important in the fusion of the virion with the host membrane. additionally, this cavity from the spike protein could work as a platform for the design or development of new drug leads against this protein (figure 3d ). such molecules might alter the trimer stability upon viral entry or upon viral coat assembly. there have been several studies performed to design drugs specific for the sars-cov-2 spike protein [20, [38] [39] [40] 42, 44] ; however, most of them are focused on the rbd domain. in addition, from our md simulation and variability analysis (figures 2 and 3) , the rbd domain is highly flexible and variable, therefore, drugging this variable site may be an obstacle in finding active hit molecules. by targeting the less variable s2 region, such as the cavity formed by the homotrimer (figures 2 and 3d) we suggest that this might be a novel approach to develop small molecule drug leads. we next investigated the targetability of the trimer cavity formed by the s2 subunit (hr1, ch, and cd domains) in the spike protein (figures 2 and 3 ) using the moe (chemical computing group inc.) package [74, 75] , before using it for high-throughput virtual screening (or sbvs) using a library of fda approved drugs. the "alpha shapes" construction [75, 77] geometric method was used to compute the possible residues that can be considered for ligand docking from this trimer cavity in the spike protein (figure 4a ). high-throughput virtual screening is a powerful computational approach that is increasingly being used in the drug discovery process, through the in silico identification of novel hits from large compound databases [78] . we applied the sbvs approach to dock the molecules to the trimer cavity and to check its feasibility as a target. ligand binding to this cavity might reduce or increase the "bouncing spring" movement in the spike protein, as observed in md simulations (figure 3 , movies s1 and s2). this perturbation might affect its interactions with the host cell receptor or the hinge movement of the rbd domain. the compounds that exhibit a relatively high binding affinity towards the sars-cov-2 spike glycoprotein trimer cavity with a binding affinity −35 to −71 kcal/mol (gbvi/wsa dg) were recorded. from the list of ligands showing best binding, the compounds that were already validated or suggested to be/can be active against the sars-cov-2 virus includes: chitosan [54] [55] [56] , rapamycin [47] [48] [49] , everolimus (rad001) [49] , paclitaxel [79] , ritonavir [48, [50] [51] [52] , selameerin (selamectin) [80] , and danoprevir [52] (table 1) . among these molecules rapamycin and everolimus drugs were previously identified as mtor pathway inhibitors [47, 50, [81] [82] [83] . the antibacterial or antiparasitic drugs from the list are chitosan [84] or selameerin (selamectin) [85] , respectively. paclitaxel, has been found to be previously target bcl-2 and microtubule associated functions [86, 87] . in addition, the fda approved drugs that target the protease are: ritonavir [88] and danoprevir (itmn-191) [89] . by docking known drugs within the trimer cavity of spike protein, the relative selectivity of the cavity suggests that the majority of higher-affinity drugs will have a molecular weight (mw) ≥~700 g/mol (table 1 and figure s3 ). however, this is with the certainty that compounds with high mw can form more interactions with the spike protein, in addition, our finding highlights the possibility that the trimer cavity can occupy large ligands deep inside the binding pocket (figure 4a ). particularly, a specific class of ligands (mostly macrolide type) were found to exhibit a better fit to the trimer cavity ( figure 4a) ; for example, rapamycin [47] [48] [49] , everolimus (rad001) [49] , paclitaxel [79] , and selameerin (selamectin) [80] (figure 4a ). the intermolecular interactions between the spike protein and the compounds suggest that residues from all three monomers (chains a, b, and c) are actively involved in binding to the drugs. in addition, placement of the compounds inside the trimer cavity suggests that they make use of the pocket space (forming different conformation) to form stable interactions with the spike protein (figure 4a ). the ligands that were found interacting with the homotrimer cavity with high binding affinity were also docked with an interface formed by the spike proteins (rbd domain; pdb id. 6lzg [90] ) that interact with the ace2 receptor. sbvs, structure-based virtual screening. in order to check the selectivity of these ligands to the trimer cavity, we docked this same subset (table 1) with the rbd domain of the viral spike protein (figure 4b ; pdb id. 6lzg [90] ). the rbd domain is involved in interacting with the ace2 host cell receptor [16, 26, 30, [38] [39] [40] [41] [42] 90, 91] . the docking suggests that all compounds from table 1 have better binding affinity to the trimer cavity compared to that of the rbd domain. in addition, chitosan [54] [55] [56] 84 ] (a linear polysaccharide; −37.29 kcal/mol) could form a linear conformation in its structure when binding tothe rbd domain (figure 4b and table 1 ), whilst the same ligand (due to its molecular structural nature) can form a slightly folded shape (as shown in 2d-diagram; figure 4a ) within the trimer cavity. by contrast, everolimus [49] (a macrolide type) exhibits high affinity for the trimer pocket, and very little selectivity for the rbd domain (table 1) . moreover, ligands (figure 4b ) that interact with the rbd domain overlap with the region bound by cr3022 (a neutralizing antibody isolated from a convalescent sars patient that, interacts with the receptor binding domain of the sars-cov-2 spike protein [92] ). the sars-cov-2 virus causing covid-19 disease uses the fusion spike glycoprotein to penetrate into the host cell, and therefore a detailed understanding of this protein forms a critical intervention point in the viral life-cycle. we interrogated the spike protein with a diversity of computational approaches. first, the variability in spike protein from 791 different viral genome sequences was evaluated. residues in the s2 subunit (residue range: 816-1141; hr1, ch, and cd domains) were found to be less evolutionarily variable compared to other regions or domains. by contrast, residues h49y, q239k, v367f, v483a, s943p, k986p, and v987p were found to be the most common amino acid substitutions in the spike protein from related viruses. secondly, md simulations revealed that residues in rbd domain (residue range: 329-521) were more flexible compared to residues in the s2 subunit, making it more complicated for drug design strategies. an examination of less variable regions revealed that the hr1, ch, and cd domains (s2 subunit) located close to the viral transmembrane formed a large cavity or pocket that is formed from three spike monomers. the md simulation traced an "up" active state and a "down" inactive state of the spike protein in its monomer form. slight movement of the trimer cavity within this structural orientation suggests that it could work as "bouncing spring" or "sarrus linkage" when interacting with the host cell receptor. the conversion between "up" and "down" states in the monomer form of spike protein using the in silico methods is defined by the md field to be relatively fast. nevertheless, there are different structural isoforms that have been identified on the spike protein using different experimental methods or virus strains. this indicates that, although conversion may be quick, there are structural endpoints which are "stable". using the recent cryo-em structure of the sars-cov-2 spike protein [18] ; an asymmetric hinge-like movement was observed in only one of the three rbd domains in the s1 subunit, which was also observed in mers-cov and sars-cov [33, 34] . however, there are also other structures where all three rbd domains are in the "up" or "down" conformation [18, 31, 33, 34, 40] . these data suggest a physiological relevance due to heterogeneous protein conformational dynamics. for example, asymmetric conformational flexibility might have a functional role, perhaps in evading the exposure of b-cell epitopes (only one rbd domain is in the "up" conformation) and/or optimized interaction with the ace2 receptor depending on virus strain. in addition, because of the "bouncing spring" mechanism (communication between the trimer pocket and the rbd domain conformation), it is possible that these different spike protein conformational isoforms provide another avenue to develop drug discovery programs that exploit and/or circumvent these dynamics. our investigation into the genomic variation within virus strains, as well as our findings from the md simulations, identified a conserved trimer cavity or pocket formed by the s2 subunit in the spike protein. these findings suggest that a novel target, "the trimer cavity formed by spike protein oligomerization", may be suitable to manipulate viruses of this class. targeting the trimer pocket might identify a new functional class of drugs against this protein. applying the sbvs approach, we docked drug libraries against the trimer cavity with the hypothesis that such a ligand might perturb the predicted "bouncing spring" movement and the homotrimer formation. protein-ligand docking identified severalhits that have already been published or proposed to inhibit the sars-cov-2 virus in cell systems. for example, our studies suggest an action mechanism for molecules such as chitosan and macrolide types (e.g., rapamycin). based on the sequence variability of the coronavirus, including our findings from md simulations of the spike protein, a conserved trimer cavity (hr1, ch, and cd domains) is a feature of the spike protein in most coronaviruses. consistent with this, previous work has shown that the molecule ek1 exhibited potent inhibitory activity against all human coronaviruses (hcovs) tested through binding to the c-terminal hr1 domain [37] . additionally, the "up" and "down" conformations of rbd domain observed during md simulations, supports that concept that the spike protein can also be a target of a possible igg therapeutic [92] . from the list of the top compounds identified that dock into the trimer cavity, some of them have already been validated or suggested as sars-cov-2 virus inhibitors in cells, including; a chitosan derivative [54] [55] [56] , rapamycin [47] [48] [49] , everolimus (rad001) [49] , paclitaxel [79] , ritonavir [48, [50] [51] [52] , selameerin (selamectin) [80] , and danoprevir [52] . among these, a modified polymeric version of the chitosan drug (a top hit in our analysis) was recently shown to inhibit cov replication with evidence that the molecule inhibits the binding of the viral spike protein to the host ace2 receptor [54] [55] [56] . the protein-protein interaction map or the network-based methodologies [14, 43] suggest that sirolimus (rapamycin) emerges as a common potential drug lead for repurposing against covid-19. this rapamycin (mtor inhibitor) drug was found previously to disrupt larp1 (la-related protein 1) and mtorc1 (mammalian target of rapamycin complex 1) binding, and has been shown to reduce mers infection by~60% in vitro [47] . the postulated geroprotectors, such as sirolimus (rapamycin) and its close derivative, the rapalog everolimus (rad001), decreased infection rates in a small sample of elderly patients [49] . moreover, the drugs sirolimus (rapamycin) and ritonavir are currently in clinical trials for repurposing against covid-19 [48, 50, 51] . sirolimus (rapamycin) is registered in a clinical trial (nct03901001 not yet recruiting) designed to evaluate adjunctive use of sirolimus (rapamycin) and oseltamivir in patients hospitalized with influenza [47, 48] . ritonavir, a hiv protease inhibitor is in an open-label trial in hospitalized adults with severe covid-19 [48, 51] . the data from this small-sample clinical study showed that danoprevir boosted by ritonavir is safe and well tolerated in all patients [52] . selamectin is a potential drug for treating covid-2019 found active against the pangolin coronavirus gx_p2v, a workable model for sars-cov-2 research [80] . the antitumor drug paclitaxel increases cellular methylglyoxal to virucidal levels, providing a rationale for repurposing doxorubicin and paclitaxel for covid-19 treatment [79] . nevertheless, whether the hit molecules we have identified that dock into the trimer cavity and impact on the virus life cycle requires orthogonal validation. we hope the findings of our study can help to understand the function of the highly conserved spike interspecies transmission and emergence of novel viruses: lessons from bats and birds severe acute respiratory syndrome coronavirus as an agent of emerging and reemerging infection middle east respiratory syndrome coronavirus: another zoonotic betacoronavirus causing sars-like disease genomic characterization of the 2019 novel human-pathogenic coronavirus isolated from a patient with atypical pneumonia after visiting 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results. key: cord-290286-3t0roikx authors: arango-lasprilla, juan carlos; zeldovich, marina; olabarrieta-landa, laiene; forslund, marit vindal; núñez-fernández, silvia; von steinbuechel, nicole; howe, emilie isager; røe, cecilie; andelic, nada title: early predictors of employment status one year post injury in individuals with traumatic brain injury in europe date: 2020-06-26 journal: j clin med doi: 10.3390/jcm9062007 sha: doc_id: 290286 cord_uid: 3t0roikx sustaining a traumatic brain injury (tbi) often affects the individual’s ability to work, reducing employment rates post-injury across all severities of tbi. the objective of this multi-country study was to assess the most relevant early predictors of employment status in individuals after tbi at one-year post-injury in european countries. using a prospective longitudinal non-randomized observational cohort (the collaborative european neurotrauma effectiveness research in tbi (center-tbi) project), data was collected between december 2014–2019 from 63 trauma centers in 18 european countries. the 1015 individuals who took part in this study were potential labor market participants, admitted to a hospital and enrolled within 24 h of injury with a clinical tbi diagnosis and indication for a computed tomography (ct) scan, and followed up at one year. results from a binomial logistic regression showed that older age, status of part-time employment or unemployment at time of injury, premorbid psychiatric problems, and higher injury severity (as measured with higher injury severity score (iss), lower glasgow coma scale (gcs), and longer length of stay (los) in hospital) were associated with higher unemployment probability at one-year after injury. the study strengthens evidence for age, employment at time of injury, premorbid psychiatric problems, iss, gcs, and los as important predictors for employment status one-year post-tbi across europe. traumatic brain injury (tbi) is one of the leading causes of disability and death worldwide [1, 2] . every year millions of individuals around the world suffer from tbi [3] . in those who survive, injuries are often associated with the presence of physical, cognitive, and emotional difficulties [4] in the short and long term. these tbi-related impairments generate problems with personal, social, family, and work reintegration following injury [4, 5] . past studies have reported employment rates that range from 8-72% in individuals with tbi during the first year after the injury [6] [7] [8] . factors influencing employment status include age at the time of trauma [9] [10] [11] [12] [13] [14] , gender [9, 12, [15] [16] [17] , ethnicity [17] , marital or partner relationship status prior to injury [9, 15, [18] [19] [20] , education level [8, 10, 13, [19] [20] [21] [22] , previous employment status [8, 10, 13, 15, 18, 21, [23] [24] [25] , occupation type [15, 18, 26] , severity of the injury [8, 11, [13] [14] [15] 18, 23, [27] [28] [29] [30] , cognitive functioning [11, 14, 16, 27, 31] , depression status [27] , and having autonomy in transportation [16, 19, 27, 32, 33] . knowledge regarding factors that are associated with employment status in individuals with tbi one year after the injury is of great importance in order to develop and implement early rehabilitation programs and design vocational rehabilitation with appropriate follow-ups to ensure successful and lasting return to the workplace. however, the majority of studies on employment status have been carried out in non-european countries [9, 20, 21, 27, 34] . european countries exhibit important differences geographically, with respect to their populations, economically and mainly, in the type of health and social care system as compared to non-european countries. the european health systems guarantee universal health care access and sickness benefits regardless of the level of income [35, 36] ; access that is not readily available for individuals with insufficient financial resources in countries outside europe. in europe, 2.5 million individuals sustain a tbi every year [1, 37] and the economic costs of the treatment of these patients exceed approximately 49.7 billion usd annually [1] , which generates high expenses for the health systems of these countries. in addition, many of these individuals never return to work, which also increases economic costs due to loss of labor productivity. it is important to study early predictive factors involved in employment status following tbi in european countries to contribute to the development and implementation of future work reintegration and employment support programs for individuals with tbi in a universal health care system setting. hence, the objective of this study was to assess early relevant predictors of employment status in individuals hospitalized with tbi one year after the injury in european countries. this multi-country study, including all tbi severities, is part of the collaborative european neurotrauma effectiveness research in tbi (center-tbi) project [38] ; thus, it should be possible to generalize the results to various geographical regions and populations. data were collected within the center-tbi project, from december 2014 to december 2019. center-tbi is a prospective longitudinal non-randomized observational study across the severity spectrum of tbi from 63 centers in 18 european countries. data were retrieved from the center-tbi database using the data access tool neurobot and core 2.0 sample (dataset frozen in may 2019). inclusion criteria for the core data set were: (1) a clinical diagnosis of tbi, (2) indication for computed tomography (ct) scan, (3) enrollment within 24 h after injury, and (4) consent for study participation. the core dataset included three strata that were differentiated according to care paths: patients seen in the emergency room (er); patients admitted to the intensive care unit (icu); and patients primarily admitted to the hospital ward (adm). patients with a severe pre-existing neurological disorder were excluded as such conditions may bias outcome assessments. for more details, see maas et al. [38] and steyerberg et al. [39] . the complete core data set consisted of n = 4509 individuals. according to the study design, patients admitted to the emergency room (er) stratum were not interviewed one-year post tbi; thus, 3661 patients admitted to the ward or the icu stratum are the subject of the current report. the present study had the following inclusion criteria: (1) potential labor market participant (excluding retired persons, students, and homemakers), (2) 16 years of age or older, and (3) participated in the study at one-year follow-up. from the core data set, n = 1015 individuals met the study inclusion criteria; for details, see the sample flow chart in figure 1 . j. clin. med. 2020, 9, x for peer review 3 of 16 in the emergency room (er); patients admitted to the intensive care unit (icu); and patients primarily admitted to the hospital ward (adm). patients with a severe pre-existing neurological disorder were excluded as such conditions may bias outcome assessments. for more details, see maas et al. [38] and steyerberg et al. [39] . the complete core data set consisted of n = 4509 individuals. according to the study design, patients admitted to the emergency room (er) stratum were not interviewed one-year post tbi; thus, 3661 patients admitted to the ward or the icu stratum are the subject of the current report. the present study had the following inclusion criteria: (1) potential labor market participant (excluding retired persons, students, and homemakers), (2) 16 years of age or older, and (3) participated in the study at one-year follow-up. from the core data set, n = 1015 individuals met the study inclusion criteria; for details, see the sample flow chart in figure 1 . employment status. employment status was assessed at baseline and at follow up at 12 months after the injury. for the purpose of analysis, employment status before injury was recoded into three groups at baseline: employed full-time (i.e., working ≥ 35 h per week); employed part-time (i.e., working 20-24 h/week, ≤ 20 h/week, currently on sick leave, or in sheltered employment); or unemployed employment status. employment status was assessed at baseline and at follow up at 12 months after the injury. for the purpose of analysis, employment status before injury was recoded into three groups at baseline: employed full-time (i.e., working ≥ 35 h per week); employed part-time (i.e., working 20-24 h/week, ≤ 20 h/week, currently on sick leave, or in sheltered employment); or unemployed (unemployed or unable to work). one year after tbi, the employment status was collapsed into two groups: employed (including return to previous job, also with less hours, change of job, or sheltered employment) and unemployed (including unemployed or unable to work). sociodemographic variables. the following sociodemographic variables were collected at the time of injury: sex (male or female), age in years, marital status (single or partnered (married, cohabitating), education (post-high school (including individuals currently in diploma or degree-oriented program, or post-high school training), secondary/high-school, or only primary school/others). the presence of premorbid psychological/psychiatric problems (yes or no) was characterized by anxiety, depression, sleep disorders, schizophrenia, drug abuse, or other psychiatric problems in the medical history of patients. injury related factors. the following injury-related variables were collected in the acute phase: cause of injury, tbi severity assessed by gcs, injury severity score (iss), loss of consciousness (loc), and length of stay in hospital (los). for the present study, recruitment occurred in two strata according to care pathways: admission stratum (adm) or intensive care (icu) stratum. the cause of injury was collapsed into three groups: road traffic accident, fall, or violent injury (violence/assault, act of mass violence, or suicide attempt) combined with other causes. tbi severity was evaluated with the glasgow coma scale (gcs [40] ). the score ranges from 3-15 (a higher score indicates a higher level of responsiveness), with 15 representing normal level. the injury severity score (iss), an indicator of overall trauma severity, was calculated as the sum of the squares of three highest values of the abbreviated injury scale score (ais) [41] from different body regions. the iss ranges from 0-75 (a higher score indicates greater severity of the trauma). loss of consciousness (loc) covered three categories: yes (including suspected loc), no, or unknown. the unknown loc was treated as missing. the assessment was based on self-report, clinical interview, or the medical chart. length of stay (los) in the hospital was determined in days beginning with enrollment. it was derived using the information of the date and time of arrival at the study hospital and date and time of hospital discharge. due to the skewed distribution, the variable was logarithmized for further analyses. the center-tbi study (ec grant 602150) has been conducted in accordance with all relevant laws of the eu if directly applicable or of direct effect and all relevant laws of the country where the recruiting sites were located. for further information on ethical approval, see https://www.center-tbi. eu/project/ethical-approval. informed consent was obtained for all patients by the patients themselves and/or the legal representative/next of kin. analyses proceeded according to the following strategy: (1) estimation of the initial theory-based model, (2) backward selection of the predictors based on a liberal p-value (p < 157) to determine factors contributing significantly to the model fit, and (3) estimation and internal validation of the final model. to examine the predictors of employment status one year after tbi, a binomial logistic regression was performed. employment status (employed/unemployed) one-year post-injury served as the dependent variable, with prediction of the probability of employment. the independent variables were derived from previously published research described in the introduction and were grouped as follows: sociodemographic and premorbid factors (sex, age, marital status, education, employment status at time of injury, and premorbid psychiatric problems), and injury related factors (stratum, injury cause, gcs, iss, presence of loc, and los in the hospital). the amount of missing values varied from <0.01% (injury cause) to 11.9% (loc). missingness at random was assumed, and values were imputed using the multiple imputation by a chained equations procedure (mice [42] ) using the mice-package in r [43] . overall model performance was assessed by performing a bootstrapping validation on imputed data and assessing optimism-corrected nagelkerke's r 2 [44] and area under the curve (auc) (e.g., reference [45] ). nagelkerke's r 2 ranges from 0-1, demonstrating the relative information gain of the estimated model compared to a null model, which contains only the intercept without any predictors. the higher the nagelkerke's r 2 , the better the model. the auc assesses how well the model can distinguish between the groups of the dependent variable. the higher the auc, the better the ability of the model to distinguish between employed and unemployed groups one year after tbi. for optimism correction, a difference in performance measures between models in bootstrap samples and models applied on original data was calculated. all analyses were conducted using r version 3.6.1 [46] under application of the psfmi-package for model estimation and validation [47] . in the stepwise procedure, a liberal alpha-value of α = 157 was applied. for all other analyses, the significance was set at 5% (α = 0.05). the data set consisted of n = 1015 individuals (25% female), with a mean age of 44.20 years (±13.70), who participated in the assessments one year after tbi and who were admitted to the study hospital either to a ward or an icu. table 1 shows sample characteristics for dependent and independent variables (including factor levels) for original and imputed data (mean of five imputed data sets). the numbers in parentheses are used for further references in the text. the majority (60.2%) experienced a mild tbi (gcs ≥ 13), followed by severe (24.7%; gcs ≤ 8), and moderate (11.3%; gcs 9-12) tbi. the mean gcs was 11.63 (sd = 4.33, iqr = 7). the average injury severity score was iss = 24.56 (sd = 15.79, iqr = 21). in total, 68% had an iss > 15, indicating a major trauma. patients were admitted for on average two and a half weeks at the study hospitals (m = 16.94 days, sd = 24.97, iqr =18.28). 100.00% -note: numbers in parentheses are used for indication in the text and further tables (except the dependent variable and the traumatic brain injury (tbi) severity groups); na = not available, missing data; a for continuous variables and the total scores, mean (m) and standard deviation (sd) are reported. iss = injury severity score; gcs = glasgow coma scale score; loc = loss of consciousness; los = length of stay in hospital; b tbi severity is computed as follows: mild tbi (gcs ≥ 13); moderate (9 ≤ gcs ≤ 12), and severe (gcs ≤ 8); c the group violent/other consisted of the following subgroups: other non-incidental injury (n = 57), violent/assault (n = 51), mass violence (n = 2), suicide attempt (n = 11), and other and unknown (n = 69); d data was imputed using the multiple imputation by a chained equations (mice) procedure, mean values from five imputed data sets were calculated to provide descriptive statistics (reported only for variables used in the model with missing values). figure 3 visualizes the relative frequencies of employed individuals at the baseline and one year after tbi by tbi severity groups. at the baseline, 10-12 percent of individuals were unemployed. while the majority of individuals after a mild or moderate tbi (71% and 57%, respectively) returned figure 3 visualizes the relative frequencies of employed individuals at the baseline and one year after tbi by tbi severity groups. at the baseline, 10-12 percent of individuals were unemployed. while the majority of individuals after a mild or moderate tbi (71% and 57%, respectively) returned to work one year after tbi, 59% of those suffering from severe tbi were unemployed. employed: individuals were in labor market or currently at sick leave split in full-time and part-time groups; and unemployed: individuals were out of labor market. at one-year post tbi, employed individuals returned to work (either with the same amount of hours or with reduced hours, or individuals who changed the work); and unemployed individuals, who are unable to work and individuals looking for work. figure 3 visualizes the relative frequencies of employed individuals at the baseline and one year after tbi by tbi severity groups. at the baseline, 10-12 percent of individuals were unemployed. while the majority of individuals after a mild or moderate tbi (71% and 57%, respectively) returned to work one year after tbi, 59% of those suffering from severe tbi were unemployed. the initial model estimation was based on the employment status at follow up as a dependent variable and factors derived from previous research (no. (1) (2) (3) (4) (5) (6) (7) (8) (9) (10) (11) (12) in table 1 ). the backward predictor selection revealed a simplified model which included the following sociodemographic, premorbid, and injury-related factors significantly influencing the probability of employment one year after tbi: age (2) , employment status at baseline (5), premorbid psychological/psychiatric problems (6), iss (9), gcs score (10) , and los at the hospital (12). according to the final model, five (2,5,6,9, and 12) out of six factors (higher age, less than full-time employment (part-time employment or unemployment at time of injury), higher iss and longer los in the hospital) were significantly associated with decreased probability of employment one year after tbi and one factor (10) (higher gcs score) significantly increased the chance of being employed (see figure 4 for visualization). for detailed results of the model estimation, see table 2 . according to the final model, five (2,5,6,9, and 12) out of six factors (higher age, less than fulltime employment (part-time employment or unemployment at time of injury), higher iss and longer los in the hospital) were significantly associated with decreased probability of employment one year after tbi and one factor (10) (higher gcs score) significantly increased the chance of being employed (see figure 4 for visualization). for detailed results of the model estimation, see table 2 . sociodemographic factors. when keeping other factors constant, the probability of employment decreased by 3% per each one-year increase in age. part-time employees and unemployed individuals (5) were also characterized by reduced likelihood of being employed one year after tbi compared to those working full-time at the time of injury. premorbid psychiatric history. individuals suffering any psychiatric problems (6) prior to tbi were less likely to be employed one year after tbi. injury related factors. a higher total iss (9) was associated with decreased probability of employment at the follow up. with one-point increase in iss, the probability of employment decreased by 2%. with one-point increase in gcs score (10) , the employment probability 12 months after tbi increased by 7%. a longer los in the hospital (12) caused a significant decrease in employment probability 12 months after tbi. corrected nagelkerke's r 2 showed the value 0.30 (optimism correction of 0.02), indicating a prediction improvement from the null model to the final fitted model. the corrected auc value was 0.79 (optimism correction of 0.01), which means that 79% of the employed individuals were classified as such in the model. the main purpose of this study was to determine early predictors associated with employment status one-year post-tbi in european countries. the results of this study emphasize the diminished employment rates among patients with tbi one-year post-injury and highlights some of the sociodemographic, premorbid, and injury-related factors associated with employment probability at this time point. indeed, older age, status of part-time employment or unemployment at time of injury, higher injury severity (as measured with higher iss, lower gcs, and longer los in the hospital), and premorbid psychiatric problems were all associated with increased unemployment probability at one year after injury. in the present study, the rate of employment (61%) one-year post-injury was higher compared to the majority of studies that have been published previously. for instance, the most recent systematic review/meta-analysis on employment outcomes world-wide found an employment rate of 35% one year following moderate to severe tbis [6]. in addition, the rate of employment was higher compared with those reported from other countries, such as the netherlands (50% complete return to work [48] ), australia (56% considering competitively employed and paid work trial [13] ), norway (50% [15] ; 55% [14] ), and the united kingdom (74% [49] ; 60-75% [50] ), and is higher compared to those reported from the usa based on the tbi model systems (range between 28% to 35% [19, 34, 51, 52] ), and lower compared to the reported rate from taiwan (79% [27] ). the higher employment rates could be explained by methodological differences between the studies regarding injury severity, the definition of employment, and timing of assessment, among others. regarding injury severity, in the current study, the majority of the sample had mild tbi (62.5%), followed by severe (25.7%), and moderate (11.8%) injury. thus, it is not surprising that the rate of employment is higher than the majority of the studies mentioned above as they had higher percentages of individuals with moderate to severe tbi [19, 34, 51, 52] . the definition and categorization of employment status vary across studies. for example, some researchers [21, 34] have included students as "unemployed", while others [14, 15, 53] have students included as "employed". additionally, some studies only include full-time competitive employment in the "employed" category [9] , whereas others also include part-time employees [15] . of course, as sigurdardottir et al. [14] indicated, systemic factors are likely to be relevant, too, such as healthcare and insurance systems, cultural values, laws, and customs-related factors. studies may also vary according economic fluctuations related to the recruitment time-window (< 1990, 1990-2000, 2000-2010 , and 2010-2020 decades). across this period of time, different economic crises and recessions occurred (e.g., early 1990s recession, which affected western countries; early 2000s recession, which impacted on the eu and the usa; 2007-2008 global financial crisis; 2009-2019 eurozone crisis) that surely affected employment rates. it is well known that, during an economic recession, individuals with disabilities are a vulnerable group that may be affected negatively by layoffs and loss of employment opportunities [54] . this is correspondingly a concern with regards to the current severe acute respiratory syndrome coronavirus 2 (sars-cov-2) pandemic with huge global economic impact. in addition, a government's passive and active employment policies and strategies may impact on the general population's employment rates, as well as on the rates of individuals with disabilities. for example, the european commission [55] developed the "european disability strategy 2010-2020" to empower individuals with disabilities to participate fully in the society and the economy. more recently, vocational rehabilitation programs are gradually implemented at rehabilitation centers, and this may improve employment rates in countries where these programs have been introduced. lastly, advancements in technology available for most individuals (e.g., memory aids, as well as augmentative and alternative communication technologies) could also support individuals with tbi in fulfilling their duties at work. pre-injury employment status is one of the most consistent factors associated with employment one year after injury and later on, with a strong level of evidence reported in various systematic reviews [10, 56] . as found within the current study, being unemployed at the time of injury has been linked to lower probability of employment one-year post-injury [13, 21, 24, 57] . furthermore, a recent study reported that the rate of job change after injury differs between individuals according to tbi severity from pre-injury to 12 months follow-up (19% of moderate/severe group; 12% of the mild group) [49] . it seems that not only the previous employment status is relevant but also prior type of occupation, with a consistent effect on employment rates regardless of educational level [26] . those patients with previous professional and managerial positions (white collar) had the greatest probability of being employed one-year post-injury compared to manual labor occupations (blue collar) [15, 18, 26] . as the present study includes both employed and unemployed individuals, the usage of type of occupation as a predictor would lead to biased results as unemployed individuals could not be arranged to any type of occupation group. the level of education has also been consistently associated with employment status in this population, with a strong level of evidence [10] . several previous studies [21, 26, 34, 51] have found that patients with less than high-school education showed a lower chance of employment post-injury. however, education was not found to be a significant predictor in the present study. in accordance with the literature, sex was not associated with employment status, and marital status has been established as having a weak level of evidence [10] . regarding age, the current study found a negative relationship with employment status, with older age increasing the probability of being unemployed. although age has been reported with higher (younger age) or lower (older age) probability for employment after a tbi [11, 13, 14, 27] , the systematic review by willemse-van son et al. [56] suggests an inconclusive level of evidence. again, this pattern of relationship with employment status is not exclusive for individuals after tbi: in general, unemployed older workers have greater difficulties in finding a new job compared to unemployed younger workers [58] . simultaneously, employment stability has been shown to be lower among younger individuals after tbi [18, 59] , perhaps due to less vocational skills and experience to aid in return to work (i.e., not yet achieved white collar level), lesser seniority if cut-backs in economic hardship, or merely due to a normal trend of increased job changes earlier in careers. a history of premorbid psychiatric problems (i.e., anxiety, depression, sleep disorders, schizophrenia, drug abuse, or other psychiatric problems) significantly reduced employment probability one year after injury. several conditions comprised in the variable in this study have been assessed as separate predictors in previous studies. for example, several studies demonstrated that pre-injury drug abuse is a significant predictor of unfavorable employment outcome [20, 22, 25, 56, 60] . having a prior psychiatric diagnosis and receiving psychological treatment pre-injury have also been associated with reduced likelihood of employment post-tbi [22, 61] . moreover, a systematic review of acquired brain injury found evidence for increased risk of developing new psychiatric disorders in individuals with a history of psychiatric problems [62] . thus, individuals who have experienced premorbid psychiatric problems may also have increased risk of psychiatric problems affecting work participation post-injury. the results of the logistic regression analyses indicate that, among injury-related factors, total iss, gcs, and los in the hospital are associated with employment status after a tbi one-year post-injury. los in the hospital has a strong level of evidence according to a systematic review [10] , with patients who have shorter los in the hospital having greater chance of being employed. this is consistent with injury severity, measured in this study with iss and gcs, given that individuals with less severe injuries usually have shorter hospital stays. most studies have used gcs, presence of post-traumatic amnesia (pta) or loc, or the disability rating scale (drs) to measure injury severity, with results suggesting that patients with less severe injury (regardless of instrument used) have higher probability of being employed [13] [14] [15] 18, 21, 27, 34] . interestingly, according to systematic reviews, while the level of evidence for gcs as a predictor of employment status in this population remains inconclusive [10, 56] , it seems that the level of evidence for drs has changed from strong [56] to weak [10] . chien et al. [27] found that iss was more accurate at predicting employment status in their sample compared to gcs, glasgow outcome scale (gos) and glasgow outcome coma scale-extended (gose) [63] . perhaps iss as an indicator of overall trauma severity is more sensitive to predict employment status at one-year post-injury, at least compared with gcs, as other injuries may affect return to work irrespective of the severity of the tbi. functional outcomes, such as autonomy for activities of daily living or independence in transportation, may be better prognostic factors after tbi, as these variables (measured most of the time with the functional independence measure) have been reported to be related with returned to work one-year post-injury [11, 19, 21, 26, 27, 49] . unlike previous tbi studies, injury cause was not significantly associated with employment status. violent cause of injury has been related with higher odds of being unemployed one-year post-injury [21, 34] . interestingly, these studies have been conducted in the usa, where assaults are the fourth leading cause of tbi (10%), after falls (35%), motor vehicle crashes (17%), and blunt impact (17%) [64] . however, in europe, the leading causes of tbi are falls and road traffic accidents [65] . in the present study, due to low prevalence, violence-related tbi was combined with other causes, which might explain the non-significant association with employment outcome. returning to employment after a tbi is a complex process that encompasses multiple individual, injury-related, and work-environment related factors. in this study, some of the factors, such as pre-injury or post-injury social support/employment support and others that have been reported previously as potential predictive factors, were not considered, such as cognitive functioning following injury [14, 57] , pre-injury health-related quality of life [49] , self-awareness and self-monitoring [53] , and occupational status [26] , among others. the majority (60.2%) of patients in this study had a mild tbi, as assessed by the gcs (13) (14) (15) . this is in line with other studies conducted across all tbi severity levels in patients admitted to trauma hospitals [65] . thus, the rates of employment may be representative for individuals with both mild and moderate/severe tbi admitted to a hospital setting. as a multi-country study across european countries, the present study is valuable as it confirms previous study results and strengthens the evidence base regarding which factors are important for the employment status at one-year post-injury. single analyses for the respective european countries were beyond the scope of this study and are a topic for future ones. it is common practice in research to exclude individuals with pre-existing psychiatric diagnoses from studies after tbi as the diagnoses may confound outcomes. however, this leads to a selection bias and a knowledge gap regarding outcome trajectories for individuals with tbi and associated premorbid psychiatric problems. inclusion of these patients is thus a strength of the present study. finally, a promising relatively new aspect that may impact probability of employment at one-year post-injury is whether individuals are participating in a vocational rehabilitation program, as an increased support in the return to work process may impact employment rates. we did not analyze data on professional help and involvement in vocational rehabilitation among the present population, and this remains an area for future study. interestingly, radford et al. [50] found that more participants who underwent a vocational rehabilitation program returned to work, had more workplace adjustments, and felt greater employer support 12 months post-injury than those in usual care. taken together, unmodifiable factors, such as age, pre-injury employment, premorbid psychiatric problems, and injury severity-related characteristics, were predictive of employment status one-year post-tbi in this large, multi-country observational european cohort study. vocational rehabilitation programs following tbi should take into account not only these factors but also possible modifiable work environment efforts, such as tailoring work tasks, supplying coaching in the workplace, ensuring workplace environmental support, flexible work hours, or graded work, etc. according to a recent systematic review on characteristics of work and workplaces that retain employees after acquired brain injury [66] , this is an under-researched area with too few high-quality studies. to increase return to work among individuals after tbi, future methodologically strong studies should focus on work environment interventions that target modifiable work-related factors and better transition from long-term sick leave to work. interestingly, the individual placement and support model has been shown effective in improving competitive employment rates among individuals with moderate-to-severe mental illness [67] and represents a relevant model for future investigation in the tbi population. as part of the center-tbi project, this study investigated some of the early predictors of employment status one year after mild-to-severe tbi among an adult population. the study found that older age, status of part-time employment or unemployment at time of injury, higher injury severity (as measured with higher iss, lower gcs, and longer los in the hospital), and premorbid psychiatric problems were associated with higher unemployment probability at one year after injury. none of these 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conditions of the creative commons attribution (cc by) license key: cord-325559-di8lljoi authors: cappello, francesco; marino gammazza, antonella; dieli, francesco; conway de macario, everly; macario, alberto jl title: does sars-cov-2 trigger stress-induced autoimmunity by molecular mimicry? a hypothesis date: 2020-06-29 journal: j clin med doi: 10.3390/jcm9072038 sha: doc_id: 325559 cord_uid: di8lljoi viruses can generate molecular mimicry phenomena within their hosts. why should severe acute respiratory syndrome coronavirus 2 (sars-cov-2) not be considered one of these? information in this short review suggests that it might be so and, thus, encourages research aiming at testing this possibility. we propose, as a working hypothesis, that the virus induces antibodies and that some of them crossreact with host’s antigens, thus eliciting autoimmune phenomena with devasting consequences in various tissues and organs. if confirmed, by in vitro and in vivo tests, this could drive researchers to find effective treatments against the virus. severe acute respiratory syndrome coronavirus 2 (sars-cov-2) induced disease (covid-19) is a planetary emergency that is urging many research groups to redirect their efforts and to channel their experience towards understanding its pathogenesis. despite many clinical reports and papers on viral genetics, detailed information on pathogenic mechanisms pertaining to covid-19 is still lacking. this type of information will no doubt help physicians in patient management and in providing treatment. the paucity of data on pathogenesis is due to a considerable extent to the very low number of autopsies that have been performed on covid-19 victims [1] . while histopathological and other data from laboratory tests and autopsies will accumulate as the pandemic persists in the next few months or so, some progress can be achieved applying bioinformatics and scientific reasoning. in this brief hypothesis paper, we have organized pertinent information available not only from the growing scientific literature but also from the chats of doctors and researchers on the web that cannot be ignored at this time, although they are not official instruments for dissemination of scientific data. these are temporarily useful channels for disclosing information as it is being generated at the "war front" (i.e., the doctors' offices and clinical departments) that under normal circumstances would be available in the form of scientific publications only many months after the fact. among the numerous articles consulted, some have caught our attention [2] [3] [4] [5] [6] [7] [8] [9] [10] [11] . by reading these and other publications, we arrived at the initial conclusion that covid-19 develops in three steps (figures 1 and 2 ). in the following considerations, we will focus on the disease caused when the virus invades the body via the upper respiratory tract disregarding the other ways of viral entry, which are considerably less frequent as per current data-nevertheless, it is very likely that the conclusions would have also applied to the latter. (1) the virus enters the body through the upper respiratory tract and invades the respiratory mucosa covering the nasal cavities, the paranasal sinuses, and the nasopharynx. here it replicates and encounters immune cells. the immune system, via the waldeyer's ring, recognizes viral antigens activating innate immunity. (2) if the virus is not eradicated at this stage, it reaches the lower airways and enters the bloodstream through the respiratory barrier. the architecture of the primary pulmonary lobules is rapidly subverted by the violent inflammatory response, including both innate and adaptive immune-systems activation (lymphocytes, macrophages, plasma cells, etc.). (3) plasma cells produce antibodies that by the bloodstream (the lung is a highly vascularized organ) can travel throughout the body. (the image of the human body is a courtesy of visible body atlas.). sars-cov-2: severe acute respiratory syndrome coronavirus 2. (1) the virus enters the body through the upper respiratory tract and invades the respiratory mucosa covering the nasal cavities, the paranasal sinuses, and the nasopharynx. here it replicates and encounters immune cells. the immune system, via the waldeyer's ring, recognizes viral antigens activating innate immunity. (2) if the virus is not eradicated at this stage, it reaches the lower airways and enters the bloodstream through the respiratory barrier. the architecture of the primary pulmonary lobules is rapidly subverted by the violent inflammatory response, including both innate and adaptive immune-systems activation (lymphocytes, macrophages, plasma cells, etc.). (3) plasma cells produce antibodies that by the bloodstream (the lung is a highly vascularized organ) can travel throughout the body. (the image of the human body is a courtesy of visible body atlas.). sars-cov-2: severe acute respiratory syndrome coronavirus 2. the first step consists of upper airway infection: the virus colonizes and multiplies in the ciliated columnar epithelial cells of the respiratory mucosa. this phase can be asymptomatic, paucisymptomatic, or symptomatic; in any case, an innate immune response against the virus is triggered. the disease can be resolved at this level (fortunately in most cases) or it can progress to the second step. . at this stage, the disease can be asymptomatic, paucisymptomatic or produce symptoms such as fever, cough, anosmia, ageusia, and shortness of breath. many subjects heal spontaneously. however, in a limited number of subjects the virus moves down to the lower airways, causing severe pneumonia. it is not clear why some patients develop pneumonia and other do not. however, cold weather, high humidity, and severe pollution can be considered prodisease factors because they may favor virus vitality outside the body and inflammatory status inside the airways. most of the patients with pneumonia manage to heal (for example, by ex juvantibus therapies, such as tocilizumab or hydroxychloroquine), however, some of them develop severe complications, i.e., a generalized activation of the immune system manifested as vasculitis, disseminated intravascular coagulation (dic), and other signs and symptoms of autoimmunity. at this point, the risk of developing a multiorgan failure (mof) is high, and the patient may die. the first step consists of upper airway infection: the virus colonizes and multiplies in the ciliated columnar epithelial cells of the respiratory mucosa. this phase can be asymptomatic, paucisymptomatic, or symptomatic; in any case, an innate immune response against the virus is triggered. the disease can be resolved at this level (fortunately in most cases) or it can progress to the second step. the second step is characterized by lung infection (bilateral interstitial pneumonia), which can be of varying severities. in the more fortunate cases, clinicians manage to contain the infection with antiviral and/or anti-inflammatory therapies (or the infection is self-limited, a possibility that cannot be excluded at this time). in more severe cases, for unknown reasons but which are probably related to a "hyperreactivity" of both innate and acquired immunities, the disease progresses towards the third step. all the pieces of information available on the internet agree in indicating that in the third step the disease is systemic (representative examples of clinical and laboratory studies are presented in table 1 ). the emerging picture is that of widespread microvascular damage, diffuse thrombosis, disseminated intravascular coagulation (dic) and, lastly, a multiorgan failure (mof), often leading to death ( figure 3 ). table 1 . examples of reports of generalized immune system activation in covid-19. highlight the association between covid-19 pathogenesis and excessive cytokine release from lungs, such as ccl2/mcp-1, cxcl10/ip-10, ccl3/mip-1a, and ccl4/mip1b. [12] clinical and laboratory compared with nonintensive care unit (icu) patients, icu patients had higher plasma levels of interleukin (il)2, il7, il10, gscf, ip-10, mcp1, mip1a, and tnfα. [13] laboratory sars-cov-2 infection significantly upregulated il6, mcp-1, cxcl1, cxcl5, and cxlc10/ip10. [14] clinical and laboratory a retrospective multicenter study of 191 patients reported more elevated il6 levels in nonsurvivors than in survivors; univariate analysis of the data revealed significant associations of elevated il6 serum levels with mortality. [2] clinical and laboratory compared to moderate cases, severe cases more frequently had dyspnea, lymphopenia, and hypoalbuminemia, with higher levels of alanine aminotransferase, lactate dehydrogenase, c-reactive protein, ferritin and d-dimer, as well as markedly higher levels of il-2r, il-6, il-10, and tnf-α. [15] clinical and laboratory elderly patients and with comorbidities showed higher plasma levels of il6, il10, lactate dehydrogenase, and c reactive protein. [16] clinical and laboratory inflammatory cytokines were more elevated in severe cases than the nonsevere ones, including il-2r, il-6, il-8, il-10, and tnf-α. immunoglobulins (ig) a, igg, and igm and complement proteins (c3 and c4) in patients with covid-19 were within normal range. there were no significant differences in the levels of iga, igg, and complement proteins c3 or c4 between the mild and severe groups, while igm slightly decreased in severe ones. [17] clinical and laboratory concentrations of alanine aminotransferase, aspartate aminotransferase, creatinine, creatine kinase, lactate dehydrogenase, cardiac troponin i, n-terminal probrain natriuretic peptide, and d-dimer were markedly higher in deceased patients than in recovered patients. [18] notes. ccl2: chemokine (c-c motif) ligand 2; mcp-1: monocyte chemoattractant protein 1; cxcl10: c-x-c motif chemokine 10; ip-10: interferon gamma-induced protein 10; ccl3: chemokine (c-c motif) ligand 3; mip-1a: macrophage inflammatory protein 1-alpha; gscf: granulocyte colony-stimulating factor; tnfα: tumor necrosis factor alpha; cxcl1: c-x-c motif chemokine 1; cxcl5: c-x-c motif chemokine 5. it is noteworthy that these patients do not show the typical features of disseminated intravascular coagulopathy (dic). typically, patients with dic present with a considerably prolonged prothrombin time and a major reduction in platelet counts. by contrast, covid-19 patients have a moderately prolonged prothrombin time and platelet counts are often in the lower range of the normal. this strongly indicates that blood-clotting activation in covid-19 is different from the standard dic clotting activation. furthermore, the moderately reduced platelet count clearly resembles an immune complex-mediated prothrombotic disorder, e.g., heparin-induced thrombocytopenia [19] . it is noteworthy that these patients do not show the typical features of disseminated intravascular coagulopathy (dic). typically, patients with dic present with a considerably prolonged prothrombin time and a major reduction in platelet counts. by contrast, covid-19 patients have a moderately prolonged prothrombin time and platelet counts are often in the lower range of the normal. this strongly indicates that blood-clotting activation in covid-19 is different from the standard dic clotting activation. furthermore, the moderately reduced platelet count clearly resembles an immune complex-mediated prothrombotic disorder, e.g., heparin-induced thrombocytopenia [19] . some reports of damage of extrapulmonary organs are listed in table 2 . in addition, at this writing there is growing evidence of autoimmune dermatitis, guillain barre syndrome, and kawasaki disease in some covid-19 patients, particularly the younger ones [20] [21] [22] . table 2 . clinical and laboratory evidence of damage to extrapulmonary organs during sars-cov-2 infection. heart blood tests on admission showed most patients had higher levels of creatine kinase isoenzyme-myocardial band (ck-mb), myohemoglobin, cardiac troponin i, and n-terminal probrain natriuretic peptide. [23] liver gastrointestinal tract sars-cov-2 rna was first detected in stool of the first reported covid-19 case in the usa, who also presented with the digestive symptoms of nausea, vomiting, and diarrhea. [4] 1 abbreviations: alt: alanine aminotransferase; ast: aspartate aminotransferase. mortality rate is very high if the patient is male, elderly, with other concomitant pathologies, especially those related to hypertension and/or diabetes [3, 5, 9, 18] . despite all the information summarized above, it is still a mystery what triggers the hyperactivation of the immune system, which is virtually always present. we have elaborated a working hypothesis [27] that we now would like to propose to the scientific community and, thus, provide food for thought and a basis to plan clinical and laboratory research. for many years our group has been studying a class of proteins highly conserved during evolution and organogenesis, the heat shock proteins (hsp), many of which are molecular chaperones. these are typically antistress proteins (asp) that have helped cells, since their origins at the beginning of life on earth, to survive environmental stresses of chemical and physical nature and have, therefore, played an important role in evolution [28] . typically, asps are overexpressed in cells exposed to various kinds of stressors including bacterial and viral infections. hsp/chaperones are essential for survival and maintenance of protein homeostasis in all organisms but, if abnormal can cause disease, the chaperonopathies [28] . understanding the role of these proteins can provide novel elements for researchers and clinicians useful in diagnosis and treatment [29] . in the course of our studies, we came to the conclusion that hsp/chaperones can be involved in molecular mimicry phenomena, most likely because of their long evolutionary history and high degree of structural conservation, which has produced a widespread sharing of various antigen within and across species. hsp/chaperones are very similar in all organisms, from the most primitive unicellular to the most complex multicellular, typically sharing many highly immunogenic epitopes. this situation sets the stage for immunological crossreactivity between hsp/chaperones from all over the spectrum of living organisms. for instance, hsp/chaperones from any organism (bacterium, virus, or protozoan) in the human skin, or gastrointestinal, respiratory, and genitourinary tracts can invade the blood and thus come in contact with the host's immune system. antibodies are formed against the foreign hsp/chaperone that will most likely crossreact with the equivalent molecule of the human host, and this would be a typical example of molecular mimicry [30] . the same can happen with other microbial and human molecules because there are epitopes shared not only between hsp/chaperones but also between them and other molecules with different functions [31] [32] [33] [34] . asps, including hsp/chaperones, are typically intracellular molecules, but following stress events that augment their intracellular levels, they undergo post-translational modifications (ptm) and translocate to the plasma-cell membrane with their antigenic epitopes exteriorized on the cell's surface [35] [36] [37] [38] . these human epitopes, in turn, can be recognized by circulating antibodies made against crossreactive microbial antigens; these antibodies behave like autoantibodies, causing the destruction of the stressed cells, representing a typical example of pathology caused by molecular mimicry and manifested as autoimmunity [30] . any asp can be affected by ptm, which may change its properties and functions and make it pathogenic against its own host, generating a chaperonopathy by mistake [39, 40] . we speculated that the progression of covid-19 from step 2 to step 3 relies on molecular mimicry phenomena, as already shown for other viruses (table 3) . table 3 . examples of molecular mimicry involving viruses in disease. alphavirus sequence alignment of structural polyproteins belonging to arthritogenic alphaviruses revealed conserved regions which share homology with human proteins implicated in rheumatoid arthritis. [41] cytomegalovirus human antibodies against ul44 (an obligate nuclear-resident, nonstructural viral protein vital for human cytomegalovirus (hcmv) dna replication) immunoprecipitated nuclear-resident systemic lupus erythematosus autoantigens (namely, nucleolin, dsdna, and ku70). [42] coronaviruses several t-cell lines isolated from multiple sclerosis patients showed cross-reactivity between myelin and coronavirus antigens. [43] enterovirus immunogenic epitopes in enterovirus capsid protein vp1 and procapsid protein vp0 have sequence similarities with diabetes-associated epitopes in tyrosine phosphatase ia-2/iar and heat shock protein 60. [44] epstein-barr virus anti-c1q in systemic lupus erythematosus (sle) patients can be induced by an ebv-derived epitope through molecular mimicry. [45] papillomavirus a potential antigenic mimicry between viral and human proteins may be causative of myalgic encephalomyelitis and chronic fatigue syndrome. [46] in active celiac disease, a subset of antitransglutaminase iga antibodies recognize the viral protein vp-7, suggesting a possible involvement of molecular mimicry in the pathogenesis of the disease. [47] varicella-zoster virus autoantibodies to protein s can induce vasculitis and direct endothelial damage. [48] west nile virus an in-silico analysis unveiled certain sequence similarities between viral antigens and receptor sequence fragments suggesting a molecular mimicry autoimmunization process. [49] zika and dengue viruses anti-non-structural protein 1 antibodies can cross-react with host platelets and endothelial cells. [50] the severe bilateral pneumonia that develops in some individuals causes a drop in the partial pressure of oxygen in the blood. this undoubtedly represents a systemic stress. all cells suffer hypoxia, and this can lead to an overexpression of stress proteins and, in turn, to their change by ptm and translocation to the plasma cell-membrane. this would trigger molecular mimicry phenomena and a pathogenic cascade leading to mof (figure 3 ). it should be clear that this cascade can be triggered also by other crossreactive proteins distinct from asp. therefore, the search for the protein responsible for molecular mimicry cannot be limited to asps but must be extended to a wider range of cellular proteins. this search, now, is really a conundrum calling for concerted efforts of many research groups worldwide (figure 4 ). it is important to bear in mind that, in addition to autoantibodies and their complexes with soluble or cell-surface antigens, other effectors of autoimmunity such as immunocompetent cells should also be sought for and characterized to obtain a comprehensive picture of the pathogenic mechanism underpinning tissue damage. , which may be irreversible without proper medical intervention. even with prompt medical intervention, the disease may follow its course and cause death. at the moment, there is no specific therapy for covid-19, but clinicians use ex juvantibus therapy based on anti-inflammatory drugs such as tocilizumab (that inhibits il6) and hydroxychloroquine (inhibits il-1 and tnf-alfa); it is noteworthy that both drugs are used with success in autoimmune diseases. we hypothesize that, at the basis of the generalized activation of the immune system, there are molecular mimicry phenomena: the antibodies produced against the virus could turn into autoantibodies against crossreactive proteins expressed on human cells, causing autoimmunity with cell destruction. what proteins? which cells? what are the predisposing factors? furthermore, can there be protective factors? all of these are open questions now, although there are several clues that show directions for research in the immediate future. for example, one possible pathogenic mechanism of tissue damage is antibody dependent enhancement (ade) of sars-cov-2 due to cross-reactivity. ade has been recently claimed as a mechanism favoring middle east respiratory syndrome coronavirus (mers-cov) entry into host cells [51] . however, in a sars-cov macaque infection model, anti-spike igg antibodies bind to the fcγr on alveolar macrophages and promote their activation with release of massive amounts of profigure 4 . working hypotheses. the establishment of generalized signs and symptoms of immune system activation indicates a serious aggravation of the covid-19, which may be irreversible without proper medical intervention. even with prompt medical intervention, the disease may follow its course and cause death. at the moment, there is no specific therapy for covid-19, but clinicians use ex juvantibus therapy based on anti-inflammatory drugs such as tocilizumab (that inhibits il6) and hydroxychloroquine (inhibits il-1 and tnf-alfa); it is noteworthy that both drugs are used with success in autoimmune diseases. we hypothesize that, at the basis of the generalized activation of the immune system, there are molecular mimicry phenomena: the antibodies produced against the virus could turn into autoantibodies against crossreactive proteins expressed on human cells, causing autoimmunity with cell destruction. what proteins? which cells? what are the predisposing factors? furthermore, can there be protective factors? all of these are open questions now, although there are several clues that show directions for research in the immediate future. for example, one possible pathogenic mechanism of tissue damage is antibody dependent enhancement (ade) of sars-cov-2 due to cross-reactivity. ade has been recently claimed as a mechanism favoring middle east respiratory syndrome coronavirus (mers-cov) entry into host cells [51] . however, in a sars-cov macaque infection model, anti-spike igg antibodies bind to the fcγr on alveolar macrophages and promote their activation with release of massive amounts of pro-inflammatory cytokines [52] . by analogy, anti-sars-cov-2/anti-asp cross-reactive antibodies may similarly mediate ade and contribute to tissue damage. this and other similar hypotheses need to be clarified. to test the proposed working hypotheses, several steps are necessary, for example: (1) in silico comparison of epitopes of viral and human proteins, considering all these as putative autoantigens; (2) screening the results from the in silico studies, using the clues provided by epidemiological and clinical data being generated as the pandemic continues, to identify the protein(s) candidates; (3) immunohistochemical and other molecular analyses of tissues obtained from autopsies of covid-19 fatalities to determine if, and where, these crossreactive molecules are expressed and are indeed reactive with pertinent antibodies. what are the main clues that epidemiology and the clinics provide to date? they are many and disparate. we have listed some of them in figure 4 and these can be classified into negative and positive prognostic factors. in brief, from an epidemiological point of view [6] , the main negative prognostic factors are the subject's advanced age, the presence of comorbidities (hypertension and dysmetabolism), and the male sex. conversely, main positive prognostic factors are young age (very few children are affected by the severe form of the disease), being female, and living in certain geographical areas. the latter might depend not only on the degree of environmental pollution or type of climate but also on genetic-driven protection that individuals might have developed by being exposed to other disease-causing agents. alongside these epidemiological indications, there are others that come from the clinic [3] [4] [5] 8, 11] . the disease, in the third step, involves endothelial cells and/or platelets and/or erythrocytes, more than other cells in the human body: this is suggested by signs of dic and anemia often found by clinicians in sars-cov-2 infected patients. furthermore, at this writing, it cannot be excluded that the renal failure that develops in many patients is not due either to the deposition of preformed circulating immune complexes or to the formation of immune complexes made by circulating antibodies bound to kidney antigens in situ. last but not least, a critical examination of the molecular mechanisms underlying the efficacy of drugs that are currently being tested with some success as ex juvantibus therapy should not be overlooked, since it may offer further cues to unveil unknown pathogenic mechanisms. all these clues, and others that may be revealed in the coming weeks from clinical and histological investigations, should guide researchers towards confirmation or exclusion of molecular mimicry as a determinant pathogenic factor. the understanding of sars-cov-2 phenotype using modern bioinformatics is critical to identify target proteins and shared epitopes between human and viral proteins. here, we want to provide some preliminary insights. many research groups have described the virus by performing structural studies or extrapolating information available pertinent to other coronaviruses. by resorting to previously known information on genome sequences and protein structures and functions as well, bioinformaticians have been successfully assisting virologists by structurally characterizing proteins of the novel virus, determining the evolutionary trajectories, identifying interactions with host proteins, and providing other important biological insights. the whole genome of sars-cov-2 was sequenced, and the sequence is available in genbank (accession number mn908947.3). structural and nonstructural proteins were identified (12 reported in genbank) and they are available in protein data bank (pdb) database and the universal protein resource (uniprot). moreover, many bioinformatics tools are currently used in the scientific literature to understand sars-cov-2 properties and in many cases are easily available online like clustal omega (embl-ebi, cambridge, uk), blast, modeller, mega-x, swiss-model (expasy, sib bioinformatics resource portal, lausanne, switzerland), just to mention a few examples. sars-cov-2 is a spherical or pleomorphic enveloped particle containing single-stranded rna associated with a nucleoprotein within a capsid comprised of matrix protein. the envelope bears club-shaped glycoprotein projections [53] . sars-cov-2, as other coronaviruses, has four structural proteins, known as the s (spike), e (envelope), m (membrane), and n (nucleocapsid) proteins. each of these proteins have a function since the n protein holds the rna genome while the s, e, and m proteins together make the viral envelope [54] . the spike protein, which has been visualized at the atomic level using cryogenic electron microscopy [55, 56] , is the protein responsible for allowing the virus to attach to and fuse with the membrane of a host cell and for this reason it has captured major interest in the scientific community [54] . experiments on the spike protein demonstrated that it has enough affinity to angiotensin converting enzyme 2 (ace2) on human cells, supporting the idea that ace2 is the cell entry receptor [10, 55, 56] . the s protein is composed of two functional subunits (s1 and s2) responsible for receptor binding and membrane fusion, respectively. the surface of the virally encoded envelope spike presents an array of host-derived glycans with each trimer displaying 66 n-linked glycosylation sites. this extensive glycosylation has important implications for vaccine design [57] . recently, a comparative analysis of sars-cov-2 proteins with human proteins was performed in search of high local homologous matches [58] . only one immunogenic epitope in sars-cov-2 had no homology to human proteins and it was concluded that, if all the parts of the epitopes that are homologous to human proteins are excluded from consideration due to risk of pathogenic priming, the remaining immunogenic parts of the epitopes may be still immunogenic and remain as potentially viable candidates for vaccine development. these results likely support our hypothesis and should prompt more investigations on this issue. covid-19 represents a global challenge for the medical community, researchers, and practitioners alike. we were not prepared from the health and social perspectives to face a pandemic, and states around the world are trying to adapt and find the best countermeasures and researchers are doing the same. at this moment, it is important not only to share the results, which may be few, but also the ideas, as these can serve as a stimulus to find solutions to the problems. with this short article, we wanted to offer our contribution, however small it might be, to face the challenge of the covid-19 pandemic, stimulating the scientific community to investigate the involvement of molecular mimicry in the pathogenesis of covid-19. this could be useful not only to reveal the pathogenetic mechanisms underpinning morbidity and mortality but also to direct the development of novel therapeutic strategies and a vaccine. acknowledgments: francesco cappello, francesco dieli and antonella marino gammazza were partially supported by the university of palermo. francesco cappello was partially supported also by iemest. everly conway de macario and alberto jl macario were partially supported by the university of maryland at baltimore-imet. this work was written under the umbrella of the agreement between iemest and imet. this is imet contribution number imet-20-011. authors declare no conflict of interest. covid-19 deaths: are we sure it is pneumonia? please, autopsy, autopsy, autopsy! 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anti-spike igg causes severe acute lung injury by skewing macrophage responses during acute sars-cov infection genotype and phenotype of covid-19: their roles in pathogenesis analysis of therapeutic targets for sars-cov-2 and discovery of potential drugs by computational methods cryo-em structure of the 2019-ncov spike in the prefusion conformation structure, function, and antigenicity of the sars-cov-2 spike glycoprotein site-specific analysis of the sars-cov-2 glycan shield pathogenic priming likely contributes to serious and critical illness and mortality in covid-19 via this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-310304-f28tjmi8 authors: alcendor, donald j. title: racial disparities-associated covid-19 mortality among minority populations in the us date: 2020-07-30 journal: j clin med doi: 10.3390/jcm9082442 sha: doc_id: 310304 cord_uid: f28tjmi8 severe acute respiratory syndrome coronavirus 2 (sars-cov-2), a betacoronavirus that causes the novel coronavirus disease 2019 (covid-19), is highly transmissible and pathogenic for humans and may cause life-threatening disease and mortality, especially in individuals with underlying comorbidities. first identified in an outbreak in wuhan, china, covid-19 is affecting more than 185 countries and territories around the world, with more than 15,754,651 confirmed cases and more than 640,029 deaths. since december 2019, sars-cov-2 transmission has become a global threat, which includes confirmed cases in all 50 states within the united states (us). as of 25 july 2020, the johns hopkins whiting school of engineering center for systems science and engineering reports more than 4,112,651 cases and 145,546 deaths. to date, health disparities are associated with covid-19 mortality among underserved populations. here, the author explores potential underlying reasons for reported disproportionate, increased risks of mortality among african americans and hispanics/latinos with covid-19 compared with non-hispanic whites. the author examines the underlying clinical implications that may predispose minority populations and the adverse clinical outcomes that may contribute to increased risk of mortality. government and community-based strategies to safeguard minority populations at risk for increased morbidity and mortality are essential. underserved populations living in poverty with limited access to social services across the us are more likely to have underlying medical conditions and are among the most vulnerable. societal and cultural barriers for ethnic minorities to achieve health equity are systemic issues that may be addressed only through shifts in governmental policies, producing long-overdue, substantive changes to end health care inequities. coronavirus disease 2019 is caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2) [1] [2] [3] [4] . to date, seven human coronaviruses (hcovs) have been identified, including two α-covs (hcov-229e and hcov-nl63) and five betacoronaviruses (β-covs) (hcov-oc43, hcov-hku1, severe acute respiratory syndrome cov [sars-cov], middle east respiratory syndrome cov [mers-cov] , and most recently β-cov sars-cov-2 ) [5] [6] [7] [8] [9] . the world health organization (who) has classified covid-19 as a β-cov of group 2b [10] . covs cause respiratory, enteric, hepatic, and neurological diseases in various animal species, including camels, cattle, cats, and bats [11, 12] . the β-cov lineages hcov-oc43 and hcov-hku1 typically are associated with self-limiting upper respiratory infections in immunocompetent hosts and occasionally lower respiratory tract infections in immunocompromised hosts and the elderly [13, 14] . examination of the viral evolution reveals that bats and rodents are gene sources for most α-covs and β-covs, whereas avian species are the proposed gene sources of most δ-covs and γ-covs [15] . covs often the health inequities in the us that impact minority communities were well in place prior to the covid-19 pandemic. these inequities have become more evident in some cities and states. in chicago, aas make up 30% of the population; yet, they represent 50% of covid-19 cases and approximately 70% of covid-19 deaths, most of which are concentrated in small numbers of the most vulnerable communities [27] . in the states of louisiana and michigan, aas are impacted disproportionately by covid-19 deaths [28] : blacks represent 32.2% and 14%, respectively, and account for 70.5% and 40% of covid-19 deaths, respectively [29] . in new york city, which had the largest number of covid-19 cases and the highest number of deaths due to covid-19 in the us, minority communities disproportionately are impacted, as aas and h/ls account for 22% and 29% of the population, respectively, and account for 28% and 34% of deaths, respectively [30] . an examination of 131 predominantly aa counties shows a covid-19 infection rate of 137.5 per 100,000 and a death rate of 6.3 per 100,000, which is three times higher than the predominant nhw counties. moreover, the death rate for the aa counties were found to be six times higher than the rate observed in predominant white counties. taken together, a disproportionate burden of covid-19 morbidity and mortality, which will require further investigation, exists among minority communities in the us. it is evident that social determinants of health play a critical role in population-level health disparities beyond the comorbidities associated with covid-19 in minority communities. in the early stages of the pandemic in the us, dismissing covid-19 as not infecting aas may have created a lack of awareness and best practices, including proper hand hygiene, use of masks in public places, and social distancing and physical isolation, likely contributing to sars-cov-2 transmission in these communities [31] . even were that not the case, among vulnerable populations with low socioeconomic status, these transmission-mitigating practices are difficult to maintain over time. racial and ethnic disparities in the prevalence of type 2 diabetes (t2d) among adult minority populations have been documented [32, 33] . more recently, the global epidemic of childhood obesity has contributed greatly to the higher prevalence of t2d among adolescents who have more progressive clinical presentations of chronic kidney disease (ckd), cvd, diabetes-related eye disease, and poor glycemic control over time [34] . higher rates of t2d are seen among minority youth when compared with nhw youth [35] . approximately 80% of youth with t2d are from minority backgrounds [36] . in addition, health disparities for t2d are present in adults and youth among racial and ethnic minorities, and this likely will impact their response to covid-19. covid-19 patients with diabetes are at increased risk of having adverse clinical complications, including death [37, 38] . maintaining glycemic control in covid-19 patients is essential, as hyperglycemia could affect pulmonary function, the immune response to infection, and the development of the pro-inflammatory cytokine storm associated with more severe clinical disease ( figure 1 ). the use of corticosteroid therapy to combat inflammation in covid-19 patients also may increase glucose levels in 80% of patients with diabetes as well as patients without diabetes [39] ( figure 1 ). . hypothetical model of uncontrolled glycemia in diabetic patients and increased risk for complications due to covid-19. patients who clinically present with normal or high blood pressure may be subject to undue complications related to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. this is an illustration of the pancreas, responsible for insulin production and regulation and the immediate surrounding tissue and organs. once infected with sars-cov-2 some patients will experience increased inflammation in the form of a cytokine storm. corticosteroids often are hypothetical model of uncontrolled glycemia in diabetic patients and increased risk for complications due to covid-19. patients who clinically present with normal or high blood pressure may be subject to undue complications related to severe acute respiratory syndrome coronavirus 2 (sars-cov-2) infection. this is an illustration of the pancreas, responsible for insulin production and regulation and the immediate surrounding tissue and organs. once infected with sars-cov-2 some patients will experience increased inflammation in the form of a cytokine storm. corticosteroids often are prescribed to suppress inflammation but also are known to induce high glucose levels in the blood of both hypoglycemic and hyperglycemic patients. high blood glucose levels have been implicated in pulmonary injury and may affect the immune response, resulting in poor or delayed viral clearance. the degree of lung injury will include angiotensin-converting enzyme 2 (ace2)-mediated infection by sars-cov-2 that leads to hypoxia, vascular leakage, and potentially fatal pneumonia. tcr (t-cell receptor), cd4 (cluster designation 4), mhc (major histocompatibility). adapted from fraussen j et al. [40] . the who concludes that hypertension is the most important risk factor for death and disability worldwide, affecting more than 1 billion people and causing an estimated 9.4 million deaths annually [41] . aa adults have the highest prevalence of hypertension in the us, affecting 40.8% of men and 41.5% of women, in contrast to significantly lower rates in nhw, non-hispanic asian (nha), and h/l men and women [42] . hypertension is the most significant factor that directly contributes to disparities in cvd and renal disease among aas compared with nhws [42] . in a study by zhou et al., the most common comorbidities associated with adverse clinical outcomes in covid-19 patients were hypertension (30%), diabetes (19%), and coronary heart disease (8%) [43] . in a separate study by wu, the most common comorbidities associated with covid-19 patients who developed acute respiratory distress syndrome were hypertension (27%), diabetes (19%), and cvd (6%) [44] . in these two major studies, hypertension was found to be the most significant comorbidity associated with the most severe complications from covid-19. hypertension is not known to be causative in covid-19 pathobiology, and elderly patients are more likely to be hypertensive and are known to be at greater risk of severe disease. it also remains unclear whether medications used to treat hypertension, such angiotensin-converting enzyme inhibitors (aceis), have a role in acquisition or progressive development of covid-19 in patients. in humans, the liver produces angiotensinogen, which is converted to angiotensin i by renin from the kidneys, and angiotensin i is converted to angiotensin ii by the action of ace ( figure 2 ). normally, angiotensin ii is converted to angiotensin-1-7 by the monocarboxypetidase ace2 ( figure 2 ). however, upon infection with sars-cov-2, the ace2 protein serves as an entry receptor for the virus and is internalized with sars-cov-2 during membrane fusion and uptake by infected cells (figure 2 ). this leads to a significant reduction in ace2 surface expression and a concomitant increase in angiotensin ii, further leading to vasoconstriction that impacts blood pressure and causes inflammation, fibrosis, and oxidative stress in infected tissues within multiple organs ( figure 2) . thus, the downregulation of ace2 leads to the upregulation of aldosterone, which increases the activity of ace1, which again leads to higher levels of angiotensin ii and an overall suppression of angiotensin-1-7, designed to mitigate the effects of angiotensin ii via vasodilation, anti-inflammatory effects, and anti-oxidation ( figure 2 ). taken together, this may result in multi-organ dysfunction (mod) or failure. however, it is known that sars-cov-2 binds to the ace2 receptor, mainly in the lung, to enter cells [45, 46] . therefore, it remains controversial whether treatment of hypertension with aceis is beneficial or counter-productive in covid-19 patients. studies have shown the aceis have a protective anti-inflammatory effect in the lung, and soluble aceis could bind free virus and serve as therapeutics to reduce virus load [47] [48] [49] . if corticosteroid therapy is recommended for covid-19 patients, glucose levels should be monitored carefully to avoid pulmonary and immune dysfunction [50, 51] (figure 1 ). controversy surrounds the discontinuation of angiotensin receptor blockers (arbs) and aceis for diabetes and hypertension treatment in covid-19 patients [52] . however, in a recent study involving renin-angiotensin-aldosterone system inhibitors, it is recommended that these drugs be continued in patients evaluated for covid-19 to avoid excess cardiovascular risk [53] . optimal care of diabetes patients with covid-19 should involve careful monitoring of corticosteroid therapy when warranted, monitoring of regular blood glucose, and avoiding inappropriate discontinuation of arbs and aceis that may increase morbidity in covid-19 patients. ace2 also is expressed in heart, kidneys, vascular endothelium, and intestinal epithelium, supporting the notion that virus interaction with several organ systems could lead to mod, which may be observed in covid-19 patients [54, 55] . treatment of hypertension with aceis is beneficial or counter-productive in covid-19 patients. studies have shown the aceis have a protective anti-inflammatory effect in the lung, and soluble aceis could bind free virus and serve as therapeutics to reduce virus load [47] [48] [49] . if corticosteroid therapy is recommended for covid-19 patients, glucose levels should be monitored carefully to avoid pulmonary and immune dysfunction [50, 51] (figure 1 ). controversy surrounds the discontinuation of angiotensin receptor blockers (arbs) and aceis for diabetes and hypertension treatment in covid-19 patients [52] . however, in a recent study involving renin-angiotensinaldosterone system inhibitors, it is recommended that these drugs be continued in patients evaluated for covid-19 to avoid excess cardiovascular risk [53] . optimal care of diabetes patients with covid-19 should involve careful monitoring of corticosteroid therapy when warranted, monitoring of regular blood glucose, and avoiding inappropriate discontinuation of arbs and aceis that may increase morbidity in covid-19 patients. ace2 also is expressed in heart, kidneys, vascular endothelium, and intestinal epithelium, supporting the notion that virus interaction with several organ systems could lead to mod, which may be observed in covid-19 patients [54, 55] . hypothetical model of uncontrolled blood pressure in patients with hypertension and increased risk for complications due to covid-19. the liver produces angiotensinogen, a peptide hormone that causes vasoconstriction that increases blood pressure. angiotensinogen is converted to angiotensin i by renin from the kidneys then is converted to angiotensin ii by the action of ace (ace-i). normally angiotensin ii (ang. ii) is converted to angiotensin-1-7 by the monocarboxypetidase acei homology ace2. however, upon infection with sars-cov-2 the ace2 protein serves as the entry receptor for the virus and is internalized in the endosome with sars-cov-2 during membrane fusion and uptake by hypothetical model of uncontrolled blood pressure in patients with hypertension and increased risk for complications due to covid-19. the liver produces angiotensinogen, a peptide hormone that causes vasoconstriction that increases blood pressure. angiotensinogen is converted to angiotensin i by renin from the kidneys then is converted to angiotensin ii by the action of ace (ace-i). normally angiotensin ii (ang. ii) is converted to angiotensin-1-7 by the monocarboxypetidase acei homology ace2. however, upon infection with sars-cov-2 the ace2 protein serves as the entry receptor for the virus and is internalized in the endosome with sars-cov-2 during membrane fusion and uptake by infected cells. this leads to significant reduction in ace2 surface expression and concomitant increase in angiotensin ii, further leading to vasoconstriction that impacts blood pressure, inflammation, fibrosis, and oxidative stress in infected cells and tissues in multiple organs. even more, the downregulation of ace2 leads to the upregulation of aldosterone, a steroid hormone produced by the zona glomerulosa of the adrenal cortex, which increases the activity of ace1. this action leads to higher levels of angiotensin ii and an overall suppression of angiotensin-1-7, which is designed to mitigate the effects of angiotensin ii via vasodilation, anti-inflammatory effects, and anti-oxidation. these levels of angiotensin ii may result in mod or failure. raasa (renin-angiotensin-aldosterone system antagonist). ace i (angiotensin-converting enzyme 1). african americans are two to three times more likely than nhws to die of preventable cvd and stroke even when accounting for the general decline in cvd mortality [56] . from 2011-2014, a study found that the prevalence of hypertension among aa adults (41.2%) was higher than among nhw (28.0%), nhw (24.9%), and hispanic (25.9%) adults [57] . medication non-adherence is one of the greatest challenges to reducing health disparities associated with cvd morbidity and mortality [58] . poor management of hypertension is linked to increased risk of cvd, stroke, and ckd; hypertension management among aas has been shown to be lower when compared with nhws [59] . cvd is a common comorbidity in patients with covid-19 and is associated with patients who have the most severe disease [60] . a recent meta-analysis of eight studies from china that included 46,248 covid-19-infected patients showed the most prevalent comorbidities were hypertension (17 ± 7%, 95% confidence interval (ci) 14-22%) and diabetes (8 ± 6%, 95% ci 6-11%), followed by cvds (5 ± 4%, 95% ci 4-7%) [61] . covid-19 interacts with the cardiovascular system, inducing myocardial injury. patients with advanced age and elevated troponin i levels have abnormal echocardiograms and are at the greatest risk for developing more severe covid-19 disease (figure 3 ). these patients have been shown to have increased blood levels of interleukin-6 (il-6), ferritin, lactic acid dehydrogenase (ldh), and fibrin degradation product (d-dimer), which are associated with the cytokine storm that likely would contribute to cardiac injury ( figure 3 ). for patients with cardiac insufficiency who have underlying heart disease, sars-cov-2 infection may lead to adverse clinical disease and death. aging, ace2 levels, waning of the immune response, and host factors that become pronounced in patients with cvd have been considered as possible explanations for the severe disease course observed in covid-19 patients. in a study in wuhan, china, which included 138 covid-19 patients, researchers observed laboratory evidence of cardiac injury indicated by elevated troponin i levels, as well as abnormal echocardiograms in 7.2% of patients (10) overall, and 22% (30) who required intensive care unit (icu) attention [62] . zheng et al., reported that 12% of covid-19 patients who died without known cvd risk clinically presented with elevated levels of troponin i or cardiac arrest during their hospitalization [63] . the connection between covid-19 and cvd remains unclear; however, it has been suggested that direct infection of the heart occurs via ace2 expression on myocardial tissue, with supporting evidence in a murine model demonstrating ace2-dependent myocardial infection of sars-cov-2 [64] (figure 3 ). in addition, sars-cov-2 rna was detected in heart tissue from 35% of patients who died during the sars-cov-2 outbreak in toronto [65] . a cytokine storm and calcium-dependent apoptosis of cardiomyocytes are among other mechanism that could link cvd and covid-19 [66] (figure 3 ). ace2-related signaling pathways also may have a role in heart injury. heart transplant patients are expected to be especially vulnerable to sars-cov-2 infection due to immunosuppression; however, in a small study performed with 87 heart transplant recipients in wuhan, china, no evidence of a higher risk of infection with sars-cov-2 was found when precautionary measures were taken [67] . recommended guidelines reported as guidance for cardiothoracic transplant and mechanical circulatory support centers regarding sars cov-2 infection and covid-19 have been established and must be followed when performing heart transplantation [68] . it is recommended that patients continue heart transplantation without changes in immunosuppression, provided the recipient has not tested positive for sars-cov-2 and has not had exposure to or symptoms of covid-19 in the prior two to four weeks [69] it is also recommended to avoid donors with known or suspected covid-19, and if donors had covid-19, they should be covid-19-free (as indicated by polymerase chain reaction) for at least 14 days, owing to the incubation period of~5 days and onset of symptoms in~11.5 days [70] . hypothetical model of patients with cvd and increased risk for complications due to covid-19. cvd patients with advanced age and elevated troponin i levels, and who have abnormal echocardiograms, are at high risk for developing more severe covid-19 disease. these patients have been shown to have increased blood levels of il-6, ferritin, ldh, and d-dimer, which are associated with the cytokine storm that likely would contribute to cardiac injury. high levels of ace2 are known to be expressed on cardiomyocytes that could result in direct infection of heart tissue by sars-cov-2, which is thought to induce hypoxia leading to increase calcium levels resulting in apoptosis and death of cardiomyocytes; this likely would contribute to myocardial injury. heart transplant patients are expected to be especially vulnerable to sars-cov-2 infection due to immunosuppression; however, in a small study performed with 87 heart transplant recipients in wuhan, china, no evidence of a higher risk of infection with sars-cov-2 was found when precautionary measures were taken [67] . recommended guidelines reported as guidance for cardiothoracic transplant and mechanical circulatory support centers regarding sars cov-2 infection and covid-19 have been established and must be followed when performing heart transplantation [68] . it is recommended that patients continue heart transplantation without changes in immunosuppression, provided the recipient has not tested positive for sars-cov-2 and has not had exposure to or symptoms of covid-19 in the prior two to four weeks [69] it is also recommended to avoid donors with known or suspected covid-19, and if donors had covid-19, they should be covid-19-free (as indicated by polymerase chain reaction) for at least 14 days, owing to the incubation period of ~5 days and onset of symptoms in ~11.5 days [70] . the 2013 american thoracic society/european respiratory society committee, and similarly healthy people 2020, published a policy statement defining disparities in respiratory health as closely linked to racial ancestry, social, economic, and/or environmental differences [71] . health disparities in respiratory diseases are 14 times higher in minority populations with the lowest socioeconomic status when compared with populations with the highest socioeconomic status [71] . globally, a disproportionate burden of chronic obstructive pulmonary disease (copd) is present among minority communities due to low socioeconomic status, differences in health behaviors, occupational and social environmental exposures, prenatal and childhood exposures, respiratory tract infections, hypothetical model of patients with cvd and increased risk for complications due to covid-19. cvd patients with advanced age and elevated troponin i levels, and who have abnormal echocardiograms, are at high risk for developing more severe covid-19 disease. these patients have been shown to have increased blood levels of il-6, ferritin, ldh, and d-dimer, which are associated with the cytokine storm that likely would contribute to cardiac injury. high levels of ace2 are known to be expressed on cardiomyocytes that could result in direct infection of heart tissue by sars-cov-2, which is thought to induce hypoxia leading to increase calcium levels resulting in apoptosis and death of cardiomyocytes; this likely would contribute to myocardial injury. the 2013 american thoracic society/european respiratory society committee, and similarly healthy people 2020, published a policy statement defining disparities in respiratory health as closely linked to racial ancestry, social, economic, and/or environmental differences [71] . health disparities in respiratory diseases are 14 times higher in minority populations with the lowest socioeconomic status when compared with populations with the highest socioeconomic status [71] . globally, a disproportionate burden of chronic obstructive pulmonary disease (copd) is present among minority communities due to low socioeconomic status, differences in health behaviors, occupational and social environmental exposures, prenatal and childhood exposures, respiratory tract infections, and tobacco use. these factors are associated with the risk of developing copd and are associated with poor clinical outcomes related to copd clinical presentations [72] . according to the who, >90% of copd deaths occur in low-income and middle-income countries [73] . social determinants of health, including economic stability, education, access to health care, and environment, play a critical role in establishing health equity that will reduce disparity-related respiratory diseases, such as copd, during a lifespan [74, 75] . copd is a condition that predisposes covid-19 patients to worse clinical outcomes [76] . smokers and individuals with copd have increased airway expression of the ace2 receptor for sar-cov-2 [77] . covid-19 patients with copd who are current smokers and have other types of lung disease, including asthma, have poor clinical outcomes (figure 4 ). sars-cov-2 infection via ace2 entry into the alveolar epithelial cells of copd patients who smoke may lead to increased surface-expression ace2 on lung epithelium, which may increase the rate of infection in the lung and contribute to higher viral loads ( figure 4 ). alveolar cells pneumocytes type ii are highly permissive for sars-cov-2 infection resulting in alveolar dysfunction, inflammation, vascular leakage, development of pulmonary emboli, and acute respiratory distress from poor gas exchange (figure 4) . progressive viral infection and the pro-inflammatory conditions lead to pulmonary vascular leakage, alveolar edema, monocyte infiltration, and pneumonia ( figure 4 ). patients often require mechanical ventilation resulting in delayed recovery, respiratory failure, septic shock, or mod or failure (figure 4 ). smokers and individuals with copd have increased airway expression of the ace2 receptor for sar-cov-2 [77] . covid-19 patients with copd who are current smokers and have other types of lung disease, including asthma, have poor clinical outcomes (figure 4) . sars-cov-2 infection via ace2 entry into the alveolar epithelial cells of copd patients who smoke may lead to increased surface-expression ace2 on lung epithelium, which may increase the rate of infection in the lung and contribute to higher viral loads (figure 4 ). alveolar cells pneumocytes type ii are highly permissive for sars-cov-2 infection resulting in alveolar dysfunction, inflammation, vascular leakage, development of pulmonary emboli, and acute respiratory distress from poor gas exchange (figure 4) . progressive viral infection and the pro-inflammatory conditions lead to pulmonary vascular leakage, alveolar edema, monocyte infiltration, and pneumonia ( figure 4 ). patients often require mechanical ventilation resulting in delayed recovery, respiratory failure, septic shock, or mod or failure (figure 4) . hypothetical model of patients with pulmonary disease and increased risk for complications due to covid-19. sars-cov-2 infection via ace2 entry into alveolar epithelial cells in patients with copd who smoke may lead to increased surface-expression ace2 on lung epithelium that may increase the rate of infection in the lung and contribute to higher viral loads. shown is the alveolar organization and resident cells that include pneumocytes type i, pneumocytes type ii and alveolar macrophages. elastic fibers are also shown and sars-cov-2 particles are shown as blue ovals. alveolar cells pneumocytes type ii are highly permissive for sars-cov-2 infection resulting in alveolar dysfunction, inflammation, vascular leakage, development of pulmonary emboli, and acute respiratory distress from poor gas exchange. progressive viral infection and potential cytokine storm leads to pulmonary vascular leakage, alveolar edema, and monocyte infiltration at sites of infection, resulting in reduced lung function. patients often require mechanical ventilation increasing the risk of delayed recovery, respiratory failure, septic shock, and mod or failure. ards (acute respiratory distress syndrome). infection via ace2 entry into alveolar epithelial cells in patients with copd who smoke may lead to increased surface-expression ace2 on lung epithelium that may increase the rate of infection in the lung and contribute to higher viral loads. shown is the alveolar organization and resident cells that include pneumocytes type i, pneumocytes type ii and alveolar macrophages. elastic fibers are also shown and sars-cov-2 particles are shown as blue ovals. alveolar cells pneumocytes type ii are highly permissive for sars-cov-2 infection resulting in alveolar dysfunction, inflammation, vascular leakage, development of pulmonary emboli, and acute respiratory distress from poor gas exchange. progressive viral infection and potential cytokine storm leads to pulmonary vascular leakage, alveolar edema, and monocyte infiltration at sites of infection, resulting in reduced lung function. patients often require mechanical ventilation increasing the risk of delayed recovery, respiratory failure, septic shock, and mod or failure. ards (acute respiratory distress syndrome). differential expression of ace2 may help to explain the discrepancy in viral pathology associated with covid-19. the production of ace2 among minority populations who smoke or have copd may partially explain the differences in covid-19 rates of morbidity and mortality among aas compared with nhws. leung et al. examined gene expression levels of ace2 in the airways of individuals with and without copd and found that active cigarette smoking and copd upregulate ace2 expression in lower airways and could contribute to the difference in disease burden observed among covid-19 patients [77] . these findings were supported in a rat model that showed smoke exposure resulted in increased expression and activity of ace2 in the airways [78] . individuals with uncontrolled asthma also appear to be at increased risk of a more severe course of covid-19 infection [79] . lung function tests and the use of nebulizers as part of copd and asthma management should be performed with caution due to the risk of virus aerosolization and potential transmission during these procedures [79] . disparities in t2d among minority adults have been pervasive in diabetes complications, glycemic control, and diabetes care [80] . glycemic control in all patients with diabetes is critical for avoiding complications such as ckd, cvd, and diabetes-related eye disease. it is projected that racial and ethnic disparities in t2d prevalence will persist until 2050 [81] . one of the most significant challenges in mitigating the effects of diabetes on the health and wellness of minority populations in the us is improving basic social determinants of health, including low socioeconomic status, poor access to educational opportunities, conditions of poverty, excess life stressors, poor health knowledge, and limited access to quality and affordable health care. strategies to mitigate diabetes among minority populations at greater risk for severe covid-19 disease would involve improving glycemic control among aas and h/ls, which is a major problem [82] . management of hypertension has been problematic among minority populations with diabetes. it has been reported that 71% of adults with diabetes have high blood pressure, and blood pressure control among diabetics is essential to reduce the risk of developing retinopathy and neuropathy associated with diabetes [83] . aas are more likely to have significantly higher, uncontrolled blood pressure than nhws [83] . obesity and a sedentary lifestyle should be avoided among diabetics [83] . the increased burden of diabetes among racial and ethnic minorities. diabetes is higher among black or african american, h/ls, and american indian individuals as compared to nhw [84] . aas experiencing household or neighborhood-level poverty are at a higher risk of developing diabetes [84] . the rates of diabetes in the southern u.s. is higher than the national average [85] . this will require overall improvements in social determinants of health that tend to predispose these populations to the worst diabetes outcomes often superimposed on the pathogenesis of covid-19 disease burden. among racial and ethnic minorities, hypertension prevalence in the us is highest among aas, who have been shown to have less control of the disease when compared with nhws [86] [87] [88] [89] . pharmacotherapy in the form of thiazide-type diuretics, calcium channel blockers, aceis or angiotensin ii receptor blockers (arbs), and lifestyle changes are essential for hypertension control [61] . medication adherence plays a critical role in the long-term management of hypertension, especially in minority communities, and is directly related to racial and ethnic disparities in cvd, stroke, and chronic kidney disease (ckd). ferdinand et al. report that strategies such as direct patient engagement, consumer-directed health care, utilization of patient portals, smart apps and text messages, digital pillboxes, pharmacist-led engagement, and cognitive-based behavior, could be important for improving medical adherence and reducing disparities in hypertension and its related complications [90] . the centers for disease control and prevention (cdc) reports that 75% of individuals who died of covid-19 disease were older than 65 years of age. age-related increase in systolic blood pressure suggests that 90% of adults in the us will develop hypertension in their lifetime. this age-related increase in blood pressure likely contributes to increased age-related mortality among the elderly with covid-19. it has been proposed that cvd is more prevalent in older patients, as these patients are more likely to have impaired immune systems that loses the ability to responds effectively to infections and the reduced levels of ace2 observed among the elderly may predispose older patients to more severe complications of covid-19 due to loss of the protective effects of ace2 to regulate and control inflammation. therefore, patients with cvd should be subjected first to preventive measures, such as monitoring for elevated cardiac biomarkers, and if necessary, should be isolated from other patient populations that would place them at higher risk. in addition, a report from the national health commission of china revealed that almost 12% of covid-19 patients without known cvd risk had elevated troponin levels or cardiac arrest during hospitalization [91] . this would suggest that sars-cov-2-mediated infection may lead directly to myocardial dysfunction. potential benefit or harm from aceis and arbs, commonly prescribed for cardiovascular disorders, is still under investigation due to lack of available evidence. individuals with copd and confirmed covid-19 are at greater risk of severe complications and death than individuals without copd. optimal testing and diagnostics, as well as management of individuals with copd in underserved communities, should be made available for early intervention and treatment. these individuals should be screened for other comorbidities that are associated with the development of copd, such as asthma, lung infections, and other related respiratory diseases. affordability and access to treatments and palliative care should be made available to low-income copd patients. furthermore, copd patients should be allowed access to optimal life-extending treatments for their disease, including medical specialists, more effective drug treatments, smoking cessation counseling, supplemental oxygen, and non-invasive ventilation that often are unaffordable for these vulnerable populations [92] . having copd and being a current smoker may greatly increase the risk of complications mortality from covid-19. interventions in copd patients who smoke should include smoking cessation, as it has been demonstrated to improve pulmonary function in younger smokers compared with older smokers who have sustained cumulative lung damage over time [92] . essential workers, who often are from minority communities and face occupational hazards, are at greater risk for developing occupation-related copd. understanding clinical risk factors and their mechanisms can benefit disparity populations by identifying at risk individuals early and developing early intervention strategies that are designed to manage these risk factors in the context of clinical trials where access to care and drug compliance can be controlled. the benefits of understanding these mechanisms could also provide information that contributes to innovative therapies that identify selective targets within these pathways among disparity populations based on unique genetic profiles. finally, an understanding of these mechanisms could lead to changes among health providers to be more aggressive in their care for these patients as clinical trial data become available, leading changes in health policy for underserved populations. changes in public policy are essential to combat the long-standing problems associated with health inequities in our health care system. these inequalities are more pronounced during a health care crisis, such as the current covid-19 pandemic. addressing these inequities would require a government-appointed race/inequity task force that is designed to implement pre-determined standards of care in minority communities at an early stage in a medical crisis. in addition, special provisions should be made for essential workers, many of whom are underserved, to be adequately equipped and compensated for vital services performed to maintain public health standards. adequate funding should be established to support these initiatives from both the public and private sectors to avoid disruptions in the readiness of our workforce, supply chains, and health care system to prevent unforeseen economic crisis. new policies must be flexible enough to accommodate changes in our scientific understanding of these emerging pathogens and the development of efficacious interventions to protect the public, including our most vulnerable populations. these policies would require a bipartisan commitment from government officials, ending a "wait and see if it goes away" strategy by replacing previous tactics with a standard plan of action, which would save lives and reduce the overall burden on our economy. policy changes would include the elimination of inequitable treatment within our health care system. doctor-patient relationships that include individuals from underserved communities in the us can be difficult due to systemic racism and implicit racial bias that has contributed to historic distrust in minority communities for health care providers. physicians could engage minority patients in ways that will help to assure them that they will receive the best possible care. for those minority populations who are essential workers that are at higher risk for covid-19 because of pre-existing health conditions, physicians should aggressively make them aware of the risks as well as precautionary measures they must take to avoid infection. aggressive strategies to help minority patients at higher risk understand the seriousness of covid-19 disease could include direct mailings, providing patients with samples of masks and sanitizers, covid-19 office placards, covid-19 infomercials in waiting areas, wellness checks, and covid-19 information for family members and caretakers. the social determinants of health (sdoh) for these individuals are directly linked to the development of risk factors that predispose them to more severe covid-19 disease. healthcare providers and patient navigators that have relationships with community based organizations (cbos) that work to improve sdoh among underserved communities could be an important option to support the long term health and wellness of disparity populations. front line health care providers in the covid-19 pandemic are being pushed to the limits of human endurance and are often overwhelmed which can trigger disparate responses to minority communities. policies need to be in place that confer oversight for when this occurs. physicians should be surveyed in ways to recognize implicit racial bias when caring for minority patients and be encouraged to take measures to constructively modify their behavior. in addition, there is a great need for more nurses and other medical practitioners to support physicians during this pandemic. some of these challenges can be met with volunteers that have past medical training as well as military medical staff. essential workers, many of whom are from minority communities are often forced to experience increased risk of virus transmission because of economic hardships and the lack of personal protective equipment (ppe). the lack of ppe is directly linked to the likelihood of virus transmission to essential workers and, therefore, should be recognized as a necessity that should be prioritized by the federal government as an emergency declaration that would be fully funded throughout this pandemic and policies put in place in this declaration for timely ramping-up of ppe supplies and development of government-owned ppe stockpiles for a future medical crisis. a commission of physicians, and nurses should be established to advocate the need for ppe on behalf of essential workers at risk for covid-19 and given the opportunity the make their case to policy makers. healthcare access is a core component of the sdoh for underserved communities and poor access to affordable healthcare is a major driver of health disparities in the us. these disparity populations are heavily impacted by this pandemic. the affordable care act (aca) represents a lifeline for the working poor and their families without health insurance. the aca should be maintained and arguably updated or revised to meet the changing needs of participants. the aca is a critical component of our existing healthcare infrastructure that directly addresses health inequities in the us and is not perfect in content and yet should be preserved. the existing political climate for legislation to directly address longstanding us racial/ethnic inequities in healthcare as well as other sdoh such as education, housing, and a living wage will require a continuance of the grass roots call for change and the acceptance by policy makers that these changes need to become law in america. longstanding health disparities such as diabetes, hypertension, cvd, and pulmonary disease among minority populations in the us may serve to predispose these communities to sars-cov-2 infection and increased risk for clinically severe covid-19. the underlying social determinants of health and standards of care in minority communities must be improved 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with chronic obstructive pulmonary disease this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license key: cord-345728-41k1bljo authors: al’joboori, yazi; massey, sarah j.; knight, sarah l.; donaldson, nick de n.; duffell, lynsey d. title: the effects of adding transcutaneous spinal cord stimulation (tscs) to sit-to-stand training in people with spinal cord injury: a pilot study date: 2020-08-26 journal: j clin med doi: 10.3390/jcm9092765 sha: doc_id: 345728 cord_uid: 41k1bljo spinal cord stimulation may enable recovery of volitional motor control in people with chronic spinal cord injury (sci). in this study we explored the effects of adding scs, applied transcutaneously (tscs) at vertebral levels t10/11, to a sit-to-stand training intervention in people with motor complete and incomplete sci. nine people with chronic sci (six motor complete; three motor incomplete) participated in an 8-week intervention, incorporating three training sessions per week. participants received either tscs combined with sit-to-stand training (stim) or sit-to-stand training alone (non-stim). outcome measures were carried out before and after the intervention. seven participants completed the intervention (stim n = 5; non-stim n = 2). post training, improvements in international standards for neurological classification of spinal cord injury (isncsci) motor scores were noted in three stim participants (range 1.0–7.0), with no change in non-stim participants. recovery of volitional lower limb muscle activity and/or movement (with tscs off) was noted in three stim participants. unassisted standing was not achieved in any participant, although standing with minimal assistance was achieved in one stim participant. this pilot study has shown that the recruitment of participants, intervention and outcome measures were all feasible in this study design. however, some modifications are recommended for a larger trial. spinal cord injury (sci) is a life-long condition, which can substantially impact the health and well-being of affected individuals. appropriate management is essential to maximise health-related quality of life. standing remains one of the only clinical interventions used both acutely and following discharge from hospital, in people with chronic sci, because it is affordable and relatively simple to do at home. regular standing has many known health benefits, including reduced muscle tone, improved blood flow in the lower limbs, beneficial effects on bladder and bowel function and improvements in quality of life [1] . active stand training, with additional electrical stimulation, may facilitate independent or minimally-assisted standing in people with chronic sci [2] . non-patterned spinal cord stimulation (scs), delivered by electrodes implanted in the epidural space of the spinal cord has been shown to elicit lower limb extensor movements in rats [3, 4] and humans [5] [6] [7] with motor complete sci, via activation of large-to-medium diameter sensory participants were recruited from the london spinal cord injury centre (royal national orthopaedic hospital) and neurokinex (specialised neurological activity-based rehabilitation facilities). inclusion criteria were (1) spinal cord injury for >1 year, (2) sci level c5-t12, (3) aged >18 years, (4) ais a-d, (5) unable to stand from a chair unaided. exclusion criteria were (1) cardiac pacemaker (2) any other musculoskeletal diagnosis affecting the lower limbs, (3) pregnancy, (4) complex regional pain syndrome, (5) implanted metal or active device at electrodes caudal to t9 (e.g., screws, contraceptive coil), (6) spinal malignancy, (7) uncontrolled autonomic dysreflexia, (8) neurological degenerative diseases, (9) peripheral nerve damage affecting the lower limbs, (10) currently on any form of anti-spasticity treatment (e.g., botox, but not including bladder botox), (11) osteoporotic-bone density t-score less than −2.5. the demographics and injury characteristics of the 9 participants assessed at baseline are provided in table 1 . this was a purposefully sampled cohort study, designed to balance training + tscs (stim) and training only (non-stim) groups by ais grade. participants were initially assessed against the inclusion/exclusion criteria, and then assigned to either the stim or non-stim group: we aimed to recruit at least two participants in each group with motor complete injuries (ais a/b), and two participants in each group with incomplete injuries (ais c/d). thirty-five participants were assessed for eligibility; of these, 26 did not participate in the trial. of these, applicants unable to meet criteria (n = 7) was primarily due to existing implanted metal work being too low and applicants being less than 1-year post-injury. others expressed that the training center locations were too far to travel 3 times a week for 8 weeks (n = 7), that they were unwilling to participate in the study after being assigned to the control arm (n = 4), or did not disclose the reason (n = 7). one applicant was willing to participate, but unable to start the study due to early termination (due to the covid-19 pandemic). therefore, nine participants were assessed at baseline; of these, 7 completed the intervention and 2 were withdrawn due to a lower limb injury (n = 1) or early termination (n = 1). see consort flow diagram ( figure 1 ). all training was carried out at neurokinex and consisted of 24 sessions (3 sessions per week for 8 weeks). during each session, participants transferred to a physiotherapy couch and were fitted with a body weight support (bws) harness. the harness was attached to a keiser power rack (keiser uk ltd., gloucestershire, uk), which incorporates a pneumatic resistance system to assist the participant in the sit-to-stand manoeuvre by partially supporting their bodyweight. during each training session, participants stood up 5 times, taking approximately one-hour. each sit-to-stand manoeuvre was initiated by increasing the bws to assist the participant into stand; two therapists were available to stabilise the participant if required. standing was then maintained for 4-5 min, during which postural exercises such as deep and shallow squats, lateral and anterior/posterior weight shifts, squat holds, single leg bends, hip thrusts, kettle bell arm presses, trunk strengthening (arms off bar and straighten posture), hip rotations and squat rotations were performed. the participants were then returned to a seated position and rested for 2-3 min before the cycle was repeated. in the stim group only, continuous tscs was applied during active standing (sit-to-stand, standing and sit-from-stand), no tscs was provided during seated rest. the self-adhesive electrodes for tscs were (5 × 5 cm axelgard, fallbrook, ca, united states), placed on the midline, the cathode at t10/11 and the anode at t12/l1. electrode placement was confirmed using single pulses (monophasic, 1 ms pulse width) applied using a digitimer ds8r constant current stimulator (digitimer, welwyn garden city, hertfordshire, uk), driven by signal software (cambridge electronic design, cambridge, uk), to test for prr threshold in lower limb muscles. stimulation current (ma) was increased until prrs were observed in all measured lower limb muscles, which was defined as motor threshold. paired pulses at an interstimulus interval (isi) of 30 ms were then used to test the presence of post-activation depression in order to confirm activation of afferent roots. previous studies have reported post-activation depression with tscs paired pulses applied at isis between 25 and 50 ms in people with sci [25] and in able-bodied subjects [37] . prrs due to single and paired tscs pulses are shown in figure 2a . optimal electrode location all training was carried out at neurokinex and consisted of 24 sessions (3 sessions per week for 8 weeks). during each session, participants transferred to a physiotherapy couch and were fitted with a body weight support (bws) harness. the harness was attached to a keiser power rack (keiser uk ltd., gloucestershire, uk), which incorporates a pneumatic resistance system to assist the participant in the sit-to-stand manoeuvre by partially supporting their bodyweight. during each training session, participants stood up 5 times, taking approximately one-hour. each sit-to-stand manoeuvre was initiated by increasing the bws to assist the participant into stand; two therapists were available to stabilise the participant if required. standing was then maintained for 4-5 min, during which postural exercises such as deep and shallow squats, lateral and anterior/posterior weight shifts, squat holds, single leg bends, hip thrusts, kettle bell arm presses, trunk strengthening (arms off bar and straighten posture), hip rotations and squat rotations were performed. the participants were then returned to a seated position and rested for 2-3 min before the cycle was repeated. in the stim group only, continuous tscs was applied during active standing (sit-to-stand, standing and sit-from-stand), no tscs was provided during seated rest. the self-adhesive electrodes for tscs were (5 × 5 cm axelgard, fallbrook, ca, united states), placed on the midline, the cathode at t10/11 and the anode at t12/l1. electrode placement was confirmed using single pulses (monophasic, 1 ms pulse width) applied using a digitimer ds8r constant current stimulator (digitimer, welwyn garden city, hertfordshire, uk), driven by signal software (cambridge electronic design, cambridge, uk), to test for prr threshold in lower limb muscles. stimulation current (ma) was increased until prrs were observed in all measured lower limb muscles, which was defined as motor threshold. paired pulses at an interstimulus interval (isi) of 30 ms were then used to test the presence of post-activation depression in order to confirm activation of afferent roots. previous studies have reported post-activation depression with tscs paired pulses applied at isis between 25 and 50 ms in people with sci [25] and in able-bodied subjects [37] . prrs due to single and paired tscs pulses are shown in figure 2a . optimal electrode location was recorded for each participant and replicated during training for participants in the stim group. during training, tscs was fixed at 30 hz (biphasic, 1 ms pulse width) and applied below motor threshold (did not elicit any visible muscle contractions), at a level that induced paraesthesia in lower limb dermatomes, or the maximum level tolerated by the participant (whichever was lower), using the chattanooga intelect mobile stimulator (chattanooga group international, chattanooga, tn, usa). stimulation intensity was initially established at the start of each session, and modified throughout each session as required, due to habituation to the stimulation. transcutaneous scs was applied tonically during each sit-to-stand manoeuvre. stimulation was present during transitions from sit-to-stand and sit-from-stand and maintained throughout standing. no stimulation was applied during rest periods between stands. and gastrocnemius (gs) muscles to record emg and electro-goniometers were placed laterally across the knee joints to synchronously record knee joint range of motion. at 0, 4 and 8 weeks of training, weekly outcome measures of bws and upper-and lower-limb loading were recorded. during sit-to-stand, the participant's feet were positioned on a force platform (wii balance board, nintendo co ltd., kyoto, japan) to record leg loading, with their hands grasping handles (each fixed to a customised s-type load cell which measured the vertical force) to record arm loading, used for additional balance, and to lift their body weight ( figure 3 ). body weight support was manually recorded from an output screen of the pneumatic resistance system. vertical ground reaction force was calculated for weeks 0, 4 and 8 from the sum of the forces measured by the load cells at the four corners of the wii boards. upper limb arm loading was measured for left and right handle load cells, previously calibrated with known weights. analogue signals were processed using a moving average (2.5 s sliding window) and the maximum load on the feet during standing at weeks 0, 4, and 8 was extracted from an isolated region of interest (roi). this roi, a time window of <1 min, was selected based on review of video footage to isolate a period during the training session where static standing is present and not during any exercises outlined in 2.3. this roi produces a less variable period for biomechanical measures to be examined. to assess changes in muscle activation patterns between baseline and final assessments compared to published patterns of neurologically intact subjects [38] , emg envelopes (semg) were produced using a root mean square algorithm (0.4 s sliding window) for both flexion and extension to demonstrate paired pulse inhibition (arrows denote the time at which the stimulus was applied). (b) experimental setup for baseline and final brain motor control assessments. participants were placed in a supine position with bilateral electromyography (emg) electrodes placed over the quadriceps (quad), hamstring (ham), tibialis anterior (ta) and gastrocnemius (gs) muscles to record emg and electro-goniometers were placed laterally across the knee joints to synchronously record knee joint range of motion. baseline and follow-up outcome measures were completed before and after the 8-week training/stimulation intervention. the international standards for neurological classification of spinal cord injury (isncsci) assessment (without the anorectal exam) was used to observe changes in 10 myotomes on both sides of the body at baseline and final assessments. the brain motor control assessment (bmca) was used to characterise previously undetected motor responses, from multi-muscle lower limb electromyography (emg). a neurophysiological protocol was developed from the original bmca procedure [38] . recordings were performed at baseline and final assessments (experimental set-up is shown in figure 2b ). pairs of surface 35 × 52 mm ag-agcl electromyography (emg) electrodes (covidien, watford, uk) were placed bilaterally over the quadriceps (qu), hamstring (ham), tibialis anterior (ta) and gastrocnemius (gs) muscles of the participant's lower limbs, according to seniam guidelines [38] . emg data were amplified (×1000) using a digitimer isolated patient preamplifier/amplifier system (d360 8-channel patient amplifier system, digitimer, welwyn garden city, hertfordshire, uk) digitised at 2 khz (power 1401, cambridge electronic design, cambridge, uk). the data were then filtered with a pass-band of 10-200 hz and stored on a personal computer for analysis. additional synchronisation of bilateral electro-goniometers (dlk800, biometrics ltd., wales, uk) recorded the range of movement (rom) of knee joints. participants lying supine, were asked to perform two bilateral voluntary manoeuvres: hip-knee flexion/extension and ankle dorsiflexion/plantar-flexion. all manoeuvres were repeated three times, cued by a light and audible tone. health-related quality of life was assessed using the sf-36 health survey [39] , which has been shown to be discriminative in the sci population [40] . the sf-36 assesses 8 health-related domains: physical functioning; physical role limitations; emotional role functioning; vitality; mental health; social functioning; bodily pain; general health perception. functional independence was assessed using the spinal cord independence measure (scim iii) [41, 42] , which assesses functional status in 3 sub-categories: self-care, respiration and sphincter management and mobility. at 0, 4 and 8 weeks of training, weekly outcome measures of bws and upper-and lower-limb loading were recorded. during sit-to-stand, the participant's feet were positioned on a force platform (wii balance board, nintendo co ltd., kyoto, japan) to record leg loading, with their hands grasping handles (each fixed to a customised s-type load cell which measured the vertical force) to record arm loading, used for additional balance, and to lift their body weight ( figure 3 ). body weight support was manually recorded from an output screen of the pneumatic resistance system. for all participants in the stim group, loading through the lower limbs increased progressively throughout the intervention, which was due to reduced bws and/or reduced lifting through the upper limbs. in the non-stim group, neither participant showed any change in bws with training; however, p8 had high initial leg loading, with little scope for improvement. participants in the stim group also reported enhanced voluntary control (ability to "actively engage" in the standing) and vertical ground reaction force was calculated for weeks 0, 4 and 8 from the sum of the forces measured by the load cells at the four corners of the wii boards. upper limb arm loading was measured for left and right handle load cells, previously calibrated with known weights. analogue signals were processed using a moving average (2.5 s sliding window) and the maximum load on the feet during standing at weeks 0, 4, and 8 was extracted from an isolated region of interest (roi). this roi, a time window of <1 min, was selected based on review of video footage to isolate a period during the training session where static standing is present and not during any exercises outlined in section 2.3. this roi produces a less variable period for biomechanical measures to be examined. to assess changes in muscle activation patterns between baseline and final assessments compared to published patterns of neurologically intact subjects [38] , emg envelopes (semg) were produced using a root mean square algorithm (0.4 s sliding window) for both flexion and extension phases for each manoeuvre (6 total). integrated emg (iemg) was then calculated to express the cumulative area under the curve over time for each phase (flexion/extension) of each manoeuvre, and this was averaged across the 3 repeat trials. simultaneous knee joint range of motion (rom) for the flexion phase was quantified by subtracting the angle ( • ) at rest from the maximum angle during the flexion. the subsequent rom during extension was quantified by subtracting the maximum flexion angle from the minimum angle in the extension phase. sf-36 and scim scores for each participant were considered significant if a change greater than the minimal detectable change (mdc) for each sub-category was found. for sf-36, mdcs were calculated from standard deviations reported in each sub-category from a cohort of 187 people with sci [43] . [44] ; these were self-care = 2.6, respiration and sphincter management = 6.1 and mobility = 3.6. of the nine participants who underwent baseline measures, seven completed the 8-week intervention, and attended all training sessions. one participant experienced symptoms of autonomic dysreflexia in the evening following a training session and was later found to have an injury to the left calf. the participant was withdrawn from participating in any further training, and the incident was reported to the trial sponsor. no further adverse events were reported. stimulation thresholds found to elicit prrs at baseline are shown in table 2 with the subsequent current amplitude used during training ranging between 40 and 110 ma in all participants. paraesthesia was experienced by all participants in the stim group during training, and tscs at this intensity was tolerated in all participants, however some reported discomfort due to the tscs current. in particular, when tscs was provided at higher intensities (>80 ma), the activation of trunk muscles caused an anterior pelvic tilt in some participants, which placed strain on the spine during standing, their stimulation intensities were reduced. 90 during training, bws was provided at the minimum level required to achieve standing for each participant, and was adjusted throughout the 8-week intervention, with the aim of progressively reducing the level of bws. the set-up and an example plot of the upper and lower limb forces, recorded during a single sit-to-stand, are shown in figure 3a ,b. for each participant, peak upper and lower limb forces and bws during standing at the first, fourth and eighth week of training is shown in figure 3c ,d. for all participants in the stim group, loading through the lower limbs increased progressively throughout the intervention, which was due to reduced bws and/or reduced lifting through the upper limbs. in the non-stim group, neither participant showed any change in bws with training; however, p8 had high initial leg loading, with little scope for improvement. participants in the stim group also reported enhanced voluntary control (ability to "actively engage" in the standing) and proprioceptive (sensory) feedback during tscs standing activities; these effects developed over several weeks and were evident in all participants in the stim group by week 5. p2 and p4 regained the ability to voluntarily activate knee flexor and extensor muscles on-command in the presence of tscs combined with standing (see p2 (ais a) in video s1); this occurred after 5 weeks of training. after 8 weeks of training, p4 (ais c) regained the ability to stand with a standing frame and minimal assistance, but only in the presence of tscs. for each participant, isncsci motor scores before and after the intervention are shown in table 3 . lower limb motor scores increased in three of the five participants in the stim group (+1 (ais a), +5 (ais c) and +7 (ais d)) and were unchanged in the other two (both ais a). p1 presented palpable trace function in his achilles tendon during ankle plantarflexion in the gravity-eliminated position. p4 showed palpable trace function in her hip flexors, ankle dorsiflexors, long toe extensors and ankle plantar flexors which were absent at baseline. p5 was able to perform full rom of his ankle dorsiflexors and knee extensors in addition to full rom of his ankle plantar flexors in the gravity-eliminated position and was able to resist moderate pressure whilst sustaining full rom of the long toe extensors. lower limb motor scores were unchanged in both participants in the non-stim group (ais a and ais c). upper limb motor scores were unchanged in all participants except for a reduction of one point recorded in one participant in the non-stim group. table 3 . international standard for neurological classification of spinal cord injury (isncsci) before (pre) and after (post) the intervention for participants in transcutaneous spinal cord stimulation (tscs) combined with sit-to-stand training (stim (s)) and sit-to-stand training alone (non-stim (ns)) groups. before and after the intervention, participants were requested to perform voluntary hip/knee and ankle flexion and extension movements to audible cues, whilst lying supine on a couch; no tscs was provided. normative data in non-injured subjects demonstrates rapid and sustained recruitment of motor units in the prime mover (agonist) muscles and smaller residual amplitude response in antagonist muscles during voluntary motor tasks [38] . in sci individuals, patterns of muscle activity range from no spinal motor output (paralysis) to appropriately sequenced reciprocal activation during controlled joint movements [38, 45, 46 ]. during the voluntary hip/knee manoeuvre, range of motion (rom) at the knee joint was unchanged at the onset of the cue, in all but one participant (p5), indicating that these participants were unable to perform the movements both before and after the intervention (all measured roms were <2 • ). p5 (ais d, stim group) was able to reliably perform hip/knee flexion but not extension before the intervention; uncontrolled coactivation of agonist and antagonist muscles was observed during both phases. although coactivation was still present after training, a more appropriate activation pattern was observed, permitting the ability to extend his left lower limb from flexed (p5 left rom extension increased from 34.4 • to 98.3 • ). this was supported by the left quadriceps emg activity increasing considerably during hip flexion (see p5 lquad in figures 4 and 5 ) followed by an increased and sustained contraction in the left hamstrings during extension (see p5 lham in figures 4 and 5) . although no change in knee rom was detected, increases in emg activity were also noted in p4 (ais c, stim group) and p6 (ais a, stim group) post training compared with pre (see p4 rquad during extension in figures 4 and 5 ; p6 lham and rham during flexion in figure 5 ). despite this activity being present, the activation pattern was inappropriate for the phase of the task. during the voluntary ankle manoeuvre, participants were asked to bring their toes up and point their toes down in response to the audio/visual cue ( figure 6 ). p5 (ais d, stim group) was unable to perform both flexion and extension of the right ankle before the intervention (figure 7c ) but after training, he regained the ability (with a delayed recruitment) to flex and extend the right foot ( figure 7c,d) . the emg quantification revealed that although this functional movement was achieved, the pattern of activity was seemingly uncoordinated (figure 6 ). p5 displayed an additional increase in volitional muscle activity with control over the left ankle movement in comparison to baseline recordings (figure 7c,d) . an increase in volitional emg activity was also present in p4 (ais c, stim group) post training compared with pre (see p4 rta and rgs during extension in figures 6 and 7a,b) showing coactivation of both agonist and antagonist muscles during the extension phase. both p6 figure 5 . emg activity recorded from the left (l) and right (r) quadriceps (q) and hamstrings (h) during hip/knee flexion (light grey) and extension (dark grey) movements without tscs, before and after the intervention. data are shown for (a,b) p4, (c,d) p5 and (e,f) p6 (all in the stim group); each movement was repeated three times, and emg data from all three movements are overlaid. during the voluntary ankle manoeuvre, participants were asked to bring their toes up and point their toes down in response to the audio/visual cue ( figure 6 ). p5 (ais d, stim group) was unable to perform both flexion and extension of the right ankle before the intervention (figure 7c ) but after training, he regained the ability (with a delayed recruitment) to flex and extend the right foot (figure 7c,d) . the emg quantification revealed that although this functional movement was achieved, the pattern of activity was seemingly uncoordinated (figure 6 ). p5 displayed an additional increase in volitional muscle activity with control over the left ankle movement in comparison to baseline recordings (figure 7c,d) . an increase in volitional emg activity was also present in p4 (ais c, stim group) post training compared with pre (see p4 rta and rgs during extension in figures 6 and 7a,b) showing coactivation of both agonist and antagonist muscles during the extension phase. both p6 all participants in the stim group, who had increased lower limb motor scores after training, showed improvements >mdc [43] , in at least one sub-category (figure 8 ). p5 showed increases in three sub-categories: role limitations due to physical health (+50), role limitations due to emotional problems (+67) and social functioning (+25). p4 increased in one sub-category: vitality (+33), and p6 increased in two sub-categories: vitality (+15) and pain (+23). in contrast, one participant in the stim group (p1) showed a reduction in one sub-category: pain (−23; i.e., the participant reported increased pain after training). in the non-stim participants, one participant (p3) showed a change >mdc in one sub-category: role limitations due to physical health (+50). no changes in scim iii scores, greater than the mdc for each sub-category [44] , were noted in any participant (figure 9 ). tscs, before and after the intervention. data are shown for (a,b) p4, (c,d) p5, (e,f) p6 (stim group) and (g,h) p8 (non-stim group); each movement was repeated three times, and emg data from all three movements are overlaid. all participants in the stim group, who had increased lower limb motor scores after training, showed improvements >mdc [43] , in at least one sub-category (figure 8 ). p5 showed increases in three sub-categories: role limitations due to physical health (+50), role limitations due to emotional problems (+67) and social functioning (+25). p4 increased in one sub-category: vitality (+33), and p6 increased in two sub-categories: vitality (+15) and pain (+23). in contrast, one participant in the stim group (p1) showed a reduction in one sub-category: pain (−23; i.e., the participant reported increased pain after training). in the non-stim participants, one participant (p3) showed a change >mdc in one sub-category: role limitations due to physical health (+50). no changes in scim iii scores, greater than the mdc for each sub-category [44] , were noted in any participant (figure 9 ). the aim of this pilot study was to assess the feasibility of adding sub-threshold transcutaneous scs to an 8-week sit-to-stand training intervention in individuals with chronic motor complete and incomplete sci. of the 35 participants assessed for eligibility, 9 participated, and 7 completed the trial. for all participants in the stim (tscs + sit-to-stand training) group, loading through the lower limbs increased progressively throughout the intervention. both participants with motor incomplete injuries in the stim group considerably improved their isncsci motor scores (by 5-7 points) and voluntary muscle activation (during the bmca), when tscs was switched off. two participants in the stim group with motor complete injuries showed small increases in volitional muscle activation in the bmca, with tscs switched off, and one of these participants also improved their isncsci motor score by one point. no changes were found in either participant in the non-stim (sit-to-stand training alone) group (ais a and c). improvements in health-related quality of life were detected in sf-36 subcategories including physical, emotional, vitality, social functioning and pain in stim group participants. there was considerable interest in the trial from people with sci worldwide. in total, 35 potential participants were assessed for eligibility. the majority (n = 28) met the study criteria. the known reasons for not participating once deemed eligible were distance to travel, and assignment to the non-stim group. to mitigate these drop-outs, a larger trial should be carried out across multiple sites, and consider adding "sham" tscs to the sit-to-stand alone intervention. the sensation caused by tscs often precludes a suitable sham intervention (i.e., participants would be aware of their group assignment); however, it may be possible to inform participants that they will receive a tscs intervention that is delivered either above or below sensory threshold. of the nine participants that carried out the intervention, none voluntarily withdrew (two participants were withdrawn for other reasons), indicating that they found the intervention acceptable. some participants (in the stim group) also reported that they would be willing to continue beyond 8 weeks, indicating that a longer intervention period may also be feasible if a suitable sham intervention can be incorporated. the outcome measures were feasible overall, however stimulation artefacts caused by the tscs precluded the aim of this pilot study was to assess the feasibility of adding sub-threshold transcutaneous scs to an 8-week sit-to-stand training intervention in individuals with chronic motor complete and incomplete sci. of the 35 participants assessed for eligibility, 9 participated, and 7 completed the trial. for all participants in the stim (tscs + sit-to-stand training) group, loading through the lower limbs increased progressively throughout the intervention. both participants with motor incomplete injuries in the stim group considerably improved their isncsci motor scores (by 5-7 points) and voluntary muscle activation (during the bmca), when tscs was switched off. two participants in the stim group with motor complete injuries showed small increases in volitional muscle activation in the bmca, with tscs switched off, and one of these participants also improved their isncsci motor score by one point. no changes were found in either participant in the non-stim (sit-to-stand training alone) group (ais a and c). improvements in health-related quality of life were detected in sf-36 subcategories including physical, emotional, vitality, social functioning and pain in stim group participants. there was considerable interest in the trial from people with sci worldwide. in total, 35 potential participants were assessed for eligibility. the majority (n = 28) met the study criteria. the known reasons for not participating once deemed eligible were distance to travel, and assignment to the non-stim group. to mitigate these drop-outs, a larger trial should be carried out across multiple sites, and consider adding "sham" tscs to the sit-to-stand alone intervention. the sensation caused by tscs often precludes a suitable sham intervention (i.e., participants would be aware of their group assignment); however, it may be possible to inform participants that they will receive a tscs intervention that is delivered either above or below sensory threshold. of the nine participants that carried out the intervention, none voluntarily withdrew (two participants were withdrawn for other reasons), indicating that they found the intervention acceptable. some participants (in the stim group) also reported that they would be willing to continue beyond 8 weeks, indicating that a longer intervention period may also be feasible if a suitable sham intervention can be incorporated. the outcome measures were feasible overall, however stimulation artefacts caused by the tscs precluded use of the emg data collected during training: this issue should be further explored prior to completing a larger trial. in addition, increased pain after training was reported in one participant in the stim group, therefore more detailed reporting of these changes during the intervention is important to distinguish the source (i.e., do they relate to the physical activity or the tscs per se). up-to 1.5 years post-trial, participants (continuing active training regimes) anecdotally reported the development of further functional changes. this highlights the importance of including long-term follow-up assessments within the study design. none of our participants were able to achieve immediate unassisted or minimally assisted standing during the first session with tscs, as has been reported by other groups [14] . this was most likely due to the tscs being delivered at an intensity below motor threshold, so the stimulation did not directly elicit lower limb extension movements or emg activity. however, all the participants in the stim group showed some evidence of motor recovery in the presence of tscs during training, which occurred after at least 4 weeks of training. this included progressive reductions in the required bws to stand up (figure 3c ), and restored ability to voluntarily activate the quadriceps to generate lower limb extension on-command during training exercises (see video s1). one participant (p4, ais c) also regained the ability to stand with a standing frame with minimal assistance after 8 weeks, which was only possible in the presence of tscs. the combination of tscs with proprioceptive feedback, due to task-specific stand training and progressive increases in lower limb loading, may have enhanced appropriate muscle activity to enable minimally assisted standing [47] . similar progressive recovery has previously been reported in the presence of either epidural [16, 17, 19, 31] or transcutaneous [14, 35] scs, but recovery is dependent on preserved functional neurons passing the lesion site, even in people diagnosed with motor complete injuries [45, 48] . of the participants in the stim group with a clinically complete diagnosis (p1, p2 and p6), none showed evidence of volitional emg activity at baseline, however p6 was known to have retained partial innervation below the level of injury [49, 50] and the other two presumably had "discomplete" injuries. it has been hypothesised that tonic scs shifts the baseline level of spinal network excitability closer to motor threshold enabling people with incomplete and discomplete sci to voluntarily generate movements by descending input via these preserved functional neurons [35, 51] . improvements in voluntary control of movement or an increase in volitional muscle activity after training (when tscs was off) were found in four of the five participants in the stim group (p1-ais a, p4-ais c, p5-ais d and p6-ais a with partial preservation). only one other study has reported functional recovery (with tscs switched off) following an intervention of non-invasive scs [35] ; that study used gravity-neutral step training combined with sub-motor threshold tscs, delivered with a 10 khz carrier frequency. no changes were observed in either participant in our non-stim group (p3-ais a and p8-ais c), indicating the importance of the additional tscs for these changes to occur. the participant in the stim group with the least severe injury (p5-ais d), and the only participant in our study who had any evidence of voluntary motor control at baseline, recovered considerable motor function after training. he was able to fully control flexion and extension in his left leg, which was absent at baseline, and regained the ability to freely move his right foot; his isncsci motor scores had improved by a total of seven points after training. similar functional recovery (with scs off) has previously been reported in four people with incomplete injuries (isncsci sores improved by between 4 and 11 points) following intensive activity-based training combined with epidural scs, applied at an intensity that enabled voluntarily-driven movements [33] . the other two participants with incomplete neurological diagnosis in our study had more extensive motor deficits (both showed little/no voluntary emg activity at baseline); one was in the stim group (p4-ais c) and the other was in the non-stim group (p8-ais c). after training, p4 (stim) showed increases in appropriate muscle activity during the bmca, and her isncsci score improved by five points; no changes were observed in p8 (non-stim), indicating the importance of additional tscs. among the three participants with motor complete injuries in the stim group (p1, p2 and p6), some recovery of motor control (with tscs off) was observed in two of them (p1 and p6). p6 had increased voluntarily driven muscle activity (during the bmca) after training, and p1 improved his isncsci motor score by one point (palpable contraction in achilles tendon). no recovery (with tscs off) was observed in p2, despite evidence of voluntary motor performance in the presence of tscs during training sessions (see video s1). previous studies using epidural scs in people with motor complete sci have also reported considerable improvements in motor control (in the presence of scs) in all participants (n = 12) [19, 32, 34] , but recovery of volitional motor control with scs off was only observed in a subset of these (5/12). pre-clinical trials, using epidural scs combined with step training, have also reported that animals with severe complete transections only show detectable improvements in stepping when stimulation is on, whereas animals with less severe injuries and greater lesion sparing were able to recover voluntary motor control when stimulation was absent [28] [29] [30] 52] . overall, in our stim participants, the amount of recovery that occurred was related to the severity of the injury; as the progressive reduction in bws had not reached a plateau after 8 weeks, a longer intervention may bring about further recovery. one factor thought to be important in the effectiveness of scs is the baseline level of excitatory support from supraspinal and peripheral systems on lumbar spinal circuity [53] : sci disrupts this process, causing an imbalance in descending and ascending transmission and dysregulated spinal activity [4, 54] . scs had been shown to regulate this with better prognosis for those with underlying excitability at baseline, for example, in spastic conditions [28, 55] . indeed, one recent study reported that the recovery of volitional control (with scs off), observed after one-month of epidural scs, was correlated with spasticity scores at baseline [34] ; those authors proposed that baseline spasticity might be a marker for preserved corticospinal tract axons [56] . given that tonic sub-threshold scs has been reported to attenuate neural hyper-excitability and recover spinal inhibitory control in people with sci [25, 26, 57] , and has been successfully used in the treatment of spasticity [58, 59] , it is also possible that acute reductions in spasticity (due to scs) enabled the participants access to these retained pathways, which were otherwise masked by hyper-excitability in the central nervous system. repeated activation of these pathways by carrying out sit-to-stand training in combination with scs, over several weeks, may have contributed to the observed improvements in voluntary function without tscs [4] . previous studies have reported optimal parameters to elicit lower limb extension movements or standing as being at 5-15 hz [5] [6] [7] . we chose a relatively higher stimulation frequency (30 hz), which is closer to the frequency shown to suppress lower limb spasticity when applied at sub-threshold intensities [25] [26] [27] . relatively higher frequencies (25) (26) (27) (28) (29) (30) have also been found to be as effective for standing as 15 hz tonic stimulation [17, 60] . another study reported that higher stimulation frequencies (80-100 hz) augmented the activity of lower limb flexor muscles specifically, whereas lower frequencies (20-30 hz) augmented extensor muscle activity [33] . guidelines reported by rejc and co-workers [60] recommend an initial stimulation frequency of 25 hz at near-motor threshold (that does not directly elicit lower limb movements) to enable standing without bws, however further investigation into the optimal frequency for standing is warranted, including patient-specific customisation approaches. in this study, we used sub-threshold scs in order to alter baseline excitability of the spinal cord, enabling movements triggered by descending inputs that remain intact after sci [54] . while lower limb extension and standing have been directly elicited by both epidural [6] and transcutaneous [13, 14] supra-threshold scs, such high stimulation intensities have been found to cause, in some people, pulsatile contractions or rhythmic bursting that interfered with standing [60, 61] , and these effects may be augmented during activities in which body position is altered such as sit-to-stand, causing inconsistency in the structures being activated during movement [15, 62, 63] . some of our participants also reported discomfort with higher stimulation intensities and, in some cases, co-activation of the trunk musculature caused discomfort or poor postural alignment. therefore, to permit adequate free range of movement during stand exercises and prevent discomfort, lower stimulation intensities were selected in this trial. the sf-36 and scim iii questionnaires were completed before and after training in order to explore whether any recovery of motor control was associated with improvements in healthy-related quality of life and functional independence. we used mdcs as a threshold to determine whether or not changes took place in each participant. all participants that showed evidence of functional changes after training (p4, p5 and p6) also had increased sf-36 in at least one sub-category, indicating that the motor recovery may have improved their health-related quality of life. in the pain sub-category, there were contradictory findings in the stim group: one participant reported an improvement (p6), and one reported more pain after training (p1). this requires further investigation, including the source of any increases in pain. one participant in the non-stim group reported an improvement in one category (role limitations due to physical health), suggesting that the changes may have been associated with the sit-to-stand training alone, or may have occurred with any intervention provided in addition to their usual activities. the main limitation in the present study was the imbalance of participants between groups. our intention was to recruit the same number of participants with a motor complete and motor incomplete injury in each group; which we did still achieve, however three of the five participants in the non-stim group were unable to complete the trial. in the stim group participants, tscs intensity may not have always been optimal during training, due to discomfort from the stimulation. while this cannot be avoided when using traditional tscs waveforms, prrs should be elicited during training to enable stimulation intensity to be defined relative to prr threshold. another limitation was stimulation artefacts in emg data when tscs was switched on. this meant that we were unable to quantify changes in emg activity due to volitional drive in the presence of tscs in out stim group participants. evidence of the motor recovery in the presence of tscs can however be viewed in one participant in video s1. this study has shown that the addition of non-invasive sub-threshold tscs to sit-to-stand training is an acceptable intervention for people with motor complete and incomplete sci. in this pilot work, we found that this intervention caused some recovery of volitional drive and control in people with clinically diagnosed complete and incomplete sci; these findings should now be verified in a larger trial. this intervention is simple and could be achieved by people living with chronic sci in their own homes i.e., using a standing frame and a commercially available stimulator. if larger trials support our early observations, the accessibility of this intervention could enable many people living with sci to achieve progressive improvements in motor recovery in the presence of tscs and, in some cases, neuroplastic change, which may also benefit health-related quality of life. future work should further explore the effects of sub-threshold scs on corticospinal excitability, focusing on the effects of different stimulation intensities, in order to determine the optimal intensity for neuroplastic change, and to improve our understanding of the underlying mechanisms. future work should also consider optimal rehabilitative interventions to combine with scs. while there is ample pre-clinical evidence to suggest that scs is more effective when combined with afferent feedback due to locomotor training [28] [29] [30] , our study, and the recent clinical studies using epidural scs [18, 34] , suggest that recovery of volitional motor control can occur when scs is combined with simple exercises (incorporating descending volitional drive), which are cheaper and more accessible to the sci population. enhancing rehabilitation and functional recovery after brain 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functional states after the loss of brain input step training reinforces specific spinal locomotor circuitry in adult spinal rats restoring voluntary control of locomotion after paralyzing spinal cord injury epidural spinal-cord stimulation facilitates recovery of functional walking following incomplete spinal-cord injury neuromodulation of lumbosacral spinal networks enables independent stepping after complete paraplegia targeted neurotechnology restores walking in humans with spinal cord injury long-term spinal cord stimulation after chronic complete spinal cord injury enables volitional movement in the absence of stimulation noninvasive reactivation of motor descending control after paralysis initiation and modulation of locomotor circuitry output with multisite transcutaneous electrical stimulation of the spinal cord in noninjured humans post-activation depression in the human soleus muscle using peripheral nerve and transcutaneous spinal stimulation appendix 1. a manual for the neurophysiological assessment of motor control: the brain motor control assessment (bmca) protoco sf-36 health survey health status rated with the medical outcomes study 36-item short-form health survey after spinal cord injury scim-spinal cord independence measure: a new disability scale for patients with spinal cord lesions the spinal cord independence measure (scim): sensitivity to functional changes in subgroups of spinal cord lesion patients comparisons of the brief form of the world health organization quality of life and short form-36 for persons with spinal cord injuries the spinal cord independence measure: how much change is clinically significant for spinal cord injury subjects clinical neurophysiological assessment of residual motor control in post-spinal cord injury paralysis neurophysiological characterization of motor recovery in acute spinal cord injury integrating multiple sensory systems to modulate neural networks controlling posture evidence of subclinical brain influence in clinically complete spinal cord injury: discomplete sci guidelines for the conduct of clinical trials for spinal cord injury as developed by the iccp panel: spontaneous recovery after spinal cord injury and statistical power needed for therapeutic clinical trials restoration of sensorimotor functions after spinal cord injury spinal cord stimulation and augmentative control strategies for leg movement after spinal paralysis in humans emergence of epidural electrical stimulation to facilitate sensorimotor network functionality after spinal cord injury targeting lumbar spinal neural circuitry by epidural stimulation to restore motor function after spinal cord injury training locomotor networks epidural electrical stimulation of posterior structures of the human lumbosacral cord: 3. control of spasticity residual descending motor pathways influence spasticity after spinal cord injury repeated transspinal stimulation decreases soleus h-reflex excitability and restores spinal inhibition in human spinal cord injury epidural spinal cord stimulation in the management of spasms in spinal cord injury: a prospective study spinal cord stimulation for the control of spasticity in patients with chronic spinal cord injury: i. clinical observations effects of lumbosacral spinal cord epidural stimulation for standing after chronic complete paralysis in humans epidural stimulation: comparison of the spinal circuits that generate and control locomotion in rats, cats and humans neuromodulation of evoked muscle potentials induced by epidural spinal-cord stimulation in paralyzed individuals modification of reflex responses to lumbar posterior root stimulation by motor tasks in healthy subjects this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors are grateful to all of the trainers at neurokinex (hemel hempstead and gatwick), particularly jane symonds, taylor omran, morgan price-king, claire ryan, stephen sims and ben smith, for their assistance with sit-to-stand training sessions, and to interns from university of bath and queen mary university. the authors also wish to express their sincere thanks to all of the participants for their time, effort and dedication to the trial. the authors declare no conflict of interest. the funders had no role in the design of the study; in the collection, analyses, or interpretation of data; in the writing of the manuscript, or in the decision to publish the results. key: cord-301157-tu3iig9o authors: felsenstein, susanna; willis, emily; lythgoe, hannah; mccann, liza; cleary, andrew; mahmood, kamran; porter, david; jones, jessica; mcdonagh, janet; chieng, alice; varnier, giulia; hughes, stephen; boullier, mary; ryan, fiona; awogbemi, olumoyin; soda, giridhar; duong, phuoc; pain, clare; riley, phil; hedrich, christian m. title: presentation, treatment response and short-term outcomes in paediatric multisystem inflammatory syndrome temporally associated with sars-cov-2 (pims-ts) date: 2020-10-14 journal: j clin med doi: 10.3390/jcm9103293 sha: doc_id: 301157 cord_uid: tu3iig9o the novel severe acute respiratory syndrome coronavirus 2 (sars-cov-2) is the pathogen responsible for coronavirus disease 2019 (covid-19). whilst most children and young people develop mild symptoms, recent reports suggest a novel paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 (pims-ts). case definition and classification are preliminary, treatment is empiric and disease-associated outcomes are unclear. here, we report 29 patients with pims-ts who were diagnosed, admitted and treated in the english north west between march and june 2020. consistent with patterns observed internationally, cases peaked approximately 4 weeks after the initial surge of covid-19-like symptoms in the uk population. clinical symptoms included fever (100%), skin rashes (72%), cardiovascular involvement (86%), conjunctivitis (62%) and respiratory involvement (21%). some patients had clinical features partially resembling kawasaki disease (kd), toxic shock syndrome and cytokine storm syndrome. male gender (69%), black, asian and other minority ethnicities (bame, 59%) were over-represented. immune modulating treatment was used in all, including intravenous immunoglobulin (ivig), corticosteroids and cytokine blockers. notably, 32% of patients treated with ivig alone went into remission. the rest required additional treatment, usually corticosteroids, with the exception of two patients who were treated with tnf inhibition and il-1 blockade, respectively. another patient received il-1 inhibition as primary therapy, with associated rapid and sustained remission. randomized and prospective studies are needed to investigate efficacy and safety of treatment, especially as resources of ivig may be depleted secondary to high demand during future waves of covid-19. since the advent of the coronavirus disease 2019 (covid-19) pandemic, dominated by respiratory disease and evolution of acute respiratory distress syndrome (ards), cardiovascular compromise, excessive systemic inflammation and coagulopathy in adults [1] [2] [3] , several countries affected by the coronavirus disease [4] pandemic have reported an unusually high number of cases of children hospitalized due to a multisystem inflammatory condition, at times requiring intensive care (table s1) . the royal college of paediatrics and child health (rcpch) defined a paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 (pims-ts) [34] , and the centers for disease control and prevention (cdc) defined a multisystem inflammatory syndrome in children (mis-c) [35] , detailed in table s2 . patients present febrile, with signs and symptoms reminiscent of systemic inflammatory responses partially resembling kawasaki disease (kd), cytokine storm (cs) or toxic shock syndrome [36] , commonly including a rash, conjunctivitis, abdominal pain and evidence of cardiac inflammation and/or injury [37] . a pathophysiological association with sars-cov-2 infection has been suggested, as a high proportion of patients are either sars-cov-2 positive by polymerase chain reaction (pcr) or seropositive for anti-sars-cov-2 igg antibodies. however, a causal relationship between sars-cov-2 and pims-ts has not been formally proven [19, 38] . to reach a consensus for evidence-based management guidance, diagnostic criteria have been defined (table s2) . while pims-ts shows similarities to kd [39] , patients tend to be older (≥5 years of age) and not all fulfil criteria for kd (table s1) . cardiac involvement appears to be more frequent in pims-ts, inotrope requirement tends to be more common and recovery, in most cases, is swift (table s1 , [18, 40] ). as in kd, inflammatory markers in pims-ts are raised, including pro-inflammatory cytokines (such as il-6), c-reactive protein (crp), ferritin, lactate dehydrogenase (ldh) and neutrophil counts. d-dimers are elevated and myocardial injury can be associated with elevated troponin t and pro-brain natriuretic peptide (pro-bnp). lymphopenia [41, 42] , thrombocytopenia and circulating immune complexes have been reported, all distinct from classical kd [41, 43] . lastly, coagulopathy associated with antiphospholipid antibodies has been repeatedly described in pims-ts and covid-19 [44, 45] and contributes to a hypercoagulable state [46] . therapeutically, the hyperinflammatory state in pims-ts and severe covid-19 has been addressed by a variety of immunomodulatory approaches, including corticosteroids, intravenous immunoglobulins (ivig), cytokine blocking strategies targeting il-6 (tocilizumab [47, 48] ) or il-1 (anakinra [45, 49] ) and adjunctive anticoagulant treatments [50] . this study focused on clinical presentations, treatment response and outcomes in a cohort of 29 patients with pims-ts diagnosed and treated in the english north west between march and june 2020. patients meeting rcpch criteria for pims-ts [34] presenting to alder hey children's nhs foundation trust, liverpool, uk, and royal manchester children's hospital, manchester, uk, between march and june 2020 were included in this study. the clinical audit department approved the study. the clinical audit department (institutional review board) approved data collection, analysis and dissemination of results, in accordance with the code for good practice (audit registration number 6417). demographic, laboratory and clinical data were collected retrospectively from hospital patient charts. laboratory and clinical parameters underlying significant physiological age-related changes (lymphocyte counts, blood pressure, respiratory rate and heart rate) were adjusted using age-specific normal ranges [51] [52] [53] . for age adjustment of lymphocyte counts, the patient's absolute lymphocyte count was divided by the 10th centile of the age-adjusted cd3 + lymphocyte count [51] and expressed as a ratio, thus allowing a comparison of age-dependent parameters across different age groups. relevant clinical and laboratory parameters were collected at admission, at their respective peak, at 48 h after symptom resolution, and at the 1-2 week follow-up visit after patient discharge. the following case definitions for determination of symptom complexes were used as comparators: the american heart association criteria for classic and incomplete kd [54] , ravelli criteria for risk assessment of secondary macrophage activation syndrome [55] and calculation of the h score [56] . published criteria by the cdc (mis-c) [35] and the rcpch (pims-ts) [34] were applied (table s2) . statistical analysis was performed using spss 22.0 (spss inc., chicago, il, usa). quantitative variables were reported as absolute numbers, percentages, means and standard deviations where following normal distribution, and by median and interquartile range (iqr) where not. comparisons of continuous variables between groups to test for equality were performed using the t test and paired t test if normally distributed (shapiro-wilk, p > 0.05), or mann-whitney and kruskal-wallis test where not. non-parametric continuous dependent samples in ≥2 groups were compared using the friedman test. tests of association between categorical variables were based on chi squared-and fisher exact tests. where applicable, holm-bonferroni correction was performed to correct for multiple comparisons, and the significance level adjusted accordingly as indicated. in all other instances, p-values reported are 2-sided and were considered statistically significant if <0.05. between march and june 2020, 29 children were admitted to tertiary paediatric centres in the english north west with a diagnosis of pims-ts (alder hey children's nhs foundation trust hospital, liverpool, n = 10; and royal manchester children's hospital, n = 19). notably, this lagged behind the peak of adult admissions for covid-19 to hospitals in the region by approximately 4 weeks and therefore occurred well into the decline of covid-19 in england ( figure 1 ) [57] ; in keeping with pims-ts/mis-c cohorts described elsewhere [9] . two-thirds of paediatric patients admitted with pims-ts (20/29; 69%) were male. twelve children were caucasian (41.4%), 6/29 (20.7%) south east asian, 2 (6.9%) east asian, 4/29 (13.8%) african/caribbean and 5 (17.2%) of unknown or multi-ethnic background. in keeping with other published reports from europe and north america, children of black, asian and other minority ethnic (bame) background were over-represented when compared to the composition of the general population in the region based on national census data ( figure 2 ) [58] . median age was 6.0 years (iqr 3.8-9.9 years). only one patient was younger than one year, five between 12 and 24 months, eight 2-5 years, eleven 5-12 years and four older than 12 years of age. on average, patients were hospitalized for 8.5 days (sd 3.1). temporal distribution of paediatric inflammatory multisystem syndrome temporally associated with severe acute respiratory syndrome coronavirus 2 (sars-cov-2) (pims-ts) cases of this cohort, in relation to covid-19 like presentations to hospitals in england. the peak of presentations of children with paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 followed the peak of presentations of patients, adult and paediatric, to english emergency departments, with a lag of 4-6 weeks (figure adapted from https://www.gov.uk/government/news/weekly-covid-19-surveillance-report-published; week 30). two-thirds of paediatric patients admitted with pims-ts (20/29; 69%) were male. twelve children were caucasian (41.4%), 6/29 (20.7%) south east asian, 2 (6.9%) east asian, 4/29 (13.8%) african/caribbean and 5 (17.2%) of unknown or multi-ethnic background. in keeping with other published reports from europe and north america, children of black, asian and other minority ethnic (bame) background were over-represented when compared to the composition of the general population in the region based on national census data ( figure 2 ) [58] . distribution of ethnicities among children presenting with paediatric multisystem inflammatory syndrome temporally associated with sars-cov-2 in relation to regional ethnic distribution in the north west of england, as per national census data (https://www.ethnicity-facts-figures.service.gov.uk/uk-population-by-ethnicity/national-andregional-populations/regional-ethnic-diversity/latest#ethnic-groups-by-area. half of the patients (14/29; 48.3%) tested positive by sars-cov-2 serology, 11 (37.9%) negative. in 13.8% (n = 4) no serological testing was undertaken. of 27 patients tested by sars-cov-2 pcr, only three were positive (11.1%). all pcr positive patients were also positive by serology. combined, 14/29 (48.3%) patients had laboratory evidence of sars-cov-2 infection, while population seroprevalence in children in the uk is reported to be much lower, at an adjusted population seroprevalence in england across all ages of 6.0-6.8% [57, 59] , and even lower among children [60] in who seroprevalence ranges between 0.7 and 10% [61] . of these, one child had pcr-proven sars-cov-2 infection in the preceding month, another had had close contact with a proven case, four had contact with a suspected case. as many as 18/29 (62.0%) patients had a history of gastrointestinal illness during the preceding month, 3/29 (10.3%) had a history of respiratory infection. in 8/29 (27.5%) children, no history of a symptomatic illness prior to the onset of pims-ts was given. overall, a laboratory, epidemiological and/or anamnestic link with sars-cov-2 was established in 24/29 (82.8%) children ( figure 3 ). males and bame were over-represented. both males (male 8/20, female 0/9; p = 0.03) and children of bame background (caucasian 1/12; bame 7/17; p = 0.04) were significantly more likely to present with clinical signs of hypoperfusion or shock. more severe courses of mis-c resp. pims-ts have repeatedly been seen in non-caucasian children in europe, and are being reported internationally [62] . pims-ts patients of this cohort exhibited significantly increased seroconversion rates in sars-cov-2 infections in the uk or paediatric population, even though lower than reported in other cohorts (table s1 ). comparable proportions, namely 4/11 (36%) of children with, and 7/18 (39%) without symptoms in keeping with classic kd were of the for kd typical age of between 1-5 years of age; and 7/19 (37%) of children who did; and 4/10 (40%) who did not fulfil criteria of either classic or incomplete kd were within the typical age-range. a total of 3/10 children (30%) who tested sars-cov-2 sero-and pcr positive, and 3/7 (42.8%) who tested negative (on pcr and/or serology) fulfilled classic or incomplete kd criteria. sars-cov-2 status was therefore not discriminatory, as suggested by the current diagnostic criteria in use in the uk and europe (table s2) . febrile illness was the presenting complaint in all 29 patients. patients had been febrile for a median of 5 days (iqr 4-7 days) before a diagnosis of pims-ts/mis-c was made. in 18/29 (62%) children, pims-ts was the initial working diagnosis, in the remaining 11 (38%), acute infectious causes including urinary tract infections, pneumonia, toxic shock syndrome, bacterial lymphadenitis, viral illness with exanthema or sepsis (n = 7); malignancy (1), systemic juvenile idiopathic arthritis (n = 1) or appendicitis (n = 2) were considered, and initial work up directed accordingly. febrile illness was the presenting complaint in all 29 patients. patients had been febrile for a median of 5 days (iqr 4-7 days) before a diagnosis of pims-ts/mis-c was made. in 18/29 (62%) children, pims-ts was the initial working diagnosis, in the remaining 11 (38%), acute infectious causes including urinary tract infections, pneumonia, toxic shock syndrome, bacterial lymphadenitis, viral illness with exanthema or sepsis (n = 7); malignancy (1), systemic juvenile idiopathic arthritis (n = 1) or appendicitis (n = 2) were considered, and initial work up directed accordingly. age-adjusted heart rates ranged between the 75-95th centile for age in 4/26 (15.3%), above the 99th centile in 17/26 (65.3%) of patients and unavailable in three. of note, all but three were febrile at ≥38 • c at the time. of 22 patients for whom blood pressure [63] was available, three had systolic blood pressures below the age-adjusted normal range [52] . pulmonary involvement was not uncommon: 6/29 (20.7%) required o2 supplementation. exanthema and gastrointestinal symptoms were common features ( figure 4 ), in keeping with other published cohorts of pims-ts patients, where gastrointestinal symptoms have been reported as common feature, often presenting as the main presenting complaint [9, 26] (table s1) . except for abdominal pain, which occurred more frequently in children over 5 years of age (3/12 in <5 y.o.a.; 12/17 in ≥5 y.o.a.; p = 0.025), clinical features did not differ significantly between age groups. duration of fever prior to diagnosis did not differ between children with changes on echocardiogram at the 2 week follow-up and those without. lastly, a composite parameter of cardiovascular involvement at diagnosis (clinical/echocardiographic evidence of functional impairment/laboratory evidence) was neither significantly associated with age nor fulfilment of classical kd criteria. lymphopenia [53] was present in 23/29 (79.3%) patients ( figure 5 ). in 17 patients, the ratio of absolute lymphocyte count and 10th centile of age adjusted normal range was lower than 0.5, indicating a lymphocyte count of <50% of the age-adjusted 10th centile, affecting the over 5s more prominently (table 1) . by comparison, only one patient had a neutrophil count below, and only 8/29 above the age adjusted normal range at diagnosis. nine patients presented with thrombocytopenia lymphopenia [53] was present in 23/29 (79.3%) patients ( figure 5 ). in 17 patients, the ratio of absolute lymphocyte count and 10th centile of age adjusted normal range was lower than 0.5, indicating a lymphocyte count of <50% of the age-adjusted 10th centile, affecting the over 5s more prominently (table 1) . by comparison, only one patient had a neutrophil count below, and only 8/29 above the age adjusted normal range at diagnosis. nine patients presented with thrombocytopenia <150 × 10 9 /l (31%), only one with thrombocytosis ≥500 × 10 9 /l (3.4%). j. clin. med. 2020, 9, x for peer review 8 of 21 figure 5 . laboratory parameters at three time points: at diagnosis, at 48 h after the patient being afebrile, at the 1-2 week follow-up, illustrating rapid normalization of thrombocytopenia, lymphopenia, c-reactive protein and brain natriuretic peptide within two weeks of presentation to a health care provider. lymphocyte count is expressed as a ratio of absolute lymphocyte count divided by 10th centile of normal range for age (see methods), 1.0 = 10th centile for age adjusted normal range. acute phase parameters were markedly elevated. twelve of 19 (63%) children had raised triacylglycerol (tag) levels at diagnosis (≥150 mg/dl), 20/25 (80%) had ferritin levels above 150 figure 5 . laboratory parameters at three time points: at diagnosis, at 48 h after the patient being afebrile, at the 1-2 week follow-up, illustrating rapid normalization of thrombocytopenia, lymphopenia, c-reactive protein and brain natriuretic peptide within two weeks of presentation to a health care provider. lymphocyte count is expressed as a ratio of absolute lymphocyte count divided by 10th centile of normal range for age (see methods), 1.0 = 10th centile for age adjusted normal range. table 1 . median (interquartile range) of complete blood count results at admission, in children presenting with paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2. included is the age-adjusted lymphocyte count, expressed as a ration of patient's absolute lymphocyte count divided by the 10th centile of age-adjusted normal lymphocyte count. acute phase parameters were markedly elevated. twelve of 19 (63%) children had raised triacylglycerol (tag) levels at diagnosis (≥150 mg/dl), 20/25 (80%) had ferritin levels above 150 ng/ml with 13/25 (52%) above 500 ng/ml (median 517 ng/ml; iqr 38-10761) ( table s3 ). all patients exhibited raised crp levels >8 mg/l (normal range: ≤8 mg/l), 22/29 (75.8%) had crp levels >100 mg/l (median 174 mg/l; iqr 102.9-232). all but one child had d-dimer levels above 500 ng/ml, with 18/22 (81.8%) children having d-dimer levels >1000 ng/ml (normal range ≤500 ng/ml), in 6 cases >5000 ng/ml (27.3%). serum transaminase levels were elevated in 12/26 (46.2%) children. a subset of patients exhibited mildly raised aptt ≥33 s (4/19; 21%). international normalized ration (inr) was only marginally elevated in one child at 1.6 (normal 0.8-1.1), and normal in all others. serum sodium, as a potential symptom of inappropriate anti-diuretic hormone (adh) release [64] , was reduced in a proportion of patients; 17/28 (60.7%) had serum sodium levels ≤135 mmol/l, 5/28 (17.8%) a "very low" sodium of 124-130 mmol/l. no significant association with clinical cardiological outcomes (below) or correlation with blood parameters suggestive of cardiac injury were identified. while some of these findings also frequently occur in kd, others are uncommon in classical kd (cytopenia, coagulopathy, hypertriglyceridemia) and may help to differentiate between pims-ts and kd [64] . in the cohort presented here, imaging followed clinical need, but no structured algorithm [65] . most pathological findings were recorded in cardiac imaging (19/27; 70 .4%), including coronary dilatation, valvular regurgitation, functional impairment and pericardial effusions (table s4) . a total of 21/29 children (72%) underwent chest x-ray or ct, with abnormal findings in half (10/21; 47%). of note, abnormal chest imaging results were not associated with sars-cov-2 seropositivity (p = 0.66). abnormalities identified included perihilar consolidation (4), lymphadenopathy (2), crazy paving pattern (1), ground glass opacities (1) and pleural and pericardial effusion (1) . with regard to abdominal imaging, pathological findings were present in 7/19 (36.8%) (table s5) . whilst gastrointestinal symptoms have been reported as a common complaints in pims-ts/mis-c [16, 66, 67] , the frequency of pathological findings on abdominal imaging in this cohort (36.8%) is striking. one patient who presented with encephalopathy and headaches underwent a brain mri and angiogram, which identified focal lesions in the deep white matter, reminiscent of inflammatory lesions seen in acute disseminating encephalomyelitis (adem). the patient, who was sars-cov-2 seropositive and had no pre-existing comorbidities, responded rapidly to ivmp and ivig, with complete resolution of neurological signs and symptoms within 48 h. similar lesions have been reported in pims-ts, both in adult and paediatric cases [68] [69] [70] . end-organ involvement occurs frequently in pims-ts, commonly including cardiovascular, gastrointestinal and respiratory involvement; and yield on medical imaging was high. there has been an international focus on classifying patients with pims-ts, and early discussions focused on similarities to kd. all patients in this cohort fulfilled rcpch criteria for pims-ts [34] , 21/29 north american mis-c criteria [71] (72.4%), and two thirds of patients criteria for kd [54] . neither classical nor incomplete kd criteria were more frequent in children <5 y.o.a. when compared to older children (5/12 in <5 y.o.a.; 6/17 in ≥5 y.o.a.; p = 1.0). as a proportion of patients exhibited laboratory (cytopenia, hyperfibrinogenemia, hypertriglyceridemia, etc., see above) and clinical (rash, fevers) features suggestive of cs or macrophage activation syndrome (mas), we calculated ravelli scores for secondary mas. only one child (15 years, necrotizing lymphadenopathy) fulfilled criteria for mas [55] , (table 2) . as neither pims-ts nor mis-c criteria are validated at present, their discriminatory value in the diagnosis of this still poorly defined symptom complex remains to be determined. while some fulfilled criteria for classical or incomplete kd, the authors argue that pims-ts remains a condition distinct from kd. some findings, such as lymphopenia and thrombopenia, are present in most pims-ts patients, but uncommon in classical kd. other common symptoms of pims-ts/mis-c are unspecific and can occur during (any viral) infection (including mucocutaneous symptoms, lymphadenopathy). lastly, cytokine storm (such as sepsis) can be associated with cardiac symptoms, such as coronary dilatation [72] . more specific criteria are required once further data becomes available [50, 73] , to facilitate an international attempt to classify inflammatory disease associated with covid-19 in children. a better understanding of clinical and laboratory features of pims-ts may allow preliminary hypotheses and conclusions on its pathophysiology and mechanisms involved. several mechanisms have been discussed, and include direct immune cell infection and activation, and antibody-dependent enhancement (ade) [74] , a phenomenon shown to contribute to damage accrual during viral infections. pims-ts, severe covid-19 in adults presents with extensive pulmonary and systemic inflammation, was linked to unfavourable outcomes [75] . intriguingly, sars-cov-2 infection takes mostly a milder course in children [76] . highest incidence of pims-ts occurs weeks after the adult cases of sars-cov-2 infection have reached their peak, sars-cov-2 pcr is commonly negative, and hence secondary immune events may play a role [77] [78] [79] . current criteria as used in europe are not distinguishing kd and pims-ts. laboratory and clinical differences are now beginning to emerge, which may enable the distinction of these two disorders as separate entities [80] . both are characterized by systemic inflammation, but differ distinctly with regard to cytokine profiles, lymphocyte subsets and markers of endothelial damage. in the absence of wider availability of these diagnostic opportunities, the discriminatory value of the currently used criteria must be re-evaluated as data becomes available. a subset of covid-19 patients reportedly develop vasculitic lesions [43] , blood vessel occlusion and infarctions [81] [82] [83] [84] . indeed, also one of the here reported patients developed iga vasculitis with renal involvement four weeks after pims-ts was diagnosed and treated (see also below). (auto-)antibody production, immune complex deposition and mucosal immune dysregulation or poor initial infection control with a delayed immune response, initially high viral loads and delayed or blunted ifn i and iii response may cause delayed hyper-inflammatory disease and co-determine the clinical picture of pims-ts [77, 78, 85] . the majority of children included in this study (25/29; 86 .2%) exhibited features of cardiac involvement. thirteen (44.8%) presented with clinical features of cardiovascular compromise, such as poor perfusion, raised lactate or required fluids and/or inotropic support. elevated biochemical markers of cardiac injury, namely brain natriuretic peptide (bnp) (≥400 pg/ml; 16/22; 72.7%; ≥2000 pg/ml 12/22; 54.5%) or troponin t (≥14 ng/ml; 11/24; 45.6%) were observed. echocardiographic results were retrieved for 27 children ( figure 6 , the majority of children included in this study (25/29; 86 .2%) exhibited features of cardiac involvement. thirteen (44.8%) presented with clinical features of cardiovascular compromise, such as poor perfusion, raised lactate or required fluids and/or inotropic support. elevated biochemical markers of cardiac injury, namely brain natriuretic peptide (bnp) (≥400 pg/ml; 16/22; 72.7%; ≥2000 pg/ml 12/22; 54.5%) or troponin t (≥14 ng/ml; 11/24; 45.6%) were observed. echocardiographic results were retrieved for 27 children ( figure 6 , table s4 ), abnormalities were documented in 19/27 (70.4%) children. where data are available for n < 29, n is specified separately. common findings included coronary artery changes in 14 patients (51.8%) (increased echogenicity, dilatation or fusiform aneurysm formation), valvular involvement in 9 (33.3%) and functional impairment in 9 (33.3%) patients. valvular regurgitation and functional impairment were classified as "mild" in most cases [86] . neither the number of days febrile before a diagnosis was made (p = 0.534), nor patient's age (p = 0.23), were significantly associated with an abnormal echocardiogram on admission (table s6) . bnp (p = 0.002) at admission as well as peak bnp (p = 0.003) were significantly associated with echocardiographic findings of impaired function, while ferritin, d-dimers and age-adjusted lymphocyte counts were not. need for inotropes (p = 0.03) and composite parameters for cardiac injury were more common composite parameters as declared. troponin-troponin t, bnp-brain natriuretic peptide. where data are available for n < 29, n is specified separately. common findings included coronary artery changes in 14 patients (51.8%) (increased echogenicity, dilatation or fusiform aneurysm formation), valvular involvement in 9 (33.3%) and functional impairment in 9 (33.3%) patients. valvular regurgitation and functional impairment were classified as "mild" in most cases [86] . neither the number of days febrile before a diagnosis was made (p = 0.534), nor patient's age (p = 0.23), were significantly associated with an abnormal echocardiogram on admission (table s6) . bnp (p = 0.002) at admission as well as peak bnp (p = 0.003) were significantly associated with echocardiographic findings of impaired function, while ferritin, d-dimers and age-adjusted lymphocyte counts were not. need for inotropes (p = 0.03) and composite parameters for cardiac injury were more common in those who were seropositive (14/14 sars-cov-2 positive had cardiac injury, 6/12 sars-cov2 negative had cardiac injury; p = 0.004) (table s6) . clinical features of peripheral hypoperfusion or shock were significantly more common in children with bame background (p = 0.03) and males (p = 0.04). abnormalities on echocardiogram were also more likely to occur in children of bame background (caucasian 5/11; bame 13/14; p = 0.02). cardiac involvement was the most frequent organ manifestation in this cohort and disproportionately affected children of bame background. a quarter of patients received inotropic support (7/29; 24.1%), and almost half received fluid boli at presentation (12/29, 41 .4%) (table s7) . admission levels of bnp but not troponin t were significantly associated with subsequent need for inotropes (troponin t: inotropes yes: 17 ng/l (iqr ; no: 6.0 ng/l (iqr 3.5-27.0); p = 0.06; bnp: inotropes yes: 6180 pg/ml (iqr 3934-46184) no: 965 pg/ml (iqr 233-5183) p = 0.02). peak bnp and troponin t also showed significantly higher levels in children who required inotropes (troponin t inotropes yes 20 ng/l (iqr 14-143); no 5 ng/l (iqr 3.75-31.75) p = 0.047, bnp inotropes yes 8902 pg/ml (iqr 3934-62009) no 1264 pg/ml (iqr 236-5183) p = 0.008). as many patients present to local hospitals prior to transfer to a tertiary unit with intensive care facilities, these findings may inform clinical practice, supporting the use of bnp levels in particular as a marker for significant cardiac involvement. administration of fluid boluses also was more common in those with higher bnp levels at diagnosis (fluid bolus yes 4615 pg/ml (iqr 965-54018); no 1367 pg/ml (iqr 222-4894) p = 0.06). as bnp is released by cardiomyocytes as a result of increased intra-cardiac volume, at least in some patients, this mechanism may have been involved and possibly attenuated cardiac function [87] . following kd protocols, and in some cases in response to coronary artery anomalies, 22 children (75.9%) received acetylsalicylic acid (asa), one patient (3.4%) did not, in six (20.6%) this information was not provided. all patients received immune modulating treatment for pims-ts (table 3) , most concomitantly with antimicrobial agents and supportive measures. reflecting the evolving guidelines and recommendations, treatment decisions were not uniform. time from hospitalization to decision to treat ranged from preceding admission to a tertiary centre (treatment commenced at admitting hospital) to 5 days thereafter (median 0 d (on admission), ranging from 48 h prior to 5 days after admission). almost all children (28/29; 96.6%) received ivig (at 2 g/kg/dose); 11/28 (39.3%) one dose, 17/28 (60.7%) two doses. six patients (6/28; 21.4%) became afebrile after receiving ivig alone. one was followed with a short course of corticosteroids after ivig. all remaining patients (23/28; 82.1%) received either (a) combined ivig and corticosteroids as initial treatment (9/28; 32.1%)) or (b) ivig alone at first, which did not result in defervescence and therefore subsequently received corticosteroids, either alone (2/28; 7.1%) or in combination with a second ivig dose (11/28; 39.2%), or a biologic agent (n = 1, infliximab; n = 1 anakinra). of 19 children who initially received ivig alone, only 6 (31.6%) became afebrile. the remaining 13/19 (68.4%) received a second dose of ivig in combination with steroids, corticosteroids alone or biologic agents (table 3) . complications attributed to treatment with ivig were not uncommon and included hypotension (n = 1), capillary leak (n = 1), respiratory complications (n = 2) and meningism (n = 3). severity of cardiac presentation may have influenced treatment decisions, as all children without cardiac manifestations at diagnosis were initially given ivig alone (n = 4), compared to 10/25 (40%) of those with cardiac manifestations. though there was a tendency to use corticosteroids at treatment initiation in patients perceived to be more unwell and those with evidence of cardiac injury, symptom resolution in these patients was quicker and echocardiographic outcome at follow-up was not different. in all but one case, corticosteroids were administered parenterally. dosing regimens of intravenous methylprednisolone (ivmp) varied widely. most patients received ivmp on 3 consecutive days at 10 to 30 mg/kg (max 1 g/d). following ivmp treatment patients rapidly became afebrile in 20/22 (90.9%). children on a treatment regimen containing corticosteroids from dose one or biologics [45] became afebrile significantly more rapidly when compared to those who received ivig alone (median 1 versus 2 days, p = 0.015); many of which thereafter required a second dose which was mostly accompanied by steroids (figure 7) . j. clin. med. 2020, 9, x for peer review 13 of 21 attributed to treatment with ivig were not uncommon and included hypotension (n = 1), capillary leak (n = 1), respiratory complications (n = 2) and meningism (n = 3). severity of cardiac presentation may have influenced treatment decisions, as all children without cardiac manifestations at diagnosis were initially given ivig alone (n = 4), compared to 10/25 (40%) of those with cardiac manifestations. though there was a tendency to use corticosteroids at treatment initiation in patients perceived to be more unwell and those with evidence of cardiac injury, symptom resolution in these patients was quicker and echocardiographic outcome at follow-up was not different. in all but one case, corticosteroids were administered parenterally. dosing regimens of intravenous methylprednisolone (ivmp) varied widely. most patients received ivmp on 3 consecutive days at 10 to 30 mg/kg (max 1 g/d). following ivmp treatment patients rapidly became afebrile in 20/22 (90.9%). children on a treatment regimen containing corticosteroids from dose one or biologics [45] became afebrile significantly more rapidly when compared to those who received ivig alone (median 1 versus 2 days, p = 0.015); many of which thereafter required a second dose which was mostly accompanied by steroids (figure 7 ). though treatment options, composition of agents and individual responses varied significantly, overall, all patients clinically improved. to be considered with caution based on small sample size, ivig alone appeared to be less effective as compared to the combination with corticosteroids and/or biologic agents. high demand of ivig during a (potentially) long-lasting pandemic may deplete though treatment options, composition of agents and individual responses varied significantly, overall, all patients clinically improved. to be considered with caution based on small sample size, ivig alone appeared to be less effective as compared to the combination with corticosteroids and/or biologic agents. high demand of ivig during a (potentially) long-lasting pandemic may deplete stocks, and whether targeted cytokine blockade may be equally or even more effective is currently unknown. optimal treatment for pims-ts is currently being evaluated in large multicentre and international trials [50] . next, we investigated short-term outcomes at 48 h after defervescence and 1-2 week follow-up. all patients remained afebrile (≤37.5 • c) at 48 h after defervescence (mean 36.7, sd 0.52, range: 36-38 • c), with the exception of two patients. one child was a 15-year-old who had fulfilled criteria for mas and was found to have necrotizing lymphadenopathy, another remained febrile despite two doses of ivig and after four days of ivmp, and defervesced following infliximab. vital signs and laboratory parameters of inflammation, crp, lymphopenia and markers of cardiac injury all normalized, in most cases within 48 h of defervescence ( figure 5 ). patients made a sustained recovery following initial defervescence. of 16 children at one week, two were again febrile after initial defervescence. they also presented with neutrophilia. one patient, a five year-old who was sars-cov2 seronegative, had presented with a high bnp (>20.000 pg/ml). the child represented with a crp of 180 mg/l, bnp and troponin t had significantly reduced, but a junctional escape rhythm was noted on ecg. the second patient, a 7 year-old represented febrile with a crp of 219 mg/l and was noted to have reduced rom in his right hip which self-resolved. neither child was re-treated, and recovered within the following week. while not febrile, one patient, a 15 year-old girl, developed iga vasculitis with purpuric skin lesions and mild renal involvement which was self-resolving. overall, 12 children (41%) still had clinical signs and symptoms at the 1-2 week follow-up (table 4 ). to monitor cardiac involvement and assess its prognosis, the "most abnormal" echocardiogram during the admission for each patient was compared to follow-up echocardiogram at 2 weeks. of 19/27 (65.5%) children with abnormal echocardiograms during admission, at fu, coronary involvement was the most frequent finding (n = 14), involving the left coronary artery in six, and both left and right coronaries eight children. severity was "mild" and ranged from coronary ectasia (n = 12) to moderate aneurysms (n = 2). nine children had valve involvement, six mild and three moderate functional impairment. mild pericardial effusion was present in n = 7.8/27 children who had a completely normal admission echocardiogram. of those, 6 were still normal at fu, one was missing fu and another had delayed development of left coronary ectasia, identified at 2 weeks following presentation (table s8) . persistent features at 2 weeks were increased echogenicity of the coronary artery wall, ectasia/moderate aneurysms. ventricular function and valvular regurgitation had almost resolved within this time frame. ct and mri of these two children with persistent dilatation of coronary artery equivalent to moderate aneurysm were carried out between 4-6 weeks follow-up. this showed normalized coronary artery caliber, and no evidence of myocardial inflammation or late gadolinium enhancement, which suggests that no scarring occurred. in keeping with these findings, a french case series of four cardiac mris in children with mis-c related myocarditis showed diffuse myocardial edema on t2-stir sequences and native-t1 mapping, with no evidence of late gadolinium enhancement suggestive of replacement fibrosis or focal necrosis, favouring post-infectious myocarditis in children and adolescents with covid-19 [88] . in summary, in almost none of the pims-ts patients with an abnormal echocardiogram during admission, echocardiographic findings at the 2 week fu had not completely normalized, but half of them showed significant improvement. at the 3-5 months follow-up, echocardiographic features continue to improve with another four now having normal echocardiograms. in only one case, right and left coronary ectasia was newly detected on fu echocardiogram. cardiac outcomes were not associated with clinical or laboratory features during admission. by 2 weeks of follow-up, the differences in echocardiographic changes seen at presentation with regard to sex and ethnic background were no longer present (abnormalities on echocardiogram at follow-up: male 4/16, female 3/8; p = 0.65 and caucasian 6/10, bame 11/13; p = 0.34). while children rarely develop severe symptoms related to sars-cov-2 infections, some experience highly inflammatory disease requiring hospital admission and treatment. current classification (pims-ts and/or mis-c) are of limited diagnostic value as not highly disease specific and, based on the recent emergence of this condition, not clinically validated. thus, international collaboration is urgently needed to collect clinical information and produce more specific diagnostic and/or classification criteria. pims-ts exhibits key features of cs with raised acute phase reactants, cytopenias and organ involvement of the heart, respiratory and/or gastrointestinal tract. while, in the absence of published laboratory studies, the pathophysiology of pims-ts remains unknown, demographic, epidemiologic and clinical data may allow for the development of hypotheses and preliminary conclusions. based on comparisons with national census data, ethnic distribution shows a noticeable over-representation of minority ethnicities. whether genetic predisposition for inflammatory disease plays the key role remains currently unknown. interestingly, both onset and peak of pims-ts was delayed by approximately 4 weeks and occurred well into the decline of covid-19 in england. this, together with the observation that seropositivity among pims-ts patients was notably higher when compared to age-matched patients experiencing mild disease, may suggest that limited early infection control and pathogen clearance in the upper airway may result in virus replication and tissue damage, contributing to tissue damage, antibody production, immune complex deposition and/or antibody-dependent enhancement, which may all trigger pro-inflammatory phenotypes. current treatment strategies are empiric and not based on evidence. they include symptomatic and supportive measures, and immune modulating treatment. the here presented data, while recognizing its limitations, may suggest that frequently used ivig may not be sufficient in all patients, and corticosteroids or cytokine blocking agents may result in more rapid defervescence. larger cohorts within prospective and controlled trials are needed to test this hypothesis, further emphasizing the importance of clinical trials and international collaboration. while currently available reports, including this study, suggest mostly favourable outcomes in pims-ts/mis-c, data on medium-to long-term outcomes are not available, and only concerted efforts to achieve a structured, comprehensive and multidisciplinary follow-up will allow evidence-based therapeutic strategies in pims/ts. the following are available online at http://www.mdpi.com/2077-0383/9/10/3293/ s1. table s1 : summary of published case series with a minimum of five cases of paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 (pims-ts). brief summary of demographic data, sars-cov-2 positive rate, key clinical findings at presentation and treatment., table s2 : summary and comparison of the case definitions for paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 (pims-ts), multisystem inflammatory syndrome in children (mis-c) by the center for disease control, us and the world health organization, table s3 : median (and interquartile range) of blood results on admission, in children with paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2, table s4 : echocardiographic and clinical evidence for cardiovascular involvement in children presenting with paediatric inflammatory multisystem syndrome temporally associated with sars-cov-2, table s5 : imaging investigation performed on patients with pims-ts, and abnormalities identified, table s6 : association of laboratory parameters with parameters reflecting cardiac injury. brain natriuretic peptide (bnp) on admission and peak bnp, and c-reactive protein (crp) were significantly associated with several parameters, and composite parameters, for cardiac injury, table s7 : supportive therapies required in children with pims-ts, table s8 : echocardiogram during admission/ at 2 week follow up. surviving sepsis campaign: guidelines on the management of critically ill adults with coronavirus disease 2019 (covid-19) the emerging spectrum of cardiopulmonary pathology of the coronavirus disease 2019 (covid-19): report of 3 autopsies from houston, texas, and review of autopsy findings from other united states cities disease and healthcare burden of covid-19 in the united states multisystem inflammatory syndrome in children and adolescents with covid-19. scientific brief an outbreak of severe kawasaki-like disease at the italian epicentre of the sars-cov-2 epidemic: an observational cohort study hyperinflammatory shock in children during covid-19 pandemic sars-cov-2-induced kawasaki-like hyperinflammatory syndrome: a novel covid phenotype in children hyperinflammatory syndrome in children associated with covid-19: need for awareness. indian pediatr. 2020, s097475591600208, epub ahead of print clinical characteristics of 58 children with a pediatric inflammatory multisystem syndrome temporally associated with sars-cov-2 multisystem inflammatory syndrome related to covid-19 in previously healthy children and adolescents multisystem inflammatory syndrome 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children: a multi-centered study in multisystem inflammatory syndrome associated with sars-cov-2 infection in 45 children: a first report from iran multisystem inflammatory syndrome in children (mis-c): report of the clinical and epidemiological characteristics of cases in santiago de chile during the sars-cov-2 pandemic multisystem inflammatory syndrome in children with covid-19 in mumbai severe clinical spectrum with high mortality in pediatric patients with covid-19 and multisystem inflammatory syndrome epidemiological and clinical profile of pediatric inflammatory multisystem syndrome-temporally associated with sars-cov-2 (pims-ts) in indian children distinct clinical and immunological features of sars-cov-2-induced multisystem inflammatory syndrome in children guidance: paediatric multisystem inflammatory syndrome temporally associated with covid-19 centers for disease control and prevention. multisystem inflammatory syndrome in children (mis-c) associated with coronavirus disease 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clinical outcomes in covid-19 patients treated with tocilizumab: an individual patient data systematic review tocilizumab for the treatment of severe covid-19 pneumonia with hyperinflammatory syndrome and acute respiratory failure: a single center study of 100 patients in use of anakinra in severe covid-19: a case report recovery-randomised evaluation of covid-19 therapy lymphocyte subsets in healthy children from birth through 18 years of age: the pediatric aids clinical trials group p1009 study advanced life support group diagnosis, treatment, and long-term management of kawasaki disease: a scientific statement for health professionals from the american heart association classification criteria for macrophage activation syndrome complicating systemic juvenile idiopathic arthritis: a european league against rheumatism/american college of rheumatology/paediatric rheumatology international trials organisation collaborative initiative development and validation of the hscore, a score for the diagnosis of reactive hemophagocytic syndrome covid-19) surveillance report antibody prevalence for sars-cov-2 in england following first peak of the pandemic: react2 seroprevalence of anti-sars-cov-2 igg antibodies in geneva, switzerland (serocov-pop): a population-based study european centre for disease prevention and control covid-19 and multisystem inflammatory syndrome in latin american children: a multinational study cell type-specific expression of the putative sars-cov-2 receptor ace2 in human hearts spectrum of imaging findings on chest radiographs, us, ct, and mri images in multisystem inflammatory syndrome in children (mis-c) associated with covid-19 sars-cov-2 productively infects human gut enterocytes intestinal ischemia secondary to covid-19 cytotoxic lesion of the corpus callosum in an adolescent with multisystem inflammatory syndrome and sars-cov-2 infection neurologic and radiographic findings associated with covid-19 infection in children pressing issues in covid-19: probable cause to seize sars-cov-2 for its preferential involvement of posterior circulation manifesting as severe posterior reversible encephalopathy syndrome and posterior strokes multisystem inflammatory syndrome in children (mis-c): interim guidance. 2020. available online coronary artery dimensions in febrile children without kawasaki disease emergency awards: radx-rad predicting viral-associated inflammatory disease severity in children with laboratory diagnostics and artificial intelligence a perspective on potential antibody-dependent enhancement of sars-cov-2 a new coronavirus associated with human respiratory disease in china covid-19 in children and adolescents in europe: a multinational, multicentre cohort study type i and type iii interferons-induction, signaling, evasion, and application to combat covid-19 understanding sars-cov-2-related multisystem inflammatory syndrome in children autoimmune and inflammatory diseases following covid-19 the immunology of multisystem inflammatory syndrome in children with covid-19 d3targets-2019-ncov: a webserver for predicting drug targets and for multi-target and multi-site based virtual screening against covid-19 potential therapeutic effects of dipyridamole in the severely ill patients with covid-19 a pathological report of three covid-19 cases by minimally invasive autopsies mapping systemic inflammation and antibody responses in multisystem inflammatory syndrome in children (mis-c) normal reference intervals for cardiac dimensions and function for use in echocardiographic practice: a guideline from the british society of echocardiography ducou le pointe, h. cardiac mri of children with multisystem inflammatory syndrome (mis-c) associated with covid-19: case series the authors wish to thank melissa pronold, for critical revision of the manuscript and statistical methods, as well as charlie pickles and saabi ahmad for their kind support with out-of-hours data collection. janet mcdonagh was supported by versus arthritis centre for epidemiology (uk grant: 21755) and the national institute for health research, manchester biomedical research centre. the authors declare no conflict of interest. key: cord-309273-gtvi37gh authors: flesia, luca; monaro, merylin; mazza, cristina; fietta, valentina; colicino, elena; segatto, barbara; roma, paolo title: predicting perceived stress related to the covid-19 outbreak through stable psychological traits and machine learning models date: 2020-10-19 journal: j clin med doi: 10.3390/jcm9103350 sha: doc_id: 309273 cord_uid: gtvi37gh the global sars-cov-2 outbreak and subsequent lockdown had a significant impact on people’s daily lives, with strong implications for stress levels due to the threat of contagion and restrictions to freedom. given the link between high stress levels and adverse physical and mental consequences, the covid-19 pandemic is certainly a global public health issue. in the present study, we assessed the effect of the pandemic on stress levels in n = 2053 italian adults, and characterized more vulnerable individuals on the basis of sociodemographic features and stable psychological traits. a set of 18 psycho-social variables, generalized regressions, and predictive machine learning approaches were leveraged. we identified higher levels of perceived stress in the study sample relative to italian normative values. higher levels of distress were found in women, participants with lower income, and participants living with others. higher rates of emotional stability and self-control, as well as a positive coping style and internal locus of control, emerged as protective factors. predictive learning models identified participants with high perceived stress, with a sensitivity greater than 76%. the results suggest a characterization of people who are more vulnerable to experiencing high levels of stress during the covid-19 pandemic. this characterization may contribute to early and targeted intervention strategies. sars-cov-2 (severe acute respiratory syndrome coronavirus 2; henceforth referred to as covid19) is a strain of coronavirus that can infect humans, attacking the lungs and causing symptoms ranging from those of the common cold to those of severe acute respiratory syndrome (sars) [1] . while approximately 80% of those who are infected recover with no special treatment (i.e., they are either asymptomatic or suffer from mild pneumonia) [2] , recent data have confirmed that older persons (60+ years old) [3] and persons with certain pre-existing medical conditions are more likely to develop serious respiratory distress that can lead to death (3-4% of the population) [4] . covid-19 spreads very easily between persons and, at the time of writing, no drugs or biologics have proven effective for preventing or treating the virus [5, 6] . covid-19 was first identified in the chinese region of wuhan in december 2019 [6] . between december 2019 and april 2020, the virus spread throughout the world, causing more than 5,000,000 infections and over 300,000 deaths [7] . on 11 march 2020, the world health organization (who) declared covid-19 a pandemic [8] . to contain the number of victims and prevent the collapse of the healthcare system, most national governments imposed strict restrictions on residents' freedom, forcing those infected with the virus to self-isolate and requiring all residents (infected or not) to stay at home and move through public areas only for reasons of absolute necessity. among western governments, the italian government was the first to apply such restrictions [9] . a few weeks after the spread of covid-19 in china, the first scientific studies investigating the psychological impact of the outbreak highlighted a mild to severe negative psychological impact of the event within a significant proportion of the chinese population (53.8%); specifically, this impact on mental health was reported to include symptoms of anxiety, depression, and stress [10, 11] . more broadly, the literature on the impact of infectious outbreaks on mental health shows that pandemics are extremely stressful events that force people to cope with totally unexpected, ambiguous, and uncertain situations [12] . specifically, two main aspects of pandemics have been found to affect people's mental state. the first relates to danger (i.e., the fear of contagion), which can increase perceived threat and sometimes lead to panic, behavioral contagion, and an emotional epidemic [13, 14] . the second regards the multiple and rapid changes to social, working, and familiar habits, due to self-isolation and social distancing measures [15] [16] [17] [18] [19] . the longer the duration of self-isolation, the more people experience frustration and boredom, along with concerns about infection [15] . well-documented psychological reactions to epidemics include emotional distress, anxiety behaviors, sleeping disorders, fear, anger, depression, health concerns, a sense of powerlessness, and uncertainty [13, 16, [20] [21] [22] [23] [24] . furthermore, studies examining the long-term consequences of infectious epidemics have shown that some individuals may even develop symptoms of post-traumatic stress disorder (ptsd) [12, 25, 26] . one review indicated that those who develop ptsd may experience the symptoms for 3 years following the end of the epidemic [15, 23] . stress is defined as an adaptive psycho-physical reaction to a physical, social or psychological stimulus, called a stressor [27] . stress-related responses may be cognitive, emotional, behavioral, or physiological. depending on the type, timing, and severity of exposure to a stressor, the resulting stress may become a risk factor for a number of illnesses, including those of a psychiatric or cardiovascular nature [28] [29] [30] [31] [32] . an emergency such as the covid-19 outbreak might rightly be considered a severe stressor, as it is a new and unexpected situation with a potentially serious impact on health (experienced both personally and through loved ones) that also involves social restrictions [13] . nevertheless, no event, in and of itself, is the precipitating cause of pathology and illness. rather, it is the perception of stress (i.e., the degree to which one considers the event stressful) that accounts for the varying physical and mental responses to the situation [33] . in this sense, it is important to detect vulnerable persons early, and to promote effective preventive programs in order to treat such persons rapidly and limit negative psychological outcomes. the identification of psycho-social risk and resilience factors for psychological distress during the covid-19 emergency comprises a significant step in this direction [34] . to date, studies on the psychological impact of covid-19 have mainly focused on the role of sociodemographic variables (e.g., gender, age, education level, and social connections) in moderating reactions to the outbreak [10, 11, 24, 35, 36] . the identified sociodemographic risk factors for psychological distress include gender (female), age (18) (19) (20) (21) (22) (23) (24) (25) (26) (27) (28) (29) (30) or 60+ years old), student status, education level, perception of the public health system, specific physical symptoms (e.g., coryza, cough, sore throat, headache), and a low reported level of health [10, 24, 35] . for chinese students, living in an urban area, having a stable family income, living with parents, and having good social support were also found to protect against anxiety [11] . in addition to investigating sociodemographic factors, many studies have also outlined the role of certain dispositional traits in modulating responses to stressful events. however, these constructs have been poorly investigated in relation to the psychological impact of epidemics and, specifically, covid-19. more generally, research has shown how individual differences, including dispositional traits, can explain life outcomes [37] . according to the theory of traits (or dispositional theory), individual differences may be explained by certain predispositions (traits), which are expressed in a relatively stable way across situations and time. traits comprise a person's manner of thinking, feeling, perceiving, and relating to others [37] . based on these considerations, dispositional traits might play a relevant role in predicting perceived stress in relation to coping is one of the most widely studied dispositional traits, and it has been found to be significant in modulating responses to stressful events. coping is defined as the effort to solve personal and interpersonal problems in an attempt to master, minimize, or tolerate stress and conflict [38] . distinct coping strategies have been found to be differentially associated with specific emotional responses [39] , physiological stress responses [40] , and self-efficacy [41, 42] . one investigation into the role of coping strategies during a virus outbreak (2009 h1n1 flu pandemic) found coping style to influence the perceived risk of contagion and vaccination intentions among canadian adults [43] . another study, based in singapore, found coping strategies to be associated with post-traumatic outcomes within visitors to community health care services during the national outbreak of sars [23] . as regards emotional self-regulation and adaptation to the world, self-control may represent a significant protective factor. there is empirical evidence that people with high dispositional self-control have better psychological adjustment and impulse control [44] ; this suggests that good self-control may mitigate the influence of a negative environment. similarly, perceived control over life outcomes has been shown to be positively associated with well-being and health-related quality of life, and negatively associated with emotional distress, in the context of stressful events [45, 46] . in this regard, locus of control [47] is a relevant concept, describing the degree to which an individual believes that they have control over the outcome of life events, as opposed to feeling that their life is subject to external forces beyond their control. finally, among the many individual difference variables that might influence reactions to covid-19, personality traits merit significant attention. several studies have highlighted an association between the big five personality traits [48] and various health behaviors, such as sedentary behavior [49] , sexual health behavior [50] , physical activity [51] , and alcohol consumption [52] . the first aim of the present study was to investigate the impact of the covid-19 pandemic and the related government-imposed restrictions on perceived stress in a western country (i.e., italy). as reported above, most studies on the psychological impact of covid-19 have related to the chinese population. however, countries differ from one another in many important aspects (i.e., social, cultural, political, and economic aspects, to name only a few); consequently, psychological responses may also vary between contexts and communities, revealing unique qualitative and quantitative psychological reactions and psychological needs. our sample of italian adults, collected during the covid-19 outbreak, would show higher levels of perceived stress compared to italian normative values. the second aim of the study was to confirm the role of certain sociodemographic factors in modulating stress responses to the covid-19 pandemic, as reported in the recent literature. (a) participants who were female, younger, and students, with a lower level of education and lower income, would report higher levels of stress, and (b) participants who were living with others would report lower levels of psychological distress. the third aim of the work was to investigate the association between certain stable psychological traits and psychological distress relating to the current situation. to this end, participants were tested for coping strategies, self-control, locus of control, and select personality traits. (a) participants with positive coping strategies, higher levels of self-control, an internal locus of control, and higher levels of emotional stability would report lower levels of stress, and (b) participants with negative coping strategies, lower levels of self-control, an external locus of control, and lower levels of emotional stability would report higher levels of stress. finally, with the goal of anticipating persons in need of treatment and improving the targeting and overall effectiveness of preventive programs, we aimed at developing machine learning models to predict individual psychological responses to the covid-19 pandemic, based on sociodemographic and psychological variables with maximal sensitivity in classifying subjects with high versus low levels of perceived stress. to summarize, the study was novel in the following two respects: first, it considered the role of not only sociodemographic variables, but also stable psychological traits, as predictors of a stressful reaction to covid-19; and second, it leveraged machine learning techniques to identify people at the greatest risk of developing severe and negative psychological outcomes due to the pandemic. to test the abovementioned hypotheses, we implemented a cross-sectional study. using google forms, we designed an ad hoc online questionnaire to collect data on participants' stress reactions to covid-19, demographical variables, and psychological traits. the questionnaire also assessed the following sociodemographic factors: gender, age, education, number of family members and/or others living in the household, monthly household income, and student status. subsequently, we administered five standardized questionnaires, as follows: • the italian version of the 10-item perceived stress scale (pss-10; cronbach's alpha = 0.74) [53] . the pss-10 is a frequently used psychological instrument to measure perceived stress [54] . respondents are asked to answer 10 questions pertaining to the frequency of experiences of stressful situations during the last month on a five-point scale ranging from 0 (never) to 4 (very often) [55, 56] . example items include "in the last month, how often have you been upset because of something that happened unexpectedly?" higher scores indicate higher levels of perceived stress. moreover, in the present study, the score corresponding to 1.5 sd above the italian normative score [53] was used as a cut-off to divide participants into two classes: low perceived stress (males: pss-10 score <24.35; females: pss-10 score <24.55) and high perceived stress (males: pss-10 score ≥24.35; females: pss score-10 ≥24.55); • the italian shortened version of the coping orientations to the problems experienced (cope-nvi-25; cronbach's alpha of factors range 0.68-0.95) [57] . the cope-nvi-25 is a multi-dimensional inventory that assesses individual differences in coping styles. it is comprised of 25 items, which are rated on a 4-point scale ranging from 1 (i usually don't do this at all) to 4 (i usually do this a lot) [58] . the instrument includes five subscales corresponding to five different coping styles: social support, avoidance strategies, positive attitude, problem solving, and turning to religion [59] . an example item is "i admit to myself that i can't deal with it, and quit trying" (avoidance strategies). a higher score on a particular subscale indicates a greater use of that specific coping strategy. the italian translation of the brief self-control scale (bscs; cronbach's alpha = 0.85) [44] . the bscs measures individual differences in dispositional capacity for self-control. the scale is comprised of 13 items that are rated on a five-point scale ranging from 1 (not at all) to 5 (very much). an example item is "i do certain things that are bad for me, if they are fun." higher scores on the bscs indicate a greater capacity for self-control, and they are also correlated with better psychological adjustment, interpersonal skills, and emotional responses [44] ; • the italian short version of the locus of control (loc) scale [60] . this 20-item questionnaire is used to measure generalized expectancies relating to an internal versus external locus of control, rated via dichotomous options ("yes" vs. "no"), similar to rotter's original internal-external locus of control scale [47] . an example item is "to do well in life, luck is more important than commitment." respondents with an internal locus of control (i.e., a high score on the internal loc scale) tend to attribute life outcomes-and general life events-to their own behavior, whereas those with a prevalent external locus of control (i.e., a high score on the external loc scale) tend to attribute life events to fate, others, or external causes beyond their control [47, 61] ; • the italian version of the 10-item big five inventory (bfi-10; spearman-brown coefficients ≥0.50) [62] . the bfi-10 assesses personality traits according to the five-factor approach [63] . it is comprised of 10 items rated on a five-point scale ranging from 1 (strongly disagree) to 5 (strongly agree), measuring five dimensions of personality, which are extraversion, agreeableness, conscientiousness, neuroticism (or, if reversed, emotional stability), and openness [64] . an example item is "i see myself as someone who is outgoing, sociable" (extraversion). the higher the score on a particular subscale, the more that specific dimension represents a characteristic trait of the respondent's personality. the complete list of 18 variables that were extracted from the responses to the questionnaire is provided in the supplementary materials. the present research was designed in accordance with the declaration of helsinki and approved by the ethics committee for psychological research at the university of padova (protocol number 3576, unique code 189b46fe116994f1a8d1077b835d83bb). data were collected during the period of 20-31 march, 2020. participants were recruited online through an invitation posted on social media (facebook and whatsapp). this approach of online recruitment was selected primarily due to the lockdown situation, which prevented us from collecting data in the laboratory. according to the aim of the study, it was necessary for us to capture the psychological state of participants at the time of the pandemic; thus an a posteriori study would not have provided useful and reliable information. participants were invited to complete an anonymous online questionnaire to report their personal experiences with the covid-19 emergency and their mental state. the inclusion criteria were the following: (a) living in italy at the time of data collection and (b) being aged 18+ years (18 years is the legal age in italy, defined by the capacity to act and be emancipated). participation was voluntary. all participants were required to read and provide informed consent before beginning the online questionnaire. they received no compensation for their participation. in total, 2072 volunteers took part in the study. of these, 19 were excluded on the basis that they responded to the questionnaire twice (we kept only their first response). thus, the final sample was comprised of 2053 participants, of whom 1555 were female, 480 were male, and 18 were reported as "other." the participants' average age was 35.81 (sd = 13.19; range: and their average education level was 15.35 years (sd = 3.43; range: 5-21). a more detailed description of the sample's demographic characteristics is provided in the supplementary materials. it has been calculated that a sample size of 2053 is sufficiently large to achieve at least a statistical power (1-β) = 0.90 in a linear multivariable regression analysis involving 18 predictors, given a significance level α = 0.05 and an effect size of 0.356 [65] . data are provided in the supplementary materials. data analysis was conducted using the jasp software [66] . a single sample t-test (t, two-sided) was performed in relation to the pss-10 score, in order to determine whether the sample's true mean (µ) was statistically different from that of the known population (m 0 ). a multivariable regression analysis was run to investigate the relationship between the pss-10 score and the independent variables that were hypothesized to impact the level of perceived stress. the collinearity assumption was checked prior to running the model, using the tolerance and variance inflation factor (vif). as a rule of thumb, if vif >10 and tolerance <0.1, the assumption is greatly violated, whereas if vif >1 and tolerance <0.2, the model may be biased [67] . the results indicated that the collinearity assumption was not violated by any of the independent variables entered in the regression model. the analysis was performed using the stepwise variable selection method, which identified predictors with a significant (p < 0.05) individual association with the outcome (pss-10 score). the results were reported using unstandardized coefficients, as recommended by friedrich [68] . recently, researchers in different scientific fields, including the clinical and social sciences, have emphasized the utility of focusing on prediction, rather than explanation, during data analysis [69] [70] [71] [72] . this increased attention to predictive models may be largely attributed to the significant spread of machine learning (ml)-a branch of artificial intelligence that trains algorithms on data samples (i.e., training sets) in order to make predictions on completely new data (i.e., test sets) without being explicitly programmed to do so [73] . as regards psychology, ml techniques have been shown to be particularly useful for predicting human behavior, including high-risk behavior; thus, they may be applied to improve the effectiveness and targeting of preventive programs and interventions [74] . in brief, ml models are capable of predicting the behavior of individual subjects, allowing greater attention to be paid to those considered most critical [69] . in the present study, ml algorithms were trained on psycho-social data to identify subjects who were more likely to present high levels of perceived stress during the covid-19 emergency, and who were consequently at the greatest risk of developing psychological symptoms, including those of ptsd. for this purpose, participants were split into two classes: high perceived stress and low perceived stress. the high perceived stress class included participants with a pss-10 score of more than 1.5 sd above the italian population mean (n = 393) for men and women, respectively. conversely, the low perceived stress class included participants whose pss-10 did not exceed 1.5 sd above the italian normative value (n = 1642). it should be noted that participants who reported their gender as "other" (n = 18) were excluded from this analysis, as the italian normative values were available for males and females only [53] . as ml models are built to fit particular data, it is important to test how each model fits new (i.e., unseen) data. for this reason, part of the data (the training set) is generally used to train and validate the model, while another part (the test set) is used to test the model's accuracy on new examples [73, 75] . this procedure guarantees the model generalization and increases the replicability of the results [76, 77] . in the present study, 20% [73, 75] of the participants were randomly chosen and retained as the test set. accordingly, the training set consisted of 1628 participants (314 with high perceived stress and 1314 with low perceived stress), and the test set consisted of 407 participants (79 with high perceived stress and 328 with low perceived stress). in the first step, feature selection was performed to remove redundant and irrelevant features and to increase model generalization by reducing overfitting and noise in the data [78] . a good strategy for feature selection is to identify the subset of features that are highly correlated with the class to predict, but not correlated with each other [78] . this procedure was performed in the present study using the correlation-based feature selector (cfs) in the weka 3.9 software [79] . the problem of class imbalance was addressed while running the classification algorithms. the ratio between participants with high perceived stress and those with low perceived stress was approximately 1:5. as ml methods work best with balanced datasets, it is necessary to account for any class imbalance, especially when training examples are limited-a condition that is frequently met by datasets in health and clinical psychology [80] . at the same time, it is equally important for ml models to be built on samples that are representative of the population, reflecting real distribution [80] . one strategy to overcome these two limitations consists of altering the relative costs associated with misclassifying the minority and majority classes, in order to compensate for the class imbalance [81] . in the present study, ml algorithms were set in such a way that any algorithmic error made in classifying the minority class (high perceived stress) was weighted four times more than any error in classifying the majority class (low perceived stress). this cost-modifying strategy has been shown to provide better results than other methods in addressing the class imbalance problem [81] . moreover, it should be noted that, for the goal of the present task, it was more beneficial to minimize false negatives than to minimize false positives (i.e., to have a model with high sensitivity rather than high specificity). in other words, it was more important to identify people who were truly at risk than to avoid misclassifying people who were not truly at risk. ml models were trained and validated on the training sample (n= 1628) through a 10-fold cross-validation procedure using the weka 3.9 software [79] . the different algorithms (i.e., logistic regression [82] , support vector machine (svm) [83] , naïve bayes [84] , random forest [85] ) were chosen as representatives of different classification strategies, to ensure that the results would be stable across classifiers and not dependent on specific model assumptions (details on the parameters of the ml classifiers are reported in the supplementary materials). k-fold cross-validation is a resampling procedure that seeks to reduce the variance in model performance relative to the performance that may be obtained from a single training set and a single test set. the procedure consists of portioning the sample into k subsets (i.e., folds; in the present study, k = 10), and using k-1 (i.e., 9) subsets to train the model and the remaining subset to validate the model's accuracy. this is repeated k (i.e., 10) times [86] . the final model metrics are obtained by averaging the metrics obtained in all validation subsets. in the present study, the models developed from the 10-fold cross-validation procedure were tested on the test sample (n = 407). the main results of the data analysis are reported in this section. a more complete descriptive analysis of each variable, including the composition of high perceived stress versus low perceived stress samples, is reported in the supplementary materials. the average pss-10 score of the entire sample was 18.81 (sd = 6.25). analyzing the responses of males and females separately (note that participants who reported a gender of "other" were excluded from this analysis due to a lack of normative data), males obtained an average score of 16.71 (sd = 6.91) and females obtained an average score of 19.44 (sd = 6.79). to determine whether the sample mean statistically differed from that of the italian normative population (males: average = 15.2, sd = 6.1; females: average = 16.3, sd = 5.5) [53] , a one-sample t-test was run separately for each gender. a first multiple regression analysis was run, including sociodemographic variables that have been shown to potentially impact the level of perceived stress during a pandemic [10, 11, 24, 35, 36] . the pss-10 score was set as the dependent variable, while gender (male), age, education, monthly income, number of family members, and student status (student) were entered as covariates. the final model accounted for a significant proportion of the variance in the level of perceived stress (r 2 = 0.103, adjusted r 2 = 0.101, f-change (1,2029) = 12.009, p < 0.001). all of the aforementioned variables, with the exception of student status, were found to contribute to the level of perceived stress. results are reported in table 1 . to better understand the role of stable psychological traits in predicting the level of perceived stress (pss-10 score), a second multiple linear regression was run, adding to the previous model the scores of the five coping styles measured by the cope-nvi-25 (cope positive, cope problem, cope avoidance, cope religion and cope support), the bscs total score, the internal loc score, and the scores for the five personality traits measured by the bfi-10 (bfi-10 agreeableness, bfi-10 conscientiousness, bfi-10 emotional stability, bfi-10 extraversion and bfi-10 openness). this second model accounted for a larger proportion of the variance in the level of perceived stress (r 2 = 0.356, adjusted r 2 = 0.352, f-change (1,2022) = 5.908, p < 0.05) compared to the previous model. bfi-10 emotional stability, cope positive, age, bcsc total score, gender (male), cope avoidance, internal loc, number of family members, cope support, monthly income, and bfi-10 conscientiousness were identified as significant predictors of the level of perceived stress during the covid-19 epidemic (see table 2 ). education, cope religion, cope problem solving, bfi-10 agreeableness, bfi-10 extraversion and bfi-10 openness were excluded. ultimately, the 18 questionnaire variables were considered predictors of perceived stress. the entire list of predictors, along with their descriptions, is provided in the supplementary materials. of these 18 variables, the following 7 were identified as the best set of predictors using correlation-based feature selection: age, monthly income, cope avoidance, cope positive, bscs total score, bfi-10 emotional stability, and bfi-10 agreeableness. using these predictors, ml algorithms were trained and tested according to the procedure described in the "data analysis" section. classification results for the test set are reported in table 3 , which quantifies predictive performance according to the following metrics: receiver operating characteristic curve (roc) area, precision, recall and f-measure (f1 score). it is worth noting that the classifiers showed an roc area ranging from 0.70 to 0.78 in the test set. however, the random forest algorithm highlighted the lower sensitivity (recall) of the high perceived stress class compared to the other classifiers, making it a weaker model for the purposes of prediction. the present study measured the impact of the covid-19 emergency on perceived levels of stress, taking into account sociodemographic variables and stable psychological traits. the results confirmed that participants perceived the covid-19 crisis as a stressful experience; in the present sample, the level of perceived stress was higher than that of the general population in a non-emergency condition. indeed, almost 20% of the sample scored above the results from the normative data on measures of perceived stress. these results are in line with the findings of recent studies on the psychological impact of covid-19 [10, 11, 87] and the international literature on epidemic outbreaks [13] . the mean values of the single items of the pss-10 suggest that, in addition to nervousness and stress, feelings of being unable to control one's personal life accounted for the majority of participants' perceived stress. this suggests that the unpredictability and uncontrollability of the pandemic may play a significant role in determining levels of perceived stress during the crisis. moreover, it may reflect participants' attitudes toward the significant lifestyle changes demanded of them due to the lockdown and other restrictive measures. as regards sociodemographic variables, the results suggest that the female gender is associated with higher levels of stress. this is consistent with the literature indicating gender differences in the psychological response to covid-19 [24, 88] and other epidemics [89] ; it is also in line with the normative data for the general population. consistent with other studies (20, 30) , the present study found an association between higher incomes and lower levels of perceived stress. one explanation for this is that higher incomes might be related to less concern about the economic effects of self-isolation and/or with more comfortable housing solutions (e.g., larger living spaces, access to outdoor spaces (such as gardens), and access to leisure activities). moreover, people with higher incomes may be more likely to perform work that can easily and fully transition to the online environment, thereby reducing some sources of stress. in the present sample, older age was found to be associated with lower levels of stress. this finding might appear surprising, since it contradicts both the results of studies on the chinese population [24, 35] and the association between older age and higher covid-19 mortality. however, the result is in line with recent italian data [87] . several studies have indicated age-related differences in coping and locus of control, with older adults presenting greater self-control and emotional self-regulation relative to younger adults [90] [91] [92] . considering the current pandemic, older people may be more used to staying at home, so their daily routines might be less impacted by mandatory self-isolation measures. data from previous investigations on age differences in stress responses to the sars epidemic reflect inconsistencies [93, 94] , but sociopolitical and cultural aspects, such as differences in elder care services and policies, might account for these discrepancies. the present study did not find education to be a significant predictor of the level of perceived stress. the large percentage of highly educated participants in the sample might partially explain this finding. however, prior research on this subject has generated mixed results-recent studies on the chinese population [24, 35] have found that education does not seem to affect mental health, while data from a spanish sample [88] and from previous studies on psychological adjustment to sars [95] have confirmed an association between a higher level of education and better mental health. the present study also found that living alone or with few family members was a protective factor against perceived stress. we might argue that this condition both conveys a sense of protection from contagion and offers continuity with pre-epidemic economic and social conditions. moreover, when cohabiting with family members, concern for loved ones might contribute to increasing perceived stress. as regards psychological variables, emotional stability was found to be an important protective factor. according to the five-factors model [63] , people with high emotional stability remain calm in response to stressful situations, and view problems in proportion to their importance. as a result, they tend to worry less about problems than do people with low emotional stability [96] . many studies have found that emotional stability is able to buffer stress responses to adverse events [97, 98] . in the present study, conscientiousness and agreeableness were found to predict psychological distress. generally speaking, individuals who score high on agreeableness tend to dislike conflict and be less suspicious of others; generally, they seek to pacify and mediate. in this sense, agreeable people might be more flexible and accepting when faced with unexpected and undesired situations, such as restrictions and changes to daily routines. conscientious people are more likely to perceive lower levels of stress (see correlation analysis). according to the literature, they are more aware of their actions and tend to exhibit more goal-oriented behavior. in this sense, in times of self-isolation, conscientious people might have a greater tolerance for frustration and imposition relative to less conscientious people, who might engage in more impulsive behavior [96] . moreover, previous research has indicated that conscientiousness may influence adaptive behavior, especially in health-related programs [99, 100] . the present study found higher levels of dispositional self-control to predict lower levels of psychological distress in response to the covid-19 emergency. personal self-control skills may play a role in determining tolerance to restrictions to personal freedom during self-isolation. this result further suggests that self-regulatory processes may have a strong influence on responses to the outbreak. the results regarding dispositional self-control are consistent with those relating to the emotional stability trait of the big five model. in fact, these dimensions are often correlated [101] , and this specific pattern may indicate the importance of personal skills, such as the ability to remain calm, and maintain emotional balance and a sense of acceptance. in this sense, practices that enhance emotional stability and acceptance, such as mindfulness, could be useful in reducing the stressful impact of the emergency [102, 103] . the results concerning coping styles are also in line with this. besides confirming the protective effect of functional coping styles and the adverse impact of dysfunctional coping styles, the results of the present study suggest that people who use a positive attitude as a coping strategy may be much less likely to experience psychological distress during the present emergency. such persons may appraise the emergency as a unique opportunity, and feel less need for psychological support. in contrast, the present findings suggest that people who use avoidance strategies may be more likely to experience higher levels of stress during the emergency. these results are consistent with the findings of previous investigations into the relation between coping style and response to an epidemic [23, 43, 104, 105] . the results relating to dispositional locus of control indicate that people with an internal locus of control may be less likely to feel stressed. again, these results suggest that the more people are inclined to confidently rely on themselves, the better they will cope with uncertainty and change. several studies have indicated an association between an internal locus of control and self-efficacy and emotional stability [106, 107] . furthermore, previous studies have found a relation between an internal locus of control and the positive appraisal of an emerging infectious disease outbreak [93] . it could be hypothesized that people with an internal locus of control interpret self-isolating as something that they determine and enact for themselves as a protective behavior, rather than something that is imposed on them; this might account for their lower levels of perceived stress. overall, the results of the present study identify some population subgroups that may be more vulnerable to experiencing stress during the covid-19 emergency. specifically, a set of seven psycho-social variables may identify a high percentage of people experiencing high stress during the covid-19 pandemic, with sensitivity approaching 0.759 (roc area of predictive models ranging between 0.70 and 0.78). according to this model, we may develop targeted preventive interventions. furthermore, self-regulatory skills (including emotional stability, an internal locus of control, and self-control) were shown to be a protective factor, indicating the importance of raising awareness of these skills during the emergency and offering training and education to increase personal abilities in these areas (e.g., mindfulness programs). the present research aimed at improving our understanding of the possible risk and protective factors for high perceived stress during the covid-19 outbreak. it is worth noting that all data were collected from an italian population. therefore, the findings were inevitably influenced by specific contextual and socio-cultural aspects. further investigations involving people of different ethnicities and residents of other countries would deepen our understanding of the generalizability of these results, and the effective influence of psychological traits. in this regard, the open access data reported in the supplementary materials may contribute to facilitating comparisons between ethnicities, countries, and specific traumatic events. some further limitations should be considered when interpreting the findings of this research. first, participants were recruited via an online link posted on social networks. while online recruitment guarantees large samples, it does not guarantee sample representativeness. for this reason, very vulnerable groups, such as homeless or low-income persons, may not be well represented in this study. similarly, the average age of the sample was young, predominantly female, and largely well educated, as indicative of a sample that is more likely to participate in an online survey. second, the use of self-report measures did not enable us to verify the reliability of the responses, or to ensure that participants correctly understood the questions. future research should aim at overcoming these shortcomings. finally, future research should also investigate the interplay and mutual interrelationship between protective and risk factors, to improve the targeting and overall effectiveness of preventive programs and interventions. indeed, the literature suggests that, during a pandemic, it is extremely important for people to sustain their use of psychological services, either online or in the context of social distancing [108, 109] . this is particularly essential for those who are more vulnerable to experiencing high levels of stress, and it is important that we ensure that such persons can access timely and high-quality psychological services in order to prevent the development of chronic outcomes, including ptsd. supplementary materials: the following are available online at http://www.mdpi.com/2077-0383/9/10/3350/s1, table s1 : descriptive statistics, table s2 : item by item analysis of the pss-10, table s3 : list of predictors, table s4 : details on ml classifiers parameters. funding: this research received no external funding. the authors declare no conflict of interest. the epidemiology and pathogenesis of coronavirus disease (covid-19) outbreak characteristics of and important lessons from the coronavirus disease 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interventions to reduce diabetes distress: the role of personal conscientiousness and diabetes self-efficacy higher-order factors of the big five let's conquer covid-19 and sustain our abilities mindfulness mediates the physiological markers of stress: systematic review and meta-analysis coping responses of emergency physicians and nurses to the 2003 severe acute respiratory syndrome outbreak the importance of coping appraisal in behavioural responses to pandemic flu relationship of core self-evaluations traits-self-esteem, generalized self-efficacy, locus of control, and emotional stability-with job satisfaction and job performance: a meta-analysis perceived behavioral control, self-efficacy, locus of control, and the theory of planned behavior 1 psychological interventions for people affected by the covid-19 epidemic the role of telehealth in reducing the mental health burden from covid-19 key: cord-283407-3zb6ufum authors: ciuti, gastone; skonieczna-żydecka, karolina; marlicz, wojciech; iacovacci, veronica; liu, hongbin; stoyanov, danail; arezzo, alberto; chiurazzi, marcello; toth, ervin; thorlacius, henrik; dario, paolo; koulaouzidis, anastasios title: frontiers of robotic colonoscopy: a comprehensive review of robotic colonoscopes and technologies date: 2020-05-31 journal: j clin med doi: 10.3390/jcm9061648 sha: doc_id: 283407 cord_uid: 3zb6ufum flexible colonoscopy remains the prime mean of screening for colorectal cancer (crc) and the gold standard of all population-based screening pathways around the world. almost 60% of crc deaths could be prevented with screening. however, colonoscopy attendance rates are affected by discomfort, fear of pain and embarrassment or loss of control during the procedure. moreover, the emergence and global thread of new communicable diseases might seriously affect the functioning of contemporary centres performing gastrointestinal endoscopy. innovative solutions are needed: artificial intelligence (ai) and physical robotics will drastically contribute for the future of the healthcare services. the translation of robotic technologies from traditional surgery to minimally invasive endoscopic interventions is an emerging field, mainly challenged by the tough requirements for miniaturization. pioneering approaches for robotic colonoscopy have been reported in the nineties, with the appearance of inchworm-like devices. since then, robotic colonoscopes with assistive functionalities have become commercially available. research prototypes promise enhanced accessibility and flexibility for future therapeutic interventions, even via autonomous or robotic-assisted agents, such as robotic capsules. furthermore, the pairing of such endoscopic systems with ai-enabled image analysis and recognition methods promises enhanced diagnostic yield. by assembling a multidisciplinary team of engineers and endoscopists, the paper aims to provide a contemporary and highly-pictorial critical review for robotic colonoscopes, hence providing clinicians and researchers with a glimpse of the major changes and challenges that lie ahead. abstract: flexible colonoscopy remains the prime mean of screening for colorectal cancer (crc) and the gold standard of all population-based screening pathways around the world. almost 60% of crc deaths could be prevented with screening. however, colonoscopy attendance rates are affected by discomfort, fear of pain and embarrassment or loss of control during the procedure. moreover, the emergence and global thread of new communicable diseases might seriously affect the functioning of contemporary centres performing gastrointestinal endoscopy. innovative solutions are needed: artificial intelligence (ai) and physical robotics will drastically contribute for the future of the healthcare services. the translation of robotic technologies from traditional surgery to minimally invasive endoscopic interventions is an emerging field, mainly challenged by the tough requirements for miniaturization. pioneering approaches for robotic colonoscopy have been reported in the nineties, with the appearance of inchworm-like devices. since then, robotic colonoscopes with assistive functionalities have become commercially available. research prototypes promise enhanced accessibility and flexibility for future therapeutic interventions, even via autonomous or robotic-assisted agents, such as robotic capsules. furthermore, the pairing of such endoscopic systems with ai-enabled image analysis and recognition methods promises enhanced diagnostic yield. by assembling a multidisciplinary team of engineers and endoscopists, the paper aims to provide a contemporary and highly-pictorial critical review for robotic colonoscopes, hence providing clinicians and researchers with a glimpse of the major changes and challenges that lie ahead. colonoscopy forms a significant portion of endoscopists' workload. however, not enough attention is given to the ergonomic aspects of conventional colonoscopy. common anatomical sites of work-related musculoskeletal pains are the back (15-57%), neck (9-46%), shoulders (9-19%) , elbows (8-15%) and hands/fingers (14-82%) [15] . however, this burden is not only limited to practicing gastrointestinal (gi) endoscopy; it seems to be also a common place among gastroenterology/endoscopy trainees or fellows [16, 17] . promoting a culture of fitness-conscious and regular-exercising professionals could reduce or delay the impact of high-volume colonoscopy workload on muscle overuse. gender and anthropomorphic features should also be considered together with basic recommendations on the monitors positioning and the examination's bed height in order to minimize endoscopy-related musculoskeletal injuries. furthermore, limited data show that injuries of the hand, wrist, forearm and shoulder are most common among colonoscopists and may derive from general overuse of the upper limbs, repetitive pinching, gripping and torqueing forces and/or awkward neck and body posturing [18] . pinch forces and forearm-muscle loads applied during routine colonoscopies represent substantial risk factors for carpal tunnel syndrome, de quervain syndrome and/or tennis elbow. although the introduction of advanced colonoscopes and insertion techniques, such as water-assisted or gasless colonoscopy, could help not only with patient comfort and/or increased polyp detection but also with effort reduction on the side of the endoscopist, their adoption is far from being "universal". therefore, a paradigm shift is required in devices and techniques to improve safety and comfort and to ensure uninterrupted, efficient and high-quality provision of endoscopy services in the face of rising demand worldwide for both screening and therapeutic colonoscopy [15] . it took almost one century before the first endoscopic attempts to visualise gi tract were performed and a few decades since 1904 when the first barium enema examination was performed [19, 20] to a full retrograde flexible colonoscopy [21] . soon after it, in 1969, endoscopic excision of colonic polyps was possible. dr. hiromi shinya, a newly qualified general surgeon at beth israel medical center (new york, ny, usa) and dr. william wolff, chairman of the general surgery department, at that time were at the forefront of a worldwide research effort to develop ways to examine the full length of the colon using a tube embedding electronic sensors [22] . in 1969, the two pioneers made a further ground-breaking advancement in collaboration with olympus corp. (tokyo, japan) by introducing a wire loop snare to cauterize a polyp as soon as it was found, thus making a second procedure unnecessary [23] . although the two advocated for their invention, they had to overcome some serious scepticism about the device's safety and efficacy. furthermore, the development of a protocol for a one-doctor technique, as the standard for performing colonoscopy, has been formally attributed to them. the first flexible endoscopes included a fibre optic bundle. approximately 250,000 glass fibres, each about 15µm in diameter, individually coated and oriented similarly at both ends, were placed in a 4 mm bundle that allowed the transmission of a visual image [24, 25] . by 1973, dr. wolff and dr. shinya had performed over 2,000 colonoscopies in the endoscopy unit at beth israel medical center, demonstrating that, in skilled hands, this procedure could be done safely [26] . since then, several steps allowed the adoption in the clinical practice of current state-of-the-art hd colonoscopes without magnification capabilities and with image enhancement modes. however, one factor that remains unaltered is that the single operator must undergo extensive practice and training to gain credentials for provision of comfortable and safe colonoscopy. nevertheless, with the advent of miniaturization, wireless control and artificial intelligence (ai) -aided digestive tract "scope" will continue to develop. a schematic illustration of history and milestones of colonoscopy is reported in figure 1 . in recent decades, the foremost general drive to develop robots was the need to drastically improve human safety in hazardous environments and/or to enhance human operator ability in medical procedures by reducing fatigue. furthermore, there was an ever-growing desire to develop products with wider potential markets aimed at improving the quality of everyday life. a common denomination of such application scenarios was the need to operate in a scarcely structured environment, which ultimately requires increased abilities, a multimodality "constellation" of sensors and a higher degree of dexterity and autonomy [27] . creating a parallelism with computer-assisted surgery, robots in colonoscopy are computerintegrated intelligent machines able to: (1) improve the safety and performances of standard healthcare provisions in diagnosis and therapy, such as precision, effectiveness, safety and reliability, (2) enhance interventional abilities of endoscopists and standardize their ability to operate, also in teleoperation, (3) reduce the daily workload with better ergonomics, and (4) augment the field of possible interventions [28] . thanks to specific regulations and standards, i.e., the new medical device regulation (european union mdr 2017/745) and ancillary directives, today classification and methodological design guidelines and functioning tests are clearly identified for guaranteeing a high level of standardization, safety and efficiency of newly introduced medical robotic devices. in order to understand the role of a robot in medicine, and in particular in colonoscopy, we need to answer a few questions, such as: "what is the difference between non-robotic instrumental colonoscopes (section 2) and robotic flexible colonoscopes (sections 3 and 4)?" although there is not a single definition that will cover all aspects, we hope-at least-that this contribution will continue the conversation on this debated issue. for the authors, the difference between them is nestled in its intrinsic capability to enable and perform controlled assisted actions or autonomous procedures in an unstructured deformable environment, such as in the colonic tract. ii. "what are the modules needed to achieve that?" not only embedded sensors, such as the vision camera into the pillcam™ capsule or ph/temperature sensors are needed but also a complex hardware and software architecture that enables computer-integrated modalities, i.e., the information collected by sensors, through wired or wireless communications, can be elaborated thanks to ai algorithms (section 5) for enabling advanced and potentially-autonomous actuation and actions (i.e., navigation of the device but also activation of mechanisms for drug-delivery and tissue sampling) in a closed-loop manner. iii. "why is now the time?" technologies are now in a mature state and thanks to the wider use of robotic systems and technologies in surgery, endoscopists are now more open in accepting and collaborating with robotic companions during their activities [29, 30] . in addition, under the current circumstances, one could in parallel to the term "social distancing" coin the term "medical distancing" (not in care, emotion or relationship but more in a physical contact during medical practices) via complex personal protective equipment or very simply reducing handshakes and consultation distance. obviously, most of us believe that the end of the sars-cov-2 pandemic will allow things to go back to normal, however, this global pandemic sets the scene for innovation in ways and speed that have not been seen before in the field of minimally-invasive surgery and/or in remote robotic diagnosis and therapy in medicine. so, our last question is "is it now the time for introducing in the medical practice a new teleoperated or even autonomous robotic colonoscope?" the paper is organized as follows: section 2 describes non-robotic colonoscopes and colonoscopy adjuncts used in the clinical practice, section 3 summarizes the robotic flexible colonoscopes, as commercially-certified instruments, whereas section 4 describes research-oriented innovative robotic colonoscopes. moving to software development, section 5 focuses on the potentialities of artificial intelligence tools in enhancing robotic colonoscopy and, finally, section 6 reports discussions and conclusions of this comprehensive review paper. thus far, standard colonoscopy (sc) is considered the most effective methodology to diagnose crc. indeed, this method represents the gold standard practice for the evaluation of a wide range of colonic pathologies, due to its ability to visualize the internal surface of the colon, to acquire tissue samples and to treat precursors and early-stage tumours. however, (1) perceived invasiveness, (2) patient discomfort and/or fear of pain, hence, need for conscious sedation, and (3) the concern of social/medical distancing at a time of a pandemic, limit (or will limit for the latest) the use of screening colonoscopy [31] . the population does not participate in screening programs because colonoscopy itself and the necessary preparation of the intestine by dietary adjustment and numerous laxatives are perceived as painful and not worth it by many people [32, 33] . the technology used for sc consists of a long semirigid insertion tube around 13 mm in diameter, with a steerable tip, but nevertheless more rigid than the colon, which is introduced through the anus and pushed forward to inspect the colonic wall. colonoscope looping may occur during insertion, considerably stretching the colon, thus generating pain and potential tissue damage, or even perforation (0.1-0.3% for diagnostic colonoscopies) [34, 35] (figure 2a ). furthermore, even well-experienced endoscopists are often limited by the lack of manoeuvrability, which can result in about 20% of missed polyps [36] . due to growing incidence of crc and of the abovementioned limitations, advanced colonoscopy techniques have been developed. the unusual shape of the colon (e.g., sigmoid stricture, stenosis, fixed sigmoid, and elongated colon), along with previous abdominal surgeries with adhesions, make colonoscopy using standard equipment extremely difficult and sometimes incomplete, diminishing its diagnostic efficiency. at least a few alternatives to the standard reusable colonoscopy technology are available in the market-as reported below-encompassing elevated diagnostic rates in comparison to sc. of note, water-assisted colonoscopy (wac) drew the attention of endoscopists, due to elevated patient comfort (reduced loop formation, no sedation, etc. [37] [38] [39] [40] ) and the quality of scoping (higher adenomas detection rate-adr-and precise muscle images due to water irrigation [41] ). however, limited educational background and time needed to perform wac stand behind this procedure nowadays for its limited use in the clinical practice [42] . a single-use (sterile) endoscope developed by ambu a/s (copenhagen, denmark) was presented in 2019 during the digestive disease week ® conference in san diego, california. the primary goal of the prototype, and in general of non-reusable endoscopes, is reducing the contamination risk [43] . however, it is likely that these devices might be not envisaged as eco-friendly, as new and more stringent polices on environmental polluters are being already announced by the eu parliament. a regulatory clearance on the device serving as duodenoscope is pending. moreover, ambu a/s declared in its website that a single-use endoscope for colonoscopy and endoscopy will also be launched in 2021. virtual colonoscopy computed tomography (cct, also called ct-colonography) is an alternative to sc. however, even if imaging systems are getting more and more accurate and high in resolution, the detection rate of polyps is limited and often lacking, since about 30% of the polyps are flat and obscured when using these techniques. furthermore, sampling and characterization of tissues are not possible because they are based solely on vision and these methods are often inconclusive [44] . all in all, if the cct visualizes a lesion, a colonoscopy still serves as diagnostic mean for further evaluation and treatment [45] . double-balloon enteroscopes were initially developed for small bowel scoping, but their specific features were utilized to design double-balloon colonoscopes (dbc) [46] , which may be the option of choice after a failed sc. dbc are about 2 m long systems including a high-resolution endoscope and two latex balloons filled with air by using pressure pumps. alternating push and pull movements place the gut sections on the overtube, resembling dbe mode of action [47] ( figure 2b ). as elegantly demonstrated in the literature, dbc following sc resulted in the discovery of advanced neoplasia [48] , colon polyps, stenosis (radiation or inflammatory) and crohn's disease that were not identified with the standard method [49] . relatively shorter time of colon examination, reduced conscious sedation and the lack of fluoroscopic evaluation, in comparison with all endoscopic interventions available, stand for this technique's effectiveness [50] [51] [52] . full spectrum endoscopy-fuse platform (endochoice inc., alpharetta, ga, usa) is equipped with extra optics at its end, allowing the medical specialist to view the gut with a 330 degrees angle ( figure 2c ). three cameras and leds snap the images and present them on three monitors. a study, comparing the effectiveness of colonoscopy instruments in adenoma detection, found that fuse platform detected a higher number of lesions in comparison to sc (missing rate 7% vs. 41%) [53] . a very recent study found that the lesion detection rate is higher in right and middle parts of the colon [54] . similarly, a study by kudo et al. [55] found diminished adenoma missing rate with fuse platform. in contrast, previous trials failed to replicate these results when compared to forward-viewing approach colonoscopy in ascending colon [55, 56] . the g-eye endoscope (naviaid g-eye, smart medical systems ltd., ra'anana, israel) has an integrated (moderately inflated) balloon serving as its bending part, which allows both the withdrawal and instrument stabilization together with flattening the haustral folds and inhibiting the slippage of the bowel ( figure 2d ). a recent study by shirin et al. [57] found that the technique yielded a higher detection rate of adenomas/polyps (adr and pdr), including well-formed, flat and sessile serrated ones, when compared to sc. when meta-analysed with a previously published paper by halpern et al. [58] , keulen et al. [59] discovered that adr by means of the g-eye endoscope is 30% higher than sc. a possible alternative to the conventional tethered colonoscopy is represented by wireless capsule endoscopes (wce), established in the last decade and representing an interesting non-invasive alternative to standard endoscopy [60] . wce allows gentle inspection of the entire gastrointestinal tract without any discomfort and therefore with no need for sedation; this may encourage patients to accept gastrointestinal tract examinations thanks to its lack of invasiveness. however, wce is a passive device moving through peristalsis and, therefore, it is not ideal for capturing images of specific areas of interest, as it cannot be stopped, oriented and navigated [61] ; this limits its key-application to the small bowel. differently, the large intestine requires adequate distension for inspection and navigation that allows visual orientation. therefore, wce for the large bowel (pillcam tm colon 1 and colon 2-medtronic inc., minneapolis, minnesota, usa) inspection so far failed to show results competitive with conventional colonoscopies [62] [63] [64] (figure 2e ). of note, lumen preparation is still necessary for the usage of wce. c-scan ® cap wireless colonic capsule (check-cap ltd., isfiya, israel) is deprived of this need, i.e., the capsule is based on x-ray technology and together with the localization data provided by means of wireless communication, it allows the creation of a 3d map of the inside colon view [65] . to increase the lesion detection rate, some adjunct tools may be placed on the top of the colonoscopes. for instance, endorings tm (endoaid ltd., caesarea, israel) are circular add-ons stretching the gut folds when removing the colonoscopic device ( figure 2f ). such adjunctive tool was found to elevate the adr as demonstrated in a clever study [66] . a randomized trial conducted by rex et al. [67] in 2018 revealed that the adr was higher in case of endorings tm usage when compared to fuse system. another similar adjunct tool is endocuff vision tm (olympus corp., tokyo, japan), a single-use device using arms instead of flaps to straighten out the mucosa ( figure 2g ). in a randomised trial, endocuff vision tm was able to significantly increase the adr if compared to sc, i.e., 35.4% vs. 20.7%, with comparable overall procedure time and without major adverse events [68] . moreover, in the same multicentre randomized study proposed by rex et al. [67] , conducted with 1188 patients, adr with endocuff vision tm (adenomas per colonoscopy-apc-mean ± standard deviation: 1.82 ± 2.58), endorings tm (1.55 ± 2.42) and standard hd colonoscopy (1.53 ± 2.33) were all higher than fuse (1.30 ± 1.96; p < 0.001 for apc). in summary, forward-viewing hd instruments that dominate the fuse system and endocuff vision tm is a dominant strategy over endorings tm , as reported by rex et al. [67] . colonoscopy assisted by a transparent cap (reveal ® distal attachment cap, steris corp., mentor, oh, usa), attached to the tip of the endoscope, was introduced to elevate the adr via mucosal folds flattening and minimizing a red-out, while preventing the mucosa to adhere to the lens. a meta-analysis by nutalapati et al. [69] found that the cap improved the adr by almost 20%, and improved the cecal intubation rate and time (cir and cit, i.e., rate and time of cecal intubation, defined as the passage of the colonoscope tip to a point proximal to the ileocecal valve, so that the entire cecal caput, including the medial wall of the cecum between the ileocecal valve and appendiceal orifice, is visible). however, as elegantly discussed by frieling [70] , it might be "[...] beneficial, especially for unexperienced endoscopists", thus associated with training commitment. on the other hand, pohl et al. [71] concluded that cap-assisted colonoscopy did not significantly improve the adr and consequently it may be beneficial only for a percentage of endoscopists. similarly to novel colonoscopy-based techniques, serving for better diagnostics and therapy of crc and elevating patient comfort during the procedure, more and more endoscopic add-on tools are being introduced in the market, such as the ones produced by ovesco endoscopy ag (tübingen, germany) [72] ( figure 2h ) that provide additional therapeutic or surgical functionalities to conventional endoscopes. table 1 reports a summary of the distinctive features, advantages, and limitations of the aforementioned non-robotic colonoscopes and colonoscopy adjuncts, used in the clinical practice, comparing them with a quantitative analysis when possible. j. clin. med. 2020, 9, x for peer review 8 of 39 lens. a meta-analysis by nutalapati et al. [69] found that the cap improved the adr by almost 20%, and improved the cecal intubation rate and time (cir and cit, i.e., rate and time of cecal intubation, defined as the passage of the colonoscope tip to a point proximal to the ileocecal valve, so that the entire cecal caput, including the medial wall of the cecum between the ileocecal valve and appendiceal orifice, is visible). however, as elegantly discussed by frieling [70] , it might be "[…] beneficial, especially for unexperienced endoscopists", thus associated with training commitment. on the other hand, pohl et al. [71] concluded that cap-assisted colonoscopy did not significantly improve the adr and consequently it may be beneficial only for a percentage of endoscopists. similarly to novel colonoscopy-based techniques, serving for better diagnostics and therapy of crc and elevating patient comfort during the procedure, more and more endoscopic add-on tools are being introduced in the market, such as the ones produced by ovesco endoscopy ag (tübingen, germany) [72] ( figure 2h ) that provide additional therapeutic or surgical functionalities to conventional endoscopes. table 1 reports a summary of the distinctive features, advantages, and limitations of the aforementioned non-robotic colonoscopes and colonoscopy adjuncts, used in the clinical practice, comparing them with a quantitative analysis when possible. long semirigid instrument (~13 mm in diameter and~1400 mm in length) with a 2-dofs cable-driven steerable tip, manually introduced through the anus and pushed forward and backward to inspect the colonic wall (+ 1-dof axial-roll). current reference standard for diagnosis and treatment; diagnosis and treatment in the same session; manual fine control of the endoscope tip. requires unpleasant laxative preparation, sedatives, and analgesia; uncomfortable procedure due to insufflation and tissue-colonoscope interaction; highly dependent of endoscopist training and ability; looping and potential risk of perforation (0.1-0.3% for diagnostic colonoscopies). [ [31] [32] [33] [34] [35] [36] virtual colonoscopy computed tomography (cct) medical imaging diagnostic procedure using x-rays to compute 3d reconstructed endoluminal views of the colon. alternative to conventional colonoscopy to diagnose disease, e.g., polyps and diverticulosis, without discomfort generally caused by colonoscope-lumen interaction. requires unpleasant laxative preparation; only ct-based morphological tissue analysis; uncomfortable procedure due to insufflation; sedatives, and analgesia often required; no tissue treatment or surgery; pdr limited (30% of the polyps are flat and obscured). [44, 45] double-balloon colonoscopy (dbc) about 2 m long system including a high-resolution endoscope and two latex balloons filled with air by using pressure pumps for easing navigation. relatively shorter time of colon examination, and reduced conscious sedation if compared to sc; used in the cases of technical difficulties, e.g., loop formation, long colonic segments, or suspected adhesions, resulting in the discovery of advanced neoplasia, colon polyps, stenosis and crohn's disease, that were not identified with sc. same of sc (often, with reduced discomfort, looping and risk of tissue damage); lack of fluoroscopic evaluation. [ minimally-invasive and painless; high-patient tolerability; negligible risk of perforation. requires unpleasant and aggressive laxatives preparation; low-accuracy and reliability for diagnosis; inability to control the capsule; inability to perform therapy and treatment. [ [60] [61] [62] [63] [64] [65] self-propelling robotic colonoscopes are already available on the market and exhaustively described in the literature; in this section, authors also report systems that were available on the market and, therefore, robotic flexible colonoscopes that obtained commercial approval, e.g., ce mark, fda or cfda. robotic flexible colonoscopes can be classified based on the actuation principle used to negotiate the deformable and unstructured colonic tract, such as: (1) electro-mechanically actuated with a "follow-the-leader" mechanism, i.e., the neoguide endoscopy system, or with an inverted-sleeve mechanism through wheels, i.e., the invendoscope, or (2) using electro-pneumatic mechanisms, i.e., aer-o-scope system, colonosight and the endotics system. an innovative robotic platform, worth mentioning in this section, even if applied so far only to the gastric tract, is navicam ® , a robotic-assisted platform to magnetically control wireless capsule endoscopes. the neoguide endoscopy system (neoguide endoscopy system inc., los gatos, ca usa) is a fda mark computer-assisted colonoscope consisting in a 16-segment insertion tube that controls the snake-like movement of the endoscope; each segment has two degrees of freedom (dofs), is independent and electromechanically controlled ( figure 3a ). thanks to positions sensors at the distal tip of the endoscope and at the external base of the device, live view of the position of the scope's tip, insertion depth and computed real-time 3d mapping of the colon, can be obtained. computerized mapping enables the insertion tube to change the segments shape at different insertion depths in a "follow-the-leader" manner to negotiate colonic flexures in order to reduce looping and unintentional lateral forces applied to the colon wall and, thus, patient discomfort during the procedure [75, 76] . neoguide endoscope showed successful cecal intubation with safety and effectiveness in 10 patients, with an overall procedure time, including therapeutic invention, of 34 min (range: 24-60 min), demonstrating a reduction in the looping rate thanks to the assistance of the computerized 3d mapping images [77] . further human studies of the neoguide endoscope were warranted in order to improve the platform and to establish its potential for notes [78] . approval of the system from the fda was obtained in 2006 and the system was acquired by intuitive surgical inc. (sunnyvale, ca, usa) in 2009. as a result of this acquisition, some of the key-technologies were translated to ion, a new robotic-assisted endoluminal platform developed by intuitive surgical inc. for minimally invasive peripheral lung biopsy. the invendoscope™ sc40 (invendo medical gmbh, weinheim, germany) is a ce and fda mark computer-assisted single-use colonoscope propelled, forward or backward, by an inverted-sleeve mechanism composed of eight drive wheels ( figure 3b) . the colonoscope has a 10-mm inner sheath; a sleeve is pulled over this inner sheath, inverted at each of the respective ends (at the biopsy port and just below the endoscope deflection) and attached to a propulsion connector. the connector is then locked into an endoscope driving unit and the examination is started. a hand-held control unit is used to activate all the endoscopic and software functions. when the forward or backward buttons on the hand-held device are pressed, eight wheels in the endoscope driving unit start to move in the selected direction. the wheels grip onto the inner side of the inverted sleeve, causing the inverted sleeve and inner sheath to drive either forward or backward. the colonoscope has a unique robotically-driven tip armed with three white light emitting diode (leds) and a complementary metal-oxide-semiconductor (cmos) vision chip with a field of view of 114 • . the colonoscope tip can be flexed electro-hydraulically through a hand-held unit to 180 • (at body temperature) in any direction and can move in circles, providing the operator with a complete view of the lumen; it also allows full retroflection for inspection of the mucosa behind colonic folds. the colonoscope has an overall diameter of 18 mm and a working length of 2000 mm (in its last version). in addition, standard functions including suction, irrigation, and insufflation are also provided along with a 3.2 mm working channel for biopsies and routine therapeutic procedures, such as polypectomy [75] . a clinical study showed a cir of 98.4% (median time: 15 min), without any pain, in 92% of patients. twenty-seven polypectomies were successfully performed in 23 patients [79] . however, this prototype has been replaced by a manually inserted single use device with standard flexibility and with a hand-held electrical control interface, namely the invendoscope™ sc200 (as part of the invendoscopy tm e200 system), that obtained the ce mark certification in 2016 and in january 2018 the fda clearance for the invendoscopy tm system e210 and for the invendoscope tm sc210. in october 2017, invendo medical gmbh was acquired by ambu a/s [75] . the aer-o-scope system (gi view ltd., ramat gan, israel) is a ce and fda mark pneumaticallyactuated self-propelling, self-steering and disposable robotic colonoscope ( figure 3c ). active locomotion is obtained through two inflatable balloons and internal pneumatic pressure. both balloons are inserted into the rectum and, by inflating them, the colon section in between is sealed. when co 2 is inflated between the two balloons, the pneumatic force pushes the frontal mobile balloon forward, minimizing the need for the operator to exert external manual pushing force, significantly facilitating its negotiation through colonic flexures. once the mobile balloon reaches the cecum, the co 2 between the balloons is vented, co 2 is inflated between the cecum and the frontal mobile balloon, so that the pneumatic force pushes the mobile balloon backward. a 360 • omni-directional high-definition vision system composed of a camera with a field of view of 57 • , a dedicated optical module and leds are carried by the frontal mobile balloon and remotely controlled by a hand-held interface by the operator to inspect the colon; the latest aer-o-scope system is equipped with two working channels dedicated to treatments. to protect the intestine from possible damages, the operating pressure is monitored through electronic sensors to not exceed 60 mbar [80] . a recent study with 58 subjects shows that the aer-o-scope colonoscope has a cir of 98.2% and a pdr (including all polyps larger than 5 mm) of 87.5% compared to sc; in addition, no mucosal damage or adverse events were reported [81] . colonosight (stryker gi ltd., haifa, israel) is a self-advancing system composed of a reusable colonoscope, named endosight, with leds and a camera at the tip, covered by a wrapped disposable multilumen sheath with working channel, named colonosleeve, to prevent the endoscope from contact with potentially infectious agents and thus to eliminate the need for disinfection [82] ( figure 3d ). the device is powered by an electro-pneumatic unit that insufflates the outer sheath to generate, by progressively unfolding it, a forward force at the distal tip enabling to pull the endoscope through the colon. this electro-pneumatic mechanism helps in reducing the overall "pushing" force required to insert the device. a multicentre trial with 178 participants showed a 90% cir in a mean time of 11.2 ± 6.5 min. biopsies were taken in some of the procedures and no complications, e.g., bleeding or perforation, were noted after a fortnight, thus showing a promising potential of this device over sc [82] . the endotics system (era endoscopy srl, pisa, italy) is a ce mark pneumatically-driven robotic disposable colonoscope able to crawl through the colon by using two mucosal clamping devices, located at the proximal and distal ends of the probe, and a soft extension/retraction central mechanism, mimicking an inchworm-like locomotion ( figure 3e ). semiautonomous locomotion occurs by a series of consecutive steps: (1) the proximal clamp attaches to the mucosal surface, next the body of the probe elongates, (2) the distal clamp attaches to the mucosa and the proximal clamp detaches, and (3) the body contracts and the process begins again. the steerable head contains leds, a cmos camera with a 140 • field of view, a water and air channel for cleaning /drying the lens and for insufflation and a 3 mm working channel. the robotic device is remotely controlled by a hand-held interface through the workstation and is able to bend up to a 180 • angle in every direction with very high precision, both in step-by-step mode (i.e., digital mode) and in continuous mode (i.e., analog mode), as well as electronic chromoendoscopy [75, 83] . a study with 40 enrolled patients evaluated the forces applied by the endotics system compared to the traditional colonoscope, showing that the stress pattern related to the rc was 90% lower than that of sc. all patients rated the rc as virtually painless compared to sc, ranking pain and discomfort as 0.9 and 1.1, respectively, on a scale of 0 to 10, versus 6.9 and 6.8, respectively, for the sc [84] . in a first study conducted on 71 subjects with clinical or familial risk of colonic polyps/carcinomas, the cecum was reached in 81.6% of examinations (94.3% with sc), and the average time was 45.1 ± 18.5 and 23.7 ± 7.2 min for the robotic and traditional colonoscopy, respectively. no patient required sedation during the robotic examination, compared with 19.7% of patients undergoing sc. finally, the sensitivity and specificity of the endotics system for detecting polyps were 93.3% and 100%, respectively, the positive predictive value 100% and the negative predictive value 97.7% [85] . in another retrospective study, senior gastroenterologists performed both traditional colonoscopy and endotics system colonoscopy without the use of sedative agents on 276 patients. one hundred and two out of 276 endotics rc examinations were performed in a series of patients who had undergone sc and had failed cecal intubation (difficult cases). overall, endotics system was successful in 93.1% of cases of incomplete sc (95% performance) [86] . recently, a single-centre prospective pilot study was performed recruiting 56 consecutive outpatients for elective rc. training progress in rc was assessed comparing the results of two consecutive blocks of 27 (group a) and 28 (group b) procedures. cir was 92.7%, reaching 100% in group b. comparing the two groups, cit significantly decreased from 55 to 22 min, whereas procedures with cit < 20 min increased. pdr was 40% (males 62.5%, females 14.3%) and adr was 26.7% (males 27.5%, females 14.3%). in addition, in this study, most of patients judged the procedure as mild or no distress, with high willingness to repeat the rc (92.7%) [83] . a noteworthy example of robotic-assisted endoscopic platform, even if applied only to the gastric district so far, is navicam ® (ankon technologies co, ltd. wuhan, shanghai, china), a robotic-assisted platform able to magnetically navigate an endoscopic wireless ingestible capsule in the stomach for gastric examinations ( figure 3f ). the external static magnetic field generated by the navicam ® platform accurately controls, with 5-dofs, a pill-size (28 × 12 mm) endoscopic capsule embedding a cmos camera with a 140 • field of view and a depth of field from 0 to 60 mm, leds and a permanent magnet [87] . the platform received the approval by the cfda mark with a class iii medical device registration certificate titled "magnetically controlled robotic capsule endoscope". table 2 reports a comparative analysis of the distinctive features and/or clinical outcomes of robotic flexible colonoscopes, that obtained approval and certification to be market-available. [83] . a noteworthy example of robotic-assisted endoscopic platform, even if applied only to the gastric district so far, is navicam ® (ankon technologies co, ltd. wuhan, shanghai, china), a roboticassisted platform able to magnetically navigate an endoscopic wireless ingestible capsule in the stomach for gastric examinations ( figure 3f ). the external static magnetic field generated by the navicam ® platform accurately controls, with 5-dofs, a pill-size (28 × 12 mm) endoscopic capsule embedding a cmos camera with a 140° field of view and a depth of field from 0 to 60 mm, leds and a permanent magnet [87] . the platform received the approval by the cfda mark with a class iii medical device registration certificate titled "magnetically controlled robotic capsule endoscope". table 2 reports a comparative analysis of the distinctive features and/or clinical outcomes of robotic flexible colonoscopes, that obtained approval and certification to be market-available. electro-mechanical actuation with a "follow-the-leader" mechanism. 16-segment insertion tube that controls the snake-like movement of the endoscope; each independent and electromechanically-controlled segment has 2-dofs; position sensors at the distal tip of the endoscope and at the external base of the device to obtain live view of the position of the scope's tip, insertion depth and computed real-time 3d mapping of the colon. computerized mapping enables the insertion tube to change the segments shape at different insertion depths to reduce looping and unintentional lateral forces and, consequently, patient discomfort; successful and safe (reduction in the looping rate) cecal intubation in 10 patients, with a cit (with therapeutic invention) of 34 min (range: 24-60 min); fda obtained in 2006, and acquisition by intuitive surgical inc. in 2009; no longer available on the market and technology translated to ion, a robotic-assisted endoluminal platform for minimally invasive peripheral lung biopsy. [ [76] [77] [78] invendoscope™ sc40 (invendo medical gmbh, weinheim, germany), then (ambu a/s, copenhagen, denmark) electro-mechanical actuation with an inverted sleeve mechanism. computer-assisted single-use colonoscope propelled, forward or backward, by an inverted-sleeve mechanism composed of eight drive wheels; robotically-driven tip with leds and a cmos 114 • camera, electro-hydraulically flexed through a hand-held control unit to 180 • in any direction with full retroflection; diameter of 18 mm and working length of 2000 mm with standard functions including: (1) suction, (2) irrigation, and (3) insufflation with a 3.2 mm working channel, also used for conventional therapeutic procedures. cir of 98.4% (median time: 15 min), without any pain, in 92% of patients. 27 polypectomies successfully performed in 23 patients; invendoscope™ sc40 replaced by a manually inserted single use device with standard flexibility and a hand-held electrical control interface, namely the invendoscope™ sc200 (as part of the invendoscopy tm e200 system); latter, obtained the ce mark in 2016 and in january 2018 the fda clearance for the invendoscopy tm system e210 and for the invendoscope tm sc210; no longer available on the market, acquisition by ambu a/s in 2017. [76, 80] aer-o-scope system (gi view ltd., ramat gan, israel) electro-pneumatic actuation. self-propelling, self-steering and disposable robotic colonoscope with navigation obtained through two sealed inflatable balloons and internal pneumatic pressure (inflation of co 2 ) for pushing the frontal mobile balloon forward and backward; hand-held control unit to teleoperate the colonoscope's tip with: (1) a 360 • omni-directional hd vision system with a 57 • fov camera, (2) leds, and (3) two working channels for conventional therapeutic procedures in the latest version; monitored pressure, through electronic sensors, ≤60 mbar. in-vivo study with 58 patients proved a cir of 98.2% and a pdr (including all polyps larger than 5mm) of 87.5% compared with sc, and no mucosal damage or adverse events were reported; fda mark obtained in 2014 (and ce mark in europe); currently available on the market. [81] electro-pneumatic actuation. self-advancing system composed of: (1) a reusable colonoscope (endosight), with leds and a camera, covered by (2) a wrapped disposable multi-lumen sheath with working channel (colonosleeve), to prevent infection and eliminate the need for disinfection; powered by an electro-pneumatic unit that insufflates the outer sheath to generate, by progressively unfolding it, a forward force at the distal tip enabling to pull the colonoscope. electro-pneumatic mechanism helps reducing the overall "pushing" force; multicentre trial with 178 participants showed a 90% cir in a mean time of 11.2 ± 6.5 min; biopsies taken in some of the procedures and no complications, e.g., bleeding or perforation, noted, thus showing promising potential over sc; fda achieved in 2008, no longer available on the market. [82] endotics system (era endoscopy srl, pisa, italy) electro-pneumatic actuation. remotely-controlled (by a hand-held control unit) disposable colonoscope able to semi-autonomously crawl the colon by using two mucosal clamping modules, located at the proximal and distal ends of the probe, and a soft extension/retraction central mechanism, mimicking an inchworm-like locomotion; steerable head, able of a 180 • bending angle and, containing: (1) leds, (2) a cmos camera with a 140 • fov, (3) a water and air channel for cleaning/drying the lens and for insufflation, and (4) a 3 mm working channel for conventional therapeutic procedures. a single-centre prospective pilot study was recently performed with 56 consecutive outpatients (two consecutive blocks of 27-group a-and 28-group b-procedures); cir was 92.7%, reaching 100% in group b; comparing the two groups, cit significantly decreased from 55 to 22 min, whereas procedures with cit < 20 min increased; pdr was 40% (males 62.5%, females 14.3%) and adr was 26.7% (males 27.5%, females 14.3%); most of patients judged it as mild or no distress, with high willingness to repeat the robotic procedure (92.7%); system available on the market with ce mark obtained in 2011. [86] in recent decades, several research institutes contributed to the development of novel robotic colonoscopes. even if results look promising for opening a new way of performing painless colonoscopy, most of them are still at the research level. in the current scientific literature, there is a consistent number of review papers describing low-trl (trl: technology readiness level) robotic colonoscopes, sometimes as a simple list of devices, sometimes classifying them based on their intrinsic features [28, 89, 90] . in this review paper, the authors decided to critically describe each of them as part of groups of robots with the same actuation principle, being it one of the most important robotic features towards a fully-automated robotic colonoscope, and in particular devices were classified as: (1) electric-, (2) hydraulic-or pneumatic-and (3) magnetic-actuated robotic colonoscopes. electric actuation produces significant forces or torques through integrated mechanisms at the cost of a high-power consumption and, in the case of a wireless device, with the need of integrating a battery, a few cm 3 in volume. however, the latter is not needed for wired robotic colonoscope in which energy, as well as other large or high-rate data and main colonoscopic services (e.g., water, air, operating channel), are provided through the tether itself. a few examples of robotic colonoscopes with electric actuation are reported in figure 4a -f. in 2014, kim et al. developed a flexible caterpillar-based robotic colonoscope actuated by an external electric motor through a flexible shaft [91] . two years later, the same research group presented an improved version of the robotic colonoscope through theoretical and experimental evaluations for the design of each component and by embedding a steering module (maximum bending angle of 178 • and minimum curvature of the radius of 20 mm-experimental/simulator average error of 5.8%) ( figure 4a ). test performed in a straight excised porcine colon demonstrated reliable locomotion performance with forward and backward velocities of 5.0 ± 0.4 mm/s and 9.5 ± 0.9 mm/s, respectively (forward velocities of 6.1 ± 1.1 mm/s and 4.7 ± 0.7 mm/s in the case of 30 • and 60 • inclination angles, respectively). further tests, performed in a 1 m long excised porcine colon, arranged to mimic the lower gi human anatomy, revealed a velocity of 3.0 ± 0.2 mm/s with a success rate (i.e., cir) of 50% and a total procedure time (i.e., cit) of 8.55 min, in case of a novice operator (#8 experiments performed). an in-vivo test performed in a live mini pig under sedation demonstrated the capability of the robotic colonoscope to arrive at the distal transverse colon, 600 mm from the anus. however, cecal intubation failed due to the mucosa structure and presence of faecal materials [92] . in 2019, lee et al. developed a legged robotic colonoscope based on simple and reliable reel-based mechanism, actuated by an external electric motor [93] ( figure 4b ). the authors demonstrated the high manoeuvrability of the colonoscopic device improved, in terms of safety, by harnessing a soft material for the six legs. in excised porcine colon the tethered robot achieved a 9.552 ± 1.940 mm/s velocity on a flat path, without any scratches or perforations on the porcine tissue [94] . starting by a first tethered robotic capsule endoscope using micropatterned treads developed in 2012 by sliker et al. [95] , formosa et al. presented in 2019 a novel design of a multi-dofs sensor-enabled treaded robotic colonoscope, named endoculus. the device presents interesting novel features such as: (1) a custom double-worm drive that removes axial gear forces while reducing radial moments, and (2) the full parameterization of gear geometries allowing size reduction via an optimization routine over design constraints [96] . two independently-controlled motors drive micro-pillared treads above and below the device allowing for 2-dofs skid-steering, even in a collapsed lumen. the proposed robotic colonoscope contains all the functionalities of a traditional endoscope: (1) a camera, (2) adjustable light emitting diodes (leds), (3) channels for insufflation and irrigation and (4) a tool port for endoscopy instruments (e.g., forceps, snares, etc.). additionally, endoculus carries an inertial measurement unit, magnetometer, motor encoders and motor current sensors to aid autonomous strategies in the future ( figure 4c ). an in-vivo preliminary test in a live pig showed endoscopic functionalities and promising results in terms of locomotion (even if it was not able to gain consistent traction in the sigmoid area, seemingly due to excessive constriction upon the non-treaded sides of the devices). ex-vivo tests demonstrated forward/reverse locomotion up to 40 mm/s on the colon mucosa (both not insufflated and distended), 2-dofs steering and the ability to traverse haustral folds and functionality of endoscopic tools [97] . in the same research group, ortega et al. in 2017 designed a soft three-modular section robot for colonoscopy with each module featured by 3-dofs, one translation and two rotations. the robotic colonoscope uses nine independently controlled shape memory alloy (sma) springs as its actuators and a novel silicone rubber skin to provide the passive recovery force to expand the springs to their original state. in addition, it also incorporates three air tubes, one for each section, to provide forced convection reducing the cooling time of the sma springs. in-depth fem analysis were performed to guarantee the required mechanical behaviour (i.e., maximize traction and provide enough recovery force) and a controller unit was designed and implemented for each of the sections allowing the robot to achieve any orientation between −90 • and +90 • in both pitch and roll in less than 4 s with near zero steady state error. the robot uses a peristaltic motion to translate, inspired by the motion generated by the bowel, and both the peristaltic motion and the orientability of the robot were tested ( figure 4d ). tests demonstrated that the robot is able to perform a peristaltic motion with a maximum and average speed of 4 mm/s and 0.36 mm/s, respectively. each section is also able to follow, with less than 2% overshoot and near zero steady-state error, periodic multi-input squared signals of 25 • of amplitude [98] . an electrically-actuated worm-like robotic endoscope, 13 mm diameter, 105 mm in length and 22.3 g in weight, was developed by wang et al. in 2017 [99] ( figure 4e ). the lightweight robot is composed of three independent segments; each segment is composed of a linear locomotor with micromotor, turbine-worm and wire wrapping-sliding mechanism. the robot is entirely covered by an external soft bellow with excellent compatibility, designed to increase the static friction and decrease the kinetic friction in the contact state. the robot was tested in-vivo in a porcine model, demonstrating an excellent locomotion capability and safety in soft tissues, with a speed ranging between 1.62 and 2.20 mm/s and passing the entire colon with an overall time of 119 s. another worm-like endoscopic robot, based on an embedded electrical cable-driven actuation system, was developed by bernth et al. in 2017 [100] ( figure 4f ). the robot consists of three segments of which the two distal segments bend, thus allowing steering, while the middle segment can only extend and contract along the axial direction. by bending the two distal segments in turn, the robot can jam or wedge itself against the folds inside the colon. when one of the segments is thus jammed, the middle segment can move the rest of the robot relative to the stationary segment by either extending or contracting. therefore, the robot itself can move forward and backward along the human colon, depending on the order in which the locomotion sequence is performed. this locomotion principle presented with this worm-like concept avoids the need for high pushing forces associated with conventional colonoscopes. additionally, fabricated in soft material, the robot is naturally compliant and flexible, which allows the robot to pass through irregular and curved sections gently; these features can help to reduce a significant amount of pain for the patient. based on the tests of the first prototype, this design enables the endoscope to pass through sharp bending radius, and the mechanism of the anchoring properly works well in complicated 3d and narrow colonic deformable environments. another locomotion strategy, explored in the design of innovative robotic colonoscopes, based its principle on hydraulic or pneumatic actuations. these actuations are typically used in soft robotics and perfectly fit in medical applications due to a few features, such as: (1) lightweight and, usually, low inertia, (2) intrinsically safety of the soft materials, (3) reduced needs to integrate sensors and high-computational control schemes (i.e., morphological computation), (4) inert materials not affected by external disturbances, and (5) low cost, usually being disposable. moreover, hydraulic or pneumatic actuations, at the cost of a wired connection with an external control unit and source, do not require integrating a battery for activation. a few recent examples of robotic colonoscopes with pneumatic actuation are reported in figure 5a ,b. magnetic locomotion can be performed using either permanent or electromagnetic sources; examples of magnetic actuation and activation means applied to medical robots and applications have been presented by sliker et al. in [104] . magnetic actuation by permanent field sources allows in 2017, dehghani et al. developed a semiautonomous colonoscopic robot for minimally invasive procedures based on an innovative pneumatically-based locomotion approach, i.e., the tip of the robot is propelled taking advantage of a longitudinal expansion of an internal latex tube ( figure 5a ). the authors performed preliminary ex-vivo experiments demonstrating that the specific robot design inherently prevents loop formation in the colon, which is recognised as the main cause of post procedural pain in patients. the robot successfully advanced for 1.5 m inside an excised porcine colon with an average speed of 28 mm/s and was capable of traversing bends up to 150 degrees. moreover, if pressurized with 90kpa, it exerted less than 6n of normal force at the tip; a maximum force generates pressure of 44.17 mmhg at the tip, which is significantly lower than safe intraluminal human colonic pressure of 80 mmhg [101] . another novel pneumatically actuated soft robot has been developed by manfredi et al. in 2019. the robotic colonoscope, named spid (i.e., soft pneumatic inchworm double balloon), consists of two inflatable distal balloons for anchorage into the colonic wall, connected by a 3-dofs central pneumatic actuator for a bio-inspired inchworm-like locomotion and bidirectional bending. spid, in the current version, has an external diameter of 18 mm, a total length of 60 mm and weighs 10 g. the soft and deformable structure is aimed at reducing the pressure applied to the colonic wall and consequently pain and discomfort during the procedure ( figure 5b ). the colonoscopic soft robot has been tested in a deformable synthetic colon phantom, mimicking shape, and dimensions of the human anatomy. it exhibited efficient locomotion by its ability to deform and negotiate flexures and bends with an average forward speed of 2.8 mm/s (a total length of 1.4 m was covered in less than 9 min). after cecal intubation, the soft robot was withdrawn by manual traction of the tether, taking about 1 min and with an average speed of 25 mm/s; no real evaluation of the colonic mucosa was performed [102] . an interesting conceptual solution has been proposed by consis medical ltd. (beer-sheva, israel), an early-stage medical device company, devoted to the development of novel, semi-disposable and self-propelling robotic colonoscopes using hydraulic-aiding internal propulsion. the proposed robotic colonoscope is composed of: (1) an inverted single-use inflatable sleeve, (2) a multiple-use electronic head, embedding a working channel, a camera, light source and air and water nozzle, and (3) an external control unit. once the electronic head is mounted and inserted into the anus, first the colon is inflated and then the device is deployed, aiding its navigation with an internal water-based hydraulic propulsion. examination is performed withdrawing the device manually from the cecum and bending the camera with 2-dofs [103] . magnetic locomotion can be performed using either permanent or electromagnetic sources; examples of magnetic actuation and activation means applied to medical robots and applications have been presented by sliker et al. in [104] . magnetic actuation by permanent field sources allows for the generation of a high strength-to-size ratio magnetic field if compared to electromagnets, i.e., they can generate lager forces than electromagnets, given a comparable size and volume. the second main feature is their permanence, i.e., they can generate an electric field indefinitely, without the need for a power supply, offering an untethered magnetic field generator. the latter can be considered an advantage but also a disadvantage in an operating room since they cannot be controlled in terms of strength and they cannot be switched off. however, they can be easily customized in terms of dimensions, shapes and magnetization directions, making them suitable for different applications. contrarily, electromagnetic field sources, if compared with permanent magnets, provide the advantage of controllability (from off to on) of the generated magnetic field, increasing safety and flexibility of the system in the operating room. nevertheless, the main disadvantages are: (1) their high size-to-strength ratio when compared to permanent magnets, (2) the need to implement control strategies, and (3) the need for a power supply to generate a magnetic field, which usually contributes to more equipment in the operating room, higher device cost and increased power demands. finally, large electromagnetic sources present a physical limit, since the larger magnetic field that is created along the n-s pole direction can be far from the external surface of the electromagnet, and thus from the medical device if placed parallel to the magnetization direction, due to the high number of windings between the centre of the electromagnet and the external surface. a few examples of robotic colonoscopes with magnetic or electromagnetic actuations are reported in figure 6a -c. magnetic-based actuation applied to endoscopic robots was first explored between 2008 and 2009 in the framework of a european fp6 project, called "versatile endoscopic capsule for gi tumor recognition and therapy (vector project)" and coordinated by novineon healthcare technology partners gmbh (tuebingen, germany) [105] . in the framework of this project, ciuti et al. proposed an active locomotion approach based on permanent magnets (outside and inside the endoscopic capsule). the robotic platform for wireless capsule endoscopy combines the benefits of magnetic field strength and limited encumbrance with accurate and reliable control through the use of an external anthropomorphic robotic arm [106] [107] [108] . even if the project focused on the development of magnetically-actuated wireless capsule robots, an interesting derivative outcome of the project consisted of a soft-tethered magnetically-driven capsule for colonoscopy. a proof-of-concept of the robotic colonoscope, presented by valdastri et al. in 2012, represents a trade-off between capsule and traditional colonoscopy combining the benefits of low-invasive propulsion (through "front-wheel" locomotion) with the multi-functional tether for treatment [109] . the system has been improved in the subsequent years in terms of modelling [110, 111] , localization [112, 113] and control [114] [115] [116] , towards autonomous locomotion [117] and other applications [118] . a novel derivative soft-tethered magnetically-driven colonoscope was designed within a european h2020 project, called "endoscopic versatile robotic guidance, diagnosis and therapy of magnetic-driven soft-tethered endoluminal robots (endoo project-2015-2019)", coordinated by the scuola superiore sant'anna (pisa, italy) [119] . the soft robotic colonoscope is featured by a high-definition stereo-camera with a custom-made optics, navigated by an external custom-made permanent magnet through a collaborative industrial anthropomorphic robot (comau spa, turin, italy) [120] . a noteworthy outcome of the eu project was the development of ai algorithms to perform vision-based closed-loop control and autonomous detection and measurement of colonic lesions, e.g., polyps [121] [122] [123] [124] (figure 6a) . a hand-guided external electromagnetic system for a wireless colonoscope was designed in the framework of a european fp7 project, called "new cost-effective and minimally invasive endoscopic device able to investigate the colonic mucosa, ensuring a high level of navigation accuracy and enhanced diagnostic capabilities (supcam project-2012-2014)", coordinated by s.e.d. srl (certaldo, italy) and supervised by dr. alessandro tozzi, inventor of the omni-vision spherical capsule concept. the external electromagnetic source navigates, through a generated static magnetic field, a colonoscopic spherical-shape capsule provided with an internal permanent magnet, able to perform a 360 • inspection through inner camera rotation [125] [126] [127] (figure 6b) . another significant example of robotic colonoscopic platform using electromagnetic fields, in this case alternated, has been presented by nouda et al. in 2018. a self-propelling capsule endoscope composed by a pillcam tm sb2 with a silicone fin, embedding a permanent magnet attached to it, has been tested for the first time in a healthy human volunteer. an external platform generates an alternating magnetic field that make the fin shake and thus propel the capsule, with an overall dimension of 45 mm in length and 11 mm in diameter and with a three-dimensional control. the capsule, inserted in the anus and transported with endoscopic forceps in the descending colon, was able to swim in the lumen in antegrade and retrograde directions without any damage to the mucosa [128] (figure 6c) . table 3 reports a comparative analysis of the distinctive technical features and/or preclinical outcomes of innovative research-based robotic colonoscopes and devices. keeping in mind current bottlenecks in the field of colonoscopy, mainly related to (1) pain management, (2) miniaturization, (3) smart actuation and (4) localization, interesting hints to face current technological and design challenges can originate from robotic systems developed for different medical purposes (e.g., cardiovascular interventions or drug delivery outside the gastrointestinal tract) or from bio-inspired robotic devices. the latter, as witnessed also by some commercial colonoscopes described before, appear particularly interesting for investigating locomotion strategies resulting efficient in unstructured environments. by taking inspiration from biological organisms benefitting from centuries of evolution to put in place power efficient locomotion paradigms at certain scales, it is possible to develop smart solutions for navigation across the gi tract. in this direction, zarrouk et al. described a single actuator robot inspired by wave-like locomotion of snakes and bacteria flagella able to move forward or backward by producing a continuously advancing wave. the peculiar modular design enables locomotion over sliding terrains (as gi tract and colon could be) and against gravity by exploiting a single electromagnetic actuator. dimensional issues (the smallest version of the robot is nearly 120 mm long and 30 mm wide) prevent at present the employment of such design inside body lumens but the principle could be inspiring for the next generation colonoscopes [129] . by taking inspiration from fungal hyphae and trailing plants, hawkes et al. developed a growing robot characterized by extension from the tip of the body and able to change its length of hundreds of percent by also actively control the growth direction. the eversion mechanisms actuation shows some similarities with the work from dehghani et al. [101] . due to the intrinsically soft structure, enabled by the constitutive materials and by the pneumatic actuation, the robot design appears suitable both for fabrication in different sizes and for safe and painless interaction with surrounding tissues. the presence of a camera on the robot tip enables one to foresee the employment of such system in future endoscopic applications [130, 131] . j. clin. med. 2020, 9, x for peer review 23 of 39 been tested for the first time in a healthy human volunteer. an external platform generates an alternating magnetic field that make the fin shake and thus propel the capsule, with an overall dimension of 45 mm in length and 11 mm in diameter and with a three-dimensional control. the capsule, inserted in the anus and transported with endoscopic forceps in the descending colon, was able to swim in the lumen in antegrade and retrograde directions without any damage to the mucosa [128] ( figure 6c) . table 3 reports a comparative analysis of the distinctive technical features and/or preclinical outcomes of innovative research-based robotic colonoscopes and devices. flexible caterpillar-based robotic colonoscope, actuated by an external electric motor through a flexible shaft, embedding a steering module (max. bending angle of 178 • and min. curvature of the radius of 20mm). reliable locomotion in ex-vivo straight excised porcine colon with forward and backward velocities of 5.0 ± 0.4 mm/s and 9.5 ± 0.9 mm/s, respectively (forward velocities of 6.1 ± 1.1 mm/s and 4.7 ± 0.7 mm/s in case of 30 • and 60 • inclination angles, respectively); ex-vivo tests, performed in a 1 m long excised porcine colon, arranged to mimic human anatomy, revealed a velocity of 3.0 ± 0.2 mm/s with a cir of 50% and a cit of 8.55 min, in case of a novice operator (#8 experiments performed); in-vivo tests, performed in a live mini pig, demonstrated the capability to reach the distal transverse colon, 600 mm from the anus, but in-vivo cecal intubation failed due to the mucosa structure and faecal materials. reel mechanism-based tethered colonoscope electric actuation. legged robotic colonoscope based on simple and reliable reel-based mechanism, actuated by an external electric motor. high manoeuvrability of the colonoscopic device improved, in terms of safety, by harnessing a soft material for the six legs; ex-vivo tests in excised porcine colon demonstrated a 9.552 ± 1.940 mm/s velocity on a flat path, without any scratches or perforations in the porcine tissue. [93, 94] formosa et al. 2019 multi-dofs sensor-enabled treaded robotic colonoscope electric actuation. two independently-controlled motors drive micro-pillared treads, above and below the device, for 2-dofs skid-steering, even in a collapsed lumen; all the functionalities of a sc, i.e., (1) camera, (2) adjustable leds, (3) channels for insufflation and irrigation and (4) a tool port for conventional therapeutic procedures; in addition, it embeds (5) an inertial measurement unit, magnetometer, motor encoders, and motor current sensors for potential autonomous navigation. in-vivo preliminary test in a live pig showed endoscopic functionalities and promising results in terms of locomotion (even if it was not able to gain consistent traction in the sigmoid area, seemingly due to excessive constriction upon the non-treaded sides of the devices); ex-vivo tests demonstrated forward/reverse locomotion up to 40 mm/s on the colon mucosa (both not insufflated and distended), 2-dofs steering, and the ability to traverse haustral folds, and functionality of endoscopic tools. [ [95] [96] [97] ortega et al. 2017 sma-based three modular section soft robotic colonoscope electric actuation. each module is featured by 3-dofs (one translation, using a peristaltic motion to translate, and two rotations); nine independently controlled sma springs as actuators and a silicone rubber skin to passively recover force to expand the springs to the original state; three air tubes, one for each section, to provide forced convection for cooling sma springs; orientation between −90 • and +90 • in both pitch and roll in less than 4 s with near zero steady state error. in-vitro tests (rigid tube and open environment) demonstrated a peristaltic motion with a maximum and average speed of 4 mm/s and 0.36 mm/s, respectively. [98] lightweight robot (13 mm diameter, 105 mm in length and 22.3 g in weight) with three independent segments, each one composed of a linear locomotor with micromotor, turbine-worm and wire wrapping-sliding mechanism; covered by an external soft bellow with excellent compatibility, designed to increase the static friction and decrease the kinetic friction in the contact state. in-vivo tests in a porcine model, demonstrating an excellent locomotion capability and safety in soft tissues, with a speed ranging between 1.62 and 2.20 mm/s and passing the entire colon with a cit of 119s. [99] bernth et al. 2017 cable-driven actuated worm-like robotic colonoscope electric actuation. worm-like endoscopic robot, based on an embedded electrical cable-driven actuation system; composed of three segments: the two distal segments bend, allowing steering, while the middle segment extends and contracts along the axial direction for forward and backward locomotion. efficient navigation through sharp bending radius curves and proper anchoring in complicated 3d and narrow colonic deformable environments; locomotion strategy avoids high pushing forces associated with sc; fabricated with soft material thus, compliant and flexible for gently passing through irregular and curved sections (potential reduced pain for patients). two inflatable distal balloons for anchorage into the colonic wall, connected by a 3-dofs central pneumatic actuator for a bio-inspired inchworm-like locomotion and bidirectional bending; external diameter of 18 mm, total length of 60 mm and weight of 10 g. soft and deformable structure aimed at reducing the pressure applied to the colonic wall and consequently pain and discomfort during the procedure; tested in a deformable in-vitro synthetic colonic phantom, mimicking shape and dimensions of the human anatomy; efficient navigation with an average forward speed of 2.8 mm/s (a total length of 1.4 m was covered in less than 9 min); manual withdrawal, pulling the tether with an average speed of 25 mm/s, in about 1 min. [102] consis medical ltd. semi-disposable and self-propelling robotic colonoscopes hydraulic actuation. semi-disposable and self-propelling robotic colonoscopes using hydraulic-aiding internal propulsion; composed of: (1) an inverted single-use inflatable sleeve, (2) a multiple-use electronic head, embedding a working channel, a camera, light source and air and water nozzle, and (3) an external control unit; once the electronic head is mounted and inserted into the anus, first the colon is inflated and then the device is deployed, aiding its navigation with an internal water-based hydraulic propulsion. hydraulic-aiding internal propulsion allows to gently approach colonic curves with a potentially-lower stress, and thus pain; examination performed withdrawing the device manually, pulling the tether and bending the camera with 2-dofs. [103] magnetic-based accurate locomotion of wireless and soft-tethered capsules; use of permanent magnets embedded into the capsule and as the external source controlled by a robotic arm; continuous upgrade of the soft-tethered system in terms of modelling, localization and control towards autonomous locomotion. wired solution represents a trade-off between capsule and sc combining the benefits of low-invasive navigation (through "front-wheel" locomotion) with the multifunctional tether for conventional treatment; ex-vivo tests in explanted porcine colon (length of 850 mm) performed by 12 users with six to eight coloured beads, measuring 5 mm in diameter, randomly installed (number and position) along the internal surface of the colon; mean percentage of identified beads of 85 ± 11% (range 64-96%) and identified beads successfully removed; mean completion time, i.e., inspection and bead removal, of 678 ± 179 s (range 384-1082 s); preliminary in-vivo tests in pigs demonstrated an average distance travelled of 800 ± 40 mm in an average time of 900 ± 195 s, including the time devoted to inserting the tool into the dedicated channel and operating the instrument. [ [105] [106] [107] [108] [109] [110] [111] [112] [113] [114] [115] [116] [117] [118] endoo european project soft-tethered stereoscopic robotic capsule colonoscope magnetic actuation (permanent magnets). soft-tethered magnetically-driven colonoscope with a full-hd 170 • fov and 3-100mm dof stereo-camera with a custom-made optics navigated by an external permanent magnet through a collaborative industrial anthropomorphic robot; advanced ai-based tools for augmented diagnosis. extensive experimental sessions in ex-vivo (preclinical outcomes under publication), and tests in human cadavers. [119] [120] [121] [122] [123] [124] supcam european project spherical-shape magnetic capsule for colonoscopy magnetic actuation (hybrid). spherical colonoscopic capsule embedding a permanent magnet and guided by an external gravity-compensated hand-guided electromagnetic system (static electromagnetic field); omni-directional view, by a single embedded camera, through 360 • rotation of an internal magnetic frame into a transparent spherical shell. reliable navigation in ex-vivo (explanted porcine colon) and in-vitro (synthetic plastic phantom) conditions; in-vitro tests performed by five novice users, completing the task (i.e., locomotion in a~900 mm long simple and rigid tube with curves) with a time of 44 ± 8 s (range 26-67 s). electromagnetic locomotion (alternating electromagnetic field through an external platform) of a 3d self-propelling capsule endoscope composed by a pillcam tm sb2 with an attached silicone fin, embedding a permanent magnet; modified capsule, 45mm in length and 11mm in diameter. in-vivo human healthy volunteer test; the capsule, inserted in the anus and transported with endoscopic forceps in the descending colon, was able to swim in the lumen in antegrade and retrograde directions without any damage to the mucosa. [128] chautems et al. recently proposed an innovative variable stiffness magnetic continuum robot eligible for different application scenarios, varying from radio-frequency cardiac ablations to interventional endoscopy in the gi tract. the device includes multiple variable stiffness modules based on a low melting point alloy, a magnetically-steerable tip and an internal working channel for an overall 2.33 mm diameter. the combination of variable stiffness polymers and magnetic tip makes it possible to accomplish complex shapes across a variety of body lumens [132] . a wide range of technologies and actuation strategies developed for smart steerable catheter, where dimension is even more relevant, can provide interesting inspiration for the development of innovative colonoscopes. extremely interesting and innovative proposals have been made also in the field of mobile robotic systems at the meso and microscale. such systems, despite being apparently far from the field of colonoscopy, could pave the way for a novel generation of wce or for innovative endoscope components by making it possible to face miniaturization, powering and painless interaction issues. a wide plethora of untethered capsule robots have been proposed in recent years not only for colonoscopy but also for biopsy and drug delivery [133] . interesting designs in this sense have been reported by don et al. by combining magnetic actuation (both for orientation and biopsy mechanism activation), soft structure and needle-based biopsy [134] . finally, inspiring in-vivo results were recently reported by abramson et al. who proposed an ingestible self-orienting capsule robot for targeted, controlled release of biomacromolecules such as insulin across the gi wall [135] . computational techniques can assist procedures in a number of ways, such as by: (1) supporting the detection and classification of disease through image analysis, (2) allowing mapping and navigation of the endoluminal environment and estimating which regions have been observed, and (3) permitting measurement of structures to support computer-aided decision making. while these application areas have been explored for several decades, it is only in recent years and through the emergence of ai systems based on data, rather than hand-crafter modelling, that the robustness of algorithms is reaching clinical translation in endoscopy. the rapid advances of ai in endoscopy in recent years have seen all major endoscopic equipment providers emerge with solutions for ai-assisted endoscopy. the most active area of ai development in endoscopy is the detection and classification of lesions, in particular colonic polyps [136, 137] but also a growing number of studies with very promising results in upper gi applications like barret's detection [138] and squamous cell neoplasia [139] . while various endoscopic image understanding methods have been explored for a long time [140] , deep-learning based techniques have shown the capability to turn algorithms into clinically valuable computer-aided diagnostic (cad) tools [141] [142] [143] . there is a growing number of studies indicating that cad systems can perform at least as well if not even better than expert endoscopists [138] , though additional validation and understanding of the clinical impact is still, without doubt, needed. such endoscopic ai systems rely on large quantities of image or video data where human observers have annotated lesions to some degree either by denoting the presence of a polyp or by delineating its position and shape within the image ( figure 7a ) [124, 144, 145] . labelling is a time-consuming task, and this is a current system bottleneck because experts have limited bandwidth to perform annotation, which is necessary to train the ai models. as a result, the majority of published studies in endoscopic ai systems utilise thousands of images for training, which is still significantly less than similar algorithms in vision applications, where datasets like imagenet contain many millions of training images [146] . some strategies around addressing this challenge are emerging in the form of open datasets, e.g., endovis-giana challenge [147] and the ead challenge [148] and the use of labelling farms or services, e.g., imertit [149] , as well as efforts to establish imagenet equivalents for gastroenterology [150] or developing unsupervised learning [151] . despite the challenges that remain around developing endoscopic ai, systems for assisting the detection of polyps are maturing into medical product lines pursued by several start-up companies, for example ai4gi [152] , odin vision [153] , shanghai wision ai [154] , as well as the major medical device and imaging manufacturers, such as gi genius™ intelligent endoscopy module [155] [156] [157] [158] [159] . despite being available on the market with regulatory approved products, quality assurance and appropriate ux for working with the clinical team are still issues that require resolution [160] . it is also likely that the next advances in cad support algorithms will be in the disease identification or classification field, with some systems already emerging [161] , where there is an opportunity to enhance clinical workflow and reduce costs/needs for histopathological analysis. some preliminary studies on the opportunities for cost saving and the potential changes to recommended clinical practice with cad are emerging [162] as are opportunities for cad to assist the standardization of services across clinical sites and units [163] . a classic but yet unsolved problem remains in the use of ai systems to enhance endoscope navigation and the clinician's localization within the gi tract by using image data or a combination of image and positional sensors. the importance of this capability is that it may enable quantitative measurement of the observed tissue and detection or regions that have not been observed and hence cleared as healthy. this is a longstanding area of research in all minimally invasive surgery and procedures [164] . the problem can be decomposed into a joint problem of estimating the shape of the gi tract during an endoscopic investigation and also estimating the pose of the endoscopic camera within. classical techniques to tackle the problem [165, 166] have now been superseded by supervised deep learning methods for endoscopy/laparoscopy [167, 168] , which estimate the 3d geometry of the anatomy. supervision is typically achieved through simulated environments or ex-vivo scenes where ground truth can be generated using another technique, such as tomographic scanning or structured light [169] . some preliminary results on deep learning using unsupervised strategies have also been reported by münzer et al. [170] . while the full navigation problem is still challenging to solve robustly, some interesting approaches have emerged to support subtasks that require monocular depth estimation ( figure 7b ) [171] and on measurements of polyp size which are used to make a decision on whether to perform polypectomy or not [172] . . (b) shows simulation data generated for a virtual colon, which can be used toward unsupervised ai models that can predict information that is not normally available such as depth. the image on the left shows examples of synthetic images from the simulation and depth maps predicted using an ai model where the darker colour illustrates further away from the image, whereas the image on the right shows examples of rendered rgb images with corresponding depth maps generated along camera path [172] . (top) original images, and (bottom) images with ai predicted regions (courtesy of prof. danail stoyanov). (b) shows simulation data generated for a virtual colon, which can be used toward unsupervised ai models that can predict information that is not normally available such as depth. the image on the left shows examples of synthetic images from the simulation and depth maps predicted using an ai model where the darker colour illustrates further away from the image, whereas the image on the right shows examples of rendered rgb images with corresponding depth maps generated along camera path [172] . since its introduction in the clinical practice, colonoscopy has been honed to a highly effective diagnostic and therapeutic modality that has transformed provision of gi healthcare and it became one of the main pillars of an entire specialty. however, the main advantage of the procedure remains one of its main drawbacks, i.e., the need to push a flexible endoscope from the anal orifice to the caecum with all that entails in terms of discomfort and complications for patients. we are living in an era of change, both in terms of fast pace developments in precision/personalised medicine, as well as technological delivery since the dawn of the millennium. we are living the "belle epoque" of start-ups, digitalisation and resurge of ai; furthermore, there is a lot of interest in ways to eliminate human impact on the environment and reduce the mistakes in healthcare services delivery together with some added efforts to "equalise" healthcare provision across the globe. what is perhaps most relevant at the moment is the fact that novel infectious agents, e.g., sars-cov-2, are disrupting our normal living conditions, the global economy and they are posing a major threat to human life either directly or indirectly through an immense strain placed on the shrivelling healthcare resources, and this calls for implementation of measures such as "social and medical distancing". in this environment, the interface between machines/robots and humans, present at the receiving end of services, is becoming smoother and their resistance of acceptance is curbed. in this era, the use of robotic adjuncts or full robotization/automatization of the procedure are major contributions to explore, such as for guaranteeing the "social and medical distancing". we should not forget though that there is a significant majority of workers, including healthcare professionals, that would like to see quotas to protect the human delivery of care in the face of increasing automation and the risk this could pose to jobs. indeed, tasks no longer needed in this area could be offset by an upswing in other areas, such as more quality time spent with patients in national health systems. nevertheless, it is envisaged that a robotic colonoscopy will allow enhanced precision and visualization enabling, therefore making possible therapeutic procedures that were otherwise considered too challenging without a robotic instrument. however, complaints of system malfunctions and reports of patient injuries may appear with widespread use and that could lead to lawsuits against stakeholders, which include the device manufacturer, the hospital or institutions and their staff, as well as the surgeons and their associates. each of these stakeholders involved in robotic surgery is responsible to uphold the highest level of training and care available to help patients achieve the desired outcomes. in addition, the senior authors would like to thank the beijing advanced innovation center for intelligent robots and systems (baicirs-beijing, china) and its robocaps team for the scientific collaboration in the field of robotic capsule colonoscopy. finally, the authors thank andrea aliperta for his contribution in preparing the original graphic of figure 1 and alexander breschi for the english proofreading. the authors declare no conflict of interest. global cancer statistics 2018: globocan estimates of incidence and mortality worldwide for 36 cancers in 185 countries global burden of colorectal cancer: 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and analysis of endoscopic images and videos: a survey implicit domain adaptation with conditional generative adversarial networks for depth prediction in endoscopy towards automated colonoscopy diagnosis: binary polyp size estimation via unsupervised depth learning this article is an open access article distributed under the terms and conditions of the creative commons attribution (cc by) license the authors would also like to thank the co-pis of the h2020 european endoo project. key: cord-333520-v2sb90rc authors: gardin, chiara; ferroni, letizia; chachques, juan carlos; zavan, barbara title: could mesenchymal stem cell-derived exosomes be a therapeutic option for critically ill covid-19 patients? date: 2020-08-26 journal: j clin med doi: 10.3390/jcm9092762 sha: doc_id: 333520 cord_uid: v2sb90rc coronavirus disease 2019 (covid-19) is a pandemic viral disease originated in wuhan, china, in december 2019, caused by the severe acute respiratory syndrome coronavirus 2 (sars-cov-2). the severe form of the disease is often associated with acute respiratory distress syndrome (ards), and most critically ill patients require mechanical ventilation and support in intensive care units. a significant portion of covid-19 patients also develop complications of the cardiovascular system, primarily acute myocardial injury, arrhythmia, or heart failure. to date, no specific antiviral therapy is available for patients with sars-cov-2 infection. exosomes derived from mesenchymal stem cells (mscs) are being explored for the management of a number of diseases that currently have limited or no therapeutic options, thanks to their anti-inflammatory, immunomodulatory, and pro-angiogenic properties. here, we briefly introduce the pathogenesis of sars-cov-2 and its implications in the heart and lungs. next, we describe some of the most significant clinical evidence of the successful use of msc-derived exosomes in animal models of lung and heart injuries, which might strengthen our hypothesis in terms of their utility for also treating critically ill covid-19 patients. the novel coronavirus 2019 (2019-ncov) has reached pandemic proportions across the world after originating in wuhan, the capital of china's hubei province, in december 2019 [1, 2] . initially called 2019-ncov, the world health organization (who) subsequently adopted the official name severe acute respiratory syndrome coronavirus 2 (sars-cov-2) for indicating the virus, and the term coronavirus disease 2019 (covid-19) for identifying the virus-associated disease [3] . as of 7 may 2020, the who has reported almost 3,634,172 confirmed cases of covid-19 with 251,446 confirmed deaths in 215 countries/areas/territories worldwide [4] . the clinical spectrum of covid-19 is highly variable-in addition to mild, severe, and critical forms, asymptomatic or paucisymptomatic infections have been described as well [5, 6] . milder clinical conditions are commonly characterized by fever, dry cough, myalgia or fatigue, headache, and mild pneumonia, whereas the severe form of the disease is associated with dyspnea, acute respiratory distress syndrome (ards), and hypoxemia (low level of oxygen in arterial blood) [1, 7] . the most critical cases experience respiratory failure requiring mechanical ventilation and support in the intensive care mice was correlated with myocardial disfunction [46] . considering the similarity with sars-cov, it has been speculated that sars-cov-2 infection might also down-regulate ace2 expression in lung and heart, thus leading to the pathological processes of lung and cardiac injuries [27] . to date, no experimental or clinical data have evidenced that using acei/arb therapy makes patients more susceptible to the virus. therefore, several leading cardiovascular societies have strongly urged to not discontinue intake of ras inhibitors in the event the patient develops covid-19 [47, 48] . j. clin. med. 2020, 9, x for peer review 4 of 28 regulated after sars-cov infection in mice, and this was accompanied by increased pulmonary vascular permeability and pulmonary edema [45] . similarly, cardiac ace2 down-regulation following sars-cov infection in mice was correlated with myocardial disfunction [46] . considering the similarity with sars-cov, it has been speculated that sars-cov-2 infection might also downregulate ace2 expression in lung and heart, thus leading to the pathological processes of lung and cardiac injuries [27] . to date, no experimental or clinical data have evidenced that using acei/arb therapy makes patients more susceptible to the virus. therefore, several leading cardiovascular societies have strongly urged to not discontinue intake of ras inhibitors in the event the patient develops covid-19 [47, 48] . , angiotensin (1-7); at1r, ang ii type 1 receptor; aceis, angiotensin-converting enzyme inhibitors; arbs, angiotensin receptor blockers; ards, acute respiratory distress syndrome. green arrows indicate that aceis/arbs increase ace2 levels in the heart, therefore increasing the susceptibility of cardiac cells to sars-cov-2 infection [42, 43] . blue dotted hammerhead indicates the hypothetical effect of sars-cov-2 on ace2 expression in lung and heart, which is based on the reported effect of sars-cov in the same body districts [45, 46] . mscs are thought to prevent or reduce the cytokine storm in covid-19 patients, owing to their powerful anti-inflammatory and immunomodulatory functions [8] . mscs exert these effects by directly interacting with different cells of innate and adaptive immunity, including t cells, b cells, dendritic cells (dcs), macrophages, and natural killer cells, and by indirectly releasing many types of inflammatory mediators by paracrine secretion [49] [50] [51] [52] . many studies have described a differential regulation by mscs on the different t cell subsets [53] [54] [55] [56] [57] . mscs inhibit effector t (teff) cell proliferation induced by mitogens or alloantigens by causing cell cycle arrest at the g1 phase [53, 58] . another explanation for this immunosuppressive capacity is the loss of cd25, the alpha-chain of the il-2 receptor, which is cleaved from the activated t cell surface by msc-secreted matrix metalloproteinases [59] . this leads to blockage of the il-2 cytokine signaling pathway required for t cells activation, expansion, and differentiation. interestingly, such t cell-suppressing properties of mscs seem to require the presence of inflammatory cytokines in the microenvironment, which provoke the production of several t cell-attracting chemokines and inducible nitric oxide synthase figure 1 . schematic diagram showing the renin-angiotensin system (ras) cascade and the effects on the cardiovascular system and lung. ace1, angiotensin-converting enzyme 1; ace2, angiotensin-converting enzyme 2; ang i, angiotensin i; ang ii, angiotensin ii; ang (1-7), angiotensin (1-7); at1r, ang ii type 1 receptor; aceis, angiotensin-converting enzyme inhibitors; arbs, angiotensin receptor blockers; ards, acute respiratory distress syndrome. green arrows indicate that aceis/arbs increase ace2 levels in the heart, therefore increasing the susceptibility of cardiac cells to sars-cov-2 infection [42, 43] . blue dotted hammerhead indicates the hypothetical effect of sars-cov-2 on ace2 expression in lung and heart, which is based on the reported effect of sars-cov in the same body districts [45, 46] . mscs are thought to prevent or reduce the cytokine storm in covid-19 patients, owing to their powerful anti-inflammatory and immunomodulatory functions [8] . mscs exert these effects by directly interacting with different cells of innate and adaptive immunity, including t cells, b cells, dendritic cells (dcs), macrophages, and natural killer cells, and by indirectly releasing many types of inflammatory mediators by paracrine secretion [49] [50] [51] [52] . many studies have described a differential regulation by mscs on the different t cell subsets [53] [54] [55] [56] [57] . mscs inhibit effector t (teff) cell proliferation induced by mitogens or alloantigens by causing cell cycle arrest at the g1 phase [53, 58] . another explanation for this immunosuppressive capacity is the loss of cd25, the alpha-chain of the il-2 receptor, which is cleaved from the activated t cell surface by msc-secreted matrix metalloproteinases [59] . this leads to blockage of the il-2 cytokine signaling pathway required for t cells activation, expansion, and differentiation. interestingly, such t cell-suppressing properties of mscs seem to require the presence of inflammatory cytokines in the microenvironment, which provoke the production of several t cell-attracting chemokines and inducible nitric oxide synthase (inos) from mscs, so that t cells migrate into proximity of these cells [60] . at the same time, mscs have been shown to induce the survival and expansion of regulatory t (treg) cells, a subset of t cells involved in the suppression of proliferation and cytokine production by teff cells [56] . therefore, treg cells foster the msc-mediated immunosuppressive effect. in addition to directly interacting with t cells, mscs also modulate the adaptive immune response by acting on antigen-presenting cells (apcs), such as dcs, monocytes, and macrophages, by shifting them to regulatory phenotypes characterized by t cell-suppressive properties [61, 62] . the spectrum of regulatory factors secreted by mscs is collectively defined as the msc secretome, and include a complex array of soluble molecules, such as anti-inflammatory cytokines, angiogenic growth factors, antimicrobial peptides, and lipid mediators. a growing body of evidence nowadays suggests that some of these molecules are packaged into cell-secreted vesicles, known as extracellular vesicles (evs) [63] [64] [65] . besides apoptotic bodies, the two main types of evs released by mscs include exosomes and microvesicles (mvs). exosomes (30-100 nm) are derived by fusion of multi-vesicular bodies with the plasma membrane, whereas mvs (100-1000 nm) are formed by cellular membrane budding, and contain cellular cytoplasm. all these evs are released into the extracellular microenvironment, where they exert biological effects in a paracrine and endocrine manner, similarly to the soluble components. for this reason, a broader definition of mscs secretome encompasses the entire spectrum of bioactive factors secreted by mscs, which consists of both the soluble and the extravesicular elements. it has now been demonstrated that mscs are also able to transfer functional mitochondria or mitochondrial dna (mtdna) to target cells, thus rescuing aerobic respiration in cells with non-healthy mitochondria or regulating t cell functions [66, 67] . following systemic injection, some mscs accumulate in the lung, where they release these soluble mediators, potentially recovering the pulmonary microenvironment, protecting alveolar epithelial cells, and counteracting pulmonary fibrosis, thus resulting in a final improvement of lung function [8] . moreover, distant injured organs, such as the cardiovascular system, can also benefit from them, by virtue of the secretory abilities of these cells. to date, two studies have investigated the employment of mscs in severely affected sars-cov-2 patients, with both reporting remarkable reversal of symptoms within a few days [10, 68] . in one of these works, the levels of biochemical indicators of liver and myocardium damage (aspartic aminotransferase, creatine kinase activity, and myoglobin) returned to reference levels 4 days after msc treatment [10] . the authors demonstrated that the cells expressed high levels of anti-inflammatory and angiogenic factors, such as transforming growth factor-beta (tgf-β), hepatocyte growth factor (hgf), leukemia inhibitory factor (lif), fibroblast growth factor (fgf), vascular endothelial growth factor (vegf), epidermal growth factor (egf), brain-derived neurotrophic factor (bdnf), and nerve growth factor (ngf), further demonstrating their potent immunomodulatory abilities. it has been reported that mscs are generally resistant to viral infections compared to their differentiated progeny, probably due to intrinsic expression of ifn-stimulated genes (isgs) [69] . among these genes, those coding for proteins of the interferon-induced transmembrane (ifitm) family prevent viruses from traversing the lipid bilayer of the cell and accessing the cytoplasm, thus impairing viral infection [70] . these antiviral proteins limit infection in cultured cells by many viruses, including sars-cov, dengue virus, ebola virus, influenza a virus, and west nile virus. however, some studies have reported that human mscs are permissive to other viruses, for example avian influenza viruses h1n1 and h9n5 and respiratory syncytial virus (rsv), losing vitality and compromising their immunomodulatory activities [71, 72] . in the case of sars-cov-2, the advantage in using mscs seems to be additionally related to the absence of ace2 receptors on the cell surface, which precludes their recognition by the virus. notably, in the study of leng and coworkers, the cells remained negative for ace2 also after transplantation in infected patients [10] . although mscs seem to be refractory to sars-cov-2 infection, in order to bypass the impact of viruses on mscs, an interesting therapeutic strategy could consider the use of the msc secretome. among the bioactive factors released by mscs, evs, exosomes in particular, have gained remarkable interest in recent years because they enable more efficient communication and targeting than soluble molecules [73] . evs, by virtue of their lipid bilayer membrane, better protect their molecular cargo of proteins and genetic material from environmental degradation (i.e., from trypsin or nuclease digestion) when compared to soluble molecules. encapsulation within evs may also facilitate delivery and targeting of these bioactive factors to distant recipient cells, mediated by binding of the ev surface proteins to cells that express appropriate receptors [74] . msc-derived exosomes offer several advantages over traditional cell-based therapies. first, exosomes are considered safer than cells, because they are biocompatible, non-immunogenic, and lack the potential for endogenous tumors and emboli formation [75] . in addition, exosomes are physiologically more stable than cells, because their multiple membrane adhesion proteins allow for efficient binding in the target tissues during transplantation. thanks to their resistant membrane, exosomes maintain their integrity during freezing and thawing procedures, making long-term storage without biological degradation possible [76] . in this context, a process has recently been proposed that combines ultrafiltration and lyophilization and is able to convert msc secretome into a freeze-dried, ready-to-use powder [77, 78] . the same research group also suggested the possibility to administer evs by inhalation in the treatment of respiratory diseases [79] . this route of administration would benefit from lower invasiveness and pain, faster onset of action, and use of lower doses to achieve the same therapeutic effect when compared to oral or injection therapies. in this regard, a pilot clinical trial (nct04276987) will be conducted in china for exploring the safety and efficiency of aerosol inhalation of msc-derived exosomes in comparison to conventional treatment in 30 severe patients with covid-19. another advantage of msc-derived exosomes over whole-cell therapy is that, to improve their therapeutic potential, exosomes could potentially be modified with various types of cargos, including mrna, microrna (mirna), and proteins, tailored to the disease process of interest [73] . in one pioneering work, exosomes incorporating the s protein have been explored as a novel vaccine approach against sars-cov infections [80] . the immunogenicity and efficacy of the s-containing exosomes were tested in mice, where they induced neutralizing antibody titers. finally, from an economical point of view, msc-derived exosome therapy might enable development of cheaper treatments other than the expansion and maintenance of individualized clonal cell populations [73] . this aspect is particularly important when a global pandemic has to be managed, as in the case of covid-19. in section 4, we provide an overview of the currently available evidence on the effects of msc-derived exosomes in pre-clinical models of lung and heart injuries, which are the body districts most affected by sars-cov-2. diabetes mellitus (dm) represents the most common inflammatory and chronic metabolic disorder worldwide, and continues to increase in number and significance-it is estimated that there will be 693 million persons with dm by 2045 [81] . type 2 diabetes mellitus (t2dm) accounts for 90-95% of all cases of diabetes and results from a progressive defect in insulin production and insensitive response of the body to insulin [82] . accumulating evidence shows that such a state of insulin resistance (ir) is closely related with obesity [83] . obesity, mainly visceral adiposity, is, indeed, one of the most important comorbidities in diabetic patients. people with diabetes have a higher overall risk of infections that result from compromised innate cell-mediated immunity; impaired phagocytosis by neutrophils, macrophages, and monocytes; and impaired neutrophil chemotaxis and bactericidal activity [84] . regarding covid-19, it is currently unknown whether patients with diabetes have a higher susceptibility to the virus; nonetheless, there is evidence of higher risk for both infection and disease severity [85] . as stated above, there is growing interest in the use of msc-derived evs as a therapeutic tool for the management of several diseases. however, because ev cargo usually reflects parent cell characteristics, and these are influenced by the metabolic state of source cells, it is reasonable to consider the risks associated with the employment of msc-derived evs from patients with coexisting metabolic disorders such as t2dm. in effect, clinical studies have found differences in the number and composition of evs isolated from the adipose tissue of obese patients and from animal models of obesity [86] [87] [88] [89] . for example, msc evs isolated from a swine model of metabolic disorder were found to be enriched with mrnas associated with inflammation, such as those coding for the integrin family proteins, or proteins of the fgf signaling [87] . these msc-derived evs also showed a distinctive mirnas cargo, being enriched in mirna-targeting genes involved in the development of metabolic disease and its complications, including diabetes, obesity, and insulin signaling [88] . apart from influencing the mrna and mirna content, metabolic disorder also alters packaging of proteins into porcine msc-derived evs, promoting the inclusion of pro-inflammatory proteins, such as those involved in acute inflammatory response, cytokine production, and leukocyte transendothelial migration [86] . the limitations of these works reside in the small sample size and short duration of metabolic disease compared to the human condition; therefore, further studies would be needed to draw clear conclusions. however, in humans also, analysis of adipose tissue-derived evs demonstrated that obesity alters their cargo of mrnas, mirnas, and proteins [90] . in particular, the differentially expressed mirnas contained in the isolated evs stimulated up-regulation of wnt/β-catenin and tgf-β signaling pathways, which are related to inflammation, into a549 lung epithelial cells. overall, these observations suggest that diabetes and metabolic disorders might alter the msc-derived ev cargo, which in turn might compromise their anti-inflammatory and immunodulatory potential both in the endogenous microenvironment and after autologous transplantation. acute lung injury (ali) and ards are major causes of respiratory failure in critically ill ventilated patients, with an estimated 60-day mortality rate of 32% [91] . ards is also one of the most common complications in severely affected covid-19 patients. the term ards is often used interchangeably with ali; nevertheless, ards should be reserved for the most severe form of the disease [92] . bacterial or viral infections are the most common causes of ali and ards; however, they can also be initiated by aspiration of gastric contents, toxic inhalation, lung contusion, or trauma [92] . the acute phase (the first 1-6 days) of the diseases is characterized by injury to both the pulmonary endothelium and the alveolar epithelium, the two barriers forming the alveolar-capillary barrier. in healthy lung microvessels, the pulmonary endothelium is maintained by vascular endothelial cadherin (ve-cadherin), an endothelial-specific adherens junction protein, whereas the alveolar epithelial barrier has e-cadherin junctions and is substantially less permeable than the endothelial counterpart [93, 94] . during lung injury, ve-cadherin bonds are destabilized by increased expression of thrombin, tnf-α, vegf, and signals from leukocytes. at the same time, e-cadherin epithelial junctions are disrupted by neutrophil migration, which causes injury, apoptosis, and membrane denudation. this ultimately results in increased epithelial permeability, leading to accumulation of protein-rich edema fluid in the alveoli, and in turn to an impairment in gas exchange and to hypoxemia [95] . dysregulated immune activation has also been implicated in the pathogenesis of ali/ards. in the air space, macrophages release pro-inflammatory cytokines and chemokines, which act locally to stimulate chemotaxis and activate neutrophils. activation of neutrophils leads to the release of numerous cytotoxic products, such as reactive oxygen species, cationic peptides, eicosanoids, and proteolytic enzymes, which may further damage the alveolar epithelium [96] . resolution of ali/ards aims at removing alveolar edema fluid, repairing the epithelial and endothelial barriers, and removing inflammatory cells and exudate from the air spaces [95] . to date, management of ali/ards includes lung protective ventilation, prone positioning, neuromuscular blockade, and extracorporeal membrane oxygenation. mechanical ventilation represents the mainstay treatment in ali/ards, and consists in the application of positive-end expiratory pressure for optimizing arterial oxygenation. it has been evidenced that ventilation with a low tidal volume (6 ml/kg) gives better results when compared to traditional tidal volume (12 ml/kg) [97] . indeed, the use of lower tidal volumes during ventilation may reduce injurious lung stretch and the release of inflammatory mediators. prone positioning enhances arterial oxygenation by improving alveolar ventilation/perfusion matching. nevertheless, this treatment should be used with caution and should be reserved for patients with critical hypoxemia, since it does not improve survival or decrease the duration of lung ventilation. all these therapeutic options remain primarily supportive; on the other hand, alternative treatments with glucocorticoids, surfactants, inhaled nitric oxide, antioxidants, protease inhibitors, or other anti-inflammatory agents had proven unsuccessful in reducing mortality or improving ali/ards outcomes [96] . in terms of promising novel strategies, msc-based approaches have been explored for the management of ali/ards. the benefit of msc therapy appears to be related to a decrease in pro-inflammatory cytokines and to an increase in anti-inflammatory cytokines, particularly il-10 [98] . mscs release prostaglandin e 2 , which in turn stimulates secretion of il-10 by monocytes and alveolar macrophages [99] . moreover, administration of mscs seems to be effective in normalizing lung endothelial and epithelial permeability to protein, as well as in reducing pulmonary edema and increasing the rate of alveolar fluid clearance [100] . recently, msc-derived exosomes have been demonstrated to have comparable and even greater effects than cells themselves in improving inflammation and injury in a variety of pre-clinical lung disease models, including ali/ards (table 1) . for the completeness of information, we have to specify that some of these works take into account the entire spectrum of evs that, in addition to exosomes, also includes mvs. this is because as of yet there are no standardized methods for isolation, quantification, and characterization of evs, or for discriminating mvs and exosomes. consequently, in the majority of these pre-clinical studies, evs, exosomes, and mvs are collectively referred to as evs. msc-derived evs have been proven to be beneficial in both bacteria-and virus-induced ali/ards. a large number of studies have employed an endotoxin-mediated in vivo model to investigate the effects of msc-derived evs for ali/ards. in one of the first works, ali was induced in c57bl/mice using the intratracheal (it) instillation of endotoxin (4 mg/kg) from escherichia coli (e. coli) [101] . mvs were isolated from the conditioned medium of human bone marrow-derived mscs with two sequential ultracentrifugations at 100,000× g for 1 h. then, 30 ul of mvs, corresponding to the vesicles released by 3 × 10 6 mscs, were administrated intratracheally or intravenously in mice. after 48 h, msc-derived mvs reduced lung inflammation and reduced edema to the same levels as mscs themselves, which were used as a positive control. furthermore, mvs also decreased the influx of neutrophils and mip-2 levels in the alveolar fluid, indicating a reduction in inflammation. surprisingly, the therapeutic effects of the mvs were comparable, regardless of route of administration. the authors suggested that the mechanism underlying the therapeutic effect of mvs might be in part mediated by the transfer of keratinocyte growth factor (kgf) mrna into the injured alveolar epithelium, with subsequent expression of the protein. kgf is an epithelial-specific growth factor released from mscs, which has been shown to reduce lung edema and inflammation in various ali models [102] [103] [104] . in the same study, the effect of mvs was additionally evaluated in raw 264.7 cells, a mouse macrophage cell line. treatment with 30 µl of msc-derived mvs to endotoxin-stimulated raw 264.7 cells reduced the levels of tnf-α and mip-2, and concomitantly increased the production of the anti-inflammatory cytokine il-10 at 6, 12, and 24 h compared with endotoxin-stimulated mouse macrophages [101] . in the work of tang and colleagues, ali was induced in c57bl/mice by the instillation of lipopolysaccharide (lps) from pseudomonas aeruginosa at 4 mg/kg intratracheally [105] . mvs released from human bone marrow mscs were isolated by two sequential ultracentrifugations at 100,000× g for 1 h, then intratracheally administrated in endotoxin-injured mice. it administration of msc-derived mvs improved the lung inflammation induced by lps in mice, including the influx of white blood cells and neutrophils, and mip-2 secretion. in that study, the authors found that the transfer of angiopoietin-1 (ang-1) mrna by mvs was essential for the reduction of inflammation and the restoration of alveolar-capillary barrier. ang-1 plays a key role in vascular stabilization, since it reduces endothelial permeability and suppresses leukocyte-endothelium interactions [106] . furthermore, msc-derived mvs showed immunomodulatory effects on raw 264.7 cells in vitro by inhibiting tnf-α mrna production and promoting the mrna levels of il-10 after 3 h [105] . collectively, these two studies suggest that the beneficial immunomodulatory effect of msc-derived mvs in ali is strongly dependent on kgf and ang-1 mrna transfer into injured endothelial cells. it has been demonstrated that, apart from mrnas, the therapeutic effect of evs is also mediated by the transfer of functional mitochondria to target cells. mscs have been reported to naturally transfer mitochondria to recipient cells through different mechanisms-incorporated within evs, via cell-to-cell contact through tunneling nanotubes, or through direct release of naked mitochondria into the extracellular microenvironment [107, 108] . in the work of phinney and colleagues, transmission electron microscopy images evidenced structures consistent with the morphology of mitochondria inside mvs over 100 nm in size, previously isolated from the conditioned medium of human mscs after centrifugation at 10,000× g for 1 h [109] . the authors found that these mitochondria were loaded in the cytoplasm into lc3-containing mvs, which migrated towards the cell periphery and were incorporated into outward budding blebs in the plasma membrane. the msc-derived mvs contained functionally active mitochondria that were taken up by macrophages and resulted in improved bioenergetics after oxidative stress increment. in particular, the transfer of human msc-derived mitochondria involved fusion with mitochondria inside macrophages, suggesting that the mitochondrial membrane was not collapsed. the same study also confirmed that mitochondria were not packaged within exosomes; rather, exosomes were able to deliver mtdna, which in mammals has an average size under 100 nm [110] . in the context of lung injury, the group of morrison and colleagues revealed that msc-derived evs protected mice against lps-induced ali by altering alveolar macrophage (am) polarization from the pro-inflammatory m1 phenotype towards the m2 anti-inflammatory phenotype [111] . in detail, evs were obtained from human bone marrow mscs after ultracentrifugation at 100,000× g for 2 h. these were used for pre-treating am, which were then intranasally administrated to lps-injured mice. the msc-derived evs increased phagocytic activity by macrophages and reduced their secretion of tnf-α and il-8, two major pro-inflammatory cytokines related to ards severity [112, 113] . the transfer of functional mitochondria contained in evs, associated with the promotion of oxidative phosphorylation, was supposed to be the mechanism responsible for the observed effects in macrophages [111] . the work of monsel and colleagues was the first to evaluate the effect of msc-derived mvs in an infectious ali model [114] . indeed, the authors instilled live e. coli bacteria into the trachea; then, they intravenously administrated 90 ul of mvs, corresponding to the vesicles released by 9 × 10 6 human bone marrow mscs. mv injection improved survival and reduced the bacterial load, as well as the influx of white blood cells, neutrophils, and mip-2 levels, in the injured alveolus of c57bl/mice. the authors reported that the effect was in part mediated by kgf mrna shuttled by the vesicles into target cells, as described in their previous study [101] . in addition to testing in mice, the effect of msc-derived mvs was also investigated in human monocytes and at2 cells. mv treatment increased the percentage of phagocytosis of human monocytes against e. coli bacteria, thus reducing the bacterial count, and decreased tnf-α secretion. furthermore, mvs showed a beneficial effect on injured human at2 cell metabolism through the restoration of intracellular atp levels to control levels. in these primary in vitro cultures, the uptake of mvs was mediated by cd44, which was essential for the observed therapeutic effects. cd44 is the hyaluronic acid receptor expressed in almost every cell type including mscs [51, 115] . the results of this work suggested that mvs, similarly to their parent cells, act through different mechanisms on the basis of anti-inflammatory, anti-microbial, and metabolomic effects. msc-derived evs have also shown reparative properties on microvascular endothelial and epithelial cells, which are often severely injured in the lung during ali, and are associated with increased mortality in ards patients. hu and coworkers investigated the effects of mvs isolated from human bone marrow mscs on human lung microvascular endothelial cells (hlmvecs) in vitro [116] . the cells were injured by cytomix, a mixture of the most biologically active cytokines found in ali pulmonary edema fluid (il-1β, tnf-α, and ifn-γ at 50 ng/ml), and simultaneously exposed to increasing doses (30 or 60 ul) of mvs using a transwell co-culture system [117] . administration of msc-derived mvs restored protein permeability of hlmvecs by preventing the reorganization of cytoskeleton protein f-actin into "actin stress fibers" and the loss of tight and adherens junction proteins (zonula occludens-1 and ve-cadherin, respectively) following inflammatory injury. the internalization of mvs via cluster of differentiation (cd)44 receptor, as well as the subsequent transfer of ang-1 mrna into injured hlmvecs, were required for the observed therapeutic effects. the study of khatri and colleagues is interesting because ards was induced in pigs after infection with a mixed swine (h3n2, h1n1) and avian (h9n5, h7n2) influenza viruses (swiv) [118] . pigs are often used as large animal pre-clinical models for several human diseases, including respiratory diseases, due to their close similarity in anatomy, physiology, and immunology to humans [119] . in addition, influenza virus pathogenesis and clinical signs are similar to those observed in humans. in that work, evs (80 µg/kg) isolated from swine bone marrow mscs with two ultracentrifugation steps at 25,000 rpm for 70 min were intratracheally administrated in pigs 12 h after swiv inoculation. msc-derived evs were found to inhibit influenza virus replication and shedding in pigs 3 days post-infection. as in other studies, evs also modulated inflammatory cytokine and chemokine production in the lungs, as demonstrated by reduction in tnf-α and cxcl10 protein levels, and increase in il-10 protein levels. unfortunately, there are not yet pre-clinical data on the effects of msc-derived ev administration in models of coronavirus respiratory infection, mostly due to the lack of an established animal model [120] . from the studies discussed above, it emerged that the rationale for using msc-derived exosomes, mvs, or evs in ali/ards is based on several processes, many of which are shared with those identified in the parent mscs. these include immunomodulation and anti-inflammatory properties on host tissue, reduction of the permeability of alveolar epithelium and endothelium, improvement of alveolar fluid clearance, enhancement of macrophage phagocytosis, and tissue repair through direct mitochondrial transfer with host cells (figure 2 ). reduction in tnf-α and cxcl10 protein levels, and increase in il-10 protein levels 3 days post-infection. [118] potential therapy in the management of other lung diseases, such as bronchopulmonary dysplasia, pulmonary arterial hypertension, idiopathic pulmonary fibrosis, and asthma, which have been recently revised in worthington [121] and behnke [122] . moreover, in these lung diseases, the most common effects of msc-derived evs were decreased inflammation and restoration of the lung architecture, achieved through the reduction of fibrosis and increase of vascularization and alveolarization. [101, 105, 114, 118] , reduction of the permeability of alveolar epithelium and endothelium [116] , improvement of alveolar fluid clearance [114] , enhancement of macrophage phagocytosis [111] , and tissue repair through direct mitochondrial transfer with host cells [111] . acute myocardial injury has been described as the most common cardiovascular complication in covid-19 patients [13] . myocardial injury is defined as an elevation in serum levels of highsensitive cardiac troponin (ctn) above the 99th percentile upper reference limit, although over the years it has also been identified through an increase in different cardiac enzymes and/or electrocardiographic abnormalities [123] . the injury is considered acute if there is a dynamic rise and/or fall of ctn values. when acute myocardial injury is caused by myocardial ischemia, it is designated as acute myocardial infarction (ami). on the contrary, myocardial injury not related to ischemic events may arise secondary to many cardiac conditions, such as myocarditis [123] . [101, 105, 114, 118] , reduction of the permeability of alveolar epithelium and endothelium [116] , improvement of alveolar fluid clearance [114] , enhancement of macrophage phagocytosis [111] , and tissue repair through direct mitochondrial transfer with host cells [111] . apart from ali/ards, there have been several investigations using msc-derived evs as a potential therapy in the management of other lung diseases, such as bronchopulmonary dysplasia, pulmonary arterial hypertension, idiopathic pulmonary fibrosis, and asthma, which have been recently revised in worthington [121] and behnke [122] . moreover, in these lung diseases, the most common effects of msc-derived evs were decreased inflammation and restoration of the lung architecture, achieved through the reduction of fibrosis and increase of vascularization and alveolarization. acute myocardial injury has been described as the most common cardiovascular complication in covid-19 patients [13] . myocardial injury is defined as an elevation in serum levels of high-sensitive cardiac troponin (ctn) above the 99th percentile upper reference limit, although over the years it has also been identified through an increase in different cardiac enzymes and/or electrocardiographic abnormalities [123] . the injury is considered acute if there is a dynamic rise and/or fall of ctn values. when acute myocardial injury is caused by myocardial ischemia, it is designated as acute myocardial infarction (ami). on the contrary, myocardial injury not related to ischemic events may arise secondary to many cardiac conditions, such as myocarditis [123] . analyzing several reports from china, a considerable proportion of patients (12-27.8%) presented elevated ctn levels, and most of them required icus and showed higher in-hospital mortality [7, [124] [125] [126] . the mechanisms of myocardial injury are not well established but likely involve direct or indirect processes and/or their combination (figure 3) . myocardial infection by sars-cov-2 resulting in cardiomyocyte death and inflammation has been proposed as a possible direct mechanism, although, to date, there are no data demonstrating the presence of sars-cov-2 within myocardial tissue [127] . nevertheless, a previous autopsy study in patients who died from sars identified the viral rna in 35% of the post-mortem human heart samples, providing evidence for direct myocardial injury by the virus [46] . in addition, patients carrying sars-cov in their hearts died considerably earlier, suggesting that viral infiltration in the myocardium was associated with a more aggressive course of illness. systemic inflammatory response or respiratory failure and hypoxemia can represent indirect mechanisms leading to increased cardiac stress and myocardial inflammation [127, 128] . in a couple of studies, biopsies taken from heart tissue of covid-19 patients evidenced mononuclear inflammatory infiltrates, mainly associated with regions of cardiomyocyte necrosis, which identifies myocarditis according to dallas criteria [129] [130] [131] . nevertheless, acute lymphocyte infiltrates were not observed in the myocardium of sars-cov-2-infected patient autopsy. died considerably earlier, suggesting that viral infiltration in the myocardium was associated with a more aggressive course of illness. systemic inflammatory response or respiratory failure and hypoxemia can represent indirect mechanisms leading to increased cardiac stress and myocardial inflammation [127, 128] . in a couple of studies, biopsies taken from heart tissue of covid-19 patients evidenced mononuclear inflammatory infiltrates, mainly associated with regions of cardiomyocyte necrosis, which identifies myocarditis according to dallas criteria [129] [130] [131] . nevertheless, acute lymphocyte infiltrates were not observed in the myocardium of sars-cov-2-infected patient autopsy. other aspects of covid-19 in cardiac involvement include blood pressure abnormalities and arrhythmias, ranging from tachycardia and bradycardia to asystole [127] . very recently, it has also been suggested that there is a link between sars-cov-2 infection and kawasaki disease (kd), especially in pediatric patients [132] . although kd is a disease of unknown etiology, infections are considered to be one of the predisposing factors [133] . the disease predominantly affects children under 5 years of age and causes inflammation in the walls of medium-sized arteries, primarily the coronary arteries, those that supply blood to the heart muscle. consequences of ami are loss of cardiomyocytes and adverse remodeling of the extracellular matrix, which contribute to the reduction of pumping of the heart and further heart failure. nowadays, the best therapeutic strategy for reducing ami is timely and effective myocardial other aspects of covid-19 in cardiac involvement include blood pressure abnormalities and arrhythmias, ranging from tachycardia and bradycardia to asystole [127] . very recently, it has also been suggested that there is a link between sars-cov-2 infection and kawasaki disease (kd), especially in pediatric patients [132] . although kd is a disease of unknown etiology, infections are considered to be one of the predisposing factors [133] . the disease predominantly affects children under 5 years of age and causes inflammation in the walls of medium-sized arteries, primarily the coronary arteries, those that supply blood to the heart muscle. consequences of ami are loss of cardiomyocytes and adverse remodeling of the extracellular matrix, which contribute to the reduction of pumping of the heart and further heart failure. nowadays, the best therapeutic strategy for reducing ami is timely and effective myocardial reperfusion. however, this treatment induces oxidative stress and inflammation, thus leading to further cardiomyocyte death, myocardial remodeling, and decreased cardiac function, a phenomenon known as myocardial reperfusion injury [134] . over the last years, management of ami using stem cell therapy was found to prevent myocardial cell apoptosis, promote local neoangiogenesis, and reduce the local inflammatory response [135] [136] [137] . similarly to what was described above for lung injuries, the beneficial effect of stem cells seems to be largely attributable to the secreted evs. since the first description of the therapeutic potential of msc-derived exosomes in a mouse model of myocardial ischemia/reperfusion (i/r) injury in 2010, several studies have subsequently reported cardio-protective effects of msc-derived evs in ami animal models (table 2 ) [138] . in one of these works, a single intravenous (iv) injection of msc-derived exosomes in a mouse ami model led to decreased infarct size, enhanced nicotinamide adenine dinucleotide (reduced form) (nadh) and atp levels, and reduced oxidative stress, which are hallmarks of reperfusion injury [139] . all these events seemed to be associated with the exosome-mediated activation of the pro-survival phosphoinositide 3-kinase/protein kinase b (pi3k/akt) signaling pathway, which resulted in an enhancement of myocardial viability and prevented adverse remodeling after myocardial i/r injury. importantly, intact but not lysed exosomes were responsible for the improved cardiac function after ami induction. another important mechanism by which msc-derived evs contribute to ischemic myocardial repair is through stimulation of neovascularization, as shown in the work of bian and colleagues [140] . neovascularization refers to processes, such as vasculogenesis, angiogenesis, and arteriogenesis, that are associated with migration and proliferation of endothelial cells. in line with these findings, ma and coworkers also demonstrated that exosomes isolated from akt-transfected mscs accelerated angiogenesis in a rat myocardial infarction model [141] . the authors suggested that platelet-derived growth factor d (pdgf-d), which was enriched in msc-derived vesicles, was mainly responsible for the akt exosome-mediated improvement of myocardial repair. a more recent study by xuan and colleagues identified notch1 as a potent modulator of angiogenesis and cardiomyocyte proliferation into ischemic mice hearts following coronary heart ligation [142] . the role of notch1 signaling in inducing cardiac angiogenesis during ischemia and enhancing survival of cardiac cells is well established [143] . the injection of msc-derived evs over-expressing notch1 intracellular domain (nicd) in ischemic myocardium led to decreased infarct size, improved cardiac function, and increased arteriole density in the peri-infarct area, 1 month after ami [142] . moreover, teng and coworkers indicated that the beneficial effect of msc-derived exosomes on infarcted rat hearts is mainly dependent on their angiogenesis-promoting activity [144] . in their study, the authors proved that exosomes also act by restraining the inflammatory response. in agreement with the results of arslan and colleagues, they also demonstrated that fresh exosomes achieved a better therapeutic effect with respect to frozen exosome preparations. several studies agree that reduced fibrosis and apoptosis of myocardial cells are other important effects of the ev-mediated ischemic cardiac repair [145] [146] [147] [148] . in particular, zhao and coworkers showed that human umbilical cord msc-derived exosomes improved cardiac function and reduced cardiac fibrosis by preventing cardiomyocyte apoptosis and promoting cell proliferation in the border zone of infarcted rats [146] . the effect mediated by exosomes was attributed to the up-regulation of the anti-apoptotic protein b cell lymphoma 2 (bcl-2) in the myocardial cells. other works have proposed that specific functional mirnas contained into evs and shuttled to target injured cells are primarily responsible for the beneficial effects. for example, feng and colleagues found that mir-22 was up-regulated in msc-derived evs, and it possibly reduced cardiac apoptosis and fibrosis in an ami mouse model via inhibition of methyl cytosine-phosphate-guanine (cpg)-binding protein 2 (mecp2) expression [145] . in that study, the authors isolated evs from mscs subjected to ischemic pre-conditioning, which is an effective approach to potentiate survival and regeneration of these cells in an ischemic environment. yu and coworkers identified mir-19a as the molecular mediator able to restore cardiac function and reduce infarct size in a rat model of ami [147] . the cardio-protective role of mir-19a was mediated by down-regulation of target genes, phosphatase and tensin homolog (pten), and bcl-2 interacting mediator of cell death (bim) in cardiomyocytes and subsequent activation of the akt and extracellular signal-regulated kinase (erk) signaling pathways. in their study, exosomes were isolated from mscs over-expressing gata binding protein 4 (gata-4), a transcription factor able to regulate mirna expression in mscs and increase their survival in an ischemic environment [149] . apart from observing reduced cardiac fibrosis and reduced inflammation in infarcted rat hearts 7 days after exosome injection, shao and colleagues identified a panel of mirnas, which were similarly up-or down-regulated in mscs and the derived exosomes [148] . on the other hand, other mirnas, such as mir-21 and mir-15, resulted as being differentially expressed between exosomes and mscs, with this potentially explaining why msc-derived exosomes demonstrated superior beneficial effects when compared with treatment with their parent cells. several studies have demonstrated that autophagy also has an important role in mediating the therapeutic effects of msc-derived exosomes. autophagy is known to be an important mechanism in cardio-protection, and dysregulated autophagy is associated with a variety of cvd [150] . in particular, it has been demonstrated that exosomes reduce apoptosis and the myocardial infarct size, as well as improve cardiac function by inducing cardiomyocyte autophagy both in vitro and in vivo [151, 152] . collectively, the described studies documented reduction in infarct size with improved recovery of cardiac function, reduction of fibrosis and apoptosis, stimulation of angiogenesis, and decreased infiltration of macrophages and other immune cells into the injured heart regions following treatment with msc-derived evs (figure 4) . when comparing the properties of exosomes recovered from different mscs sources, those isolated from adipose tissue samples exhibited the strongest cardio-protective effects [153] . [151] 1 ischemia/reperfusion; 2 left coronary artery; 3 intravenous; 4 glucogen synthase kinase-3; 5 c-jun nterminal kinase; 6 acute myocardial infarction; 7 left anterior descending; 8 platelet-derived growth factor d; 9 cardiac mscs over-expressing notch1 intracellular domain; 10 methyl cytosine-phosphateguanine binding protein 2; 11 b cell lymphoma 2; 12 phosphatase and tensin homolog; 13 phosphatebuffered saline; 14 microtubule-associated protein 1 light chain 3 beta. collectively, the described studies documented reduction in infarct size with improved recovery of cardiac function, reduction of fibrosis and apoptosis, stimulation of angiogenesis, and decreased infiltration of macrophages and other immune cells into the injured heart regions following treatment with msc-derived evs (figure 4) . when comparing the properties of exosomes recovered from different mscs sources, those isolated from adipose tissue samples exhibited the strongest cardioprotective effects [153] . [139, 144, 148] , reduction of cardiac fibrosis, reduction of cardiomyocyte apoptosis [145, 146, 148, 149, 151] , promotion of angiogenesis [140] [141] [142] 144] , and induction of cardiomyocytes autophagy [151] . [151] 1 ischemia/reperfusion; 2 left coronary artery; 3 intravenous; 4 glucogen synthase kinase-3; 5 c-jun n-terminal kinase; 6 acute myocardial infarction; 7 left anterior descending; 8 platelet-derived growth factor d; 9 cardiac mscs over-expressing notch1 intracellular domain; 10 methyl cytosine-phosphate-guanine binding protein 2; 11 b cell lymphoma 2; 12 phosphatase and tensin homolog; 13 phosphate-buffered saline; 14 microtubule-associated protein 1 light chain 3 beta. the recent coronavirus covid-19 global pandemic has driven the need for novel urgent therapies. mscs and their derivatives are being evaluated for the treatment of a number of diseases that currently have limited or no therapeutic options. msc-derived evs (exosomes and mvs) have recently attracted great attention because, similarly to their parent cells, they possess strong anti-inflammatory, immunomodulatory, and pro-angiogenic abilities, just to name a few. however, compared to mscs themselves, evs hold many biological and technological advantages. ev administration is considered safer than msc transplantation, lacking some of their negative side-effects, and they are more stable than mscs themselves, allowing for easier handling and storage. over the last years, a plenty of pre-clinical studies in animal models have demonstrated that the administration of msc-derived evs significantly reduced lung inflammation and pathological impairment subsequent to different types of lung injury, as well as resulted in improved cardiac function after acute myocardial injury. however, several challenges still need to be overcome to make the transition from animal models to humans possible. for example, standardized techniques for isolation, characterization, and quantification, as well as criteria for establishing dose, quality control, and storage conditions of msc-derived evs, are required before these can be advanced to the clinic. to date, it is difficult to compare and analyze studies employing msc-derived evs since there is a large degree of heterogeneity in ev preparations, and because msc-derived evs differ depending on tissues and donors from which the cells are isolated. regarding covid-19, the lack of an established animal model of coronavirus-induced lung injury requires a more prudent and careful use of msc-derived evs. in this context, a significant issue is to establish under what circumstances and with what criteria to administer msc-derived evs. for example, which population among covid-19 patients to target and when to start ev administration. moreover, there remains the challenge to clarify the optimal route of ev administration that, in the case of lung diseases, mostly occurs through it instillation or iv injection, although the possibility of ev inhalation has recently been explored. to date, no studies have investigated the biodistribution and the in vivo metabolic fate of evs following it instillation. on the other hand, systemic iv injection has been shown to deliver evs primarily to the spleen and liver, then to the gastrointestinal tract and lungs, followed by renal and hepatic clearance in mice [138, 154] . apart from the administration route, another major issue concerns the optimal ev therapeutic dose. considering that the average therapeutic dose of mscs for treating lung injuries is 10 × 10 6 cells/kg per body weight, the amount of cells required to generate enough evs to achieve the equivalent effect of mscs is generally 5-10 times higher [155] . this necessitates large scale production of msc-derived evs. although this could be implemented with the use of bioreactors for msc expansion, different bioreactor culture conditions would result in alterations of ev content, which in turn may impact on the therapeutic efficacy. another challenge to consider for the administration of msc-derived evs to covid-19 patients is the need to manufacture a safe and reproducible therapeutic product. since the production of evs requires the use of living cells, these have to be cultured under good manufacturing practice (gmp)-compliant procedures to preserve the quality and safety standards criteria. therefore, ev production must follow the same rigorous scientific and ethical guidelines that apply to mscs, and any therapy based on msc-derived evs needs to be approved by the national regulatory agencies to demonstrate its safety and efficacy. in light of these observations, in our opinion, the use of msc evs could be contemplated to treat critically ill patients with ards requiring mechanical ventilation or icu support, or patients with recognized risk factors, such as pre-existing cvd, or cardiovascular complications, for whom standard therapeutic approaches have not proven resolutive. the authors declare no conflict of interest. a pneumonia outbreak associated with a new coronavirus of probable bat origin characteristics of and important lessons from the coronavirus disease 2019 (covid-19) outbreak in china: summary of a 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