Заразный, симптоматичный и довольно живучий
Канадцы определили, что больные ковидом активно выделяют коронавирус, и он обнаруживается в больших количествах в слюне, каплях при кашле, выделениях из носа, выдыхаемом воздухе, мазках с поверхностей окружения (телефоны, кнопки звонков вызова), рук больных, их принадлежностей, итд. Остается заразным после высыхания, и для того, чтоб заражать- нужны его маленькие количества (у них было от 14 заражающих единиц на 1 мл, или примерно 150 тыс вирусных частиц). Почти все их пациенты были симптоматичные в той или иной степени (97%). Из них 87% имели 1 или более основных респираторных симптомов, характерных для ковида, а у тех, у кого не было респираторных, все равно были другие симптомы, вроде аносмии-авгезии, проблем с жкт, миалгии, температуры, бессилия итп итд. Вариативность и выраженность симптомов ковида была очень большая, но, в целом, у 88% больных было минимум 3 симптома, которые можно отнести к ковидным.
3% были пресимптоматичны на момент теста, симптомы у них появились спустя 24-72 часа.
Вирус культивировался, в основном, у тех, у кого в ПЦР было 25 циклов или меньше. Заразность больных (по заражению культур клеток) варьировала очень сильно (6 порядков). В общем, среди их пациентов примерно треть была заразными. И после того, как прошло 8 дней, в среднем, с первых симптомов, культуры клеток не заражались (если образцы были от людей сн ормальным иммунитетом, а не с иммунодефицитами). Иммунодефицитным хорошо помог ремдесивир, вкупе с отменой или снижением дозы имумносупрессанта (у кого были).
Из мазков из горла, а также образцов, собранных при разговоре, вирус у них не культивировался (но ПЦР был положителен)- а вот из слюны тех же самых, и даже из образца "поцелуев"- заражал. В группе больных была беременная, которая как раз и родила- ее плацента тоже была ПЦР положительна, и вирус из нее культивировался.
Хотя с рук вирус культивировался, образцы, взятые с оксиметра, в основном, не заразили культуру.
Так что у вируса большой потенциал заразности, который реализуется, обычно, до 8 дня после первых симптомов.
После пребывания 2 часа при комнатной температуре на открытом воздухе и сопутствующего этому высыхания образцы не теряли способности к заражению культур клеток. Салфетка для промокания носа, которую случайно вовремя не проверили на заразность, как оказалось, имела на себе заразный вирус и через 9 часов, в тех же условиях. Вирус с мазков клавиатуры, мембраны стетоскопа, кнопки вызова экрана телефона, этс, сохранял способность заражать спустя несколько часов. С поверхности респиратора и медицинских СИЗ- через час. Вирус заражал не только культуры клеток, но и хомячков (2 изолята проверили и так), причем в невысоких "дозах".
Авторы обращают внимание на то, что вирус может передаваться через фомиты, и неплохо схраняется в окружающей среде, и заразность его- очень высокая. И так же, на что, что большинство больных- симптоматичны, нужно внимательнее собирать симптомы. Разброс симптомов очень большой, и заразность больных тоже варьирует очень сильно, и между больными, и по времени, потому на основании 1 мазка, допустим, выводы про заразность-незаразность, сделать неверно.
Few studies have assessed for infectious SARS-CoV-2 in multiple types of clinical and environmental samples. In almost 500 samples from 75 hospitalized and community cases, we detected infectious virus with quantitative burdens varying from 5.0 plaque-forming units/mL (PFU/mL) up to 1.0×106 PFU/mL in clinical specimens and up to 1.3×106 PFU/mL on fomites including facial tissues, nasal prongs, call bells/cell phones, dentures, and sputum deposits with confirmation by plaque morphology, PCR, immunohistochemistry, and sequencing. We demonstrated infectious virus stability in clinical samples, including those dried for prolonged periods of time. Infectious virus correlated with time since symptom onset with no detection after 7-8 days in immunocompetent hosts and with N-gene based Ct values ≤25 significantly predictive of yielding plaques in culture. One PFU was associated with ~105 copies of N gene RNA across a diversity of samples and times from symptom onset. n line with the recommendations on systematic symptom assessment and serial follow up (Meyerowitz et al. 2020), our comprehensive interviewing strategy and serial follow upfound that over 97% of all patients hadsymptoms and/or signs although our dataset was predominantly adults with only 2 pediatric cases. There was no significant difference whether the affected persons were hospital inpatients or from the community. Our findings are congruent with a recent systematic review of higher quality studies (Byambasuren et al. 2020)which used a fixed effect meta-analysis and found asymptomatic cases represented only 17% (95% CI 14% to 20%) of COVID-19 cases. Arecent studyof household transmission where a comprehensive captureof symptoms was done with use of a daily symptom monitoring tool, review of classic and non-classic symptoms plus initially daily RT-PCR testing, found 100% (12/12) of COVID-19patients were symptomatic and is very consistent with our data(Lewis et al. 2021). Our results are not congruent with earlier studies suggesting as many 40-45% of patients with COVID-19 are asymptomatic(Oran and Topol 2020)but much of the early literature was based on rapid and incomplete cross-sectional studies which were likely subject to significant bias. The presence ofrelatively high viral titers in the NP, TS, sputum, nasal secretions,and saliva samples provides valuable insights intothe potential modes of transmission. We found infectiousvirus in cough samplesin 28% of the patients where the “control”NP or TSor saliva waspositive for infectious virus, but not onecontinuous “speech” sample was found to have infectious virus.Although we were unable to recover any virus from the samples collected within the limits of our methods, we cannot rule out the possibility that infected persons might shedinfectious SARS-CoV-2 with shoutingor singing(Katelaris et al. 2021) We didfind infectious virus in high quantities ranging up to 2.2×104 PFU/mLon hands, kisses, nasal prongs, and occasionally cell phones, call bells, gowns, dentures, and a specimen of placenta. We also demonstrated the virus can be transferred from one contaminated hand to a previously cleansed hand. Our observationssuggest thatkisses and human hands could be important for direct contact transmission given the high frequency of habitual human behaviours such as nose, lips and eye touching (up to 15.7 times per hour) and nose-picking (up to one of every three subjects(Nicas and Best 2008; Hendley, Wenzel, and Gwaltney 1973). Thiswould allow inoculation of a relatively high virus burden directly onto sites bearing ACE2 receptors. This mode of transmission has previously been documentedin the seminal studies(Hendley, Wenzel, and Gwaltney 1973)with reported attack rates of 36.4 % in human challenge experiments, when susceptible volunteers were asked to touchtheir nasal or conjunctival mucosa with fingers previously contaminated witha dried drop of Type 39 rhinovirus. SARS-CoV-2 also appears to be environmentally stable under real world circumstancesin clinical specimens. We detected very littledecay in virus titers usingdesiccatedsalivaacquired from apatient who was able to provide multiplereplicate specimens. The virus titer changed little relative to the baseline samples An abundance of previousstudies hasdocumented transfer of infectious respiratory viruses from inanimate objects(fomites) that have been contaminated with nasalor respiratory secretionsinvolving transfers onto the fingertipsand then to the mucous membranes of the nose, mouth,and eyes. These investigations have employed an array of human challenge studies, epidemiological studies, virologic studies and intervention studies(Boone and Gerba 2007).Rhinoviruses are responsible for the majority of human cases of the “common cold”and in one such study attack rates of 50% and 56% was observed when recipients handledcoffee cups and plastic tiles, respectively, that had been previously contaminated with a clinical strain of rhinovirus byinfected donors(Gwaltney and Hendley 1982). Infectious rhinoviruses are also found on high-touch surfaces in home settings, providing further support for the hypothesis that contact transmission is an important mode of rhinovirus transmission (Winther et al. 2011; Gwaltney and Hendley 1978). Human CoVs (229E, OC43, NL63, and HKU1) are the second most frequent cause of the “common cold” after rhinoviruses and may be responsible for up to 30% of common colds. Where the inactivation coefficients have been calculatedand compared directly ithasbeen reportedthatHcoVsOC43 and 229E exhibit a stability comparable to rhinovirus and are muchmore stablethan influenza virus (Boone and Gerba 2007).Respiratory syncytial virus (Hall, Douglas, and Geiman 1980)has been recovered from multiple fomites and infectious virus has been demonstrated to be transferred to hands from touching these contaminated surfaces.Other studies have shown that when preparations of SARS-CoV-1, MERS and otherHCoVaresuspended in a matrix resemblinglung cell debristo mimic natural respiratory secretions,they can persist up to nine dayson inanimate surfaces,further implicating this as atransmission mode(Sizun, Yu, and Talbot 2000; Otter et al. 2016; Kampf et al. 2020). A recent study on the survival of SARS-CoV-2 mixed with mucus from the upper respiratory tractrevealed a survival time analysis of 10.2-12 hours on human skin which corroborates our findings(Hirose et al. 2020).InfectiousMERS was also isolated from fomites in a hospital setting including bedrails, bedsheets, an anteroom table and an IV hanger(Bin et al. 2016). Our findings of extensive environmental contamination and the strong supportive evidence for fomite transmission do not align with other studies and commentaries (Zhou et al. 2020; Goldman 2020; Ben-Shmuel et al. 2020)which reported no infectious SARS-CoV-2in the environment and discountedfomites as a risk for transmission. However, unless the sampling is designed to accommodate the limited period when patients are typically infectious (up to 7-8 days post-symptom onset) andcan capture sufficient material needed to detect an infectious particle (~160,000 PCR templates, Ct≤25), such studies would be highly compromisedto detect any SARS-CoV-2 32Our findingsprovide unique insights into understanding the contagiousness of this virus. Some of the most infectious saliva and cough specimens contained >105PFU/mLof virus, suggesting that a sourcedroplet of 10-100 μL might have contained 106to 107PFU/mLor 1to 10 PFU/μL. This is comparable to the best titersof virus that can be obtainedthrough cellculturein a controlled laboratory setting. Saliva was used as a typical “fomite” seed and the fact that purifiedclones of SARS-CoV-2 derived from this sourcecausedinfection in the Syrian hamster model provides direct evidence of the transmissibilityof the virus to another mammalian host. It thus fulfills both Koch’s postulates and the Gwaltney-Hendley postulates of viral causation(Gwaltney and Hendley 1978; Fredricks and Relman 1996).More critically we could produce disease with doses of only 14-to-30 PFU with these isolates and the minimum infectious dose in Syrian hamstershas been reported to be as low as 1 TCID50with other stocks (Rosenke et al. 2020). It is quite conceivable that the minimal infectious dose in humans is in the range of 1-5PFUwhich is extraordinarily low. Experiments in human challenge studies in the Common Cold Unit from the 1960s showed that as little as100.6-1.5TCID50(~3-20PFU) of a HCoV could causeinfection with attack rates ranging from 17-67% of inoculated volunteers (Bradburne, Bynoe, and Tyrrell 1967). Thus,very fewparticles (Macnaughton et al. 1980)ofa relatively stable virusmay be capable of transmitting SARS-CoV-2 betweenhumans, which would favour multiple routes of transmission andcontribute to its relative contagiousness. Our study has several strengths including the large number of patients acquired in a prospective manner and the ability to carefully establish the timing of symptom onset through a detailed chart and record review of the medical interviewsby experiencedhealthcare providers. We could detect, titerand identify infectious virus with relative ease in manysamples from a diverse group of patients including immunocompromised hosts.
Clinical salivary isolates caused illness in a hamster model with a minimum infectious dose of ≤14 PFU/mL. Our findings of high quantitative burdens of infectious virus, stability even with drying, and a very low minimal infectious dose suggest multiple modes of transmission are exploited by SARS-CoV-2, including direct contact, large respiratory droplet, and fomite transmission and in the context of a high binding avidity to human cellular receptors, offer an explanation of the high contagiousness of this virus.
3% были пресимптоматичны на момент теста, симптомы у них появились спустя 24-72 часа.
Вирус культивировался, в основном, у тех, у кого в ПЦР было 25 циклов или меньше. Заразность больных (по заражению культур клеток) варьировала очень сильно (6 порядков). В общем, среди их пациентов примерно треть была заразными. И после того, как прошло 8 дней, в среднем, с первых симптомов, культуры клеток не заражались (если образцы были от людей сн ормальным иммунитетом, а не с иммунодефицитами). Иммунодефицитным хорошо помог ремдесивир, вкупе с отменой или снижением дозы имумносупрессанта (у кого были).
Из мазков из горла, а также образцов, собранных при разговоре, вирус у них не культивировался (но ПЦР был положителен)- а вот из слюны тех же самых, и даже из образца "поцелуев"- заражал. В группе больных была беременная, которая как раз и родила- ее плацента тоже была ПЦР положительна, и вирус из нее культивировался.
Хотя с рук вирус культивировался, образцы, взятые с оксиметра, в основном, не заразили культуру.
Так что у вируса большой потенциал заразности, который реализуется, обычно, до 8 дня после первых симптомов.
После пребывания 2 часа при комнатной температуре на открытом воздухе и сопутствующего этому высыхания образцы не теряли способности к заражению культур клеток. Салфетка для промокания носа, которую случайно вовремя не проверили на заразность, как оказалось, имела на себе заразный вирус и через 9 часов, в тех же условиях. Вирус с мазков клавиатуры, мембраны стетоскопа, кнопки вызова экрана телефона, этс, сохранял способность заражать спустя несколько часов. С поверхности респиратора и медицинских СИЗ- через час. Вирус заражал не только культуры клеток, но и хомячков (2 изолята проверили и так), причем в невысоких "дозах".
Авторы обращают внимание на то, что вирус может передаваться через фомиты, и неплохо схраняется в окружающей среде, и заразность его- очень высокая. И так же, на что, что большинство больных- симптоматичны, нужно внимательнее собирать симптомы. Разброс симптомов очень большой, и заразность больных тоже варьирует очень сильно, и между больными, и по времени, потому на основании 1 мазка, допустим, выводы про заразность-незаразность, сделать неверно.
Few studies have assessed for infectious SARS-CoV-2 in multiple types of clinical and environmental samples. In almost 500 samples from 75 hospitalized and community cases, we detected infectious virus with quantitative burdens varying from 5.0 plaque-forming units/mL (PFU/mL) up to 1.0×106 PFU/mL in clinical specimens and up to 1.3×106 PFU/mL on fomites including facial tissues, nasal prongs, call bells/cell phones, dentures, and sputum deposits with confirmation by plaque morphology, PCR, immunohistochemistry, and sequencing. We demonstrated infectious virus stability in clinical samples, including those dried for prolonged periods of time. Infectious virus correlated with time since symptom onset with no detection after 7-8 days in immunocompetent hosts and with N-gene based Ct values ≤25 significantly predictive of yielding plaques in culture. One PFU was associated with ~105 copies of N gene RNA across a diversity of samples and times from symptom onset. n line with the recommendations on systematic symptom assessment and serial follow up (Meyerowitz et al. 2020), our comprehensive interviewing strategy and serial follow upfound that over 97% of all patients hadsymptoms and/or signs although our dataset was predominantly adults with only 2 pediatric cases. There was no significant difference whether the affected persons were hospital inpatients or from the community. Our findings are congruent with a recent systematic review of higher quality studies (Byambasuren et al. 2020)which used a fixed effect meta-analysis and found asymptomatic cases represented only 17% (95% CI 14% to 20%) of COVID-19 cases. Arecent studyof household transmission where a comprehensive captureof symptoms was done with use of a daily symptom monitoring tool, review of classic and non-classic symptoms plus initially daily RT-PCR testing, found 100% (12/12) of COVID-19patients were symptomatic and is very consistent with our data(Lewis et al. 2021). Our results are not congruent with earlier studies suggesting as many 40-45% of patients with COVID-19 are asymptomatic(Oran and Topol 2020)but much of the early literature was based on rapid and incomplete cross-sectional studies which were likely subject to significant bias. The presence ofrelatively high viral titers in the NP, TS, sputum, nasal secretions,and saliva samples provides valuable insights intothe potential modes of transmission. We found infectiousvirus in cough samplesin 28% of the patients where the “control”NP or TSor saliva waspositive for infectious virus, but not onecontinuous “speech” sample was found to have infectious virus.Although we were unable to recover any virus from the samples collected within the limits of our methods, we cannot rule out the possibility that infected persons might shedinfectious SARS-CoV-2 with shoutingor singing(Katelaris et al. 2021) We didfind infectious virus in high quantities ranging up to 2.2×104 PFU/mLon hands, kisses, nasal prongs, and occasionally cell phones, call bells, gowns, dentures, and a specimen of placenta. We also demonstrated the virus can be transferred from one contaminated hand to a previously cleansed hand. Our observationssuggest thatkisses and human hands could be important for direct contact transmission given the high frequency of habitual human behaviours such as nose, lips and eye touching (up to 15.7 times per hour) and nose-picking (up to one of every three subjects(Nicas and Best 2008; Hendley, Wenzel, and Gwaltney 1973). Thiswould allow inoculation of a relatively high virus burden directly onto sites bearing ACE2 receptors. This mode of transmission has previously been documentedin the seminal studies(Hendley, Wenzel, and Gwaltney 1973)with reported attack rates of 36.4 % in human challenge experiments, when susceptible volunteers were asked to touchtheir nasal or conjunctival mucosa with fingers previously contaminated witha dried drop of Type 39 rhinovirus. SARS-CoV-2 also appears to be environmentally stable under real world circumstancesin clinical specimens. We detected very littledecay in virus titers usingdesiccatedsalivaacquired from apatient who was able to provide multiplereplicate specimens. The virus titer changed little relative to the baseline samples An abundance of previousstudies hasdocumented transfer of infectious respiratory viruses from inanimate objects(fomites) that have been contaminated with nasalor respiratory secretionsinvolving transfers onto the fingertipsand then to the mucous membranes of the nose, mouth,and eyes. These investigations have employed an array of human challenge studies, epidemiological studies, virologic studies and intervention studies(Boone and Gerba 2007).Rhinoviruses are responsible for the majority of human cases of the “common cold”and in one such study attack rates of 50% and 56% was observed when recipients handledcoffee cups and plastic tiles, respectively, that had been previously contaminated with a clinical strain of rhinovirus byinfected donors(Gwaltney and Hendley 1982). Infectious rhinoviruses are also found on high-touch surfaces in home settings, providing further support for the hypothesis that contact transmission is an important mode of rhinovirus transmission (Winther et al. 2011; Gwaltney and Hendley 1978). Human CoVs (229E, OC43, NL63, and HKU1) are the second most frequent cause of the “common cold” after rhinoviruses and may be responsible for up to 30% of common colds. Where the inactivation coefficients have been calculatedand compared directly ithasbeen reportedthatHcoVsOC43 and 229E exhibit a stability comparable to rhinovirus and are muchmore stablethan influenza virus (Boone and Gerba 2007).Respiratory syncytial virus (Hall, Douglas, and Geiman 1980)has been recovered from multiple fomites and infectious virus has been demonstrated to be transferred to hands from touching these contaminated surfaces.Other studies have shown that when preparations of SARS-CoV-1, MERS and otherHCoVaresuspended in a matrix resemblinglung cell debristo mimic natural respiratory secretions,they can persist up to nine dayson inanimate surfaces,further implicating this as atransmission mode(Sizun, Yu, and Talbot 2000; Otter et al. 2016; Kampf et al. 2020). A recent study on the survival of SARS-CoV-2 mixed with mucus from the upper respiratory tractrevealed a survival time analysis of 10.2-12 hours on human skin which corroborates our findings(Hirose et al. 2020).InfectiousMERS was also isolated from fomites in a hospital setting including bedrails, bedsheets, an anteroom table and an IV hanger(Bin et al. 2016). Our findings of extensive environmental contamination and the strong supportive evidence for fomite transmission do not align with other studies and commentaries (Zhou et al. 2020; Goldman 2020; Ben-Shmuel et al. 2020)which reported no infectious SARS-CoV-2in the environment and discountedfomites as a risk for transmission. However, unless the sampling is designed to accommodate the limited period when patients are typically infectious (up to 7-8 days post-symptom onset) andcan capture sufficient material needed to detect an infectious particle (~160,000 PCR templates, Ct≤25), such studies would be highly compromisedto detect any SARS-CoV-2 32Our findingsprovide unique insights into understanding the contagiousness of this virus. Some of the most infectious saliva and cough specimens contained >105PFU/mLof virus, suggesting that a sourcedroplet of 10-100 μL might have contained 106to 107PFU/mLor 1to 10 PFU/μL. This is comparable to the best titersof virus that can be obtainedthrough cellculturein a controlled laboratory setting. Saliva was used as a typical “fomite” seed and the fact that purifiedclones of SARS-CoV-2 derived from this sourcecausedinfection in the Syrian hamster model provides direct evidence of the transmissibilityof the virus to another mammalian host. It thus fulfills both Koch’s postulates and the Gwaltney-Hendley postulates of viral causation(Gwaltney and Hendley 1978; Fredricks and Relman 1996).More critically we could produce disease with doses of only 14-to-30 PFU with these isolates and the minimum infectious dose in Syrian hamstershas been reported to be as low as 1 TCID50with other stocks (Rosenke et al. 2020). It is quite conceivable that the minimal infectious dose in humans is in the range of 1-5PFUwhich is extraordinarily low. Experiments in human challenge studies in the Common Cold Unit from the 1960s showed that as little as100.6-1.5TCID50(~3-20PFU) of a HCoV could causeinfection with attack rates ranging from 17-67% of inoculated volunteers (Bradburne, Bynoe, and Tyrrell 1967). Thus,very fewparticles (Macnaughton et al. 1980)ofa relatively stable virusmay be capable of transmitting SARS-CoV-2 betweenhumans, which would favour multiple routes of transmission andcontribute to its relative contagiousness. Our study has several strengths including the large number of patients acquired in a prospective manner and the ability to carefully establish the timing of symptom onset through a detailed chart and record review of the medical interviewsby experiencedhealthcare providers. We could detect, titerand identify infectious virus with relative ease in manysamples from a diverse group of patients including immunocompromised hosts.
Clinical salivary isolates caused illness in a hamster model with a minimum infectious dose of ≤14 PFU/mL. Our findings of high quantitative burdens of infectious virus, stability even with drying, and a very low minimal infectious dose suggest multiple modes of transmission are exploited by SARS-CoV-2, including direct contact, large respiratory droplet, and fomite transmission and in the context of a high binding avidity to human cellular receptors, offer an explanation of the high contagiousness of this virus.